Cancer Microenvironment (2015) 8 (Suppl 1):S1 S141 DOI 10

Cancer Microenvironment (2015) 8 (Suppl 1):S1–S141DOI 10.1007/s12307-015-0175-9

http://crossmark.crossref.org/dialog/?doi=10.1007/s12307-015-0175-9&domain=pdf

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The International Cancer Microenvironment Society

Officers

& PresidentIsaac P. Witz, Tel Aviv, Israel

& SecretarySmadar Fisher, Tel Aviv, Israel

& Treasurer—Western HemisphereMenashe Bar-Eli, Houston, TX, USA

& Treasurer—Eastern HemisphereEitan Yefenof, Jerusalem, Israel

& Ron N. Apte, Beer Sheva, Israel& Yona Keisari, Tel Aviv, Israel& Benjamin Sredni, Ramat Gan, Israel& Fernando Vidal Vanclocha, Leioa, Vizcaya, Spain

Charter Members

& Ron N. Apte, Beer Sheva, Israel& Frances R. Balkwill, London, United Kingdom& Jan Bubenik, Prague, Czech Republic& Isaiah J. Fidler, Houston, TX, USA& Wolf H. Fridman. Paris, France& Robert C. Gallo, Baltimore, MD, USA& Ian R. Hart, London, United Kingdom& Ronald B. Herberman, Pittsburgh, PA, USA& Claude Jasmin, Villejuif, France& Hynda K. Kleinman, Bethesda, MD, USA& Daniela Männel, Regensburg, Germany& Alberto Mantovani, Milan, Italy& Avraham Raz, Detroit, MI, USA& Volker Schirrmacher, Heidelberg, Germany& Benjamin Sredni, Ramat Gan, Israel& Eiichi Tahara, Hiroshima, Japan& Fernando Vidal Vanaclocha, Leioa, Vizcaya, Spain& Israel Vlodavsky, Jerusalem, Israel& Theresa L. Whiteside, Pittsburgh, PA, USA& Isaac P. Witz, Tel Aviv, Israel& Eitan Yefenof, Jerusalem, Israel& Jan Zeromski, Poznan, Poland& Dov Zipori, Rehovot, Israel

Organizing Committee

& Isaac P. Witz, Tel Aviv University, Tel Aviv (Chairman)& RonN.Apte,Ben-GurionUniversity of theNegev, Beer Sheva& Yona Keisari, Tel Aviv University Tel Aviv& Varda Rotter, Weizmann institute, Rehovot& Yuval Shaked, Technion, Haifa& Benjamin Sredni, Bar Ilan University, Ramat Gan& Eitan Yefenof, Hebrew University, Jerusalem& Smadar Fisher, Conference Coordinator, Tel Aviv& Conference Secretariat- DIESENHAUS UNITOURS,

Tel Aviv

International Advisory Committee

& Heike Allgayer, DKFZ, Heidelberg, Germany& Menashe Bar-Eli, University of Texas MD Anderson

Cancer Center, Houston, TX, USA& Joan Brugge, Harvard Medical School, Boston, MA,

USA& Peter Carmeliet, Vesalius Research Center, Leuven,

Belgium& Hans Clevers, Hubrecht Institute, Utrecht, Netherlands& Carlo M Croce, Ohio State University, Columbus, OH,

USA& Janine Erler, University of Copenhagen, Copenhagen,

Denmark& Hervé Fridman, University Paris Descartes, Paris,

France& Peter Krammer, DKFZ, Heidelberg, Germany& Luyuan Li, Nankai University, Tianjin, China& Alberto Mantovani, Humanitas Clinical and Research

Center, Milan, Italy& SureshMohla, National Cancer Institute, NIH, Rockville,

MD, USA& Jacques Pouyssegur, Centre Antoine Lacassagne, Nice,

France& Arne Östman, Karolinska Institutet, Stockholm, Sweden& Avraham Raz, Wayne State University, Detroit, MI,

USA& Blanka Rihova, Academy of Sciences, Prague,

Czech Republic& Fernando Vidal Vanaclocha, San Pablo University,

Madrid, Spain& Robert Weinberg, Massachusetts Institute of Technology,

Cambridge, MA, USA

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Dear Friends and Colleagues,

It gives me great pleasure to welcome you to the 7th International Conference on Tumor Microenvironment: Progression,Therapy & Prevention.

This year we celebrate the twentieth anniversary of the International Cancer Microenvironment Society. We have come a longand fruitful way since the first Tumor Microenvironment Conference that took place on the shore of the Sea of Galilee in 1995.PubMed data indicate that the number of “tumor microenvironment”-related articles has grown from 83 in 1994 to 3080 articlesin 2014.

The identification of cells and molecules in the tumor microenvironment and the nature of their interactions with tumor cellsenabled the development of drugs that promote and enhance malignancy-suppressing interactions and drugs that antagonizemalignancy-promoting ones.These advances and many others will be reported within the frame of the exciting scientific program at hand.

I do hope that in addition to the excellent science and networking offered by the people who actively participate in the scientificprogram, you will enjoy the unique social activities which are a landmark of our conferences and the dynamic atmosphere of“The City that Never Sleeps”. We pride ourselves with, 24/7 cultural events, a beautiful beach and a lively nightlife. Tel Aviv’sWhite City, comprising the world’s largest concentration of Bauhaus buildings, was designated a UNESCOWorld Heritage Sitein 2003.

I wish all of us an exciting, stimulating and enjoyable conference.

Isaac P. WitzPresident ICMS

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Acknowledgments

The Members of the Organizing Committee of the 7th International Conference on Tumor Microenvironment: Progression,Therapy & Prevention express their gratitude and acknowledge the following institutions and companies for their generoussupport

Sponsors

The assistance ofMs. Linda Brand andMs. Adena Turk of Tel Aviv University, Ms. Yael Kfir of the TAUGraphic Design Studio,Ms. Malka Ben-Haim, and Mr. Oren Wassersprung is highly appreciated.

The Israel Cancer Association

DKFZ (German Cancer Research Center)

Tel Aviv University

C.A.I.R. Research Institute Bar Ilan University The Dr. ToviComet-Wallerstein Endowment for Cancer Research

Ben-Gurion University of the Negev

“Cancer Microenvironment” the official journal of theInternational Cancer Microenvironment Society

The Hebrew University of Jerusalem

Teva Pharmaceutical Industries Ltd.

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Exhibitors

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Sunday, October 11:Dan Tel Aviv Hotel, 99 Hayarkon Street, Tel Aviv

Monday–Thursday, October 12–15:Tel Aviv University, Ramat Aviv, Tel AvivSmolarz Auditorium (Gate 4) and Dan David Building, Halls 001, 002 & 003

SUNDAY, OCTOBER 11, 2015

15:30–19:00 RegistrationDan Tel Aviv Hotel

18:30–19:45 OPENING SESSIONDan Tel Aviv HotelGreetings:Isaac P. Witz – President, ICMSMahrata Baruch Ron – Deputy Mayor, Tel Aviv Municipality

19:00 Opening Lecture:Hans Clevers, Utrecht, NetherlandsLgr5 Stem Cells in Self-Renewal and Cancer

19:45 WELCOME RECEPTIONDan Tel Aviv Hotel

MONDAY, OCTOBER 12, 2015

08:30–10:35 PLENARY SESSION 1: Regulatory Pathways in the Tumor Microenvironment (I)Smolarz Auditorium

Chair: Yona Keisari, Tel Aviv, Israel

08:30 Carlo M. Croce, Columbus, OH, USARole of Non-Coding RNAs in Cancer

08:55 Jacques Pouyssegur, Nice & Monaco, FranceHypoxic Microenvironment and Tumor Metabolism

09:20 Luyuan Li, Tianjin, ChinaModulation of Vascular and Lymphatic Endothelial Functions by TNFSF15

09:45 Arne Östman, Stockholm, SwedenMarker-Defined Perivascular Cells Predict Prognosis and Response to Treatment

10:10 Raghu Kalluri, Houston, TX, USABiology and Function of Tumor Stroma and Exosomes in Cancer

LOCATION OF SESSIONS

Sunday, October 11:Dan Tel Aviv Hotel, 99 Hayarkon Street, Tel Aviv

Monday–Thursday, October 12–15:Smolarz Auditorium (Gate 4)Dan David Building - Halls 001, 002 & 003Josef & Raya Jaglom Auditorium, Senate Building



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MONDAY, OCTOBER 12, 2015 (cont’d)

10:35–11:00 Coffee Break & Poster Viewing

11:00–13:05 PLENARY SESSION 2: Targeting the Tumor Microenvironment—Novel Approaches (I)Smolarz Auditorium

Chair: Eitan Yefenof, Jerusalem, Israel

11:00 Drew Pardoll, Baltimore, MD, USAEstablishment of Immunotherapy as the Fourth Pillar of Cancer Treatment

11:25 Blanka Rihova, Praha, Czech RepublicImmune System as a Vital Partner in Cancer Treatment

11:50 Peter H. Krammer, Heidelberg, GermanyAnnexin-Mediated Regulation of the Peripheral Immune Response

12:15 Isaiah Fidler, Houston, TX, USATargeting Astrocyte Endothelin Axis for Therapy of Experimental Breast and Lung Cancer Brain Metastasis

12:40 Michael Shurin, Pittsburgh, PA, USAMyeloid Regulatory Cells and Nano-Delivery of Chemotherapeutic Agents

13:05–14:30 Business Meeting followed by Light LunchSmolarz Auditorium

14:30–16:10 PLENARY SESSION 3: Inflammatory & Immune Reactions in the Tumor Microenvironment (I)Smolarz Auditorium

Chair: Benjamin Sredni, Ramat-Gan, Israel

14:30 Adit Ben-Baruch, Tel Aviv, IsraelMicroenvironmental Factors Shape Tumor Heterogeneity and Routes of Metastatic Dissemination in Breast Cancer

14:55 Elizabeth Grimm, Houston, TX, USAMolecular Mechanisms of Inflammation Affecting Human Melanoma

15:20 Viktor Umansky, Heidelberg, GermanyImmunsuppressive Pathways inMelanoma

15:45 Neta Erez, Tel Aviv, IsraelBreaking Bad – Cancer Associated Fibroblasts are Reprogrammed from Growth Inhibitory to Pro-Inflammatory and Tumor-Promoting in Breast Cancer


16:40–18:20 PLENARY SESSION 4: Inflammatory & Immune Reactions in the Tumor Microenvironment (II)Smolarz Auditorium

Chair: Ronnie Apte, Beer-Sheva, Israel

16:40 Wolf Herman Fridman, Paris, FranceCancer Phenotypes and their Immune Contextures

17:05 Catherine Sautès-Fridman, Paris, FranceCharacterization and Prognostic Impact of the Intra-Tumoral Immune Response in Human Cancers


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17:30 Angel Porgador, Beer-Sheva, IsraelExtracting Tumor Tissue Immune Status from Expression Profiles: Correlating Prognosis with Tumor-Associated Immunome

17:55 Michal Baniyash, Jerusalem, IsraelInsights into Chronic Inflammation-Induced Immunosuppression in Cancer: from Mouse to Human

20:00 CONFERENCE DINNER—Lauren’s Gallery in Old JaffaDeparture:18:30 From Smolarz Auditorium to Conference hotels – Dan, Renaissance, Art Plus, Prima City,

Metropolitan & Deborah hotels.19:45 From above hotels to the Lauren's Gallery. Transportation will be provided after the event.

TUESDAY, OCTOBER 13, 2015

08:30–10:45 PARALLEL SESSIONS:Halls 001, 002 & 003—Dan David Building, Tel Aviv University

08:30–10:45 SYMPOSIUM 1: Regulatory Pathways in the Tumor Microenvironment (I)Hall 001—Dan David Building

Chair: Michael Grusch, Vienna, Austria

08:30 Michael Grusch, Vienna, AustriaOptically Activated Receptor Tyrosine Kinases (Opto-RTKs) for Investigating Tumor-Stroma Interactions

08:50 Ursula Kees, Subiaco Perth & Crawley Perth, Western Australia, AustraliaHigh Expression of Connective Tissue Growth Factor (CTGF/CCN2) Modifies the Bone MarrowMicroenvironment and Accelerates Dissemination of Leukaemia

09:10 Karlheinz Friedrich, Jena, GermanyMalignancy of Bladder Cancer Cells is Enhanced by Tumor Associated Fibroblasts through aCytokine-Chemokine-Loop

09:30 Moshe Elkabets, New York, NY, USA & Beer-Sheva, IsraelKRAS Mutation in Colorectal Cancer is Associated with a Stromal-Derived Gene Signature

09:45 Eli Breuer, Jerusalem, IsraelCarbamoylphosphonates Inhibit Extracellular Zinc Enzymes in the Tumor Microenvironment

10:00 Sabina Pucci, Rome, ItalyLOX-1: a Novel Metabolic Target in Human Breast Cancer

10:15 Yoav Binenbaum, Haifa, IsraelExosomes Secreted byMacrophages Facilitate Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma

10:30 Rajkumar Savai, Bad Nauheim & Giessen, GermanyMacrophage and Cancer Cell Crosstalk via CCR2 and CX3CR1 is a Fundamental Mechanism Driving LungCancer



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TUESDAY, OCTOBER 13, 2015 (cont’d)

08:30–10:45 SYMPOSIUM 2: The Inflammatory and Immune Tumor MicroenvironmentHall 002—Dan David Building

Chair: Zvi Fridlender, Jerusalem, Israel

08:30 Zvi Fridlender, Jerusalem, IsraelTumor Related Neutrophils—A New Challenge in Cancer Immunology and Immunotherapy

08:50 Zvi Granot, Jerusalem, IsraelNeutrophil Subpopulation Ratio Determines Overall Pro- or Antitumor Contribution

09:10 Luca Vannucci, Prague & Libechov, Czech RepublicChanges in the Colon Mucosal Scaffold under Activation of the Local Immunity: Lessons from Germ-Freeversus Conventional Animal Pro-Inflammatory Induction

09:30 Anton Keskinov, Pittsburgh, PA, USAPeripheral Neuroglia Forms the Inflammatory-Like Tumor Microenvironment

09:45 Tali Feferman, Rehovot, IsraelOxygen Shapes CTL Function: Insights from Live Intratumoral Imaging

10:00 Nongnit Laytragoon Lewin, Jönköping, SwedenSingle Nucleotide Polymorphisms, Proteins and Cytokines Related to Inflammatory Microenvironment:Biomarkers for Cancer Risk and Clinical Outcome of Head and Neck Squamous Cell Carcinoma Patients

10:15 Nirit Mor-Vaknin, Ann Arbor, MI, USAThe Pro-Inflammatory Role of Vimentin in the Colon

10:30 Gilli Galore Haskel, Ramat-Gan & Tel Aviv, IsraelA Novel Immune Resistance Mechanism of Melanoma Cells Controlled by the ADAR1 Enzyme

08:30–10:45 SYMPOSIUM 3: Targeting the Tumor Microenvironment—Preclinical and Clinical Trials

Hall 003—Dan David Building

Chair: Reuven Reich, Jerusalem, Israel

08:30 Rachel Bar-Shavit, Jerusalem, IsraelG-Protein Coupled Receptors; GPCRs in Epithelial Cancers and Novel TargetedPH-Domain Dinding Motifs

08:50 Albrecht Reichle, Regensburg, GermanyAnakoinosis: Communicative Reprogramming of Tumor Systems—for Rescuing ChemorefractoryNeoplasia

09:10 Israel Vlodavsky, Haifa, IsraelRoneparstat, a Novel Heparanase Inhibitor for Multiple Myeloma Therapy

09:30 Milada Sirova, Prague, Czech RepublicPolymer Drug Delivery Dystems for Effective Therapy and Modulation of Tumor Microenvironment


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09:45 Gabi van Pelt, Leiden, NetherlandsStromal Lymph Node Involvement Adds to the Prognosis of Stage III Colon Cancer Patients

10:00 Roni Oren, Rehovot, IsraelUtilizing Fibroblasts Recruitment for Detection and Targeted Therapy of Ovarian Tumors

10:15 Erez Persi, Bethesda & College Park, MD, USAHarnessing Cancer’s pH-Vulnerability to Identify an Optimal Two-Step Therapeutic Strategy

10:30 Jai Prakash, Enschede, NetherlandsNovel MicroRNA Targets and Targeting Technology to Modulate Pancreatic Stellate Cells inPancreatic Tumor

10:45–11:15 Coffee Break

11:15–13:30 PARALLEL SESSIONS:Halls 001, 002 & 003—Dan David Building, Tel Aviv University

11:15–13:30 SYMPOSIUM 4: Regulatory Pathways in the Tumor Microenvironment (II)Hall 001—Dan David Building

Chair: Reuven Stein, Tel Aviv, Israel

11:15 Maty Tzukerman, Haifa, IsraelTumor Specific Recruitment and Reprograming of Mesenchymal Stem Cells in Tumorigenesis

11:35 Ben-Zion Katz, Tel Aviv, IsraelNovel Signaling Pathway in Chronic Lymphocytic Leukemia—Pathophysiology and New Target for Therapy

11:55 Reuven Stein, Tel Aviv, IsraelTargeting CD38 Inhibits Glioma and Melanoma Progression in Vivo

12:15 Imad Shams, Haifa, IsraelFrom Hypoxia Resistance to Cancer Tolerance: the Adaptations Evolved in the Blind Subterranean Mole Rats

12:30 Ruth Scherz-Shouval, Cambridge, MA, USACell Autonomous and Non-Cell-Autonomous Activities of Heat Shock Factor 1 in Cancer

12:45 Tatiana Pavlova, Stockholm, SwedenImmortalized Fibroblasts with Decreased RhoGTPase Activity Suppress Tumor Cell Growth in Vitro and in Vivo

13:00 Adam Weinstock, Tel Aviv, IsraelA Joint Analysis of Transcriptomic and Metabolomic Data Uncovers Increased Metabolic TranscriptionalRegulation in Breast Cancer

13:15 Carolina Ilkow, Ottawa, Ontario, CanadaReciprocal Cellular Cross-Talk within the Tumor Microenvironment Promotes Oncolytic Virus Activity


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11:15–13:30 SYMPOSIUM 5: The Tumor Microenvironment and Progression Towards MalignancyHall 002—Dan David Building

Chair: Karin Joehrer, Innsbruck, Austria

11:15 Irwin Gelman, Buffalo, NY, USAMetastasis Promotion: Control of the Src/STAT-Regulated Senescent Stromal Secretome by SSeCKS/AKAP12 Scaffolding Protein

11:35 Karin Joehrer, Innsbruck, AustriaMyeloma-Derived CCL27 Induces Stroma-Dependent Resistance Against Bortezomib via Regulationof IL-10

11:55 Elena Voronov, Beer-Sheva, IsraelThe Effects of IL-1 Molecules on the Different Stages of Colon Cancer Development

12:15 Assaf Menachem, Tel Aviv, IsraelIntercellular Transfer of Small RNAs fromAstrocytes to Lung Tumor Cells Induces Resistance to Chemotherapy

12:30 Saran Kumar, Jerusalem, IsraelStem Cell-Like Properties of Glioma Tumor-Initiating Cells: Microenvironmental Inputs

12:45 Amit Benbenishty, Tel Aviv, IsraelBrain Metastasis: the Impact of Surgical Stress, Immune Stimulation and NK Cells

13:00 Francesca Caccuri, Brescia, ItalyHHV-6 U94 Inhibits Motility, Migration and Invasiveness of Human Breast Cancer Cells by Modulation ofSrc Signaling Pathway

13:15 Bettina Couderc, Toulouse, FranceChemoresistance Acquisition by Ovarian Adenocarcinoma Cells due to the Crosstalks between MSCs andMacrophages

11:15–13:30 SYMPOSIUM 6: Targeting the Tumor Microenvironment & the Immune SystemHall 003—Dan David Building

Chair: Robert Hoffman, San Diego, CA, USA

11:15 Michael Goldberg, Boston, MA, USASurface Modification of PLGA Nanoparticles Improves Drug Loading, Sustains Drug Release and EnhancesEnzyme Eficiency in Vivo

11:35 Robert Hoffman, San Diego, CA, USAHighly-Effective Fluorescence-Guided Surgery Enabled by Color-Coding Cancer Cells and the Tumor Mi-croenvironment with Genetic Reporters in a Patient-Derived Orthotopic Xenograft (PDOX) Model of Pan-creatic Cancer

11:55 Andrei Bakin, Buffalo, NY, USADefining a Novel Pathway Promoting Tumor Progression and Metastasis


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12:15 Valery Khramtsov, Columbus, OH, USAIn Vivo Profiling the Chemical Tumor Microenvironment for Diagnosis and Therapy

12:30 David J. Waxman, Boston, MA, USAMetronomic Chemo-Immunotherapy: Schedule-Dependent Activation of Immune-Induced Tumor Regression

12:45 Yaron Carmi, Stanford, CA, USATumor-Binding IgG Combined with Dendritic Cell Stimuli Induces Anti-Tumor T Cell Immunity

13:00 Orit Itzhaki, Ramat-Gan, IsraelAdoptive Cell Therapywith Tumor Infiltrating Lymphocytes: Clinical Results and Correlation to Clinical Success

13:15 Martina Seiffert, Heidelberg, GermanyTargeting Dysfunctional Myeloid Cells Delays Disease Development and Improves Immune Function in aMouse Model of Chronic Lymphocytic Leukemia

13:45 FREE / OPTIONAL SIGHTSEEING TOURS13:45—Departure for the Jerusalem tour from the Dan David Building, Tel AvivUniversity, and to Conference hotels for those who do not take part in the tour—Dan, Renaissance, Art Plus,Prima City, Metropolitan and Deborah hotels.

08:30–10:50 PARALLEL SESSIONS:Smolarz Auditorium and Jaglom Auditorium, Senate Building, Tel Aviv University

08:30–10:50 SYMPOSIUM 7: Regulatory Pathways in the Tumor Microenvironment (III)Smolarz Auditorium

Chair: Jonathan Sleeman, Mannheim & Karlsruhe, Germany

08:30 Jonathan Sleeman, Mannheim & Karlsruhe, GermanyMatrix-Assisted Autocrine Signaling Regulates Stemness Properties in Melanoma Cells through Induction ofId1 and Id3 Expression

08:50 Georg Bauer, Freiburg, GermanyExtracellular Superoxide Anion Generation and Expression of Membrane-associated Catalase During TumorProgression: Dynamic Interplay and Consequences for Tumor Cell Survival

09:10 Reuven Reich, Jerusalem, IsraelExosomes as Modulators of Tumor Microenvironment

09:30 Edna Cukierman, Philadelphia, PA, USAClinical Relevance, Pathological Details andMechanistic Functions of Desmoplastic 3D-Adhesion Structures

09:50 Charlotta Dabrosin, Linköping, SwedenFlaxseed and TamoxifenAlters the Expression of ExtracellularmicroRNA inNormalHumanBreast Tissue in Vivo

10:05 Yan Stein, Rehovot, IsraelMutant p53 Modulates the Signal of Stromal-Secreted Hepatocyte Growth Factor (HGF) to Endow CancerCells with Drug Resistance


WEDNESDAY, OCTOBER 14, 2015

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WEDNESDAY, OCTOBER 14, 2015 (cont’d)

10:20 Michelino Di Rosa, Catania, ItalyOPN/MMP-9 Pathway Activation in B-Cell Non-Hodgkin Lymphoma

10:35 Federica Marchesi, Rozzano, ItalyDual Prognostic Significance of Tumor Associated Macrophages in Human Pancreatic AdenocarcinomaTreated or not with Chemotherapy

08:30–10:45 SYMPOSIUM 8: Metastasis and the Metastatic Microenvironment (I)Jaglom Auditorium, Senate Building

Chair: Dario Marchetti, Houston, TX, USA

08:30 Shelly Peyton, Amherst, MA, USASynthetic Environments to Understand Cancer Metastasis and Drug Resistance

08:50 Dario Marchetti, Houston, TX, USADissecting CTC Phenotypes: Insights into Mechanisms of Breast Cancer Dormancy

09:10 Lubor Borsig, Zurich, SwitzerlandSelectin-Mediated Recruitment of Leukocytes Contributes to Metastatic Niche Formation and ChemokinesOrchestrate this Process

09:30 Yael Nemlich, Ramat-Gan, IsraelADAR1-Mediated Regulaton of Melanoma Invasion

09:45 Valéry L. Payen, Brussels, BelgiumA Mitochondrial Switch Promotes Tumor Metastasis

10:00 Chen Rachman, Haifa, IsraelLysyl Oxidase Overexpressed in Mice that Undergo Surgery may Promote Metastasis

10:15 Hila Schwartz, Tel Aviv, IsraelAstrogliosis is Instigated in a Novel Mouse Model of Spontaneous Melanoma Brain Metastasis

10:30 Asaf Spiegel, Cambridge, MA, USANeutrophil-Mediated Survival and Extravasation of Disseminated Carcinoma Cells


11:15–13:45 PARALLEL SESSIONS:Smolarz Auditorium and Jaglom Auditorium, Senate Building, Tel Aviv University

11:15–13:45 SYMPOSIUM 9: Targeting the Tumor Microenvironment—Angiogenesis and Blood VesselsSmolarz Auditorium

Chair: Adriana Haimovitz-Friedman,NewYork, NY, USA

11:15 Martin Pruschy, Zurich, SwitzerlandCombined Treatment Strategies for Microtubule Interfering Agent-Resistant Tumors


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11:35 Adriana Haimovitz-Friedman, New York, NY, USAEnhanced Radiosensitization Using Anti-Angiogenic Therapy in Sarcoma Tumors

11:55 Eugenie S. Kleinerman, Houston, TX, USAHypoxia Stimulates Vascular Mimicry by Inducing Ewing’s Sarcoma StemCells to Differentiate into VascularPericytes that Express EWS-FLI-1

12:15 Michal Rahat, Haifa, IsraelAn Epitope-Specific Novel Anti-EMMPRIN Polyclonal Antibody Inhibits Tumor Progression

12:30 Tomer Itkin, Rehovot, IsraelDistinct Bone Marrow Blood Vessels Differentially Regulate Normal and Malignant Hematopoiesis

12:45 Lukas Hawinkels, Leiden, NetherlandsTargeting Endoglin Inhibits TumorAngiogenesis and Strongly ReducesMetastatic Spread of Breast Cancer Cells

13:00 Iker Badiola, Leioa, SpainExperimental Hepatic Metastasis Treatment with Antiangiogenic microRNA-Encapsulated Nanoparticles

13:15 Gerold Untergasser, Innsbruck, AustriaThe Aplidin Analogs PM01215 and PM02781 Inhibit Angiogenesis in Vitro and in Vivo

13:30 Miriam Gross-Cohen, Haifa, IsraelHeparanase 2 Attenuates Head and Neck Tumor Vascularity and Growth

11:15–13:40 SYMPOSIUM 10: Metastasis and the Metastatic Microenvironment (II)Jaglom Auditorium, Senate Building

Chair: Marcelo Ehrlich, Tel Aviv, Israel

11:15 Marcelo Ehrlich, Tel Aviv, IsraelATF-1, Retinoic Acid Induced 14 (RAI14) and C-Jun form a Novel Regulatory Axis of Invasive-ness and Proliferation

11:35 Zhi-Xiong Jim Xiao, ChengDu, Chinap53 Family, Oncogenic Signaling and Cancer Metastasis

11:55 Sivan Izraely-Bino, Tel Aviv, IsraelThe Microenvironment of Melanoma Brain Metastasis

12:10 Daphne Weihs, Haifa, IsraelMaking Holes: Identifying How Metastatic Cancer Cells Apply Force to Invade their Microenvironment

12:25 Dalit Barkan, Haifa, IsraelPreventing the Recurrence of Breast Cancer at the Metastatic Niche Using Resolution-Phase Macrophages

12:40 Lara Perryman, Copenhagen, DenmarkLysyl Oxidase as a Potential Target for Infiltrative Growth in Glioblastoma


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12:55 Cheryl Jorcyk, Boise, Idaho, USANew Insights on How an Inflammatory Tumor Microenvironment Promotes Invasion and Early Stage BreastCancer Metastasis

13:10 Itay Barnea, Tel Aviv, IsraelLabel-Free Morphological Discrimination between Different Cancer Cell Lines and Leukocytes by DigitalHolographic Microscopy: a Model for Enumeration of Circulating Tumors Cells in Blood

13:25 Shelly Tartakover Matalon, Kfar Saba, IsraelBreast Cancer Cell Detachment from Placenta Conditioned ECM Activates Integrin-TGFβ/JNK Axis andEnhances their Metastatic Potential

13:45–15:10 Lunch Break

15:15–16:55 PLENARY SESSION 5: Targeting the Tumor Microenvironment—Novel Approaches (II)Smolarz Auditorium

Chair: Varda Rotter, Rehovot, Israel

15:15 Peter Carmeliet, Leuven, BelgiumAngiognesiss Revisited: Role and (Therapeutic) Implications of Endothelial Metabolism

15:40 Heike Allgayer, Mannheim/Heidelberg, GermanyATranslational Approach to the Metastatically Relevant MicroRNA-Landscape

16:05 Bernd Groner, Frankfurt, GermanyReprogramming Tumor Cells and their Paracrine Interactions: A New Approach to Cancer Therapy

16:30 Ronit Satchi-Fainaro, Tel Aviv, IsraelIdentifying Molecular Signatures for Tumor Dormancy as a Basis for the Rational Design ofPrecision Nanomedicines


17:15–18:30 PLENARY SESSION 6: Metastasis & the Metastatic Microenvironment (I)Smolarz Auditorium

Chair: Isaac P. Witz, Tel Aviv, Israel

17:15 Patrizia Paterlini-Brechot, Paris, FranceCirculating Tumor Cells and Tumor Microenvironment

17:40 Avraham Raz, Detroit, MI, USAOn the role of Galectin 3 in the Tumor Microenvironment

18:05 Menashe Bar-Eli, Houston, TX, USARNA Editing and Melanoma Metastasis

18:30–20:30 FAREWELL EVENT: Tel Aviv the White City - Walking Tour & Farewell ToastDeparture: 18:30 From the Smolarz Auditorium, Tel Aviv University


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THURSDAY, OCTOBER 15, 2015

08:30–10:10 PLENARY SESSION 7: Metastasis & the Metastatic Microenvironment (II)Smolarz Auditorium

Chair: Yuval Shaked, Haifa, Israel

08:30 Robert Kerbel, Toronto, Ontario, CanadaVessel Co-Option and Impact of the Metastatic Tumor Microenvironment on Antiangiogenic Drug Efficacy

08:55 Janine T. Erler, Copenhagen, DenmarkECM Remodelling during Cancer Progression

09:20 Fernando Vidal-Vanaclocha, Madrid, SpainLiver Metastasis-Related Genes at Primary and Metastatic Sites from Patients with Colon Cancer

09:45 Dave Hoon, Santa Monica, CA, USAMicroenvironmental Influences on Splicesome Factor Expression Contribute to Melanoma Brain MetastasisProgression


10:10–10:40 Coffee Break

10:40-13:15 CLOSING PLENARY SESSIONSmolarz Auditorium

Chair: Isaac P. Witz, Tel Aviv, Israel

10:40 Presentation of Late-Breaking Abstracts:

10:40 Valerie LeBleu, Houston, TX, USA and Boston, MA, USAThe Role of Perivascular Heterogeneity in Metastasis

10:55 Meenakshi Upreti, Lexington, KY, USATumor Models Incorporating Aspects of Tumor Microenvironment for Novel Cancer Nanotherapies

11:10 Sofia Avnet, Bologna, ItalyTumor-Elicited Secretion of IL6 and of IL8 from Reactive Mesenchymal Stroma is Enhanced Under theAcidic Microenvironment of Glycolytic Cancer Cells and Promotes Osteosarcoma Stemness

11:25 Poster Session:Presentation of Best Posters and Awarding of Poster Prizes

12:45 Conference Summary:The 7th International Conference on Tumor Microenvironment - Take Home Messages

13:15 Adjourn

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LIST OF POSTERS

Posters should be displayed throughout the meeting, i.e., from Monday morning, October 12th to the end of the sessions onWednesday, October 14th.Poster presenters should place their posters on their respective board number listed below.

1. Tumor Microenvironment Interactions—Novel Aspects and Technologies

P1 Martha Alvarez, Haifa, IsraelMigration and Mechanical Invasiveness in Breast Cancer Cells, are they Related?

P2 Racheli Blau, Tel Aviv, IsraelSynthesis and Characterization of Polymeric Nanotheranostics for Real-Time Non-Invasive Optical Imaging

P3 Michael David, West Hollywood, CA, USASerum BCMA: A Novel Prognostic Indicator in Patients with Multiple Myeloma

P4 Alessandro Marturano, New York, NY, USABiomechanical Stimuli Modulate the Expression of Osteolytic Lesion-Related Proteins in an Engineered HumanTumor

P5 Sonbula Massalha, Haifa, IsraelAdherence Rates, Morphology and Force Depend on Microenvironment Stiffness and Metastatic Potential inBreast Cancer Cells

P6 Paula Ofek, Tel Aviv, IsraelMulti-Modal Nanomedicine for Glioblastoma

P7 Fares Qeadan, Albuquerque, NM, USAStatistical Visualization for the Association between Soil Transmitted Helminth Infection and Changes inImmune Microenvironments at the Cervix

P8 Orit Uziel, Petah-Tikva, IsraelCirculating hTERT (human telomerase) mRNA in Exosomes Derived from T-Cell Leukemia Cells Promotes theAggressiveness of Microenvironment Related Primary Fibroblasts

P9 Aranzazu Villasante, New York, NY, USAMicroenvironmental Regulation of Tumor-Derived Exosomes using a Tissue-Engineered Model

2. Tumor Microenvironment Interactions that Resist or Promote Tumor Progression

P10 Ali Abed El Wahad, Haifa, IsraelThe Role of Pregnancy-Induced Hormonal Milieu in Lymphoma Progression

P11 Oshrat Attar-Schneider, Kfar-Saba & Tel Aviv, IsraelSecretome of Bone Marrow Mesenchymal Stem Cells: an Emerging Player in Lung Cancer Progression andMechanisms of Translation Initiation

P12 Uri Barash, Haifa, IsraelHighly Effective Heparanase-Based Therapy for Mesothelioma

P13 Bar Ben Baruch, Tel Aviv, IsraelCD38 Inhibition Decreases Melanoma Expansion and Ameliorates Metastatic Burden

LIST OF POSTERS

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P14 Yaacov Ben-David, Guiyang, ChinaErythropoietin Enriches Specific iNOS Producing Myeloid Cells within the Spleen Tumor MicroenvironmentCapable of Inhibiting Leukemogenesis

P15 Hamutal Ben-Dov, Tel Aviv, IsraelPPM1A the Guardian of Homeostasis in the Microenvironment: PPM1A Deficiency in SKPs AffectsFunction and Fate of Immune and Endothelial Cells in the Tumor Microenvironment

P16 Sivan M. Bokobza, Macclesfield, UKStromal Content in Prostate Cancer Correlates with Responses to Docetaxel and Prognosis

P17 Ilanit Boyango, Haifa, IsraelHeparanase Promotes Mammary Gland Branching Morphogenesis and Tumor Growth

P18 Almut Brand, Regensburg, Bavaria, GermanyLactic Acid Promotes Tumor Growth via Inhibition of IFN-γ Expression

P19 Anna Brichkina, Singapore, SingaporeTargeting Tumor Microenvironment for Lung Cancer Treatment

P20 Noam Cohen, Tel Aviv, IsraelFibroblast-Secreted CHI3L1 Enhances Tumor Growth and Angiogenesis

P21 Mahmoud Dabbah, Kfar Saba & Tel Aviv, IsraelMultiple Myeloma Cells Reprogram Bone Marrow Mesenchymal Stem Cells’ Translation Initiation therebyPromoting their Migration

P22 Shani Doron, Rehovot, IsraelHuman RNASET2 Derivatives as Potential Cancer Therapeutic Agents

P23 Liat Drucker, Kfar Saba & Tel Aviv, IsraelMultiple Myeloma and Bone Marrow Mesenchymal Stem Cells’ Crosstalk: Effect on Translation Initiation

P24 Moran Frig, Tel Aviv, IsraelDo Metastatic Melanoma Cells Utilize CD40-CD40L Axis in the Post-Stroke Regenerative Niche for theEstablishment of Brain Metastasis?

P25 Marleen Gloger, Berlin, GermanyInfluence of the Lymph Node Stromal Microenvironment on the Development of Malignant B Lymphocytes

P26 Esther Hermano, Jerusalem, IsraelHeparanase Links Obesity-Associated Inflammation and Breast Cancer Via Modulation of MacrophageResponses

P27 Tal Hirschhorn, Tel Aviv, IsraelDifferential Regulation of Processing of Type I BMP Receptor Isoforms: Implications for Signaling inOvarian Cancer

P28 Anat Klein, Tel Aviv, IsraelAstrocytes Facilitate Melanoma Brain Metastasis via Secretion of IL-23

P29 Yulia Liubomirski, Tel Aviv, IsraelRegulatory Roles of the Notch Signaling Pathway in Inflammation-Driven Tumor-MSCNetworks in Breast Cancer

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P30 Shelly Loewenstein, Tel Aviv, IsraelThe Crosstalk between Omental Fat and Tumor Cells in Gastrointestinal Malignancies

P31 Avi Maimon, Jerusalem, IsraelThe Anticoagulant Protein S Mediates Tumor—Microenvironment Interactions Impacting Tumor Metastasis

P32 Shelly Maman, Tel Aviv, IsraelThe Beta Subunit of Human Hemoglobin Inhibits Lung and Bone Marrow Metastasis

P33 Anatolii Mamchur, Haifa, IsraelAnti-Angiogenic Activity of Factors Secreted by Normal Fibroblasts of Hypoxia Tolerant and Cancer ResistantBlind Mole Rat Spalax: in Vitro Investigation

P34 Artur Mezheyeuski, Stockholm, Sweden & Minsk, BelarusPerivascular Cell Status is Associated with Survival in Metastatic Colorectal Cancer

P35 Noam Rudich, Tel Aviv, IsraelErythropoietin and Its Receptor Play a Role in Melanoma Brain Metastasis and the Brain Microenvironment

P36 Catherine Sem Wegner, Oslo, NorwayCharacterization of the Microenvironment of Cervical Carcinoma Xenografts by Magnetic Resonance Imaging

P37 Yoray Sharon, Tel Aviv, IsraelCancer Associated Fibroblasts Breaking Bad—from a Growth Inhibitory to Tumor Promoting Phenotype

P38 Polina Weitzenfeld, Tel Aviv, IsraelCCR7-Driven Metastatic Spread is Shaped by the Microenvironment of Luminal Breast Tumors

P39 Guili Yang, Tianjin, ChinaMolecular Mechanism of TNFSF15-Induced Inhibition of VEGF-Stimulated Vascular Hyperpermeability

P40 Bella Zamlin-Vizel, Tel Aviv & Ramat Gan, IsraelDeconstructing the Intracellular Events Leading to Enhanced Melanoma Cell Proliferation byCEACAM1 Protein

P41 Kun Zhang, Tianjin, ChinaTumor Necrosis Factor Superfamily-15 Inhibits VEGF Gene Expression via microRNA-29b

P42 Qiang-Zhe Zhang, Tianjin, ChinaTumor Necrosis Factor Superfamily-15 Facilitates Lymphangiogenesis via Upregulation of VEGFR3 GeneExpression in Lymphatic Endothelial Cells

3. Metabolic Pathways in the Tumor Microenvironment

P43 Bratko Filipic, Ljubljana, SloveniaThe Antiproliferative, Proapoptotic and Antitumour Activity in Vitro of 10% PBS Holocene Grain WashoutAgainst CaCo-2 CELLS can be Enhanced with Different Human Alfa Interferons

4. The Immune and Inflammatory Tumor Microenvironment

P44 Mikhail Buldakov, Tomsk, RussiaAmount of CD68+ Tumor Associated Macrophages in Gaps of Ductal Tumor Structures Negatively Correlateswith Lymphatic Metastases in Human Breast Cancer

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P45 Charlotta Dabrosin, Linköping, SwedenCorrelation between Vascular Endothelial Growth Factor and Leptin in Normal Human Breast Tissue in Vivo

P46 Shiri Davidi, Haifa, IsraelThe Role of IL-31 in Tumorigenesis

P47 Naamit Deshet-Unger, Tel Aviv, IsraelImmuno-Skeletal Effects of Erythropoietin in Multiple Myeloma Mice—a Friend and Foe

P48 Margalit Efrati, Tel Aviv, IsraelEfficient Treatment of Mouse Breast Adenocarcinoma by Ablation with Intratumoral Alpha Irradiation Com-bined with Inhibitors of Immunosuppressor Cells and Immunostimulation with CpG

P49 Irit Gil-Ad, Petah-Tiqva, IsraelEvidence for Differential Effect of Two SSRIs Antidepressants on Murine Breast Cancer (4T1) in-Vitro andTumor Grafted Female Mice under Normal and Stress Conditions

P50 Dafna Gilboa, Tel Aviv, IsraelErythropoietin-Associated Increase in Liver Monocyte Derived Macrophages; a Role for Kupffer Cells asMediators?

P51 Lilach Gutter-Kapon, Haifa, IsraelImpact of Heparanase Residing in the Tumor Microenvironment on Cancer Progression

P52 Irena Kaplanov, Beer-Sheva, IsraelHost-Derived IL-1b Regulates Immune Response during 4T1 Breast Carcinoma Tumor Growth

P53 Yasmine Kayal, Tel Aviv, IsraelEnhancement of the Anti-Tumor Immunity Following Tumor Ablation by Electrochemical Treatment

P54 Liran Komov, Haifa, IsraelModulation of the HLA Peptidome of Cancer Cells by Cytokines

P55 Tal Leibovich-Rivkin, Tel Aviv, IsraelCooperative Pathways Regulating Metastasis: The Inflammatory Cytokine Tumor Necrosis Factor α Brings theEvil Out of Rasα

P56 Shalom Lerrer, Tel Aviv, IsraelBreast Cancer: Co-Inflammatory Roles of TGFβ are Revealed in the Presence of TNFα and Amplify Pro-Tumoral Traits of Mesenchymal Stem/Stromal Cells

P57 Yaron Meirow, Jerusalem, IsraelMyeloid Derived Suppressor Cells (MDSCs): the Key to Understanding Chronic Inflammation and its Compli-cations in Cancer

P58 Hadar Reichman, Tel Aviv, IsraelEosinophils Promote Colorectal Cancer through Expression of S100a8 and S100a9

P59 Milan Reinis, Prague, Czech RepublicIFNγ in the Tumour Microenvironment as a Potential Senescence Inducer and Epigenetic Modifier

P60 Vladimir Ryabov, Mannheim, Germany & Tomsk, RussiaStabilin-1 Supports Breast Cancer Growth by Silent Clearance of SPARC

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P61 Lee Shaashua, Tel Aviv, IsraelSurgical Excision of a Primary Tumor Enhances Spontaneous Metastasis of Breast Cancer Through COX-2 andβ-adrenergic Pathways

P62 Preeti Singh, Haifa, IsraelInvolvement of Heparanase in the Pathogenesis of Pancreatitis: Potential Therapeutic Target

P63 Morad Zayoud, Tel Aviv, IsraelThe Role of the IL-22/IL-22R1 Axis in Pancreatic Ductal Adenocarcinoma

5. Circulating Tumor Cells, Dormant Micrometastasis and Cancer Stem Cells

P64 Jo Hantae, Suwon, South KoreaResearch of Cytokine Expression Pattern when Endometrial Cancer Co Culture with Stroma

P65 Young-Chul Kim, Hwasun, Jeonnam, South KoreaDetection of Epidermal Growth Factor Receptor (EGFR) Mutations in Circulating Tumor DNA during EGFR-Tyrosine Kinase Inhibitor (EGFR-TKI) Treatment

P66 Michael Timaner, Haifa, IsraelHost Response to Chemotherapy Induces Mesenchymal Stem Cells Activity Towards Preserving Cancer StemCells Niche

P67 Galia Tiram, Tel Aviv, IsraelMicroRNA Expression Analysis in a Model of Osteosarcoma Dormancy Reveals Novel Regulators of Osteo-sarcoma Progression and Tumor-Host Interactions

6. Metastasis and the Metastatic Microenvironment

P68 Itay Barnea, Tel Aviv, IsraelMonitoring Changes in Morphological Parameters of Primary and Metastatic Melanoma Cell-Lines followingExposure to Taxol by Digital Holographic Microscopy

P69 Anat Eldar-Boock, Tel Aviv, IsraelPrevention of Metastasis Development by RGD-Bearing PGA-Paclitaxel Nanoconjugate: Adjuvant Treatmentfor Breast Cancer

P70 Nour Ershaid, Tel Aviv, IsraelCancer-Associated Fibroblasts: Co-Migrating from Primary to Metastatic Tumor Sites

P71 Luba Hunakova, Bratislava, SlovakiaEffects of Selected Organotin Halides on HumanBreast Cancer Cell LineMDA-MB-231 Growth andMigration

P72 Ho Young Kim, Anyang-si, Gyeonggi-do, South KoreaThe Clinical Utility of FDG PET-CT in Evaluation of Bone Marrow Involvement by Lymphoma

P73 Sandra Klusmeier, Eggenstein-Leopoldshafen & Mannheim, GermanyHigh Systemic VEGF-C Levels Lead to a Specific Neutrophil Accumulation in the Lung and Oromote Metas-tasis in a Syngeneic Rat Breast Cancer Model

P74 Lucia Kucerova, Bratislava, SlovakiaFacilitation of Breast Cancer Cell Engraftment by Human Mesenchymal Stromal Cells in Model of Experimen-tal Metastasis

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P75 Osnat Naftali, Tel Aviv, IsraelPHOX2B: a Regulator of Micro-Metastasis in Neuroblastoma

P76 Sookwhan Sung, Seoul, South KoreaPredicive Factors of Early Recurrence after Curative Surgery in Stage 1 and 2 Pulmonary Adenocarcinoma

P77 Valtteri Tulkki, Cambridge, UKThe Role of Oncostatin M Receptor Over-Expression in Tumour Microenvironment of Cervical Squamous CellCarcinoma

7. Novel Approaches to Target the Tumor Microenvironment

P78 Avraham Dayan, Tel Aviv, IsraelPhotoactivation of a Nanobiocomplex: a Novel Photodynamic Therapy of Cutaneous Melanoma

P79 Yana Epshtein, Tel Aviv, IsraelProtease-Degradable Polymeric Nanomedicine Bearing Paclitaxel and a Turn-On Fluorescent Probe for CancerTheranostics

P80 Ruowen Ge, Singapore, SingaporeThe Secreted Protein Isthmin Selectively Triggers Apoptosis in Cancer Cells and Cancer Endothelial Cellsthrough Cell-Surface GRP78 Mediated Mitochondria Targeting

P81 Hadas Gibori, Tel Aviv, IsraelTargeting Pancreatic Ductal Adenocarcinomawith a Polymeric Nanocarrier and anAnticancer miRNA Polyplex

P82 Rachel Hamri, Rehovot, IsraelTumorMicroenvironment-Targeted Bacteriochlorophylls as Theranostic Agents for Triple Negative Breast Cancer

P83 Victoria Jennings, Ottawa, ON, CanadaOncolytic Rhabdoviruses Expressing Immunomodulatory miRNA Enhance Anti-Tumour Efficacy

P84 Seong-Geun Kim, Yangsan, Gyeongnam, South KoreaPhase 2 Trial of Pexa-Vec (pexastimogene devacirepvec; JX-594), an Oncolytic and Immunotherapeutic Vac-cinia Virus, in Patients with Metastatic, Refractory Renal Cell Carcinoma (RCC)

P85 Philip Lazarovici, Jerusalem, IsraelVimocin and Vidapin, Cyclic KTS Peptides, are Dual, Partial Antagonists of α1β1/α2β1 Integrins withAntiangiogenic Activity

P86 Yaeli Lebel-Haziv, Tel Aviv, IsraelInhibition of the Pro-Malignancy Chemokine CCL5 by “40s Loop Mimetics”

P87 Ondřej Lidický, Prague, Czech RepublicPolymer System for Controlled Drug Delivery and Release of All-Trans Retinoic Acid

P88 Arthur Machlenkin, Tel Aviv, IsraelComputational Discovery and Experimental Validation of Novel Drug Targets in Immuno-Oncology

P89 Keri Schadler, Philadelphia, PA & Houston, TX, USATumor Vessel Normalization through Increased Shear Stress by Exercise Enhances Chemotherapeutic Efficacy

P90 Zohar Shatsberg, Tel Aviv, IsraelmicroRNA-Based Polymer Therapeutics for Combating Glioblastoma

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P91 Cornelis F.M. Sier, Leiden, NetherlandsStromal Cells as Targets for Image-Guided Surgery of Breast Cancer

P92 Shelly Soffer, Tel Aviv, IsraelPolymer Therapeutics Rationally-Designed for a Combination Therapy of Paclitaxel and Doxorubicin

P93 Ran Stein, Rehovot, IsraelDevelevopment of a New Therapeutic Avenue for Basal Cell Carcinoma Eyelid Tumors

P94 Mary Taub, Buffalo, New York, USAActivation of the Prostaglandin E2 EP1 Receptor Antagonizes the Growth Stimulatory Effects of ProstaglandinE2, a Component of the Cancer Microenvironment

P95 Zvi Yaari, Haifa, IsraelTheranostic Barcoded Nanoparticles

P96 Ayelet Zlotogorski-Hurvitz, Tel Aviv, IsraelOral Fluids-Derived Exosomes from Oral Cancer Patients as delivering Vehicles of Natural Anti-Cancer Sub-stances to the Tumor Microenvironment

8. The Tumor Microenvironment and Drug Resistance

P97 Seungmin Bang, Seoul, South KoreaConcomitant Statins can be the Favorable Factor for Gemcitabine and Erlotinib Combination Chemotherapy inAdvanced Pancreatic Cancer

P98 Nancy Gordon, Houston, TX, USAHeat Shock Protein 27 as a Biomarker Predicting the Role of Chemotherapy-Induced Autophagy on DrugSensitivity

P99 Ipsa Jain, Bengaluru, IndiaMolecular Insights into Mechanisms of Drug Resistance using Doxorubicin Resistant Hela Cells

P100 Karin Kfir, Ramat-Gan & Tel Aviv, IsraelRegulation of CEACAM1 Protein Expression in Braf-Mutant Human Metastatic Melanoma Cells

P101 Shlomit Kfir-Erenfeld, Jerusalem, IsraelmiR-103 Sensitizes Hemopoietic Tumor Cells for Glucocorticoid Induced Apoptosis

P102 Ho-Suk Oh, Gangneung, South KoreaDaurinol, a Catalytic Inhibitor of Topoisomerase IIα, Suppresses SNU-840 Ovarian Cancer Cell Proliferationthrough Cell Cycle Arrest in S Phase

P103 Jianwei Qi, Tianjin, ChinaTNFSF15 Inhibits Vasculogenesis by Regulating Relative Levels of Membrane-Bound and Soluble Isoforms ofVEGF Receptor 1

P104 Hitam Saadi, Haifa, IsraelThe Source of the Stroma Affects the Trafficking Potential of Mantle Lymphoma Cells and Alters the Effect ofIbrutinib on Their Survival

P105 Martina Sboarina, Brussels, BelgiumA High Rate of Glycolysis Participates in the Resistance of Glioblastoma to Temozolomide Chemotherapy

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P106 Ladislav Sivák, Prague, Czech RepublicOvercomingMultidrug Resistance with Inhibitor of ABC Transporters Bound to HPMACopolymer Carrier as aPotential Therapeutic Approach in Cancer Treatment

P107 Inna Zubrilov, Tel Aviv, IsraelVemurafenib Resistance Selects for Highly Malignant Brain and Lung-Metastasizing Melanoma Cells

Late-Breaking Abstracts

P108 Ophir Shani, Tel Aviv, IsraelChitinase 3-like 1 Mediates Tumor-Promoting Interactions between Fibroblasts and Macrophages in the Micro-environment of Breast Tumors

P109 Dalit Barkan, Haifa, IsraelIntegrin Alpha V Beta 3 Expression in Luminal Breast Cancer Cells Promotes their Reversion to Acinar-LikeStructure in Conjunction with their Relevant Microenvironment

P110 Yona Kalechman, Ramat Gan, IsraelRedox Modulation of Adjacent Thiols in VLA-4 by AS101 Converts Myekoid Leukemia Cells from a Drug-Resistant to Drug-Sensitive State

P111 Gabriela Vazquez Rodriguez, Linköping, SwedenNeutrophils Promote Breast Cancer Progression and Metastasis via LFA-1 Integrin

P112 Lucia Knopfova, Brno, Czech Republicc-Myb Inhibits Opening of Lung Endothelium by Breast Tumor Cells Thereby Dictates Metastasis Pattern

P113 Petr Benes, Brno, Czech RepublicTrop-2 in Breast Cancer Adaptation to Specific Conditions of Tumor Microenvironment

P114 Ehud Shahar, Kiryat Shmona, IsraelRe-Modulation Innate Immune Activity in the Tumor Microenvironment by a Combination of Inducer onMicro-Particles

P115 Noa Cohen Anavy, Haifa, IsraelNano-Ghosts for Selective Drug Delivery to and Across the Blood Brain Barrier as a Potential Therapy forGlioblastoma Multiforme

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18:30–19:45

OPENING SESSION

O1

Lgr5 Stem Cells in Self-Renewal and Cancer

Hans CleversHubrecht Institute, Royal Netherlands Academy of Arts andSciences & University, Medical Centre Utrecht, Princes Max-ima Center for Pediatric Oncology, Utrecht, Netherlands

The intestinal epithelium is the most rapidly self-renewing tissuein adult mammals. We originally defined Lgr5 as a Wnt targetgene, transcribed in colon cancer cells. Two knock-in allelesrevealed exclusive expression of Lgr5 in cycling, columnar cellsat the crypt base. Using lineage tracing experiments in adultmice, we found that these Lgr5+ve crypt base columnar cells(CBC) generated all epithelial lineages throughout life, implyingthat they represent the stem cell of the small intestine and colon.Lgr5 was subsequently found to represent an exquisitely specificand almost ‘generic’ marker for stem cells, including in hairfollicles, kidney, liver, mammary gland, inner ear tongue andstomach epithelium. Single sorted Lgr5+ve stem cells can initiateever-expanding crypt-villus organoids, or so called ‘mini-guts’in 3D culture. The technology is based on the observation thatLgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D cultures systems have been developed forthe Lgr5+ve stem cells of stomach, liver, pancreas and kidney.Using CRISPR/Cas9 technology, the CFTR locus has beencorrected in intestinal organoids of cystic fibrosis patients.


08:30–10:35

PLENARY SESSION 1—Regulatory Pathwaysin the Tumor Microenvironment (I)

O2

Role of Non-Coding RNAs in Cancer

Carlo M. CroceMolecular Virology, Immunology andMedical Genetics, TheOhio State University, Columbus, Ohio, USA

MicroRNAs (miRNAs) are small non-codingRNAs, 19–24 nucle-otides in length, which regulate gene expression, and are aberrantlyexpressed in most types of cancer. MiRNAs have also been detect-ed in the blood of cancer patients, and can serve as circulatingbiomarkers. It has been shown that secreted miRNAs withinexosomes can be transferred from cell to cell, and can regulate geneexpression in the receiving cells by canonical binding to their targetmessenger RNAs. Here we show that tumor secreted miR-21 andmiR-29a can also function by a novel mechanism, by binding asligands to receptors of the Toll-like receptor family, murine TLR7and human TLR8, in immune cells, triggering a TLR-mediatedprometastatic inflammatory response, which ultimately may leadto tumor growth, spreading and metastasis. Thus, by acting asperegrine agonists of TLRs, secreted miRNAs are key regulatorsof the tumor microenvironment. This mechanism of action ofmiRNAs is implicated in the tumor-immune system communica-tion, and is important in tumor growth and spread, thereforerepresenting a new possible target for cancer treatment.

O3

Hypoxic Microenvironment and Tumor Metabolism

Jacques Pouyssegur1, Jérome Durivault2, Yann Cormerais2,Ibtissam Marchiq31Centre A Lacassagne, IRCAN, University of Nice & CentreScientifique (CSM) de Monaco, Nice & Monaco, France2Biomedical Division, Centre Scientifique de Monaco (CSM),Monaco, Monaco3Centre A lacassagne, IRCAN, University of Nice, Nice, France

In metazoans, sensing the availability of oxygen and key nutrientssuch as glucose and amino acids is integrated with growth factorand hormone signaling. Therefore, rapidly growing cells havedeveloped sophisticated regulatory systems to rapidly respondto fluctuations in oxygen and nutrients in the microenvironment.Early on in evolution, oxygen sensing emerged as a centralcontrol mechanism of energy metabolism and vasculogenesis.At the heart of this regulatory system are the Hypoxia-Inducible Factors, HIFs, which control the expression of nu-merous gene products including VEGF-A, Angiopoïetin-2 andNotch-ligand, three key angiogenic factors in vertebrates. Thisfinding has placed the hypoxia-signaling pathway at the fore-front of nutritional control. HIF-1 and HIF-2 can also induce avast array of gene products controlling import of nutrients(GLUT1, LAT1), glycolysis, intracellular pH (pH), angiogen-esis, cell migration and invasion, and so are recognized asstrong promoters of tumor growth. It is therefore not surpris-ing that HIF-1 also promotes access to another source ofnutrients by inducing via BNIP3 macro-autophagy.In this presentation we will highlight two anticancer targetsinduced by HIFs and highly expressed in rapidly growing

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tumors: the plasma membrane transporters LAT1 (SLC7A5)importing essential amino acids and MCT4 (SLC16A4), alactate/H+ symporter regulating pHi by efficient lactic acidextrusion. The second target, MCT4, is particularly interestingin the context of this symposium. Lactic acid generated fromtumors is emerging as a major metabolite that impacts on thetumor microenvironment not only as a signaling molecule, butalso as an acid stressor compromising immune surveillance.Thus an approach that blocks lactic acid export could have adual effect as an anticancer strategy.

O4

Modulation of Vascular and Lymphatic EndothelialFunctions by TNFSF15

Jianwei Qi, Guili Yang, TingTing Qin, Qiangzhe Zhang,Zhisong Zhang, Luyuan LiCollege of Pharmaceutical Science, Nankai University, Tian-jin, Tianjin, China

Vasculogenesis and lymphangiogenesis are important aspectsof cancer development, progression, and metastasis. We havefound that tumor necrosis factor superfamily-15 (TNFSF15,also known as VEGI or TL1A), a cytokine predominantlyproduced by endothelial cells, is able tomodulate the activitiesof VEGF receptor-1, -2, and -3. TNFSF15 negatively modu-lates endothelial progenitor cell-supported vasculogenesis bysimultaneously promoting degradation of membrane-boundform of VEGFR1 (mFlt1) that mediates VEGF-stimulatedvasculogenesis, while upregulating the expression of solubleform of VEGFR1 (sFlt1) that scavenges VEGF and thus in-hibits its activities. In addition, TNFSF15 is part of a molec-ular mechanism that prevents VEGF-induced vascularhyperpermeability by inhibiting VEGF-induced VEGFR2-phosphorylation, resulting in diminished VEGFR2-mediatedsignals and reduced endothelial permeability. Examined inanimal models, the ability of topically applied VEGF to in-duce vessel leakage is markedly lowered in the skin ofTNFSF15-overexpressing transgenic mice compared withthat in the transgene-negative littermates. Similar results areobtained when wild-type mice are treated with recombinantTNFSF15 by intraperitoneal injection. Moreover, TNFSF15can promote lymphatic endothelial cell (LEC) growth andmigration, stimulate lymphangiogenesis, and facilitate lym-phatic function. Treatment of mouse LEC with TNFSF15 re-sults in upregulation of VEGFR3 expression. TNFSF15-overexpressing transgenic mice exhibit a marked enhance-ment of lymph drainage. Death domain-containing receptor-3 (DR3), a cell-surface receptor of the TNF receptor super-family (TNFRSF25), mediates the abovementioned TNFSF15activities. These findings are consistent with the view that, via

intervention with the signaling pathways of VEGF and itsreceptors VEGFR1, VEGFR2, and VEGFR3, TNFSF15 playsa key role in the modulation of the functions of both vascularand lymphatic endothelia.

O5

Marker-Defined Perivascular Cells PredictPrognosis and Response to Treatment

Arne ÖstmanOncology-Pathology, Karolinska Institutet, Stockholm,Sweden

Experimental studies suggest that perivascular cells affect tumorgrowth, metastasis and treatment response. Characterization ofperivascular cell in clinical samples, and analyses of impact onprognosis and response to treatment, is therefore warranted.We have developed an integrated set of procedures for analy-ses of perivascular cells using IHC-stainings with one endo-thelial cell marker (CD34) and markers for perivascular cells(PDGFaR, PDGFbR, ASMA and desmin). Digital image-analyses are used to determine fraction of covered vessels,intensity of marker expression and heterogeneity of markerexpression. Studies focus on breast, colorectal, ovarian andkidney cancer.Comparative analyses identified striking differences betweentumor types including a higher fraction of covered vessels,and higher abundance of PDGFbR positive perivascular cells,in colorectal cancers. In the case of ovarian cancer, perivascularstatus in matched primary tumors and metastases was com-pared. A large degree of dis-concordance of most vascularcharacteristics was detected.Concerning prognostic relevance, significant associations wasdetected between high perivascular PDGFbR cells shorter sur-vival in breast, kidney and ovarian cancer. These associationswere also significant in multi-variate analyses. High heteroge-neity in perivascular PDGFbR expression was also associatedwith shorter survival in ovarian and kidney cancer.To analyze impact on response to treatment, groups ofchemotherapy-treated or non-treated metastatic colorectal can-cer were analyzed. Interestingly, this analysis demonstrated anassociation between low perivascular PDGFbR expression, par-ticularly strong in the chemo therapy-treated group. This findingsuggests novel links between perivascular PDGFbR status andchemo-treatment response. Additional analyses also identifiedan association between shorter survival and low perivascularPDGFbR expression in bevacizumab-treated patients.Collectively these studies demonstrate clinically relevant var-iation in tumor perivascular cells, suggestion continued explo-ration of these cells as biomarkers and treatment targets.

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O6

Biology and Function of Tumor Stromaand Exosomes in Cancer

Raghu KalluriCancer Biology, The University of Texas MD Anderson Can-cer Center, Houston, Texas, USA

Tumors contain many different host cell types and non-cellularconstituents such as ECM, apart from cancer cells. The centralgoal of our laboratory is to evaluate the functional role of thesenon-cancer cells/constituents in cancer progression and metas-tasis. Cancer progression significantly depends on the influenceof many different host cells on the genetically unstable cancercells. Whether such host responses are recruited to control can-cer progression or further aid in tumor growth (or both) is stillunclear. Additionally, chronic tissue fibrosis involves fibroblastactivation and inflammation that leads to deposition of type Icollagen and eventual organ failure. There is a strong experi-mental and clinical correlation between tissue fibrosis and in-cidence of cancer. But it remains unclear how fibrosis maycontribute to the emergence of cancer. This lecture will high-light the functional role of mesenchymal cells in tumor immu-nity in PDAC. Additionally, the utility of exosomes in diagno-sis and treatment of PDAC will be discussed.

11:00–13:05

PLENARY SESSION 2: Target ing the TumorMicroenvironment—Novel Approaches (I)

O7

Establishment of Immunotherapy as the FourthPillar of Cancer Treatment

Drew PardollOncology, Johns Hopkins, Baltimore, Maryland, USA

Major advances have been made in the immune-based therapy ofcancer by antibody blockade of immune inhibitory pathwayssuch as CTLA-4 and PD-1. Anti-PD-1 antibodies have producedobjective responses in one third to one half of patients with ad-vanced, chemotherapy refractory melanoma and renal cancer andone quarter of patients with non-small cell lung cancer. Theseresponses are highly durable, the majority lasting significantlygreater than 1 year and beyond cessation of therapy. Further,expression by tumor cells of ligands for PD-1 is associated withhigher response to anti-PD-1 therapy. In exploring the basis for

up-regulation of the major PD-1 ligand, PD-L1, on tumor cells,we found that its expression is not constitutive, but rather, ishighly associated with lymphocytic infiltration. We identifiedIFN-g as an immune signal sensed by the tumor cell that inducesPD-L1 expression. In addition to IFN-g, genes associated withTh1 responses, CTL responses and other inhibitory molecules,such as LAG-3, are up-regulated in lymphocytic infiltrates asso-ciated with PD-L1+ tumor cells. These findings indicate thatmultiple counterbalancing immune effector and inhibitory path-ways are operative in the immunemicroenvironment. They led usto hypothesize a newmechanism of PD-L1 expression in tumors,termed adaptive resistance, distinct from a constitutive mecha-nism of PD-L1 expression in tumors. The adaptive resistancemechanism implies that other therapies such as vaccines mayinduce antitumor responses that in turn induce up-regulation ofPD-L1. In such a circumstance, vaccination and PD-L1 blockademight produce synergistic anti-tumor activity. Using a novel vac-cine formulation termed STINGVAX, we indeed demonstrateIFN-g dependent induction of PD-L1 on tumors and synergisticactivity such that the vaccine/anti-PD-1 combination.

O8

Immune System as a Vital Partner in CancerTreatment

Blanka Rihova1, Tomas Etrych2, Milada Sirova1, VladimirSubr2, Marek Kovar1, Karel Ulbrich21Tumor Immunology, Institute of Microbiology Academy ofSciences of the Czech Republic, Praha, Czech Republic2Biopolymers, Institute of Macromolecular Chemistry Acade-my of Sciences of the Czech Republic, Praha, Czech Republic,Czech Republic

The potential for treating cancer patients by immunologic ap-proaches is currently a great promise for oncologists and immu-nologists. One of the hallmark features of an effective immuno-therapy is its ability to stimulate lasting tumor-specific immunity.Immunopharmacological agents or immunomodulators canconventionally be divided to immunosuppressive orimmunostimulating drugs which correspond to the therapeuticneed. The medical purpose of immunostimulators is, amongothers, to support rejection of cancer cells. The field of pharma-cological immunostimulationwithmonoclonals targeting controlcheckpoints of the immune system is still at a rather early stageof development. Another modern approach is represented bytargeted synthetic nanotherapeutics based on N-(2-hydroxypropyl)methacrylamide copolymers (HPMA) with dual,i.e., cytostatic and immunomodulating activity. We have shownthat HPMA nanomedicines bearing anthracycline antibiotics(doxorubicin; DOX) or taxanes have an exceptional anticancereffect based on the direct cytotoxicity and therapy-activated

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anticancer-immune response. HPMA-DOX conjugates kill tu-mor cells by immunogenic apoptosis which stimulates compo-nents of the immune system including dendritic cells. Exposureto HPMA-DOX induces calreticulin (CRT) translocation andrelease of high mobility group box-1 protein (HMGB1) in EL4T cell lymphoma cells. Specific systemic anti-cancer immuneresponse is mainly mediated by CD8+ cytotoxic T cells. Suchan approach represents a complementary treatment based onsynergistic effect between immunotherapy and chemotherapy.It protects cancer-bearing animals against minimal residual dis-ease and second cancer attack. The well-defined structure, con-trolled molecular weight, in situ drug release, successful in vivotreatment and the ability to induce treatment-dependent long-lasting tumor-specific immunity predestine the nanotherapeuticsbased on HPMA for efficient anti-cancer therapies.

O9

Annexin-Mediated Regulation of the PeripheralImmune Response

Peter H. Krammer, Björn Linke, Lucie Abeler-Dörner,Veronika Jahndel, Alexandra Kurz, Andrea Mahr, SandraPfrang, Linda Linke, Heiko WeydD030 Immunogenetics, DKFZ, Heidelberg, Germany

Peripheral tolerance against self-antigens derived from apoptoticcells is essential for the organism. Apoptotic cells continuouslyevolve in the course of tissue turnover and are rapidly removed byphagocytes, e.g., dendritic cells (DC). Apoptotic cells have beenshown to actively suppress DC in vitro, and engulfment of apo-ptotic cells is associated with inhibition of immune responses andthe development of peripheral tolerance in vivo. However, spe-cific molecules that mediate such tolerogenic effects of apoptoticcells have been largely elusive. Annexins are a family of evolu-tionary well conserved cytosolic proteins differentially expressedin various tissues. All annexins bind to negatively charged phos-pholipids in a calcium-dependent manner; however, the physio-logical function of the twelve vertebrate annexins has not beenfully elucidated yet. We have shown that annexins, located in thecytosol of living cells, become translocated to the outer surface ofapoptotic cells. Via their conserved core domain, annexins interactwith DC and impose a tolerogenic phenotype on these DC, char-acterized by low expression of costimulatory surface moleculesand inflammatory cytokines, reduced pro-inflammatory signaltransduction and resistance to maturation. Such tolerogenic DCare able to actively induce so called “deleterious tolerance” bydepleting autoreactive T cells. Thus, during the physiologic turn-over of tissue cells the exposure of annexins on the surface ofapoptotic cells acts as an immune checkpoint, which ensurestolerance to self-antigens derived from apoptotic cells.

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Targeting Astrocyte Endothelin Axis for Therapyof Experimental Breast and Lung Cancer BrainMetastasis

Isaiah Fidler, Ho-Jeong Lee, Hyun Kyung Yu, Sun-Jin KimDepartment of Cancer Biology, The University of Texas M.D.Anderson Cancer Center, Houston, Texas, USA

Brain metastasis is associated fatal outcome. The median sur-vival of patients with brain metastasis is 2 to 4 months. Ra-diotherapy and chemotherapy can increase the overall survivalto 6 months. Development of novel therapeutic approaches toimprove clinical outcomes is urgently needed.Recently, we discovered that brain endothelial cells and astro-cytes protect breast cancer cells and lung cancer cells from pac-litaxel through an endothelin-dependent signaling mechanismthat leads to the upregulation of anti-apoptotic proteins in cancercells. The chemoprotective effect can be abolished by combinedantagonism of ETAR and ETBR signaling or by siRNA targetingof both endothelin receptors on cancer cells. In this study, wedetermined the extent that endothelin signaling is critical for thesurvival of experimental breast and lung cancer brainmetastases.Nude mice bearing established MDA-MB-231 breast cancer orPC-14 non-small cell lung cancer brain metastases were treatedwith vehicle, a dual endothelin receptor antagonist, macitentan,paclitaxel, ormacitentan plus paclitaxel. Cell division, apoptosis,tumor vasculature, and expression of survival-related proteinswere assessed by immunofluorescent microscopy.Cancer cells and tumor-associated endothelial cells expressedactivated forms of AKTandMAPK in vehicle- and paclitaxel-treated groups, but these proteins were downregulated in me-tastases of mice that received macitentan. The survival-relatedproteins BCL2L1, GSTA5, and TWIST1 in cancer cells andtumor-associated endothelial cells were suppressed bymacitentan. Macitentan combined with paclitaxel produced asignificant reduction in cancer cell division and marked apo-ptosis of both cancer cells and tumor-associated endothelialcells and produced complete responses in 35/35 mice. In con-clusion, dual antagonism of ETAR and ETBR signaling sensi-tizes brain metastases to paclitaxel and may represent a newtherapeutic option for patients with brain metastases.

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Myeloid Regulatory Cells and Nano-Deliveryof Chemotherapeutic Agents

Michael ShurinPathology, University of Pittsburgh, Pittsburgh, PA, USA

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The use of nanovehicles for drug delivery is a promising andwidely investigating approach to cancer therapy. However, thebehavior of nanomaterials and carried chemotherapeutic agentsin the local tumor microenvironmental conditions has not beeninvestigated. We have revealed that chemotherapeutic agents,such as doxorubicin, can lose the antitumor activity in themyeloperoxidase-catalyzed and peroxynitrite-mediated oxidativeconditions reflecting the activity of tumor-activated myeloid-de-rived suppressor cells (MDSC). Our data also suggests that thechemotherapeutic agents delivered by the nanocarrier, whichconstitutes an oxidized single-walled nanotube and a branchedphospholipid-polyethylene glycol, may be protected from theenzymatic inactivation associated with myeloid cells in the tu-mor microenvironment while exhibiting the constant doxorubi-cin release rate. Additional development of nano-delivery sys-tem allowed generating unique carbon nanotube cups, i.e.,nanoscale containers, that can be loaded with chemotherapeuticagents, corked with gold nanoparticles and be opened byMDSC-derived myeloperoxidase-catalyzed reactive intermedi-ates and sodium hypochlorite. This results in local release ofchemotherapeut ic drugs in the MDSC-enr ichedmicroenvironment, i.e., the tumor milieu, and effective inhibi-tion of MDSC activity. Thus, understanding the biology ofmyeloid regulatory cells in the tumor microenvironment opensnew opportunity for development of effective and controllablenanocarrier for safe delivery of therapeutic agents to thetumor site.

14:30–16:10

PLENARY SESSION 3: Inflammatory & ImmuneReactions in the Tumor Microenvironment (I)

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The Microenvironment of Breast Tumors Re-ShpaesCCR7-Driven Metastatic Spread

Polina Weitzenfeld1, Olga Kossover2, Cindy Körner3, TsipiMeshel1, Stefan Wiemann3, Dror Seliktar2, Daniel Legler4,Adit Ben-Baruch11Cell Research and Immunology, Tel Aviv University, Tel Aviv,Israel2Faculty of Biomedical Engineering, Technion-Israel Instituteof Technology, Haifa, Israel3Division of Molecular Genome Analysis, German CancerResearch Center (DKFZ), Heidleberg, Germany4Biotechnology Institute Thurgau, University of Konstanz,Konstanz, Germany

Introduction: The axes CCR7-CCL21 and CXCR4-CXCL12determine organ-specific metastatic spread in lymph node (LN)and distant organs. Here, we investigated the regulation ofchemokine-induced metastatic dissemination by TME factors,focusing on luminal-A breast tumors.Methods: Using the METABRIC dataset, we determinedthe expression patterns of CCR7 and CXCR4, and theassociation between CCR7 and LN metastasis, in differentbreast cancer subtypes. In parallel, luminal-A MCF-7 cellsover-expressing CCR7 or CXCR4 were stimulated by acombination of factors typically residing in the TME ofluminal-A tumors (=TME stimulation). Downstream sig-naling and migratory abilities in response to chemotacticsignals were assessed in vitro using migration assays and anovel 3D hydrogel system in which directional protrusionstowards chemokines were determined. In vivo mousemodels were used to examine the metastatic spread.Results: METABRIC analyses indicated that CCR7 andCXCR4 expression levels were lower in luminal-A tumorsthan in more aggressive subtypes of breast cancer. TME stim-ulation down-regulated the migration of CCR7- and CXCR4-expressing MCF-7 cells in response to their respective li-gands, CCL21 and CXCL12, and inhibited the formation ofdirectional protrusions in response to CCL21. TME stimula-tion has shut-off CCL21-induced PI3K and MAPK signalingin CCR7-expressing cells (not by inducing receptor internal-ization). In vivo, TME stimulation alone elevated LN metas-tasis but in its presence the signals delivered by CCR7 couldnot come into effect, in terms of LN spread. Rather, when theTME stimulus was imposed on CCR7-expressing cells, themetastatic spread in bones was considerably increased.Conclusions: Microenvironmental signals induce an aggres-sive metastatic phenotype in luminal-A breast tumor cells,providing a personalized context to the potential use ofchemokines in targeting metastasis.

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Molecular Mechanisms of Inflammation AffectingHuman Melanoma

Elizabeth GrimmMelanoma Medical Oncology, The University of Texas M.D.Anderson Cancer Center, Houston, Texas, USA

Many cancers contain the products of inflammation, believed tobe a result of both intrinsic cancer cell processes, and those fromthe microenvironment. Data in human melanoma suggests thatthe unique cancer-related form of “para-inflammation” leadsto the production of reactive oxidants, which support tumorcell survival, proliferation and other functions. Molecules from the

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microenvironment driving melanoma include responses to theIL-1 s [1] and IFNg [2], leading to poor patient prognosis asso-ciated with expression of inducible nitric oxide synthase(iNOS or NOS2), macrophage migration inhibitory factorand additional downstreammediators to be presented. Inflam-matory molecules associating with good survival of melano-ma patients are rare, but a strong association of CD-74 (MHCClass II invariant chain) protein expression in both the mela-noma cells and also tumor-infiltrating lymphocytes has beenrecently confirmed by us (Ekmekcioglu, submitted). At thebiochemical level, nitric oxide leads to the oxidation of impor-tant molecules, and identification of these post-translationallyoxidized proteins in cancer cells mediating various effects hasbeen studied, with both nitro-tyrosine (NT) and cysteinyl-S-nitrosylation (S-NO) now identified as providing insight into anovel mechanism of potentially targetable pathways [3].1. Qin, Y., et al., Constitutive aberrant endogenous interleukin-1facilitates inflammation and growth in human melanoma. MolCancer Res, 2011. 9(11): p. 1537–50.2. Tanese, K., E.A. Grimm, and S. Ekmekcioglu, The role ofmelanoma tumor-derived nitric oxide in the tumor inflammatorymicroenvironment: its impact on the chemokine expression pro-file, including suppression of CXCL10. Int J Cancer, 2012.131(4): p. 891–901.3. Grimm, E.A., A.G. Sikora, and S. Ekmekcioglu, Molecularpathways: inflammation-associated nitric-oxide production as acancer-supporting redox mechanism and a potential therapeutictarget. Clin Cancer Res, 2013. 19(20): p. 5557–63.

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Immunsuppressive Pathways in Melanoma

Viktor Umansky, Carolin Blattner, Huanhuan Jiang,Christoffer Gebhardt, Jochen UtikalSkin Cancer Unit, German Cancer Research Center (DKFZ),and Department of Dermatology, Venereology and Allergology,University Medical Center Mannheim, Ruprecht-Karl Universityof Heidelberg, Heidelberg, GermanyMelanoma is known for its poor response to current immuno-therapies. Insufficient anti-tumor reactivity could be due to theformation of a chronic inflammation, leading to a profoundimmunosuppression. Using the ret transgenic murine melano-ma model that mimics human melanoma, we found in skintumors andmetastatic lymph nodes increased levels of chronicinflammatory factors (IL-1beta, IL-6, IFN-gamma, TNF-alpha etc.) associated with the accumulation of myeloid-derived suppressor cells (MDSC) known to inhibit tumor in-filtrating T cells. The accumulation of CD11b+Gr1+ MDSCin melanoma lesions was associated with upregulation ofCCR5 expression on these cells. Concentration of CCR5

ligands chemokines CCL3, CCL4 and CCL5 was significant-ly elevated in melanoma lesions as compared to serum oftumor-bearing mice. CCR5-positive MDSC displayed a sig-nificantly higher immunosuppressive activity than theirCCR5-negative counterparts. MDSC were also shown to ex-press ectonucleotidases CD39 and CD73 stimulating the pro-duction of adenosine that strongly inhibits T cell functions.Tumor-infiltrating T cells expressed not only markers of acti-vated or memory cells but also CD39 and CD73 that were up-regulated by tumor-derived soluble factors (like TGF-beta).This suggests that adenosine producing by effector T cellscan inhibit their anti-tumor reactivity via autocrine signalingas a part of the negative feedback loop.In melanoma patients, we found that the level of serum IL-1beta,IFN-gamma, CXCL10 and the frequency of circulating mono-cytic MDSC were strongly increased in advanced melanomapatients that significantly correlated with a decreased progressionfree survival of these patients. Our data suggest that chronicinflammatory mediators, MDSC and adenosine metabolism inmelanoma patients are of importance for the pathogenesis andprognosis of melanoma progression and could help to identifypatients with high risk of disease progression.

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Breaking Bad—Cancer Associated Fibroblasts areReprogrammed from Growth Inhibitoryto Pro-Inflammatory and Tumor-Promotingin Breast Cancer

Yoray Sharon1, Noam Cohen1, Yael Raz1,2, Amir BenShmuel1, Ophir Shani1, Metsada Pasmanik-Chor3, NetaErez1, Tamar Geiger41Department of Pathology, Sackler School of Medicine, TelAviv University, Tel Aviv, Israel2Department of Obstetrics and Gynecology, LIS MaternityHospital, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel3Bioinformatics Unit, Faculty of Life Sciences, Tel Aviv Uni-versity, Tel Aviv, Israel4Department of Human Molecular Genetics and Biochemis-try, Sackler School of Medicine, Tel Aviv University, Tel Aviv,Israel

Breast tumors are characterized by an extensive desmoplasticstroma, abundantly populated by fibroblasts. Nevertheless,there is no detailed analysis of the dynamic changes inCancer-Associated Fibroblasts (CAFs) during tumor progres-sion.We therefore characterized the transcriptome of mamma-ry CAFs isolated from distinct stages of mammary carcino-genesis in a transgenic mouse model of human breast cancer.We found unique CAF gene signatures that correspond to

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different tumor stages, with only partial overlap between thestages, suggesting co-evolution of the microenvironment withtumorigenic progression. Interestingly, the gene signature offibroblasts isolated from hyperplastic lesions is inverse to thatin CAFs from neoplastic stages, and has a growth inhibitoryphenotype, while CAFs from mammary tumors express pro-inflammatory and tumor promoting gene signatures. A prote-omic screen of breast cancer cells secretome identified Osteo-pontin (OPN) in reprogramming of normal mammary fibro-blasts to pro-inflammatory CAFs. Knockdown of OPN in tu-mor cells in vivo resulted in attenuated stromal activation,reduced immune cell recruitment and inhibition of tumorgrowth. Furthermore, we show that immune cell recruitmentand pro-inflammatory signaling by mammary CAFs is medi-ated by the pro-inflammatory secreted glycoprotein CHI3L1,highly upregulated in CAFs isolated from invasive mammarytumors. Knockdown of Chi3L1 in fibroblasts duringorthotopic breast cancer transplantations resulted in attenuatedtumor growth, functionally implicating CAF-derived Chi3L1in breast cancer progression. Moreover, CHI3L1 enhancedtumor vascularization and increased macrophage migrationand upregulation of an M2-associated gene signature, impli-cating fibroblast-derived CHI3L1 as a key player in thecrosstalk between cancer-associated fibroblasts and macro-phages in the microenvironment of breast tumors. Taken to-gether, our findings reveal a reciprocal crosstalk between fi-broblasts, cancer cells and macrophages, which mediates theco-evolution of fibroblasts during breast cancer progression.

16:40–18:20

PLENARY SESSION 4: Inflammatory & ImmuneReactions in the Tumor Microenvironment (II)

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Cancer Phenotypes and their Immune Contextures

Wolf Herman Fridman1, Etienne Becht1, Giraldo Nicolas1,de Reynes Aurelien2, Laurent-Puig Pierre2, BeuselinckBenoit2, Zucman-Rossi Jessica2, Sautes-Fridman Catherine11Cancer, Immunology, Immunopatology, Cordeliers ReseachCentre, Paris, France2Carte d’Identité des Tumeurs, Ligue Nationale FrancaiseContre le Cancer, Paris, France

In the vast majority of cancer types, as demonstrated and ex-emplified in colorectal cancer (CRC), a high density of CD8+ Tcells in the tumor microenvironment correlates with a goodprognosis for the patient. However, an opposite correlation

has been reported in primary clear cell Renal Cell Carcinoma(RCC) and several other cancers. Our team studied the immuneinfiltrates of pulmonary metastases from CRC and RCCmetas-tases. As in the primary tumors, a high density of CD8+ T cellscorrelated with good prognosis for CRC metastases, while itcorrelated with a bad prognosis for RCC metastasis. Theseresults suggest that the identity of the tumor cells, rather thanthe organ where they grow, is critical for shaping the immunecontexture of a given tumor. We therefore investigated molec-ular characteristics of the tumor cells, which could be respon-sible for this difference in prognostic value for RCC and CRC.Subgroups of both RCC and CRC tumors exhibited similarimmune contextures, identifying patients with high risk ofdeath, either with low T and B cells infiltrations or, alternative-ly, high lymphocyte infiltration in the context of a high expres-sion of genes related to inflammation, immunosuppression, andangiogenesis. In CRC, additionally, the MSI-enriched sub-group identifies patients with high cytotoxic T cells infiltrationand favorable prognosis. Based on their immune contextures,cancers can therefore be better classified accordingly to theirmolecular characteristics than their origin.

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Characterization and Prognostic Impactof the Intra-Tumoral Immune Response in HumanCancers

Catherine Sautès-Fridman, Nicolas A Giraldo, ClaireGermain, Etienne Becht, Jeremy Goc, Wolf-Hervé Fridman,Marie Caroline Dieu-NosjeanCancer, Immunologie et Immunopathologie, Centre deRecherche des Cordeliers, INSERMU1138, Paris, France

In contrast to most cancers, infiltration of the primary tumorby memory CD8+ T cells is associated with poor prognosis inclear cell Renal Cell Carcinoma (ccRCC). We investigated thecharacteristics and the organization of the local immune re-sponse in Non Small Cell Lung Cancer (NSCLC) and inccRCC. We showed that high density of mature DC presentin Tertiary Lymphoid Structures (TLS-DC) correlates withgood prognosis in both cancers. In NSCLC, the TLS-B-cellfollicles are characterized by Ki-67+ proliferating germinalcenter B cells, surrounded by CD138 positive plasma cells.Follicular B cell density correlates with long term survival,both in patients with early- and advanced-stage NSCLC.IgG and/or IgA specific for different tumor-associated anti-gens (TAA) were produced by tumor-infiltrating B cells cul-tured in vitro. We further characterized the microenvironmentof ccRCC primary tumors and revealed associations betweenan extensive CD8+ T cell infiltrate, high expression of

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immunoregulatory molecules and poor prognosis. Interesting-ly, most of the DC were located outside TLS, and were MHCClass II low and CD83 negative, suggesting that they corre-spond to not fully mature DC. Their density correlated withpoor prognosis. Altogether our findings highlight the pivotalfunction of DC and TLS in shaping an effector and memoryimmune response mediated by T and B cells against cancer.

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Extracting Tumor Tissue Immune Statusfrom Expression Profiles: Correlating Prognosiswith Tumor-Associated Immunome

Angel Porgador, Omri Teletsh, Eitan RubinThe Shraga Segal Dept of Microbiology, Immunology and Ge-netics, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Investigating the expression of genes in cancer-associated im-mune cells (immunome) is imperative for prognosis prediction.However, evaluating the expression of immune-associatedgenes within cancer biopsy is subject to significant inconsis-tencies related to the sampling methodology. We presentimmFocus, a method for extracting immune signals from totalRNA sequencing of tumor biopsies, intended for immunitydepiction and prognosis evaluation. It is based on reducingthe variation which biopsy preparation adds to the apparentexpression levels of immune genes. We employed immFocusto normalize gene expression with an immune index using dataobtained from kidney renal clear cell carcinoma biopsies. Genesthat became less variable due to normalization were found to bepreferentially immune-related. Moreover, immune-relatedgenes tended to become more prognostic due to the normaliza-tion. These results demonstrate, for the first time, that wholetranscriptome sequencing can be used for interrogation of acancer immunome and for advancing immune-based prognosis.

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Insights into Chronic Inflammation-InducedImmunosuppression in Cancer: from Mouseto Human

Michal Baniyash1, Moshe Sade-Feldman1, JuliaKanterman1, Yaron Meirow1, Ayala Hubert2, Michal Lotem2

1The Lautenberg Center for Immunology and Cancer Re-search, IMRIC, Faculty of Medicine, The Hebrew Universityof Jerusalem, Jerusalem, Israel2Sharett Institute of Oncology, Hadassah University MedicalCenter Ein Kerem, Jerusalem, Israel

Chronic inflammation is considered as the secret killer as it canlead to various life threatening complications including cancer.In addition, developing tumors by themselves can induce mi-cro and macro chronic inflammatory environments. Underboth circumstances, an immunosuppressivemilieu ensues, me-diated primarily by myeloid derived suppressor cells, whichenable escape of the tumor from immune surveillance. In thecourse of our studies we explored the underlying mechanismsfor the observed immunosuppression and demonstrated thatsuch an environment suppresses not only the host’s immunesystem but also newly administered immune cells andvaccination-based therapies thus, limiting success of cancerimmunotherapies. In addition, we discovered that chemother-apeutic drugs have adverse effects on the immunosuppressiveenvironment and thus, dictate tumor regression or progression.We are currently analyzing modalities/drugs that can neutral-ize the immunosuppressive environment and increase effica-cies of given chemo- and immune-based therapies. We arealso establishing a high-fidelity detection system using uniquebiomarkers for monitoring the host’s immune status modifiedby chronic inflammation that could predict success of giventherapies and disease regression/progression. To this end, weare using various mouse models such as a pathology freemodel, inflammatory bowel disease and inducible colorectalcancer, all driven by chronic inflammation. The obtained re-sults are then beeing “translated” to the clinic.Our recent retrospective studies show the first proof of conceptin humans; analyses of blood samples from cancer patients forthe expression of the biomarkers that sense the host’s immunestatus prior to and following chemo- and immune-based thera-pies could predict therapy efficacies and success. Such studiescould facilitate designing of innovative combinatorial strategiesfor cancer therapies and immune system monitoring towardsestablishing optimal personalized cancer treatments.


08:30–10:45

SYMPOSIUM 1: Regulatory Pathways in the TumorMicroenvironment (I)

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Optically Activated Receptor Tyrosine Kinases(Opto-RTKs) for Investigating Tumor-StromaInteractions

Michael Grusch1, Alvaro Ingles-Prieto2, Karin Schelch1, EvaReichhart2, Stephanie Kainrath1,2, Harald Janovjak2

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1Institute of Cancer Research, Comprehensive Cancer CenterVienna, Medical University of Vienna, Vienna, Austria2Synthetic Physiology Research Group, IST Austria (Instituteof Science and Technology Austria), Klosterneuburg, Austria

Optogenetic methods are based on genetically encodedsignaling molecules that can be rapidly and non-invasively switched on or off by light. Optogenetic ap-proaches had a big impact in the neurosciences by en-abling control of neuronal activity with supreme spatio-temporal resolution. In the last few years the optogenetictoolkit underwent a massive expansion and now enablesthe optical control of fundamental cell signaling pathwayswith high precision (1). Different research groups havecreated approaches for the optical activation of, for in-stance, the mitogen-activated protein kinase (MAPK),the phospatidylinositol-3 kinase (PI3K) and the Wnt path-way. Deregulation of these signaling pathways in tumorand stromal cells plays a huge role in malignancy. Ourgroup has recently engineered receptor-tyrosine kinasesthat can be activated by light-induced dimerization(Opto-RTKs)(2). Light thus enabled fine-tuned control ofthe MAPK, PI3K and phospholipase Cγ (PLCγ) path-ways and in consequence regulation of cell behavior rel-evant for tumors and their microenvironment: cell prolif-eration, epithelial to mesenchymal transition (EMT) andsprouting of endothelial cells. Current Opto-RTKs fromour group include Opto-mFGFR1, Opto-hEGFR andOpto-hRET. The corresponding endogenous receptorsare hyperactivated in various tumors and are also relevantin the immune system and for neoangiogenesis. Advan-tages of optogenetic methods are the ease, specificity,speed and spatial precision with which stimuli can beapplied and withdrawn. This unique combination of fea-tures can be exploited for a multitude of applicationsranging from dissection of intracellular signaling dynam-ics to cell-type specific and fine-tuned interventions intumor-stroma co-culture models (3).(1) Zhang et al., Trends Biotechnol 33:92–100, 2015; (2)Grusch et al., EMBO J 33:1713–26, 2014; (3) Ingles-Prietoet al., Mol Cell Oncol 2: e964045-2, 2014

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High Expression of Connective Tissue GrowthFactor (CTGF/CCN2) Modifies the Bone MarrowMicroenvironment and Accelerates Disseminationof Leukaemia

Ursula Kees1,2, Julia Wells1,2, Meegan Howlett1,2, LaurenceCheung1,2, Sajla Singh1, Jasmin Heng1, Jette Ford1, CatherineCole1,2

1Telethon Kids Institute, The University of Western Australia,Subiaco Perth, Western Australia, Australia2School of Paediatrics and Child Health, The University ofWestern Australia, Crawley Perth, Western Australia, Australia

International cooperative clinical trials for childhood leukae-mia have successfully increased the overall cure rate to over85 %. The current focus is to improve outcome for patientsthrough discovery of less toxic targeted therapies. The bonemarrow microenvironment plays an important role in leukae-mogenesis and minimal residual disease, facilitating drug re-sistance and disease relapse. A target not yet explored in leu-kaemia is the extracellular matrix (ECM).Our laboratory aims to identify ECM-associated drug targetsin B-cell precursor acute lymphoblastic leukaemia (pre-BALL). We identified connective tissue growth factor (CTGF/CCN2) as over-expressed in approximately 75 % of patientsamples in childhood pre-B ALL (Boag et al. Leukemia,2006,20:1731; Boag et al. BJH 2007,138:740). CTGF is amatricellular protein and is significantly involved in aggres-sive cancers (Wells et al. Int J Cancer 2015,137:504; Wellset al. J Cell Commun Signal. 2015 Epub May 31). To testwhether CTGF plays a functional role in disease outcomewe engrafted NOD/SCIDmicewith pre-BALL cells that werelentivirally transduced to express increased or reduced levelsof CTGF. Increased CTGF expression in PER-371 cells re-duced survival by 11 days (p=0.003). Reduced CTGF expres-sion extended mean survival by 14 days (p<0.0002) in PER-371, and 38 days (p=0.0015) in PER-377 xenografted mice.Increased CTGF expression was associated with increasedlysyl oxidase (LOX) expression in non-leukaemic cells at anearly stage of disease development. At later stages of diseasedevelopment increased CTGF was associated with increasedECM deposition.Further studies are in progress to explore targeting theoverexpressed CTGF and LOX proteins in the bone marrow,shown to be associated with increased ECM synthesis and anaccelerated spread of disease.

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Malignancy of Bladder Cancer Cells is Enhancedby Tumor Associated Fibroblaststhrough a Cytokine-Chemokine-Loop

Karlheinz Friedrich1, Susanne Grimm1, Susanne Jennek1,Rajan Singh1, Vrinda Mohta1, Astrid Enkelmann2, KerstinJunker21Institute of Biochemistry II, University Hospital Jena, Jena,Germany2Department of Urology, University Medicine Saarland,Homburg, Germany

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The tumor microenvironment plays an important role in can-cer progression. In particular, stromal fibroblasts have pro-found influence on migration and invasiveness of tumor cells.We employed urinary bladder carcinoma (UBC) cell lines ofdifferent de-differentiation grade to pursue the hypothesis thattumor cell malignancy correlates with the cells’ potential torecruit support by tumor associated fibroblasts (TAFs).UBC cell lines RT112 and Cal-29 were used as models fortumor cells of low or high de-differentiation grade, respective-ly. They were grown separately or in co-culture with TAFsisolated from bladder carcinomas. Moreover, conditioned me-dium (CM) fromUBC cell lines or TAFs, respectively, as wellas recombinant cytokines were analysed for their effects onmalignancy-related parameters.Migration and invasiveness of UBC cell lines RT112 and Cal-29 was enhanced by co-cultivation with TAFs. Low grade cellRT112 cells, however, were much more dependent on TAF-derived signals to enforce malignancy parameters. CM fromtumor cells induced the secretion of Matrix Metalloprotein-ases (MMPs) and the release of Interleukin- (IL-) 8, Hepato-cyte Growth Factor (HGF), and Monocyte Chemotactic Pro-tein 1 (MCP-1, CCL2) by TAFs.We could identify tumor cell-derived IL-1α as well as TransformingGrowth Factor- (TGF-)β as stimuli responsible for the upregulation of IL-8, HGF andMCP-1 in in fibroblasts. While HGF and MCP-1 promotedthe migration/invasiveness of UBC cells, IL-8 induced down-regulation of E-cadherin expression in low-grade RT112 cells,a central aspect of epithelial-mesenchymal transition (EMT).These results support a model of reciprocal, cytokine/chemokine-mediated crosstalk between UBC cells and stro-mal TAFs which favors the development of malignant tumorproperties.Supported by: Interdisziplinäres Zentrum für KlinischeForschung (IZKF) Jena, European Regional DevelopmentFund ERDF.

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KRAS Mutation in Colorectal Cancer is Associatedwith a Stromal-Derived Gene Signature

Moshe Elkabets2,3, Raphael Pelossof2, Oliver S. Chow1,Lauren Fairchild1, Chin-Tung Chen1, Manu Setty1, JesseJoshua Smith1, Kevin P. O’Rourke1, Scott W. Lowe1, Chris-tina S. Leslie1, Jose Baselga2, Julio Garcia-Aguilar11Department of Radiation Oncology, Memorial Sloan Ketter-ing Cancer Center, New York, New York, USA2HOPP, Memorial Sloan Kettering Cancer Center, New York,USA3Immunology, Microbiology and Genetics, Ben-Gurion Uni-versity of the Negev, Beer-Sheva, Israel

Background: KRAS mutation in colorectal cancer (CRC) ischaracterized by an altered transcriptional profile when com-pared towild-type (WT)KRAS tumors. The list of differentiallyexpressed genes between KRAS mutant and WT CRC tumorsis associated with a stromal fibroblast activation (SFA) signa-ture. Here we confirm the variation of the SFA signature withKRASmutation and infer its origin in the stromal component ofthe tumor using experimental models and in clinical samples.Methods: The SFA signature was assessed in an inducible-KRAS murine CRC model using RNA-sequencing, and in aCRC cell line with and without a transduced KRAS mutant vec-tor by microarray analysis. Moreover, RNA-sequencing of CRCpatient-derived xenografts (PDXs) was used to determine wheth-er the SFA signaturewas being expressed in the tumor epitheliumor the surrounding stroma. Finally, we validated the expression ofPOSTN (SFA–gene) in large cohort of CRC cancer patients.Results: The SFA signature was identified in the inducible-KRAS mouse model, matching human cohort observations ofdecreased SFA gene expression in KRAS mutant CRC. On theother hand, KRAS transduction did not recapitulate the SFAsignature in a CRC cell line. RNA-seq reads for SFA signaturegenes in CRC PDXs data suggest that the SFA transcriptionalprogram is associated with the stroma rather than the epithelialtumor. Finally, expression of POSTNwas associated with KRASWT in CRC cancer patient. cells.Conclusions: KRAS mutation in CRC is associated with a geneexpression signature derived from the tumor stroma. These find-ings suggest that KRAS mutation in the epithelial tumor cellsmay impact the tumor microenvironment in CRC.

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Carbamoylphosphonates Inhibit Extracellular ZincEnzymes in the Tumor Microenvironment

Eli BreuerInstitute for Drug Research, The Hebrew University of Jeru-salem, Jerusalem, Israel

The tumor microenvironment contains certain extracellular zincenzymes that safeguard the cancer and support its proliferationand dissemination. These water soluble enzymes are (a) matrixmetalloproteinase-2 (MMP-2), that degrades basement mem-brane and opens the way to the dissemination of metastases;(b) carbonic anhydrases (CAIX & XII) that regulate tumormicro-environment pH to support tumor survival; and finally(c) autotaxin, (ATX) that converts lysophosphatidylcholine(LPC) to lysophosphatidic acid (LPA) which supports cellularproliferation, tumor growth andmetastasis. In view of this hostilemicroenvironment, scientists are making immense efforts to de-velop inhibitors to the abovementioned enzymes.We developedwater soluble carbamoylphosphonic acid (CPO) inhibitors to

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maximize their chances to encounter and inhibit the enzymes inthe extracellular space, away from cancer or healthy cells.Our compounds inhibit all three types of enzymes mentioned atsimilar concentrations. Inhibition of MMP-2 prevents the dis-semination ofmetastases.[1] Inhibition of CAIX or CAXIImod-ulates the pH of the extracellular fluid disabling the advantageof tumor cells,[2] or inhibition of ATX stops the generation ofLPA and tumor proliferation.[3] Successful inhibition of theseenzymes by CPOs disables cancer and results in a new kind ofextracellular, nontoxic antimetastatic & anticancer therapy.References[1] J. Frant, A. Veerendhar, T. Chernilovsky, S. Nedvetzki, O.Vaksman, A. Hoffman, E. Breuer, R. Reich, ChemMedChem,2011, 6, 1471.[2] R. Reich, A. Hoffman, A. Veerendhar, A. Maresca, A.Innocenti, C.T. Supuran, E.Breuer, J.Med.Chem. 2012,55, 7875.[3] Reich, R. Hoffman, A. Suresh, R.R. Shai, O., Frant, J.Maresca, A., Supuran, C.T., Breuer, E. J Enzyme Inhib.Med. Chem., doi:10.3109/14756366.2014.968146.

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LOX-1: a Novel Metabolic Target in Human BreastCancer

Sabina Pucci, Chiara Polidoro, Francesca Amati, MichelaBiancolella, Elena Morini, Barbara Rizzacasa, MichelaMurdocca, Francesca Mastrangeli, Augusto Orlandi, FedericaCarla Sangiuolo, Giuseppe NovelliBiomedicine and Prevention, University of Rome Tor Vergata,Rome, Italy

Targeting the metabolic pathways of cancer is a hot topic fordrug discovery. OLR1 gene, encodes the cell membrane re-ceptor LOX-1 (Lectin-like oxidized low density lipoproteinreceptor). We recently identifies two novel alternative Olr1isoforms, whose expression is modulated in mouse duringdevelopmental stages of cardiogenesis. Hormones such as es-trogens and growth factors in the tumor microenvironmentcontribute to the transcriptional regulation of metabolic geneLOX-1 also through the activation of downstream signalingand a cross-talk among diverse transduction pathways. Ouraim is to delineate the role of the metabolic gene codifyLOX-1 in breast cancer . A strong up-regulation of LOX-1was observed in about 70 % of breast cancer tissues positivelycorrelated to the tumor stage and grade. Interestingly, LOX-1expression in tumoral tissues was related to TGFβ expressionin the microenvironment. On the contrary a faint LOX-1expression was observed in the normal counterpart of thesame patients. A peculiar and atypical nuclear localizationwas noted only in highly aggressive tumors correlated toHER2 amplification. A positive correlation between the

metabolic oncogene FASN and LOX-1 was found in tumorsHER2 positive suggestinga direct cross talk among EGFR,FASN and LOX. OLR1 Knock down by shRNAi inMCF7(ER+) and SKBR3 (Her2+) breast cancer cells resultsin down-regulation of genes necessary for oncogenic transfor-mation and maintenance of transformed phenotypeThe over-expression of LOX-1 specific isoforms in vitro strongly in-crease MCF12 normal cell proliferation,in cancer cells mark-edly enhanced proliferation, migration, affecting the epigenet-ic regulation of cancer pro-survival, and tumor invasion path-ways. Altogether, these results suggest that LOX-1 may actsas a molecular link among metabolism, inflammation andcancer, indicating its potential role as biomarker and new mo-lecular target, representing an attractive and concrete opportu-nity to improve current strategies for breast cancer therapy.

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Exosomes Secreted by Macrophages FacilitateGemcitabine Resistance in Pancreatic DuctalAdenocarcinoma

Yoav Binenbaum1, Jacob T. Cohen1, Eran Fridman1, TomerShlomi2, Gil Ben-David1, Ziv Gil1,31Clinical Research Institute at Rambam Health Campus,Laboratory of Applied Cancer Science, Haifa, Israel2Department of Computer Science, Lokey Center for Life Sci-ence and Engineering, Technion-Israel Institute of Technolo-gy, Haifa, Israel3Research in the Medical Sciences, Ruth and BruceRappaport Faculty of Medicine, Technion-Israel Institute ofTechnology, Haifa, Israel

Gemcitabine is the drug of choice for Pancreatic DuctalAdenocarcinoma(PDAC), albeit resistance to the drug is al-most uniform. We recently demonstrated that tumor-associatedmacrophages(TAMs) cause Gemcitabine resistanceby inducing Cytidine-deaminase(CDA) in the PDAC cell, anenzyme that metabolizes Gemcitabine to its inactive form,dFdUridine1. The goal of the present work was to elucidatethe mechanism by which TAMs communicate with PDACcells, inducing CDA expression.Electron microscopy of TAM condition medium showed thatits abundant with micro-vesicles expressing molecularmarkers of exosomes. Application of purified macrophage-derived-exosomes(MDEs) on PDAC cells doubledGemcitabine’s LD50% from 4.7–2 μM to 8.62 μM. TAMMDEs were enriched with miR-365 as compared withexosomes from M1 macrophages. Transfection of TAMs co-cultured with PDAC cells with 365-antagomiR led to reversalof resistance, in contrast to Rab 27a-/-b-/- TAMs lackingexosome secretion2. Treatment of PDAC cells with miR-365

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mimic or with MDEs, caused elevation in CDA expressionfollowed by elevation in dFdUridine.In an in-vivo adoptive immune-transfer experiment, micewere treated with Gemcitabine and either Rab 27a-/-b-/- mac-rophages; WT macrophages transfected with mock miR; orWT macrophages transfected with 365-antagomiR. By63 days all mice in the WT transfer group died, but only halfin the 365-antagomiR transfer group (P=0.03). Survival in theRab 27a-/-b-/- control was similar to 365-antagomiR group.Tumors exhibited minimal CDA expression in the Rab 27a-/-b-/- group, moderate expression in the 365-antagomiR group,and high expression in the WT transfer group.Our results demonstrate that Gemcitabine resistance is medi-ated by MDEs, shuttling miRNAs between TAMs and tumorcells. 365-antagomiR augments Gemcitabine efficacy in vitroand in vivo by mitigating CDA expression in the cancer cell,thus preventing inactivation of the drug.1. Weizman etal. Oncogene (2013). doi:10.1038/onc.2013.3572. Ostrowski etal. NatCellBiol. (2009). doi:10.1038/ncb2000

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Macrophage and Cancer Cell Crosstalk via CCR2and CX3CR1 is a Fundamental Mechanism DrivingLung Cancer

Anja Schmall1, Susanne Herold2, Marian Kampschulte2,Andreas Weigert3, Werner Seeger1,2, Soni Pullamsetti1,2,Rajkumar Savai1,21Department of Lung Development and Remodeling, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim,Germany2Department of Internal Medicine, Justus-Liebig University,Giessen, Germany3Institute of Biochemistry I, Goethe-University Frankfurt,Frankfurt, Germany

Rationale:Recent studies indicate that tumor associated mac-rophages with anM2 phenotype can influence cancer progres-sion and metastasis, but the regulatory pathways remain poor-ly characterized.Objectives: This study investigated the role of tumor-associated macrophages (MΦ) in lung cancer.Methods and results: Co-culturing of MΦ with mouse LewisLung Carcinoma (LLC1) and 10 different human lung cancercell lines (adenocarcinoma, squamous and large cell carcinoma)caused upregulation of CCR2/CCL2 and CX3CR1/CX3CL1 inboth the cancer cells and theMΦ. In theMΦ-tumor cell system,IL-10 drove CCR2 and CX3CR1 upregulation, while CCL1,G-CSF and MIP1a were required for the upregulation of CCL2and CX3CL1. Downstream phenotypic effects included en-hanced LLC1 proliferation and migration and MΦ M2

polarization. In vivo, MΦ depletion (clodronate, MaFIA mice)and genetic ablation of CCR2 and CX3CR1 all inhibited LLC1tumor growth and metastasis, shifted tumor-associated MΦ to-wards M1 polarization, suppressed tumor vessel growth andenhanced survival (metastasis model). Furthermore, mice treat-ed with CCR2 antagonist mimicked genetic ablation of CCR2by reducing tumor growth and metastasis. In human lung can-cer samples, tumor MΦ infiltration and CCR2 expression cor-related with tumor stage and metastasis.Conclusions: Tumor-associated MΦ play a central role in lungcancer growth and metastasis, with bidirectional crosstalk be-tween MΦ and cancer cells via CCR2- and CX3CR1-signalingas an central underlying mechanism. These findings suggestthat the therapeutic strategy of blocking CCR2 and CX3CR1may prove beneficial for halting lung cancer progression.

08:30–10:45

SYMPOSIUM 2: The Inflammatory and ImmuneTumor Microenvironment

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Tumor Related Neutrophils—A New Challengein Cancer Immunology and Immunotherapy

Zvi Fridlender1, Inbal Mishalian1, Jitka Sagiv2, JannaMichaeli1, Liran Levy1, Rachel Bayuch1, Simaan Assi2, RonitSionov2, Zvi Granot21Institute of Pulmonary Medicine, Hadassah-HebrewUniversity Medical Center, Jerusalem, Israel2Department of Developmental Biology and Cancer Research,Institute for Medical Research Israel-Canada, HebrewUniversity Medical School, Jerusalem, Israel

Asignificant portion of the inflammatory cell infiltrate in cancer isconsist of neutrophils. During the last years, we and others havepublished several studies challenging the limited view of neutro-phils as short-acting phagocytic cells. We demonstrated that Tu-mor associated neutrophils (TAN) can have a dual role in tumorbiology, polarized by the tumor microenvironment to have eitheranti-tumorigenic (‘N1’) or pro-tumorigenic (‘N2’) functions. Cir-culating neutrophils in cancer have been shown to be capable ofdirect tumor cytotoxicity. N1/N2 TAN differ from each other,from naïve neutrophils (NN) and from the granulocytic fractionof MDSC (G-MDSC), with many immune-related genes/pathways up-regulated in TAN. In a recent study, we identifieda heterogeneous subset of circulating low-density neutrophils(LDN) that accumulate continuously with cancer progression.LDN display impaired neutrophil function and immunosuppres-sive properties, in stark contrast to those of mature, high-density

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neutrophils (HDN). Importantly, LDN consist of both immatureMDSC and mature cells derived from HDN. In additional workwe found several links between tumor neutrophils and the adap-tive immune system affecting the immune microenvironment.TAN are capable of recruiting regulatory T-cells into the tumorby secretion of CCL17, and currently we are investigating theircapability to induce apoptosis of CD8+ T-cells. Our findings pro-vide a mechanistic explanation to mitigate the controversy sur-rounding neutrophil functions in cancer. Proper understanding ofthe effect of tumors on neutrophils, as well as the way these cellssupport or fight cancer and affect tumor immune microenviron-ment, will help us develop strategies to direct the immune systemagainst the tumor. Our long term goal is to promote cancertreatment using manipulated neutrophils as a novel toolto treat advanced cancer and metastases.

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Neutrophil Subpopulation Ratio Determines OverallPro- or Antitumor Contribution

Zvi Granot1, Jitka Y. Sagiv1, Janna Michaeli2, Simaan Assi1,Inbal Mishalian2, Liran Levy2, Pazzit Damti2, LolaPloyansky1, Ronit V. Sionov1, Avi-Hai Hovav3, Erik Henke4,Zvi . Fridlender21Developmental Biology and Cancer Research, Institute forMedical Research Israel Canada, Hebrew University, Jerusa-lem, Israel2Institute of Pulmonary Medicine, Hadassah-Hebrew Univer-sity Medical Center, Jerusalem, Israel3Institute of Dental Sciences, Hebrew University-HadassahSchool of Dental Medicine, Jerusalem, Israel4Institut for Anatomy and Cell Biology, Universität Würzburg,Würzburg, Germany

In recent years it has become apparent that the non-malignantstroma surrounding tumors plays a critical role in the processes oftumor initiation, growth and metastatic progression. In this con-text the part neutrophils play has been a matter of debate. Neu-trophils are the most abundant leukocytes in the human circula-tion and are usually associated with inflammation and with fight-ing infections. In cancer, neutrophils were shown to provide avariety of pro-tumor functions including secretion of tumor pro-moting cytokines, degradation of the ECM, immune suppressionand more. In contrast, neutrophils were also shown to have thecapacity to kill disseminated tumor cells either through directcytotoxicity or via antibody dependent cytotoxicity. These con-flicting reports suggest that although neutrophils are largelyviewed as a homogeneous population they may consist of dis-tinct subsets with significantly different properties. Indeed, weidentified a heterogeneous subset of low-density neutrophils(LDN) that appears transiently in self-resolving inflammation

but accumulates continuously with cancer progression. Whilehigh-density neutrophils (HDN) maintain a pro-inflammatory,anti-tumor phenotype, LDN present with a reduced inflammato-ry profile, impaired neutrophil function and acquire immunosup-pressive properties. In early tumor development HDN are thepredominant neutrophil subpopulation giving neutrophils, ingeneral, an anti-tumor phenotype. However, with tumor progres-sion, LDN are preferentially propagated to the extent that theybecome the dominant circulating neutrophil subpopulation.When this happens the overall neutrophil contribution switchesfrom anti- to pro-tumor. Our observations identify dynamicchanges in neutrophil subpopulations and provide a mechanisticexplanation to mitigate the controversy surrounding neutrophilfunction in cancer.

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Changes in the Colon Mucosal Scaffoldunder Activation of the Local Immunity: Lessonsfrom Germ-Free versus Conventional AnimalPro-Inflammatory Induction

Luca Vannucci1,6, Jiří Křížan1, Dmitry Stakheev1, FabiánČaja1, Lenka Rajsiglová1, Helena Tlaskalová-Hogenová2,Miloslav Kverka2, Oleksandr Chernyavskiy3, TomášHudcovic4, Renata Štěpánková4, Hana Kozaková4, JiříDvořak2, Peter Makovicky5, Pavel Rossmann2, MonikaČervinková6, Petr Šima11Laboratory of Immunotherapy, Institute of Microbiology ofthe Academy of Sciences of the Czech Republic, v.v.i., Prague,Czech Republic2Laboratory of Cellular and Molecular Immunology, Instituteof Microbiology of the Academy of Sciences of theCzech Republic, v.v.i., Prague, Czech Republic3Department of Biomathematics, Institute of Physiology of theAcademy of Sciences of the Czech Republic, v.v.i., Prague,Czech Republic4Laboratory of Gnotobiology, Institute of Microbiology of theAcademy of Sciences of the Czech Republic, v.v.i., NovyHradek, Czech Republic5Czech Centre for Phenogenomics, Institute of Molecular Ge-netics of the Academy of Sciences of the Czech Republic, v.v.i.,Prague, Czech Republic6Laboratory of Tumor Biology, Institute of Animal Physiologyand Genetics of the Academy of Sciences of theCzech Republic, v.v.i., Libechov, Czech Republic

The immune system is involved in modelling of tissue struc-tures. Constant immune stimulation by microbiota can impacton colon development, immunity, and health of individuals.Highly dynamic stromal structure remodeling is demonstratedby 2-photon microscopy second-harmonic generation (SHG)

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imaging in the mucosa of mice transferred from germ-free(GF) to conventional (CV) conditions (microbiota coloniza-tion). In rat model, healthy colon mucosa of CV rats expressesmore pro-inflammatory cytokines (IFNγ, IL1, TNFα) andTGFβ than GF rat mucosa. This more active network can alsojustify differences in stromal structures revealed by SHG.Analysis of SHG images proved significant differences inGF vs CV collagen architecture, suggesting direct effects ofimmunological network on it. Similarly, structural changes arewell documented in both induced chronic colitis and carcino-genesis models. They show very similar trend in tissue fibrosisthat starts early. Early peaks of INFγ and TGFβ1 expressionwere noted in both models. Azoxymethane carcinogenesisdetermined early increase of IL-6 and TGFβ1. This resultssuggest possible conditions for Th17 cell maturation with fos-tering carcinogenesis. Our results indicate a role of local pro-inflammatory/regulatory cytokines in remodelling the colla-gen scaffold under chronic inflammatory conditions. Stromalchanges appears a suitable new marker reflecting early immu-nological conditions accompanying the tumor microenviron-ment establishment. Acknowledgements: grants GAAVIAA500200917, CZ.1.05/2.1.00/03.0124 (Project ExAM),RVO 61388971, RVO 67985904 (CZ), Rusconi Foundation,Varese (IT), ENI Czech Republic, Prague (CZ), ManghiCzech Republic s.r.o, Prague (CZ), Unicredit Bank s.r.o.,Prague (CZ), Torino-Praga Invest and Rivergate s.r.o Prague(CZ), SIAD Czech spol. s.r.o., Prague (CZ)

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Peripheral Neuroglia Forms the Inflammatory-LikeTumor Microenvironment

Anton KeskinovPathology, University of Pittsburgh Medical Center, Pitts-burgh, PA, USA

Various divisions of the nervous system can affect tumor growthand progression in vivo. However, far less is known about themechanisms by which it is executed. Our program aims to in-vestigate how the sensory division of the peripheral nervoussystem acts in the formation of the tumor microenvironment atthe initial stage of carcinogenesis. Recently, we have reportedsensory DRG (dorsal root ganglia) neurons to support tumorgrowth in vivo in experimental animal models. Our new dataconfirm these findings and provide new insights into the poten-tial mechanisms of the phenomenon. We have found thatmelanoma-activated neuroglia expresses high levels of a pro-inflammatory S100A9 protein that attracts myeloid-derived sup-pressor cells (MDSC). Additionally, tumor-activated DRG neu-rons up-regulate expression of pro-nodal/nodal protein, knownto induce polarization of conventional dendritic cells (DC) into

tolerogenic regulatory DC. These data support the concept of theearly interaction between initial malignant cells and the sensory(afferent) nervous system to result in the neuroglia-mediatedformation of the inflammatory-like tolerogenic tumorimmunoenvironment. Therefore, the cross-talk between cancer-ous, neuronal and immune cells plays a significant role in creat-ing the unique tumor milieu resulting in antitumor immunityinhibition and tumor growth progression.

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Oxygen Shapes CTL Function: Insights from LiveIntratumoral Imaging

Tali Feferman, Yoav Manaster, Yael Gruper, Guy ShakharDepartment of Immunology, Weizmann Institute of Science,Rehovot, Israel

Poor tumor vascularization is an obstacle to immunotherapyby CTLs. It impairs tumor infiltration but also introduces hyp-oxia, known to interfere with T cell migration. It is yet un-known how suboptimal vascularization affects CTLmigrationand function within tumors.To study this question, we combined immunohistochemistry ofhuman melanoma samples with two-photon imaging in livemice. Orthotopically implanted B16-OVA tumors were studiedafter adoptive transfer of in-vitro matured antigen-specific OT-ICTLs. In patients, CD8 T cells concentrated around peripheralvessels in the tumor and sparsely infiltrated avascular areas. Inmice, CTLs crawled rapidly in oxygenated areas within 50 μmof flowing blood vessels. Occluding intratumoral blood vesselstriggered immediate arrest of CTL motility, which was quicklyreversed when flow was resumed. Immunohistology indicatedthat CTLs avoided hypoxic tumor areas. Live CTL imagingin vitro showed deceleration under hypoxic conditions andwhenoxidative phosphorylation was blocked.We employed several strategies to circumvent intratumoralCTL dysfunction. To change the vascular density we im-planted tumors in matrices containing or devoid of bFGF.bFGF-laced tumors were more easily rejected after transferof CTLs and displayed delayed growth in untreated mice butwere not affected in mice deficient in CD8 T-cells. To accli-matize them to a harsh hypoxic environment, OT-I CTLs wereexpanded and cultured in Oxygen-depleted conditions.In vitro, such CTLs killed tumor cells more efficiently underboth normoxic and hypoxic conditions. In-vivo, they sup-pressed tumor progression more effectively.We propose that CTLs depend on Oxygen and normal vascu-larization for intratumoral migration and cytolysis, Tumor im-munotherapy using hypoxia-resilient CTLs or combined withpro-angiogenic treatment with may thus improve clinicaloutcome.

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O33

Single Nucleotide Polymorphisms, Proteinsand Cytokines Related to InflammatoryMicroenvironment: Biomarkers for Cancer Riskand Clinical Outcome of Head and Neck SquamousCell Carcinoma Patients

Nongnit Laytragoon Lewin1, Lena Cederblad2, Bengt-ÅkeAndersson1, Mattias Olin3, Mats Nilsson4, Lars-ErikRutqvist5, Jan Lundgren6, Sture Löfgren1, Freddi Lewin31Laboratory Medicine, Ryhov Hospital, Jönköping, Sweden2Oncology, Uppsala University, Uppsala, Sweden3Oncology, Ryhov Hospital, Jönköping, Sweden4Futurum, Ryhov Hospital, Jönköping, Sweden5North Europe, Swedish Match AB, Stockholm, Sweden6ENT, Karolinska Hospital, Stockholm, Sweden

Smoking or human papilloma virus (HPV) was suggested tobe the causative agents of head and neck squamous cell car-cinoma (HNSCC). Genetic are also likely to play a role sinceonly a proportion of smokers or HPV carriers get cancer. Re-cently, we found that cigarette smoke induced massive celldeath, altered cellular phenotype and gene expression profileand these were related to single nucleotide polymorphisms(SNPs). A possible role of the chronic inflammatory microen-vironment, individual genetic variation and its associationwith cancer risk, tumour recurrence or clinical outcome ofHNSCC was investigated. Circulating plasma proteins or cy-tokines and SNPs in characterized genes were used as bio-markers for this purpose.Statistically significant differences in frequency of SNPs re-lated to cell death and inflammation were found between 174HNSCC patients and 245 healthy controls (p0,05). The levelof circulating plasma C-reactive protein and tumour necrosisfactor alpha at the diagnosis was both clinically and statisti-cally significantly related to tumour recurrence and survivaltime of these patients. Thus, SNPs and circulating plasmaprotein or cytokine related to inflammatory microenvironmentmight be used as biomarker for cancer risk and clinical out-come of HNSCC patients.

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The Pro-Inflammatory Role of Vimentinin the Colon

Nirit Mor-Vaknin, Maureen Legendre, Yu Yue, John Y Kao,Grace Y Chen, David M MarkovitzInternalMedicine, The University ofMichigan School ofMed-icine, Ann Arbor, MI, USA

Inflammatory Bowel Disease (IBD) is most frequently mani-fested by ulceration and chronic inflammation of the gastroin-testinal tract and is associated with increased risk of colorectalcancer. IBD is believed to be caused by an abnormal immuneresponse to commensal bacteria, as well as environmental fac-tors and genetic predisposition. Recent treatments for IBD in-clude anti-inflammatory drugs, which may increase the risk ofsecondary infections and cancer, thereby increasing the needfor preventative care and improved treatment modalities.Two distinct studies of human tissue of gene expression duringthe course of IBD have shown increased vimentin, expressionespecially in ulcerative colitis. In addition, interaction betweenvimentin and nucleotide binding oligomerization domain-containing protein 2 (NOD-2) has been found. Despite thesefindings, vimentin’s function and its role in IBD remain elusive.We now suggest a new role for the vimentin protein in intes-tinal homeostasis. Using E. coli and dextran sodium sulfate(DSS)-induced colitis mouse models, we have found that Vim-/- mice show significant resistance to the development ofacute and chronic colitis, while under steady state conditionsVim -/- mice have a remarkable normal phenotype and verysubtle abnormalities. Moreover, in a model of colitis-associated tumors induced by azoxymethane (AOM) andDSS, vimentin -/- mice developed significantly less tumorsthan wild-type mice under the same conditions. Our resultssuggest that vimentin suppress the production of reactive ox-ygen species (ROS) and autophagy. In addition, vimentin caninduce production of pro-inflammatory cytokines such as IL-1β and IL-6. Thus, we concluded that vimentin support chron-ic inflammation of the colon and promote tumorogenesis.

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A Novel Immune Resistance Mechanismof Melanoma Cells Controlled by the ADAR1Enzyme

Gilli Galore Haskel1,3, Yael Nemlich1, Eyal Greenberg1,3,Shira Ashkenazi1,3, Motti Hakim4, Orit Itzhaki1, NoaShoshani1, Ronnie Shapira-Fromer1, Eytan Ben-Ami1, EfratOfek2, Liat Anafi2, Michal J. Besser1,3, Jacob Schachter1, GalMarkel1,3,51Sheba Medical Center, Ella Lemelbaum Institute of Melano-ma, Ramat-Gan, Israel2Sheba Medical Center, Institute of Pathology, Ramat-Gan,Israel3Tel Aviv University, Department of Clinical Microbiologyand Immunology, Tel Aviv, Israel4cCAM Biotherapeutics, cCAM Biotherapeutics, Misgav,Israel5ShebaMedical Center, TalpiotMedical Leadership Program,Ramat-Gan, Israel

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The blossom of immunotherapy in melanoma highlights theneed to delineate mechanisms of immune resistance. Recently,we have demonstrated that the RNA editing protein, adenosinedeaminase acting on RNA-1 (ADAR1) is down-regulated dur-ing metastatic transition of melanoma, which enhances mela-noma cell proliferation and tumorigenicity. Here we investi-gate the role of ADAR1 in melanoma immune resistance.Importantly, knockdown of ADAR1 in human melanomacells induces resistance to tumor infiltrating lymphocytes ina cell contact-dependent mechanism. We show that ADAR1,in an editing-independent manner, regulates the biogenesis ofmiR-222 at the transcription level and thereby IntercellularAdhesion Molecule 1 (ICAM1) expression, which conse-quently affects melanoma immune resistance. ADAR1 thushas a novel, pivotal, role in cancer immune resistance. Cor-roborating with these results, the expression of miR-222 inmelanoma tissue specimens was significantly higher in pa-tients who had no clinical benefit from treatment withipilimumab as compared to patients that responded clinically,suggesting that miR-222 could function as a biomarker for theprediction of response to ipilimumab.These results provide not only novel insights on melanomaimmune resistance, but also pave the way to the developmentof innovative personalized tools to enable optimal drug selec-tion and treatment.

08:30–10:45

SYMPOSIUM 3: Targeting the Tumor Microenvi-ronment—Preclinical and Clinical Trials

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G-Protein Coupled Receptors; GPCRs in EpithelialCancers and Novel Targeted PH-Domain DindingMotifs

Rachel Bar-Shavit1, Myriam Maoz1, Arun Kancharla1,Mohammad Jaber1, Ariel Sebag-Shimoni1, DanielAgranovich1, Sorina Grisaru-Granovsky2, Beatrice Uziely11Department of Oncology, Hadassah-Hebrew UniversityMedical center, Jerusalem, Israel2Department of Obstetrics and Gynecology, Shaare ZedekHospital, Jerusalem, Israel

Despite the fact that GPCRs emerge as oncogenes thatregulate cancer-associated signaling networks, their rolein tumor biology is not well understood. Indeed, severalcancer “driver” GPCR-genes are becoming now recog-nized as playing a central role in tumor biology. Amongthese are protease-activated receptors; PARs, members of

chemokine receptors such as CXCR4 and in bone metas-tasis, the parathyroid hormone receptor; PTHR. We dem-onstrate novel signal-binding motifs in PAR1&2 C-tailsthat are critical for breast cancer growth. This is manifest-ed via the association of Akt/PKB pleckstrin-homology(PH)-domain as a key-signaling event of PARs. OtherPH-domain signal-proteins such as Etk/Bmx and Vav3 al-so associate with PAR1 and PAR2 through their PH-do-mains. Priority however, is allocated to Etk/Bmx. A pointmutation in PAR2, H349A, but not mutant R352A, abro-gated PH-protein association and was sufficient to mark-edly reduce PAR2-instigated breast tumor growth in vivoand placental extravillous trophoblast (EVT) time-limitedinvasion in vitro. Similarly, PAR1 mutant hPar1-7A, de-void of PH domain binding, markedly reduced mammarygland tumors and EVT invasion, endowing these motifswith physiological significance and underscoring the im-portance of novel PAR1 and PAR2 PH-domain bindingmotifs in both pathological and physiological invasionprocess. In the PTHR but not in CXCR4 we find similar-ly, a PH-domain binding motif located in its C-tail that iscritical for PTHR instigated invasion. This novel approachbroadens our prospect for identifying translational meansand suitable platform in cancer models.Reference: Kancharla A, Maoz M, Jaber M, Agranovich D,Peretz T, *Grisaru-Granovsky S, Uziely B, and Rachel Bar-Shavit . Nature Commun, in revision, June 2015

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Anakoinosis: Communicative Reprogrammingof Tumor Systems—for Rescuing ChemorefractoryNeoplasia

Christina Hart1, Martin Vogelhuber1, DanielWolff1, SebastianKlobuch1, Lina Ghibelli3, Jürgen Foell2, Selim Corbacioglu2,Klaus Rehe2, Guy Haegemann4, Simone Thomas1, WolfgangHerr1, Albrecht Reichle11Hematology and Oncology, University Hospital Regensburg,Regensburg, Germany2Pediatrics, University Hospital Regensburg, Regensburg,Germany3Biology, Universita di Roma Tor Vergata, Rome, Italy4Legest, University Gent, Gent, Belgium

Disruptive technologies, such as communicativereprogramming (anakoinosis) with cellular therapies insitu for treating refractory metastatic cancer allow patient careto accelerate along a totally new trajectory and highlight whatmay well become the next sea change in the care of patientswith many types of advanced neoplasia (Reichle A et al., Can-cer Microenvironment 2008, 2009). Cellular therapy in situ

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consisted of repurposed drugs, pioglitazone plus all-transretinoic acid or dexamethasone or interferon-alpha (dual tran-scriptional modulation) combined with metronomic low-dosechemotherapy or low-dose 5-azacytidine, plus/minus classictargeted therapy. The novel therapeutic tools for specificallydesigning communication processes within tumor diseases fo-cus on redirecting (1) rationalizations of cancer hallmarks(constitution of single cancer hallmarks), (2) modular events,(3) the ‘metabolism’ of evolutionary processes (the sum oftherapeutically and intrinsically inducible evolutionary pro-cesses) and (4) the holistic communicative context, whichdetermines validity and denotation of tumor promoting com-munication lines. Published data on cellular therapies in situ (6histologic tumor types, 144 patients, age 0.9 to 83 years) incastration-resistant prostate cancer (Vogelhuber M et al., Can-cer Microenvironment 2014), pretreated renal clear cell carci-noma (Biomark Insights 2007), chemorefractory acute mye-locytic leukemia (Thomas S et al., Haematologica 2014), mul-tiple myeloma 2nd-line (Reichle A et al., Blood 2012),chemorefractory Hodgkin lymphoma (Ugocsai U, Br. J.Haematol 2015) or multivisceral Langerhans cell histiocytosis(Reichle A et al., Br. J. Haematol 2005), outline the possibilityfor treating refractory metastatic cancer with the hope that thistype of reprogrammed communication will be scalable withminimal toxicity. Accessibility to anakoinosis is a tumor in-herent feature, and cellular therapy in situ addresses extrinsicand intrinsic drug resistance, by redirecting convergent orga-nized communication tools, while been supported by quitedifferent pattern of (molecular-)genetic aberrations.

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Roneparstat, a Novel Heparanase Inhibitorfor Multiple Myeloma Therapy

Israel Vlodavsky1, Paola Barbieri2, Alessandro Noseda2,Annamaria Naggi3, Ralph D. Sanderson41Cancer & Vascular Biology Research Center, Technion, Haifa,Israel2Sigma Tau Research Switzerland SA, Sigma Tau Pharmaceu-ticals, Mendrisio, Switzerland3Carbohydrate Research, Ronzoni Institute for Chemical andBiochemical Research, Milano, Italy4Department of Pathology, University of Alabama atBirmingham, Birmingham, USA

Heparanase cleaves heparan sulfate (HS) chains and therebyregulates multiple biological activities that enhance tumorgrowth, angiogenesis and metastasis. Much of the impact ofheparanase on tumor progression is related to its function inmediating tumor host crosstalk, priming the tumor

microenvironment to better support tumor growth.Heparanase represents a druggable target because there is onlya single active heparanase in humans, heparanase null miceexhibit no obvious deficits, and the enzyme is poorlyexpressed in normal tissues but elevated in human cancerwhere it is associated with poor prognosis and short postop-erative survival.Development of heparanase inhibitors focused on carbohydrate-based compounds with heparin-like properties. Among these isSST0001 (Proposed INN Roneparstat), a reduced oxidized N-acetylated heparin, a potent inhibitor devoid of significant anti-coagulant effect. In preclinical testing, SST0001 profoundly in-hibits multiple myeloma (MM) tumor growth, angiogenesis andbone metastasis. Moreover in a model of dexamethasone resis-tant MM, the combination of SST0001 with dexamethasoneinhibited tumor growth. This prompted a First in Man, multi-center phase I clinical study, currently ongoing in advancedheavily pre-treated refractory MM patients who have exhaustedthe available anti-MM therapies.Heparanase in MM has been extensively studied, suggestingthat it may play a role in affecting the tumor microenviron-ment as well as in determining resistance following chemo-therapy. Thus, the ability of SST0001 to affect tumor burdenin MM was further explored showing that SST0001 in pre-clinical models when combined with either bortezomib ormelphalan significantly improved the effect of either of thesedrugs alone.The outcome of the phase I clinical study together with thesestrong preclinical data will provide the rationale for furtherSST0001 development as combination therapy in MM.

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Polymer Drug Delivery Dystems for EffectiveTherapy and Modulation of TumorMicroenvironment

Milada Sirova1, Martin Studenovsky2, Tomas Etrych2,Veronika Horkova1, Ladislav Sivak1, Blanka Rihova11Laboratory of Tumor Immunology, Institute of Microbi-ology, The Czech Academy of Sciences, Prague,Czech Republic2Department of Biomedical Polymers, Institute of Macromo-lecular Chemistry, The Czech Academy of Sciences, Prague,Czech Republic

Polymer drug delivery systems represent a promising strategyof tumor treatment leading to complete control over the tumornot compromised by significant systemic toxicity. Conjuga-tion of a low-molecular-weight drug to a synthetic polymercarrier enables targeted drug delivery to tumor tissue/cells viaEnhanced Permeability and Retention (EPR) effect. Water-

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soluble N-(2-hydroxypropyl)methacrylamide (HPMA) is asuitable drug carrier, allowing creation of conjugates with var-ious structures, molecular weight, and with tuneable drug con-tent and release. The conjugates show significantly extendedcirculation time, preferential tumor accumulation, and limitedside toxicity of the drug. HPMA-based conjugates carryingcancerostatic drugs proved excellent anti-tumor capacity inexperimental murine models. Complete tumor regression to-gether with development of anti-tumor immune response, en-suring long-term resistance against the original tumor, wasdocumented in EL4 T cell lymphoma model following thetreatment with HPMA copolymer conjugates carrying doxo-rubicin or docetaxel. The anti-tumor immune response, whichis co-responsible for the therapeutic outcome, was demon-strated as a treatment-dependent activation of dendritic cells,by the presence of specific CD8+ T cells in the spleen, andimmune infiltrate within the tumor.Vasodilators such as nitric oxide (NO) enhance the EPR effectby increasing the blood flow. Three HPMA-based conjugatesof nitroesters were prepared as NO donors with the aim toachieve their tumor-selective accumulation and generation ofNO, resulting in EPR enhancement, thereby potentiating ac-cumulation of co-administered macromolecular cancerostatics.The NO donors release NO upon endocytosis by tumorcells, and can synergize with the polymer-boundcancerostatics. Together, we demonstrate that HPMAcan be successfully explored as a drug delivery systemfor targeted chemotherapy of tumors as well as modu-lation of tumor microenvironment.Supported by Czech Science Foundation, Projects 14-12742Sand 301/12/1254.

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Stromal Lymph Node Involvement Addsto the Prognosis of Stage III Colon Cancer Patients

Gabi van Pelt1, TorbenHansen2, Sanne Kjaer-Frifeldt3, EstherBastiaannet1, Vincent Smit4, Han van Krieken5, RobTollenaar1, Flemming Sorensen3, Wilma Mesker11Surgery, LeidenUniversityMedical Center, Leiden, Netherlands2Oncology, Vejle Hospital, Vejle, Denmark3Clinical Pathology, Vejle Hospital, Vejle, Denmark4Pathology, Leiden University Medical Center, Leiden,Netherlands5Pathology, Radboud University Medical Center, Nijmegen,Netherlands

Background: The tumor microenvironment has ample impacton the behavior of the malignant process in colon cancer (CC).Patients with a high percentage of stromal cells within the pri-mary tumor, determined by the tumor-stroma ratio (TSR), show

a poor prognosis. Analysis of this parameter in lymph nodes ofstage III CC patients was observed to be a heterogeneous pro-cess and may have impact on patients’ prognosis and treatment.Methods: Primary tumor (PT) and lymph node (LN) tissueslides from 187 patients with stage III colon cancer were an-alyzed for their TSR. Stroma-high (50 % stroma) and stroma-low (≤50 % stroma) groups were evaluated with respect todisease free survival (DFS).Results: Of 187 analyzed primary tumors, 84 (44.9 %) werescored as stroma-high and 103 (55.1 %) as stroma-low. In total53 patients had at least one stroma-high LN, and 134 patientshad one or more stroma-low LNs. Interestingly, 44 (23.5 %)patients had stroma-high as well as stroma-low LNs, and inanother 83 cases the TSR between PT and LNs differed; 57patients had a stroma-high PT but stroma-low LNs, and 26patients vice versa. As a result of the combination of the TSRanalysis of the PTand the involved LNs, 109 patients (58.3 %)were stroma-high and 78 (41.7 %) stroma-low, restaging13.4 % patients to stroma-high, with a significantly worse 5-year DFS compared to stroma-low patients [48 % versus66 %, p=0.014, HR=1.69 (95%CI 1.11–2.55)].Conclusion: The analysis of the TSR in LNs revealed themetastasizing process to be very heterogeneous. The presenceof abundant stroma in lymph nodes of stage III colon cancerpatients adds to the prognostic information of the primarytumor and supports selective treatment.

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Utilizing Fibroblasts Recruitment for Detectionand Targeted Therapy of Ovarian Tumors

Roni Oren1, Lian Narunsky1, Yoseph Addadi1, Gila Meir1,Ami Fishman2, Michal Neeman11Department of Biological Regulation, Weizmann Institute ofScience, Rehovot, Israel2Oncogenetic Laboratory, Meir Medical Center, Kfar Saba,Israel

Ovarian cancer is the most lethal gynecologic malignancy. It isoften diagnosed at late stage when metastases are widelyspread. Currently, patients diagnosed with ovarian cancer aretreated by debulking surgery and chemotherapy and survivalrate remains low. We have previously demonstrated specifichoming of labeled fibroblasts to solid ovarian tumors inmousexenograft model. In this work, we demonstrate the feasibilityof using naive fibroblasts for detection and therapy of ovarianmetastasis in mouse model. We used fresh human and mouseascites to demonstrate that fibroblasts are recruited to tumorcells in vitro. Using an in vivo metastatic model for ovariancancer in mice, we demonstrated that fluorescently labeledfibroblasts injected intra-peritoneally, were specifically

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recruited to tumor nodules (resulting in 93–100 % co-locali-zation). We further used fibroblasts recruitment for targetedtherapy. Fibroblasts cells over expressing the solublereceptor variant of VEGFR1 (s-Flt1) were injected tomice bearing tumors. The mice received two doses(day 7 and 14) of control or s-Flt1 expressing fibro-blasts. The injection of s-Flt1 expressing fibroblasts re-sulted in significant reduction in the ascites volume inthe mice and in reduced vascularization of adherent me-tastases. These results suggest fibroblasts could be anovel tool for targeting ovarian tumor metastases fordetection and therapy. Fluorescently labeled fibroblastscould serve as a beacon for surgeons to identify other-wise invisible metastases in ovarian patients using avail-able intra-operative fluorescence imaging tools. The fi-broblasts could assist in directed biopsies, advancingsurgical efforts and in delivery of anti angiogenic oranti tumor molecules.

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Harnessing Cancer’s pH-Vulnerability to Identifyan Optimal Two-Step Therapeutic Strategy

Erez Persi1,4, Miquel Duran-Frigola2, Mehdi Damaghi3, Pat-rick Aloy2, Robert Gillies3, Eytan Ruppin41Computational Biology, NIH/NCBI/NLM, Bethesda, Mary-land, USA2Structural Bioinformatics and Network Biology group, Insti-tute for Research in Biomedicine (IRB), Barcelona, Spain3Cancer Imaging and Metabolism, Moffitt Cancer Center,Tampa, Florida, USA4Center of Bionformatics and Computational Biology(CBCB), University of Maryland, College Park, Maryland,USA

The initiation and development of cancer is associated withmajor metabolic alterations. An important aspect and oftenoverlooked of cancer metabolism is the acidification of itsextracellular environment and the concomitant alkalizationof the cytoplasm, generating a reverse pH-gradient. Althoughmuch effort has been devoted to studying the consequences ofextracellular acidification of cancer’s microenvironment, therole and importance of intracellular alkalization remains poor-ly understood. Here we provide for the first time a systemsbiology comprehensive understanding of how changes in in-tracellular pH (pHi) are coupled to network-wide cancer met-abolic alterations, by integrating enzymatic pH-dependent ac-tivity profiles into human genome-scale metabolic models ofcancer and normal cells. We show that lowering pHi renderscancer cells vulnerable for disruption and contributes to re-versing its “Warburg” nature. This vulnerability is further

exploited to identify optimal metabolic targets whose inhibi-tion selectively kills cancer at low pHi. The results unravel anunprecedented role of intracellular pH in cancer metabolismand put forward a ground for novel combinatorial efficienttherapy.

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Novel MicroRNATargets and Targeting Technologyto Modulate Pancreatic Stellate Cells in PancreaticTumor

Praneeth Kuninty1, Jonas Schnittert1, Linda Bojmar2, PerSandström2, Gert Storm1, Jai Prakash11Targeted Therapeutics- Biomaterials Science and Technology,University of Twente, Enschede, Netherlands2Department of Clinical and Experimental Medicine,Linköping University, Linköping, Sweden

Pancreatic stellate cells (PSCs) are the main producers ofthe pancreatic tumor stroma, which in turn, promote tumorgrowth and metastasis. During carcinogenesis microRNA(miRNA) dysregulation is not limited to the cancer cellsbut also occurs in PSCs. We hypothesized that inhibitionof the upregulated miRNA in PSCs might inhibit the PSC-mediated tumor-promoting effects. In this study, we foundmiR-199a and -214 as novel targets in TGF-ß-activatedhuman PSCs. Transfection of anti-miR-199a or -214 sig-nificantly inhibited the TGFß-induced differentiation, asconfirmed by the inhibition of PSC markers (α-SMA, col-lagen, PDGFßR). Also, transfection of these antagomiRssignificantly reduced migration and proliferation of PSCs.To study the paracrine effect of PSCs on Panc-1 tumorcells, we generated spheroids of Panc-1 and PSCs usingthe hanging-drop method. We found that spheroids contain-ing PSCs transfected with anti-miR-199a or -214 weresmaller in size compared to that with control PSCs. Next,PSC-conditioned medium induced tumor cells proliferationand endothelial tube formation and these paracrine effectswere abrogated with PSCs transfected with anti-miR-199aor 214. These data confirmed that miR-199a and 214 arepotential therapeutic candidates in PSCs. Furthermore, todeliver antagomiRs preferably to PSCs, we developed acell-penetrating peptide based delivery system. This pep-tide makes NanoComplexes with anti-miR oligonucleotidesand delivers them into PSCs with a high efficiency (80 %).In contrast, Panc-1 cells showed a weaker uptake. Hightransfection of NanoComplexes in PSCs was likely due tothe abundance of syndecans (proteoglycans) in PSCs com-pared to Panc-1, as shown with mRNA expression. In con-clusion, we have found novel therapeutic miRNA targets inPSCs and also developed a technology to deliver anti-

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miRNA oligonucleotide to PSCs, which in combinationcan be applied for inhibiting PSC activity to treat pancre-atic tumor.

11:15-13:30

SYMPOSIUM 4: Regulatory Pathways in the TumorMicroenvironment (II)

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Tumor Specific Recruitment and Reprogramingof Mesenchymal Stem Cells in Tumorigenesis

Maty Tzukerman, Liron Berger, Yeela Shamai Shamai, KarlSkoreckiMolecular Medicine, Rapport Faculty of Medicine, Technion-Israel Institute of Technology and Rambam Medical Center,Haifa, Israel

Non-neoplastic stromal cells harvested from patient tumorswere identified as tumor-derived Mesenchymal Stem Cells(MSC) by their multi-potential capacity to differentiateinto adipocytes, osteoblasts and chondrocytes and by theexpression of MSC specific cell surface markers. Theseprocedures yielded also epithelial cancer cells and theircounterpart MSC from gastric carcinoma (GSC1) and lungcarcinoma (LC2). While the LC2 cancer cell growth isindependent of their LC-MSC, the GSC1 cancer cellgrowth is critically dependent on the presence of theircounterpart GSC-MSC or their conditioned medium(CM). The fact that none of the various other tumor-derived MSC were able to restore the specific effect ofGSC-MSC on GSC1 cancer cell growth suggests specific-ity of tumor-derived MSC, which are specifically recruitedand ‘educated’/reprogrammed by the cancer cells to sup-port tumor growth. Using cytokine array analysis, we wereable to demonstrate that GSC1 cell growth is mediatedthrough HGF/c-MET signaling pathway which is activatedexclusively by HGF secreted from GSC-MSC. An innova-tive approach demonstrates GSC1-mediated specific tro-pism of ‘naïve’ MSC from the adjacent tissue in a tumorspecific manner to support tumor progression. The resultssuggest that specific tumor tropic ‘naïve’ MSC arereprogrammed in a tumor-specific manner to support gas-tric tumor progression. Understanding the mechanisms in-volved in the interactions of the tumor cancer cells andtumor-derived MSC will constitute the basis for develop-ing multimodal anti-cancer therapeutic strategies that willalso take into account the specific tumor tropism proper-ties of MSC and their reprogramming.

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Novel Signaling Pathway in Chronic LymphocyticLeukemia—Pathophysiology and New Targetfor Therapy

Ben-Zion Katz1,2, Nili Dezorella1,2, Mika Shapiro1, IritAvivi1,2, Chava Perry1, Yair Herishanu1,21Hematology, Tel-Aviv Medical Center, Tel Aviv, Israel2Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Chronic lymphocytic leukemia (CLL) is the most common leu-kemia in the Western world. The clinical course varies amongpatients with CLL: in some it is indolent and prolonged whileothers experience an aggressive disease with a short life expec-tancy. In the last decade, the B-cell receptor (BCR) has emergedas a pivotal stimulus in CLL pathogenesis. The BCR responsive-ness in CLL cells is heterogeneous among patients and correlateswith disease aggressiveness. Our understanding of the criticalrole of the BCR pathway in CLL pathogenesis made it a centraltherapeutic target in this disease. Small inhibitory molecules,such as ibrutinib (directed against Btk), induce dramatic clinicalresponses in CLL patients. These are particularly prominentwithin the tissue compartments, and characterized by rapidshrinkage of enlarged lymph nodes and splenomegaly, and over-come classical poor prognostic factors such as genomic aberra-tions in the p53 gene. We have recently identified ectopic ex-pression of several proteins in CLL cells that are involved in theBCR signaling. Their expression levels correlate with ZAP70expression as well as time to disease progression and treatment.Herein, we provide a novel paradigm for BCR signaling in CLL,where an alternative signaling cascade exits in addition to theclassical pathway. Our data may further suggest that combinedtargeting of the classical and alternative BCR pathways could beamore effective approach to treat CLL, and perhaps define noveltherapeutic targets within the BCR pathway, as well as novelprognostic tools in CLL. These findings may also be applicablein future research of other lymphoproliferative diseases whichdepend on microenvironmental interactions.

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Targeting CD38 Inhibits Glioma and MelanomaProgression in Vivo

Reuven Stein1, Eran Blacher1, Bar Ben Baruch1, HilaShwartz2, Hananya Vaknine3, Neta Erez21Neurobiology, Tel Aviv University, Tel Aviv, Israel2Department of Pathology, Tel Aviv University, Tel Aviv, Israel3Department of Pathology, Wolfson Medical Center, Holon,Israel

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It is well established that in many cases tumor microenvironmentpromotes tumor progression. Among the tumors where their mi-croenvironment supports tumor expansion are glioma andmelanoma.CD38 is a proinflammatory ectoenzyme that catalyzes the for-mation of the calcium-mobilizing metabolites adenosinediphosphoribose (ADPR), cyclic ADPR (cADPR), and nicotinicacid adenine dinucleotide phosphate from its substrates nicotin-amide adenine dinucleotide (NAD) and NAD phosphate. CD38is also the major constitutively active NAD-degrading enzymeexpressed in mammalian cells, thereby regulating NADhomeostasis.In previous studies using CD38 deficient mice (Cd38-/-) weshowed that loss of CD38 in the glioma microenvironment at-tenuated glioma progression in vivo in a mouse model of gliomaprogression (GL261 glioma cells implanted into C57BL/6J micebrains). This effect was probably mediated via an inhibitory ef-fect on microglia, the main constitute of the glioma microenvi-ronment. Having demonstrated the effect of loss of CD38 onglioma we next examined whether such condition can inhibitalso melanoma progression. To this end we used two differentmouse models of melanoma progression: B16 or Ret melanomacells implanted subcutaneously into C57BL/6J mice. Our resultsshowed that loss of CD38 also inhibited melanoma progressionin these mouse models. Furthermore, fibroblasts seem to be in-volved in the effect of loss of CD38 on the melanoma microen-vironment. Collectively these results suggest that targeting CD38may be a new potential therapeutic approach for treating tumorsin which tumor microenvironment promotes tumor progression.To further test this possibility we identified a new small moleculeCD38 inhibitor, K-rhein. Treatment of glioma- or melanoma-bearing WT mice with K-rhein substantially inhibited tumor ex-pansion of both tumor types and this effect was CD38 dependentsince the inhibitor did not affect tumors progression in Cd38-/-

mice.

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From Hypoxia Resistance to Cancer Tolerance:the Adaptations Evolved in the Blind SubterraneanMole Rats

Imad Shams, Vered Domankevich, Hossam Eddini, IrinaZotkin, Anatolii Mamchur, Aaron Avivi, Irena ManovInstitute of Evolution & Department of Evolutionary and En-vironmental Biology, University of Haifa, Haifa, Israel

Biodiversity that evolved on Earth created solutions for variousbiological challenges. In this regard, the study of natural resis-tance to cancer observed in some wild animals is a promisingdirection in the fight against cancer. We recently reported that thehypoxia-resistant long-lived subterranean blindmole rat (Spalax)

possesses an outstanding cancer- tolerance to spontaneous andin vivo-induced carcinogens, and a direct inhibition of cancer cellgrowth by Spalax’s fibroblasts. Here we demonstrate that senes-cence with subsequent caspase-independent apoptosis is the pre-dominant mode of cancer cell death in response to factors re-leased by Spalax fibroblasts. Viability, colony formation andinvasion assays were used to monitor cancer growth of cell ofdifferent species (MDA-MB-231, PC-3, Hep3B and HepG2cells, as well as mouse- and Spalax-derived fibrosarcomas).Transwell migration assay was employed to evaluate chemotac-tic activity. In addition, effects of conditionedmedia generated bySpalax, rat or mouse fibroblasts on viability, nuclei and mito-chondria integrity and cell cycle distributions were investigated.Spalax fibroblasts monolayer strongly reduced tumor growthwhile mouse, rat and Acomys fibroblasts had no effects or stim-ulated colony formation. Similarly, Spalax fibroblasts suppressedcancer cells migration. Medium conditioned by Spalax fibro-blasts induced arrest of proliferation in different cancer cells.Protein expression profiling in Hep3B cells exposed to Spalaxfibroblast conditioned medium revealed alterations in extracellu-lar matrix signaling molecules and apoptotic proteins. Cancermicroenvironment is an integral part of the tumorigenesis wherecancer cells grow and invade. The scenario of cancer progressionincludes dynamic repression of natural host tolerance. We pro-pose Spalax as a natural cancer-resistant model in which cancer-ous cells do not receive support from adjacent stroma, andwherein tumors fail to proliferate and metastasize.

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Cell Autonomous and Non-Cell-AutonomousActivities of Heat Shock Factor 1 in Cancer

Ruth Scherz-Shouval1, Sandro Santagata2, Marc L.Mendillo1, Lynette M. Sholl2, Irit Ben-Aharon3, Andrew H.Beck2, Dora Dias-Santagata2, Martina Koeva1, Solomon M.Stemmer3, Luke Whitesell1, Susan Lindquist1,41Lindquist lab, Whitehead Institute, Cambridge, MA, USA2Pathology, Harvard Medical School, Boston, USA3Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv,Israel4Lindquist Lab, Howard Hughes Medical Institute, Cam-bridge, USA

For tumors to expand, invade and metastasize, the recruitmentand reprogramming of non-malignant stromal cells is required.Yet surprisingly little is known about the factors that drive thetranscriptional reprogramming of stromal cells within tumors.Our work shows that Heat-shock Factor 1 (HSF1), an evolution-arily conserved transcriptionmaster regulator, plays a crucial rolein this process. Across a broad range of human cancers, HSF1 isactivated not only in the malignant cells themselves, but in

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cancer-associated fibroblasts (CAFs). HSF1 drives a transcrip-tional program in CAFs that complements, yet is completelydifferent from, the program it drives in adjacent cancer cells.Recently we have shown that this CAF program is uniquelystructured to support the malignant potential of cancer cells in anon-cell-autonomous way. In early stage breast and lung cancer,high stromal HSF1 activation is strongly associated with poorpatient outcome. Here we further explore the HSF1-dependentcross talk between cancer and stroma.We dissect the mechanismof stromal HSF1 activation, identify key components of theHSF1-dependent stromal transcriptional program and highlightthe prognostic implications of cell-autonomous and non-cell-autonomous activation of HSF1.

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Immortalized Fibroblasts with Decreased RhoGTPase Activity Suppress Tumor Cell Growthin Vitro and in Vivo

Tatiana Pavlova, Pontus Aspenström, Benedek Bozoky,Twana Alkasalias, Hayrettin Guven, Georg Klein, Annica GadDepartment of Microbiology, Tumor and Cell Biology (MTC),Karolinska Institutet, Stockholm, Sweden

Cancer-associated fibroblasts (CAFs) are important factors forthe growth and spread of tumors. Fibroblasts that initiallyinhibit tumor growth may lose their inhibitory capacity andeven become stimulatory during tumor development. Usinghigh-throughput imaging system and field live cell microsco-py of tumor-fibroblast co-cultures, we showed, that the inhib-itory capacity of human hTERT-immortalized fibroblastsBjhTERT correlates with morphology and of their growinglayer. The organized “whirly” growth pattern is linked tostrong inhibition of tumor cell proliferation andmotility. Com-pared to disorganized “crossy” sub-clone, “whirly” fibroblastshad lower activity of RhoA, one of the major governor ofmechanical properties. To check, if RhoA activity in fibro-blasts correlates with tumor inhibitory capacity, we knockedout RhoA in BjhTERT. RhoA knocked out (KO) fibroblastshad “whirly” phenotype with significantly down-regulatedexpression of alpha-SMA and actin stress fibers, decreasedin width. They were less spread and elongated and showedpronounced peripheral actin protrusions and diffuse, feather-like, adhesions. Moreover the RhoA(KO)-BjhTERT demon-strated increased tumor inhibitory capacity in vitro andin vivo. After 80 days, only 13 % of SCID mice injected withPC3 cells and RhoA(KO)-BjhTERT had formed a tumor. Incontrast, 88 % of animals injected with a mixture of prostatecancer cells PC3 and BjhTERT with active RhoA haddeveloped tumor.

Our findings confirm, that mechanical properties of fibroblastsplay important role in tumor growth. Activation of RhoAleads to changes of the cytoskeleton and cell-matrix adhesionthat reduces the ability of fibroblasts to inhibit tumor growth.1. Flaberg E, Guven H, Savchenko A, Pavlova T, Kashuba V,Szekely L, Klein G. (2012) The architecture of fibroblastmonolayers of different origin differentially influences tumorcell growth. Int J Cancer.

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A Joint Analysis of Transcriptomic andMetabolomicData Uncovers Increased Metabolic TranscriptionalRegulation in Breast Cancer

AdamWeinstock1, Keren Yizhak1,3, Anuradha Budhu4, XinWang4, Perwez Hussain4, Wei Tang4, Stefan Ambs4, EytanRuppin1,2,51Blavatnik School of Computer Science, Tel Aviv University,Tel Aviv, Israel2Sackler school of medicine, Tel Aviv University, Tel Aviv,Israel3Getz Lab, The Broad Institute of MIT and Harvard,Cambridge, MA, USA4Center for Cancer Research, National Cancer Institute,Bethesda, MD, USA5Center for Bioinformatics and Computational Biology,University of Maryland, College Park, MD, USA

Recent years have witnessed a growing interest in the study ofcancer metabolism, facilitated by genome scale omics mea-surements that uncover the metabolic alterations occurring incancer versus normal cells. Here we harness machine learningtools to analyze metabolomic and transcriptomic profilesjointly collected from breast cancer patients to identify meta-bolic reactions controlled by transcriptional regulation andconstruct predictors of metabolite levels from the much moreubiquitous gene expression measurements. Analyzing theexisting measured data we find that cancer cells exhibit agreater number of significant transcriptional-metabolite asso-ciations than normal cells, testifying to an overall markedincrease in the transcriptional regulation of cancer metabo-lism. Following, we show that we can predict these associa-tions on unseen data with an accuracy exceeding 90%. Build-ing on these results we further construct genome-wide predic-tors of metabolite levels from gene expression data, whichcorrectly evaluate the levels of ~60 % of the measured metab-olites across samples based on their associated expression dataalone. Finally, we apply our pipeline to a large cohort of breastcancer samples to predict their corresponding metabolitelevels and examine their association with patients’ survival.We find that low levels of key known cancer related

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metabolites, including glucose, glycine, serine and acetate aresignificantly associated with improved survival time, andidentify new predicted metabolites of interest. Overall, thisanalysis is the first to identify and chart a global increase inmetabolic transcriptional regulation in cancer, and the first toprovide means for inferring metabolite levels from transcrip-tional data in breast cancer on a genome wide level. We arecurrently working to extend our analysis to liver cancer (HCC)and pancreatic cancer (PDAC).

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Reciprocal Cellular Cross-Talk within the TumorMicroenvironment Promotes Oncolytic VirusActivity

Carolina Ilkow1,2, Monique Marguerie1,2, Cory Batenchuk1,2, Victoria Jennings1,2, Theresa Falls1, John Bell1,21Centre for Innovative Cancer Therapeutics, Ottawa HospitalResearch Institute, Ottawa, Ontario, Canada2Department of Immunology and Microbiology, University ofOttawa, Ottawa, Ontario, Canada

Signalling between cancer cells and the normal stromalcells that support them is known to negatively impactthe activity of a variety of chemically based anti-cancertherapies1–4. In this regard, we have recently shown that,in the case of oncolytic virus based therapeutics, crosstalkbetween cancer- associated fibroblasts (CAFs) and cancercells leads to enhanced infectivity. TGFβ produced bytumor cells re-programs supporting cancer-associated fi-broblasts dampening their steady state level of anti-viraltranscripts and rendering them sensitive to virus infection.We found that in turn, CAFs produce high levels of fibro-blast growth factor 2 (FGF-2) initiating a signaling cas-cade in cancer cells that leads to a decrease in RIG-Iexpression and impedes the ability of the malignant cellto sense and respond to an invading virus therapeutic. Inpancreatic cancer patient explants, the expression of FGF-2 correlated with susceptibility to infection with oncolyticviruses and resistant patient samples could be sensitizedby the addition of FGF-2 in vitro and in vivo.1. Chung, A.S., et al. An interleukin-17-mediated paracrinenetwork promotes tumor resistance to anti-angiogenic thera-py. Nature medicine 19, 1114–1123 (2013).2. Nakasone, E.S., et al. Imaging tumor-stroma interactionsduring chemotherapy reveals contributions of the microenvi-ronment to resistance. Cancer cell 21, 488–503 (2012).3. Straussman, R., et al. Tumour micro-environment elicitsinnate resistance to RAF inhibitors through HGF secretion.Nature 487, 500–504 (2012).

4. Sun, Y., et al. Treatment-induced damage to the tumor mi-croenvironment promotes prostate cancer therapy resistancethrough WNT16B. Nature medicine 18, 1359–1368 (2012).

11:15–13:30

SYMPOSIUM5: The TumorMicroenvironment andProgression Towards Malignancy

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Metastasis Promotion: Control of the Src/STAT-Regulated Senescent Stromal Secretomeby SSeCKS/AKAP12 Scaffolding Protein

Irwin Gelman1, Masashi Muramatsu2, Jennifer Peresie1,Lingqiu Gao1, Ben Balderman11Cancer Genetics, Roswell Park Cancer Institute, Buffalo,New York, USA2Molecular Biology, Kyushu University, Kyushu, Fukuoka,Japan

There is growing appreciation that the pre-metastatic niche(pre-MN) results from crosstalk between secreted factors fromprimary-site tumor cells, tumor-associated macrophages(TAM), stromal and endothelial cells in the pre-MN, and my-eloid precursor (BMMP) and mesenchymal stem cells(BMSC) recruited to the pre-MN from the bone marrow.Moreover, pro-oncogenic genetic changes in the pre-MN areassociated with increased incidence of the senescence-associated secretory phenotype (SASP), noted for the localsecretion of inflammatory mediators that promote metastasisby activating local cells and by increasing recruitment ofBMMP and BMSC. The downregulation of SSeCKS/AKAP12, a scaffolding protein for PKC, Src and PKA, instromal cells has been linked to increased incidence of cancermalignancy including metastasis. Moreover, SSeCKS defi-ciency induces a senescence-associated secretory phenotype(SASP) in fibroblasts, a phenomenon known to promote met-astatic progression. Here, we show that B16F10-luciferasemelanoma cells injected s.c. or i.v. into SSeCKS-null mice(KO) induce higher levels of peritoneal metastasis comparedto WT hosts, whereas primary-site s.c. tumors grow at thesame rate. The increased metastasis correlated with increasedtumor cell chemotaxis, dependent on tumor cell CXCR3 andCXCL9/10 ligands secreted by KO peritoneal myofibroblasts(PMF). Increased PMF SASP was controlled by the loss ofscaffolding of Src and PKCα, correlating with increased ex-pression and secretion of CXCL9/10 induced by enhancedSrc-STAT1 signaling. Our data are consistent with the notionthat SSeCKS normally suppresses pre-MN formation by

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attenuating Src/PKCα signaling in stromal cells, therebyinhibiting SASP-induced metastatic chemotaxis and coloniza-tion of pre-MN. These data further the concept that metastaticprogression can be prevented or treated by the therapeutictargeting Src-family kinases.

O53

Myeloma-Derived CCL27 InducesStroma-Dependent Resistance Against Bortezomibvia Regulation of IL-10

Karin Joehrer1, Shanmugapriya Thangavadivel1, AngelikaOlivier1, Benno Postert1, Claudia Zelle-Rieser1, JohannKern2, Gerold Untergasser1,2, Andrea Brunner3, WolfgangWillenbacher4, Rainer Biedermann5, Richard Greil61Tumor Microenvironment Group, Tyrolean Cancer ResearchInstitut, Innsbruck, Austria2Laboratory for Tumor Biology and Angiogenesis, Depart-ment of Internal Medicine V, Medical University Innsbruck,Innsbruck, Austria3Department of Pathology, Medical University Innsbruck,Innsbruck, Austria4Department of Internal Medicine V, Medical University Inns-bruck, Innsbruck, Austria5Department of Orthopedic Surgery, Medical University Inns-bruck, Innsbruck, Austria6Laboratory for Immunological and Molecular Cancer Re-search, IIIrd Medical Department of Internal Medicine, Par-acelsus Medical University, Salzburg, Austria

Multiple Myeloma is a still incurable plasma cell malignancyand drug-resistance is the major cause for relapse and early deathof the patients. The bone marrow microenvironment plays afundamental role in shaping tumor progression. Chemokinesare soluble mediators of cell migration, proliferation and survivaland myeloma-derived chemokines might also play an importantrole in drug-resistance. Here we discovered that the chemokineCCL27 can trigger bortezomib-resistance of myeloma cells.Briefly, myeloma cells secreted CCL27 and high levels corre-lated with poor survival in vivo. These cells as well as stromalcells expressed the respective receptor CCR10. Ligand bind-ing led to enhanced adhesion and migration of the malignantcells. In coculture with stromal cells but not in single culture,CCL27 protected myeloma cells from bortezomib-inducedcell death but not from melphalan-induced cell death. Thiswas also confirmed in vivo. We found that myeloma-derivedCCL27 was further upregulated by bortezomib-treatment andmodulated stroma-derived IL-10 levels. In this setting, stromalIL-10 acted as a survival protein for myeloma cells. Knock-down of CCR10 on stromal cells as well as blocking IL-10and the IL-10 receptor reversed bortezomib resistance in

myeloma cells. Modulation of proteasomal activity and im-munoglobulin expression levels in the myeloma cells ap-peared to be involved in this escape mechanism.In conclusion, from our data we suggest that CCL27 is a novelplayer involved in bortezomib resistance in multiple myelomaand targeting this system could convert drug resistance andcontribute to better therapeutic response in this cancer.

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The Effects of IL-1 Molecules on the Different Stagesof Colon Cancer Development

Sami Stalin, Marina Bersudsky, Makka R. White, IrenaKaplanov, Ron N. Apte, Elena VoronovThe Shraga Segal Department od Microbiology, Immunologyand Genetics, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Interleukin-1 is a major, pro-inflammatory, upstream cytokinethat initiates and propagates inflammation.Interleukin-1α (IL-1α) has been found mainly in intestinalepithelial cells (IECs), both under homeostatic conditions andduring inflammation; whereas Interleukin-1β (IL-1β) was pri-marily detected in inflammatory cells upon stimulation. Inareas of severe tissue damage, both IL-1α and IL-1β werefound in myeloid infiltrating cells. Previously, we showed thatIL-1α, mainly from IECs is responsible for the initiation andpropagation of acute and chronic colon inflammation. Further-more, chronic colon inflammation is associated with colorectalcancer (CRC) development and progression.In this study, we assessed the role of IL-1α and IL-1β indifferent stages of colon cancer development and carcinogen-esis. For this aim, mice deficient in IL-1 molecules were sub-jected to a two step carcinogen model, where AOM was usedas an initiator of carcinogenesis and DSS-induced inflamma-tion, as a promotor. Both IL-1α and IL-1β deficient mice wereless susceptible to inflammation; however, it was mainly IL-1β deficiency that conferred tumor protection. Similar resultswere found when using surgical orthotopic implantation of themouse colon cancer line. Histological evaluation and FACsanalyses found differences in the cellular infiltrate in develop-ing tumors and their microenvironment. For example, lessinflammation was observed in IL-1β deficient mice. In con-trast, a deficiency in IL-1α led to an inefficient immune re-sponse against cancer and thus, increased tumor progression.In this study, we found that IEC-derived and myeloid-derivedIL-1α played a differential role in inflammation and cancerprogression and invasiveness, whereas IL-1β from myeloidcells served as a promotor of tumor invasiveness by inducingpro-inflammatory molecules and recruiting pro-inflammatorymyeloid cells to the site of the tumor.

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Intercellular Transfer of Small RNAsfrom Astrocytes to Lung Tumor Cells InducesResistance to Chemotherapy

Assaf Menachem1, Victoria Makovski1, Or Bodner1,Metsada Pasmanik-Chor2, Reuven Stein1, Noam Shomron3,Yoel Kloog11Department of Neurobiology, The George S. Wise Faculty ofLife Sciences, Tel Aviv University, Tel Aviv, Israel2Bioinformatics Unit, Faculty of Life Sciences, Tel Aviv Uni-versity, Tel Aviv, Israel3Department of Cell and developmental Biology, Sackler Fac-ulty of Medicine, Tel Aviv University, Tel Aviv, Israel

Brain metastases are highly resistance to chemotherapy andhave a poor prognosis for cure. Prior studies have shown thattumor cells are surrounded by activated astrocytes and exploittheir cyto-protective properties for protection from apoptosisinduced by chemotherapy. The mechanism by which astrocytesprotect tumor cells is poorly understood. An important non-mutational mechanism of chemotherapy resistance that has re-ceived an extensive attention in the last year is regulation ofgene translation mediated by small non-coding RNAs(sRNAs), in particular, microRNAs (miRNAs). Here we stud-ied the role of astrocytic sRNAs in promoting resistance of thehuman lung tumor PC14 cells to apoptosis induced by chemo-therapy. To this end we compared the sRNA profile of humanlung tumor cells that were cultured with or without astrocytesby miRNA microarray. The results show that sRNAs are trans-ferred from astrocytes to PC14 cells in a contact-dependentmanner. This transfer is fast and reached plateau already aftersix hours of co-culturing. Carbenoxolone, a broad-spectrumgap junction antagonist, inhibited the sRNAs transfer indicatingthat the sRNAs are transferred via gap-junction. Among thesRNAs that were transferred were several sRNAs that wereimplicated in survival pathways. Enforced expression of thesesRNA in PC14 cells increased their resistance to the chemo-therapy agent Taxol. In light of these results, our research pro-vides novel findings that can be clinically relevant to the treat-ment of patients with brain metastases.

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Stem Cell-Like Properties of GliomaTumor-Initiating Cells: Microenvironmental Inputs

Saran Kumar, Libat Bar-Lev, Eli KeshetDevelopmental Biology and Cancer Research, Faculty ofMedicine, Institute for Medical Research Israel-Canada, TheHebrew University of Jerusalem, Jerusalem, Israel

A superior tumor-initiation capacity of a relatively small sub-population of tumor cells has earned them the name of cancerstem cells (CSCs). CSCs identification and experimentalsorting is primarily based on expression of certain surfacemarkers rather than on the basis of unique properties sharedby all other stem cells in adult organs. Such a property is theirslow cell division rate compared to the rapid amplification oftheir descendants.Here we examined brain grafts of glioblastomamultiforme (GBM) cells pre-labeled with a membrane-bound fluorescent dye to follow cell division rates, rea-soning that CSCs will be distinguished as label-retainingcells due to their relative quiescence. Label-retainingcells were indeed shown to possess CSC hallmarks suchas expression of stem cell surface marker CD133 andhigher tumor initiation potential upon implantation intoNOD/SCID mice.To examine the notion that microenvironmental cues, ingeneral, and that proximity to blood vessels, in particular,impact acquisition of CSC properties, we analyzed spatialpatterns of CSC distribution and show that label-retainingglioma cells are indeed preferentially distributed in prox-imity to blood vessels. Further, RNA-seq analysis of tu-mor cells sorted according to their relative proximity toblood vessels reveals differences in multiple genes relatedto ‘stemness’ control, cell cycle regulation, metabolicchanges and oncogenic signaling.

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Brain Metastasis: the Impact of Surgical Stress,Immune Stimulation and NK Cells

Amit Benbenishty1,2,3, Alisa Lubart1,3, Hagr Lavon2, AriellaGlasner4, Pablo Blinder1,3, Shamgar Ben-Eliyahu2,31Neurobiology, Tel Aviv University, Tel Aviv, Israel2School of Psychological Sciences, Tel Aviv University, TelAviv, Israel3Sagol School of Neuroscience, Tel Aviv University, Tel Aviv,Israel4The Lautenberg Centre for General and Tumor Immunology,The Hebrew University of Jerusalem, Jerusalem, Israel

Brain metastasis (BrM) have poor prognosis, and prophy-lactic approaches are scarce. Surgical stress responseshave been shown to promote metastasis in peripheral or-gans through their immune-suppressive impacts andthrough direct effects on the malignant tissue and hostphysiology. However, BrM have not been studied inthese respects, and the unique brain immune milieu,blood supply, and BBB, may react differently to suchneuroendocrine challenges. Thus, we studied the effect

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of laparotomy, role of NK cells, and CpG-C immune-stimulation—a TLR-9 agonist having minimal adverseeffects in humans—in early stages of brain and lung me-tastasis. Two syngeneic animal models were used: 3LL/D122 in C57BL/J6 mice, and MADB106 mammary ade-nocarcinoma in F344 rats. Tumor cells were injected ei-ther through the tail vein or employing a novel internalcarotid injection approach we have developed, whichgenerates BrM with high efficacy and minimalinjection-related interferences to cerebral blood flow.Employing both models and inoculation approaches innaïve and NK-depleted animals, our results indicate thatNK cells surprisingly have no impact on BrM, whileprofoundly controlling lung metastases. On the otherhand, laparotomy significantly enhanced brain and lungstumor infiltration, and CpG-C reduced it, overcoming theeffects of laparotomy. Thus, surgery is a significant riskfactor for BrM through yet unknown mechanisms, andCpG-C treatment may be used prophylactically in cancerpatients.

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HHV-6 U94 Inhibits Motility, Migrationand Invasiveness of Human Breast Cancer Cellsby Modulation of Src Signaling Pathway

Francesca Caccuri, Federica Campilongo, Pietro Mazzuca,Arnaldo CarusoSection of Microbiology, Molecular and Translational Medi-cine, University of Brescia, Brescia, Italy

Cancer metastasis is the most life-treatening aspect of hu-man cancer, being responsible for around 90 % of cancerpatient mortality. The central, defining process of metasta-tic disease is the ability of tumor cells to mobilize, invadeand cross normally non-permissive tissue barriers. Tointravasate, tumors also promote local angiogenesis andlymphangiogenesis by secreting active molecules in thetumor microenvironment capable of inducing endothelialcell (EC) migration and consequently, the pathological forma-tion of new blood vessels. HHV-6 infects ECs and its latencygene U94 inhibits angiogenesis and lymphangiogenesis. Be-cause of its potent anti-migratory activity on ECs, wetested the capability of HHV-6 U94 to interfere withthe individual steps of the metastatic cascade, includingcancer cell detachment, migration and invasion. We ex-amined the HHV-6 U94 biological activity on the hu-man breast cancer cell line MDA-MB-231 as a modelof highly aggressive cancer cell. Here we show thatexpression of HHV-6 U94 delivered by an HSV-1-based amplicon vector strongly inhibit cell motility and

migration, thus affecting cell invasiveness. Moreover, inthis study, we show that HHV-6 U94 effects on humanbreast cancer cells are mediated by the down-modulation of Src signal leading to activation of β-catenin and to a better cell-cell adhesion. We investigat-ed the effects of HHV-6 U94 on cell-cell interactions ina three-dimensional rotary cell-culture system (RCCS).Analysis of MDA-MB-231 cell expressing HHV-6 U94showed, as expected, a greater aggregation and an in-creased expression of β-catenin on the cell membrane,compared to control cells.The capability of HHV-6 U94 to block migratory and invasiveproperty of cancer cells may lead to the development of newtherapeutic approaches.

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Chemoresistance Acquisition by OvarianAdenocarcinoma Cells due to the Crosstalksbetween MSCs and Macrophages

Bettina Couderc1, Augustin Le Naour1, Benoit Thibault1,Magali Castells1, Melissa Prat3, Alejandra Martinez2,Delphine Milhas1, Julie Guibert-Guillermet4, Jean PierreDelord1, Gwenael Ferron2, Agnes Coste3117, CRCT UMR1037 INSERM, Toulouse, France2Surgery, IUCT-Oncopole, Toulouse, France3MRN2i, UMR152 UPS-IRD, Toulouse, France417, CRCT UMR1037 INSERM, Toulouse, France

It is well established that the tumor microenvironment plays arole in modulating the sensitivity of tumor cells to cytotoxicagents. We have shown that cancer associated –mesenchymalstromal cells (CA-MSC) are involved not only on the tumorprogression but also in the acquisition of drugs resistance ofovarian adenocarcinoma cells via, among others, secretedmolecules in vitro and in vivo. Our aim is to identify themechanisms bywhich CA-MSC activate tumor cells signalingpathway for both effects. First we showed that factors releasedby CA-MSC are able to induce angiogenic cytokines (IL-6,IL-8 and VEGF) synthesis by tumor cells in a cell line specificway. Second, CA-MSC are able to attract and activate macro-phages into a M2 TAM like phenotype and allow them tosecrete a huge quantity of pro-angiogenic cytokines, favorableto tumor progression of all the associated ovarian adenocarci-noma cells tested. Third, factors released by CA-MSC protectadenocarcinoma cells from carboplatin-induced apoptosis byinhibiting the activation of effector caspases 3 and 7, inducingthe activation of PI3K/Akt pathway signalling and the phos-phorylation of the downstream target XIAP (caspase inhibitorfrom IAP family). XIAP depletion by siRNA strategy or in-hibitors permitted to restore carboplatin-induced apoptosis in

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ovarian cancer cells stimulated by CA-MSC conditionedmedium.This work has already served as the basis for the estab-lishment of a clinical trial using inhibitors of cIAP. Ourresults suggested targeting factors released by CA-MSCsuch as IL-6 or IL-8 in combination with conventionaltherapies could be of interest in ovarian cancer therapy.

11:15–13:30

SYMPOSIUM 6: Targeting the Tumor Microenvi-ronment & the Immune System

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Surface Modification of PLGA NanoparticlesImproves Drug Loading, Sustains Drug Releaseand Enhances Enzyme Eficiency in Vivo

Michael Goldberg, Zohreh AmoozgarCancer Immunology & AIDS, Dana-Farber Cancer Institute,Boston, MA, USA

Drug resistance is a central challenge to the treatment of ovar-ian cancer. Metronomic chemotherapy decreases the extent ofdrug-free periods, thereby hindering the development of drugresistance. Intraperitoneal chemotherapy allows for treatmentof tumors confined within the peritoneum, but achievingsustained tumor-localized chemotherapy remains difficult.We hypothesized that modulating the surface properties ofpoly(lactic-co-glycolic acid) (PLGA)-based nanoparticlescould enhance their drug retention ability and extend theirrelease profile, thereby enabling metronomic localized che-motherapy in vivo. Chemotherapeutic drugs were encapsulat-ed in particles coated with a layer of polydopamine and asubsequent layer of polyethylene glycol (PEG). These parti-cles achieved a 3.8-fold higher loading content compared tonanoparticles formulated from traditional linear PLGA-PEGco-polymers. In vitro release kinetic studies and in vivo drugdistribution profiles demonstrate sustained drug release.Whereas free drug conferred no survival advantage, low-dose intraperitoneal administration of drug-laden, surface-coated nanoparticles to mice bearing drug-resistant ovariantumors resulted in significant survival benefits in the absenceof any apparent systemic toxicity. We have additionally inves-tigated the possibility of conjugating biologics to the surfaceof these biodegradable particles to concentrate these mole-cules at the desired site of action. Excitingly, it was observedthat such immobilization can enhance the activity of enzymesin vivo.

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Highly-Effective Fluorescence-Guided SurgeryEnabled by Color-Coding Cancer Cellsand the Tumor Microenvironment with GeneticReporters in a Patient-DerivedOrthotopic Xenograft(PDOX) Model of Pancreatic Cancer

Robert Hoffman2, Yukihiko Hiroshima1, Ali Maawy2, YasuoUrata3, Michael Bouvet2, Toshiyoshi Fujiwara4, Shuya Yano11., AntiCancer Inc., San Diego, California, USA2Department of Surgery, University of California San Diego,San Diego, California, USA3., Oncolys Biopharm Inc., Tokyo, Japan4Department of Gastroenterological Surgery, Okayama Uni-versity Graduate School of Medicine, Dentistry and Pharma-ceutical Sciences, Okayama, Japan

Precise fluorescence-guided surgery (FGS) for pancreatic can-cer has the potential to greatly improve outcome. In order toachieve this goal, we have used genetic reporters to color codecancer cells and stroma cells in a patient-derived orthotopicxenograft (PDOX) model. The telomerase-dependent greenfluorescent protein (GFP) containing adenovirus OBP401was used to label the cancer cells of the pancreatic cancerPDOX. The PDOXwas previously grown in a red fluorescentprotein (RFP) transgenic mouse that stably labeled the PDOXtumor microenvironment bright red fluorescent. The color-coded PDOX model enabled FGS to completely resect thepancreatic tumors including the tumor microenvironment.Dual-colored FGS significantly prevented local recurrence,which bright-light surgery (BLS) or single color surgery couldnot. FGS, with color-coded cancer and the tumor microenvi-ronment has important potential for improving the outcome ofrecalcitrant cancer.

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Defining a Novel Pathway Promoting TumorProgression and Metastasis

Andrei Bakin, Michelle Limoge, Justin Zonnenville, AlexGruievskiCancer Genetics, Roswell Park Cancer Institute, Buffalo, NewYork, USA

Breast cancer (BC) is the second leading cause of cancer-related death in women. A significant proportion of BC pa-tients develop recurrent disease that progresses to the metasta-tic stage and becomes refractory to therapies. Our goals are todefine what drives metastatic progression and to develop

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novel therapies that would eliminate the mortality associatedwith metastatic BC.The interaction of tumor cells with tumor microenvironmentplays a key role in cancer progression, response to therapy,and progression to metastatic disease. The anti-inflammatoryTGF-beta and pro-inflammatory TNF and IL1 cytokines areabundant in the tumor microenvironment and promote tumorinvasion and metastatic progression. Our laboratory hasshown that TAK1 is essential for TGF-beta promotion of me-tastasis by stimulating expression of pro-invasive, pro-angio-genic, and pro-survival factors including matrix metallopro-teinase 9 (MMP9), VEGF, and cellular inhibitors of apoptosis(cIAPs). To define the molecular targets for anti-cancer thera-py, we undertook a rigorous investigation of how TGF-betaand TNF/IL1 cytokines cooperate in the regulation of MMP9and other pro-metastatic factors.The study assessed how genetic modulation of signal transduc-tion pathways by RNA interference and dominant-negativeapproaches would impact the biochemical markers of TAK1responses and tumorigenic capacity of tumor cells. Knock-down of signaling components of cytokine pathways revealedmajor signaling molecules that play essential role in the regu-lation of MMP9 and other pro-metastatic factors. To comple-ment this genetic analysis, we explored how experimentalcompounds targeting critical kinases in the pathways wouldimpact tumor invasive and metastatic capacity. Our preclinicalstudies identified novel strategies for treatment of metastaticbreast cancers, especially triple-negative breast cancers that arefrequently irresponsive to current therapeutic regiments. Wewill present details of these novel findings and outline ourrecent advancements on the role of TAK1 in breast cancer.

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In Vivo Profiling the Chemical TumorMicroenvironment for Diagnosis and Therapy

Valery KhramtsovDepartment of Internal Medicine, The Ohio State University,Columbus, Ohio, USA

We hypothesized Janus-faced properties of chemical tumormicroenvironment (cTME) [1], proposing that specific pat-terns of tissue oxygenation (pO2), extracellular pH (pHe), re-dox and intracellular glutathione (GSH) homeostasis act toutilize an orchestrated mechanism to promote cancer cell sur-vival while at the same time being highly toxic and mutagenicfor normal cells. Therefore, noninvasive in vivo assessment ofthese parameters provides important knowledge for advancedTME-targeted anticancer therapies. We utilized electron para-magnetic resonance (EPR)-based spectroscopy and imagingfor cTME profiling in mouse models of various cancers

including in vivo assessment of tissue redox, GSH, pO2,pHe, and concentration of interstitial phosphate (Pi) usingfunctional paramagnetic probes [2–4]. cTME profiling wereperformed as tumor progresses to malignancy and during ap-plication of metabolically active drugs. The in vivo studiesperformed in breast tumor-bearing mice show that all the mea-sured parameters, pO2, pH, Pi, redox and GSH, tend to deviatefrom the pattern characteristic of normal tissue upon progres-sion to malignancy. Normalizing the cTME parameters corre-lated with partial inhibition of tumor growth, therefore provid-ing a tool for TME-targeted anticancer therapy. A capacity ofspecific cTME pattern to be used as a prognostic factor intumorigenesis and the approaches for normalizing chemicaltumor microenvironment for anticancer TME-targeted thera-peutic interventions will be discussed. Supported by NIHgrant EB014542 and CA194013.[1] Khramtsov VV; Gillies RJ. Janus-Faced Tumor Microen-vironment and Redox. Antioxid Redox Signal., 2014, 21, 723–729.[2] Bobko AA et al. Magn. Reson. Med., 2012, 67, 1827–1836.[3] Dhimitruka I et al. J. Am. Chem. Soc., 2013, 135, 5904–5910.[4] Bobko AA et al. Angew. Chem. Int. Edit. 2014, 53, 2735–2738.

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Metronomic Chemo-Immunotherapy:Schedule-Dependent Activation of Immune-InducedTumor Regression

David J. Waxman, Junjie Wu, Marie E. Jordan, Bin DuDepartment of Biology, Boston University, Boston, Massachu-setts, USA

Clinical strategies that target cancer cells for destructionare frequently confounded by responses of tumor-associated stromal cells, which can lead to increased tumorgrowth, angiogenesis, invasion and metastasis, and conferan immune suppressive environment. Chemotherapy givenas a single dose can stimulate anti-tumor immune re-sponses, but this finding has not translated well to theclinic, where many conventional cancer chemotherapy reg-imens are toxic to immune cells, leading to immunosup-pression. Using preclinical models of glioma, we identifieda novel approach that combines direct tumor cell cytotox-icity with repeated disruption of the tumor microenviron-ment. Cyclophosphamide (CPA) treatment on an intermit-tent, 6-day repeating metronomic schedule (‘metro-CPA’)activates potent anti-tumor immune responses that inducemajor regression of large, implanted gliomas, with

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functional contributions from both the innate and the adap-tive immune systems and the activation of long-term anti-tumor immunity. These immune responses require drug-free breaks for recovery of drug-sensitive immune cellsfrom drug-induced cytotoxicity, suggesting a need to opti-mize current, daily clinical metronomic treatment sched-ules in order to maximize anti-tumor immune responses.We also characterized several tumor models that are intrin-sically sensitive to CPA cytotoxicity but do not exhibitdrug-induced immune responses, and we identified genesand upstream regulators associated with this tumor-differential responsiveness to metro-CPA treatment. Thegenes and regulators identified may include diagnosticand prognostic markers that facilitate translation to theclinic. The high potency of these immune-based responsesto metro-CPA treatment may result from synergistic ac-tions on tumor cells, by increasing tumor cell antigenicityand/or tumor cell susceptibility to immune attack, and onthe immune system, by activation of both innate and adap-tive immune effectors, and by inactivation of immune sup-pressors. Grant support: NIH-R01CA049248 (to DJW).

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Tumor-Binding IgG Combined with Dendritic CellStimuli Induces Anti-Tumor T Cell Immunity

Yaron CarmiImmunology Department, Stanford University, Stanford,California, USA

While cancers grow in their hosts and evade host immunitythrough immunoediting and immunosuppression, tumors arerarely transmissible between individuals. Much liketransplanted allogeneic organs, allogeneic tumors are reliablyrejected by host T cells, even when the tumor and host sharethe samemajor histocompatibility complex (MHC) alleles, themost potent determinants of transplant rejection. How suchtumor-eradicating immunity is initiated remains unknown,though elucidating this process could provide a roadmap forinducing similar responses against naturally arising tumors.We found that allogeneic tumor rejection is initiated by natu-rally occurring tumor-binding IgG antibodies, which enabledendritic cells (DC) to internalize tumor antigens and subse-quently activate tumor-reactive T cells. We exploited thismechanism to successfully treat autologous and autochtho-nous tumors. Either systemic administration of DC loadedwith allogeneic IgG (alloIgG)-coated tumor cells orintratumoral injection of alloIgG in combination with DCstimuli induced potent T cell mediated anti-tumor immuneresponses, resulting in tumor eradication in mouse models ofmelanoma, pancreas, lung and breast cancer. Moreover, this

strategy led to eradication of distant tumors and metastases, aswell as the injected primary tumors. To assess the clinicalrelevance of these findings, we studied antibodies and cellsfrom patients with lung cancer. T cells from these patientsresponded vigorously to autologous tumor antigens after cul-ture with alloIgG-loaded DC, recapitulating our findings inmice. These results reveal that tumor-binding alloIgG can in-duce powerful anti-tumor immunity that can be exploited forcancer immunotherapy.

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Adoptive Cell Therapy with Tumor InfiltratingLymphocytes: Clinical Results and Correlationto Clinical Success

Orit Itzhaki1, Ronnie Shapira1, NaamaDror1, Daphna Levy1,Yishai Pickman3, Gideon Gross5, Gal Markel1,2, RamitMehr3, Shai Shen Orr4, Jacob Schachter1, Michal J. Besser1,21The Ella Lemelbaum Institute, Sheba Medical Center, RamatGan, Israel2Department of Clinical Microbiology and Immunology, TelAviv University, Tel Aviv, Israel3Computational Immunology Lab, The Mina and EverardGoodman Faculty of Life Sciences, Bar-Ilan University,Ramat-Gan, Israel4Department of Immunology, Rappaport Institute ofMedical Research, Faculty of Medicine and Faculty ofBiology, Technion-Israel Institute of Technology, Haifa,Israel5Laboratory of Immunology, MIGAL, Kiryat Shmona, Israel

The presence of immune cells in the tumor microenvironment,in particular T cells, strongly correlates with favorable clinicaloutcome. Adoptive cell transfer (ACT) of ex vivo expandedTumor Infiltrating Lymphocytes (TIL) followed by tumor mi-croenvironment preconditioning, is an effective treatment formetastatic melanoma, as demonstrated by multiple cancercenters. Over 100 patients were treated at our institute andabout 40 % experienced objective responses, including 12 %complete remissions. The therapy was also effective in pa-tients who were refractory to immune checkpoint inhibitors.Searching for parameters of clinical response, we examined a21 different cytokines in the serum of patients and in theirmelanoma and TIL cultures. In addition, we analyzed TILinfusion products in order to define specific phenotypic T-cell subsets which correlate to tumor regression. Usingbioinformatical tools, we evaluated 600 subpopulations. Cy-totoxic T cell, co-expressing CD28 and CD33 were positivelycorrelated to clinical response. By FASC sorting or geneticengineering, we were able to enrich CD8+CD28+CD33+ cellswith superior in vitro and in vivo activity in comparison to

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their CD8+ counterparts. Enrichment of this specific T cellssubset by rational manipulation or selection may further im-prove the clinical response to TIL-ACT. Using new multi-marker cytometry by Time-Of-Flight (CyTOF) enables themeasurement of 39 extra- and intra-cellular expressionmarkers simultaneously for rapid and robust identification ofnew cell populations of interest. This may significantly enableefficient anti-tumor reactive T cell selection to improve theclinical outcome of patients treated with TIL-ACT.

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Targeting Dysfunctional Myeloid Cells DelaysDisease Development and Improves ImmuneFunction in a Mouse Model of Chronic LymphocyticLeukemia

Bola Hanna1, Fabienne McClanahan1,2, Alexander Egle3,John Gribben2, Peter Lichter1, Martina Seiffert11Molecular Genetics, German Cancer Research Center(dkfz), Heidelberg, Germany2Centre for Haemato-Oncology, Barts Cancer Institute,London, UK3Laboratory for Immunological and Molecular CancerResearch (LIMCR), Paracelsus Medical University, Salzburg,Austria

Chronic lymphocytic leukemia (CLL) is a B-cell malignancythat is stringently associated with a tumor-supportive micro-environment and defective anti-tumor immunity. T cells fromCLL patients show features of exhaustion, including expres-sion of PD-1, and are highly impaired in immune synapseformation, which is mediated by aberrant expression of sever-al inhibitory receptors like PD-L1 on CLL cells. Our previouswork showed that immune checkpoint blockade using anti-PD-L1 effectively prevents disease and restores T-cell activityin a CLL mouse model (McClanahan et al., 2015).To investigate the role of myeloid cells in the CLL microen-vironment, we analyzed their composition and function in theEμ-TCL1 mouse model of CLL, and observed a severeskewing of monocytes and macrophages along with CLL de-velopment. This included an accumulation of M2-like macro-phages and Ly6Clow patrolling monocytes and a drop ofMHC-IIhigh dendritic cells (DCs). Associated with that, sever-al inflammatory serum factors like IL-10, TNFα and CXCL9were upregulated in leukemic mice. Myeloid cell depletionusing liposomal clodronate resulted in significant control ofCLL development, repair of innate immune cell phenotypesand partial resolution of systemic inflammation. Also, CLL-induced T-cell defects were resolved. Gene expression profil-ing of CLL-associated monocytes revealed aberrantly highPD-L1 expression which was confirmed on DCs, suggesting

their contribution to defective T-cell responses and treatmentsuccess of PD-L1 blockade. Therefore, targeting myeloid cellsurvival and immunosuppressive activity can serve as a novelstrategy for CLL immunotherapy.McClanahan, F., Hanna, B., Miller, S., Clear, A.J., Lichter, P.,Gribben, J.G., and Seiffert, M. (2015). PD-L1 CheckpointBlockade Prevents Immune Dysfunction and LeukemiaDevelopment in a Mouse Model of Chronic LymphocyticLeukemia. Blood Mar 23. [Epub ahead of print].

WEDNESDAY, OCTOBER 14, 2015

08:30–10:50

SYMPOSIUM 7: Regulatory Pathways in the TumorMicroenvironment (III)

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Matrix-Assisted Autocrine Signaling RegulatesStemness Properties in Melanoma Cellsthrough Induction of Id1 and Id3 Expression

Jonathan Sleeman1,2, Justyna Krachulec1, Vanessa Kuch1,Georg Sedlmeier1, Julia Philipp1, Ruolin Wu11Centre for Biomedicine and Medical Technology Mannheim(CBTM), Medical Faculty Mannheim of the Universityof Heidelberg, Mannheim, Germany2KIT Karlsruher Institut für Technologie, Campus Nord,Institut für Toxikologie und Genetik, Karlsruhe, Germany

Cancer stem cells are thought to drive tumor growth and me-tastasis through their stemness properties, and are character-ized by the ability to initiate tumor growth. Tumor initiationin vivo is strongly increased by coinjecting tumor cells withECM components such as Matrigel, suggesting a critical rolefor the extracellular matrix microenvironment in determiningstemness properties. Using gene expression profiling and sub-sequent validation, we found that melanoma cells grown in3D ECM microenvironments such as Matrigel, laminin andcollagen exhibited strongly increased expression of Id1 andId3 in comparison to tumor cells cultivated on 2D surfaces.Id1 and Id3 are transcriptional regulators that are implicated ingoverning stemness properties, and play a key role in regulat-ing the ability of tumor cells to initiate primary tumors andmetastatic growth. Using loss of function approaches (treat-ment with noggin or inhibition of BMP receptor activation),we observed with a variety of melanoma and breast cancercells that several types of 3D ECM microenvironment as wellas artificial 3D matrices can induce Id1 and Id3 expression by

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promoting autocrine BMP signalling. Mechanistically, theseand other data collectively suggest that 3D ECM microenvi-ronments can act as a mechanical barrier that inhibits diffusionof endogenously produced BMP protein, thereby increasinglocal BMP concentrations. Subsequent autocrine BMP signal-ling leads to increased expression of Id1 and Id3, and modu-lates tumor cell properties associated with stemness. Com-pounds that target Id1/Id3 expression or function may there-fore prove useful as effective cancer therapies.

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Extracellular Superoxide Anion Generationand Expression of Membrane-associated CatalaseDuring Tumor Progression: Dynamic Interplayand Consequences for Tumor Cell Survival

Georg BauerInstitute of Virology, University Medical Centre Freiburg,Freiburg, Germany

Oncogenic transformation depends on the activation ofmembrane-associated NADPH oxidase. The resultant extra-cellular superoxide anions and H2O2 control the proliferationof malignant cells, but they also drive two intercellular signal-ing pathways that cause selective apoptosis induction in ma-lignant cells, i. e. the HOCl and the NO/peroxynitrite signalingpathway.Tumor progression requires the establishment of resistanceagainst these apoptosis-inducing pathways through expres-sion of membrane-associated catalase that interferes withHOCl signaling through decomposition of H2O2 and withNO/peroxynitrite signaling through oxidation of NO and de-composition of peroxynitrite. Therefore bona fide tumor cellsregularly express a substantial concentration of membrane-associated catalase. As catalase is inhibited by superoxideanions, optimal catalase-mediated protection against intercel-lular apoptosis signaling requires the colocalization of SOD.NOX1-derived superoxide anions/H2O2 control the expres-sion of membrane-associated catalase in a dynamic mode.Inhibition of NOX1 therefore causes a dramatic decrease inthe concentration of protective catalase and in parallel inter-feres with the stimulation of tumor cell proliferation leading toa state that resembles tumor cell dormancy.Whereas nontransformed cells do not express sustainedNOX1 activity, transformed cells, tumor cells and met-astatic cells are characterized by strong NOX1 activitythat increases with their stage related to tumor progres-sion. The counterbalance by catalase is, however, notoptimal at all stages.Inhibition of membrane-associated catalase by neutralizingantibodies or its inactivation by exogenous or cell-derived

singlet oxygen bears novel chances for therapy, based on re-activation of intercellular ROS signaling. Interestingly, a vari-ety of natural plant compounds modulate the endogenous NOlevel in tumor cells and thus trigger the onset of anamplificatory pathway that culminates in extracellular singletoxygen generation, catalase inactivation and tumor cellapoptosis.

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Exosomes as Modulators of TumorMicroenvironment

Reuven ReichInstitute for Drug Research, The Hebrew University ofJerusalem, Jerusalem, Israel

The metastatic process involves the manipulation of the tumormicroenvironment to optimize the conditions for local growthand to enable dissemination of the tumor cells through out thepatient’s body.Recent studies point to the exosomes, small (30–100 nm)vesicles secreted continuously by both, normal and dis-eased cells, as important components of tumor microenvi-ronment involved in intercellular communication.Exosome cargo reflects the molecular and cellular contentof the cell of origin such as growth factors, receptors,proteases, coding and non-coding RNA and certain lipids.They also reflect dynamic changes in the cellular compart-ments that are occurring in health and at different stages ofa disease.Ovarian cancer (OC) is the most lethal and second mostcommon gynecological malignancy in the western world.Current knowledge of the biological factors that drive OCmetastasis as solid lesions or effusions is incomplete atbest. In the present study we analyzed the expression pro-file of miRNAs in exosomes isolated from the effusionfluid of OC patients and compared them to the cellularprofile of miRNAs in those patients. Our results indicatedon specific miRNA population that correlated with thesurvival of the patients. Further, the same population ofmiRNAs increased the invasive potential of OC cellsin vitro and in vivo models.Examination of the secretory mechanism of exosomes re-vealed the expression of nSMmase2, Tsap6 and Rab27amRNA in our tumor samples. Clinical analysis shows thatelevated nSmase2 and Tsap6 mRNA expression correlateswith poor survival (p0.036) and less favorable response tochemotherapy, respectively (p0.027).Understanding the precise mechanism of exosome secretionsand analysis of their cargo opens a new avenue for potentialtreatment of this yet incurable disease.

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Clinical Relevance, Pathological Detailsand Mechanistic Functions of Desmoplastic3D-Adhesion Structures

Janusz Franco-Barraza1, Tiffany Luong1, Neelima Shah1, RajMadhani1, Gil Cukierman2, Essel Dulaimi1, KarthikDevarajan1, Brian Egleston1, Katherine Alpaugh1, JohnHoffman1, Robert Uzzo1, Edna Cukierman11Cancer Biology, Fox Chase Cancer Center, Philadelphia,PA, USA2Computer Science, University of North Carolina, ChapelHill, NC, USA

We have demonstrated that desmoplastic extracellular ma-trices (ECMs) effectively induce an active myofibroblasticphenotype upon naive fibroblasts. Here, we aim to repro-gram the desmoplastic microenvironment back to tumorsuppressive and query its clinical relevance. Using synge-neic human fibroblasts harvested from pancreatic or renalcancer patient-matched normal and tumor surgical sam-ples, we prompted cells to produce a human mimetic 3Dstroma system and attempted reprograming. Approachesincluded the use of specific inhibitor and activator drugsas well as genetic manipulations. Pathophysiological val-idations were conducted via simultaneous multi-channelimmunofluorescence labeling of the original surgical tis-sue samples distinguishing tumoral from stromal compart-ments and questioning the localization and activity levelsof α5β1-integrin with regards to desmoplastic 3D-adhesion structures. To establish clinical relevance, thesame approach was combined with a novel computationalcode applied as a batch analysis on human renal and pan-creatic tissue cohorts.We show that, while TGFβ is needed for desmoplasticfibrillogenesis, it is dispensable for desmoplastic ECM-induced myofibroblastic activation. Data point to a mecha-nism whereby αvβ5-integrin triggers the redistribution ofFAK-independent α5β1-integrin activity away fromdesmoplastic 3D-adhesion structures. This in turn preventsα5β1-integrin from inhibiting the desmoplastic ECM-induced phenotype. Results from the pathological samplessuggest that levels of stromal α5β1-integrin activity localizedaway from desmoplastic 3D-adhesions could foretellrecurrence.We posit that this desmoplastic mechanism not only com-prises a clinically important occurrence and novel outcome-predictive and desmoplastic index bio-marker, but it also con-stitutes a possible new therapeutic aimed at normalizing-desmoplasia. Results from this study consolidate some currentcontroversies regarding the tumorigenic value of desmoplasia.

Funding: Pennsylvania’s Commonwealth, Bucks CountyChapter Board of Associates, PanCan Greenberg FamilyFund, Keystone Program in Personalized Kidney Cancer,NCI/NIH and DOD.

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Flaxseed and Tamoxifen Alters the Expressionof Extracellular microRNA in Normal HumanBreast Tissue in Vivo

Charlotta Dabrosin, Annelie AbrahamssonOncology, Linköping University, Linköping, Sweden

Exposure to sex steroids is key in the development of breastcancer with to date limited knowledge of the mechanisms in-volved. Studies of sex steroid dependent alterations in normalbreast tissue are needed for the development of novel preventivestrategies. Diet modifications may be among the means for breastcancer prevention as women with Eastern diet habits includingingestions of phytoestrogens exhibit a lower risk of developingbreast cancer. One of the riches sources of phytoestrogens inWestern diet is flaxseed.We have previously shown that hormoneexposure alter extracellular levels several proteins and extracellu-lar microRNAs in vivo in normal human breast tissue. Here, wesampled extracellular microRNAs locally in situ using microdi-alysis in normal human breast tissue and subcutaneous abdominalfat. Different cohorts of women were included; premenopausalwomen investigated during the menstrual cycle before and afterthe addition of 25 g of ground flaxseed every day for 4weeks andpostmenopausal women investigated in their normal healthybreast and subcutaneous fat before and after 6weeks of tamoxifentherapy. Samples were screened using TaqMan array cards withsubsequently absolute quantification. Over 100 miRNA were

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expressed in microdialysates from breast tissue. Absolute quanti-fications revealed that several extracellular miRNAs were signif-icantly altered in normal human breast tissue after flaxseed addi-tion to the diet or tamoxifen therapy. None of these changes werefound in plasma or microdialysates from subcutaneous fat. Ourdata revealed breast tissue specific changes of extracellularmiRNAs after diet interventions and tamoxifen therapy thatwould be otherwise unraveled using blood samples alone.Dietarymodifications may be used to prevent breast cancer, but possiblemechanisms involved in the effects of flaxseed requires furtherstudies before any general recommendations can be given.

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Mutant p53 Modulates the Signalof Stromal-Secreted Hepatocyte Growth Factor(HGF) to Endow Cancer Cells with Drug Resistance

Yan Stein, Adi Jacob, Naomi Goldfinger, Ravid Straussman,Varda RotterDepartment of Molecular Cell Biology, Weizmann Institute ofScience, Rehovot, Israel

Drug resistance is a major obstacle in cancer therapy. Recently, aco-cultivation screen identified a major role of the microenviron-

ment in promoting drug resistance in various solid tumors1. Mu-tations in the p53 tumor suppressor protein are highly frequent intumors and often endow cells with tumorigenic capacities. How-ever, the role of mutant p53 in the context of tumor-stroma in-teraction has not been thoroughly studied yet. In this study, weperformed a cytokine screen, to identify potential secreted mole-cules which endow the cancer cells with drug resistance, in amutant p53 dependent manner. We established isogenic lungcancer sub-lines, harboring different p53 statuses and labeledwith tRFP. After treating these sub-lines with EGFR inhibitorand a cytokine library, we identified several potential cytokineswhich endow the mutant p53 expressing sub-line with enhancedresistance compared to mutant p53-knocked down sub-line. Re-markably, themost significant factor endowing drug resistance ina mutant p53 dependent manner was hepatocyte growth factor(HGF, see attached figure), which was previously shown to be amajor contributor to innate resistance to RAF inhibitors1. Wetherefore demonstrate a potential role for mutant p53 in modu-lating stromal-derived signals to promote drug resistance in thetumor cells. In a broad sense, we demonstrate how a cancer celldeterminant can manipulate a stromal signal for the tumor’sbenefit.Reference1. Straussman, R. et al. Tumour micro-environment elicitsinnate resistance to RAF inhibitors through HGF secretion.Nature 487, 500–4 (2012).

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OPN/MMP-9 Pathway Activation in B-CellNon-Hodgkin Lymphoma

MichelinoDi Rosa1, Saverio Candido1, Jerry Polesel2, GraziaMalaponte1, Massimo Libra1

1Biomedical & Biotechnological Sciences, University ofCatania, Catania, Italy2Epidemiology, National Cancer Institute, Aviano, Italy

Non-Hodgkin’s lymphomas (NHL) are a heterogeneous groupof lymphoproliferative malignancies with variable patterns ofbehaviour and responses to therapy. NHL development and

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invasion depend on multiple interactions between tumor cellsand non-neoplastic cells. Such interactions are usually modulat-ed by several cytokines. Accordingly, it was demonstrated that inhuman lymphoid cell lines interleukin-6 (IL-6) activates matrix-metalloproteinase (MMP)-2 andMMP-9. The activation of theseenzymes is associated with tumor invasion and metastasis inhuman cancers. MMPs are also activated in several cancers byosteopontin (OPN), a secreted glycoprotein that regulates celladhesion, migration, and survival. However, it is still unclear ifMMPs play a role in NHL development and if their activation isdetermined byOPN and/or IL-6. In the present study, two groupsof 78 NHL patients and 95 healthy donors were recruited for theanalysis of OPN, MMP-2, MMP-9 and IL-6 using both experi-mental and bioinformatics approaches.Compared to healthy donors, NHL cases reported significant-ly higher concentrations of MMP-2 (median: 1041 and659 ng/mL in NHL cases and controls, respectively; p0.01),MMP-9 (median: 114.0 and 17.1 ng/mL; p0.01), OPN (medi-an: 147.0 and 27.3 ng/mL; p0.01), and IL-6 (median: 12.5 and1.7 ng/mL; p0.01). The multivariate regression model indi-cates that, in both NHL cases and healthy donors, OPN isassociated with the increase of MMP-2 and MMP-9 levelsindependently of IL-6. Similar data were obtained byanalysing Hummel and Rosenwald datasets.These data suggest that the activation of MMPs in NHL de-velopment is mediated by OPN and not by IL-6. However, IL-6 may play an important role in the lymphomagenesis throughthe activation of other molecular pathways.

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Dual Prognostic Significance of Tumor AssociatedMacrophages in Human PancreaticAdenocarcinoma Treated or not with Chemotherapy

Nina Cortese1, Giovanni Francesco Castino1, Giuseppe DiCaro1, Fabio Grizzi1, Giovanni Capretti2, Cristina Ridolfi2,Francesca Gavazzi2, Alessandro Zerbi2, Paola Allavena1,Alberto Mantovani1, Federica Marchesi11Department of Immunology and Inflammation, HumanitasClinical and Research Center, Rozzano, Italy2Section of Pancreatic Surgery, Department of Surgery,Humanitas Clinical and Research Center, Rozzano, Italy

Tumor-associated macrophages (TAMs) play key roles in tu-mor progression. Recent evidence suggests that TAMs criti-cally modulate the efficacy of anticancer therapies, raising theprospect of their targeting in human cancer. In a large retro-spective cohort study involving 110 pancreatic ductal adeno-carcinoma (PDAC) patients, we addressed the clinical rele-vance of TAMs and the mechanisms regulating their function

in PDAC. Density of CD68-TAM immune reactive area(IRA%) was evaluated at the tumor-stroma interface. Prog-nostic relevance was evaluated in relation to post-surgical ad-juvant chemotherapy treatment (CTX). The synergism of che-motherapy and TAMs was dissected in vitro. In humanPDAC, TAMs predominantly exhibited an immunosuppres-sive profile, characterized by expression of scavenger recep-tors (CD206, CD163) and production of IL-10. Surprisingly,while the density of TAMs associated to worse prognosis anddistant metastasis, CTX restrained their protumor prognosticsignificance. High density of TAMs at the tumor margin pos-itively dictated prognostic responsiveness to CTX indepen-dently of T cell density. Accordingly, in vitro, Gemcitabine-treated macrophages became tumoricidal, activating a cyto-toxic gene expression program. In human PDAC patients,neoadjuvant CTX was associated to a decreased density ofCD206-TAMs at the tumor margin. Overall, our data highlightTAMs as critical determinants of prognostic responsiveness toCTX and provide clinical and in vitro evidence that CTXdirectly reeducates TAMs to an anti-tumor phenotype. Theseresults suggest that the quantification of TAMs could beexploited to select patients more likely to respond to CTXand provide basis for novel strategies aimed at reeducatingmacrophages in the context of chemotherapy treatment.

08:30-10:45

SYMPOSIUM 8: Metastasis and the Metastatic Microen-vironment (I)

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Synthetic Environments to Understand CancerMetastasis and Drug Resistance

Shelly Peyton, Lauren Barney, Thuy Nguyen, Lauren Jansen,Elizabeth Brooks, Alyssa Schwartz

Chemical Engineering, University ofMassachusetts, Amherst,Massachusetts, USA

Metastasis is the leading cause of fatality for women diag-nosed with breast cancer. The most common anatomicalsites of distant tumor growth include the brain, lung, liver,and bone, and it is well known that this metastatic spread inbreast cancer is not random. Rather, different clinical sub-types of breast cancer exhibit unique patterns of metastaticsite preference, called tissue tropism. Given the physicaland chemical diversity of these secondary tissue sites,my lab hypothesizes that there is a relationship between the

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biophysical and biochemical properties of the tissue, and theability of cells within a particular subtype of breast cancer toadhere, migrate, grow, and respond to chemotherapeutics atthese secondary sites. We created biomaterial microenviron-ments, which capture some of the key physical and biochem-ical elements of the secondary site tissues often recipient ofbreast cancer spread (brain, lung, and bone). Our approach isrevealing how cell-material interactions are predictive of met-astatic spread and non-canonical signaling pathways involvedin drug resistance at these tissue sites. First, we can use a cell-ECM screening method in vitro to predict where a cell willmetastasize in vivo (Barney et al. 2015). Second, we havedemonstrated that a stiff tumor microenvironment reduces so-rafenib treatment efficacy, which can be abrogated via JNKinhibition (Nguyen et al. 2014). We will discuss these andcurrent efforts toward biomaterial capture of dormant metasta-tic cells, rapid tumor spheroid formation, and the role of mes-enchymal stem cells in drug resisatnce. We propose that thesetypes of biomaterial environments can be used to predicttissue-specific metastasis, and may serve as a system thatpharmaceutical companies can use to rule out false positivesand potentially save billions of dollars in the drug develop-ment pipeline.

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Dissecting CTC Phenotypes: Insightsinto Mechanisms of Breast Cancer Dormancy

Dario Marchetti1, Monika Vishnoi1, Sirisha Peddibhotla1,Wei Yin1, Antonio Scamardo2, David Hong21Biomarker Research Program, Methodist Hospital ResearchInstitute, Houston, Texas, USA2Investigational Cancer Therapeutics, MD Anderson CancerCenter, Houston, Texas, USA

Uncovering phenotypes of patient-derived Circulating TumorCells (CTCs) offers the promise to dissect CTC heterogeneityin relation to metastatic competence, and to determine bio-markers of therapeutic utility.

We hypothesized that breast cancer CTC subsets possessingmarkers of pluripotency avoid organ arrest with extreme effi-ciency by the concomitant presence of quiescence and stemcell properties; and that expression of urokinase plasminogenactivator receptor (uPAR) and beta-1 integrin (β1int) are rel-evant in controlling the recurrence of breast cancer brain me-tastasis (BCBM). First, we captured EpCAM-negative/CD45-/ALDH1+/CD44+/CD24- breast cancer CTC subsets thatpossessed combinatorial uPAR and β1int expression, andcharacterized these subsets using DEPArray™, a CTC plat-form able to dissect CTC heterogeneity at a single-cell level.Second, CTC subsets generated in vitro tumorspheres thatwere characterized for human embryonic stem cell markersby RT2 PCR arrays. Gene expression profiling was consistent-ly distinct among uPAR+/β1int+ vs. uPAR-/β1int- CTC sub-set ratios and dependent upon patients’ BCBM clinical status.Expression of genes implicated in cell cycle progression, an-giogenesis, and pluripotency was 30-fold higher than controlsin uPAR/β1int ratios of CTC subsets from patients diagnosedwith BCBM. Of note, expression of RIF-1, a protein thatcounteracts actions of the breast cancer suppressor BRCA1,was highest (50-fold) with presence of RIF-1 nuclear foci inBCBM CTC subsets. Lastly, BRCA1 gene was found to bewild-type in CTC subsets following single-cell whole genomeamplification and DNA sequencing.In summary, we have linked EpCAM-negative uPAR/β1intCTC subsets and their properties to clinical BCBM; and willassess the therapeutic inhibition of uPAR/β1int CTC bio-markers on BCBM development and RIF-1/BRCA1 interplaysin cell cycle-dependent mechanisms of DNA repair as keybiomarkers delineating dormancy vs. BCBM competence.

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Selectin-Mediated Recruitment of LeukocytesContributes to Metastatic Niche Formationand Chemokines Orchestrate this Process

Lubor Borsig, Irina Häuselmann, Darya Protsyuk, MarkoRoblekInstitute of Physiology, University of Zurich, Zurich,Switzerland

Metastasis is the primary cause of cancer-related mortality andthe adopted metastatic microenvironment significantly contrib-utes this process. During hematogenous metastasis tumor cellsinteract with blood constituents and these contacts enhance thecapacity to colonize distant organs. Selectins are vascular re-ceptors that facilitate tumor cell interactions with platelets, leu-kocytes and the endothelium. At sites of metastatic initiationactivated endothelium is always detected, which is associatedwith E-selectin expression. Here we provide evidence that E-

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selectin contributes to the recruitment of leukocytes and therebypromotes metastasis. We observed reduced metastasis in theabsence of E-selectin using several mouse models. Tumorcell extravasation was dependent on endothelial activa-tion, which in turn contributed to increased chemokinelevels in metastatic tissues. Next we tested the hypothesisthat local inhibition of chemokine CCL2 at metastaticsites can interfere with tumor cell extravasation and there-by reduce metastasis. The use of two different targetingstrategies to the lungs showed an effective inhibition ofmetastatic seeding and thereby metastasis. Taken together,we present a new mechanism how selectin promotes earlymetastatic niche formation and thereby metastasis.

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ADAR1-Mediated Regulaton of Melanoma Invasion

Yael Nemlich1, Jacob Schachter1, Gal Markel1,21Sheba Medical Center, Ella Institute of Melanoma, Ramat-Gan, Israel2Sackler Faculty of Medicine, Department of Clinical Micro-biology and Immunology, Tel Aviv, Israel

The main RNA-editing enzyme, Adenosine Deaminase Act-ing on RNA-1 (ADAR1), is silenced in many metastatic tu-mors, including melanoma. We have recently shown thatADAR1 suppresses several cancer features, as its downregu-lation alters cell morphology, facilitates cell-cycle, prolifera-tion, and dramatically enhances the tumorigenicity in-vivo.We further demonstrated that ADAR1 controls the expressionof 100 microRNAs, which regulate hundreds of genes thataccount for the observed phenotype.Cutaneous melanoma is a highly metastasizing neoplasticdisease, and its malignant potential has been previouslyassociated with integrin beta-3 (ITGB3) expression, aknown oncogene, strongly linked to the acquisition of in-vasive properties of many tumors. However, only little isknown about the regulation of ITGB3 expression in cancercells. ITGB3 is upregulated during the transition from dys-plastic nevi to tumorigenic melanomas, inversely to thesubstantial reduction in ADAR1 expression. We show inseveral cell lines that silencing of ADAR1 directly en-hances melanoma cell invasiveness and ITGB3 expression.The enhanced invasion is corrected when ITGB3 isblocked with monoclonal antibodies. Experiments with aseries of melanoma cell lines transfected with wild type orcatalytically inactive ADAR1 mutants show that this phe-nomenon is independent of RNA-editing. Mechanistically,we found that ADAR1 controls ITGB3 expression both atthe post transcriptional and transcriptional level, via miR-22 and PAX6 transcription factor, respectively, which are

described here as direct regulators of ITGB3. The novelADAR1-dependent and RNA-editing-independent regula-tion of invasion presented here, mediated by ITGB3, aswell as the ADAR1-controlled regulation of ITGB3 ex-pression, strongly points on a central involvement ofADAR1 in cancer progression and metastasis. These find-ings provide novel insights on the process of cancer devel-opment with potential implications for future translationalmedicine.

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AMitochondrial Switch Promotes TumorMetastasis

Valéry L. Payen, Paolo E. Porporato, Pierre SonveauxPole of Pharmacology and Therapeutics, University of Lou-vain (UCL) Medical School, Brussels, Belgium

Cancers evolve a subpopulation of tumor cells that metaboli-cally rely on glycolysis uncoupled from oxidative phosphor-ylation irrespectively of oxygen availability (aerobic glycoly-sis). Given that most metastases are abnormally avid for glu-cose and because clinical data show a positive correlationbetween lactate production and tumor metastasis, we reasonedthat cells performing aerobic glycolysis could constitute apopulation of metastatic progenitor cells that would remainglycolytic in the blood stream. We found a different metabolicphenotype, though. Indeed, using serial rounds of in vitro se-lection of highly invasive tumor cells (starting from wild-typeSiHa human cervix adenocarcinoma cells) and in vivo selec-tion of supermetastatic tumor cells (starting from B16-F10mouse melanoma cells), we identified a mitochondrial switchcorresponding to an overload of the tricarboxylic acid cyclewith preserved mitochondrial functions (including ATP pro-duction) but increased mitochondrial superoxide production.The switch provided a metastatic advantage that wasphenocopied by moderate oxidative phosphorylations(Oxphos) inhibition associatedwith mildmitochondrial super-oxide increase. Thus, two different events, Oxphos overloador moderate Oxphos inhibition, promote superoxide-dependent tumor cell migration, invasion, clonogenicity, andmetastasis; demonstrating the central role of mitochondrialsuperoxide generation in the pathogenesis of metastasis. Con-sequently, we report that mitochondria-specific superoxidescavenging (using mitoTEMPO or mitoQ) inhibits metastaticdissemination from primary mouse and human tumors, whichopens a new avenue for the therapeutic prevention of tumormetastasis.Supported by ERC Starting Grant 243188 TUMETABO, IAPgrant #UP7-03 from Belspo, the Communauté Française deBelgique (ARC 09/14-020), the Belgian F.R.S.-FNRS, and theBelgian Fondation contre le Cancer.

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Lysyl Oxidase Overexpressed in Mice that UndergoSurgery may Promote Metastasis

Chen Rachman1, Michael Timaner1, Dror Alishekevitz1,Valeria Miller1, Sheli Eilot1, Moran Grossman2, Irit Sagi2,Peleg Hasson1, Yuval Shaked11Medicine, Technion-Israel Institute of Technology, Haifa,Israel2Biological Regulation, Wiezmann Institute of Science,Rehovot, Israel

Tumor resection is one of the major treatment modalitiesfor cancer. It is sometimes combined with chemo-radiation in order to reduce the risk of tumor re-growthor metastasis spread from existing residual disease. Yet,patients who undergo surgery may exhibit metastaticspread. Here we show that the host in response to radicalsurgery is vulnerable to metastasis seeding. Non-tumorbearing mice, which undergo surgery, succumb to LLCor EMT/6 lung metastasis earlier than control mice in anexperimental lung metastasis assay. Similarly, miceinjected with plasma from mice which underwent surgerywere prone to metastasis seeding more than mice injectedwith plasma from control mice. Changes in primary tu-mor growth, angiogenesis and the colonization of bonemarrow derived cells at the primary tumor site were doc-umented following surgery. Importantly, increased LOXactivity in the lungs of mice that underwent surgery re-sulted in lung extracellular matrix modulation. Conse-quently, the blockade of LOX family members by BAPNor by specific neutralizing antibodies reduced metastasisspread in the lungs of mice following surgery and in-creased their survival. Taken together, our results empha-size the modulation of the extracellular matrix in the pre-metastatic microenvironment induced by surgery, andsuggest that LOX may contribute to surgery-induced me-tastasis. The study also offers new therapeutic interven-tion in combination with surgery to reduce risks of me-tastasis.

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Astrogliosis is Instigated in a Novel Mouse Modelof Spontaneous Melanoma Brain Metastasis

Hila Schwartz1, Eran Blacher2, Malak Amer1, Nir Livneh1,Anat Klein1,3, Dikla Ben-Shushan4, Shelly Soffer4, MeikeMueller5, Karin Mueller-Decker5, Reuven Stein2, GaliaTsarfaty6, Ronit Satchi-Fainaro4, Viktor Umansky7,8, TobiasPukrop9, Neta Erez

1Department of Pathology, Sackler School of Medicine, TelAviv University, Tel Aviv, Israel2Department of Neurobiology, George S. Wise Faculty of LifeSciences, Tel Aviv University, Tel Aviv, Israel3Department of Cell Research and Immunology, George S.Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv,Israel4Department of Physiology and Pharmacology, SacklerSchool of Medicine, Tel Aviv University, Tel Aviv, Israel5Tumor Models Unit, DKFZ, Heidelberg, Germany6Department of Diagnostic Imaging, Chaim Sheba MedicalCenter, Ramat Gan, Israel7Skin Cancer Unit, German Cancer Research Center (DKFZ),Heidelberg, Germany8Department of Dermatology, Venereology and Allergology,University Medical Center Mannheim, Ruprecht-Karl Univer-sity of Heidelberg, Mannheim, Germany9Department of Hematology and Internal Oncology, Univer-sity of Regensburg, Germany, Germany

Malignant melanoma is the deadliest of all skin cancers.Melanoma frequently metastasizes to the brain, resultingin dismal survival. One of the major obstacles for charac-terizing mechanisms of brain metastasis is the lack oftractable pre-clinical models. Utilizing a Ret-melanoma-derived cell line we have established and characterized anovel mouse model of spontaneous melanoma brain me-tastasis in immunocompetent mice that gives rise to bothmicro- and macrometastases. We show that 3–4 monthsafter surgical excision of primary tumors, 50 % of themice develop brain macrometastases. By utilizing aunique ex-vivo modeling system we detected brainmicrometastases in 50–60 % of the mice and quantifiedthe metastatic load. Moreover, we established tools forintravital diagnosis of brain micrometastases by analyzingblood and cerebrospinal fluid (CSF). We next demonstrat-ed that astrogliosis is instigated early during metastasesformation: astrocytes were recruited to metastatic brainlesions, and paracrine signaling by melanoma cells acti-vated a ‘wound healing program’ in astrocytes, reflectedin up-regulation of Cxcl10, Timp1, Lcn2, Serpine1 andSerpina3n. Finally, we show that co-injection of astro-cytes with melanoma cells intra-cranially resulted in astriking increase of tumor volume. Our findings suggestthat astrogliosis, physiologically instigated as a brain tis-sue damage response, is hijacked by tumor cells to sup-port metastatic growth. This novel model enables the in-tegrative study of tumor cells, immune cells and brainstromal cells and can be utilized as a platform for pre-clinical trials. Since brain metastases are currently incur-able, elucidating molecular mechanisms operative in earlymetastatic growth and signaling pathways that governmetastatic dormancy is the key to developing new

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therapeutic approaches that may prevent brain metastaticrelapse.

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Neutrophil-Mediated Survival and Extravasationof Disseminated Carcinoma Cells

Asaf Spiegel1, Samin Houshyar1, Mary Brooks1, FerencReinhardt1, Michele Ardolino2, Evelyn Fessler1, JasmineDecock1, Ioannis Zervantonakis3, Alexandre Ianello2,Yoshiko Iwamoto4, Virna Cortez-Retamozo4, Mikael Pittet4,David Raulet2, Robert Weinberg11Whitehead Institute, MIT, Cambridge, MA, USA2Molecular and Cell Biology, University of California atBerkeley, Berkeley, CA, USA3Mechanical Engineering, MIT, Cambridge, MA, USA4Center for Systems Biology, Harvard Medical School, Bos-ton, MA, USA

Distant site metastases represent the leading cause ofcancer-associated mortality. As the biological complexityof tumor growth and metastasis is explored more fully, ithas become apparent that key aspects of tumor biologycan only be explained by a detailed understanding of thecontributions to tumor growth by host cell components.While the role of local immune-neoplastic interactions inthe microenvironment has been the subject of intensiveinvestigations, relatively little is known about the functionof circulating immune cells on the more dynamic andtransient phases of the later steps of the invasion-metastasis cascade following entrance of cancer cells intothe general circulation. Here we define novel functions ofneutrophils in promoting intraluminal survival and extrav-asation at sites of metastatic dissemination. We show thatG-CSF released by primary tumors enhances the mobili-zation of CD11b+/Ly6G+ neutrophils into the circulation.The newly mobilized neutrophils proceed to enhance me-tastasis formation via two distinct mechanisms. First, neu-trophils inhibit natural killer cell function, which leads toa significant increase in the intraluminal survival of tumorcells. Thereafter, neutrophils operate to facilitate extrava-sation of tumor cells through the secretion of IL-1β andmatrix metalloproteinases. These results identify a net-work of host cell interactions between innate immunecells, tumor cells and endothelial cells, and positions neu-trophils at the core of this network. This dynamiccrosstalk regulates the intravascular stages of theinvasion-metastasis cascade—intraluminal survival fol-lowing metastatic dissemination and subsequent extrava-sation, i.e., entrance of cancer cells into the parenchymaof target tissues.

11:15–13:45

SYMPOSIUM 9: Targeting the Tumor Microenviron-ment—Angiogenesis and Blood Vessels

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Combined Treatment Strategies for MicrotubuleInterfering Agent-Resistant Tumors

Angela Broggini-Tenzer, Ashish Sharma, Sabine Bender,Katarzyna Nytko-Karouzakis, Martin PruschyDepartment of Radiation Oncology, University Hospital Zu-rich, Zurich, Switzerland

Treatment resistances to specific anticancer agents and ra-diotherapy are often linked to the mutated genetic back-ground of tumor cells. Their activities towards co-targeted tumor microenvironment-related cells are therebyalso abrogated but could be reestablished as part ofrationally-designed combined treatment modalities. Herewe mechanistically investigate in two clinically relevantmicrotubule-interfering agent (MIA)-refractory tumormodels the potency of combined treatment modalities ofMIAs, inhibitors of angiogenesis and ionizing radiation toovercome MIA-resistance.Single and combined treatment regimens of ionizing ra-diation, microtubule stabilizing (taxol, patupilone) andde-stabilizing (BAL101553) agents and anti-angiogeniccompounds (everolimus, bevacizumab) were investigatedin genetically defined MIA-sensitive and MIA-resistantlung and colon adenocarcinoma carcinoma cell systems(A549, SW480) and in the corresponding tumorxenografts.MIAs potently inhibited A549wt and endothelial cell pro-liferation, while no anti-proliferative effect was observedin the corresponding MIA-resistant tumor cells. More im-portant, MIAs did not downregulate anymore HIF-1alphatranscriptional activity and subsequent secretion of HIF-1alpha-mediated growth factors and cytokines from theseMIA-resistant tumor cells. Continuous pro-survival auto-and paracrine signaling from these resistant tumor cellsresulted in an additional level of treatment resistance to-wards MIAs and ionizing radiation as determined in tu-mor xenografts derived from MIA-resistant tumor cells.However rationally-designed combined treatment ofMIAs with mTOR-signaling- or VEGF-antagonistsstrongly re-sensitized treatment-resistant tumors to thecorresponding MIA.These data demonstrate that the interaction between thetumor cell compartment and the tumor microenviron-ment strongly determines the treatment response to

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different anticancer treatment modalities. A combinedtreatment modality of MIAs with antiangiogenic agentsis potent to overcome tumor cell-linked MIA-resistanceand should be considered as clinical strategy for MIA-refractory tumor entities.

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Enhanced Radiosensitization Using Anti-AngiogenicTherapy in Sarcoma Tumors

Adriana Haimovitz-Friedman1, Feng Wang1, Hongyan Li1,Nian-Hong Chen1, Ryan Glass1, Elisa De Stanchina2, GarySchwartz31Radiation Oncology, Memorial Sloan Kettering Cancer Cen-ter, New York, New York, USA2Anti-tumor Core Facility, Memorial Sloan Kettering CancerCenter, New York, New York, USA3Division of Hematology and Oncology, Columbia UniversityMedical Center, New York, New York, USA

Recent data in our laboratory indicates that engagement ofhost-derived microenvironmental elements impact tumor re-sponse to single high dose radiation therapy (SDRT). In thesestudies we showed that microvascular endothelial damageplays a critical role in tumor response as regulator of directlethal damage of SDRT. We used a genetic model of AcidSphingomyelinase (ASMase)-deficient mice to prove that ac-tivation of this enzyme by RT-induced damage in the endo-thelium is mandatory for tumor cure. ASMase activation trig-gers ceramide-mediated apoptosis, and therein microvasculardysfunction, which increased the vulnerability of tumor cellsto lethal damage by radiation. In vitro, VEGF prevented RT-induced ASMase activation and apoptosis in cultured endo-thelium, whilst anti-VEGF/VEGFR2 acts conversely enhanc-ing ASMase-mediated apoptosis. In vivo, anti-VEGF/VEGFR2 de-represses ASMase activation only when deliv-ered immediately prior to SDRT, thus synergistically increas-ing endothelial apoptosis and tumor cure in a fibrosarcomatumor model. Similar results were obtained using SDRTin combination with Pazopanib (anti-VEGFR-1, VEGFR-2, VEGFR-3, PDGF-α/β and c-kit) in two animal modelsof human sarcoma. A single dose of Pazopanib mimics theanti-VEGF/VEGFR impact on SDRT, increasing ASMaseactivity in the serum and endothelial dysfunction, enhanc-ing SDRT tumor cure, and exhibiting critical dependenceon timing relative to RT exposure, suggesting a mechanismof action identical to that demonstrated for anti-VEGF/VEGFR2 antibodies. These results demonstrate the abilityof Pazopanib to shift the response towards tumor cure andcould therefore have a significant impact on clinical trialdevelopment.

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Hypoxia Stimulates Vascular Mimicry by InducingEwing’s Sarcoma Stem Cells to Differentiateinto Vascular Pericytes that Express EWS-FLI-1

Eugenie S. Kleinerman, Zhichao ZhouPediatrics, The University of Texas M.D. Anderson CancerCenter, Houston, Texas, USA

Vasculogenesis and angiogenesis are required for expan-sion of the Ewing’s sarcoma vasculature. We demonstratedthat pericytes and the DLL4 Notch signaling pathway arecritical to the formation of these new tumor vessels. Usingdouble fluorescent staining, we discovered that a subset oftumor vascular pericytes (Desmin+, NG2+) expressed EWSin TC-71 tumor tissue and patient tumor samples suggest-ing that these pericytes originated from Ewing’s tumorcells. These cells were in areas of hypoxia defined byHIF-1α staining. This area also had increased CD133+

cells. Culturing TC-71 cells under hypoxic conditions in-duced sphere formation, and expression of the stem cellmarkers CD133, Sox-2, Oct 3/4 and Nanog. Hypoxia alsoled to the upregulation of DLL4 and the pericyte markersDesmin, SMA and PDGF-BB. To determine whether TC-71 stem cells can contribute to the pericyte pool, CD133+

and CD133− TC-71 cells were isolated following hypoxicculture and incubated with 5 μg/ml DDL4. Desmin andNG2 expression were upregulated by DLL4 in theCD133+ cells but not the CD133− cells. This up-regulation was blocked by the γ-secretase inhibitor DAPT.To confirm that TC-71 cells had differentiated intopericytes, TC-71 cells were transduced with a Desmin-driven promoter vector linked to GFP. Culturing thesetransduced cells under hypoxic conditions resulted in GFPexpression confirming differentiation into a pericyte line-age. While solid tumors are known to contain subsets ofundifferentiated embryonic-like cells with plasticity toserve an endothelial function, this is the first demonstrationthat the hypoxic tumor microenvironment can trigger con-version of Ewing’s sarcoma stem cells into pericytes thatcontribute to the expansion of the tumor vascular network.

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An Epitope-Specific Novel Anti-EMMPRINPolyclonal Antibody Inhibits Tumor Progression

Michal Rahat1,3, Miriam Walter1, Elina Simanovich1, VeraBrod2, Nitza Lahat1,3, Haim Bitterman2,31Immunology Research Lab, Carmel Medical Center, Haifa,Israel

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2Ischemia-Shock Research Laboratory, Carmel Medical Cen-ter, Haifa, Israel3The Ruth and Bruce Rappaport Faculty of Medicine,Technion-Israel Institute of Technology, Haifa, IsraelExtracellular matrix metalloproteinase inducer (EMMPRIN/CD147) mediates tumor cell-macrophage interactions, andhas been shown to induce both MMPs and VEGF. However,the epitope responsible for MMP induction is controver-sial, and the epitope responsible for VEGF induction isyet unknown. We generated a novel anti-EMMPRIN an-tibody directed against a specific epitope that successful-ly inhibited the production of both MMP-9 and VEGF intumor cell-macrophage in vitro co-culture systems,exhibiting a U-shaped dose response. Furthermore, thisantibody efficiently inhibited in vivo tumor progressionin both the RENCA renal cell carcinoma and CT26 coloncarcinoma tumor models. This was achieved byinhibiting angiogenesis as assessed by immunohisto-chemical staining for the endothelial marker CD31, byinhibiting tumor cell proliferation as assessed by thestaining for Ki-67, and by enhancing tumor cell apopto-sis as assessed in the TUNEL assay. Moreover, adminis-tration of the antibody recruited more macrophages intothe tumor, and skewed the tumor microenvironment fromTGFβ-dominated anti-inflammatory microenvironment,to a less immunosuppressive one. The antibody improvedthe ability of stimulated macrophages to performantibody-dependent cell cytotoxicity (ADCC) and kill tu-mor cells. Thus, our antibody maps a novel epitopewhich is capable of inducing both MMPs and VEGF,and places EMMPRIN as a good target for cancertherapy.

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Distinct Bone Marrow Blood Vessels DifferentiallyRegulate Normal and Malignant Hematopoiesis

Tomer Itkin1, Shiri Gur Cohen1, Joel Spencer2, AmirSchajnovitz2, Saravana Ramasamy3, Anjali Kusumbe3, GuyLedergor1, Yookyung Jung2, Idan Milo1, Michael Poulos4,Alexander Kalinkovich1, Aya Ludin1, Orit Kollet1, GuyShakhar1, Jason Butler4, Shahin Rafii4, Ralf Adams3, DavidScadden2, Charles Lin2, Tsvee Lapidot11Department of Immunology, Weizmann Institute of Science,Rehovot, Israel2Department of Stem Cell and Regenerative Biology, HarvardMedical School, Boston, USA3Department of Tissue Morphogenesis, Max Planck Institutefor Molecular Biomedicine, Munster, Germany4Department of Genetic Medicine, Weill Cornell Medical Col-lege, NY, USA

Bone marrow (BM) endothelial cells (BMECs) form a net-work of blood vessels (BVs) that regulate both leukocyte traf-ficking and hematopoietic stem and progenitor cell (HSPC)maintenance. However, it is not clear how do BMECs balancebetween these dual regulatory roles and if these events occurat the same vascular sites. BM arteries were found to be most-ly encircled by αSMA+ pericytes whereas the ensuing small-diameter endosteal arterioles were mostly surrounded by stemcell-niche supporting stromal precursor cells. These endostealarteriole BVs exhibited reduced permeability and high levelsof adhesion- and tight-junction molecules. Primitive HSPCslocated in peri-arteriole regions were found in a non-activated,low reactive oxygen species (ROS) state. Live imaging studiesrevealed that immature and mature leukocyte trafficking oc-curred exclusively through the more permeable sinusoids, lo-cated downstream to the endosteal arterioles. The BM sinu-soids also contain a higher prevalence of ROShigh cells in theirmicroenvironment. In line with these results we show thatduring conditions favoring BM HSPC expansion, endothelialintegrity is enhanced along with reduced HSPC bi-directionaltrafficking. Conversely, disruption of endothelial barrier aug-mented ROS levels in HSPC, enhancing their bi-directionaltrafficking while reducing their BM maintenance. In the con-text of malignancy, engrafted human pre-B ALL cells facili-tated enhanced endothelial barrier integrity and reduced BMpermeability. Clinical studies reported that pre-BALL patientsexhibit higher levels of angiogenic factors alongside with in-creased prevalence of BM BVs density (Perez-Atayde et al.,Am J Pathol. 1997). Moreover, pre-B ALL cells occupyand modify the vascular niche on the expense of normalHSPC (Colmone et al., Science 2008). Taken together,we suggest that malignant cells modify the endothelialbarrier to facilitate a protective and leukemia-supportivemicroenvironment.

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Targeting Endoglin Inhibits Tumor Angiogenesisand Strongly Reduces Metastatic Spread of BreastCancer Cells

Madelon Paauwe1,2, Renier Heijkants2, Charlotte Oudt2, Gabivan Pelt3, Chao Cui2, Charles Theuer4, James Hardwick1,Cornelis F.M. Sier1,3, Lukas Hawinkels1,21Gastroenterology-Hepatology, Leiden University MedicalCenter, Leiden, Netherlands2Molecular Cell Biology, Leiden University Medical Center,Leiden, Netherlands3Surgery, Leiden University Medical Center, Leiden,Netherlands4Tracon Pharmaceuticals, CA, San Diego, California, USA

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Endoglin, a transforming growth factor-β co-receptor, is high-ly expressed on angiogenic endothelial cells in solid tumors.Therefore, targeting endoglin is currently being explored inclinical trials for anti-angiogenic therapy. In this project, theredundancy between endoglin and vascular endothelialgrowth factor (VEGF) signaling in angiogenesis and the ef-fects of targeting both pathways on breast cancer metastasiswere explored.In patient samples, increased endoglin signaling after VEGFinhibition was observed. In vitro TRC105, an endoglin neu-tralizing antibody, increased VEGF signaling in endothelialcells. Moreover, combined targeting of the endoglin andVEGF pathway, with the VEGF receptor kinase inhibitorSU5416, increased anti-angiogenic effects in vitro and in azebrafish angiogenesis model. Next, in a mouse model forinvasive lobular breast cancer, the effects of TRC105 andSU5416 on tumor growth and metastasis were explored. Al-though TRC105 and SU5416 decreased the tumor vasculardensity, tumor volume was unaffected. Strikingly, in micetreated with TRC105, or TRC105 and SU5416 combined, astrong inhibition in the number of metastases was seen. More-over, upon resection of the primary tumor, strong inhibition ofmetastatic spread by TRC105 was observed in an adjuvantsetting. To confirm these data, we assessed the effects ofendoglin-Fc (an endoglin ligand trap) onmetastasis formation.Similar to treatment with TRC105 in the resection model,endoglin-Fc expressing tumors showed strong inhibition ofdistant metastases.These results show, for the first time, that targeting endoglin,either with neutralizing antibodies or a ligand trap, stronglyinhibits metastatic spread of breast cancer in vivo.

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Experimental Hepatic Metastasis Treatmentwith Antiangiogenic microRNA-EncapsulatedNanoparticles

Iker Badiola1, Joana Marquez1, Ariane Schaub1, MaiteUnzurrunzaga2, Ines Fernandez-Piñeiro3, AlejandroSanchez3, Gaskon Ibarretxe1, Fernando Unda11Department of Cell Biology and Histology. Faculty of Med-icine, University of the Basque Country, Leioa, Spain2Centro de Salud Elorrio, Osakidetza, Elorrio, Spain3Department of Pharmaceutical Technology, University ofSantiago de Compostela, Santiago de Compostela, Spain

Colorectal cancer is one of the leading causes of cancerdeath worldwide as a result of its considerable risk ofdevelopment of metastases. When metastasic disease isconfined to the liver, partial liver resection is the onlycurative therapeutic option. For this reason, it is

important to study new antimetastasic treatments. Cancermetastasic cells have many strategies to survive, colonizeand growth in the hepatic microenvironment. One of themost important strategies is known as angiogenesis. Dur-ing angiogenesis, tumor cells recruit the endothelial cellsand create new blood vessels to support the tumorgrowth. In this work, we studied the transformation ofliver sinusoidal endothelial cells (LSEC) duringmetastasic process and their post-transcriptional regula-tion by microRNAs (miRNA). In addition, we designeda strategy to block LSEC transformation and thereforeangiogenesis and metastasis progression. To perform thisassay, CT26 cells were intrasplenically inoculated tomice and after 2 weeks, once the cells metastasized theliver, tumor microenvironment LSECs were isolated byliver perfusion and gradient separation. Then, a microar-ray was carried out to compare gene and miRNA expres-sion pattern of healthy LSECs with respect to LSECsthat received the metastasic tumor. Bioinformatic toolsselected E2F1 and VEGF genes as proteins involved intumor progression and miRNA-20 and miRNA-652 wererespectively found as regulators of the mentioned genesexpression. Both genes and microRNAs were in vitrovalidated by qPCR and western blot. Finally, chondroitinsulphate-conjugated sorbitan ester based nanoparticlesloaded with selected miRNAs were designed to guidethem directly to LSECs and repress the angiogenesisand tumor progression in vitro and in vivo.

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The Aplidin Analogs PM01215 and PM02781 InhibitAngiogenesis in Vitro and in Vivo

Gerold Untergasser1,4, Normann Steiner1, Silvia Carbon1,Johann Kern3, Andrés Francesch2, Wolfgang Willenbacher1,Eberhard Gunsilius11Medical University Innsbruck, Internal Medicine V, Inns-bruck, Austria2Pharmamar, R&D Department, Madrid, Spain3Oncotyrol GmbH, R&D Department, Innsbruck, Austria4Tyrolean Cancer Research Institute, ExperimentalOncogenomics, Innsbruck, Austria

Background: Novel synthesized analogs of Aplidin,PM01215 and PM02781, were tested for antiangiogenic ef-fects on primary human endothelial cells in vitro and for inhi-bition of angiogenesis and tumor growth in vivo.Methods and Results: Both derivatives inhibited angiogeniccapacities of human endothelial cells (HUVECs) in vitro at lownanomolar concentrations, as determined by real-time cell prolif-eration and migration, capillary tube formation and vascular

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endothelial growth factor (VEGF)-induced spheroid sproutingassays. Antiangiogenic effects of both analogs were observedin vivo in chicken chorioallantoic membrane (CAM) assays. Inaddition, growth of human multiple myeloma xenografts in theCAMwas significantly reduced after application of both analogs.On the molecular level, both derivatives induced cell cycle arrestin G1 phase, as determined by flow cytometric analysis of endo-thelial cells. This growth arrest correlated with induction of thecell cycle inhibitor p16INK4A and increased senescence-associated beta galactosidase activity. In addition, Aplidin ana-logs did not promote ER-stress, but induced oxidative stress anddecreased production of the vascular maturation factorsVasohibin-1 and Dickkopf-3.Conclusions: From these findings we conclude that both ana-logs are promising agents for the development of antiangiogenicdrugs acting independent on classical inhibition of VEGF signal-ing.

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Heparanase 2 Attenuates Head and Neck TumorVascularity and Growth

Miriam Gross-Cohen1, Sari Feld1, Ilana Doweck2, GeraNeufeld1, Peleg Hasson3, Gil Arvatz1, Uri Barash1, InnaNaroditsky4, Neta Ilan1, Israel Vlodavsky11Cancer and vascular Biology Research Center, the BruceRappaport Faculty of Medicine, Technion-Israel Institute ofTechnology, Haifa, Israel2Department of Otolaryngology, Head and Neck Surgery,Carmel Medical Center, Haifa, Israel3Department of Cell Biology, the Bruce Rappaport Faculty ofMedicine, Technion-Israel Institute of Technology, Haifa,Israel4Department of Pathology, Rambam Health Care Campus,Haifa, Israel

Heparanase is an endoglycosidase that specifically cleavesheparan sulfate (HS) side chains of proteoglycans, activity thatis highly implicated in tumor metastasis and angiogenesis.Heparanase 2 (Hpa2) is a close homolog of heparanase thatlacks intrinsic HS-degrading activity but retains the capacityto bind HS with high affinity. In head and neck cancer pa-tients, Hpa2 expression was markedly elevated correlatingwith prolonged time to disease recurrence and inversely cor-relating with tumor cell dissemination to regional lymphnodes, suggesting that Hpa2 functions as a tumor suppressor.The molecular mechanism associated with favorable progno-sis following Hpa2 induction is unclear.Here, we provide evidence that Hpa2 over expression in headand neck cancer cells markedly reduces tumor growth. Re-strained tumor growth was associated with a prominent

decrease in tumor vascularity (blood and lymph vessels), like-ly due to reduced Id1 expression, a transcription factor highlyimplicated in VEGF-A and VEGF-C gene regulation. Hpa2also induces the expression of lysyl oxidase (LOX), an en-zyme that crosslinks collagen and elastin, thereby affectingECM remodeling and tissue fibrosis. Expression of lysyloxidase-like 2 (LOXL2) was not induced but assumed nuclearlocalization in tumor xenografts produced by Hpa2 over ex-pressing cells.Notably, heparanase enzymatic activity was not impairedin cells over expressing Hpa2, suggesting that reducedtumor growth is not due to heparanase regulation. More-over, growth of tumor xenografts produced by Hpa2over-expressing cells was not affected by a monoclonalantibody that targets the heparin binding domain ofHpa2, implying that Hpa2 functions in heparanase-,and HS-independent manner.

11:15–13:40

SYMPOSIUM 10: Metastasis and the MetastaticMicroenvironment (II)

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ATF-1, Retinoic Acid Induced 14 (RAI14) and C-Junform a Novel Regulatory Axis of Invasivenessand Proliferation

Marcelo Ehrlich, Karin EytanCell Research and Immunology, Tel Aviv University, Tel Aviv,Israel

Unraveling the molecular mechanisms which transduce mi-croenvironmental cues and integrate such signals with intrin-sically aberrant regulation of proliferation and invasiveness ofcancer cells, and metastatic cells in particular, should furtherthe development of effective therapeutic strategies. Over-activation of Jun N-terminal Kinase (JNK)/c-Jun signalingwas shown to mediate tumorigenic attributes in different tu-mor models. However, the molecular basis of such aberrantactivation and its integration into the tumor cell context meritfurther study. Proteins which dynamically localize both to thecytoskeleton/membrane interface and the cell nucleus poten-tially integrate excessive signaling input at the cell surface andmodified transcriptional control of gene expression. We iden-tify retinoic acid induced 14 (RAI14) as a candidate for suchregulatory function. We opted for glioblastoma cells as a mod-el of invasive and aggressive tumor. The RAI14 message isoverexpressed in glioblastoma clinical samples, and this over-expression correlates with poor survival. Glioblastoma cell

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lines expressed the RAI14 protein, which localized to plasmamembrane, actin cytoskeleton and cell nucleus. Antibody-mediated pull-down of RAI14 identified its interaction withmultiple cellular components, including the phosphorylatedform of the transcription factor ATF-1. Knockdown ofRAI14 or ATF-1 reduced proliferation and invasiveness ofglioblastoma cells. Moreover, siRNA-mediated knockdownstudies also revealed the regulation of c-Jun and phospho-c-Jun expression by both ATF-1 and RAI14, and the regulationof RAI14 expression by ATF-1. Inhibition of JNK activityreduced the expression of phospho- and total c-Jun, and theproliferation and invasiveness of U87-MG cells; whilesiRNA-mediated knockdown of c-Jun attenuated their prolif-eration. Together, our data identify an ATF-1-RAI14-c-Junregulatory circuit in glioblastoma cells, and suggest that suchcircuit contributes to the invasive and proliferative characterof this malignancy.

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p53 Family, Oncogenic Signaling and CancerMetastasis

Zhi-Xiong Jim Xiao, Linshan Hu, Shan Liang, Yujun Zhang,Johann BergholzCollege of Life Sciences, Sichuan University, ChengDu,China

Inactivation of the tumor suppressor protein p53 plays a criticalrole in tumorigenesis, whereas p53-related p63 gene is essentialin epithelial development and stem cell biology. Emerging evi-dence indicates that mutant p53 proteins acquire gain of func-tions in promoting cancer metastasis and that p63 is important inregulation of cell migration/invasion. We have recently shown anovel pathway with which the p53 hotspot point mutant, p53-R273H, in promoting cancer metastasis. In addition, we havedemonstrated that p63 modulates ERK signaling in regulatingcell migration/invasion. Our recent studies also indicate an essen-tial role for p63 in mediating PI3K-induced cancer progression.References:1. J Bergholz1, Y Zhang, J Wu, L Meng, EM Walsh, A Rai,MY Sherman and Z-X Xiao*.(2014) ΔNp63α regulates Erksignaling via MKP3 to inhibit cancer metastasis. Oncogene33, 212–224.2. Junfeng Wu, Shan Liang, Johann Bergholz, Hanbing He,Erica M. Walsh, Yujun Zhang, Zhi-Xiong Xiao* (2014)ΔNp63α activates CD82 metastasis suppressor to inhibit cancercell invasion. Cell Death and Disease 5, e1280; doi:10.1038/cddis.2014.2393. Chenghua Li, Donny L.F. Chang, Zemin Yang, Jin Qi,Ruihong Liu, Hanbing He, Decai Li, Zhi-Xion Xiao*(2013) Pin1modulatesΔNp63α protein stability in regulation

of cell survival, proliferation and tumor formation. Cell Deathand Disease 4, e943; doi:10.1038/cddis.2013.468.4. Johann Bergholz andZhi-Xiong Xiao*, (2012) Role of p63in Development, Tumorigenesis and Cancer Progression.Cancer Microenvironment 5:311–322.

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The Microenvironment of Melanoma BrainMetastasis

Sivan Izraely-Bino1, Orit Sagi-Assif1, Shlomit Ben-Menachem1, Maya Rappaport-Saban1, Moran Frig1, TsipiMeshel1, Metsada Pasmanik-Chor2, Roshini Prakash3, S.Thomas Carmichael3, Dave S.B. Hoon4, Isaac P. Witz11Department of Cell Research and Immunology, Tel Aviv Uni-versity, Tel Aviv, Israel2Bioinformatics Unit, Tel Aviv University, Tel Aviv, Israel3Department of Neurology, University of California LosAngeles, Los Angeles, California, USA4Department of Molecular Oncology, John Wayne CancerInstitute, Saint John’s Health Center, Santa Monica,California, USAThe progression of cancer towards metastasis is driven byautonomous traits of the tumor cells as well as by interactionsof tumor cells with non-tumor cells in their vicinity and withsoluble factors released by them. Brain metastases occur fre-quently in melanoma patients with advanced stage disease.Yet, understanding the mechanisms underlying developmentof brain metastasis is far from complete. Our study aims touncover these mechanisms and the interactions between mel-anoma and brain cells.

For our study we have used two experimental systems:1. We developed human melanoma xenograft modelsencompassing cutaneous, brain macro-metastatic and brainmicro-metastatic melanoma variants, originating from singlemelanoma tumors. Using these models we identified a set ofmelanoma brain metastasis signature genes, includingClaudin1 (CLDN1). We have found that CLDN1 functionsas melanoma brain metastasis inhibitor.2. Neuro-repair processes occurring after stroke involvemicroglia, astrocytes and endothelial cells (BECs), andcontrol the localization, survival and differentiation of im-mature neurons. We hypothesize that melanoma cells uti-lize post-stroke repair mechanisms for the establishment ofbrain metastasis. An oxygen-glucose depravation (OGD)treatment of microglia, astrocytes and BECs was used asan in-vitro stroke model. OGD promoted pro-metastaticinteractions between brain cells and melanoma, and in-duced secretion of inflammatory cytokines from braincells. Inoculation of melanoma cells into mice after

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http://dx.doi.org/10.1038/cddis.2014.239



transient middle cerebral artery occlusion (MCAO)showed preferential localization of metastatic melanomacells to areas of tissue regeneration, and recruitment ofastrocytes and microglia to metastatic region.

These results demonstrate that brain microenvironment has acrucial role in melanoma brain metastasis formation.Uncovering the involved mechanisms could enable the detec-tion of novel therapeutic targets.This study was supported by the Dr. Miriam and Sheldon G.Adelson Medical Research Foundation (Needham, MA,USA).

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Making Holes: Identifying How Metastatic CancerCells Apply Force to Invade their Microenvironment

Daphne WeihsFaculty of Biomedical Engineering, Technion-Israel Instituteof Technology, Haifa, Israel

The process of invasion is of special importance in cancermetastasis, the main cause of death in cancer patients.Cells typically penetrate a matrix by degrading it or bysqueezing through pores. However, cell mechanics andforces applied by cells especially during the initial stagesof metastatic penetration, as metastatic cells indent a sub-strate, are still unknown. We use a specialized technologyto measure the strength of the cells, specifically the forcesthat cells apply to an impenetrable, synthetic 2-dimensional gel-matrix to focus only on mechanical-interactions; gels are non-degradable polyacrylamide withsub-micron pores. We show that single metastatic breast-cancer cells will apply force to an impenetrable gel, andindent it in attempted invasion, when the gel is in theappropriate stiffness range; benign cells do not indentthe gels. The metastatic cells require gel-substrates to besoft enough to indent, yet stiff enough to grip and gener-ate force on. Cells develop grip handles and pull the un-derlying gels inwards and upwards bringing the nucleusinto the indentation concavity. We reveal a special coor-dinated role for the nucleus and the cytoskeleton when asingle cell attempts to invade the impenetrable barrier.The actin, nucleus, and microtubules reorganize in se-quence, with the actin at the leading edge of the cell. Cellsrepeatedly attempt penetration over several hours and thenrelocate, indicating an advanced mechano-transductionfeedback loop. The systems and analysis approachesshown here reveal cell adaptation, force application mech-anisms, and can potentially serve as a diagnostic/prognostic platform.

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Preventing the Recurrence of Breast Cancerat the Metastatic Niche Using Resolution-PhaseMacrophages

Odelya Gilon, Sagie Schif, Amiram Ariel, Dalit BarkanDepartment of Human Biology, University of Haifa, Haifa,Israel

Breast cancer that recurs as metastatic disease many years afterprimary tumor resection and adjuvant therapy appears to arisefrom tumor cells that disseminated early in the course of thedisease but did not develop into clinically apparent lesions.These long-term surviving, disseminated tumor cells maintaina state of dormancy and are resistant to conventional therapiesthat target actively dividing cells. The mechanisms responsiblefor maintaining the survival and outgrowth of dormant tumorcells remain largely unknown. Recently, we found that fibrotic-like microenvironment with extensive deposition of Type I col-lagen (Col-I) and fibroectin established at the site of the residingdormant tumor cells promoted the outbreak of residing dormanttumor cells. Therefore, we hypothesized that promotingresolution- the endogenous mechanism that terminates inflam-mation and fibrotic responses and actively directs tissue return tohomeostasis- at the permissive site will ‘normalize’ it and pre-vent metastatic outbreak. Here we demonstrate that soluble fac-tors secreted by ex-vivo generated pro-resolving CD11blowmac-rophages can inhibit the establishment of a fibrotic microenvi-ronment by inhibiting TGFß1-induced differentiation of fibro-blasts to myofibroblasts and consequently myofibroblasts ex-pression of Col-I. Furthermore, these soluble factors preventedthe metastatic outbreak of dormant tumor cells co-cultured withmyofibroblasts in an in vitro 3D system that models tumor dor-mancy and metastatic outgrowth. Interestingly, our data alsodemonstrate that the blockade of fibroblast differentiation tomyofibroblasts is not mediated by inhibition of the canonicalsignaling of TGFß1. Taken together our results demonstrate apioneering conceptual approach to ‘normalize’ the microenvi-ronment that supports the outgrowth of disseminated dormanttumor cells by promoting the tissue resolution axis.

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Lysyl Oxidase as a Potential Target for InfiltrativeGrowth in Glioblastoma

Lara Perryman1, Anette M. Høye1, Thomas R. Cox1, JanStrøbech1, Lidia Leonte1, Lukram B. Singh1, Sergey Popov3,Hans Skovgaard Poulsen2, Chris Jones3, Janine T. Erler11University of Copenhagen, Biotech Research and InnovationCentre (BRIC), Copenhagen, Denmark

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2Copenhagen University Hospital,, Department of RadiationBiology, The Finsen Center, Copenhagen, Denmark3Glioma Team, The Institute of Cancer Research, Sutton, UK

Glioblastoma (GBM) is the most common primary brain can-cer. It is highly aggressive and unlike many other cancers types,the median survival for patients after treatment (14.6 months)has barely improved in the last 20 years. Infiltrative growth intothe surrounding brain parenchyma is thought to be responsiblefor tumour recurrence and ultimately the death of the patient.Novel therapies are desperately needed to prevent this infiltra-tive growth and the subsequent recurrence.GBM infiltrative cells invade by migrating along the region im-mediately adjacent to blood vessels, known as the perivascularniche. The perivascular niche is composed of a specialisedlaminin-rich basement membrane, and is a knownchemoattractant for GBM cells. Here, we investigated a role forthe enzyme Lysyl Oxidase (LOX) in GBM infiltration. LOX is atumour-secreted hypoxia regulated enzyme previously shown topromote invasion of other solid tumour cancers.Analysis of publicly available gene expression data fromGBM patient samples revealed a strong association betweenLOX expression and poor survival, specifically in the morehypoxic and invasive mesenchymal GBM subtype. We havedemonstrated that invasive phenotype in patient-derivedGBM stem cell lines is associated with high LOX expression,and invasion is dependent on LOX activity through blockingexperiments. Furthermore, over expression of LOX in non-invasive lines drives infiltrative growth in vivo and ex vivo,and exhibits increased chemotaxis towards extracellular lam-inin. We have shown that a MMP-7 is associated with the overexpression of LOX to drive invasive growth in GBM.Our findings suggest that LOX up regulates MMP-7 that frag-ments laminin creating a chemotactic gradient, which attractsGBM cells enabling infiltration of the perivascular niche.Targeting LOX may represent an effective therapy againstinfiltrative GBM.

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New Insights on How an Inflammatory TumorMicroenvironment Promotes Invasion and EarlyStage Breast Cancer Metastasis

Celeste Bolin1, Ken Tawara1, Jordan Koncinsky1, Hunter Co-vert1, Danielle Hedeen1, Robin Anderson2, Joel Garbow3,Cheryl Jorcyk11Biological Sciences/Biomolecuar Sciences, Boise State Uni-versity, Boise, Idaho, USA2Oncology, The Sir Peter MacCallum Cancer Centre, Mel-bourne, Victoria, Australia

3School of Medicine, Washington University, St. Louis, Mis-souri, USA

The propensity of primary breast cancer to invade and metas-tasize has been partially explained by high levels of endoge-nous inflammation. However, it is not fully understood howinflammatory mediators, expressed either by the primarybreast cancer cells or cells of the tumor microenvironment(TME), specifically contribute to metastasis. Our lab has beenstudying how oncostatin M (OSM), an interleukin-6 (IL-6)-family inflammatory cytokine, promotes the development ofearly metastatic properties distinct from IL-6 and other familymembers. Human breast tissue microarray analysis shows thatOSM is expressed at highest levels in the precancerous epi-thelial cells of ductal carcinoma in situ (DCIS), suggesting arole for autocrine-produced OSM in invasive potential.Tumor-associated neutrophils (TANs) release large amountsof OSMwhen they come into contact with breast cancer cells,and this paracrine-produced OSM induces invasive capacity.Employing human breast and mouse mammary orthotopicxenograft and syngeneic mouse models, OSM promotes me-tastasis to bone, lung, and other organs. Furthermore, OSMincreases circulating tumor cell (CTC) numbers that are re-duced in an OSM knockout background, demonstrating theimportance of paracrine-produced OSM in this process. Whensupported by in vitro results, where OSM induces breast tu-mor cell epithelial-mesenchymal transition (EMT), proteasesecretion, and detachment, our findings suggest a role forOSM in early stage metastasis. Finally, the importance ofOSM relative to IL-6 in these processes will be discussed,and as anti-IL-6 cancer therapies (i.e., siltuximab) are failingin clinical trials, our results suggest that OSMmay be themorerational target. Collectively, these data suggest that OSM,whether TME- or autocrine-produced, is a critical factor driv-ing breast cancer invasion and tumor cell dissemination withsubsequent metastasis.

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Label-Free Morphological Discriminationbetween Different Cancer Cell Lines and Leukocytesby Digital Holographic Microscopy: a Modelfor Enumeration of Circulating Tumors Cellsin Blood

Itay Barnea1, Alex Barbul1, Pinhas Girshovitz2, NathanShaked2, Rafi Korenstein11Department of Physiology and Pharmacology, Tel Aviv Uni-versity, Tel Aviv, Israel2Department of Biomedical Engineering, Tel Aviv University,Tel Aviv, Israel

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The isolation and enumeration of circulating tumor cells(CTCs) in blood are used to monitor metastatic disease pro-gression and guide cancer therapy. The common accepted ap-proach of enriching and enumeration of CTCs from the largecell population of leukocytes is based on immune-labeling ofepithelial markers such as the epithelial cell adhesion molecule(EpCAM) and cytokeratins (CK), as well as the absence of theleukocyte marker, CD45. We suggest an alternative label-freemethodology for discriminating between CTCs and leuko-cytes, based on label-free 2D and 3D morphological parame-ters acquired by Digital Holographic Microscopy (DHM).DHM is based on measuring an interference pattern composedof a superposition of the light field which has interacted withthe cell and a mutually-coherent reference field. From the re-corded complex field, it is possible to digitally reconstruct thequasi-three-dimensional distribution of the cell quantitativephase image, which is proportional both to the spatial cellthickness (with nanometer accuracy) and its refractive index.Thus DHM provides label-free quantitative information on aspectrum of parameters defining cell morphology and content.Several types ofmetastatic cell-linesWM-266-4,MCF-7, SW-640, and A549, isolated from different organs (skin, breast,colon and lung, respectively), were used as cellular modelsfor CTC. Three main subsets of leukocytes (monocytes, neu-trophils and T cells) were isolated from venous human blood,using negative selection. About 50 cells of each cell type wereimaged by DHM. The morphological parameters of the differ-ent cells were analyzed and compared using ANOVA. Resultsdemonstrate statistically significant differences in several mor-phological parameters, consisting of the area, mass and surfaceroughness of the cells, demonstrating the ability of DHM todiscriminate the model CTC types from leucocytes.

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Breast Cancer Cell Detachment from PlacentaConditioned ECM Activates Integrin-TGFβ/JNKAxis and Enhances their Metastatic Potential

Shelly Tartakover Matalon1,5, Gali Epstein Shochet1,5,Oded Komemi1,5, Meir Pomeranz2,5, Ami Fishman2,5,Metsada Pasmanik-Chor4, Liat Drucker1,5, Michael Lishner1,3,5

1Oncogenetic Laboratory, Meir Medical Center, Kfar Saba,Israel2Obstetrics & Gynecology, Meir Medical Center, Kfar Saba,Israel3Internal Medicine A, Meir Medical Center, Kfar Saba, Israel4Bioinformatics Unit G.S.W., Faculty of Life Sciences, Tel AvivUniversity, Tel Aviv, Israel5Sackler Faculty of Medicine, Tel Aviv University, Kfar Saba,Israel

Background: Extracellular matrix (ECM) affects cell charac-teristics. Detachment from the ECM induces cell apoptosis,termed anoikis. Cancer cells can develop anoikis resistance, amandatory step for metastasis, by switching integrins, over-expressing EGFR members and inducing epithelial mesen-chymal transition (EMT). The placenta is described in theliterature as a non-supportive microenvironment for cancercells. We showed that breast cancer cells (BCCL) were elim-inated from placental implantation sites. During implantation,the placenta manipulates its surrounding matrix, which mayinduce the BCCL elimination. Aim: Exploring the effect ofplacental induced ECM manipulations on BCCL.Methods: BCCL (MCF-7/T47D) were cultured on placenta/BCCL conditioned ECM (matrigel used for first trimesterplacenta/BCCL culture and cleared by NH4OH). Followingculture we analyzed BCCL phenotype (death, count, aggrega-tion, MMP) and signaling (microarray analysis and validation(qPCR, western-blot)).Results: BCCL migrated away from previous placental im-plantation sites, displayed elevated MMP activity and mRNA,formed aggregates in distant areas and enhanced proliferation(all p<0.05). MCF-7 were significantly more affected thenT47D, their cell death was modestly elevated (p<0.05), theyunderwent EMT, expressed elevated level of CD44+\CD24low

(stem-cell markers) and developed drug resistance.Microarrayanalysis of the MCF-7 highlighted changes in integrin, estro-gen, EGFR and TGFβ pathways. Indeed, placental ECM re-duced ERα, induced Smad3/JNK phosphorylation and elevat-ed integrin-α5 expression (RGD dependent integrin) in theBCCL (all p<0.05). Addition of RGD or TGFβR/JNK inhib-itors reversed some of the phenotype observations.Conclusions: BCCL detachment from the placental ECM in-duces cell characteristics that facilitate metastasis as EMTanddrug-resistance. ITGα5, TGFβ/JNK and RGD dependentpathways are activated in these cells. These molecules maybe activated in cells that detached from the primary tumorsand serve as therapeutic targets.

15:15-16:55

PLENARY SESSION 5: Targeting the Tumor Microenvi-ronment—Novel Approaches (II)

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Angiognesiss Revisited: Role and (Therapeutic)Implications of Endothelial Metabolism

Peter CarmelietVesalius Research Center, Laboratory of Angiogenesis andNeurovascular Llink, Leuven, Belgium

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Angiogenesis, the growth of new blood vessels, plays acrucial role in numerous diseases, including cancer.Anti-angiogenesis therapies have been developed tostarve cancer cells from nutrients. Clinically approvedanti-angiogenic drugs prolong the survival of cancer pa-tients, but their success is limited by intrinsic refractori-ness and acquired resistance. New strategies are thusneeded to block tumor angiogenesis via alternativemechanisms. We recently reported that PFKFB3-drivenglycolysis regulates the endothelial tip cell function dur-ing vessel sprouting, even capable of overruling the po-tent pro-stalk activity of Notch, and that its loss in en-dothelial cells causes vascular hypobranching defects.Moreover, partial and transient reduction of glycolysisby blocking PFKFB3 reduced pathological angiogenesisin several disease models. Ongoing studies explore therole of lipid and amino acid metabolism in vesselsprouting, and assess the therapeutic potential oftargeting these metabolic pathways for anti-angiogenictherapy.

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ATranslational Approach to the MetastaticallyRelevant MicroRNA-Landscape

Heike AllgayerExperimental Surgery, Ruprecht-Karls University of Heidel-berg, Mannheim Medical Faculty, Mannheim/Heidelberg,Germany

This lecture will present our most actual data on systemat-ically defining the metastasis-associated microRNA land-scape in a translational research approach. We profiled andvalidated microRNA and mRNA expression in a uniqueseries of human colorectal metastasis tissues together withtheir matched primary tumors and corresponding normaltissues, using these data to guide functional studies. Weidentified a miR-signature exclusively differentiallyexpressed in metastases with three of these miRs identifiedas key drivers of an EMT-regulating network acting thougha number of novel targets. These include SIAH1, SETD2,ZEB2, and especially FOXN3, which we demonstrated forthe first time as a direct transcriptional suppressor of N-cadherin. This had significant impact on migration, inva-sion, and metastasis in two different in vivo models. Thesignificant deregulation of all players of our network wasconfirmed in an independent own patient set, and in 6000patients in a large database of diverse malignancies. Ourdata define a novel metastasis-orchestrating network withpotential impact on the metastatic process in general, based

on systematic hypothesis generation from metastasistissues.In addition, the lecture will present latest data of thegroup on single-molecule, single-cell laser superresolutionmicroscopy of micro RNAs in comparison between low-versus highly metastatic cells, as well as first preliminaryresults on genomic changes found in the same set ofresected metastasis and further tissues of the patient seriesabove.

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Reprogramming Tumor Cells and their ParacrineInteractions: A New Approach to Cancer Therapy

Bernd Groner, Chul Min YangInstitute for Tumor Biology and Experimental Therapy, GeorgSpeyer Haus, Frankfurt, Germany

Cellular transformation is initiated by the activation ofoncogenes and a closely associated developmentalreprogramming of the epigenetic landscape. Transcriptionfactors, regulators of chromatin states and microRNAsinfluence cell fates in development and stabilize the phe-notypes of normal, differentiated cells. The same molec-ular mechanisms provide transforming properties to can-cer, determine their paracrine cellular interactions in thetumor microenvironment and the potential of primarytumor cell to metastasize. We investigated if the stateof transformation can be reversed to achieve the normal-ization of cellular properties. We used the geneticallyhighly aberrant glioblastoma cells and investigated theeffects exerted by the expression of the miR-302/367cluster. The miR-302/367 cluster can promote the cellu-lar reprogramming of human and mouse cells and con-tribute to the generation of iPSC. We found that theepigenetic reprogramming potential of the miR-302/367cluster is able to “de-program” tumor cells, i.e., shifttheir gene expression pattern towards an alternative pro-gram associated with more benign cellular phenotypes. Itsuppressed transformation related proteins, e.g., thereprogramming factors OCT3/4, SOX2, KLF4 and c-MYC, and the transcription factors POU3F2, SALL2and OLIG2, required for the maintenance of glioblastomastem like tumor propagating cells. It also diminishedPI3K/AKT and STAT3 signaling, impeded colony forma-tion in soft agar and cell migration. At the same time,the miR-302/367 cluster restored the expression of neu-ronal markers of differentiation. The suppression of pro-inflammatory cytokine secretion drastically affected thetumor microenvironment. miR-302/367 cluster express-ing cells lose their ability to form tumors and to establish

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liver metastasis in nude mice. The process of “de-pro-graming” of tumor cells could potentially become anew concept for cancer therapy.

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Identifying Molecular Signatures for TumorDormancy as a Basis for the Rational Designof Precision Nanomedicines

Ronit Satchi-FainaroDepartment of Physiology and Pharmacology, Sackler Schoolof Medicine, Tel Aviv University, Tel Aviv, Israel

Tumor progression is dependent on a number of sequentialsteps, including interactions of tumor cells with their sur-rounding microenvironment and its different immune, en-dothelial and connective cellular and extra-cellular compo-nents. Failure of a microscopic tumor, either primary, re-current or metastatic, to complete one or more of theseearly stages may lead to delayed clinical manifestation ofthe cancer. Micrometastasis, dormant tumors, and residualtumor cells—referred to as minimal residual disease, con-tribute to the occurrence of relapse, and constitute funda-mental clinical manifestations of tumor dormancy that to-gether are responsible for the vast majority of cancerdeaths. However, although the tumor dormancy phenome-non has critical implications for early detection and treat-ment of cancer, it is one of the most neglected areas incancer research and the associated biological mechanismsare still mostly unknown.We have created several models of patient-derived xeno-grafts mimicking pairs of dormant vs fast-growing, prima-ry vs metastatic and drug-sensitive vs resistant cancers.We investigated the molecular and cellular changes intumor-host interactions that govern tumor dormancy.Those led to the discovery of novel targets and providedimportant tools for cancer theranostics. Based on the ac-quired knowledge, we designed a new strategy to improvetreatment outcomes of patients with bone neoplasms, glio-blastoma, brain metastases, melanoma, breast and prostatecancers. We have identified molecular signatures whichfollowing their selective delivery into target cells, canpotentially induce a dormant-like phenotype. This goalwas achieved by utilizing polymeric nanomedicines andguidance by high resolution, intravital non-invasive imag-ing techniques.A better understanding of tumor dormancy and the availabilityof relevant markers will most likely change the way we diag-nose and treat the disease using novel combined theranosticnanomedicines.

17:15–18:30

PLENARY SESSION 6: Metastasis & the MetastaticMicroenvironment (I)

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Circulating Tumor Cells and TumorMicroenvironment

Patrizia Paterlini-Brechot1Faculte de Medecine Paris Descartes, University Paris Des-cartes and INSERM, Paris, France

Circulating Tumor Cells (CTC) and Circulating TumorMicroemboli (CTM) are cancer seeds in transit from primaryto secondary soils. They thus experience a changing microen-vironment from primary tumor, to blood and to metastaticsites. We review the interaction between CTC/CTM and thechanging CTC/CTM microenvironments and discuss the newdevelopments shedding light on the impact of this interactionon tumor cells growth, detachment, invasion, circulation, sur-vival, seeding, proliferation and colonization.The relevance of CTC/CTM stems from their key role in tumorinvasion. We thus review the major issues involved in CTC andCTM tumor identity and biological characteristics, the relatedtechnical challenges for their extraction from blood, diagnosticidentification and discuss their potential for clinical benefit. Weanalyze CTC/CTM-related methodological bias and critical is-sues to use CTC for bringing clinical benefit to patients, andhighlight recent developments and burning questions whichshould be addressed to improve our understanding of this domain.We discuss the conceptual difference between CTC/CTM andCirculatingCancerCells (CCC)/CirculatingCancerMicroemboli(CCM) and the role of CCC/CCM in tumor invasion.Finally, we review the challenges related to the study of CCC/CCMmolecular code and the relevance of their molecular char-acterization for a better understanding of the invasion process.CCC/CCM are poised to bring a major improvement in thefield of personalized, non-invasive predictive oncology. Wewill review the scientific background and developmentswhich allow predicting this change.

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On the role of Galectin 3 in the TumorMicroenvironment

Avraham RazOncology, Wayne Stae University, Karmanos Cancer Insti-tute, Detroit, Michigan, USA

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It was reported that the level of secreted Galectin 3 (Gal-3)could be as high as 31- fold in the serum of cancer patientswith breast, colorectal, lung, bladder, head and neck, thyroid,prostate, and melanoma, and that patients with metastatic dis-ease have higher concentrations of circulating Gal-3 thanthose with localized tumors. Thus Gal-3 was suggested to bean emerging promoter of cancer metastasis. Gal-3 was shownto recognize and bind to nearly all cell-surface and extracellu-lar matrix glyco-components that contain terminal galactoseresidues, including growth factor receptors such as EGFR,VEGFR, and TGF-βR to name a few/ This wide array ofGal-3 interactions, mediated solely by sugar recognition, in-dicates that Gal-3 is involved in regulating a myriad of activ-ities during cancer development, progression, and metastasis,leading to its characterization as a “jack-of-all-trades in can-cer”. A lthough the initial studies demonstrating the role ofgalectin-3 in tumor angiogenesis were reported more than adecade ago, in the recent years an increasing interest in thisfield confirms its significance. More studies are in order usingdifferent models to analyze the significance of galectin-3 onvarious steps of angiogenesis to get a more comprehensiveknowledge of this potentially important and exciting field. Inthe bone micro environment will show the presence of a com-plex network of multiple soluble factors drives vicious cyclesof highly destructive bone remodeling, and will report thatGal-3 a may be targeted. In bone metastasis patient samples.

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RNA Editing and Melanoma Metastasis

Menashe Bar-EliCancer Biology, M.D. Anderson Cancer Center, Houston,Texas, USAEinav Shoshan1, Aaron K. Mobley1, Russell R. Braeuer1,Takafumi Kamiya1, Li Huang1, Mayra Vasquez1, AhmadSalameh2, Ho Jeong Lee1, Sun Jin Kim1, Cristina Ivan3,George A. Calin4, Anil K. Sood1,3, Patrick Hwu5, Jeffrey EGershenwald6, Gal Markel7,8, Isaiah J. Fidler1, and MenasheBar-Eli1

Although recent studies have shown that adenosine-to-inosine(A-to-I) RNA editing occurs in microRNAs, its effects on tumorgrowth and metastasis are not well understood. We present evi-dence of CREB-mediated low expression of ADAR1 in meta-static melanoma cell lines and tumor specimens. Re-expressionof ADAR1 resulted in the suppression of melanoma growth andmetastasis in vivo. Consequently, we identified 3 miRs undergo-ing A-to-I editing in the low-metastatic melanoma cell lines butnot in highly metastatic. One of these miRs, miR-455 has twoA-to-I RNA editing sites. The biological function of edited miR-455 is different from the unedited form. Indeed, w.t. miR-455

promotes melanoma metastasis via inhibition of the tumor sup-pressor gene CPEB1. Moreover, w.t. miR-455 enhances mela-noma growth and metastasis in vivo while the edited form in-hibits these features. TCGA analysis confirmed accumulation ofwild-typemiR-455 inmetastatic melanoma lesions. These resultsdemonstrate a previously unrecognized role of RNA editing inmelanoma progression.Shoshan et al.…Menashe Bar-Eli, Nat Cell Biol. 2015Mar;17(3):311–21. doi:10.1038/ncb3110. Epub 2015Feb 16. PMID:25686251


08:30–10:10

PLENARY SESSION 7: Metastasis & the MetastaticMicroenvironment (II)

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Vessel Co-Option and Impact of the MetastaticTumor Microenvironment on Antiangiogenic DrugEfficacy

Robert KerbelBiological Sciences, Sunnybrook Research Institute, Toronto,Ontario, Canada

There are currently nine antiangiogenic drugs approved aroundthe world for over ten different types of cancer. These drugsinhibit angiogenesis primarily, or only, by targeting the VEGFpathway. They include antibodies and/or TKIs. The indicationsinclude colorectal, lung, breast, ovarian, cervical, liver, renal andgastric carcinomas. However, the survival benefits of these drugsare incremental. These transient benefits are thought largely aconsequence of innate and acquired resistance. Many mecha-nisms have been proposed to account for resistance to suchagents, such as increased hypoxia and thus induced angiogenesisgrowth factor redundancy.We have developed several new models of localizedorthotopic primary tumors or postsurgical advancedmetastaticdisease to study antiangiogenic drug therapeutics. Using onesuch model, three different antiangiogenic drugs showed clearefficacy when treating primary breast tumors, but failed to doso when treating advanced visceral metastatic disease—whichrecapitulated prior phase III clinical trial outcomes. Analysisof the tumor vasculature revealed the likely reason for thediscrepant outcomes: the primary tumors showed evidenceof neoangiogenesis, whereas the metastases did not. Insteadthey appeared to ‘hijack’ the existing lung vasculature—a phe-nomenon known as “vessel co-option”. We also have found

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evidence in another model that liver tumors responding tosorafenib can become invasive and then switch to a co-option phenotype—and thus stop responding to the drug—aform of acquired or evasive resistance.Vessel co-option should now be assessed as a potential targetfor ‘anti-vascular’ therapy.

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ECM Remodelling during Cancer Progression

Janine T. ErlerBiotech Research & Innovation Centre (BRIC), University ofCopenhagen, Copenhagen, Denmark

The extracellular matrix (ECM) is one of the most importantregulators of cellular and tissue function in the body. The ECMis known to play a critical role in driving cancer progression, andyet we lack knowledge of how ECM is altered during tumourprogression to promote cancer metastasis. We have found thatan enzyme secreted by tumour and stromal cells called lysyl ox-idase (LOX) is responsible for altering the ECM at primary andmetastatic sites to greatly enhance metastasis. LOX is an amineoxidase that catalyses the crosslinking of collagens and elastin inthe ECM. LOX activity alters the structural and biochemical prop-erties of the ECM, to drive cell proliferation, invasion and angio-genesis. Importantly, LOX modifies pre-metastatic tissue micro-environments prior to tumour cell arrival, enhancing metastaticcolonisation and outgrowth. We are expanding our studies to fur-ther investigate how ECM is remodelled during cancer progres-sion and how this impacts on cell behaviour to drive metastasis.

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Liver Metastasis-Related Genes at Primaryand Metastatic Sites from Patients with ColonCancer

Fernando Vidal-VanaclochaInstitute of AppliedMolecularMedicine (IMMA), Ceu San PabloUniv and Hm-Hospital School of Medicine, Madrid, Spain

Colorectal cancer (CRC) frequently metastasizes to the liver,but the genetic and phenotypic properties of specific cancercells able to implant and grow in this organ have not yet beenestablished. Neither is it known the contribution of the pa-tient’s genetic, physiologic and pathologic backgrounds tohepatic CRC metastasis. DNA microarray and RT-PCR werefirst used to determine hepatic metastasis signature genes inbiopsies from primary and metastatic CRCs. Our resultsshowed that hepatic metastasis occurrence was in part

associated with marked gene expression changes that originat-ed in the primary CRC tumors. However, other CRC genechanges were liver-dependent and were regulated by hepaticparenchymal and non-parenchymal cells, which represent thenew microenvironment for metastatic CRC cancer cells. Con-versely, several prometastatic genes were also induced in theliver by both CRC-derived soluble factors and systemic fac-tors from the patient, suggesting that the hepatic metastasismicroenvironment is acting as a functional interconnector be-tween liver pathophysiology and metastatic cell biology inpatients with CRC. Next, new surgical biopsies were obtainedfrom early and advanced stage CRC patients, and TaqMan-low density arrays were used to quantify prometastatic geneexpression level at primary/metastatic sites and the liver fromCRC patients with and without metastases. We verified thattogether with metastasis-related gene profiles, which sug-gested the existence of a prometastatic potential in primarytumors, other signature genes, representing the pro-metastatic microenvironment supported by the liver reactionto CRC factors, were also detected in liver biopsies from pa-tients with no liver metastases, suggesting their potential forindividual assessment of hepatic metastasis risk in CRC pa-tients. Knowledge on hepatic metastasis genes and their reg-ulation by the hepatic microenvironment open new opportu-nities for therapeutic intervention during CRC metastasis.

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Microenvironmental Influences on SplicesomeFactor Expression Contribute to Melanoma BrainMetastasis Progression

Dave Hoon, Diego MarzeseMolecularOncology, JohnWayneCancer Institute at ProvidenceSaint John’s Health Center, Santa Monica, California, USA

With the advent of targeted therapies and the associated longersurvival, brain metastasis is a complication with increasingincidence for melanoma patients. Due to melanoma’s highpropensity to metastasize to the brain and the resulting poorprognosis, there is an urgent need to identify potentialtheranostic targets on melanomas and the metastatic niches.The dynamics and phenotypic plasticity of melanoma to thrivein diverse microenvironments are mainly governed by epige-netic mechanisms. Another epistasis mechanism that confersphenotypic plasticity is the alternative RNA splicing (AS).The AS is determined by the combination of ribonucleopro-teins denominated spliceosome factors (SF). In this study, weevaluated the gene and isoform expression of melanoma cellsin different tissue contexts, including primary melanoma(PRM), regional lymph node metastasis (LNM), and brainmetastasis (MBM). We observed that CD44 variant 6-

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expressing (CD44v6+) PRMs favor homing to the brain.Functionally CD44v6+ cells exhibited increased migration inthe presence of two particular molecules that are released dur-ing acute brain inflammation: hyaluronic acid (HA) and hepa-tocyte growth factor (HGF). To identify regulating factors inthe expression of CD44v6 isoform, we evaluated the geneexpression and DNA methylation of 190 SF in the progres-sion to MBM. Importantly, our findings divulge that theAS of CD44 in PRM was regulated by ESRP1 and ESRP2factors; however, ESRP1 was epigenetically silenced inMBM. Interestingly, within the brain’s microenvironment,we found CD44v6 being promoted by PTBP1 and U2AF2factors. Consistently, PTBP1 knockdown resulted in sig-nificant reduction of CD44v6+ phenotype and migratoryability in brain’s microenvironment. Our results reveal anew avenue towards understanding melanoma cells’ highaffinity for the brain, as well as the prospect of CD44v6and SF as potential MBM-specific targets with theranosticutility.

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The Role of Perivascular Heterogeneity in Metastasis

Doruk Keskin1,3, Jiha Kim1,5, Vesselina G. Cooke3, Chia-Chin Wu2, Hikaru Sugimoto1,3, Chenghua Gu5, Michele DePalma4, Raghu Kalluri1,3, Valerie LeBleu1,31Cancer Biology, University of Texas MD Anderson CancerCenter, Houston, Texas, USA2Genomic Medicine, University of Texas MD Anderson Can-cer Center, Houston, Texas, USA3Matrix Biology, Beth Israel Deaconess Medical Center andHarvard Medical School, Boston, Massachusetts, USA4Ecole Polytechnique Federale de Lausanne (EPFL), TheSwiss Institute for Experimental Cancer Research (ISREC),Lausanne, Switzerland5Neurobiology, Harvard Medical School, Boston, Massachu-setts, USA

Targeting tumor angiogenesis also inhibits the vessel-stabilizing properties of vascular pericytes. Pericyte targetingin early stages of tumor growth suppresses nascent angiogene-sis and limits both tumor growth and metastatic dissemination.In contrast, pericyte targeting in tumors with pre-establishedvasculature resulted in enhanced intra-tumoral hypoxia, leadingto cancer cell invasion and promoting metastasis. Our studiesunraveled angiopoietin signaling as a key regulatory pathwayassociated with pericyte targeting and metastatic spread. Spe-cifically, controlling Angiopoeitin-2 mediated signaling re-stored vascular stability associated with pericyte coverage lossin tumor angiogenesis, and suppressed tumor growth metasta-sis. These studies highlight the complexity and heterogeneity of

pericytes in tumor angiogenesis and inform on novel combina-tion therapy to control metastasis.

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Tumor Models Incorporating Aspects of TumorMicroenvironment for Novel Cancer Nanotherapies

Meenakshi Upreti1, Amar Jyoti1, Pallavi Sethi1, EldenSwindell3, Jonathan Feddock2, Kyle Fugit1, Ryan Chan1,Ronald McGarry2, Bradley Anderson1, Thomas O’Halloran31Pharmaceutical Sciences, College of Pharmacy, Universityof Kentucky, Lexington, Kentucky, USA2Radiation Medicine, College of Medicine, University of Ken-tucky, Kentucky, Lexington, USA3Department of Chemistry, Chemistry of Life Processes Insti-tute, Northwestern University, Chicago, Illinois, USA

Several studies, including our own, have demonstrated thatgenetic alterations of tumor cells are not the sole driving forcebehind tumor development and that tumor growth, metastasisand response to treatment are intimately controlled by thetumor microenvironment. An appropriate representation ofthe tumor microenvironment in tumor models can thus havea pronounced impact on directing combinatorial treatmentstrategies and cancer nanotherapeutics. This presentation willdemonstrate a multimodal nanotherapeutic system that func-tions via the biological response to radiation in the tumorendothelial cells using an in vitro/ in vivo tumor model incor-porating characteristics of tumor microenvironment. This pre-sentation will also elucidate upon the utility of 3D tumormodels in vitro to understand the sustained release of activedrug from liposomal formulations at the tumor site over aprolonged period of time, simulating the action of localizedmetronomic therapy in cancer, a treatment modality that tar-gets the tumor microenvironment. Such tumor models thatlend themselves to controlled experimental manipulation in acost-effective and reproducible manner are expected to betterpredict treatment response and accelerate drug development.

References:1. Sethi P, Jyoti A, Swindell E, Langner UW, Feddock JM,Nagarajan R, O’Halloran TVand Upreti M. 3D Tumor Tis-sue Analogs and their Orthotopic implants for UnderstandingTumor-targeting of Microenvironment-responsive NanosizedChemotherapy and Radiation. Accepted for publication inNanomedicine: Nanotechnology, Biology & Medicine.2. Jyoti A, Fugit K, Sethi P,McGarry RC,AndersonBD andUpreti M. An in vitro assessment of liposomal topotecansimulating metronomic chemotherapy in combination withradiation in tumor-endothelial spheroids. Resubmitted withminor edits to Scientific Reports.

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Tumor-Elicited Secretion of IL6 and of IL8from Reactive Mesenchymal Stroma is EnhancedUnder the Acidic Microenvironment of GlycolyticCancer Cells and Promotes Osteosarcoma Stemness

Sofia Avnet1, Gemma Di Pompo2, Margherita Cortini1, Glo-ria Bonuccelli2, Tokuhiro Chano3, Nicola Baldini1,21Orthopaedic Pathophysiology and Regenerative MedicineUnit, Rizzoli Orthopaedic Institute, Bologna, Italy2Department of Biomedical and Neuromotor Sciences, Uni-versity of Bologna, Bologna, Italy3Department of Clinical Laboratory Medicine, Shiga Univer-sity of Medical Science, Otsu, Shiga, JapanOsteosarcoma (OS) is an aggressive malignancy of childhoodwith a high relapse rate. Mesenchymal stromal cells (MSC) inOS microenvironment concur to tumor metabolicreprogramming1. Recently, we have also reported that OS ishighly glycolytic, thereby resulting into extracellular acidosis2

that, in turn, may favor the release of pro-tumorigenic para-crine factors. Here, we evaluated if OS-elicited activation ofMSC impacts on the stem-like tumor fraction, both under neu-tral and acidic conditions.WemaintainedMSC at pH 6.8, or inco-culture with OS cells (Saos-2 and HOS). By deep-sequencing analysis, and ELISA, we found in stressed MSCthe activation of NF-kB pathway, and the consequential secre-tion of cytokines (IL6 and IL8), and chemokines (CCL5,CXCL5, CXCL1), especially under acidosis. The secreatomefrom reactive MSC induced chemoattraction of other MSC,clone formation of OS cells, and, most importantly, an in-creased number of floating spheres and stem-related gene ex-pression (Sox2, Nanog) in OS cells. Such enhanced stemnesswas associated with higher chemoresistance and a remarkableincrease in migratory potential. In conclusion, we demonstrat-ed that, once attracted at the tumor site, especially in acidicregions, reactive MSC enhance OS aggressiveness and pro-gression through an auto-feeding process. Indeed, reactiveMSC induce the expansion of tumor stem-like fraction, mean-while attracting circulating MSC, eventually dramatically ac-celerating progression. Altogether, our data highlight the needof a comprehensive knowledge of the interplay between tumorand stroma for effective anticancer therapies in OS.

ACKNOWLEDGMENTSSupported by AIRC (15608) and by Ministry of Health andEducation (FIRB RBAP10447J) to NB.

REFERENCES[1] Bonuccelli et al. Oncotarget 2014;5:7575–88[2] Perut et al. Exp Cell Res 2014;320:21–32

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P1

Migration and Mechanical Invasiveness in BreastCancer Cells, are they Related?

Martha Alvarez, Daphne WeihsBiomedical Engineering, Technion-Israel Institute of Technol-ogy, Haifa, Israel

Tumor cell migration and invasion are critical steps in metas-tasis, and their study has provided many targets for cancertherapy. Different transmigration assays have been designedto evaluate invasiveness of cancer cells through their ability tocross small pores in, such as Boyden chambers. Previously,we have used mechanical interactions, or forces applied bycells distinguish between benign and breast cancer cells withdifferent metastatic potential (MP). Specifically, we measurethe normal forces applied by breast cancer cell lines on a soft,impenetrable, polyacrylamide gel-substrate, resulting in gel-indentation during attempted cell penetration; about 50 % ofcancer cells indented gels, while no benign cells indent. Here,we correlate the populations of cells that are able to transmi-grate through a Boyden chamber with those that apply normalforces and indent gels, to show a correlation between transmi-gration and mechanical invasiveness. When high MP cells areseeded on the gel 50 % will indent. However, we have foundthat 45 % of high MP cells transmigrate through a Boydenchamber (8 μm pore size), and if that population is seeded onthe gel, 70 % of cells indent the gels. Thus the Boyden cham-ber effectively “concentrates” the more invasive cell popula-tion. Furthermore, our results show that migration and me-chanical invasiveness are directly correlated in breast cancercells. Most importantly, our results validate the gel-based as-say for mechanical interactions as an invasiveness assay andin contrast to Boyden chamber, our assay provides quantita-tive measurements (e.g., normal force, indentation depth) toevaluate invasiveness.

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Synthesis and Characterization of PolymericNanotheranostics for Real-Time Non-InvasiveOptical Imaging

Racheli Blau1, Hemda Baabur-Cohen1, Shiran Ferber1, YanaEpshtein1, Orit Redy-Keisar2, Einat Kisin-Finfer2, DoronShabat2, Ronit Satchi-Fainaro11Physiology and Pharmacology, Tel Aviv University, SacklerSchool of Medicine, Tel Aviv, Israel2Organic Chemistry, Tel Aviv University, School of Chemistry,Tel Aviv, Israel

Theranostics is a relatively new field, introduced in 2002 (1)that describes any material for applications combining boththerapy and diagnostics. The great challenge for future per-sonalized therapy in oncology is exploring improved method-ology for (i) early detection of localized and disseminatedtumor cells in patients and (ii) monitoring drug release at thetarget site in order to evaluate the treatment’s efficacy. Thedetermination of both is critical to the success of cancer ther-apy and improvement of patients’ survival rates. A theranosticnanosystem composed of nanocarrier, drug and Turn-ONprobe is an ideal platform to address these challenges.In this study, we designed, synthesized and characterized atheranostic nanomedicine based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. The diagnostic systemconsists of self-quenched Cy5 and the therapeutic system isbased on the anticancer agent paclitaxel (PTX). Both systemswere conjugated to HPMA copolymer through a Gly-Phe-Leu-Gly (GFLG) linker, cleaved by cathepsin B, a lysosomalcysteine protease overexpressed in several tumor types such aslung, colon, prostate, melanoma and breast cancers. Our sys-tems enable site-specific release of the drug concomitantlywith the fluorophore activation to its Turn-ON state upon en-zymatic degradation (2). HPMA copolymer-PTX conjugateinhibited the proliferation of endothelial and breast cancercells.Our preliminary results with the diagnostic nano-conjugateHPMA copolymer-Cy5 present its potential use as a novelprobe for sensing real-time drug release from the polymericnanocarrier. This approach of co-delivery of two complemen-tary systems serves as a proof-of-concept for non-invasivereal-time deep tissue intravital orthotopic monitoring thatmay potentially be exploited as a theranostic nanomedicinein the clinic.References:1. S. S. Kelkar, T. M. Reineke, Bioconjugate chemistry 22,1879 (Oct 19, 2011).2. S. Ferber et al., Cancer letters, (Mar 12, 2014).

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Serum BCMA: A Novel Prognostic Indicatorin Patients with Multiple Myeloma

Michael Ghermezi, Suzie Vardanyan, Nika Harutyunyan,Michael David, Berenson Ariana, Berenson JamesHematology and Oncology, Institute for Myeloma and BoneCancer Research, West Hollywood, California, USA

Background: B-cell maturation antigen (BCMA) is a tumornecrosis factor receptor family member that is expressed onnormal and malignant B-cells, including those from patients(pts) with multiple myeloma (MM).

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Objective: We have analyzed the relationship between serumBCMA levels and monoclonal (M)-protein levels as well asthe relationship of BCMA to response status, progression-freesurvival (PFS) and OS in a cohort of MM pts.Methods of Use: Two hundred thirty-three MM pts wereevaluated. Kaplan-Meier survival of MM pts was determinedfrom time of initial serum BCMA to death/last follow-up.Cox-proportional hazards regression was utilized to determinethe predictive influence of serum BCMA. ELISAwas used tocorrelate serum BCMA and M-protein levels.Results: Serum BCMA levels correlated with patient’s clini-cal status at the time of its determination (P 0.0001). Pts withPR had significantly lower serum BCMA levels (median,11.69 ng/mL) than those with stable and progressive disease(median, 64.17 ng/mL; P 0.001). Changes in serum BCMAlevels correlated with changes in M-protein levels. PFS waslonger among pts with serum BCMA levels below thanamong those with levels above the median (P.0001). OS ofpts whose serum BCMA levels were above the median(≥32.6 ng/mL) was significantly shorter than among thosewhose levels were below the median (P.0001). In the multi-variate analyses, serum BCMA levels significantly correlatedwith OS (P=0.0003). In contrast, age, bone disease status,creatinine, hemoglobin, and ISS staging did not correlate withOS.Conclusion: The results demonstrate that BCMA is a novelserum marker that can be used to follow the course of disease,clinical status, and predict survival for MM pts.

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Biomechanical Stimuli Modulate the Expressionof Osteolytic Lesion-Related Proteins in anEngineered Human Tumor

Alessandro Marturano1, Aranzazu Villasante1, KeithYaeger1, Gordana Vunjak-Novakovic1,21Department of Biomedical Engineering, Columbia Universi-ty, New York, NY, USA2Department of Medicine, Columbia University, New York,NY, USA

Biomechanical forces, either generated by cell contractions orsensed from the microenvironment, have pronounced effectson cell differentiation, growth and survival, playing a key rolein the development of many organs. Recent studies have sug-gested that biomechanical stimuli resulting from tissue stiff-ening, matrix deformation and interstitial fluid flow regulatepivotal processes in cancer. For instance, increased cytoskel-etal tension can arise in response to increased extracellularmatrix stiffness, leading to oncogene (Ras)-drivenextracellular-signal-regulated kinase (ERK) activation.

Notably, RUNX2 transcription factor is regulated byERK1/2 phosphorylation and is aberrantly expressed at highlevels in many cancers invading the bone.The aim of this study was to investigate the involvement ofbiomechanical stimuli in the expression of RUNX2 and someof RUNX2 downstream targets associated with osteolytic le-sions. To this end, we have developed an in vitro biomimetic3D human model of Ewing’s Sarcoma (ES) along with a bio-reactor platform that allows delivery of biologically relevantmechanical signals.RUNX2 mRNA and protein were expressed in ES tumors butwere down-regulated in ES cell lines cultured in 2D plasticsubstrates, supporting the importance of the tumor microenvi-ronment. Biomechanical loading of the ES cells within thebioengineered tumor model induced re-expression of RUNX2and RUNX2 downstream targets OPN, BSP, PTHrP andMMP13. Biomechanical stimuli also induced rapid phosphor-ylation of ERK1/2, while ERK inhibition completely blockedthe expression of OPN and BSP mRNAs. Our findings sug-gest that biomechanical stimuli are important for the expres-sion of RUNX2 in ES and that they can modulate, upon acti-vation of the ERK signaling cascade, the expression of pro-teins related to osteolytic disease.

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Adherence Rates, Morphology and Force Dependon Microenvironment Stiffness and MetastaticPotential in Breast Cancer Cells

Sonbula Massalha, Daphne WeihsFaculty of Biomedical Engineering, Technion-Israel Instituteof Technology, Haifa, Israel

Cell adhesion plays an important role in the normal functionsof cells, including contraction, spreading, crawling, and inva-sion. In cancer cells, adhesion regulates migration and inva-siveness, and following biochemical attachment, dependsmainly on the stiffness of the matrix surrounding a tumor. Inthe current work we show that the rate of adhesion, dynamiccell morphology and forces applied during adhesion vary be-tween benign and cancer cells. Thus, through measurementsof those differences and while varying substrate-stiffness wecan determine malignancy and metastatic potential (MP) ofcancer cells. We evaluate the rate of attachment from suspen-sion on a 2D collagen-coated polyacrylamide gel, concurrent-ly with changes in cell morphology and the strength of adher-ence. We compare high and low MP breast cancer cells anduse benign cells as a control. We monitor the time-dependentforce applied by the cells to gels in a stiffness range 2–10 kPa,using traction force microscopy; applied forces are measuredthrough cell-induced displacement of particles embedded in

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the gel surface. We observe that both high and low MP cancercells apply significantly higher lateral traction forces than be-nign cells on stiffer gels (4.3±0.1 kPa and above), while nodifference in lateral forces is observed on the softer gel (2.4±0.04 kPa). In conclusion, we have shown that there is a directcorrelation between the forces applied by cancer cells and themicroenvironment (gel) stiffness during the adhesion process.

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Multi-Modal Nanomedicine for Glioblastoma

Paula Ofek1, Marcelo Calderon2, Fatemeh Sheikhi-Mehrabadi2, Shiran Ferber1, Rainer Haag2, Ronit Satchi-Fainaro11Physiology and Pharmacology, Sackler School of Medicine,Tel Aviv University, Tel Aviv, Israel2Department of Chemistry and Biochemistry, Freie Universityof Berlin, Berlin, Germany

Glioblastoma multiforme (GBM) is an aggressive prima-ry neoplasm of the brain that exhibit notable refractivityto standard treatment regimens. Recent large-scale mo-lecular profiling has revealed deregulated molecular net-works as potential targets for therapeutic development.MicroRNAs (miRNAs) play major roles in normal de-velopmental processes, and their deregulation signifi-cantly contributes to various aspects of carcinogenesis.Nevertheless, in vivo delivery of small interfering RNA(siRNA) and miRNA remains a crucial challenge fortheir therapeutic success.We have recently developed a cationic carrier system, whichcan strongly improve its stability, intracellular trafficking andsilencing efficacy. Polyglycerol (PG)-Amine, a water-solublepolyglycerol-based hyperbranched polymer accumulates inthe tumor microenvironment due to the enhanced permeabilityand retention (EPR) effect, and therefore, represents an idealnanocarrier for antitumor oligonucleotides.Using our novel nanocarrier, we have studied the expres-sion targets and functional effects of miR-34a in severalhuman glioblastoma cell lines and human tissue samples.miR-34a levels inversely correlated to their target genelevels measured in the same cell lines or tissue. Transienttransfection of PG-NH2-miR-34a polyplex into glioblasto-ma cells strongly inhibited cell proliferation, cell cycle pro-gression, and cell migration. Consequently, we performedan in vivo experiment and achieved a significant tumorgrowth inhibition following treatment with PG-NH2-miR-34a polyplex in a human glioblastoma mouse model. Wefurther characterized the synergistic effect of combiningPG-NH2-miR-34a polyplex with chemotherapy andachieved promising results.

Together, our findings show that PG-NH2 efficiently deliversanticancer miRNAs to glioblastoma cells and their microen-vironment and suppresses brain tumor growth. These resultssuggest that our polyplex could serve as a potentialnanomedicine for glioblastoma.

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Statistical Visualization for the Associationbetween Soil Transmitted Helminth Infectionand Changes in Immune Microenvironmentsat the Cervix

Fares Qeadan1, Patti Gravitt21Department of Internal Medicine, Division of Epidemiology,Biostatistics, & Preventive Medicine, University of New Mex-ico Health Sciences Center, Albuquerque, New Mexico, USA2Pathology, University of New Mexico Health Sciences Cen-ter, Albuquerque, New Mexico, USA

Investigation of the immunologic microenvironment inthe initiation of tumor development requires relativelynon-invasive sample collection methods, such as measure-ment of soluble immune markers (e.g., cytokines/chemokines) in lumina l f lu id samples such ascervicovaginal secretions. Traditionally, these markershave been compared between groups based solely on con-centration differences, despite the complexity of immunesystem interactions in tissues. We explore alternativemethods for analyzing large panels of immune proteinmarkers, in a study [1] evaluating the role of soil trans-mitted helminth (STH) infection on oncogenic humanpapillomavirus (HPV) persistence as an early microenvi-ronmental interaction predisposing to the development ofcervical cancer.Data on 90 women from Peruvian Amazon reveal asignificantly increased correlation between IL-4, the ca-nonical Th2 cytokine, and other cytokines andchemokines linked to the Th2 immune responses amongSTH-infected compared with STH-uninfected women.To capture this finding, in one presentation, we providea lower triangular correlation matrix (Figure 1) in whichwe use coloring to highlight the Spearman correlation’sSidak adjusted significance of the p-value betweenSTH-infected and STH-uninfected women for every cy-tokine with all other cytokines. The generated matrix isordered based on the observed hierarchical clustering inthe STH-infected women to emphasize the role of IL-4and hence introduces the new concept of second/horizontal chain correlation among a panel of immunemarkers. Dendrograms and cluster analysis plots areused for further illustration.

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[1] Gravitt PE et al. Intestinal parasitic infection is associatedwith increased HPV prevalence in the Peruvian Amazon. 28thInternational Papillomavirus Conference, Puerto Rico, De-cember 2012.Funding source: R21 CA156537-02 (Gravitt, PI), NIH/NCI.“HPV Persistence: An overlooked consequence of helminthinfection?”

P8

Circulating hTERT (human telomerase) mRNAin Exosomes Derived from T-Cell Leukemia CellsPromotes the Aggressiveness of MicroenvironmentRelated Primary Fibroblasts

Orit Uziel1, Anna Gutkin1, Einat Beery2, JardenaNordenberg1, Steven Henick2, Meir Lahav31The Felsenstein Medical Research Center, Rabin MedicalCenter and Tel Aviv University, Petah-Tikva, Israel2The Felsenstein Medical Research Center, Rabin MedicalCenter, Petah-Tikva, Israel3Institute of Hematology, Davidoff Cancer Center, RabinMedical Center and Tel Aviv University, Petah-Tikva, Israel

The importance of the cancer microenvironment to the estab-lishment and aggressiveness of tumors has been wellestablished. One of the mediators of the intensive crosstalkbetween cancer microenvironment and the tumor mass areexosomes. These are small vesicles (30–100 nm in size) se-creted from all types of cells into the body fluids and cell

surroundings. They contain a variety of proteins, lipids andamino acids including DNA, RNA and micro RNAs. In thepresent study we explored whether hTERT, the mRNA tran-script of human telomerase is secreted from cancer cells viaexosomes. We present data which describes the secretion ofhTERT transcript into exosomes derived from Jurkat (T-cellleukemia) cells. We show that exosomal hTERT is transferredto recipient fibroblasts where it is translated to a fully activeenzyme. Upon translation, telomerase is able to change thephenotype of these primary non-telomerase expressing fibro-blasts by increasing the primary fibroblasts’ proliferation andprotecting them from DNA damage imposed on these cells.These results suggest that the transfer of hTERT from thetumor to the surrounding microenvironment contributes tothe capability of the cancer microenvironment to becomemore tumors supportive by transforming fibroblast cells intocancer associated fibroblasts. Overall, understanding of thesemechanisms may have a strong impact on deciphering metas-tases formation.

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Microenvironmental Regulation of Tumor-DerivedExosomes using a Tissue-Engineered Model

Aranzazu Villasante1, Alessandro Marturano-Kruik1,Srikanth R. Ambati2, Zen Liu1, Hesam Parsa1, MalcolmA.S. Moore3, Gordana Vunjak-Novakovic11Department of Biomedical Engineering, Columbia Universi-ty, New York, New York, USA2Department of Pediatrics, Memorial Sloan-Kettering CancerCenter, New York, New York, USA3Department of Cell Biology, Memorial Sloan-Kettering Can-cer Center, New York, New York, USA

Cancer cells are typically studied in monolayers that do notprovide the 3-dimensional microenvironment and signalingpresent in native tumors. The lack of specific cell-matrix in-teractions and stiffness of the substrate lead to changes inmany cancer cell processes, including endo- and exocytosis.In recent years, there has been an increased interest in under-standing how exosomes modulate the cancer cell microenvi-ronment, after the discovery of their role in the pre-metastaticniche formation in the lung. However, despite the well-established importance of the microenvironment for cancercell behavior, supernatants from monolayer cultures still rep-resent the major source for isolation of tumor-derivedexosomes, such that their microenvironmental regulation re-mains largely unknown.Bioengineering methods that have transformed the stem cellresearch and regenerative medicine are just starting to enterthe field of cancer research. Our lab is making progress in

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replicating the physiological landscape of human tumorsusing 3-dimensional in vitro tissue models that contain theessential components necessary for recapitulating in vivo con-ditions. Here, we report the effects of the tumor microenviron-ment on exosomes, using a bioengineered tumor model ofEwing’s sarcoma as a clinically relevant example. To thisend, we cultured cancer cells on 3-dimensional scaffolds thatwere designed to mimic the biological and mechanical prop-erties of the native tumor. Our data confirm the role of theextracellular matrix in regulating tumor-derived exosomesand provide compelling evidences for a link between the stiff-ness, 3-dimensionality and composition of the environmentand the size of exosomes. Notably, the size of exosomes re-leased from the bioengineered tumor models were indistin-guishable from those secreted into the plasma of patients,and significantly smaller than those secreted from cancer cellmonolayers.

P10

The Role of Pregnancy-Induced Hormonal Milieuin Lymphoma Progression

Ali Abed El Wahad1, Noam Bettman2, Shoham Arad2, IritAvivi2, Tamar Katz1,2, Netanel Avraham Horowitz1,21Bruce Rappaport Faculty of Medicine, Technion-Israel Insti-tute of Technology, Haifa, Israel2The Department of Hematology and Bone Marrow Trans-plantation, Rambam Health Care Campus, Haifa, Israel

Introduction: Lymphoma is the most common hematologicalcancer during pregnancy. Recent data suggest that pregnancy-associated lymphoma is characterized by an excessive in-volvement of reproductive organs, advanced disease stage atdiagnosis and an aggressive course, potentially leading to ahigh death rate of mothers.Objective: To determine the role of estrogen and progesteronein lymphoma progression.Materials and Methods: Estrogen and progesterone receptorswere evaluated by qPCR and flow-cytometry in human lym-phoma cell lines (Raji and BL2) and lymph node stromal cells(HK). The hormones effect on Raji/BL2 proliferation wasdetermined using trypan blue exclusion and CFSE dye, andtheir viability, either when cultured alone or in the presence ofHK cells, was assessed by Annexin-V staining. Raji migratorycapacity towards estrogen-treated HK cells conditioned medi-um was examined by Transwell® inserts. In vivo, lymphomaprogression and dissemination was compared in pregnant vs.non-pregnant balb/c mice, injected (SC or IV) with A20 micelymphoma cells.Results: Lymphoma and HK cells express estrogen and pro-gesterone receptors. Estrogen-treated lymphoma cells

demonstrated reduced proliferation and cell viability in a con-centration dependent manner and progesterone further aug-mented this phenomenon when co-administered with estro-gen. However, BL2 cells that were in direct contact with HKcells were protected from estrogen toxic effects. Interestingly,estrogen-containing conditioned medium inhibited Raji mi-gration. In vivo, pregnant mice showed shorter overall surviv-al and enhanced tumor growth.Conclusion: Lymphoma cells express pregnancy-related hor-mone receptors. Indeed, estrogen induces cell death and re-duces proliferation and migration. However, the in vivo stud-ies, suggest a supportive effect of pregnancy on lymphomaprogression. These contradicting results imply that otherpregnancy-related factors affect the microenvironment to fa-cilitate lymphoma progression. The underlying mechanismsshould be studied.

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Secretome of Bone Marrow Mesenchymal StemCells: an Emerging Player in Lung CancerProgression and Mechanisms of TranslationInitiation

Oshrat Attar-Schneider1,3,4, Victoria Zismanov1,4, LiatDrucker3,4, Maya Gottfried1,2,41Meir Medical Center, Lung Cancer Research Laboratory,Kfar-Saba, Israel2Meir Medical Center, Oncology Department, Lung CancerUnit, Kfar-Saba, Israel3Meir Medical Center, Oncogenetic Laboratory, Kfar-Saba,Israel4Tel Aviv University, Sackler Faculty of Medicine, Tel-Aviv,Israel

Introduction: Non-small cell lung cancer (NSCLC) re-mains the most common cause of cancer-related death. Pa-tients with advanced stage have poor prognosis while met-astatic spread accounts for >70 % of patients deaths. Themajor advances in treatment of NSCLC have brought onlyminor improvements in survival; therefore novel strategictreatment approaches are needed. Accumulating data allo-cate a central role for the cancer microenvironment in-cluding mesenchymal stem cells(MSCs) in acquisition ofdrug resistance and disease relapse. Furthermore, studiesindicate that translation initiation factors are over-expressed in NSCLC and negatively impact its prognosis.Importantly, translation initiation is highly modulated bymicroenvironmental cues. Therefore, we decided to exam-ine the effect of bone marrow (BM)-MSCs on NSCLCcell lines with special emphasis on the role of translationinitiation.

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Methods: NSCLC cell lines were treated with normal BM-MSCs’ conditioned medium(secretome) and assesed forchanges in the cells’ viability/ proliferation/death/migrationand immunoblotted for translation initiation factors (eIF4E,eIF4GI), and their targets/regulators.Results: Our results demonstarted deleterious effects on thecells’ proliferation, viability, death and migration. We alsodemonstrated reduced levels of translation initiation factorsimplicated in cancer progression eIF4E and eIF4GI, their tar-gets, and regulators. Finally, we outlined a mechanism bywhich BM-MSCs’ secretome affected NSCLCs’ MAPK sig-naling, downredulated the cells’ migration and diminshedtranslation initiation factors’ levles.Conclusions: We showed a direct dialogue between BM-MSCs’ secretome and NSCLC cells that manipulates transla-tion initiation and critically affects cell fate. We showed inhib-itory effect on the lung cancer cells’migration that is regulatedboth by MAPK signaling and by translation initiationmechanism.We suggest that theraputic approach that will sabotage thedialoge, espacially in the BM microenviornment, may dimin-ish metastatic spread and improve the patients life quality.

P12

Highly Effective Heparanase-Based Therapyfor Mesothelioma

Uri Barash1, Moshe Lapidot2, Yaniv Zohar3, Neta Ilan1, Is-rael Vlodavsky11Cancer and Vascular Biology Research Cente, RappaportFaculty of Medicine, Technion-Israel Insitute of Technology,Haifa, Israel2Department of General Thoracic Surgery, Rambam HealthCare Campus, Haifa, Israel3Department of Pathology, Rambam Health Care Campus,Haifa, Israel

Malignant mesothelioma is a highly aggressive form of cancerthat develops from cells of the mesothelium—the protectivelining that covers many internal organs of the body. It has apoor prognosis because of the lack of markers for early diag-nosis, and resistance to conventional therapies, underscoringthe need for novel treatments. Mesothelioma tumors expresshigh levels of heparanase , the so le mammal ianendoglycosidase degrading heparan sulfate (HS) side chainsof HS-proteoglycans in the extracellular matrix (ECM) andcell surface. Heparanase activity facilitates cell invasion andreleases growth- and angiogenesis-promoting factors that arestored as a complex with HS in the ECM and tumor microen-vironment. We demonstrate that heparanase is expressed andenzymatically active in several pleural mesothelioma cell

lines. Moreover, AE17 mouse mesothelioma cells developedsignificantly smaller tumors when inoculated subcutaneouslyinto heparanase knockout (Hpa-KO) vs control mice, empha-sizing the contribution of the host microenvironment. Immu-nostaining revealed lower proliferation and higher apoptosisrates in tumors developed in Hpa-KO mice associating withreduced c-Jun phosphorylation and VEGF expression. Like-wise, Heparanase gene silencing diminished cell invasionin vitro and tumor xenograft growth in-vivo. In addition,heparanase inhibitor (PG545) attenuated cell invasion and an-chorage independent growth of mesothelioma cell linesin vitro, and reduced mesothelioma tumor xenografts devel-opment by inhibiting angiogenesis and Akt phosphorylation.Notably, PG545 significantly increased the survival of miceimplanted with mesothelioma cells, beyond conventional che-motherapy (cisplatin). In conclusion, heparanase plays an im-portant role in mesothelioma tumor progression, thus encour-aging further development of heparanase inhibitors (e.g.,PG545) for this incurable malignancy.

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CD38 Inhibition Decreases Melanoma Expansionand Ameliorates Metastatic Burden

Bar Ben Baruch1, Eran Blacher1, Hila Shwartz2, HananyaVaknine3, Neta Erez2, Reuven Stein11Department of Neurobiology, George S. Wise Faculty of LifeSciences, Tel Aviv University, Tel Aviv, Israel2Department of Pathology, Sackler School of Medicine, TelAviv University, Tel Aviv, Israel3Department of Pathology, Wolfson Medical Center andSackler Faculty of Medicine, Tel Aviv University, Holon,Israel

Melanoma has the highest propensity to metastasize to thebrain and in many reports is the third most frequent cause ofmetastatic brain tumors.The tumor microenvironment contains different types of cellsincluding cancer-associated fibroblasts (CAFs). Numerousstudies showed that CAFs support the growth and angiogen-esis of various types of cancers including melanoma. CD38 isa multifunctional ectoenzyme that uses NAD+ as a substrate togenerate second messengers. We have recently shown thattargeting CD38 in the tumor microenvironment may serve asa novel approach to treat glioma.Using the syngeneic B16-F10 model of melanoma progres-sion in wild-type and CD38 null mice, we show that CD38deficiency or treatment with the novel CD38 inhibitor K-rhein, which was identified by us, significantly attenuatesmel-anoma expansion both subcutaneous and in the brain, andprolonged the survival of melanoma bearing mice. This

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reduction in tumor growth was associated with decreased ne-crotic area and angiogenesis in Cd38-/- tumors. CD38 defi-ciency inhibited also primary tumor volume and pulmonaryand brain spontaneous metastases in Ret melanoma injectedmiceWhen B16-F10 cells were co-injected with WT or Cd38-/-

primary fibroblasts tumor volumes were affected mostly andsignificantly by the genotype of the co-injected fibroblasts,regardless to the hosts genotype; i.e., the tumors grown inCd38-/--enriched microenvironment were significantly small-er. Our results thus suggest that CD38 participates in thetumor-supporting action of the tumor microenvironment andthat targeting CD38 might be a potential therapeutic approachfor melanoma treatment.

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Erythropoietin Enriches Specific iNOS ProducingMyeloid Cells within the Spleen TumorMicroenvironment Capable of InhibitingLeukemogenesis

Yaacov Ben-David, Xiao XiaoCancer biology devision, The Key Laboratory for NaturalProducts of Guizhou and Chinese Academy of Science, Gui-yang, Guizhou, China

It is well established that the tumor microenvironment plays acritical role in the induction and progression of hemopoieticmalignancies. We have previously demonstrated that thespleen provides a “tumor-promoting niche” that supports leu-kemic growth. Specific treatments, such as cytokine therapyand genetic manipulation, can lead to dramatic changes in thesplenic microenvironment and reverse a tumor-promoting out-come to an inhibitory effect. For example, we were first todemonstrate that two-fold overexpression of systemic levelsVEGF levels in mice heterozygous for a VEGF-Ahypermorphic allele, markedly decelerates the progression ofFriend virus-induced leukemia. Moreover, in a unique mousemodel of familiar polycythemia, in which erythropoietin isconstitutively activated, we have identified the abnormal ex-pansion of unique hematopoietic progenitors capable of leu-kemia inhibition through the secretion of iNOS and additionalfactors. These results implicate the tumor microenvironmentas a pivotal factor in the control of cancer growth and progres-sion. In a recent study, we have shown that treatment of nor-mal mice with Epo resulted in a polycytemic phenotype asso-ciated with enrichment of iNOS-producing myeloid cells.Moreover, treatment of leukemic mice with Epo delayed can-cer progression associated with enrichment of specific c-Kit-/Sca1+ myeloid cells within the spleen, expressing high levelsof iNOS. Further characterization suggested that these cells

may be identical to the previously identified Myeloid derived,suppressor M1 macrophages cells with potent tumor inhibito-ry activity. Overall, this study demonstrates for the first timethat Epo can enrich specific myeloid suppressor cells capableof inhibiting cancer progression. Thus, Epo could be a candi-date for microenvironmental therapy of leukemia as well asother cancers.

P15

PPM1A the Guardian of Homeostasisin the Microenvironment: PPM1A Deficiencyin SKPs Affects Function and Fate of Immuneand Endothelial Cells in the TumorMicroenvironment

Hamutal Ben-Dov1, Libat Backal1, Rona Ben-Dov1,Daniella Ben Meir1, Galina Weingarten2, Efrat Wertheimer2,Aviv Barzilai3, Sara Lavi11Cell Research and Immunology, Tel Aviv University, Tel Aviv,Israel2Department of Pathology, Sackler School of Medicine, TelAviv University, Tel Aviv, Israel3Department of Dermatology, Sheba Medical Center, Tel Aviv,Israel

Using Protein phosphatase metal dependent 1 A (PPM1A)knockout mice, created in our laboratory, we identifiedPPM1A as a major regulator of the microenvironment,down regulating the inflammation response (Dvashiet al., 2014). In the PPM1A-ablated mice, wound-healingprocess goes awry and culminates in uncontrolled inflam-mation and angiogenesis. As these features are comprisedamong the Hallmarks of Cancer, reflecting the microenvi-ronment response to tumor cells’ growth, we investigatedthe role of PPM1A in cancer using multiple mouse models.Surprisingly, we found that the absence of PPM1A couldbe either tumor promoting or tumor suppressive dependingon the tumor initiation protocol.When cultured in non-adherent conditions and with specificgrowth factors, primary dermal fibroblasts generate three-dimensional sphere of skin-derived precursor cells (SKPs).These cells are characterized by their capacity for self-renew-al, pluripotency and expression of specific SKP markers. Wegenerated 3D-spheres from dermal fibroblasts of wild-type(WT) and PPM1A-ablated (KO) mice, and functionallyproved they have SKP properties. Interestingly, PPM1A-KOSKPs showed differences in gene expression as well as phys-ical and functional properties compared to WT cells. A bioin-formatics analysis of the gene expression in PPM1A-KOSKPs predicts a significant role in affecting, via aberrant func-tions and signaling processes, tumor microenvironment—be it

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directly or indirectly, by affecting surrounding cells such asendothelial cells and immune cells.Here we will demonstrate the effects of PPM1A-ablation onSKP functionality and their signaling effect. Usingconditioned-medium we will demonstrate SKP effects on en-dothelial cells functionality, hematopoietic stem cell differen-tiation and immune cells activation. We will argue thatPPM1A activity in SKPs is crucial for maintaining homeosta-sis of the microenvironment in response to pathology andtumor development.

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Stromal Content in Prostate Cancer Correlateswith Responses to Docetaxel and Prognosis

Sivan M. Bokobza1, Emma J. Davies1, Kenneth Hiew2, Mi-chael Brown2, Noel Clarke2, Simon T. Barry1, Barry Davies1,Neil R. Smith11Translational Science, AstraZeneca, Macclesfield, UK2Genito Urinary Cancer Research Group, Cancer ResearchUKManchester Institute, The University of Manchester, Man-chester, UK

Docetaxel can deliver survival benefit to patients with meta-static castrate resistant prostate cancer (mCRPC). However,50 % of patients do not respond, and responders will eventu-ally progress. Improving response rates or duration to doce-taxel will ensure greater benefit can be achieved for mCRPCpatients. We have previously shown that tumours stratify intotwo phenotypes based on tumour-stromal architecture;stromal-vessel (SV; high stromal content, complex) andtumour-vessel (TV; low stromal content, simple)1. The major-ity of prostate tumours exhibit a SV phenotype, so we ex-plored whether this dense stromal compartment contributesto tumour insensitivity to chemotherapy by impeding drugpenetration, delivery, and efficacy. To evaluate this, 24 PDXprostate cancer models (LuCaP) were phenotyped for stromalcontent using immunoflourescent (IF) staining for α-Smoothmuscle actin (αSMA) and CD31. Those with a SV phenotype(4/5) were resistant to docetaxel, while those with a TV phe-notype (14/16) were highly responsive. In order to test theeffect of stromal modulating agents on the response to doce-taxel, four of these LuCaP models have been optimised forex vivo tissue slice culture, acting as a 3D model to study thetumour microenvironment. Furthermore, immunohistochemi-cal (IHC) staining of αSMA/CD31 was used to phenotype acohort of FFPE prostate cancer biopsies and with the use ofimage analysis software HALO, stromal content and architec-ture was shown to significantly impact on prognosis, and inthis cohort, was a better prognostic biomarker than Gleason,(p0.05). Collectively, this data suggests that tumour stroma

plays an important role in prostate cancer progression and thatstromal modulation may be a means of improving drug deliv-ery and response in this disease setting.(1) Smith et al., Clin Cancer Res 2013 19(24):6943–6956.

P17

Heparanase Promotes Mammary Gland BranchingMorphogenesis and Tumor Growth

Ilanit Boyango, Uri Barash, Liat Fux, Neta Ilan, IsraelVlodavskyCancer and Vascular Biology Research Center, The BruceRappaport Faculty of Medicine, Technion-Israel Institute ofTechnology, Haifa, Israel

Recently, we have shown that over expression of heparanaseor its C-terminal domain (8C) that mediate signaling proper-ties of heparanase, by human breast MCF10A cells results inlarger and asymmetrical acinar-like structures. In order to ex-plore the role of heparanase in mammary gland developmentwe have targeted heparanase and 8C expression to the mam-mary gland of transgenic mice utilizing the regulatory ele-ments of the mouse mammary tumor virus (MMTV). Specifictargeting of heparanase or the 8C variant to the mammaryepithelium at relatively low levels was sufficient to enhancemammary gland development evident by thicker end-budsand more branch alveolar structures that densely fill the mam-mary fat pad. Enhanced mammary branching morphogenesiswas associated with increased STAT5 phosphorylation inheparanase transgenic strains, while decreased STAT5 phos-phorylation was detected in Hpa-KO mice. Furthermore, highlevels of heparanase/8C expression are maintained during theinvolution phase, associating with delayed involution. Nota-bly, orthotopic implantation of EMT6 cells in the mammarygland of MMTV-HPA mice resulted in bigger tumors vs con-trol. Likewise, Hpa-Tg mice are more susceptible to chemicalcarcinogen and die at a higher rate. Taken together, these re-sults show that over-expression of heparanase in the mamma-ry gland of transgenic mice enhance mammary gland devel-opment, delay involution and support tumor growth.

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Lactic Acid Promotes Tumor Growth via Inhibitionof IFN-γ Expression

Almut Brand1, Katrin Singer1, Marina Kreutz1,21Department of Internal Medicine III, University Hospital Re-gensburg, Regensburg, Bavaria, Germany2Regensburg Center for Interventional Immunology, Univer-sity of Regensburg, Regensburg, Bavaria, Germany

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Tumor-derived lactic acid polarizes tumor-associatedmacrophages but its impact on tumor-infiltrating T cellsis unknown. For this purpose we generated B16.SIYmurine melanoma cells with low lactate secretion(Ldhalow) by means of Ldha shRNA. Tumor growth ofLdhalow cells was significantly delayed in immune com-petent C57BL/6 mice but not in Rag2-/- mice lackinglymphocytes. Ldhalow tumors exhibited significantly in-creased numbers of IFN-γ expressing cells and lacticacid inhibited IFN-γ expression in T cells in vitro.The difference in the tumor growth rate was abolishedwhen tumor clones were injected in IFN-γ-/- mice. Ourfindings show that lactic acid is a potent modulator ofIFN-γ in tumor-infiltrating T cells.

P19

Targeting Tumor Microenvironment for LungCancer Treatment

Anna Brichkina1, Hui Mun Loh1, Thi Minh NguyetNguyen1, Maria Antipina3, Dmitry Bulavin1,21Cancer Genetics and Therapeutics, Institute of Molecularand Cell Biology, A-STAR, Singapore, Singapore2Stress Response and Oncogenesis, Institute for Research onCancer and Ageing of Nice, Nice, France3Patterning and Fabrication, Institute of Materials Researchand Engineering, A-STAR, Singapore, Singapore

Chemotherapy as the widely used regimen of lung can-cer treatment is designed to kill dividing cancer cells. Inmost cases, it does not affect non-cancer stromal cellsthat in turn are required to sustain tumor growth. Thedevelopment of novel approaches for cancer therapybased on targeting tumor microenvironment includingtumour vasculature, cancer-associated fibroblasts andimmune cells is gaining significant interest as a direc-tion to improve cancer treatment.To understand further the role of tumor microenviron-ment in the development of a Non-Small-Cell-Lung-Cancer and thus to assess novel potential strategies fortreatment, we used a mouse model of NSCLC withsomatic expression of mutated K-ras. We monitored K-ras-induced lung tumor development in multiple mousestrains with inactivated or conditional deleted p38αMAPK which has been suggested to be involved inlung tumorigenesis.We found that activity of p38α MAPK in lung stromalfibroblasts and in hematopoietic cells, mainly in macro-phages, is essential to control lung tumor developmentin mouse model of NSCLC. We suggest that targetingof tumor-associated macrophages and lung stromal

fibroblasts could be used as an efficient complementarytherapy for the treatment of NSCLC with mutated K-ras.

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Fibroblast-Secreted CHI3L1 Enhances TumorGrowth and Angiogenesis

Noam Cohen, Ophir Shani, Yael Raz, Yoray Sharon, DanielHoffman, Neta ErezDepartment of Pathology, Sackler School of Medicine, TelAviv University, Tel Aviv, Israel

Breast cancer continues to be one of the leading causesof cancer related death in women, and the requirementfor better therapies is an unmet clinical need. Breasttumors are characterized by an extensive desmoplasticstroma, abundantly populated by fibroblasts. Cancer-Associated Fibroblasts (CAFs) are an activated sub-population of stromal fibroblasts, which have differentcharacteristics in different tumor types and tissue lo-cales. CAFs were shown to facilitate tumor growth bysupporting tumor cell growth, enhancing angiogenesisand remodeling the extracellular matrix (ECM).During breast carcinogenesis, fibroblasts express various fac-tors that facilitate tumor progression, but many of them remainunknown. We found that CHI3L1 is highly upregulated inCAFs isolated from invasive mammary tumors and from theirpre-metastatic lungs, as compared with normal mammary andlung fibroblasts.Although CHI3L1, a secreted glycoprotein, plays a piv-otal role in exacerbating the inflammatory processes,promoting angiogenesis and remodeling of the ECM,its functional role in cancer-related inflammation is stilllargely unknown.We found that CHI3L1 induced expression of pro-inflammatory and pro-tumorigenic pathways and en-hanced cell migration in breast cancer cells. In vivo,supplementing tumor cells with CHI3L1 induced angio-genesis in matrigel plugs. Furthermore, orthotopic trans-plantation of breast cancer cells mixed with fibroblasts inwhich the expression of CHI3L1 was knocked-down re-sulted in attenuated tumor growth. Interestingly, CHI3L1also increased macrophage migration and upregulation ofM2-like gene signature. Taken together, our findings im-plicate fibroblast-derived CHI3L1 as a key player in thecrosstalk between tumor cells and their microenviron-ment, and deepen our understanding of the contributionof CAFs to tumor progression and metastasis.

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P21

Multiple Myeloma Cells Reprogram Bone MarrowMesenchymal Stem Cells’ Translation Initiationthereby Promoting their Migration

Mahmoud Dabbah1,2, Liat Drucker1,2, Oshrat Attar1,2,Shelly Matalon1,2, Michael Lishner1,2,31Oncogentic Laboratory, Meir Medical Center, Kfar Saba,Israel2Sackler Facoulty Of Medicine, Tel Aviv University, Tel Aviv,Israel3Internal Medicine Department, Meir Medical Center, KfarSaba, Israel

Background and objectives: Previously, we showed co-modulation of translation initiation (TI) in BM-MSCsfrom normal donors (ND) co-cultured with MM cells.Here, we characterized the time line of MSCs condi-tioning by multiple myeloma (MM) cells, the persis-tence of this effect, and the consequences on cellphenotype.Methods: ND-MSCs were co-cultured with MM celllines (U266, ARP1) (MMcond-MSCs)(1–3 days), count-ed and assayed for death (trypan blue), viability (WST1),migration (scratch assay, inhibitors), expression of TIfactors, regulators and targets (immunoblotting). Involve-ment of MAPKs in MSCs conditioning and altered mi-gration was tested (immunoblotting, inhibitors).MMcond-MSCs were re-cultured alone (1–7 days) andassayed for TI. qPCR of extracted RNA was tested formicroRNAs levels.Results: MMcond-MSCs increased TI (↑150–200 %,p0.05) and proliferation (↑130 %, p0.01) is evident after2 days of co-culture and persists on the 3rd day. Theregulators are increased after 1 day and persist on the2nd day (↑220 %, p0.05), whereas the targets peak onthe 3rd day (↑250 %; p0.05). MAPKs are activated with-in 1.5 h of co-culture and responsible for MMcond-MSCs TI status (↑~200 %, p0.05) and elevated migration(16 h, ↑~400 %, p0.05). The BM-MSCs conditioning byMM cells is reversible after only 1 day of MMcond-MSCs culture alone. Decreased expression of MIR199and MIR125a (↑~140 %, p0.05) in MMcond-MSCs sup-ports elevated migration.Discussion: Time and proximity dependent conditioningof ND-MSCs underscores the dynamic co-evolution ofMM and BM-niche. MAPK/TI induced MMcond-MSCsmigration may contribute to disease progression and ad-ditional studies are warranted to establish its potential asan effective therapeutic target.

P22

Human RNASET2 Derivatives as Potential CancerTherapeutic Agents

Shani Doron1, Liron Nesiel2, Betty Schwartz1, OdedShoseyov21The Institute of Biochemistry, Food Science and Nutrition,Faculty of Agricultural, Food and Environmental Quality Sci-ences, Hebrew University of Jerusalem, Rehovot, Israel2The Robert H. Smith Institute of Plant Science and Geneticsin Agriculture, Faculty of Agricultural, Food and Environ-mental Quality Sciences, Hebrew University of Jerusalem,Rehovot, Israel

Huma n RNASET 2 h a s b e e n im p l i c a t e d i nantiangiogenic and antitumorigenic activities via amechanism associated with specific binding to actinand inhibition of the cell motility, independent of itsribonuclease capacities. We have constructed a trun-cated version of human RNASET2, with an incom-plete RNase active site (trT2-50) which maintainedits ability to bind actin, inhibit angiogenesis and con-sequently tumorigenesis in vitro and in vivo. Weidentified the region that is involved in the ability tobind actin, a 26 amino acid sequence derived from thehRNASET2 protein (peptide K108-K133). This pep-tide was tested for its ability to bind actin using theimmobilized actin solid phase assay (ELISA) and theBIAco re sy s t em . These a s s ay s r evea l ed tha thrRNASET2, trT2-50 and peptide K108-K133 boundactin with similar affinities. We continued by demon-strating that peptide K108-K133 has the ability toinhibit angiogenesis using the in vitro HUVEC tubeformation assay and the ex ovo CAM assay. Previousstudies have shown that migrating cells display higherlevels of actin polymerization as compared to station-ary cells. We therefore suggest that endothelial andtumor cells may be sensitive to trT2-50 or peptideK108-K133 treatment. The focus of my study is toshed light on the putative mechanism of action bywhich the hRNASET2 associated proteins inhibit an-giogenesis. In conclusion, we present the actin-binding sequence of the hRNASET2 protein. We iden-tified a 26 amino acid short peptide that is a deriva-tive of the hRNASET2, which is able to bind actinand consequently inhibit angiogenesis. The effect ofth is newly des igned pept ide is s imi lar to thehrRNASET2 and trT2-50 and therefore may offer anovel antiangiogenic therapeutic agent that will pre-vent tumor progression.

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P23

Multiple Myeloma and Bone Marrow MesenchymalStem Cells’ Crosstalk: Effect on TranslationInitiation

Liat Drucker1,3, Oshrat Attar Schneider1,3, MahmoudDabbah1,3, Victoria Zismanov1,3, Shelly TartakoverMatalon1,3, Michael Lishner1,2,31Oncogenetic Laboratory, Meir Medical Center, Kfar Saba,Israel2Internal Medicine A, Meir Medical Center, Kfar Saba, Israel3Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv,Israel

Multiple myeloma (MM) malignant plasma cells residein the bone marrow (BM) and convert it into a special-ized pre-neoplastic niche that promotes the proliferationand survival of the cancer cells. BM resident mesenchy-mal stem cells (BM-MSCs) are altered in MM andin vitro studies indicate their transformation by MMproximity is within hours. The response time frame sug-gests that protein translation may be implicated. Thus,we assembled a co-culture model of MM cell lines withMSCs from normal donors (ND) and MM patients totest our hypothesis. MM cell lines (U266, ARP1) andBM-MSCs (ND, MM) were harvested separately after3 days of co-culture and assayed for proliferation, death,levels of major translation initiation factors (eIF4E,eIF4GI), their targets, and regulators.Significant changes were observed: ND and MM BM-MSCs co-cultured with MM cell lines displayed elevat-ed proliferation, death, and expression/activity of eIF4E/eIF4GI; MM cell lines co-cultured with MM-MSCs alsodisplayed higher proliferation, death and eIF4E/eIF4GI;in contrast, MM cell lines co-cultured with ND-MSCsdid not display elevated proliferation, only death andhad no changes in eIF4GI levels/activity and un-uniform levels of eIF4E. We also determined that theMM induced changes in ND-MSCs’ eIF4E/eIF4GI arereversible. Lastly, we showed that MM cell lines prox-imity to ND-MSCs caused elevated migration of MSCs.We depict a flowchart of our observations.Our study demonstrates that there is direct dialoguebetween the MM and BM-MSCs populations that in-cludes translation initiation manipulation and criticallyaffects cell fate. Future research should be aimed atidentifying therapeutic targets that may be used to min-imize the collateral damage to the cancer microenvi-ronment and limit its recruitment into the malignantprocess.

P24

Do Metastatic Melanoma Cells Utilize CD40-CD40LAxis in the Post-Stroke Regenerative Nichefor the Establishment of Brain Metastasis?

Moran Frig1, Sivan Izraely-Bino1, Orit Sagi-Assif1, TsipiMeshel1, Shlomit Ben-Menachem1, Maya Rappaport1,Roshini Prakash2, S. Thomas Carmichael2, Isaac P. Witz11Cell Research and Immunology, Tel Aviv University, Tel Aviv,Israel2Neurology, UCLA, Los Angeles, California, USA

Stroke and metastasis are devastating diseases with sig-nificant mortality and morbidity. Post-stroke regenera-tion and brain metastasis could consist of overlappingcellular and molecular processes, including: angiogene-sis, inflammation and secretion of inflammatory media-tors. Therefore, further investigation of the molecularpathways shared by the regenerative niche after strokeand brain metastasis is required.CD40-CD40L signaling has been shown to play a major rolein inducing the pro-inflammatory phenotype after stroke. Inthe CNS, CD40 is expressed on a variety of cells, includingbrain endothelial cells (BECs), astrocytes and microglia. Inaddition, plasma levels of CD40L in patients after stroke issignificantly increased. Thus, CD40 and CD40L constituteimportant molecular targets for therapeutic intervention ofdiseases.We found that brain metastatic melanoma cells express CD40.Also, we found that “stroked” BECs and astrocytes secrethigher levels of CD40L than “non-stroked” cells. In Addition,brain metastatic melanoma cells migrate significantly betterthrough BECs stimulated by recombinant CD40L (rhCD40)than through unstimulated BECs. Stimulation of BECs, andbrain metastatic melanoma cells with rhCD40L elevated thelevels of phosphorylated JNK, compared to the basic phos-phorylation level.In view of these data, the fact that CD40 is expressed byBECs, secreted CD40L from these cells could induce, by anautocrine mechanism, the expression of adhesion moleculeson BECs. This in turn could mediate adhesion of brain meta-static melanoma cells to BECs. The activation of JNK signal-ing in brain metastatic melanoma cells could increase trans-endothelial migration and subsequently the formation of brainmetastasis.The therapeutic options for both melanoma metastasisand stroke are extremely limited. Uncovering commonmechanisms may lead to a novel druggable systemthat could manipulate cancer progression and neuralrepair.

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P25

Influence of the Lymph Node StromalMicroenvironment on the Development ofMalignantB Lymphocytes

Marleen Gloger1, Georg Lenz3, Michael Grau3, Uta E.Hoepken2, Armin Rehm1

1Hematology, Oncology and Tumorimmunology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany2Tumor Genetics and Immunogenetics, Max-Delbrück-Centerfor Molecular Medicine, Berlin, Germany3Translational Oncology, University Hospital Muenster,Muenster, Germany

In lymphoma pathogenesis, homeostatic chemokine receptorsand their cognate ligands gain particular interest because of theirability to organize lymphoid structures. Thus, the crosstalk be-tween tumor cells and their stromal microenvironment via theseengagements could give rise to the infiltration and subsequentproliferation of malignant lymphocytes. However, a detailedcharacterization of the molecular mechanisms involved in lym-phoma cell survival is warranted. More specifically, it remainsunclear which cellular stromal elements collaborate with lym-phoma cells and how the tumor cells get access to their putativeanti-apoptotic and pro-survival niche.Here, we apply an oncogene-driven transgenic mouse model,the Eμ-Mycmouse strain, which mimics an aggressive type ofB cell lymphoma. Upon adaptive transfer, Eμ-Myc tumor cellshome to the lymph node paracortex, where they find a growthpromoting microenvironment. To better understand the mo-lecular interactions between benign or malignant lymphoidcells and stromal cells, we focus in this project onlymphoma-induced changes in the stromal compartment, asdetermined by transcriptional profiling. Furthermore, we aimto identify the influence of a tumor-induced alteration of thevasculature on tumor cell trafficking and the formation of apro-survival niche for lymphoma B cells.

P26

Heparanase Links Obesity-Associated Inflammationand Breast Cancer Via Modulation of MacrophageResponses

Esther Hermano1, Rachel Goldberg1, Ariel Rubinstein1, Is-rael Vlodavsky2, Tamar Peretz1, Michael Elkin11Sharett Oncology Institute, Hadassah-Hebrew UniversityMedical Center, Jerusalem, Israel2Cancer and Vascular Biology Research Center, TheRappaport Faculty of Medicine, Technion-Israel Institute ofTechnology, Haifa, Israel

Obesity profoundly affects breast cancer progression and in-flammatory responses are critical contributors to the obesity-breast cancer link. While function of immunocytes (largely,macrophages) and their derived cytokines in coupling obesity-associated inflammation and breast cancer is well character-ized, the involvement of extracellular matrix (ECM) and itsenzymatic remodeling in these phenomena remains under-in-vestigated. The purpose of our study was to elucidate the roleof heparanase, mammalian endoglycosidase that cleaves hep-aran sulfate (chief ECM polysaccharide) in the obesity-breastcancer link.Utilizing murine model of high fat diet-induced obesitywe detected elevated heparanase levels in orthotopicbreast tumors growing in obese mice. Furthermore, ap-plying heparanase-deficient (Hpse-KO) mice we foundthat obesity accelerates breast tumor progression in wildtype (wt) but not in Hpse-KO mice. Importantly, while2-fold increase in infiltration of macrophages was ob-served in tumors grown in wt obese vs. wt lean mice,no difference in macrophage infiltration was detected intumors growing in Hpse-KO obese vs. Hpse-KO leanmice. Moreover, in vitro heparanase enzyme conferredpro-cancerous phenotype to macrophages stimulated bysaturated fatty acids (sFA), which are typically elevatedunder obesestate .In conclusion, our data underscore previously unknown roleof heparanase in promoting breast tumorigenesis under obeseconditions. It appears that the enzyme (which is induced inbreast carcinoma tissue by estrogen-dependent mechanism),creates tumor-promoting inflammatory microenvironment bydirecting pro-cancerous action of macrophages.Given intensive development and ongoing clinical testing ofheparanase inhibitors our findings are expected to offer futurestrategies toward a more efficacious therapy in rapidly grow-ing fraction of breast carcinoma patients with increased bodyweight.

P27

Differential Regulation of Processing of Type I BMPReceptor Isoforms: Implications for Signalingin Ovarian Cancer

Tal Hirschhorn, Michal Levi-Hofman, Nechama I.Smorodinsky, Marcelo EhrlichDepartment of Cell Research and Immunology, Tel Aviv Uni-versity, Tel Aviv, Israel

The transforming growth factor-β (TGF-β) superfamilyof ligands includes bone morphogenic proteins (BMPs),and plays crucial roles in tumor development and me-tastasis. TGF-β superfamily signals also regulate the

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differentiation and proliferation of normal tissues, andare interpreted with dependence on cellular context.This duality of effects complicates the targeting ofTGF-β superfamily receptors and ligands in therapeuticsettings. Moreover, the processing, trafficking and func-tion of TGF-β superfamily receptors also depend oncellular context, and changes to such context are in-duced by different chemotherapy drugs.The type I BMP receptors (BMPRIa and BMPRIb) dis-play extensive sequence similarity; however, theirknockout in mouse models yield different outcomes,suggesting that they perform different functions. Ourresults show that the prevalent isoform of BMPR1b(isoform 1) differs from its alternatively spliced isoform2 and from BMPRIa in the mode of its ER transloca-tion. Moreover, we show that only BMPR1a is N-glycosylated and that receptor glycosylation is importantfor the folding and plasma membrane localization oftype I BMP receptors. Together, these data raise thepossibilities of differential regulation of the processingof BMPR1a and BMPR1b, and of their differential sus-ceptibility to agents that alter the cellular context. Wetest these scenarios through the examination of the ef-fects of 2-Deoxy-D-Glucose (2-DG), an anti-cancer ther-apy agent, in ES-2 mesenchymal-like human ovariancancer cell model. Our data point to the importance ofBMP signaling in the induction of the aggressive meta-static phenotype of ES-2 cells. We showed that 2-DGaltered BMPRIa-dependent responses through perturba-tion of BMPRIa glycosylation. These findings point tothe possibility of manipulating the amount, localization

and function of TGF-β superfamily signals as a futuretherapeutic approach.

P28

Astrocytes Facilitate Melanoma Brain Metastasis viaSecretion of IL-23

Anat Klein1, Hila Shwartz2, Orit Sagi-Assif1, Tsipi Meshel1,Sivan Izraely-Bino1, Isaac P. Witz1, Neta Erez21Department of Cell Research & Immunology, Tel Aviv Uni-versity, Tel Aviv, Israel2Department of Pathology, Tel Aviv University, Tel Aviv, Israel

The major cause of melanoma mortality is metastasis todistant organs, frequently to the brain. The microenviron-ment plays a critical role in tumourigenesis and metastasis.In order to treat or prevent metastasis, the interactions ofdisseminated tumor cells with the microenvironment at themetastatic organ have to be elucidated. However, the role ofbrain stromal cells in facilitating metastatic growth is poorlyunderstood. Astrocytes are glial cells that function in repairand scarring of the brain following injury, in part via medi-ating neuroinflammation, but the role of astrocytes in mel-anoma brain metastasis is largely unresolved. Here we showthat astrocytes can be reprogrammed by human brain-metastasizing melanoma cells (HBMMCs) to express pro-inflammatory factors, including the cytokine IL-23, whichwas highly expressed by metastases-associated astrocytesin vivo. Moreover, we show that the interactions betweenastrocytes and melanoma cells are reciprocal: paracrine

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signaling from astrocytes up-regulates the secretion of thematrix metalloproteinaseMMP2 and enhances the invasive-ness of HBMMCs. IL-23 was sufficient to increase melano-ma cell invasion, and neutralizing antibodies to IL-23 couldblock this enhanced migration, implying a functional rolefor astrocyte-derived IL-23 in facilitating the progression ofHBMMCs. Knocking down the expression of MMP2 inmelanoma cells resulted in inhibition of IL-23-induced in-vasiveness. Thus, our study demonstrates that bidirectionalsignaling between melanoma cells and astrocytes results inthe formation of a pro-inflammatory milieu in the brain, andin functional enhancement of the metastatic potential of dis-seminated melanoma cells.

P29

Regulatory Roles of the Notch Signaling Pathwayin Inflammation-Driven Tumor-MSC Networksin Breast Cancer

Yulia Liubomirski1, Shalom Lerrer1, Tsipi Meshel1, DavidSprinzak2, Adit Ben-Baruch11Department of Cell Research and Immunology, George S.Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv,Israel2Department of Biochemistry and Molecular Biology, GeorgeS. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv,Israel

Breast tumor cells interact with mesenchymal stem cells(MSC) that are recruited to the tumor and promote pro-cesses aggravating disease course. Such tumor-MSC in-teractions may be mediated by cell-to-cell contacts andmay be promoted by inflammatory cytokines that pre-vail at the tumor microenvironment (TME), leading toelevated angiogenesis, tumor growth and metastasis.Our recent findings indicate that the release of the in-flammatory and metastasis-promoting chemokinesCXCL8, CCL2 and CCL5 by breast cancer cells(BCC) and bone-marrow-derived MSC is amplifiedwhen the two cell types are grown in co-cultures. Therelease of the inflammatory chemokines was further po-tentiated by stimulation by the inflammatory cytokinesTNFα and IL-1β, known to be highly prevalent at theTME in breast tumors. This amplified release ofchemokines was partly mediated by soluble factors ex-changed between the two cell types, but it also requireddirect tumor-MSC cell-to-cell contacts. Accordingly,confocal studies demonstrated that BCC position them-selves in close proximity to MSC and make intimatecontacts with them.

Using specific inhibitors and RT-qPCR analyses we identifiedcomplex regulatory roles of Notch pathway in the cytokine-induced, contact-dependent processes of chemokine up-regu-lation. Our results suggest potential roles of Notch-1 andJagged-2 expressed by BCC and Notch-3 and Delta-like-1expressed by MSC, in induction of CXCL8 and CCL2 (butnot CCL5) in tumor-MSC co-cultures. Direct roles for BCC-expressed Notch-1 were found to play key roles in theseevents.Overall, our findings indicate that inflammatory net-works regulate the tumor-promoting associations be-tween tumor cells and stroma cells in their vicinity.By further elucidating the impact of such networks ontumor progression, we will provide a better basis for thedesign of new therapeutic strategies in breast cancer.

P30

The Crosstalk between Omental Fat and TumorCells in Gastrointestinal Malignancies

Shelly Loewenstein1, Valerya Feygenzon1, Olga Kersy1, Jo-seph M Klausner1,2, Guy Lahat1,21Division of General Surgery, Tel-Aviv Sourasky MedicalCenter, Tel Aviv, Israel2Sackler School of Medicine, Tel Aviv University, Tel Aviv,Israel

Gastrointestinal (GI) malignancies are a common cause ofcancer- related mortality. While surgery is the only poten-tial curative treatment the majority of patients are deemedinoperable at presentation, resected tumors usually recur,and most patients die due to distant metastasis. Omentalspread is a relatively common and untreatable event,representing advanced disease stage that harbors verypoor prognosis. This crucial event has been studied tosome extent in ovarian cancer, however there is few ex-perimental data regarding omental metastasis of GI can-cers, and the exact molecular mechanisms involved in theprocess remain obscure. The omentum include an abun-dant amount of adipocytes and to a lesser extent fibro-blasts, endothelial cells, various immune cells, and stemcells; therefore, it is reasonable to assume that the omen-tum which constitute the immediate microenvironment ofthe metastatic cancer cell plays an active role in the de-velopment and progression of omental metastasis. Ourresults show that factors secreted by the varied cellularcomponents of the omentum enhance gastric and pancre-atic tumor proliferation, invasiveness, and survival. Uti-lizing liquid chromatography tandem mass spectrometry,we identified several cancer related cytokines secreted in-to the omental fat conditioned medium (CM). A

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comprehensive mRNA array of pancreatic cancer cellsco-cultured with omental fat CM identified specificomental induced molecular alternations that are associ-ated with cancer progression and metastasis. In addition,we conducted several in vivo experiments using differ-ent animal models supporting the premise that omentalfat increase tumor growth and aggressiveness. Our re-sults provide novel data defining the role of the omen-tum in GI cancer omental metastasis. Understanding themechanisms of omental metastasis may improve patientdiagnosis and hopefully enable to explore new potentialtargets for therapy.

P31

The Anticoagulant Protein S MediatesTumor—Microenvironment Interactions ImpactingTumor Metastasis

Avi Maimon, Ziv Talmi, Avishai Segev, Ray Fodor, TaliBurstyn-CohenDental Medicine, The Hebrew University of Jerusalem, Jeru-salem, Israel

Tumor development is critically impacted by inflamma-tion created by distant and/or infiltrating immune cellsin the tumor microenvironment (TME). A dynamic crosstalk between cancer cells and the immune system isknown to produce signaling pathways that mediate eithera pro- or antitumorigenic effect. With respect to cancer,the receptor tyrosine kinases Tyro3, Axl, and MerTK(TAM family), are proto-oncogenes, found to beoverexpressed and activated in various cancers, leadingto increased tumor cell proliferation, migration, survivaland drug - resistance. TAMs are also expressed by manyimmune cells, including macrophages, dendritic cells, nat-ural killer (NK) cells and T cells, where they are impor-tant negative regulators of innate immunity. Here we in-vestigate the role of the TAM agonist Protein S (PROS1)expressed by myeloid cells, in TME interactions, and itsimpact on metastasis. Our data indicate that mice lackingPROS1 in myeloid cells are susceptible to lung metasta-sis, suggesting a protective role for myeloid-derivedPROS1. Moreover, in-vitro stimulation of Lewis LungCarcinoma (LLC) cells with conditioned medium fromPROS1—deficient macrophages significantly increasedthe metastatic potential of LLC cells. These results reveala mechanism by which macrophages affect tumor cellaggressiveness, and that this switch is regulated byPROS1. Interestingly, Pros1−deficient macrophages ex-hibit a baseline pro-inflammatory profile.

Taken together, we speculate that deletion of myeloid—de-rived PROS1 contributes to increased LLC tumor cell aggres-siveness and at the same time to an elevated inflammatorymilieu, rendering host cells permissive to metastaticcolonization.These studies warrant further investigation into the on-going clinical implementation of TAM inhibitors forcancer therapy.

P32

The Beta Subunit of Human Hemoglobin InhibitsLung and Bone Marrow Metastasis

Shelly Maman1, Orit Sagi-Assif1, Weirong Yuan3, RavitGinat1, Tsipi Meshel1, Inna Zubrilov1, Yona Keisari2,Wuyuan Lu3, Isaac P. Witz11The Department of Cell Research and Immunology, TheGeorge S. Wise Faculty of Life Sciences, Tel Aviv University,Tel Aviv, Israel2The Department of Clinical Microbiology and Immunology,Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel3The Department of Biochemistry and Molecular Biology, In-stitute of Human Virology, University of Maryland School ofMedicine, Baltimore, Maryland, USA

Background. Metastatic disease may occur years or evendecades after a successful treatment of the cancer patient. Thisdisease progression is due to the propagation ofmicrometastases to clinically detectable metastasis. We previ-ously showed that soluble factors derived from mouse lungskeep lung micrometastatic cells in check, inhibiting their via-bility and thus their progression to overt metastasis. Here weisolate and characterize the lung-derived inhibitory factor.Methods. Reversed-phase HPLC followed by LC-MS/MS isolated and identified the lung-derived inhibitoryfactor to be the beta subunit of murine hemoglobin(HBB2). Synthesis of 14 peptide segments of 15 aminoacids each of the beta subunit of human hemoglobin(HBB) enabled the identification of a short C-terminalregion of HBB (designated Metox) as being responsiblefor the inhibitory activity.Results.HBB2 and HBB exert growth arrest and apoptosis ofhuman lung-metastasizing tumor cells in-vitro. In-vivo thera-py experiments employing Metox which exerts growth arrestand apoptosis of tumor cells in-vitro, inhibits the developmentof adrenal neuroblastoma tumors in xenografted mice and thedevelopment of spontaneous lung and bone marrowmetastasis.Conclusions. In addition to its known functions, the betasubunit of hemoglobin operates as an innate anti-tumor resis-tance factor restraining the proliferation of cancer cells and

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keeping them in a state of dormancy. HBB2 is expressed bymouse lung cells and is up-regulated in mice bearinghuman neuroblastoma micrometastases. HBB may thusbe developed into a novel drug and may serve as a bio-marker signaling the presence of clinically undetectablemicrometastasis.

P33

Anti-Angiogenic Activity of Factors Secretedby Normal Fibroblasts of Hypoxia Tolerantand Cancer Resistant Blind Mole Rat Spalax:in Vitro Investigation

Anatolii Mamchur, Irina Zotkin, Imad Shams, Irena ManovInstitute of Evolution, University of Haifa, Haifa, IsraelBlind subterranean mole rat Spalax evolved adaptivestrategies to cope with hypoxia (1). Recent investigationfrom our laboratory demonstrated unusual resistance ofSpalax to cancer either naturally occurring or inducedby chemical carcinogens (2). Moreover, normal fibro-blasts isolated from Spalax inhibit growth and kill broadspectrum of cancer cells either via direct co-cultivationor via conditioned medium (2). The vasculature is oneof the tumor microenvironment components, and angio-genesis plays a pivotal role in tumor progression anddevelopment of metastatic lesion. We thus addressedthe question whether secreted factors of Spalax fibro-blasts may influence angiogenesis in vitro. Tube forma-tion assay was used to compare effects of conditionedmedium (CM) of Spalax and mouse fibroblasts onHUVEC (human umbilical vein endothelial cells) migra-tion and formation of tube-like structures in 3D extra-cellular matrix. Tube formation was stimulated bymouse CM; nonetheless, it was significantly inhibitedwhen HUVEC cells were exposed to Spalax CM.Secretome analysis of Spalax fibroblasts CM revealedincreased level of mult iple proteins with anti-angiogenic activity. Since mesenchymal stem cells maycontribute to tumor angiogenesis, we investigated theeffect of CM of adipose-derived stromal cells (ADSC)isolated from Spalax adipose tissue on HUVEC tubeformation. Preliminary results did not show effect ofSpalax ADSC CM on tube formation compared to un-treated HUVEC. We conclude that Spalax fibroblastsinhibit tube formation in vitro in paracrine manner. Fu-ture studies will include the role of Spalax fibroblasts orits CM on intratumoral vessel formation by usingxenographt model.1. Shams, I., et al., 2005. FASEB J 19, 307–309.2. Manov, I., et al., 2013. BMC Biol 11, 91.

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Perivascular Cell Status is Associated with Survivalin Metastatic Colorectal Cancer

Artur Mezheyeuski1,2, Tormod Kyrre Guren4,5, InaHrynchyk14, Maja Bradic Lindh1, David Edler3, AncaDragomir6, Per Pfeiffer7, Elin H. Kure8, Tone Ikdahl9, EvaSkovlund10, Kristian Pietras11, Fredrik Ponten6, JanMulder12,Marja Hallström1, Camilla Qvortrup7, Katarina Öhrling1, An-na Portyanko2, Kjell Magne Tveit4, Peter Ragnhammar1,Bengt Glimelius6, Halfdan Sorbye13, Arne Östman11Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden2Department of Pathology, Belarusian State Medical Univer-sity, Minsk, Belarus3Department of Molecular Medicine and Surgery, KarolinskaUniversity Hospital Solna, Stockholm, Sweden4Department of Oncology-Pathology, Oslo University Hospi-tal, Oslo, Norway5K.G.Jebsen Colorectal Cancer Research Center, Oslo Uni-versity Hospital, Oslo, Norway6Department of Immunology, Genetics and Pathology, Upp-sala University, Uppsala, sweden7Department of Oncology-Pathology, University of SouthernDenmark, Odense, Denmark8Department of Cancer Genetics, Institute for Cancer Re-search, Oslo, Norway9Akershus University Hospital, Akershus University Hospital,Lørenskog, Norway10School of Pharmacy, University of Oslo and the NorwegianInstitute of Public Health, Oslo, Norway11Division of Translational Cancer Research, Lund Universi-ty, Lund, Sweden12Department of Neuroscience, Science for Life Laboratory,Karolinska Institutet, Stockholm, Sweden13Department of Oncology-Pathology, Haukeland UniversityHospital, Bergen, Norway14Department of General Pathology #2, State Clinical Bureauof Pathology, Minsk, Minsk, Belarus

Background: Perivascular cells (PC) regulate multiple as-pects of tumor biology. PC status in larger series of clinicalsamples remains poorly characterized. The role of PC as pre-dictors of survival or response to the therapy in colorectalcancer has not been analyzed. This study investigates the var-iation in PC status and the prognostic and response-predictivesignificance of PC markers and vessel characteristics in coloncancer (CC) and metastatic colorectal cancer (mCRC).Methods: Tumor sections of CC from a randomized phase-IIItrial of adjuvant treatment and tissuemicroarrays with primarytumors from two independent cohorts of mCRC were subject-ed to 2–4 double-staining with endothelial marker (CD34) and

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one of pericyte markers: platelet-derived growth factorreceptor-alpha and -beta (PDGFR-α, PDGFR-β), smoothmuscleα-actin (ASMA) and desmin. A novel dedicated meth-odological pipeline for digital image-analyses was used toquantitate characteristics of vessels and PC.Results: Analyses of associations of vascular characteristicsuncovered previously un-recognized independent expressionof the analyzed perivascular markers.Concerning prognostic relevance, we observed significant as-sociations between perivascular PDGFR-β status and DFS inCC, and between perivascular PDGFR-α or PDGFR-β andsurvival in metastatic disease in mCRC.The associations between survival endpoints and perivascularPDGFR-β were stronger in the treatment group of the adju-vant study suggesting impact on response to treatment. Ex-plorative analyses of the population-based mCRC cohort in-dicated reduced benefit of bevacizumab-treatment in thegroup of cases with low perivascular PDGFR-β expression.Conclusion: Perivascular PDGFR-α and -β in primary tu-mors are novel independent markers predicting survival inmetastatic CRC. Analyses also imply novel relationships be-tween PC and response to treatment. Collectively, the resultsimply previously un-recognized potential of PC as candidatebiomarkers and targets for therapy.

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Erythropoietin and Its Receptor Play a Rolein Melanoma Brain Metastasis and the BrainMicroenvironment

Noam Rudich1, Sivan Izraely-Bino1, Orit Sagi-Assif1, ShlomitBen-Menachem1, Maya Rappaport-Saban1, Dave S.B. Hoon2,Roshini Prakash3, S. Thomas Carmichael3, Isaac P. Witz11Department of Cell Research and Immunology, Tel Aviv Uni-versity, Tel Aviv, Israel2Department of Molecular Oncology, John Wayne Cancer Insti-tute, Saint John’s Health Center, Santa Monica, California, USA3Department of Neurology, University of California LosAngeles, Los Angeles, California, USAErythropoietin (Epo) is a hormone, known to regulate eryth-ropoiesis via stimulation of red blood cell production. Fewtrials have reported negative outcomes of anemia treatmentwith Epo in cancer patients, suggesting a direct responsemechanism on tumor cells. Indeed, Epo and Erythropoietin-receptor (EpoR) were found to be expressed and functional intumors cells, including melanoma.Melanoma commonly complicates in brain metastasis. Thera-peutic options for these patients are limited, and the prognosis isusually poor. The Epo-EpoR axis was shown to contribute inmelanoma cell survival, and to modulate neuroinflammation inastrocytes and microgl ia , which form the bra in

microenvironment. Therefore, here we focus on the role ofEpo-EpoR axis in the interactions of melanoma brain metastasiswith the brain microenvironment.To characterize the relevance of the Epo-EpoR axis in mela-noma brain metastasis, we first validated EpoR expression incutaneous and brain metastatic variants of three melanomamodels, as well as in brain microenvironment cells: astrocytesand brain endothelial cells (BEC). All these cells express thereceptor at the mRNA and protein level.Next, we asked whether Epo-EpoR axis regulation is depen-dent on the malignancy stage of melanoma. Surprisingly, wehave noticed that EpoR is downregulated in melanoma brainmetastases compared to cutaneous melanoma. Therefore,EpoR expression is locally regulated.Last, we noticed an upregulation of EpoR in BEC induced bymelanoma supernatants, and vice-versa, the supernatants ofBEC upregulate EpoR in melanoma cells.Our results suggest that Epo-EpoR axis may play a role in theinteraction between melanoma brain metastasis and its microen-vironment, and may serve as a therapeutic target in this disease.

This study was supported by the Dr. Miriam and Sheldon G.AdelsonMedical Research Foundation (Needham, MA, USA).

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Characterization of the Microenvironmentof Cervical Carcinoma Xenografts by MagneticResonance Imaging

Catherine Sem Wegner, Jon-Vidar Gaustad, Trude G.Simonsen, Christine Ellingsen, Lise Mari K. Andersen, EinarK. RofstadDepartment of Radiation Biology, Institute for Cancer Re-search, Norwegian Radium Hospital, Oslo University Hospi-tal, Oslo, Norway

The outcome of radiation therapy of patients with locally ad-vanced squamous cell carcinoma of the uterine cervix is deter-mined primarily by characteristic features of the tumor microen-vironment. For these patients, there is a dual problem of over-treatment and highmortality rates of 30–50%. On the one hand,the addition of chemotherapy to radiation therapy increases thesurvival rates with only 10 % while inflicting all patients withadded severe side effects. On the other hand, a large group ofpatients would probably benefit from additional or alternativetreatments. There fore, novel biomarkers predicting the outcomeof radiation therapy would be highly beneficial for personalizedtreatment of locally advanced cervical cancer. Magnetic Reso-nance Imaging (MRI) is an established method for anatomicalcharacterization of cervical carcinomas andmay be developed tobe an attractive method for characterization of the tumor micro -environment and, hence, for prognostic evaluation.

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In this study, several cervical carcinoma xenograft lineswere investigated by dynamic contrast enhanced MRIand diffusion weighted MRI. The radioresponsivenessof the tumors and their propensity to metastasize tolymph nodes were also determined. Moreover, character-istics of the microenvironment of the tumors wereassessed by probe measurements and immunohistochem-istry. Statistically significant correlations were found be-tween MRI-derived parametric images (Ktrans, the volumetransfer con stant of the MR contrast agent and ADC, theapparent diffusion coefficient), response to radiationtreatment, metastatic propensity, and characteristic fea-tures of the tumor microenvironment, including tumoroxygenation, fraction of hypoxic tissue, lactate concen-tration, glucose concentration, interstitial fluid pressure,tumor cell density, and the density of the tumor stroma.

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Cancer Associated Fibroblasts Breaking Bad—froma Growth Inhibitory to Tumor Promoting Phenotype

Yoray Sharon1, Yael Raz1,2, Noam Cohen1, Amir BenShmuel1, Hila Schwartz1, Lina Alon1, Tamar Geiger3, Neta Erez11Department of Pathology, Sackler School of Medicine, TelAviv University, Tel Aviv, Israel2Obstetrics and Gynecology, LIS Maternity Hospital, Tel Aviv,Israel3Human Molecular Genetics and Biochemistry, SacklerSchool of Medicine, Tel Aviv University, Tel Aviv, Israel

Breast tumors are characterized by an extensivedesmoplastic stroma, abundantly populated by fibro-blasts. Fibroblasts, among other stromal cell types popu-lating the neoplastic microenvironment have an importantrole in facilitating tumorigenesis. Cancer Associated Fi-broblasts (CAFs) are an activated sub-population of stro-mal fibroblasts, which can have different characteristicsin different tumor types. CAFs promote tumor growth bydirectly stimulating tumor cell proliferation and by en-hancing angiogenesis and inflammation. However, notmuch is known about how normal fibroblasts co-evolveduring tumor progression to become CAFs.To address this question, we have profiled the tran-scriptome of mammary CAFs isolated from distinctstages of mammary carcinogenesis. We found that fibro-blasts express unique gene signature in each tumorigen-ic stages, supporting the hypothesis that the stroma co-evolves with tumor development. Interestingly, fibro-blasts derived from hyperplasic tissues (HAFs) exhibita growth inhibitory gene expression signature, while fi-broblasts isolated from mammary tumors expressed pro-

inflammatory and growth promoting genes. Moreover,we show that this growth-inhibitory phenotype is exhib-ited also by fibroblasts isolated from lactating mammaryglands, which is a physiologic, non-tumorigenic,hyperplasic tissue. Furthermore, we show that normalmammary fibroblasts (NMFs) are reprogrammed toCAFs by tumor cell-secreted Osteopontin (OPN). Acti-vation of mammary fibroblasts by secreted OPN de-pends on two of its known receptors: CD44 andαVβ3 integrin. Strikingly, knockdown of OPN in tumorcells in vivo resulted in attenuated stromal activationand significant inhibition of tumor growth.Thus, we revealed a crosstalk between fibroblasts residing inthe tumor microenvironment and cancer cells which mediatethe co-evolution of fibroblasts during tumor progression.Maintaining the HAFs phenotype as well as blocking signal-ing by tumor-derived OPN may be an effective way to inhibittumor development.

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CCR7-Driven Metastatic Spread is Shapedby theMicroenvironment of Luminal Breast Tumors

Polina Weitzenfeld1, Olga Kossover2, Cindy Körner3, TsipiMeshel1, Stefan Wiemann3, Dror Seliktar2, Daniel Legler4,Adit Ben-Baruch11Department of Cell Research and Immunology, Faculty ofLife Sciences, Tel Aviv University, Tel Aviv, Israel2Faculty of Biomedical Engineering, Technion-Israel Instituteof Technology, Haifa, Israel3Division of Molecular Genome Analysis, German CancerResearch Center (DKFZ), Heidelberg, Germany4Biotechnology Institute Thurgau, University of Konstanz,Kreuzlingen, Switzerland

Metastatic spread is a complex process, regulated both byinternal characteristics of breast tumor cells, and by externalstimuli from the tumor microenvironment (TME) and the dis-tant pre-metastatic niches. Chemokine gradients in remote or-gans dictate homing of tumor cells to these sites. The CCR7-CCL21 axis mediates lymph node (LN) infiltration, while theCXCR4-CXCL12 pair is involved in bone metastases. Patientdata revealed that luminal-A breast tumors express lowerlevels of CCR7 and CXCR4 compared to more aggressivesubtypes. As the TME and tumor-regulating mechanisms dif-fer amongst disease subtypes, we sought to evaluate the reg-ulation of CCR7- and CXCR4-mediated metastatic spread bythe TME in luminal-A tumors.Our previously published study showed that stimulatingluminal-A tumor cells by factors typical of the luminalTME—inflammatory mediators, hormones and growth

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factors—induced high metastatic potential. In our currentstudy, we over-expressed CCR7 or CXCR4 in luminal breasttumor cells, endowing the cells with potent migratory capabil-ities in response to the chemokines, CCL21 and CXCL12,respectively. However, stimulating the cells with the “TMEstimulation” has shut-off CCL21- and CXCL12-induced mi-gration and inhibited chemokine-induced PI3K and MAPKactivation. Furthermore, the “TME stimulation” shifted themetastatic balance in vivo: while naive CCR7-over-expressing luminal-A cells infiltrated LN efficiently, mice in-oculated with TME-stimulated cells exhibited lower LN in-volvement, while demonstrating increased incidence of me-tastases in remote organs. Accordingly, CCR7was found to bea poor indicator of LN metastases in luminal-A breast cancerpatients.This study demonstrates that the TME of luminal-A breasttumors dominates the activities of chemokine receptors in reg-ulating site-specific metastases, and shifts the metastatic bal-ance from LN-infiltration to remote metastases and thereforeto a more aggressive phenotype.

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Molecular Mechanism of TNFSF15-InducedInhibition of VEGF-Stimulated VascularHyperpermeability

Guili Yang1,2, Zhisong Zhang2, Zilong Zhao1, Tingting Qin2,Dong Wang1, Rongcai Jiang1, Jianning Zhang1, Luyuan Li21Tianjin Neurological Institute, Tianjin Medical UniversityGeneral Hospital, Tianjin, China2State Key Laboratory of Medicinal Chemical Biology andCollege of Pharmacy, Nankai University, Tianjin, China

Vascular hyperpermeability is critical in ischemic diseasesincluding stroke and myocardial infarction, as well as ininflammation and cancer. VEGF-VEGFR2 signaling path-ways play a central role in facilitating vascular permeabil-ity. Counter-balancing mechanisms that negatively controlvascular permeability, although important to the mainte-nance of the integrity of blood vessels, are not yet fullyunder s tood . We repor t he re tha t TNFSF15 , anendothelium-derived cytokine and a specific inhibitor ofvascular endothelial cell proliferation, can inhibit VEGF-induced vascular permeability in vitro and in vivo. DR3,the receptor of TNFSF15, mediates TNFSF15 induced de-phosphorylation of VEGFR2 by utilizing phosphataseSHP-1, which is recruited to DR3 upon TNFSF15 interac-tion with the latter. A protein complex consisting ofVEGFR2, DR3 and SHP-1 is formed in response toTNFSF15 and VEGF actions on endothelial cell. This pro-tein complex plausibly provides a structural basis to the

molecular mechanism in which TNFSF15 induces the in-hibition of VEGF-stimulated vascular hyperpermeability.

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Deconstructing the Intracellular Events Leadingto Enhanced Melanoma Cell Proliferationby CEACAM1 Protein

Bella Zamlin-Vizel1,2, Rona Ortenberg2, Jacob Schachter2,Gal Markel1,2,31Clinical Microbiology and Immunology, Sackler Faculty ofMedicine, Tel Aviv University, Tel Aviv, Israel2Ella Lemelbaum Institute of Melanoma, Sheba Medical Cen-ter, Ramat Gan, Israel3Talpiot Medical Leadership Program, ShebaMedical Center,Ramat Gan, Israel

CEACAM1 protein expression in primary cutaneous melano-ma lesions predicts poor survival. We have previously foundthat CEACAM1 facilitates cell cycle and net proliferation ofmelanoma cells. CEACAM1 is a transmembrane glycoproteincontains extracellular, transmembrane and intracellular do-main. Previously we showed that the entire protein is crucialfor proliferation. In order to deconstruct the intracellularevents that lead to enhanced proliferation, we created a seriesof point mutations in CEACAM1. The constructs were stablytransfected into CEACAM1-negative melanoma cells andtested for proliferation. Remarkably, mutation analysis iden-tifies the ITIM residues (tyrosine 493 and 520) as well asserine508 to hold a critical role in CEACAM1-mediated pro-liferation. Indeed, mutation in these amino acids entirely ab-rogates the proliferative effect of CEACAM1 on melanomacells both in vitro and in a xenograft animal model. Accord-ingly, the role of SHP phosohatases. that known to interactwith CEACAM1 in lymphocytes. was studied. Co-IP assaysshowed more interactions of SHP-1 with CEACAM1 in mel-anoma cells transfected with wild type compare to mutatedCEACAM1, unlike SHP-2 that showed no difference. Theresults of the Co-IP assays were supported by knockdownassays with siRNA that showed only the effect of SHP-1 onproliferation but not SHP-2. This implies that the effect ismediated by SHP-1.Finally, we found that CEACAM1 affect important cellcycle inhibitors: mir-34 and p21. Cells transfected withCEACAM1 showed lower levels of p21 (RNA and pro-tein) and mir-34 (RNA) compare to cells with mocktransfection.In conclusion, we propose a new pathway that facilitatingmelanoma cell proliferation, mediated by recruitment ofSHP-1 to key residues in CEACAM1 protein that leads todown regulation of cell cycle inhibitors.

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Tumor Necrosis Factor Superfamily-15 InhibitsVEGF Gene Expression via microRNA-29b

Kun Zhang, Hong-Xing Cai, Qiang-Zhe Zhang, Luyuan LiState Key Laboratory of Medicinal Chemical Biology, NankaiUniversity, Tianjin, China

Tumor necrosis factor superfamily-15 (TNFSF15; VEGI), acytokine largely produced by endothelial cells, specifically in-hibits endothelial cell proliferation and endothelial progenitorcell differentiation. Vascular endothelial growth factor (VEGF)plays a pivotal role in the regulation of blood vessel growth andfunction. We report here that TNFSF15 is able to downregulateVEGF gene expression. Treatment of mouse endothelial cellline bEnd3 with TNFSF15 results in a decrease of VEGF atboth mRNA and protein levels, and at the same time an in-crease of microRNA-29b (miR-29b). Overexpression of miR-29b in bEnd3 leads to suppressed VEGF expression. Elimina-tion of miR-29b gives rise to increased VEGF expression.Blocking TNFSF15-activated signals by using either siRNAagainst death domain-containing receptor-3 (DR3;TNFRSF25), which is the receptor of TNFSF15 on endothelialcells, or a neutralizing antibody 4-3H against TNFSF15 leadsto a suppression of TNFSF15-induced miR-29b expression.We have determined the intracellular signaling pathwaysresulting from TNFSF15-stimulation and leading to miR-29bupregulation. These findings are consistent with the view thatTNFSF15 is a critical regulator of VEGF activities.

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Tumor Necrosis Factor Superfamily-15 FacilitatesLymphangiogenesis via Upregulation of VEGFR3Gene Expression in Lymphatic Endothelial Cells

Qiang-Zhe Zhang, Ting-Ting Qin, Guo-Ce Xu, Jian-Wei Qi,Gui-Li Yang, Kun Zhang, Hai-Lin Liu, Luyuan LiState Key Laboratory of Medicinal Chemical Biology andCollege of Pharmacy, and Tianjin Key Laboratory of Molec-ular Drug Research, Nankai University, Tianjin, China

Lymphangiogenesis is essential in embryonic developmentbut is rare in adults. It occurs, however, in many disease con-ditions including cancers. Vascular endothelial growth factor-C/D (VEGF-C/D) and VEGF receptor-3 (VEGFR3) play acritical role in the regulation of lymphangiogenesis. We inves-tigated how the VEGF-C/VEGFR3 signaling system is regu-lated by tumor necrosis factor superfamily-15 (TNFSF15), anendothelium-derived cytokine. We report here that TNFSF15,

which is known to induce apoptosis in vascular endothelialcells, can promote lymphatic endothelial cell (LEC) growthand migration, stimulate lymphangiogenesis, and facilitatelymphatic circulation. Treatment of mouse LEC withTNFSF15 results in upregulation of VEGFR3 expression; thiscan be inhibited by gene-silencing of death domain–contain-ing receptor-3 (DR3; TNFRSF25), a cell surface receptor forTNFSF15, with siRNA, or by blocking TNFSF15-DR3 inter-action with a TNFSF15 neutralizing antibody 4-3H. Addition-ally, TNFSF15/DR3 signaling pathways in LEC include acti-vation of NF-κB. TNFSF15-overexpressing transgenic miceexhibit a marked enhancement of lymph drainage; this is con-firmed by treatment of wild type mice with intraperitonealinjection of recombinant TNFSF15. Moreover, systemic treat-ment of pregnant TNFSF15 transgenic mice with 4-3H leadsto inhibition of embryonic lymphangiogenesis. Our data indi-cate that TNFSF15, a cytokine produced largely by endothe-lial cells, facilitates lymphangiogenesis by upregulatingVEGFR3 gene expression in LEC, contributing to the main-tenance of the homeostasis of the circulatory system. Thisfinding also suggests that TNFSF15 be of potential value asa therapeutic tool for the treatment of lymphedema.

P43

The Antiproliferative, Proapoptotic and AntitumourActivity in Vitro of 10 % PBS Holocene GrainWashout Against CaCo-2 CELLS can be Enhancedwith Different Human Alfa Interferons

Bratko Filipic1, Lidija Gradišnik2, Adriana Pereyra3, JanaPotokar3, Hrvoje Mazija41Laboratory for Interferon research, Institute for Microbiolo-gy and Immunology, Ljubljana, Slovenia2Institute for Pharmacology, Medical Faculty, Maribor,Slovenia3Control & Research department, MEDEX D.o.o., Ljubljana,Slovenia4Institute for Translational oncology, Koledinećka 3, Zagreb,Croatia

Holocene sands near river Drava (Croatia) contain Holo-cene minerals which, in grained form, exhibit unusualbiological/microbiological activity. The grained Holoceneminerals were analysed : SiO2 (88,71 %), Al2O3(5,37 %),Fe2O3 (1,08 %) and gold (Au) (12,1 %) among elements.Other elements were present in smaller range. It wasfound that a 10 % Holocene grain Phosphate Buffer Sa-line (PBS) washout and HuIFN—αN3 affect the growthof CaCo-2 cells . The aim was to measure the effect ofdifferent Human Alfa IFNs on antiproliferat ive,proapoptotic and antitumour activity in vitro of 10 %

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PBS washout of Holocene grain on CaCo-2 cells . Thefollowing interferons were used: HuIFN-αN3 (IMZ—Za-greb, Croatia), Alfaferon (Alfa Wasserman, Italy),Egiferon (Trigon, Hungary); Human recombinant αA In-terferon (EMD Chemicals Inc., San Diego,Ca 92121USA), Roferon-A (IFN Alfa-2a), Roche and Intron A(IFN Alfa-2b) (MSD Italia Srl, Italy). The interferonswere analysed by RP-HPLC at 280 nm. CaCo-2 cellswere treated as: (a) cell control, (b) interferon + 10 %PBS (1:1,1:2,2:1), (c) interferon, (d) 10 % PBS. Compar-ison with the interferon-untreated cells was calculated.The apoptotic cells were isolated by BioVision: Apoptoticcell isolation kit. The 10 % Holocene grain washoutshowed an antiproliferative activity. This can be enhancedup to three times by HuIFN-αN3 and not with theHuIFN-α2a or HuIFN-α2b. In case of proapoptotic activ-ity, the 10 % Holocene grain washout shows 26,52 % ofapoptotic cells. It was increased to 49,85 % withHuIFN-αN3. With HuIFN-α2a and HuIFN-α2b it was22,80 % and 42,60 %, respectively. It can be concludedthat 10 % PBS Holocene grain washout synergizes theeffect of the HuIFN-αN3 in highest level of AP activity,while this is not the case with HuIFN-α2a or HuIFN-α2b.

P44

Amount of CD68+ Tumor Associated Macrophagesin Gaps of Ductal Tumor Structures NegativelyCorrelates with Lymphatic Metastases in HumanBreast Cancer

Mikhail Buldakov1,2, Marina Zavyalova1,2,3, NadejdaKrakhmal2,3, Nadejda Telegina3, Sergey Vtorushin2,3, Vladi-mir Riabov4, Shuiping Yin4, Nadezhda Cherdyntseva1,2, JuliaKzhyshkowska1,41Laboratory for translational cellular and molecular biomed-icine, Tomsk State University, Tomsk, Russia2Laboratory of mollecular oncology and immunology, TomskCancer Research Institute, Tomsk, Russia3Department of Pathological Anatomy, Siberian State Medi-cal University, Tomsk, Russia4Department of Innate Immunity and Tolerance, Institute ofTransfusion Medicine and Immunology, Medical FacultyMannheim, University of Heidelberg, Mannheim, Germany

Tumor associated macrophages (TAM) support tumorgrowth and metastasis in several animal models of breastcancer, and TAM amount is predictive for efficient tumorgrowth and metastatic spread via blood circulation. How-ever, limited information is available about intratumoralTAM heterogeneity and functional role of TAM subpopu-lations in tumor progression. The aim of our study was to

examine correlation of TAM presence in various morpho-logical segments of human breast cancer with clinical pa-rameters. Patients with nonspecific invasive breast cancerT1-4N0-3M0 from Tomsk Cancer Research Institute wereincluded in the study. Morphological examination waspe r fo rmed us ing Car l Ze i s s Ax io Lab .A1 andM i r a xM i d i Z e i s s . Immun o h i s t o c h em i c a l a n dimmunofluorescence/confocal microcopy analysis wasused to detect CD68 and stabilin-1 in 5 different tumorsegments: 1) areas with soft fibrous stroma; 2) areas withcoarse fibrous stroma; 3) areas of maximum stromal-and-parenchymal relationship; 4) parenchymal elements; 5)gaps of ductal tumor structures. The highest expressionof CD68 was in areas with soft fibrous stroma or areasof maximum stromal-and-parenchymal relationship(79 %). The lowest expression of CD68 was in areas withcoarse fiber stroma (23 %). Inverse correlation of tumorsize and expression of CD68 in gaps of tubular tumorstructures was found (R=−0.67; p=0.02). In case of thelymph node metastases the average score of CD68 expres-sion in ductal gaps tumor structures was lower (1.4±0.5)compared to negative lymph nodes case (3.1±1.0; F=10.9; p=0.007). Confocal microscopy identified 3 pheno-type of TAM: CD68+/stabilin-1−; CD68+/ stabilin-1+ (over50 %); and CD68−/ stabilin-1+. However, expression ofstabilin-1 did not correlate with lymph node metastasis.We concluded, that increased amount of CD68+TAM ingaps of ductal tumor structures is protective against met-astatic spread in regional lymph nodes.

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Correlation between Vascular Endothelial GrowthFactor and Leptin in Normal Human Breast Tissuein Vivo

Charlot ta Dabros in1 , Viv ian Morad1 , Anne l ieAbrahamsson1, Preben Kjölhede21Oncology, Linköping University, Linköping, Sweden2Ob/Gyn, Linköping University, Linköping, Sweden

Introduction: Events in the microenvironment are importantfor carcinogenesis of the breast. Adipocytes, which produceadipokines with paracrine effects, are the most abundant celltype in breast tissue. Exposure to sex steroids affects the riskof breast cancer. It has previously been shown that estrogenregulates the extracellular levels of leptin, adiponectin, andVEGF in normal human breast tissue in vivo.Objective:We aimed to determine if there were any relation-ships between leptin, adiponectin, and/or VEGF in normalhuman breast tissue in vivo and to elucidate the role of adipo-cytes in the regulation of these factors.

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Design and methods: Microdialysis was used to sampleproteins of normal human breast tissue and abdominalsubcutaneous (s.c.) fat in situ in pre-and postmenopaus-al women. An in vitro co-culture model of breast can-cer cells and primary mature human adipocytes wasused.Results: In vivo, in normal breast tissue, significant posi-tive correlations between VEGF and leptin, and VEGFand leptin/adiponectin ratio were detected. No correla-tions were found in s.c. abdominal fat tissue. Co-cultureof adipocytes and breast cancer cells per se increased thesecretion of VEGF and leptin and enhanced the effects ofestradiol compared to culture of either cell type alone.In vitro, inhibition of VEGF diminished the release ofleptin while inhibition of leptin had no influence onVEGF secretion.Conclusions: Our results suggest that VEGF regulatesleptin rather than a leptin regulation of VEGF. More-over, co-culture between adipocytes and breast cancercells, induces phenotypic changes and enhances the ef-fects of estradiol. These mechanisms may be involvedin breast cancer progression.

P46

The Role of IL-31 in Tumorigenesis

Shiri Davidi, Ella Fremder, Tal Kan, Dror Alishkevitz, Mi-chael Timaner, Valeria Miller, Ofrat Beyar Katz, YelenaBarbarov, Ziv Raviv, Ami Aronhiem, Yuval ShakedDepartment of Cell Biology and Cancer Science, The BruceRappaport Faculty of Medicine, Technion-Israel Institute ofTechnology, Haifa, Israel

The development of new drugs against cancer has been agreat challenge in recent years. While most of the drugsare designed to kill tumor cells, a growing body of liter-ature suggests that the modulation of host cells e.g., theimmune system against tumor cells may also act as apotential target for therapy. Based on a screening tech-nique in which we identified host factors which are up-regulated in the plasma following chemotherapy, we iden-tified IL-31 as a possible factor with immune modulationproperties. IL-31 has been shown to promote an acuteimmune response by activating macrophages and T-cytotoxic cells leading to dermatitis in mouse and human.However, the role of IL-31 in tumorigenesis is not known.Here we generated a construct of IL-31 fused with a sta-ble protein (IL-31-IgG) in order to maintain high levels ofIL-31 in the blood. IL-31-IgG directly inhibits the prolif-eration and viability of tumor cells which express thereceptors for IL-31. Consequently, mice bearing 4T1 or

MC-38, murine breast and colon carcinomas, respectively,that have been treated with IL-31-IgG resulted in reducedtumor growth, inhibition of angiogenesis, modulation ofmacrophages towards pro-inflammatory phenotype, andreduced number of metastatic lesions in the lungs of micebearing 4T1 tumors. Our results therefore suggest that IL-31 is a multi-targeted agent that can affect both tumor andhost-derived protumorigenic cells. This study will pavethe way toward the development of a novel anti-cancermodality affecting primary tumors and their metastasis.

P47

Immuno-Skeletal Effects of Erythropoietinin Multiple Myeloma Mice—a Friend and Foe

Naamit Deshet-Unger1, Sahar Hiram-Bab1,2, Dafna Gilboa1,Yasmin Haim-Ohana1, Moshe Mittelman3, Yankel Gabet2,Drorit Neumann11Department of Cell and Developmental Biology, Tel AvivUniversity, Tel Aviv, Israel2Department of Anatomy&Anthropology, Tel Aviv University,Tel Aviv, Israel3Department of Medicine, Tel Aviv University, Tel Aviv, Israel

Clinical introduction of recombinant human erythropoietin(Epo) has been a breakthrough in treating patients sufferingfrom anemia associated with chronic kidney failure. We havepreviously reported that Epo treatment in multiple myeloma(MM) patients and mouse models was associated with im-proved immunological functions. We have recently found thatEpo treatment or overexpression, stimulates bone resorptionin mice (Hiram-Bab et al., 2015). Here we address Epo actionon the immune and skeletal systems in the 5T33 MM mousemodel.Epo administration to MM mice attenuated disease progres-sion as demonstrated by a significant decrease in MM patho-logical κ light chain, expression of IL-6 and RORγt (a Th17hallmark). Interferon-γ transcript levels (activator cytokine ofmacrophages) and number of macrophages (F4/80+CD11b+)in the bone marrow (BM) were increased 1.7 and 1.4-fold,respectively in the Epo- versus vehicle-treated MM mice(p0.05). In vitro, activation of purified BM-derived macro-phages with Epo, led to a 33 % increase (p=0.01) in phago-cytosis of 5T33 MM cells.High-resolution μCT analysis of the distal femurs revealedEpo-associated bone loss in both healthy and MM mice. Bcells are a significant source of RANKL. Epo led to an in-crease (1.8-fold, p=0.0003) in membrane bound RANKL onB cells (B220+CD19+) isolated from BM. These cellsexpressed Epo receptors, suggesting that they are direct targetsof Epo.

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Taken together, these findings portray Epo as a double-edgedsword, which stimulates immune response while acceleratingbone resorption in MM. The current study highlights admin-istration of targeted bone protective treatment alongside Epoin MM patients, to attenuate the anemia and MM progression,while preventing bone damage.Supported by the MMRF and by the FP7 European commis-sion grant; 282551 EpoCan.

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Efficient Treatment of Mouse BreastAdenocarcinoma by Ablation with IntratumoralAlpha Irradiation Combined with Inhibitorsof Immunosuppressor Cells and Immunostimulationwith CpG

Margalit Efrati1, Hila Confino1, Michael Schmidt2, IlanHochman1, Viktor Umansky3, Itzhak Kelson2, Yona Keisari11Department of Clinical Microbiology and Immunology,Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv,Israel2Department of Physics and Astronomy, Sackler Faculty ofExact sciences, Tel Aviv University, Tel Aviv, Israel3Skin Cancer Unit, German Cancer Research Center, Heidel-berg and Department of Dermatology, Venereology andAllergology, Ruprecht-Karl University of Heidelberg, Mann-heim, Germany

Background: Our team developed a potent tumor ablationtechnique, Diffusing Alpha-emitters Radiation Therapy(DaRT), which is based on insertion of 224Ra–loaded wire(s)into the tumor. Short-lived atoms are released from the wires,spread in the tumor and emit lethal alpha particles, which killthe malignant cells. The destruction provoked T cell depen-dent anti-tumor immune reactions, which conferred protectionagainst a tumor challenge, and killed malignant cells in theprimary tumor and distant metastases.In this study we attempted to enforce the anti-tumor immunereaction by combining DaRT with neutralization of immuno-suppressive regulatory T cells (Tregs) and myeloid derivedsuppressor cells (MDSCs), and immunostimulation by theimmunoadjuvant CpG.Results: Mice bearing DA3 mammary adenocarcinoma withmetastases were treated with DaRTwires in combination withan MDSC inhibitor (sildenafil), a Tregs inhibitor (low dosecyclophosphamide), and the immunostimulant, CpG. Combi-nation of DaRT with the three immunomodulating agents re-sulted in tumor growth retardation, complete rejection of pri-mary tumors (1/9 tumor-bearing mice) and retardation of lungmetastases. Only 30 % of such treated animals carried lungmetastases compared with over 50 % of the mice treated with

an inert wire and the three reagents. Treatment with DaRT,Treg and MDSC inhibitors (without CpG) also diminishedsignificantly tumor growth and reduced the lung metastaticburden. In 60 % of such treated mice lung metastases weredetected compared to 77 % of the animals treated by a non-radioactive wire and the inhibitors.Conclusions: Therapy with DaRT combined with the inhibi-tion of immunosuppressive cells and immunostimulation withCpG enforced both local and systemic anti-tumor immuneresponses. This treatment can be applied in patients whensurgery is not an option or before surgical removal of tumors.

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Evidence for Differential Effect of Two SSRIsAntidepressants on Murine Breast Cancer (4 T1)in-Vitro and Tumor Grafted Female Miceunder Normal and Stress Conditions

Irit Gil-Ad2, Michal Taler1,2, Iris Brener1,2, WeizmanAbraham1,2

1Felsenstein Institute, Tel Aviv University, Petah-Tiqva, Israel2Lab. Biological Psychiatry, Felsenstein Institute, Petah-Tiqva, Israel

Background Breast cancer diagnosis and treatment arestressful events for most women, which often lead to severedepression. Antidepressants are widely prescribed to treatdepression in cancer patients, However, no general consid-eration is applied regarding antidepressant selection. Previ-ous results showed that some serotonin reuptake inhibitors(SSRIs), mainly Sertraline and Paroxetine demonstrated an-tiproliferative and apoptotic activity against several cell-lines in-vitro and colorectal tumor in xenografted mice,whereas other agents e.g., Citalopram were not effective.Aims 1. Evaluation of the comparative effect of Sertralineand Citalopram on murine 4T1 breast cancer cell prolifera-tion in- vitro and in-vivo 2. Determine the effect of stressand antidepressants therapy on 4T1 cell viability as well ason immune cells (splenocytes) viabil i ty and pro-inflammatory cytokine secretion. 3. Evaluate the compara-tive effect of the two SSRIs on tumor growth as well as onanxiety parameters in mice. Results In vitro, we found amarked difference between Sertraline and Citalopram. Ser-traline induced a dose dependent inhibition of cell viabilityand splenocytes viability and proliferation. Whereas,Citalopram did not alter cell viability. In-vivo, chronic mildstress (CMS) induced anxiety in 4T1 grafted Balb/c micemodel, however, it did not modify tumor growth. Sertralinebut not Citalopram (10 mg/kg/d each), deteriorated theanimal’s physical condition inducing shortening of survivaland increase in tumor growth. Conclusion Our results

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demonstrated a dissociation between the in vitro and thein vivo results, with Sertraline but not Citalopram, showinginhibitory effect in-vitro, and tumor accelerating activity athigh dose in- vivo. Moreover, we could not demonstrate adeteriorating effect of our stress condition on tumor growth.We suggest that a careful approach should be taken regard-ing Sertraline therapy in patients.

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Erythropoietin-Associated Increase in LiverMonocyte Derived Macrophages; a Role for KupfferCells as Mediators?

Dafna Gilboa1, Yasmin Ohana Haim1, Nathali Ben-Califa1,Naamit Deshet-Unger1, Sahar Bab Hiram1, Max Gassman2,Moshe Mittelman3, Drorit Neumann11Cell and Development Biology, Tel Aviv University, Tel Aviv,Israel2Institute of Veterinary Physiology, University of Zurich, Zu-rich, Switzerland3Department of Medicine A, Tel Aviv Sourasky Medical Cen-ter, Tel Aviv, Israel

Erythropoietin (EPO) is the major hormone that drives mam-malian erythropoiesis, via its surface receptor, EPO-R. It ismainly used for treating anemia associated with chronic renalfailure, and certain malignancies. EPO-Rs were also found innon-erythroid cells, including dendritic cells and bone marrowmacrophages (Lifshitz, 2008; 2010). Here we addressed theeffect of EPO on hepatic-macrophages, namely resident livermacrophages (Kupffer cells) and liver monocyte-derived mac-rophages (MFs). Utilizing the rat Kupffer cell line (KCL-2) wedemonstrate that these cells express EPO-R transcripts and cellsurface EPO-R, as detected by our novel EPO-R antibody(GM1012; Maxwell 2015). EPO treatment of the KCL-2 cellsled to a 1.5±0.06 fold increase in EPO-RmRNA levels and a 2±0.01 fold decrease in surface EPO-R levels. Stimulation of thecells with EPO induced a 3±0.5 fold increase in transcriptlevels of CCL-2 (a chemo attractant for monocytes) and a15 %±0.6 increase in the levels of the secreted chemokine.EPO treatment also enhanced cellular activity of the KCL-2cells as manifested in a 50 %±0.13 increase in cell migration,an increase in phagocytosis of microbeads (40 %±0.08) and ofE.coli (13 %±0.05).Finally, in vivo experiments demonstrated a specific EPO-induced selective increase (19.5 % ±0.01) in MFs, but not inKupffer cells. Elevated CCL-2 in sera of EPO-injected mice(2.1 fold increase) supports a mechanism by which EPO stim-ulates Kupffer cells to increase secretion of CCL-2 whichenhances recruitment of monocytes to the liver and their sub-sequent differentiation into MFs.

The present study points to a new EPO action on two separateliver macrophages populations, thus calling for future studieson EPO effects on inflammation in the liver and in other tissues.

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Impact of Heparanase Residing in the TumorMicroenvironment on Cancer Progression

Lilach Gutter-Kapon, Neta Ilan, Israel VlodavskyCancer & Vascular Biology Research Center, Technion-IsraelInstitute of Technology, Haifa, Israel

Heparanase is an endoglycosidase that cleaves heparan sulfateside chains. In many cases, heparanase induction correlateswith increased tumor growth and metastasis, thus encouragingthe development of heparanase inhibitors as anti-cancer drugs.We investigated the impact of host- vs. tumor- derivedheparanase on cancer progression, emphasizing the cross talkbetween the epithelial, stromal and immune compartments ofthe tumor and its microenvironment. To this end, we utilizedheparanase knockout (Hpa-KO) and transgenic (Hpa-Tg)mice to investigate tumor development following inoculationwith cancer cells. We found that cancer cells produced biggertumors in Hpa-Tg vs. control mice. Likewise, smaller tumorswere developed by those cells when inoculated in Hpa-KOmice. Notably, we found that reduced tumor growth in Hpa-KO mice was associated with accumulation of macrophages(MФ) in the tumor periphery. Moreover, over expression ofMIP2 in LLC cells results in reduced tumor weight whenimplanted in control but not in Hpa-KO mice. In KO mice,the recruited MФ arrest at the tumor periphery and are signif-icantly less activated. Implantation of control, but not KOMФ, together with LLC cells eradicates tumor growth. Fur-thermore, the number and activity of immune cells (i.e., MФ,T-cells, NK and dendritic cells) recruited to the tumors waselevated only when control MФ were implanted. In vitro, theinvasion and migration capacity of the Hpa-KO MФ was re-duced. Real time PCR analyses reveal that most of the cyto-kines are expressed at lower levels in Hpa-KOMФ vs control.

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Host-Derived IL-1b Regulates Immune Responseduring 4T1 Breast Carcinoma Tumor Growth

Irena Kaplanov, Yaron Carmi, Ziv Shavshevitz, RachelKornetsky, Peleg Rider, Shahar Dotan, Elena Voronov, RonN. ApteMicrobiology and Immunology Faculty of Health Sciences,Ben-Gurion University of the Negev, Beer-Sheva, Israel

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We have studied the effects of host-derived Interleukin-1β(IL-1β) on breast cancer progression, using 4T1 tumor model.Cells were injected orthotopically into IL-1b KO mice andtheir wild-type (WT) counterparts. During the first 2 weeks,tumors in both WT and IL-1b KO mice developed similarly,whereas later, deficiency of microenvironmental IL-1b leadsto complete tumor regression, which is CD8+ T cell de-pendent. This response is impaired in WT mice, whereaccumulation of monocytic myeloid suppressor cellsMDSC (M-MDSC), dependent on monocyte chemotacticprotein 1 (MCP-1), was more abundant than in mice withIL-1β deficiency. Host-derived IL-1b at the tumor sitedirects M-MDSC differentiation to tumor promoting anti-gen presenting cells (APCs), while in IL-1b KO mice, M-MDSCs differentiate into LY6ClowCCR2− myeloid cells.Overall, the results demonstrate that IL-1β is an importantfactor in recruitment and differentiation of myeloid cellsand plays a crucial role in determining the balance be-tween immunity and inflammation.

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Enhancement of the Anti-Tumor ImmunityFollowing Tumor Ablation by ElectrochemicalTreatment

Yasmine Kayal1, Margalit Efrati1, Sarah Eckstat1, RafiKorenstein2, Yona Keisari11Department of Clinical Microbiology and Immunology,Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv,Israel2Department of Physiology and Pharmacology, SacklerFaculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Background: Tumor ablation is a nonsurgical techniquethat is used to eradicate solid tumors. This strategyresults in stimulating anti-tumor immunity against dis-tant tumor cells by exposing the body to large amountsof tumor antigen. Pulsed Electric Current Tumor Abla-tion (PECTA) is an intratumoral treatment developed inour lab which destroys the tumor tissue, mainly byforming low and high pH areas and free radicals for-mation. In this study we examined the effects ofPETCA treatment on primary tumor destruction, tumorrecurrence, stimulation of anti-tumor immunity and sur-vival. Moreover, we examined the ability to promotethe ant i - tumor immune response by ei ther theimmunoadjuvant, CpG, or the suppressor cells inhibi-tors; APCP (CD73 inhibitor), Sildenafil (PDE-5inhibitor) and Cimetidine.Methods: Tumors of breast adenocarcinoma (DA3), or fibro-sarcoma (BLC25) cells, in Balb/c mice, were treated by

PECTA or surgery. PECTAwas applied by intratumoral elec-trodes delivering 75–100 coulombs per electrode per cm3 oftumor tissue (C/E/cm3). The efficacy of ablation and the de-velopment of anti-tumor responses were evaluated by survivalmonitoring and challenging the mice with secondary tumorcells.Results: The charge of 100 C/E/cm3 was found as optimal formaximal elimination of primary tumors inDA3model, where-as in BLC25 the optimal charge was 75 C/E/cm3. In addition,DA3 bearing mice cured by PECTA combined with CpG,APCP or Sildenafil were more resistant to the growth of atumor cell challenge and survived longer than mice treatedby surgery.Conclusions: The findings indicate that PECTA efficientlyeradicates breast adenocarcinoma and fibrosarcoma tumors.Furthermore, when combined with CpG, APCP or Sildenafilthe treatment stimulates a protection against secondary tumorand distant metastases development and thus improves thesurvival.

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Modulation of the HLA Peptidome of Cancer Cellsby Cytokines

Liran Komov, Eilon Barnea, Arie AdmonDepartment of Biology, Technion-Israel Institute of Technolo-gy, Haifa, Israel

The human leukocyte antigen (HLA) class I presentationof peptide to T cells is a part of the immunosurveillancemechanism that enables the elimination of infected anddiseased cells by the immune system. One of the hall-mark of cancer is down-regulation of genes involved inthe HLA class I expression such as TAP1/2 andLMP2/7. This is caused by the immunosuppressive tu-mor microenvironment and hinders immune response,thus contributing to the survival of the tumor cells. In-terferons (IFNs) are a family of immune stimulatorycytokines, which up-regulates many components of theantigen presenting pathway, this way increasing both thequantity and the diversity of peptides presented by theHLA class I and therefore induce a strong immune re-sponse that can be directed against the cancer cells. Theproblem with the use of IFNs for cancer immune ther-apy relates to their high toxicity. Identifications of IFNsinduced HLA peptides can be exploited to combine spe-cific peptide immunization with low concentrations ofIFNs treatment of cancer patients.Our research strategy is based on treating human cancercell lines with IFN-β or IFN-γ and characterizing thechanges induced in the HLA class I peptidomes.

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Treating the cells with IFN-β and IFN-γ affected dif-ferently the cells’ proteome and HLA peptidome andinduce the presentation of many unique HLA peptides.Therefore, treating the cells with the specific IFNs cantrigger the HLA presentation of tumor specific antigenswhich are potential candidates for development of can-cer vaccines.

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Cooperative Pathways Regulating Metastasis:The Inflammatory Cytokine Tumor Necrosis Factorα Brings the Evil Out of Rasα

Tal Leibovich-Rivkin1, Yulia Liubomirski1, Tsipi Meshel1,Anastasia Abashidze1, Daphna Brisker1, Hilla Solomon2,Varda Rotter2, Miguel Weil1, Adit Ben-Baruch11Cell Research and Immunology, Tel Aviv University, Tel Aviv,Israel2Molecular Cell Biology, Weizmann Institute of Science,Rehovot, Israel

Breast cancer is a multi-factorial disease in which interactionsbetween tumor microenvironment (TME) and tumor cells dic-tate disease development and progression.In this study we determined the relative contribution of in-flammatory cytokines highly expressed in TME of breast can-cer patients, tumor necrosis factor α (TNFα) and interleukin1b (IL-1β), vs. intrinsic oncogenic alterations in Ras and p53to malignant processes of the breast.To this end, we used two model systems. In the first model ofnon-transformed cells we found that oncogenic RasG12Vcould not induce expression of pro-cancerous chemokinecluster in the absence of cooperation with down-regulatedp53 activities, and that TNFα and IL-1β had a stronger effecton the acquisition of the malignancy phenotype than alter-ations in the intrinsic cellular components.In the second model of breast tumor cells we found that incontrast to the non-transformed cells, RasG12V induced thepro-malignant angiogenic chemokine CXCL8 without needfor accompanying down-regulation of p53. Moreover, TNFαand IL-1b synergized with RasG12V, together amplifyingCXCL8 expression. WT-Ras, which is the prevalent form ofRas in breast cancer patients, was not active in promotingCXCL8; however, TNFα stimulation turned WT-Ras into anactive and oncogenic entity and the joint activities of TNFα+WT-Ras have led via MEK activation to increased CXCL8release. The additive effects of TNFα+Ras, mediated byNF-kB and AP-1, increased angiogenesis and elevated dis-semination of tumor cells to lymph nodes.Overall, our findings propose that in breast cancer pa-tients, where TME is enriched with TNFα, the cytokine

may rescue the oncogenic potential of WT-Ras, togetherleading to a more aggressive disease. These findings shedlight on novel therapeutic strategy targeting TME to treatbreast cancer patients.

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Breast Cancer: Co-Inflammatory Roles of TGFβ areRevealed in the Presence of TNFα and AmplifyPro-Tumoral Traits of Mesenchymal Stem/StromalCells

Shalom Lerrer1, Alexander Bott2, Yulia Liubomirski1, NinoOren1, Tsipi Meshel1, Stefan Wiemann2, Adit Ben-Baruch11Department of Cell Research and immunology, George S.Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv,Israel2Devision of Molecular Genome Analysis, German CancerResearch Center (DKFZ), Heidelberg, Germany

Accumulating evidence substantiates potent pro-malignancyroles for bone marrow-derived mesenchymal stem/stromalcells (MSC) in breast cancer. In parallel, the breast tumormicroenvironment (TME) is enriched with inflammatory cy-tokines such as TNFα, having causative roles in promotingdisease course. The breast TME also possesses manyimmune-suppressive properties that promote tumor growthand progression, including the cytokine TGFβ.In this study, we investigated the impact of the inflammatorybalance between TNFα and TGFβ on pro-malignancy traitsof MSC and their functional implications in dictating breastcancer progression. Based on microarray analysis, that wasvalidated at the protein level, we have identified genes thatare significantly and prominently up-regulated by both TNFαand TGFβ. Focusing on those genes known as having tumor-promoting activities, we found that TGFβ amplified the abil-ity of TNFα to induce the expression of tumor-promotingmediators in MSC. Our findings propose that TAK1 is anup-stream master-regulator involved in the activation ofNF-κB, p38, JNK and SMAD-3, and that the joint activitiesof TNFα+TGFβ are channeled to activation of p65, then in-ducing the up-regulation of the tumor-promoting genes.By applying the conditioned-media of stimulated-MSC tobreast cancer cells, we examined the effects of MSC-derivedsecreted factors on cancer cell-remodeling and motility. Ourresults indicate that following combined stimulation of MSCby TNFα+TGFβ, the MSC-secreted factors amplify migrato-ry and invasive properties of breast tumor cells.Our findings indicate that cytokines with seemingly opposingroles, TNFα and TGFβ, act together to promote inflammatoryand pro-malignancy traits in MSC and breast tumor cells, andreveal inflammation-supporting activities for TGFβ. In future

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studies, we will investigate inflammation-MSC interactions,determining their functional implications on disease course.

P57

Myeloid Derived Suppressor Cells (MDSCs): theKeyto Understanding Chronic Inflammation and itsComplications in Cancer

Yaron Meirow, Julia Rifman, Nira Twaik, Hadas Ashkenazi,Kerem Ben-Meir, Hana Boocholez, Ivan Mikula, LeonorDaniel, Lynn Wang, Michal BaniyashThe Lautenberg Center of Immunology, The Hebrew Univer-sity Medical School, Jerusalem, Israel

Chronic inflammation is typical to various chronic diseasesand is associated with immunosuppression. In such condi-tions, a variety of inflammatory factors induce the accumula-tion of highly suppressive myeloid-derived suppressor cells(MDSCs), thus manipulating the host’s immune functionand suppressing the innate and adaptive arms of the immunesystem. The immunosuppression is reflected by down-regulation of CD247 expression in T and NK cells, resultingin their dysfunction. Such chronic inflammation-induced im-munosuppressive conditions are also found in many caces ofcancer, generating a suppressive—MDSC enriched tumormicro- and macro-environments. MDSCs act as critical bar-riers inpreventing beneficial anti-tumor responses and severe-ly reducing the efficacy of anti cancer therapies. Our researchfocuses on the mechanisms underlying the biology ofMDSCssuch as chemokine receptors, epigenetics and exosome pro-duction in the various MDSC-related complications. We dem-onstrate that the environment generated under non-cancerousand cancerous chronic inflammatory settings is harmful to thehost, leading to a broad spectrum ofcomplications:1) Imparedimmune responses and increased susceptibility to oportunisticifections. 2) Deleterious changes in the cross-talk between gutinflammation and micro-biota, predisposing the host to colo-rectal cancer development. 3) Injurious changes in bone ho-meostasis leading to bone lose.Understanding the function of MDSCs at the molecularlevels and the physiological and immunological conse-quences could offer new and novel candidate immunetargets for therapeutic interventions. Combination ofsuch treatments with chemotherapy and other conven-tional approaches in cancer targeted therapy, may pre-vent chronic inflammation and MDSC associated com-plications and increase treatment’s efficacy. A betteridentification of environmental and tumor-specific in-flammatory mechanisms will allow directing the clinicalmanagement of cancer toward a more personalizedmedicine.

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Eosinophils Promote Colorectal Cancerthrough Expression of S100a8 and S100a9

Hadar Reichman1, Michal Itan1, Chen Varol2, MetsadaPasmanik-Chor3, Ariel Munitz11Department of Clinical Microbiology and Immunology, TelAviv University, Tel Aviv, Israel2Research Center for Digestive Tract and Liver Diseases, TelAviv SouraskyMedical Center & Sackler Faculty of Medicine,Tel Aviv University, Tel Aviv, Israel3Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

Eosinophils are bone marrow-derived cells that have beenlargely implicated in Th2-associated diseases. However, re-cent data highlight key roles for eosinophils in non-classicalTh2 settings (e.g., metabolism, thermogenesis and tissue re-generation). Quite surprisingly despite the fact that the gastro-intestinal (GI) tract is the largest eosinophil reservoir in thebody, the roles of GI eosinophils have been largelyunderstudied especially in chronic GI inflammation and sub-sequent tumorigenesis.We now report that eosinophils are a bona-fide cellular com-partment of the tumor microenvironment in colorectal cancer(CRC). Substantial eosinophilic infiltration was observed inthree independent models of CRC, representing the genetical-ly driven and inflammation-driven CRCmodels (i.e., Apcmin/+

model and AOM+DSS treatment, respectively), as well as incolonic orthotopic injection of a tumor epithelial cell line.Eosinophil recruitment was accompanied with decreasedblood eosinophilia, suggesting active recruitment to the colon.AOM+DSS-treated eosinophil-deficient mice (ΔdblGATAmice) displayed decreased epithelial cell proliferation, de-creased collagen deposition and decreased expression of ma-trix metaloproteinases and TNF-a. Consequently, tumor loadwas dramatically reduced in the colons of ΔdblGATA mice.Microarray analysis of primary colonic eosinophils sorted atvarious stages of the tumorigenic process (naïve-, inflammato-ry-, tissue repair- and tumor associated-eosinophils) revealeddynamic regulation of colonic eosinophil mRNA expressionduring carcinogenesis. Interestingly, the pro-tumorigenic andclinically relevant genes s100a8 and s100a9 were strikinglyand kinetically increased in colonic eosinophils (250-fold and90-fold, respectively). Indeed, local and systemic expression ofs100a8 and s100a9 were nearly diminished in AOM+DSS-treated ΔdblGATA mice, and re-constituted upon adoptivetransfer of eosinophils into the colon.These data establish a key and unforeseen pro-tumorigenicrole for eosinophils in CRC. Furthermore, our results suggestthat eosinophils can promote carcinogenesis via expressionand secretion of s100a8 and s100a9.

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P59

IFNγ in the Tumour Microenvironmentas a Potential Senescence Inducer and EpigeneticModifier

Milan Reinis1, Veronika Pollakova1, Georg Michlits1, IvanStepanek1, Sona Hubackova2, Oleksandr Korolov1, JanaSimova1, Marie Indrova1, Veronika Hruskova1, Jiri Bartek2,Zdenek Hodny21Laboratory of Immunology, CCP, Institute of Molecular Ge-netics of the ASCR, v.v.i., Prague, Czech Republic2Laboratory of Genome Intergrity, Institute of Molecular Ge-netics of the ASCR, v.v.i., Prague, Czech Republic

IFNγ, a crucial cytokine mediating anti-tumour immunity, alsoexerts direct effects on tumour cells; some of them may be re-lated to oxidative stress. First, we focused on a potential role ofIFNγ as an epigenetic modifier. We have investigated the mech-anisms by which IFNγ restores expression of the antigen-presenting machinery genes, located in the MHC genomic locus(TAP-1, TAP-2, LMP-2, LMP-7), in several MHC class I-deficient murine tumour cell lines. Activation of these genesand MHC class I upregulation on the cell surface was stronglyassociated with DNA demethylation and increased histone H3acetylation in the promoter regions of the APM genes. So far,our data suggest that this IFNγ-induced DNA demethylation isan active process. In another set of experiments, we demonstrat-ed in vitro that Th1 cytokines IFNγ and TNFα were able toinduce cellular senescence in B16 melanoma but not in TC-1tumour cells. This cytokine-induced senescence was associatedwith ROS production and NOX4 expression in B16 cells in-duced via the TGFβ/SMAD pathway. Collectively, our resultsdemonstrate some novel mechanisms by which IFNγ can medi-ate tumour cell interactions with the immune system within thetumour microenvironment and cast more light on the role of theIFNγ signalling pathway in tumour cell proliferation regulation.This workwas supported by the NT 14461 grant from theGrantAgency of the Ministry of Health of the Czech Republic, grantNo. 15-24769S from the Czech Science Foundation (GACR)and by the Institutional Grant (Project RVO 68378050).

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Stabilin-1 Supports Breast Cancer Growth by SilentClearance of SPARC

Vladimir Ryabov1,5, Shuiping Yin1, Bin Song1, Aida Avdic2,Kai Schledzewski2, Alexei Gratchev1,5, Katja Simon-Keller3,Frederick Pfister3, Alexander Marx3, Bernd Arnold4, SergijGoerdt2, Julia Kzhyshkowska1,5

1Institute for Transfusion Medicine and Immunology, MedicalFaculty Mannheim, Ruprecht-Karls University of Heidelberg,Mannheim, Germany2Department of Dermatology, Medical Faculty Mannheim,Ruprecht-Karls University of Heidelberg, Mannheim,Germany3Department of Pathology, Medical Faculty Mannheim,Ruprecht-Karls University of Heidelberg, Mannheim,Germany4Division of Molecular Immunology, German Cancer Re-search Center, Heidelberg, Germany5Laboratory for Translational Cellular and Molecular Bio-medicine, Tomsk State University, Tomsk, Russia

Stabilin-1 is a multifunctional scavenger receptor expressedon alternatively-activated macrophages. Stabilin-1 mediatesphagocytosis of “unwanted-self” components, transcytosis,intracellular sorting, and endocytic clearance of extracellularligands including SPARC known to modulate breast cancergrowth. Recently, the expression of stabilin-1 was found ontumor-associated macrophages (TAM) in several types ofmouse and human cancers such as melanoma, lymphoma,glioblastoma, and pancreatic insulinoma. Despite its tumor-promoting role in mouse models of melanoma and lymphomathe expression and functional role of stabilin-1 in breast cancerwas unknown. In this study we demonstrated that stabilin-1 isexpressed on TAM in human breast cancer biopsies, and itsexpression is most pronounced on stages I and II of disease.Uisng stabilin-1 knock-out mice we showed that stabilin-1facilitates primary tumor growth in mouse TS/A mammaryadenocarcinoma. Functional endocytosis assay on ex vivoTAM demonstrated that attenuated tumor growth in stabilin-1 knockout mice was associated with impaired SPARC clear-ance by TAM. Affymetrix microarray analysis of TAM puri-fied fromwild type and stabilin-1 knockout mice, and reporterassays in stabilin-1 expressing cell lines demonstrated that theabsence of stabilin-1 did not have significant effect on TAMtranscriptional programs or induction of intracellular signal-ling. We concluded that stabilin-1 affects tumor growth bysilent clearance of extracellular tumor growth-regulating fac-tors such as SPARC. Silent clearance function of stabilin-1makes it an attractive candidate for delivery of immunomod-ulatory anti-cancer therapeutic drugs to TAM.

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Surgical Excision of a Primary Tumor EnhancesSpontaneous Metastasis of Breast CancerThrough COX-2 and β-adrenergic Pathways

Lee Shaashua1, Ronit Satchi-Fainaro2, Erica Sloan3,Shamgar Ben-Eliyahu1

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1Department of Psychobiology, Sagol School of Neuroscience& School of Psychological Sciences, Tel-Aviv University, Tel-Aviv, Israel2Department of Physiology and Pharmacology, SacklerSchool of Medicine, Tel-Aviv University, Tel-Aviv, Israel3Department of Drug Discovery Biology, Monash Institute ofPharmaceutical Sciences, Monash University, Melbourne,Australia

Evidence suggests that the surgical removal of a primary tumorin cancer patients elicits processes that promote the outbreak ofpre-existing micrometastases and the initiation of new metasta-ses. Indeed, the peri-operative period has been suggested to bepivotal in determining long-term cancer outcomes, despite of itsrelatively short duration in the course of cancer progression.Recent findings have pinpointed peri-operative surgical stressresponses and inflammation as potential pro-metastatic media-tors. Specifically, the excess secretion of catecholamines andprostaglandins was shown to suppress immunity and to affectthe tumor and its microenvironment to acquire pro-metastaticcharacteristics. In this study we aimed at assessing the simulta-neous blockade of catecholamines and prostaglandins duringthe perioperative period, as well as elucidating underlyingmechanisms. To this end, we injected orthotopically theMDA-MB-231 breast cancer cell line to nude mice. Primarytumor and metastatic progression were monitored in vivoemploying bioluminescent imaging. Once metastatic foci weredetected, the primary tumor was excised, and half of the micewere subjected to laparotomy, simulating a more extensive sur-gical procedure. Additionally, mice were treated peri-operatively for 30 h with both a β-adrenergic blocker and aCOX-2 inhibitor, or with vehicle. Our results indicate that anextensive surgical procedure significantly increases metastasis,and that our combined drug treatment completely abolish thisdeleterious effect of surgery. We are now examining the role ofnatural killer (NK) cells in this model, as surgery was shown toreduce their cytotoxicity through elevating catecholamines andprostaglandins, and as NK-depletion in nude mice significantlyelevated tumor lung colonization as well as the growth ofestablished micrometastases. Based on these and previous find-ings, a preliminary clinical trial is now conducted.

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Involvement of Heparanase in the Pathogenesisof Pancreatitis: Potential Therapeutic Target

Preeti Singh1, Iyad Khamaysi2, Neta Ilan1, IsraelVlodavsky1, Hoda Awad3, Niroz Abu-Saleh3, YehudaChowers2, Zaid Abassi3,41Cancer and Vascular Biology, Rappaport Faculty of Medi-cine, Technion-Israel Institute of Technology, Haifa, Israel

2Department of Gastroenterology, Rambam Health CareCampus, Haifa, Israel3Department of Physiology, Rappaport Faculty of Medicine,Technion-Israel Institute of Technology, Haifa, Israel4Research Unit, Rambam Health Care Campus, Haifa, Israel

Heparanase (Hpa) is the sole mammalian endoglycosidasethat selectively degrades heparan sulphate. Extensivelystudied for its capacity to promote cancer progression,heparanase has been recently implicated as an importantdeterminant in several inflammatory disorders as well.Present study examine whether Hpa is involved in thepathogenesis of cerulein-induced experimental acute pan-creatitis (AP) in mice. AP is defined as an acute inflam-matory process developing within the pancreatic glandwith lesser or greater involvement of adjacent tissuesand/or other organs.Heparanase over-expressing transgenic mice (hpa-TG), theirwild-type (wt) BALB/c mice and heparanase knockout mice(hpa-KO), their wt C57BL mice were intraperitoneallyinjected with Cerulein (50 mg/kg, 5 times, at 1 h apart) orvehicle. Cerulein-induced pancreatitis in wt mice was associ-ated with significant rise in serum levels of amylase and li-pase, accompanied by enhanced heparanase activity and in-flammation in the pancreas. The elevation in amylase andlipase levels as well as pancreatic edema/inflammation in re-sponse to cerulein, were profoundly exaggerated in hpa-TGvs. wtmice. In contrast severity of pancreatitis was attenuatedin hpa-KO mice as compared with their wt controls.Heparanase over-expressing mice showed increased infiltra-tion of neutrophils and increased expression of cathepsin Land phospho IκB in pancreas as compared to wt mice, aftercerulein administration. Importantly, pretreatment withPG545 (400 μg, 1-day prior to cerulein), a potent inhibitorof heparanase enzymatic activity, resulted in pronounced ame-lioration of inflammatory response. Altogether, results clearlydemonstrate an important role of heparanase in the pathogen-esis of AP. The protective effect of heparanase inhibition pro-vides a rational basis for therapeutic application of heparanaseinhibitors (i.e., PG545) in AP and possibly other inflammatorydisorders.

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The Role of the IL-22/IL-22R1 Axis in PancreaticDuctal Adenocarcinoma

Morad Zayoud1, Victoria Marcu-Malina1, Dikla Atias2,Chani Stossel2, Talia Golan3, Itamar Goldstein11Sheba Cancer Research Center; Chaim Sheba Medical Cen-ter, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv,Israel

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2Institute of Oncology, Sheba Medical Center, Ramat Gan,Israel3Institute of Oncology, ShebaMedical Center, Sackler Facultyof Medicine, Tel Aviv University, Tel Aviv, Israel

Pancreatic ductal adenocarcinoma (PDAC) remains one ofthe most lethal types of cancer with poor prognosis de-spite extensive efforts. JAK-STAT3 signaling plays a sig-nificant role in the development and progression of pan-creatic cancer. IL-22 is a cytokine, which belongs to theIL-10 family, acts via activation of Jak/Stat3-dependentsignaling cascades and is a well-described growth factorfor epithelial cells.To study the role of IL-22 in the tumor microenvironment ofPDAC patients, focusing on the reciprocal interactions be-tween IL-22-producing lymphocytes and PDAC cells we an-alyzed the following parameters: the expression of IL-22RA1chain on both primary tumor cells isolated from ascites ofPDAC patients and various PDAC cell lines; the pro-proliferative effect of IL-22 on these various PDCA cells in-vitro; the capacity of ascites-derived supernatants from meta-static PDAC patients to polarize naïve T cells, in vitro, to-wards the Th22 phenotype; and detection by intracellularstaining the percentage of IL-22 secreting lymphocytes inthe ascites of metastatic PDAC patients.We found high expression of IL-22RA1 on all the primaryPDAC cells and cell lines tested coupled with a significantincrease in their proliferation after the addition of IL-22 tothe culture medium. Moreover, isolated mononuclear cellsfrom PDAC patients’ ascites showed higher percentage ofIL-22+ lymphocytes, and importantly factors found in PDACascites induced significant increase on the polarization of na-ïve T cells into Th22 cells.In summary, we show that IL-22 has a positive effect on thegrowth of PDAC that uniformly express the IL-22RA1, whilereciprocally the PDAC environment contains factors that canpotentially instruct tissue infiltrating lymphocytes to producethe pro-proliferative and immunosuppressive cytokine IL-22to create a positive feedback loop.

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Research of Cytokine Expression Pattern whenEndometrial Cancer Co Culture with Stroma

Jo Hantae, Churl K. MinBiological Sciences, Ajou University, Suwon, South Korea

Recently tumor micro-environment study has researchvery much. cancer usually origin epithelial cells. so can-cer surrounded by stroma cell, immune cells. And cancergive and take signals such as growth factor, chemokine,

cytokines. These action called ‘cross-talk’. cancer crosstalk with stroma, normal epithelial cell, and even immunecell. It provide cancer has more malignancy. so we didcytokine assay via endometrial cancer line co culture withendometrial stroma cells. and we perform Reverse tran-scription PCR. our result show that cytokine from co-culture with endometrial cancer and endometrial stromarelease more than culture alone. and another pattern ofexpression observed in co-culture medium.

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Detection of Epidermal Growth Factor Receptor(EGFR) Mutations in Circulating Tumor DNAduring EGFR-Tyrosine Kinase Inhibitor(EGFR-TKI) Treatment

Young-Chul Kim1, Cheol-Kyu Park1, In-Jae Oh1, Hyun-JuCho1, Yoo-Duk Choi2, Kye-Young Lee3, Tae-Won Jang41Lung Cancer Clinic, Pulmonology, Chonnam National Uni-versity Hwasun Hospital, Hwasun, Jeonnam, South Korea2Pathology, Chonnam National University Hwasun Hospital,Hwasun, Jeonnam, South Korea3Pulmonology, Konkuk University Hospital, Seoul, Seoul,South Korea4Pulmonology, Kosin University Gospel Hospital, Pusan, Pu-san, South Korea

BackgroundKnowledge of EGFR mutation status is a crucial step to selectthe best treatment in first line as well as second line settingafter the development of acquired resistance. Obtaining Tu-mor tissue or cytology samples are not always available, andrebiopsy is not easy to perform. Recently, studies showed thatcirculating tumor DNA can be used as a suitable substitute formutation analysis. We compared the sensitivity of EGFR mu-tation detection techniques from tumor tissue and circulatingtumor DNA in patients being treated with EGFR-TKIs.MethodsWe collected plasma samples before treatment with EGFR-TKI and after acquired resistance in 11 patients with activatingEGFRmutations (5 cases with exon 19 deletion and 6 cases ofL858R) in DNA extracted from paraffin-embedded tissuesusing PNA Clamp EGFR Mutation Detection kit (Panagen,Korea). The extraction of circulating tumor DNA (CtDNA)from plasma was performed using QIAamp circulatingnucleic acid kit (Qiagen, Germany). EGFR mutation analysisfor CtDNA was performed by pyrosequencing and PANAMutyper R EGFR kit (Panagene, Korea).ResultsThe EGFR mutation detection sensitivity of pyrosequencingfrom CtDNA was 33.3 %, and that of PANA Mutyper was

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72.7 %. The degree of agreements between tissue DNA andCtDNA were better in PANA Mutyper (k=0.429, p=0.033)than pyrosequencing (k=0.194, p=0.087). After the develop-ment of acquired resistance, same EGFR mutations were de-tected in 63.6 % by PANA Mutyper from CtDNA. One ofthem showed newly developed T790M mutation.ConclusionPANA Mutyper test were better than pyrosequencing in thesensitivity and the strength of agreement between CtDNA andtissue DNA. The detection sensitivity of T790M resistancemutation from CtDNA should be validated with more sensi-tive technique.

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Host Response to Chemotherapy InducesMesenchymal Stem Cells Activity TowardsPreserving Cancer Stem Cells Niche

Michael Timaner, Ruslana Kotsofruk, Ofrat Beyar-Katz,Chen Rachman, Dror Alishekevitz, Shiri Davidi, MadeleineRose Benguigui, Yuval ShakedDepartment of Cell Biology and Cancer Science, The BruceRappaport Faculty of Medicine, Technion-Israel Institute ofTechnology, Haifa, Israel

One of the main obstacles in clinical oncology is that tumorsacquire resistance to therapy leading to their relapse and out-growth. Numerous studies suggest that a subset of tumor cellsat the tumor microenvironment, called cancer stem cells(CSCs) can substantially resist conventional therapy and wasfound to play a crucial role in initiating tumorigenesis andmetastasis. In addition to intrinsic tumor resistance to therapy,growing body of literature shows that the host may also con-tribute to therapy resistance by generating pro-tumorigenicand pro-metastatic effects. In this study, we focused on mes-enchymal stem cells (MSCs), since they were found to con-tribute to therapy resistance. MSCs home to the treated tumorsand secrete a variety of factors which protect tumor cells fromeffects of chemotherapy. We hypothesized that MSCs maycontribute to tumor resistance to therapy by preserving theCSC niche therefore allowing tumor re-growth and expansion.Our results show that MSCs massively home to PANC1 (pan-creatic adenocarcinoma) tumors following Gemcitabine che-motherapy, and reside in close proximity to CSCs. MSCsexposed to Gemcitabine promote PANC1 CSC enrichmentand viability in a paracrine mechanism. Furthermore,Gemcitabine-activated MSCs promote resistance to therapyand accelerate tumor growth, when co-implanted in mice withpancreatic cancer cells, in comparison to tumors co-implantedwith naïve MSCs. The factors mediating the protumorigenicactivity of MSCs are currently studied, with the goal to further

develop drugs which can block activated MSCs thereby im-proving chemotherapy outcome. Overall, our study provides apossible mechanism which can explain how MSCs promotetumor resistance to therapy, and also widens our understand-ing of the changes occurred in the tumor microenvironmentfollowing therapy.

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MicroRNA Expression Analysis in a Modelof Osteosarcoma Dormancy Reveals NovelRegulators of Osteosarcoma Progressionand Tumor-Host Interactions

Galia Tiram1, Ehud Segal1, Rony Shreberk-Hassidim1,Shiran Ferber1, Paula Ofek1, Taturo Udagawa2, Liat Edry3,Noam Shomron3, Maayan Roniger4, Batsheva Kerem4, YuvalShaked5, Sarit Aviel-Ronen6, Iris Barshack6, MarceloCalderon7, Rainer Haag7, Ronit Satchi-Fainaro11Department of Physiology and Pharmacology, SacklerSchool of Medicine, Tel Aviv University, Tel Aviv, Israel2Vertex, Vertex Pharmaceuticals, Cambridge, Massachusetts,USA3Cell & Developmental Biology, Sackler School of Medicine,Tel Aviv, Israel4Department of Genetics, The Life Sciences Institute, EdmondJ. Safra Campus, The Hebrew University of Jerusalem, Jeru-salem, Israel5Department of Molecular Pharmacology, Rappaport Facultyof Medicine, Technion-Israel Institute of Technology, Haifa,Israel6Department of Pathology, Sheba Medical Center, TelHashomer, Israel7Institut für Chemie und Biochemie, Freie Universität Berlin,Berlin, Germany

The presence of dormant, microscopic cancerous lesions pos-sesses a major obstacle for the treatment of metastatic andrecurrent cancers.While it is well-established that microRNAsplay a major role in tumorigenesis, their involvement in tumordormancy has yet to be fully elucidated. We developed a hu-man osteosarcoma dormancy model of a pair of cells originat-ing from the same parental tissue; one that remains avascularand non-palpable a year following inoculation into mice andanother that generates vascularized palpable tumors 1 monthfollowing inoculation. Using this model of cell lines generat-ing dormant or fast-growing osteosarcomas, we identifiedthree novel regulators of osteosarcoma dormancy: miR-34a,miR-93 and miR-200c. This is the first time to show that lossof these three microRNAs occurs during the switch from dor-mant avascular into fast-growing angiogenic phenotype. Fur-thermore, we validated their downregulation in patients’ tumor

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samples compared to normal bone. Reconstitution of thesemicroRNAs into Soas-2 and MG-63 cells, which generatefast-growing osteosarcomas, reduced the levels of their targets,MET proto-oncogene, hypoxia-inducible factor 1α, andmoesin, critical to cancer angiogenesis and cancer cells’ mi-gration. We further demonstrate that these miRNAs attenuatethe angiogenic capabilities of fast-growing osteosarcomain vitro and in vivo. Moreover, treatment with each of thesemicroRNAs using our novel polyglycerol dendritic nanocarriersignificantly prolonged their dormancy period. Taken together,these findings suggest that miR-34a, miR-93 and miR-200chave a key role in osteosarcoma progression, and provide therationale for the development of novel diagnostic and thera-peutic tools for osteosarcoma and other malignancies.

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Monitoring Changes in Morphological Parametersof Primary and Metastatic Melanoma Cell-Linesfollowing Exposure to Taxol by Digital HolographicMicroscopy

Itay Barnea1,2, Pinhas Girshovitz1, Rafi Korenstein2, NathanShaked11Department of Biomedical Engineering, Tel Aviv University,Tel Aviv, Israel2Department of Physiology and Pharmacology, Tel Aviv Uni-versity, Tel Aviv, Israel

Fast determination of the sensitivity of cancer cells towardschemotherapeutic agents is of high importance for effectivechoice of the chemotherapeutic treatment. We suggest a newapproach for monitoring various morphological parameters ofthe cancer cells to a chemotherapeutic agent, by using digitalholographic microscopy (DHM). DHM or interferometricphase microscopy can be used to simultaneously record thequantitative spatial profiles of both the amplitude and thequantitative phase of mostly transparent biological samples.The quantitative phase profile is proportional, at each pixel, tothe product of the cell thickness and its refractive index. Theaccuracy of this product is sub-nanometric. Thus, DHM pro-vides label-free quantitative information on various conven-tional and new parameters related to the cell morphology andintracellular content.We compared the morphological changes induced by taxol ina primary (WM-115) and metastatic (WM-266-4) melanomacell lines isolated from the same patient. Dose dependent via-bility of both cell lines was determined following 48 h expo-sure to taxol by XTT assay. Cell viability following exposureto 50nM taxol was determined by employing PI and YO-PRO®-1 probes, at shorter exposure periods of 1–6 h. In par-allel, we used DHM to measure the change in the

morphological parameters of the two cell lines. The resultsindicate that melanoma cells that were exposed to taxol un-dergo morphological changes including in the area, thicknessand dry mass of the cells. The original morphological param-eters and their taxol-induced changes were different for theprimary and metastatic cells.These findings demonstrate the possibility of DHM tomonitordifferential sensitivity of primary and metastatic cell lines to-wards the toxicity of taxol, solely based on label-free, quanti-tative imaging.

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Prevention of Metastasis Developmentby RGD-Bearing PGA-Paclitaxel Nanoconjugate:Adjuvant Treatment for Breast Cancer

Anat Eldar-Boock1, Joaqiun Sanchis2, Ruth Lupu3, Maria J.Vicent2, Ronit Satchi-Fainaro11Department of Physiology and Pharmacology, SacklerSchool of Medicine, Tel Aviv University, Tel Aviv, Israel2Polymer Therapeutics Laboratory, Centro de InvestigaciónPríncipe Felipe, Valencia, Spain3Mayo Medical Laboratories, Mayo Clinic, Rochester, MN,USA

Prevention of metastasis growth presents an unmet clinicalneed. Anti-angiogenic therapy might provide an alternativeway to manipulate cancer, yet it did not materialize into clin-ical practice. Therefore, combination of anti-angiogenic ther-apy with cytotoxic therapy directed to the metastatic cancercells, offers a promising therapeutic approach. Paclitaxel(PTX) is a widely-used potent cytotoxic drug, however, itsuse is limited by severe side effects, caused by the hydropho-bic drug and its solubilizing agents.We designed and synthesized a novel polyglutamic acid(PGA)-PTX-E-[c(RGDfK)2] nanoconjugate. Polymer conju-gation converted PTX to a water-soluble macromolecule,which passively targeted the tumor tissue exploiting the en-hanced permeability and retention (EPR) effect, while extrav-asating via the leaky tumor neovasculature. PGA isenzymatically-degradable by cathepsin B, leading to PTX re-lease. The E-[c(RGDfK)2] serves as an active targeting toαvβ3 integrin. Integrins play a key role in cell matrix interac-tions. The highly restricted integrin αvβ3 is overexpressed ontumor endothelial and some epithelial cells, during tumorgrowth, invasion, and metastasis. PGA-PTX-E-[c(RGDfK)2]displayed a potent anti-angiogenic therapy. Mice bearingorthotopic mammary tumors demonstrated preferential tumoraccumulation of the RGD-bearing conjugate, leading to en-hanced antitumor efficacy and a marked decrease in toxicitycompared with free PTX[1].

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We used a mouse model that mimics the clinical setting, of mam-mary cancer metastases following resection of the primary tumor.Integrin αvβ3 expression was detected on circulating mCherry-labeled cancer cells ofmice. Adguvant treatmentwith PGA-PTX-E-[c(RGDfK)2] conjugate prevented metastases formation.Taken together, our conjugate alters the pharmacokinetics offree PTX. Inclusion of an active targeting moiety to integrinexpressing-cells, have the potential to prevent breast cancermetastasis development as an anti-angiogenic and anticanceradjuvant therapy.1. Eldar-Boock et al. :2011;32(15):3862–74.

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Cancer-Associated Fibroblasts: Co-Migratingfrom Primary to Metastatic Tumor Sites

Nour Ershaid, Yael Raz, Neta ErezDepartment of Pathology, Sackler School of Medicine, TelAviv University, Tel Aviv, Israel

Breast tumors are characterized by an extensive desmoplasticstroma, abundantly populated by fibroblasts. Cancer-associated fibroblasts (CAFs) are the most abundant cell typein tumor stroma. CAFs are an essential component of thetumor microenvironment involved in many processes thatpromote tumor progression, including growth promoting,pro-angiogenic and pro-inflammatory signaling. In addition,CAFs were shown to support cancer cell invasion and metas-tasis via their extracellular matrix remodeling capacities.However, a direct active participation of CAFs in supportingthe dissemination of cancer cells to secondary organs andsupporting metastatic colonization is still largely obscure.We show that fibroblasts from mammary tumors are capable ofco-migrating with disseminated tumor cells to the lungs and areincorporated into ensuing pulmonary metastases. We utilizednovel multi-transgenic mouse models of spontaneous breast car-cinoma and lung metastasis (MMTV-PyMT) combined withtransgenic mice in which fibroblasts are genetically labeled withfluorescent reporter genes, thus allowing their unbiased trackingand isolation during tumor progression and metastasis. Byorthotopic transplantations of fluorescently labeled tumors wewere able to detect CAFs derived from primary tumors in lungmetastases. We plan to characterize the functional role of co-migrating CAFs by analyzing the transcriptional changes thatmigrating CAFs undergo in the process.Wewill seek to uncoverspecific molecular pathways which contribute to the reciprocalinteractions between migrating CAFs and cancer cells whichserve to promote and enhance the metastatic process.In summary, we were able to uncover a novel role for mam-mary CAFs in which they co-migrate with cancer cells fromthe primary tumor to metastatic sites in the lungs.

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Effects of Selected Organotin Halides on HumanBreast Cancer Cell Line MDA-MB-231 Growthand Migration

Luba Hunakova1, Margita Sulikova1, Julius Brtko21Department of Oncology, Cancer Research Institute, Brati-slava, Slovakia2Department of Molecular Endocrinology, Institute of Exper-imental Endocrinology, Bratislava, Slovakia

Background and aims. Organotins represent a group of or-ganic pollutants with potent endocrine-disrupting properties.The aim of the study was to assess the cytotoxicity of tribu-tyltin chloride (TBT-Cl), tributyltin bromide (TBT-Br), tribu-tyltin iodide (TBT-I), and non-halide tributyltin hydride (TBT-H) in human triple-negativeMDA-MB-231 cell line as well astheir ability to influence migration of cancer cells.Methods.MTTassay and the INCUCYTE™Kinetic ImagingSystem were used to measure cytotoxicity of tested com-pounds and growth characteristics of cells. The migration as-say used the 96-well WoundMaker to generate a cell-free zonein amonolayer of cells with subsequent cell imaging set to scanthe experiment every hour using “Scratch Wound” experimenttype. CD44 expression was determined by Flow cytometry.Results. The IC50 values determined by MTT assay revealedthe toxicity order of tested endocrine-disrupting organic pollut-ants, which was confirmed also by observation of growth inhi-bition based on high-quality phase-contrast imaging confluenceassessment. Their cytotoxicity was in this order: tributyltin chlo-ride ≥ tributyltin bromide ≥ tributyltin iodide tributyltin hydrid.TBT-Cl was able to slow migration of MDA-MB-231 cells,which was accompanied by the surface CD44 down-regulation.Conclusions.Our work suggests that observed cytotoxicity oftributyltin halides could depend on type of halogen atom in theorganotin molecule and TBT-Cl may modulate the metastaticproperties of MDA-MB-231 cells.This work was supported by the APVV-0160-11, VEGA2/0080/15 and VEGA 2/0171/14 as well as by RfL2012 pro-gram funded by the Cancer Research Foundation, Slovakia.

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The Clinical Utility of FDG PET-CT in Evaluationof Bone Marrow Involvement by Lymphoma

Ho Young Kim1, JI Sun Song21Hemato-Oncology, Hallym University Medical Center, An-yang-si, Gyeonggi-do, South Korea

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2Pathology, Catholic Kwandong University International ST.Mary’s Hospital, Incheon Metropolitan City, South Korea

Purpose:Bone marrow biopsy is a standard method for the evaluationof bone marrow infiltration by lymphoma; however, it is aninvasive and painful procedure. Fluorodeoxyglucose positronemission tomography–computed tomography (FDG PET-CT)is a noninvasive imaging technique with the potential to detectbone marrow involvement by lymphoma.Materials and Methods:We retrospectively reviewed medical records of lymphomapatients. All patients were examined by FDG PET-CT andiliac crest bone marrow biopsy for initial staging work-up.Results:The study population comprised 94 patients (median age,60 years; 56 males) with Hodgkin’s lymphoma (n=8) ornon-Hodgkin’s lymphoma (n=86). Maximum standardizeduptake values on the iliac crest of patients with lymphomainfiltrated bone marrow were significantly higher than thoseof patients with intact bone marrow (2.2±1.2 g/mL vs. 1.3±0.4 g/mL; p=0.001). The calculated values for FDG PET-CTduring evaluation of bone marrow involvement were as fol-lows: sensitivity 50 %, specificity 96 %, positive predictivevalue 80 %, negative predictive value 85 %, and positive like-lihood ratio (LR+) 11.7. The value of LR+ was 16.0 in patientswith aggressive subtypes of non-Hodgkin’s lymphoma (NHL).Conclusion:FDG PET-CTcould not replace bone marrow biopsy due to thelow sensitivity of FDG PET-CT for detection of bone marrowinfiltration in lymphoma patients. Conversely, FDG PET-CThad high specificity and LR+; therefore, it could be a usefultool for image-guided biopsy for lymphoma staging, especiallyfor aggressive subtypes of NHL. In addition, unilateral bonemarrow biopsy could be substituted for bilateral bone marrowbiopsy in lymphoma patients with increased FDG uptake onany iliac crest.

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High Systemic VEGF-C Levels Lead to a SpecificNeutrophil Accumulation in the Lung and OromoteMetastasis in a Syngeneic Rat Breast Cancer Model

Sandra Klusmeier1,2, Luca Quagliata1,2, Melanie Rothley1,2,Diana Plaumann-Ziegler1,2, Wilko Thiele1,2, Jonathan P.Sleeman1,21Institute of Toxicology & Genetics, Karlsruhe Institute ofTechnology, Eggenstein-Leopoldshafen, Germany2Centre for Biomedicine &Medical Technology, Medical Fac-ulty Mannheim University of Heidelberg, Mannheim,Germany

VEGF-C (Vascular endothelial growth factor-C) and itsr e c e p t o r V EG FR - 3 a r e k e y r e g u l a t o r s o flymphangiogenesis. Tumors that express VEGF-C caninduce lymphangiogenesis peritumorally. Enhancedperitumoral lymphatic vessel density induced by VEGF-C is thought to increase the probability that invasivetumor cells enter the vasculature, disseminate and formmetastases. Nevertheless, we have found high levels ofVEGF-C in blood from breast cancer patients comparedto healthy volunteers, and here provide evidence thattumor-derived VEGF-C may not only act locally but alsosystemically to promote metastasis. By intravenouslyinjecting recombinant VEGF-C or adeno-associated vi-ruses that induce VEGF-C expression (AAV-VEGF-C),we artificially increased the circulating levels of VEGF-C in healthy experimental animals to mimic the situationin breast cancer patients. This led to an accumulation ofCD11b+ myeloid cells in the lungs, but not liver orspleen. These CD11b+ cells virtually all co-expressedGr-1 and Ly6G, identifying them as neutrophils. Accord-ingly, enhanced circulating VEGF-C levels resulted inincreased numbers of neutrophils in the blood, with aconcomitant reduction in the number of circulating lym-phocytes. In the lungs, VEGF-C-induced myeloid cellclusters localized to the periphery of terminal bronchi-oles, sites associated with pre-metastatic niche formationand lung metastasis. Pre-conditioning of experimental an-imals with daily injections of recombinant VEGF-C for5 days prior to injection of poorly metastatic breast can-cer cells strongly stimulated lung metastasis formation.Furthermore, in a highly metastatic VEGF-C-secretingbreast cancer model, neutrophilia in the circulation wasobserved, and clusters of CD11b+ myeloid cells accumu-lated in the pre-metastatic lung. Together these data sug-gest that increased systemic VEGF-C induces neutrophilaccumulation in the lung, thereby contributing to the for-mation of metastatic niches and the development of lungmetastases.

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Facilitation of Breast Cancer Cell Engraftmentby Human Mesenchymal Stromal Cells in Modelof Experimental Metastasis

Lucia Kucerova1, Svetlana Skolekova1, Lucia Demkova1,Miroslava Matuskova1, Michal Mego21Department of Molecular Oncology, Cancer Research Insti-tute SAS, Bratislava, Slovakia22nd Department of Oncology, Faculty of Medicine, Comeni-us University and National Cancer Institute, Bratislava,Slovakia

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Increasing body of evidence has confirmed stromalinfuence on cancer stem cells (1–3). In our work we fo-cused on evaluation of the effect of human mesenchymalstromal cells (MSC) on breast cancer cells (BCC) in directadherent cocultures and 3D models in vitro. We examinedalso the role of the MSC on BCC engraftment in experi-mental metastatic model in vivo. In direct cocultures wedetected cell fusions, but these cells were not able to prop-agate. Direct cell cocultures were evaluated by flow cy-tometry, viability assays and kinetic assays to determinethe alterations provoked in BCC by the MSC (4). Impor-tantly, MSC and BCC coinjection resulted in higher en-graftment of the BCC in mouse lungs as detected by bothimmunohistochemistry and PCR. Moreover, we haveestablished the protocol for the immunomagnetic separa-tion of the circulating tumor cells (CTC) from the breastcancer patients and evaluated the capability of these cellsto initiate xenograft growth and metastatic capabilitiesin vivo on SCID mouse model. Based on the models withthe BCC, we have coinjected the CTC and MSC to exam-ine whether the MSC facilitated engraftment of patient-derived CTC in vivo. The data and outcomes from theseexperiments will be presented. Taken together we intend toexploit the established models for further exploration ofthe critical signals, which facilitate engraftment of the me-tastasis initiating cells.1. Pattabiraman DR, Weinberg RA. Nat Rev Drug Discov.2014 Jul;13(7):497–512.2. Kucerova L, Zmajkovic J, Toro L, Skolekova S, DemkovaL, Matuskova M. Cancer Microenviron. 2015 Apr;8(1):1–14.3. Kucerova L, Skolekova S. Neoplasma. 2013;60(1):1–10.4. Kucerova L, Skolekova S, Matuskova M, Bohac M,Kozovska Z. BMC Cancer. 2013 Nov 9;13:535. doi:10.1186/1471-2407-13-535.

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PHOX2B: a Regulator of Micro-Metastasisin Neuroblastoma

Osnat Naftali, Tsipi Meshel, Shelly Maman, Orit Sagi-Assif,Ravit Ginat, Isaac P. WitzCell Research and Immunology, Tel Aviv University, Tel Aviv,Israel

An orthotopic mouse model for human neuroblastoma metas-tasis, comprising local and metastatic variants originatingfrom single tumors, was developed in our lab. Inoculation ofthese variants into a new set of nude mice generated two typesof variants: lung macro and micro-metastatic cells, the latternot generating overt lung metastases. In previous work, thesecells were characterized for tumorigenic and metastatic

abilities, indicating a more malignant phenotype of themacro-metastatic cells.PHOX2B is a transcription factor used as a minimal residualdisease marker in neuroblastoma patients. Higher expressionlevels of PHOX2B were identified through qRT-PCR andwestern-blot analyses in micro as compared to macro-metastatic neuroblastoma cells.Having a common genetic background, we hypothesizedthat an epigenetic event had led to the differential expres-sion of PHOX2B in the micro and macro-metastatic neu-roblastoma cells. Indeed, examination of the methylationpattern of the PHOX2B promoter revealed that 25 CpGdinucleotides in the promoter region are 31 % more meth-ylated in the macro than in the micro-metastatic cells. Fur-ther on, luciferase assay examining 1100 bp region of thePHOX2B promoter, showed that in vitro methylation ofthat region lowered transcription by 93 %. These resultsstrongly imply that hyper-methylation could be the causeof PHOX2B lack of expression in the macro-metastaticcells.Larger primary tumors were seen in mice inoculatedwith the micro-metastatic variant where PHOX2B wasdown-regulated, and 20 fold more human cells weredetected in the lungs and bone marrow of these mice.These results strongly support the hypothesis postulatingthat PHOX2B can function as an inhibitor of tumorprogression and metastasis.

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Predicive Factors of Early Recurrence after CurativeSurgery in Stage 1 and 2 PulmonaryAdenocarcinoma

Sookwhan SungThoracic Surgery, Seoul St Mary Hospital, Seoul, SouthKorea

Back groundAlthough surgery is the most effective treatment of early stagenon-small cell lung cancer, postoperative recurrence has beenoccurred occasionally. The purpose of this study is to find thepredictive factors for early recurrence after curative surgery instage I and II pulmonary adenocarcinoma.MethodsBetween 2011 and 2013, 279 patients underwent cura-tive surgery for stage I or II pulmonary adenocarcino-ma. Early recurrence was defined as recurrence within2 years after surgery. Recurrence-free survival and clin-icopathological factors associated with recurrence wereanalysed using the Kaplan-Meier method and Coxpropotional hazards model.

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ResultsOf the 279 patients, 30 patients experienced early recurrence.Locoregional recurrence was 19 cases and distant recurrencewas 11 cases. Mean follow up duration was 760.9 days. 3-years recurrence free survival was 88.4 % in stage I and64.0 % in stage II. Multivariate analyses showed thatmicropapillary component (5 % of tumor) was significantlyassociated with early recurrence (p=0.043, HR 3.110), espe-cially locoregional recurrence (p=0.006, HR 7.406).ConclusionMicropapillary component had a significant effect on earlyrecurrence especially locoregional recurrence after curativesurgery for pulmonary adenocarcinoma.

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The Role of OncostatinMReceptor Over-Expressionin TumourMicroenvironment of Cervical SquamousCell Carcinoma

Valtteri Tulkki, Justyna Kucia, Maria Caffarel, CinziaScarpini, Nick ColemanDepartment of Pathology, University of Cambridge, Cam-bridge, UK

An important feature of cervical squamous carcinogene-sis is genomic instability caused by deregulated expres-sion of human papillomavirus oncogenes in proliferatingepithelial cells. One common change is gain of the re-gion of chromosome 5p that encodes, amongst others,the Oncostatin-M receptor (OSMR; located at 5p13.1).OSMR is often over-expressed in cervical SCC and thisover-expression is associated with a significantly worseclinical outcome (1). Cervical SCC cells that over-express OSMR show enhanced responsiveness to its ma-jor ligand Oncostatin-M (OSM), which in turn, mediatesmultiple pro-malignant effects, including a pro-angiogenic phenotype and increased cell migration andinvasiveness (2). Furthermore, OSM induces expressionof genes associated with hypoxia, epithelial to mesenchy-mal transition, metastasis and immune cell recruitment(2). In vivo, OSM promotes lung colonisation andgrowth of OSMR over-expressing cervical SCC cells inmouse xenografts (unpublished data). We are currentlystudying the effects of OSMR over-expression on theSCC tumour microenvironment in vivo as well as theinteractions between OSMR over-expressing cells andmacrophages in vitro, to ultimately better understandthe activation of pro-tumorigenic hypoxic responses andtheir role in immune cell recruitment, angiogenesis andepithelial to mesenchymal transition.

1. Ng G, et al. (2007) Gain and overexpression of theoncostatin M receptor occur frequently in cervicalsquamous cell carcinoma and are associated with ad-verse clinical outcome. Journal of Pathology 212:325–34.2. Winder DM, et al. (2011) Overexpression of theoncostatin M receptor in cervical squamous cell carci-noma cells is associated with a pro-angiogenic pheno-type and increased cell motility and invasiveness. Jour-nal of Pathology 225:448–623. Caffarel MM, Coleman N. (2014) Oncostatin M receptor isa novel therapeutic target in cervical squamous cell carcino-ma. Journal of Pathology 232:386–90

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Photoactivation of a Nanobiocomplex: a NovelPhotodynamic Therapy of Cutaneous Melanoma

Avraham Dayan1, Gideon Fleminger1, Osnat Ashur-Fabian21Department of Molecular Microbiology and Biotechnology,Tel Aviv University, Tel Aviv, Israel2Department of Human Molecular Genetics and Biochemis-try, Tel Aviv University, Tel Aviv, Israel

Cutaneous melanoma (CM) is the most aggressive and dead-liest form of skin cancer worldwide.The cytotoxic effect of photo-excited titanium dioxide(TiO2) by UV illumination, creating reactive oxygenspecies (ROS), has been examined in several cancermodels in vitro. However, serious damage to the sur-rounding healthy tissue limits the applicability of thisapproach. Our group has discovered a unique proteinthat strongly binds TiO2. This protein, dihydrolipoamidedehydrogenase (DLDH), is critical for energy and redoxbalance in the cell. It has been reported in the literaturethat ROS are generated as a result of its oxidative ac-tivity. CM cancer cells overexpress the cell surface re-ceptor avb3 integrin, which interacts with proteins of theextra cellular matrix (ECM) through an RGD (Arg-Gly-Asp) recognition site. We bio-engineered the humanDLDH with RGD tails on both sides (DLDH-RGD2),thus generating a protein capable of serving as a bridgebetween the integrin expressing cancer cells and TiO2

n a n o s t r u c t u r e f o r m s . A c t i v a t i o n o f t h i snanobiocomplex by UVA illumination produces asynergistic toxic effect leading to enhanced cancercell death (Fig 1).We believe that the understanding gained from thiswork will also be beneficial to other integrin-expressing tumor models that have not been tested sofar.

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Protease-Degradable Polymeric NanomedicineBearing Paclitaxel and a Turn-On Fluorescent Probefor Cancer Theranostics

Yana Epshtein1, Hemda Baabur-Cohen1, Orit Redy-Keisar2,Einat Kisin-Finfer2, Doron Shabat2, Ronit Satchi-Fainaro11Physiology and Pharmacology, Sackler School of Medicine,Tel Aviv, Israel2Exact sciences, School of Chemistry, Tel Aviv, Israel

The tumor microenvironment plays a crucial role in cancerprogression and metastasis as well as in the way cancer re-sponds to therapy. In particular, it is known that several lyso-somal Cysteine proteases such as Cathepsin B, which partic-ipate in ECM degradation, are overexpressed in some cancertypes.Early detection of cancer and real-time monitoring of therapyresponse could provide a paramount opportunity for individ-ualized therapy. Among different imaging modalities, opticalimaging holds several advantages, although clinically avail-able fluorescence-based imaging agents suffer from manylimitations.

Theranostics is a relatively new term, introduced in2002 (1), that describes any material for applicationscombining both therapy and diagnostics. Combining anactivatable, fluorescent imaging probe with a therapeuticagent on a delivery platform such as polymers therebyforms a theranostic nanomedicine. This novel precisionnanomedicine may overcome the fluorescence imagingprobe limitations while providing real-time diagnosticand monitoring of drug release.In our study, we designed and characterized a noveltheranostic nanomedicine based on our widely studied, non-toxic polymeric nanocarrier Polyglutamic Acid (PGA) bear-ing the chemotherapeutic drug paclitaxel (PTX) and Self-Quenched Cyanine NIR fluorophore (SQ-Cy5) as a diagnosticprobe. PGA backbone is biodegradable by cathepsin B. Oursystem enabled site-specific drug release concomitantly withthe activation of the fluorophore to its Turn-ON state. Further-more, PGA-PTX-SQ-Cy5 conjugate inhibited the prolifera-tion, migration and formation of tubular-like structures of en-dothelial and different types of cancer cells.Our platform described here can be an appropriate candidateto be used as research tool for real-time, intravital, deep tissuemonitoring and may have clinical utility as a potentialtheranostic nanomedicine.1. S. Ferber et al., Cancer letters, (Mar 12, 2014).

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The Secreted Protein Isthmin Selectively TriggersApoptosis in Cancer Cells and Cancer EndothelialCells through Cell-Surface GRP78 MediatedMitochondria Targeting

Mo Chen Jiajie Wu Ruowen GeDepartment of Biological Sciences, National University ofSingapore, Singapore, Singapore

Isthmin (ISM) is a secreted protein of 60 kDa that inhibitsangiogenesis and induces endothelial cell apoptosis. Cell-surface glucose-regulated protein of 78 kDa (GRP78) is ahigh-affinity receptor for ISM, mediating its pro-apoptoticfunction. Through screening a large number of human cancercell lines, we found that the cell-surface GRP78 level corre-lates with the invasiveness and metastatic potential of the can-cer cell line. Correspondingly, ISM selectively induces apo-ptosis only in cancer cells that harbor high level cell-surfaceGRP78. Hence, ISM is a dual targeting agent, targeting bothcancer cells and the cancer stromal endothelial cells.Mechanistic investigation revealed that ISM-GRP78 interac-tion initiates clathrin-dependent endocytosis and ISM inter-nalization. The internalized ISM-GRP78 complex are co-targeted to mitochondria via endosomes. Inside mitochondria,ISM blocks ATP release to cytosol through interacting withADP/ATP carriers on the mitochondria inner membrane, caus-ing mitochondrial dysfunction without affecting its membraneintegrity. We discovered a novel protein transport pathwayfrom extracellular environment to mitochondria. We demon-strate that this pathway is not unique to ISM, and other secret-ed antiangiogenic proteins may also use similar pathways toreach mitochondria and induce apoptosis.

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Targeting Pancreatic Ductal Adenocarcinomawith a Polymeric Nanocarrier and an AnticancermiRNA Polyplex

Hadas Gibori, Shay Eliyahu, Ronit Satchi-FainaroPhysiology and Pharmacology, Tel Aviv University, Tel Aviv,Israel

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggres-sive malignancy and currently the fourth leading cause ofcancer death in the United States. The high mortality and poorprognosis are due to extensive metastasis and lack of earlydiagnostic markers and symptoms. In spite of advances inchemo therapies, the 5-year survival rate is still less than

5 %, indicating the ineffectiveness of current approaches totreatment.MicroRNAs, after being demonstrated as deregulated in can-cer, are now being explored as therapeutic targets for cancertreatment. Reversion of miRNA expression to normal levelscan restore perturbed cellular homeostasis. Importantly, theheterogeneity and complexity of cancer suggests that the onlyway to successful treatment might lie with the simultaneoustargeting of multiple genes, further emphasizing the therapeu-tic potential of miRNAs. miR-34a, a master regulator of tumorsuppression, is downregulated in numerous cancers includingPDAC and inhibits malignant growth by repressing oncogenicgenes.Efficient delivery of miRNA for therapeutic purposes is ex-tremely challenging due to low cellular uptake, RNases deg-radation and rapid renal clearance. Therefore, we synthesizeda novel polymeric nanocarrier to deliver microRNAs to tu-mors and their vasculature. Our nanocarrier was able to com-plex electrostatically with miR-34a and form therapeuticallyactive nano-scaled polyplexes. The polymer enhanced the in-ternalization of miR-34a in its active form into human PDACcells, as the miR was capable of downregulating four of itsdirect target genes. miR-34a delivered by the nanocarriercould also inhibit growth, clonogenicity and cell cycle ofPDAC cells. Systemic administration of polymer-siRNAnanoplexes to orthotopically-inoculated pancreatic tumorsshowed no toxicity and accumulated selectively at the tumorsite warranting its potential as a novel therapeutic for PDAC.Key words: microRNA, pancreatic cancer, polymer therapeu-tics, nanomedicine.

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Tumor Microenvironment-TargetedBacteriochlorophylls as Theranostic Agentsfor Triple Negative Breast Cancer

Rachel Hamri1, Lilach Agemy1, Tamar Yechezkel1, RonnyUzana1, Alexander Brandis1, Yoram Salomon2, AvigdorScherz11Department of Plant Science, Weizmann Institute of Science,Rehovot, Israel2Department of Biological Regulation, Weizmann Institute ofScience, Rehovot, Israel

New compounds comprising light activated bacteriochloro-phyll (Bchl) conjugates with the tri-peptide Arg-Gly-Asp(Bchl-RGD, e.g., STL-6014) are shown to target several cellpopulations in the tumor microenvironment of triple negativebreast cancer. The compounds uptake enables imaging, prog-nosis, primary tumor eradication and provocation of anti-tumor immunity.

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STL-6014, selectively accumulates in the primary tumor andlung metastases of murine 4T1 mammary and human MDA-MB-231 breast cancer tumors in mouse models and resides inthe tumor necrotic domains for 9 days. FACS analysis, NIRfluorescence microscopy and co-administration of Bchl freeRGD analogues, showed integrin dependent uptake by cancerand endothelial cells, cancer associated fibroblasts (CAFs),myeloid-derived suppressor cells (MDSC), and tumor-associated macrophages (TAMs).Within each cell population,high, low and null STL-6014 uptakes were observed. Thetumor cell composition was modified following STL-6014administration. TAMs and neutrophils numbers were elevat-ed, while the population of cancer cells was reduced at 24 hpost administration and then proliferated at a slower rate.Continuous i.v loading of STL-6014 retarded tumorgrowth. β3 integrin’s expression correlated well with thepercentage of high uptake cell population in each subset,supporting active transport mechanism. Drug uptake andaccumulation is mediated by recruited neutrophils that ag-gregate with tumor associated platelets. Illumination of 4T1tumors by NIR diode laser (15 min, 755 nm) at 6 h postSTL-6014 administration resulted in a complete ablation ofthe treated tumors in 63 % of the animals 21 days laterthrough necrotic and apoptotic pathways. Preliminary re-sults using combinations of immune modulators and PDTprovide indications for increased animal survival throughsystemic, anti-metastatic effect.In conclusion, STL-6014 provides new means for imagingand treatment of TNB cancers in both the local and metastaticsettings.

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Oncolytic Rhabdoviruses ExpressingImmunomodulatory miRNA Enhance Anti-TumourEfficacy

Victoria Jennings1,2, Carolina Ilkow1,2, Brian Keller1,2, Lau-ra Evgin1,2, Thersea Falls1,2, Meaghan Boileau1,2, IsabelleSimonne St-Hilaire1,2, Donald Bastin1,2, Harry Atkins1,2, JohnBell1,21Cancer Therapeutics, Ottawa Hospital Research Institute,Ottawa, ON, Canada2Department of immunology and microbiology, University ofOttawa, Ottawa, ON, CanadaBackground: Tumour-induced immunosuppressive mecha-nisms in the tumour microenvironment (TME) is a major rea-son for the limited current success of therapeutic cancer vac-cines. Reversing this immunosuppressive milieu will be criti-cal to generate effective anti-tumour immunity. Oncolytic vi-ruses (OV) specifically replicate in and lyse tumour cells. In

addition, OV stimulate the immune system and facilitate thegeneration of anti-tumour immune responses.MiRNAs are small endogenous non-coding RNAs implicatedin the post-transcriptional control of gene expression. Modu-lating the activity of miRNAs provides opportunities for novelcancer interventions. However, low bioavailability and poorcellular uptake are major challenges for delivering miRNAmimetics specifically to cells within theTME.We hypothesise that expressing immunomodulatorymiRNAs from OVs will lead to enhanced anti-tumourimmune response by targeting immunosuppressive ele-ments within the TME.Results: Specific 22 nt mature miRNA sequences targetingkey immunosuppressive molecules, or a control miRNA se-quence targeting GFP were constructed into the pre-miR30cassette and cloned into oncolytic Rhabdoviruses, VSVΔ51andMG-1. Here, we show that the mature 22 nt sequences areefficiently processed from the pre-miR30 cassette followingVSVΔ51/MG-1 infection of melanoma and ovarian cancercells, without impairing cytotoxicity or replication. Moreover,in vitro knockdown of immunosuppressive molecules in tu-mour cells and M2 macrophages are observed following in-fection. Intravenous delivery to B16-F10 tumour-bearingmice resulted in knockdown of predicted targets, in both theTME and within the spleen. This led to reduced tumour bur-den and an increased anti-tumour T cell response compared tocontrol treated mice.Conclusion: Expression of immunomodulatory miRNA fromoncolytic Rhabdoviruses results in knockdown of immuno-suppressive targets in both the TME and spleen leading toenhanced anti-tumour immunity and reduced tumour burdenof B16-F10 bearing mice.

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Phase 2 Trial of Pexa-Vec (pexastimogenedevacirepvec; JX-594), an Oncolyticand Immunotherapeutic Vaccinia Virus, in Patientswith Metastatic, Refractory Renal Cell Carcinoma(RCC)

Seong-Geun Kim1, Tae-Ho Hwang21Hemato-Oncology, Pusan National University School ofMedicine, Yangsan, Gyeongnam, South Korea2Clinical Pharmacology, Pusan National University School ofMedicine, Yangsan, Gyeongnam, South KoreaBackground: Pexa-Vec is a vaccinia virus engineered to ex-press granulocyte-macrophage colony stimulating factor(GM-CSF), thereby stimulating direct oncolysis, tumor vas-cular disruption and anti-tumor immunity (Nat Rev Cancer2009). Pexa-Vec was shown to replicate in metastatic tumors

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following intratumoral (IT) or intravenous (IV) administration(Lancet Oncol 2008; Nature 2011).Methods: Treatment-refractory RCC patients received fiveweekly intravenous Pexa-Vec infusions. Starting at Week 6,patients exhibiting disease control or otherwise clinicallybenefitting from Pexa-Vec treatment could continue to receiveIV Pexa-Vec infusions every 3 weeks. The primary objectiveof the study was to determine the radiographic response ratebased on modified Response Evaluation Criteria in Solid tu-mors (RECIST) 1.0. Secondary objectives include diseasecontrol rate, progression free survival and safety.Results: Seventeen (17) RCC patients having failed at leastone prior VEGF/R-targeted therapy were enrolled. Allpatients received the initial 5 weekly Pexa-Vec infu-sions. Twelve patients (71 %) went on to receive atleast one additional infusion (median Pexa-Vec infu-sions=8; range 5–12). The treatment regimen waswell-tolerated. Transient influenza-like illness (n=17;100 %), asthenia (n=8; 47 %), anemia (n=5; 29 %)and nausea (n=5; 29 %) were the most common ad-verse events. All patients were evaluable radiographical-ly at Week 6. The RECIST response rate was 6 % (1partial response which was classified as a complete re-sponse at Week 36). The RECIST disease control ratewas 76 % at Week 6.Conclusions: Pexa-Vec was well-tolerated and associatedwith one complete RECIST response and 76% disease controlat Week 6 in patients with advanced RCC. Further trials ofPexa-Vec in RCC patients are warranted.

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Vimocin and Vidapin, Cyclic KTS Peptides, areDual, Partial Antagonists of α1β1/α2β1 Integrinswith Antiangiogenic Activity

Philip Lazarovici1, Momic Tatjana1, Jehoshua Katzhendler1,Ofra Benny1, Efrat Noy2, Hanoch Senderowitz2, JohannesEble3, Cezary Marcinkiewicz41Institute for Drug Research School of Pharmacy, The He-brew University of Jerusalem, Jerusalem, Israel2Department of Chemistry, Bar-Ilan University, Ramat-Gan,Israel3 I n s t i t u t e f o r P h y s i o l o g i c a l C h em i s t r y a n dPathobiochemistry, University of Munster, Munster, Germany4Department of Bioengineering,College of Engineering, Tem-ple University, Philadelphia, USAReciprocal interactions between tumor and stromal cells withthe extracellular matrix (ECM) collagens are involved in can-cer progression and metastasis through interactions withintegrins. Snake venom disintegrins are important tools incancer research because they inhibit integrins with relative

selectivity. The short disintegrins Viperistatin, is a 41 aminoacids polypeptide, containing the characteristic three aminoacid KTS motif, which is present in the loop conformationrequired for integrin binding. This disintegrin binds selective-ly α1β1integrin, in contrast to partial selectivity of RGDdisintegrins. Viperistatin served as lead compound for the syn-thesis of linear and cyclic peptides containing the KTS motifin conformational constraint by cyclization via disulfide brid-ges. Vimocin and Vidapin showed a high potency (IC50=0.17nM) and intermediate efficacy (20 and 40 %) in inhibition ofadhesion of α1/α2 integrin overexpressor cells to respectivecollagens. Vimocin was more active in inhibition of the woundhealing (53 %) and corneal micropocket vascularization(17 %), whereas Vidapin was more potent in inhibition ofmigration in the Matrigel tube formation assay (90 %). Bothcompounds similarly inhibited proliferation (50–90 %) of en-dothelial cells, and angiogenesis induced by VEGF (80%) andglioma (55 %) in the CAM assay. These peptides were nottoxic to endothelial cells, acutely tolerated upon intravenousinjection in mice, and were stable for 10–30 h in human serum.Vimocin and Vidapin can be further exploited for drug devel-opment and may also serve as tools for investigations intoα1β1/α2β1 biological function in angiogenesis and cancer.* P.L. holds the Jacob Gitlin Chair in Physiology, and isaffiliated and partially supported by the Hebrew Universityof Jerusalem Grass Center for Drug Design and Synthesis ofNovel Therapeutics.

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Inhibition of the Pro-Malignancy Chemokine CCL5by “40s Loop Mimetics”

Yaeli Lebel-Haziv1, Tsipi Meshel1, Adva Yeheskel2, AditBen-Baruch11Department of Cell Research and immunology, George S.Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv,Israel2Bioinformatics Unit, George S. Wise Faculty of Life Sciences,Tel- Aviv University, Tel Aviv, Israel

The inflammatory microenvironment of tumors, comprised ofcells, chemokines and cytokines, was recently shown to havea major role in cancer development and progression. The in-flammatory chemokine CCL5 is secreted by tumor cells or byother cells in the tumor milieu and acts as a tumor-promotingfactor in many malignancies. Its pro-cancerous activities aremediated by the binding of CCL5 to glycosaminoglycans(GAGs) and to CCL5 receptors, mainly through positively-charged amino acid sequence of 44-RKNR-47 located at the40s loop of the chemokine. Our overall goal in this study is to

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develop new therapeutic modalities that would prevent can-cerous processes that are based on CCL5 activities.To this end, we have designed a CCL5-loop mimetic, calledherein RKNR-CCL5-peptide. This peptide is a unique inhib-itory technique of CCL5-driven inflammatory conditionswhich is based on the artificial looping of the 40s domain ofCCL5. Preliminary analyses that we have performed indicatethat the RKNR-CCL5-peptide inhibits the binding of WTCCL5 to GAGs and the migration of monocytic cells in re-sponse to GAG-presented WT CCL5 in vitro.Overall, our results provide initial support to the ability of theRKNR-CCL5-pepide to inhibit activities induced by full-lengthWT CCL5. These findings may have clinical relevancein all malignancies in which CCL5 is involved. Therefore, abetter identification of the RKNR-CCL5-peptide inhibitoryeffects is necessary and may set the basis for the future designof innovative strategy to treat malignant diseases.

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Polymer System for Controlled Drug Deliveryand Release of All-Trans Retinoic Acid

Ondřej Lidický1, Milada Šírová2, Tomáš Etrych11Institute of Macromolecular Chemistry, Academy of Sciencesof the Czech Republic, v.v.i., Prague, Czech Republic2Institute of Microbiology, Academy of Sciences of theCzech Republic, v.v.i., Prague, Czech Republic

All-trans retinoic acid (ATRA) is used as an anti-cancer che-motherapeutic to treat patients with acute promyelocytic leu-kemia. Due to ATRA ability to drive differentiation of imma-ture granulocytes into mature ones, ATRAwas also tested as adrug capable of restricting the suppressive capacity ofmyeloid-derived suppressor cells (MDSC) in tumormicroenvironment.Hydrophobicity of ATRA is one of the major disadvantages ofits medicinal use. With the aim to increase solubility and en-able delivery and controlled release of ATRAwithin the placeof action, we have designed and synthesized several water-s o l u b l e p o l y m e r c a r r i e r s b a s e d o n N - ( 2 -hydroxypropyl)methacrylamide copolymers. Various keto-group-containing polymer carriers were prepared and usedfor the attachment of hydrazide derivative of ATRA via pH-sensitive hydrazone bond. This bond is rather stable in neutralpH (pH 7.4, model of blood stream) but it is hydrolyzed inmild acidic condition with pH 5–6 (model of tumormicroenvironment).In vitro tests in a cell line HL-60, corresponding to immaturegranulocytes, showed that hydrazide form of ATRA keeps itsbiological properties. The HPMA-based conjugates, theATRA and its hydrazide derivative induced an increase in

expression of CD11b granulocyte marker on the surface ofthe treated cells and restricted their proliferation in aconcentration-dependent manner as evidenced by 3H-thymi-dine incorporation method and detection of overall viability ofcells by MTT. The preliminary data showed that this conju-gates could be used as an immunomodulator to decrease thesuppressive activity ofMDSC.Moreover, the conjugates werealso able to decrease proliferation of human T cell lymphomaJurkat, murine T cell lymphoma EL4 or murine 4T1 breastcarcinoma cell lines.This work was supported by Czech science foundation (grantNO.P301/11/0325).

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Computational Discovery and ExperimentalValidation of Novel Drug Targetsin Immuno-Oncology

Arthur Machlenkin1, Ofer Levy1, Galit Rotman1, AmirToporik1, Gady Cojocaru1, Yair Benita1, Liat Dassa1, TalFridman1, Ilan Vaknin1, Shirley Sameah-Greenwald1, InbalBarbiro1, Spencer Liang2, John Hunter2, Eyal Neria1, ZuritLevin11Research & Discovery, Compugen Ltd, Tel Aviv, Israel2Antibody R&D, Compugen USA Inc, South San Francisco,USA

The past few years have witnessed a renaissance in thefield of immuno-oncology largely due to the clinical suc-cess in targeting the immune checkpoints CTLA-4 andPD-1. Towards identification of novel immune check-point drug targets we developed a dedicated predictivediscovery platform.The B7/CD28 discovery platform is a predictive modelbased on genomic and protein features along with ex-pression patterns of known B7/CD28 proteins. The plat-form has been tested and validated extensively and hasdemonstrated its validity by identifying non-novel im-mune checkpoints such as TIGIT and VISTA, whichwere not used in the design stage.The B7/CD28 predictive platform was employed to identify11 new immune checkpoint candidates which are currently indifferent validation stages. In this poster, we will describe ourdiscovery approach as well as our validation path. In addition,we will present experimental data demonstrating the immuno-modulatory function and expression patterns of several of ournovel immune checkpoints. These experimental results serveas an additional confirmation to the accuracy of our B7/CD28predictive discovery platform and shed light on the therapeuticpotential of the novel immune checkpoints identified usingthis unique discovery approach.

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Tumor Vessel Normalization through IncreasedShear Stress by Exercise EnhancesChemotherapeutic Efficacy

Keri Schadler1,2, Nicholas Thomas1, Peter Galie4, DonghaBhang1, Kerry Roby1, Christopher Chen3, EugenieKleinerman2, Sandra Ryeom1

1Department of Cancer Biology, The University of Pennsylva-nia, Philadelphia, Pennsylvania, USA2Pediatrics Research, M.D. Anderson Cancer Center Chil-dren’s Cancer Hospital, Houston, Texas, USA3Department of Biomedical Engineering, Boston University,Boston, Massachusettes, USA4Department of Physiology, The University of Pennsylvania,Philadelphia, Pennsylvania, USA

Chemotherapy is the standard of care for most cancersbut has limited efficacy and associated adverse side ef-fects. Targeted therapies are now utilized in combinationwith chemotherapy to improve outcome. Anti-angiogenicagents are targeted therapies that enhance chemothera-peutic efficacy by normalizing tumor vasculature to im-prove delivery of chemotherapy. Tumor vessels are dis-organized and largely dysfunctional, with disrupted bloodflow impeding drug delivery to cancer cells. Althoughpharmacologic anti-angiogenic therapy can remodel tu-mor vessels, there is a limited window of efficacy andthese drugs are associated with severe side effects neces-sitating alternatives for vascular normalization. Shearstress, the force exerted on endothelial cells by bloodflow, regulates vessel sprouting and permeability. In-creasing vascular shear stress through aerobic exercisecan alter blood vessels in normal tissues. We found thatincreasing shear stress in mice using a clinically relevantprogram of moderate aerobic exercise normalized tumorvasculature, increasing the number of functional vesselsand ultimately drug delivery, in melanoma, pancreaticductal adenocarcinoma, and Ewing sarcoma models. Im-portantly, combining exercise with chemotherapy causeda significantly greater decrease in tumor growth thanchemotherapy alone in all tumor models. Further, wedemonstrate that shear stress activates the transcriptionfactor Nuclear Factor of Activated T cells (NFAT) toinduce expression of Thrombospondin-1 (Tsp-1) in endo-thelial cells. Our data indicate that endothelial activationof calcineurin-NFAT-Tsp-1 signaling plays a critical rolein exercise-induced, shear stress-mediated tumor vesselremodeling. Our work suggests that the vascular normal-izing effects of aerobic exercise can be an effective che-motherapy adjuvant.

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microRNA-Based Polymer Therapeuticsfor Combating Glioblastoma

Zohar Shatsberg1, Xuejiao Zhang2, Marcelo Kalderon2,Rainer Haag2, Paula Ofek1, Ronit Satchi-Fainaro11Department of Physiology and Pharmacology, SacklerSchool of Medicine, Tel Aviv University, Tel Aviv, Israel2Institute for Chemistry and Biochemistry, Freie University ofBerlin, Berlin, Germany

Glioblastoma Multiforme (GBM) is one of the most aggres-sive cancers. The median survival with current standard-of-care radiation and chemotherapy is about 14 months. GBMcan be difficult to treat due to heterogeneity in cancer cellpopulation. MicroRNA-based drugs have rapidly become avast and burgeoning field. Besides being potent, microRNA(miR) can be used to target many genes involved in cellularsignaling pathways. In this work, we focused on miR-34a,which is known for its key role in important oncogenic path-ways and its tumor suppression capability. In vivo delivery ofmiR remains a crucial challenge for its therapeutic success. Tobypass these shortcomings, we developed polymeric nanogelswhich are based on a polyglycerol-scaffold.We evaluated the capability of several nanogel deriva-tives (NGs) to encapsulate miR-34a and neutralize itsnegative charge in a dose-dependent manner. A substan-tial silencing and anticancer effect was accomplishedin vitro with our miR-nanogel polyplexes, as measuredby luciferase reporter assay and proliferation assays.Systemic administration of miR-34a nanoplexes to hu-man GBM tumors subcutaneously inoculated in SCIDmice, showed no significant toxicity. We expect that thistherapeutic approach will be the basis for a new prom-ising drug to treat GBM.Key words: microRNA, Glioblastoma, nanogels, polymertherapeutics.

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Stromal Cells as Targets for Image-Guided Surgeryof Breast Cancer

Cornelis F.M. Sier1, Shadhvi Bhairosing1, Marieke Prevoo1,Ronald van Vlierberghe1, Lukas Hawinkels2,WilmaMesker1,Cornelis van de Velde1, Peter Kuppen1, Alex Vahrmeijer11Surgery, Leiden University Medical Centre, Leiden,Netherlands2Gastroenterology, Leiden UniversityMedical Centre, Leiden,Netherlands

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The distinction between malignant and normal tissue is notalways evident. Image-guided surgery (IGS) is a techniqueusing near-infrared fluorescent (NIRF) probes to visualize tu-mor tissue during the operation. NIRF light offers a relativelydeep tissue penetration in combination with low tissue auto-fluorescence. NIRF light is invisible for the human eye anddoes not interfere with the surgeons view. Special camerasystems and software visualize the NIRF signal on a monitor.The ‘contrast agents’ or probes consist generally of a NIRFdye conjugated to an antibody directed against a target protein.Most of the target proteins presently under evaluation for IGSare cell membrane proteins, over-expressed on malignant,epithelial-derived cells. Next to these malignant cells, manycancers consist for a substantial part of stromal cells and ex-tracellular matrix. Stromal cells are a physiological reaction ofthe body and the expression of their cell membrane proteinrepertoire will not directly be influenced by genetic alter-ations, like for malignant cells.To determine whether stromal cells could be an alterna-tive target for IGS of breast cancer, we have evaluatedthe presence of stromal cells in lobular and ductal tumorsusing immunohistochemistry on sequential sections fromparaffin embedded tissues. Lobular tumors consisted for26–72 % of stromal tissue and lobular tumors slightlyless (26–43 %). Antibodies against pan-cytokeratin,vimentin, CD31, CD105, CD45, CD68, CD163, and α-SMA identified the major stromal cell types: endothelialcells, from which approximately 50 % stained withCD31 as well as CD105, indicating a neo-angiogenicstate; immune cells, from which the majority appearedto be type 1 macrophages; and fibroblasts with a sub-stantial part of the activated α-SMA expressing pheno-type. Our results underline the potential of stroma cells,as target for IGS.

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Polymer Therapeutics Rationally-Designedfor a Combination Therapy of Paclitaxeland Doxorubicin

Shelly Soffer, Hemda Baabur-Cohen, Ela Markovsky, RonitSatchi-FainaroDepartment of Physiology and Pharmacology, Sackler Schoolof Medicine, Tel Aviv University, Tel Aviv, Israel

Conjugation of chemotherapeutic drugs to a polymer allowsspecific delivery to the tumor by the enhanced permeabilityand retention (EPR) effect and provides an ideal platform for acombination treatment, since both drugs are given simulta-neously in one injection and share the same pharmacokineticprofile (1, 2).

We have found that the combination of paclitaxel (PTX) anddoxorubicin (DOX) displays a synergistic cytotoxic effect oncancer cell lines, such as MDA-MB-231 human mammaryadenocarcinoma and ES-2 human ovarian carcinoma.PTX and DOX were bound to polyglutamic acid (PGA), awater-soluble, biocompatible polymer, which is biodegrad-able by enzymes overexpressed in tumor tissue, particularlycathepsin B. PTX was bound directly to the PGA backboneand DOX was coupled via an acid-labile hydrazone linker.We show here that PGA-PTX-DOX exhibited a significantanti-proliferative and anti-migratory effect on MDA-MB-231 and ES-2 cells. More importantly, our novel conjugatewas highly effective in inhibiting the growth of mammarytumors inoculated orthotropically into the mammary fat padof nu/nu female mice, compared to combination of free drugsand drugs conjugated to polymer separately. Furthermore,PGA-PTX-DOX had no toxic effects in vivo and did not in-duce immune response in human peripheral blood mononu-clear cells in contrast to the free drugs.Our results with PGA-PTX-DOX nano-conjugate present itspotential use as a novel combination therapy for breast andovarian cancer.Keywords: Polymer therapeutics, nanomedicines, combina-tion therapy, EPR effectReferences1. E. Markovsky et al., J Control Release 161, 446 (Jul 20,2012).2. A. Eldar-Boock, D. Polyak, A. Scomparin, R. Satchi-Fainaro, Curr Opin Biotech in press, (2013).

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Develevopment of a New Therapeutic Avenuefor Basal Cell Carcinoma Eyelid Tumors

Ran Stein, Zeev Dvashi, Asher Milstein, Ayala PollackDepartment of Ophthalmology, Kaplan Medical Center affil-iated to The Hebrew University of Jerusalem, Rehovot, Israel

Purpose: Basal Cell Carcinoma (BCC) is the most commonperiocular malignancy. BCC is usually managed surgically,with subsequent aesthetic and functional implications. Re-cently, innovative treatment with smoothened inhibitors wasthoroughly investigated, but substantial side-effects limit itsuse. As the search for cure continues, the common availableBCC models, conducted by UV or ionizing radiation (IR) inknocked out mice, pose their own limitations. Our study aimsto generate a novel model system of eyelid BCC in mice,through which to investigate the tumor’s microenvironmentand to suggest novel treatments for BCC tumors.Methods:Murine BCC cells were injected into the eye-lids of C57black mice, followed by injection of PBS

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solution, bevasizumab, or anti- platelet-derived growthfactor (PDGF). The mice were monitored for tumorgrowth and sacrificed. The tumors underwent patholog-ical and histological examinations. The cells secretion ofmatrix metalloproteinases (MMPs) was investigated byzymography assay.Results:Murine BCC cells were injected into the eyelidsof C57black mice. The mice were then randomly divid-ed into three groups; control and treated with eitherbevasizumab or anti-PDGF. Tumors were seen in 90,80 and 44 % of the PBS, anti-PDGF and bevasizumabtreated mice, respectively. Bevasizumab decreased therate of tumor progression compared to anti-PDGF andcontrol groups. In the zymography assay tumor necrosisfactor-α (TNFα) demonstrated increase in the secretionof MMP-2.Conclusions:Murine BCC model can be generated by directinjection of tumor cells. Development of BCC is abrogated bydirect bevasizumab injection. In contrast, the use of anti-PDGF does not affect tumor initiation and progression. Tumorinvasiveness is suggested to be related to MMP-2 secretion.The results may indicate bevasizumab as adjuvant treatmentfor BCC tumors.

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Activation of the Prostaglandin E2 EP1 ReceptorAntagonizes the Growth Stimulatory Effectsof Prostaglandin E2, a Component of the CancerMicroenvironment

Mary Taub, Robert Parker, Paramala Mathivanan, UlasKaplan, Muhamad Ariff, Trina RudraBiochemistry, University at Buffalo, Buffalo, New York, USA

Prostaglandin E2 (PGE2) is overproduced in a numberof tumors, where it promotes tumor cell growth, angio-genesis and metastasis. The actions of PGE2 are medi-ated by G protein-coupled E-prostanoid (EP) receptors,including EP1, coupled to Gq, EP2 and EP4, coupled toGs, as well as EP3, coupled to Gi. This report is con-cerned with epithelial derived tumors, and thus, exam-ines the role of EP receptors in mediating the effects ofPGE2 on the growth of a kidney epithelial cell line,MDCK. Our results indicate that activation of EP2 andEP4 by PGE2 results in increased growth, unlike EP1,whose activation is growth inhibitory. Indeed, two EP1antagonists (ONO-8711 and SC51089) stimulate, ratherthan inhibit, growth, an effect that is lost following anEP1 knockdown. ONO-8711 stimulates growth in theabsence of exogenous PGE2, presumably due to its abil-ity to prevent the binding of endogenously produced

PGE2 to EP1. The involvement of Akt and the EGFReceptor (EGFR) in EP1 signaling is indicated by 1)the stimulatory effect ONO-8711 and SC51089 on thephosphorylation of Akt and the EGFR, 2) the ability ofboth MK2206, an Akt inhibitor, and AG1478, an EGFRkinase inhibitor, to prevent the increased growth causedby ONO-8711 and SC51089. Similarly, an EGFRknockdown, and a dominant negative EGFR both pre-vent the increased growth caused by EP1 antagonists.Of particular interest in these regards, our co-immunoprecipitation studies indicate a direct interactionbetween EP1 and the EGFR. These results support thehypothesis that 1) signaling via the EP1 receptor in-volves its interaction with the EGFR and 2) EP1 recep-tor pharmacology can be employed to retard the growthof carcinomas.

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Theranostic Barcoded Nanoparticles

Zvi Yaari, Dana da Silva, Avi SchroederChemical Engineering, Technion-Israel Institute of Technolo-gy, Haifa, Israel

Selecting the proper drug, that addresses each patient’s uniquedisease presentation, is the primary goal of personalized med-icine. We describe here a nanoparticle-based barcoded systemfor predicting the therapeutic potency of drugs against cancer-ous lesions. The screen is performed inside the patient’s bodyusing extremely low drug doses, and grants insights regardingdrug potency with single-cell sensitivity. This approach wastested on BALB/c mice bearing metastatic breast cancer tu-mors (4T1).The diagnostic system is based on 100-nm liposomes loadedwith a drug and a corresponding unique DNA barcode.Once a tumor is detected, a cocktail of DNA-barcoded nano-particles, each containing a different drug, is injected intrave-nously. The particles accumulate in the various cells that com-pose the tumor microenvironment, utilizing the enhanced per-meability and retention (EPR) effect. Two days later, enablingeach of the drugs to take action, a biopsy is taken from thetumor and the tissue is homogenized, to form a single-cellsuspension. The cells are sorted by FACS according to celltype and to their live/dead viability state (potency screen).Then, the DNA barcodes are extracted from the cells andexpanded using RT-PCR. The cell viability data is correlatedwith the type of drug/s found inside each of the cells, therebyidentifying which drug or drug combination is optimal fortreating the lesion.Based on the screen, a treatment protocol was selected,generating a successful outcome in vivo. Interestingly,

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we found that barcodes can be detected even inside asingle cell.This approach, in which nanoparticles act as theranosticgauges for examining the therapeutic potency of a drug ordrug combination may prove effective for personalizing med-icine.

P96

Oral Fluids-Derived Exosomes from Oral CancerPatients as delivering Vehicles of NaturalAnti-Cancer Substances to the TumorMicroenvironment

Ayelet Zlotogorski-Hurvitz1, Dan Dayan1, GavrielChaushu2, Tuula Salo3,4, Marilena Vered1,51Oral Pathology and Oral Medicine, Tel Aviv University, TelAviv, Israel2Oral and Maxillofacial Surgery, Rabin Medical Center,Petah Tikva, Israel3Diagnostics and Oral Medicine, University of Oulu, Oulu,Finland4Oral Pathology, Institute of Dentistry, University of Helsinki,Helsinki, Finland5Institute of Pathology, The Chaim Sheba Medical Center, TelHashomer, Ramat-Gan, Israel

Oral cancer (OC) has a low 5-year survival rate of ~50 %despite advances in molecular biology and improvementsin treatment. The tumor microenvironment (TME) con-tributes to the poor prognosis in OC. Curcumin, a naturalproduct with proved anti-tumor potential, showed encour-aging results in OC preclinical studies; however poor bio-availability limits its clinical efficacy. Exosomes, especial-ly tumor-derived, can be absorbed by neoplastic or TME–related cells, and therefore can be utilized as a new strat-egy for curcumin delivery. Aim: to load curcumin intooral fluids (OF)-derived exosomes and compare betweenhealthy individuals (HI) and OC patients. Methods: 1 mlaliquots of pooled OF from OC patients (n=36) or HI (n=25) were incubated with 40/80 μM curcumin. Exosomalpellets were isolated by ExoQuick-TC™ or ultracentrifu-gation (120,000 g, 90 min X2). Supernatants were sepa-rated from the pellets, which were submitted for sonica-tion. The concentrations of curcumin from the superna-tants and that released from the exosomal pellets werespectrophotometrically assessed (420 nm). Pooled OFfrom OC was assessed by nanoparticle tracking analysis(NTA) before/after incubation with 50 μM curcumin.Results: Curcumin-loaded pellets could not be obtainedfrom microvesicles/exosome-free supernatants. The hy-drophobic ExoQuick-TC™ competed with the exosomes

for binding curcumin, therefore results were inconsistent.OC-pellets were constantly larger than HI-pellets. OC-pellets bound more curcumin than HI-pellets at 80 μM(p0.05). Simultaneously, supernatants of OC-pelletscontained less curcumin than HI-pellets at 80 μM(p0.05). NTA showed larger nanoparticles in OC-OF in-cuba t ed w i th cu rcumin than wi t hou t (p0 .05 ) .Conclusions: Exosomes bound curcumin from a hydro-philic medium such as OF. OC-OF-derived exosomeswere able to load more curcumin than HI-OF-derivedexosomes, thus creating a novel platform to combat bothOC and TME cells.

P97

Concomitant Statins can be the Favorable Factorfor Gemcitabine and Erlotinib CombinationChemotherapy in Advanced Pancreatic Cancer

Seungmin Bang, Hee Seung Lee, Moon Jae Chung, SeungWoo Park, Si Young SongDivision of Gastroenterology, Department of Internal Medi-cine, Yonsei University College of Medicine, Seoul, SouthKorea

Background : Combination chemotherapy of erlotinib withgemcitabine is considered as a standard treatment forunresectable pancreatic cancer. However, which clinical fac-tors are related to the response of this combination is stillunknown. This study was aimed to find out the clinical factorsfor the response of gemcitabine/erlotinib chemotherapy.Materials andMethods : Between Oct., 2006 and Jan., 2014,a total of 180 patients with unresectable pancreatic cancer, whoreceived at least 2 cycles of gemcitabine/erlotinib combinationtherapy as a first line palliative chemotherapy, were included.Medical records including medication histories of statins andaspirins were retrospectively collected and analyzed. For thisstudy, we defined “long-term response” which showed tumorstabilization more than 6 cycles of chemotherapy.Results :Median progression free survival and overall surviv-al were 3.9 months and 8.1 months respectively. In univariateanalysis, liver metastasis (p=0.023) had negative correlationwith “long-term response”. Locally advanced stage (p=0.017), medication history of statins (p=0.01) and CEA 4.5(p=0.029) had favorable effect on “long-term response”. Inmultivariate analysis, medication history of statins was iden-tified the only independently favorable factor for “long-termresponse” (OR 4.11, p=0.017). Prognostic factor for OS andPFS showed a significant correlation with liver metastasis (OS: HR 1.46, p=0.038; PFS : HR 1.50, p=0.013)Conclusion : These results suggest that statins had favorableeffect on “long-term response” in gemcitabine/erlotinib

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chemotherapy for unresectable pancreatic cancer. It would besignificant that statins have the chemoadjuvant role to stabilizetumor growth for long term, as almost equal to median sur-vival times.

P98

Heat Shock Protein 27 as a Biomarker Predictingthe Role of Chemotherapy-Induced Autophagyon Drug Sensitivity

Nancy Gordon1, Janice Santiago O’Farrill1, MarioHollomon2, Eugenie Kleinerman11Division of Pediatrics, UT MD Anderson Cancer Center,Houston, Texas, USA2Biology, Texas Southern University, Houston, Texas, USA

Lung metastasis constitutes the main cause of death inosteosarcoma (OS) patients. We reported that aerosolGemcitabine (GCB) has a significant therapeutic effectagainst OS lung metastases [1]. However, a subset of cellsfailed to respond to GCB resulting in persistent smallisolated lung metastases. Autophagy [2], a catabolic pro-cess involved in cellular homeostasis, can either be pro-tective or promote cell death. Our in vitro and in vivostudies demonstrated that autophagy plays a role in OSresponse to GCB. GCB induces autophagy in several dif-ferent OS cell lines including the human LM7 and CCH-OS-D. Blocking autophagy using Hydroxychloroquineand/or the downregulation of Beclin 1 increased the sen-sitivity of LM7 cells to GCB but decreased the sensitivityof CCH-OS-D cells. This dual role of autophagy has beendescribed in other tumor types. However, predictingwhether blocking autophagy will increase or decreasedrug-sensitivity has not been described. We found thatthe induction of phosphorylated heat shock protein 27(pHsp27) following drug exposure with camptothecin orGCB correlated with the role of autophagy in drug sensi-tivity. Blocking autophagy in cells whose pHsp27 wasincreased following chemotherapy resulted in enhanceddrug sensitivity whereas blocking autophagy in cellswhere pHsp27 was decreased resulted in reduced drugsensitivity. In conclusion, induction of pHsp27 followingchemotherapy predicts whether blocking autophagy willincrease or decrease drug sensitivity.1. Gordon, N. and E.S. Kleinerman, Aerosol therapy for thetreatment of osteosarcoma lung metastases: targeting the Fas/FasL pathway and rationale for the use of gemcitabine. Jour-nal of aerosol medicine and pulmonary drug delivery, 2010.23(4): p. 189–96.2. Chen, N. and J. Debnath, Autophagy and tumorigenesis.FEBS Lett, 2010. 584(7): p. 1427–35.

P99

Molecular Insights into Mechanisms of DrugResistance using Doxorubicin Resistant Hela Cells

Ipsa Jain, Naseer Maliyakkal, Meera Saxena, AnnapoorniRangarajanMolecular Reproduction Development and Genetics, IndianInstitute of Science, Bengaluru, India

Drug resistance poses a challenge to cancer therapy. In thiswork, we have modelled drug-induced changes in cancercells, as observed in the clinical settings, by giving chronicdoxorubicin treatment to cervical cancer derived cell line,HeLa. The cells so obtained are multi-drug resistant. Wefound the drug resistant cells to have higher levels of the drugtransporter ATP Binding Cassette transporter B1 (ABCB1).These cells show low drug retention, which can be reversedusing an ABCB1 inhibitor, suggesting ABCB1 is sufficient toconfer the drug resistance phenotype to these cells. Recentliterature has shown a correlation between drug resistanceand stemness properties of cancer cells. Consistent with this,we observed increased anchorage independent colony forma-tion and have higher levels of Bmi1, a stem cell marker. Bmi1is also known to be involved in maintaining Epithelial to mes-enchymal transition. Accordingly, we found a loss of E-cadherin expression in these cells. Expectedly, these cellsshow more metastatic lung nodules. A microarray analysisperformed between parental HeLa and the drug resistant cellsrevealed changes in integrin and focal adhesion pathways.Integrin β1 (ITGB1) was up-regulated in the drug resistantcells. ITGB1 is reported to sense modified extra-cellular ma-trix in the tumor and lead to activation of Mitogen activatedprotein kinase (MAPK) and Akt signalling pathways, confer-ring drug resistance. Indeed, we found the levels of activatedMAPK and Akt molecules to be up-regulated in drug resistantcells compared to parental cells. This model system allows usto dissect the molecular mechanisms which confer drug resis-tance and cause ABCB1 gene up-regulation.

P100

Regulation of CEACAM1 Protein Expressionin Braf-Mutant Human Metastatic Melanoma Cells

Karin Kfir1,2, Rona Ortenberg1,2, Michal J. Besser1,2, JacobSchachter1, Gal Markel1,2,31Sheba Medical Center, Ella Lemelbaum Institute of Melano-ma Research, Ramat-Gan, Israel2Sackler School of Medicine, Clinical Microbiology and Im-munology, Tel Aviv, Israel

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3ShebaMedical Center, TalpiotMedical Leadership Program,Ramat-Gan, Israel

Melanoma is among the most aggressive types of cancers andis responsible for 80 % of skin cancer–related deathsworldwide.BRAF, a key component of MAPK pathway, is mutated in50–70 % of melanoma cases, with V600E responsible forthe majority of cases.FDA approved BRAF inhibitor achieves high and rapid re-sponse rate. However, secondary resistance develops withinvarying periods of time.CEACAM1 is a transmembrane glycoprotein that protectsmelanoma cells from immune attack. CEACAM1 expressionin primary lesions strongly predicts the development of me-tastasis and poor survival.Our results show strong correlation between BRAF mutationstatus and CEACAM1 expression in melanoma cells derivedfrom patients. Cells treated with MAPK inhibitors (MAPKi)showed down-regulation in CEACAM1 expression, onlyamong BRAFV600E melanoma lines in a dose- and exposuretime-dependent manner. Furthermore, mRNA of CEACAM1decreased following MAPKi treatment, suggesting a tran-scriptional regulation. MAPKi-resistant cell system showedan increase of membrane CEACAM1 expression and mRNA,compared to control. Investigation of CEACAM1 transcrip-tion regulation show significant reduction in pCEACAM1activity following treatment with MAPKi. ETS1 is a down-stream effector of the MAPK pathway and a regulator of thepCEACAM1. Our results indicate a reduction ofpCEACAM1 activity following ETS1 binding site deletionfrom pCEACAM1. ETS1 overexpression enhanced the activ-ity of pCEACAM1 and mutant ETS1T38A showed dominantnegative effect. Thus we hypothesize that CEACAM1 is reg-ulated at transcriptional level through MAPK pathway acti-vated by BRAFV600E mutation via ETS1. Finally, CEACAM1down-regulation by MAPKi rendered the cells more sensitiveto immune attack. These results provide a new view on apotential immunological mechanism of action of MAPKi inmelanoma, as well as on the aggressive phenotype observed indrug-resistant cells.

P101

miR-103 Sensitizes Hemopoietic Tumor Cellsfor Glucocorticoid Induced Apoptosis

Shlomit Kfir-Erenfeld, Noa Haggiag, Nathalie Asherie,Eitan YefenofThe Lautenberg Center for Immunology and Cancer Re-search, The Hebrew University-Hadassah Medical School,Jerusalem, Israel

Glucocorticoid (GC) hormones are an important ingredi-ent of leukemia therapy since they are potent inducersof lymphoid cell apoptosis. However, the developmentof GC resistance remains an obstacle in GC based treat-ment. In the present investigation we used deep se-quencing analysis to detect miRNAs that interplay inthe apoptotic response of leukemic cells to GC. Themost significantly upregulated miRNA having the stron-gest influence on GC induced apoptosis (GCIA) wasmiR-103. Transfection of GC resistant cells with miR-103 sensitized them to GCIA, while miR-103 spongingof GC sensitive cells rendered them partially resistant.miR-103 is encoded within the fifth intron of PANK3gene. We demonstrate that the GC receptor (GR)upregulates miR-103 by direct interaction with GC re-sponse element (GRE) in the PANK3 enhancer. Conse-quently, miR-103 targets the c-Myc activators c-Myband DVL1, thereby reducing c-Myc expression. Sincec-Myc is a transcription factor of the miR-17∼92apoly-cistron, all six miRNAs of the latter are alsodownregulated. Of these, miR-18a and miR-20a are in-volved in GCIA, as they target GR and BIM, respec-tively. Consequently, GR and BIM expression are ele-vated, thus advancing GCIA. Furthermore, miR-103 re-duces the expression of cycline dependent kinase(CDK2) and its cycline E1 target, as well as cellularproliferation. Altogether, this study highlights miR-103as a useful prognostic biomarker and drug for leukemiamanagement in the future.

P102

Daurinol, a Catalytic Inhibitor of TopoisomeraseIIα, Suppresses SNU-840 Ovarian Cancer CellProliferation through Cell Cycle Arrest in S Phase

Ho-Suk OhDepartment of Internal Medicine, Gangneung Asan Hospital,Gangneung, South Korea

Daurinol, a lignan from the ethnopharmacological plantHaplophyllum dauricum, was recently reported to be anovel topoisomerase II inhibitor and an alternative to theclinical anticancer agent etoposide based on a colorectalcancer model. In the present study, we elucidated the de-tailed biochemical mechanism underlying the inhibition ofhuman topoisomerase IIα by daurinol based on a molec-ular docking study and in vitro biochemical experiments.The computational simulation predicted that daurinolbinds to the ATP-binding pocket of topoisomerase IIα.In a biochemical assay, daurinol (10–100 μM) inhibitedthe catalytic activity of topoisomerase IIα in an ATP

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concentration-dependent manner and suppressed the ATPhydrolysis activity of the enzyme. However, daurinol didnot inhibit topoisomerase I activity, most likely becausetopoisomerase I does not contain an ATP-binding domain.We also evaluated the anti-proliferative activity ofdaurinol in ovarian, small cell lung and testicular cancercells, common target cancers treated with etoposide.Daurinol potently inhibited SNU-840 human ovarian can-cer cell proliferation through cell cycle arrest in S phase,while etoposide induced G2/M phase arrest. Daurinol in-duced the increased expression of cyclin E, cyclin A andE2F-1, which are important proteins regulating S phaseinitiation and progression. Daurinol did not induce abnor-mal cell and nuclear enlargement in SNU-840 cells, incontrast to etoposide. Based on these data, we suggest thatdaurinol is a potential anticancer drug candidate for thetreatment of human ovarian cancer with few side effects.

P103

TNFSF15 Inhibits Vasculogenesis by RegulatingRelative Levels of Membrane-Bound and SolubleIsoforms of VEGF Receptor 1

Jianwei Qi1,2, TingTing Qin1, LiXia Xu1, Kun Zhang1, GuiLiYang1, Jie Li1, HuaiYuan Xiao1, ZhiSong Zhang1, Luyuan Li11College of Pharmacy, Nankai University College of Pharma-cy, State Key Laboratory of Medicinal Chemical Biology, andTianjin Key Laboratory of Molecular Drug Research, Tianjin,China2Experimental Hematology, State Key Laboratory of Experi-mental Hematology, Institute of Hematology and Blood Dis-eases Hospital, Chinese Academy of Medical Sciences & Pe-king Union Medical College, Tianjin, China

Mouse bone marrow-derived Lin−-Sca-1+ endothelial progen-itor cell (EPC) has pluripotent abilities such as supportingneovascularization. Vascular endothelial growth factor(VEGF) receptor 1(VEGFR1) (Flt1) recognizes variousVEGF isoforms and is critically implicated in a wide rangeof physiological and pathological settings, includingvasculogenesis. Mouse EPC expresses two isoforms ofVEGFR1: mFlt1, which transmits ligand-induced signals;and sFlt1, which acts as a negative regulator by sequesteringligands of VEGF receptors. How the relative levels of mFlt1and sFlt1 are regulated is not yet clear. We report here thattumor necrosis factor superfamily 15 (TNFSF15) (also knownas VEGI or TL1A), an endothelial cell-secreted cytokine, si-multaneously promotes mFlt1 degradation and up-regulatessFlt1 expression in EPC, giving rise to disruption of eNOSand MAPK p38 and effective inhibition of VEGF-driven,EPC-supported vasculogenesis in a murine Matrigel implant

model. TNFSF15 treatment of EPC cultures facilitates Aktdeactivation-dependent, ubiquitin-assisted degradation ofmFlt1 and stimulates sFlt1 expression by activating thePKC, Src, and Erk1/2 signaling pathway. These findings in-dicate that TNFSF15 is a key component of a molecular mech-anism that negat ively modulates EPC-supportedvasculogenesis through regulation of the relative levels ofmFlt1 and sFlt1 in EPC.

P104

The Source of the Stroma Affects the TraffickingPotential of Mantle Lymphoma Cells and Altersthe Effect of Ibrutinib on Their Survival

Hitam Saadi1,2, Shoham Shivtiel-Arad1, Tamar Katz1,2,Netanel Horowitz1,21Department of Hematology and Bone Marrow Transplanta-tion, Rambam Health Care Campus, Haifa, Israel2Bruce Rappaport Faculty of Medicine, Technion-Israel Insti-tute of Technology, Haifa, Israel

Introduction: Mantle cell lymphoma (MCL) is characterizedby dissemination of malignant B-lymphocytes in differentcompartments, mainly lymph-nodes (LN) and bone-marrow(BM). The interaction between lymphoma cells and their mi-croenvironment is mediated by adhesion molecules and sig-naling cues regulating malignant cell behavior. This study isaimed to explore the relations between MCL cells and differ-ent stromal cells, comparing their effects on lymphoma celltrafficking and response to ibrutinib, a Bruton’s tyrosine ki-nase inhibitor.Results: Co-culture of MCL cells with BM- and LN-derivedstromal cells increased the expression of CD44 and CXCR4.Ibrutinib treatment resulted in robust apoptosis of lymphomacells. However, co-culture with stromal cells demonstrated anapoptosis-protection effect, where BM stromal cells showedhigher effect than LN. Ibrutinib-resistance was also demon-strated under non-contact conditions, suggesting that secretedfactors are involved.Stromal barrier increased the migration potential of MCLthrough BM stroma and LN cells compared to non-adherentconditions. Ibrutinib decreased cell migration through BMstromal cells; however, resulted in opposite effect when cellsencountered LN stromal cells. Moreover, conditioned medi-um obtained from LN stroma facilitated extensive lymphomacell migration that was impaired following AMD3100 treat-ment. In contrast, BM-derived stromal conditioned mediumbarely promoted lymphoma cell migration with no effect ofAMD3100 treatment.Conclusions:Our results point to substantial differences inlymphoma behavior and ibrutinib response depending on

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stromal cell source. These differences may be mediated bythe unique adhesion interaction and specific survival sig-nals provided by each stromal source. The different adhe-sion constellation and modulations in the lymphoma en-vironment had a diverse effect on Ibrutinib outcome.Therefore, in MCL patients overall response to therapymay be determined by lymphoma dissemination patternsand organ involvement.

P105

A High Rate of Glycolysis Participatesin the Resistance of Glioblastoma to TemozolomideChemotherapy

Martina Sboarina1, Florence Lefranc2, Pierre Sonveaux11Pole of Pharmacology and Therapeutics, University of Lou-vain (UCL) Medical School, Brussels, Belgium2Department of Neurosurgery, Université libre de Bruxelles(ULB), Erasme Hospital, Brussels, Belgium

Glioblastomas (GBM) account for more than 50 % ofall brain tumors in humans. Temozolomide (TMZ) is anorally available alkylating agent that was initially devel-oped for brain cancer. It is a prodrug and its conversionis entirely pH dependent. Methyltransferase MGMT canfurther modulate the activity of TMZ by specificallydemethylating O6-MeG. Because a glycolytic metabo-lism can induce extracellular acidification and therebypotentially interfere with the activation mechanism ofthe drug, we aimed to understand whether metabolicchanges could provide resistance to TMZ in GBM. Cellviability was measured using a SpectraMax Mini-Max300 analyzer, glucose consumption and lactate re-lease with a CMA600 enzymatic analyzer, and extracel-lular acidification with a Seahorse bioenergetic analyzer.We generated isogenic TMZ-resistant and sensitiveT98G and U373 human GBM cell lines. Metabolic anal-yses revealed an increased glycolytic rate in resistantcells. Enhanced glycolysis was associated to higher ex-pression of transcription factor HIF-1a and to an in-creased extracellular acidification rate. TMZ-resistantcells also overexpressed MGMT, but MGMT silencingwith siRNAs did not restore sensitivity to TMZ. Inter-estingly, extracellular acidification rate was maintainedwhen MGMT was silenced, indicating that glycolysisper se plays an important role in TMZ resistance. Ourfindings indicate that resistance to TMZ in glioblastomais linked to increased glycolysis independently fromMGMT expression.Study supported by ERC Starting Grant 243188 TUMETABO,FRS-FNRS and Télévie.

P106

Overcoming Multidrug Resistance with Inhibitorof ABC Transporters Bound to HPMA CopolymerCarrier as a Potential Therapeutic Approachin Cancer Treatment

Ladislav Sivák1, Vladimir Subr2, Karel Ulbrich2, MarekKovar1, Blanka Rihova11Institute of Microbiology, v.v.i, The Czech Academy of Sci-ences, Prague, Czech Republic2Institute of Macromolecular Chemistry, v.v.i, The CzechAcademy of Sciences, Prague, Czech Republic

A significant problem in cancer chemotherapy is that even orig-inally sensitive tumors become resistant to the effects of cytostaticdrugs. This phenomenon has been described as multidrug resis-tance (MDR). MDR can develop in several ways, with the pre-dominant mechanism being the overexpression of ATP-bindingcassette (ABC) transporters, such as P-glycoprotein (P-gp).The aim of the study was to examine the potential ofnove l po l yme r i c t h e r a peu t i c s ba s ed on N - ( 2 -hydroxypropyl)methacrylamide (HPMA) copolymers bearingeither anticancer drug, inhibitor of ABC transporters or both,for overcomingMDRmediated by P-gp in doxorubicin (Dox)resistant cell line P388/MDR.Series of low-molecular weight derivatives of inhibitorreversin 121 (R121) were tested for their ability to block theP-gp efflux pump. 5-methyl-4-oxohexanoyl R121 (MeOHe-R121) showed the highest inhibitory activity out of all thetested derivatives. This derivative was consequently conjugat-ed to the HPMA copolymer carrier. Conjugate P-Ahx-NH-N=MeOHe-R121 showed superior ability to sensitizeP388/MDR cells in a dose-dependent manner and inducedan almost 50-fold increase of cytostatic activity of Dox at24 μM. Moreover, P-Ahx-NH-N=MeOHe-R121 exertedgradually increasing intracellular accumulation of Dox inP388/MDR cells and thus increased cytotoxic activity ofDox in concentration dependent manner. Additionally, the cy-tostatic activity of HPMA copolymer conjugate P-Ahx-NH-N=MeOHe-R121(Dox) bearing both Dox and P-gp inhibitorMeOHe-R121 bound via pH-sensitive hydrazone bond wasalmost 45 times higher than that of the conjugate bearing onlyDox in P388/MDR cells.Finally, the in vivo evaluation of anti-tumor activity of conju-gate P-Ahx-NH-N=MeOHe-R121(Dox) or mixture of theconjugates P-Ahx-NH-N=MeOHe-R121 and P-Ahx-NH-N=Dox in mouse P388/MDR model showed significant inhi-bition of tumor growth with the best effects of P-Ahx-NH-N=MeOHe-R121(Dox). The treatment with conjugate P-Ahx-NH-N=Dox alone exerted similar growth as obtained inthe control group.

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P107

Vemurafenib Resistance Selects for HighlyMalignant Brain andLung-MetastasizingMelanomaCells

Inna Zubrilov1, Orit Sagi-Assif1, Sivan Izraely-Bino1, TsipiMeshel1, Shlomit Ben-Menachem1, Ravit Ginat1, MetsadaPasmanik-Chor2, Clara Nahmias3, Pierre-Olivier Couraud4,Dave Hoon5, Isaac P. Witz11Cell Research and Immunology, Tel AvivUniversity, Tel Aviv, Israel2Bioinformatics Unit, Tel Aviv University, Tel Aviv, Israel33 Inserm U981, Institut Gustave Roussy, Villejuif, France4Inserm U1016, Institut Cochin, Paris, France5Department of Molecular Oncology, John Wayne CancerInstitute, Saint John’s Health Center, Santa Monica, CA, USA

V600E being the most common mutation in BRAF, leads toconstitutive activation of the MAPK signaling pathway. Themajority of V600E BRAF positive melanoma patients treatedwith the BRAF inhibitor vemurafenib showed initial good clin-ical responses but relapsed due to acquired resistance to thedrug. The aim of the present study was to identify possiblebiomarkers associated with the emergence of drug resistantmelanoma cells. To this end we analyzed the differential geneexpression of vemurafenib-sensitive and vemurafenib resistantbrain and lung metastasizing melanoma cells. The major find-ing of this study is that the in vitro induction of vemurafenibresistance in melanoma cells is associated with an increasedmalignancy phenotype of these cells. Resistant cells expressedhigher levels of genes coding for cancer stem cell markers(JARID1B, CD271 and Fibronectin) as well as genes involvedin drug resistance (ABCG2), cell invasion and promotion ofmetastasis (MMP-1 and MMP-2). We also showed that drug-resistant melanoma cells adhere better to and transmigratemoreefficiently through lung endothelial cells than drug-sensitivecells. The former cells also alter their microenvironment in adifferent manner from that of drug-sensitive cells. Biomarkersand molecular mechanisms associated with drug resistancemay serve as targets for therapy of drug-resistant cancer.This study was supported by the Dr. Miriam and Sheldon G.AdelsonMedical Research Foundation (Needham, MA, USA).

P108

Chitinase 3-like 1 Mediates Tumor-PromotingInteractions between Fibroblasts and Macrophagesin the Microenvironment of Breast Tumors

Ophir Shani Noam Cohen, Yael Raz, Yoray Sharon, DanielHoffman, Neta Erez

Department of Pathology, Sackler School of Medicine, TelAviv University, Tel Aviv, Israel

Cancer-Associated Fibroblasts (CAFs) are an activated sub-population of stromal fibroblasts, which have different char-acteristics in different tumor types and tissue locales. CAFswere shown to facilitate tumor growth by supporting tumorcell growth, enhancing angiogenesis and remodeling the ex-tracellular matrix (ECM). In some tumor types, includingbreast cancer, fibroblasts are the most abundant cell type,and their abundance correlates with worse prognosis. Breastcancer continues to be one of the leading causes of cancerrelated death in women, and the requirement for better thera-pies is an unmet clinical need.During breast carcinogenesis, fibroblasts are reprogrammed toexpress various factors that facilitate tumor progression, butmany of them remain unknown. We found that Chi3L1, alsoknown as YKL-40 in humans, is highly upregulated in CAFsisolated from invasive mammary tumors as compared with nor-mal fibroblasts. Chi3L1 is a secreted heparin-binding glycopro-tein, induced specifically during the course of inflammation.Although Chi3L1 plays a pivotal role in exacerbating the in-flammatory processes and in promoting angiogenesis and re-modeling of the extracellular matrix, its functional role incancer-related inflammation is still largely unknown. We foundthat Chi3L1 plays a role in the communication between CAFsand macrophages: Chi3L1 increased macrophage migration andupregulation of a pro-tumorigenic gene signature in macro-phages. Furthermore, orthotopic transplantation of breast cancercells mixed with fibroblasts in which the expression of Chi3L1was knocked-down resulted in attenuated tumor growth. Takentogether, our findings implicate fibroblast-derived Chi3L1 as akey player in the crosstalk between tumor cells and their micro-environment, and deepen our understanding of the contributionof CAFs to tumor progression and metastasis.

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Integrin Alpha V Beta 3 Expression in LuminalBreast Cancer Cells Promotes their Reversionto Acinar-Like Structure in Conjunction with theirRelevant Microenvironment

Hanan Abu-Tayeh1, Keren Weidenfeld1, Alisa Zhilin-Roth1,Sagie Schif-Zuck1, Sonja Thaler, Cristina Cotarelo3, Tuan ZeaTan4, Jean Paul Thiery5, Geula Klorin7, Jeffrey Green6, Ed-mond Sabo7, Jonathan P. Sleeman8, Maty Tzukerman9, DalitBarkan11Department of Human Biology, University of Haifa, Haifa,Israel2Campus Nord, Institut für Toxikologie und Genetik, Karlsru-he Institute of Technology (KIT), Karlsruhe, Germany

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3Department of Pathology, University Medical Center Mainz,Langenbeckstr, Mainz, Germany4National University of Singapore, Cancer Science Institute ofSingapore, Singapore, Singapore5A*STAR, Institute of Molecular and Cell Biology, Singapore,Singapore6Laboratory of Cancer Biology and Genetics, National Can-cer Institute, Bethesda, MD, USA7Department of Pathology, Rambam Medical Center, Haifa,Israel8Medical Faculty Mannheim, Centre for Biomedicine andMedical Technology Mannheim (CBTM), University of Hei-delberg, Mannheim, Germany9Molecular Medicine Laboratory, Rapport Faculty of Medi-cine, Technion-Israel Institute of Technology, Haifa, Israel

Re-establishing tissue organization of breast cancer cells intoacini was shown previously to override their malignant phe-notype. Here we demonstrate that expression of Integrinαvβ3(Int- αvβ3) in luminal A breast cancer cell lines reverts themback to a normal-like acini when cultured in their relevant 3-Dmicroenvironment. These acini like structures expressed themilk protein beta casein thus resembling normal alveolar cells.Importantly, this reversion promoted their growth arrest andwas mediated by Int-αvβ3 expression and activation on can-cer luminal progenitor-like cells (CLPC). Furthermore, down-regulation of NOTCH-4 expression and downstream signal-ing was shown to mediate this reversion. Intriguingly, by uti-lizing a humanized tumor microenvironment model based onthe potential of human embryonic stem cells to generate tera-tomas in immunodeficient mice, we could demonstrate thatInt-αvβ3 expression in luminal A breast cancer cells promot-ed their differentiation when placed in mature teratomas. Fur-thermore, Int-αvβ3 expression was mostly detected in benignstage of human breast tissues.Hence, this data proposes a novel strategy to ‘normalize’ themalignant phenotype by reprogramming CLPC to differenti-ate via the expression of Int-αVβ3. Therefore, promotingsuch differentiation of luminal breast cancer cells in conjunc-tion with their microenvironment may serve as a novel ap-proach to combat local recurrences of breast cancers that resistconventional therapies, thus keeping them on halt.

P110

Redox Modulation of Adjacent Thiols in VLA-4by AS101 Converts Myekoid Leukemia Cellsfrom a Drug-Resistant to Drug-Sensitive State

Yona Kalechman1, Itai Skornick1, Adi Layani-Bazar, AlainBerrebi2, Moran Pauker1, Elad Noy1, Michael Albeck1, DanLongo3, Benjamin Sredni1

1Life Sciences, Bar-Ilan University, Ramat Gan, Israel2Hematology, Kaplan Medical Center, Rehovot, Israel3Biomedical Research Center, National Institute on Aging,Baltimore, MD, USA

Interaction between the integrin VLA-4 on acute mye-logenous leukemia (AML) cells with stromal fibronectinis a decisive factor in chemotherapeutic resistance. Inthis study, we provide a rationale for a drug reposi-tioning strategy to blunt integrin activation in AMLcells and restore their sensitivity to chemotherapy. Spe-cifically, we demonstrate that the nontoxic telluriumcompound AS101, currently being evaluated in clinicaltrials, can abrogate the acquired resistance of AML.Mechanistic investigations revealed that AS101 causedredox inactivation of adjacent thiols in the exofacialdomain of VLA-4 after its ligation to stromal fibronec-tin. This effect triggered cytoskeletal conformationalchanges that decreased PI3K/Akt/Bcl2 signaling, anobligatory step in chemosensitization by AS101. In amouse xenograft of AML derived from patient leukemiccells with high VLA-4 expression and activity, we dem-onstrated that AS101 abrogated drug resistance andprolonged survival in mice receiving chemotherapy. De-creased integrin activity was confirmed on AML cellsin vivo. The chemosensitizing activity of AS101persisted in hosts with defective adaptive and innateimmunity, consistent with evidence that integrin deacti-vation was not mediated by heightening immune attack.Our findings provide a mechanistic rationale to reposi-tion the experimental clinical agent, AS101, to degradeVLA-4–mediated chemoresistance and improve clinicalresponses in patients with AML.

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Neutrophils Promote Breast Cancer Progressionand Metastasis via LFA-1 Integrin

Gabriela Vazquez Rodriguez1, Annelie Abrahamsson1,Lasse Jensen2, Charlotta Dabrosin11Oncology, Linköping University, Linköping, Sweden2Cardiovascular Medicine, Linköping University, Linköping,Sweden

Cancer is considered an inflammatory condition whereimmune cells play an important role in progression andmetastasis. Neutrophils may be pro- or antitumorigenic,depending on their phenotype or the number of infiltrat-ing neutrophils in the tumor microenvironment. Massiveinfiltration of neutrophils in cancer tissue may elicit acytotoxic effect, leading to tumor regression, whereas a

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low-grade neutrophil gradient is tumor progressive.Chemokines, cytokines, and growth factors present inthe tumor microenvironment, as well as cell-cell inter-actions mediated by integrins have shown to be deter-minant steps for cancer cells to break through the en-dothelial wall and establish metastatic niches.In this work we evaluated the role of lymphocyte function-associated antigen 1 (LFA-1) integrin in neutrophils-mediatedmetastasis of estrogen receptor positive breast cancer cells(MCF-7) cells in a tumor xenograft model in zebrafish andin neutrophil infiltration in MCF-7 mammospheres. The met-astatic capability of MCF-7 cells was evaluated in presence orabsence of human neutrophils and with/without estradioltreatment. Two days old zebrafish embryos were injected intothe perivitelline space with labeled MCF-7 cells and humanneutrophils, an anti-human LFA-1 antibody (CD11a) wasincluded.We show that estradiol treatment significantly increased theinfiltration of neutrophils intoMCF-7mammospheres and thisinfiltration was significantly reduced by the presence of ananti-human CD11a antibody. Co-injection of MCF-7 cellswith neutrophils significantly increased the migration ofMCF-7 cells to distant sites in zebrafish and this effect wasinhibited by using an anti-human CD11a antibody.We conclude that neutrophils affect the dissemination ofbreast cancer cells via LFA-1 integrin. Although estradiol in-creased the number of infiltrating neutrophils intomammospheres exposure to estradiol seemed to have minoreffects on the dissemination in the zebrafish.

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c-Myb Inhibits Opening of Lung Endotheliumby Breast Tumor Cells Thereby Dictates MetastasisPattern

Lucia Knopfova1,2, Petr Benes1,2, Michal Masarik3, LuborBorsig4, Jan Smarda11Department of Experimental Biology, Faculty of Science,Masaryk University, Brno, Czech Republic2International Clinical Research Center, CBCE, St. Anne'sUniversity Hospital, Brno, Czech Republic3Department of Pathological Physiology, Faculty of Medi-cine, Masaryk University, Brno, Czech Republic4Institute of Physiology, University of Zurich, Zürich,Switzerland

Metastasis is a multistep process that involves dissemi-nation of tumor cells to distant sites in body and adap-tation to foreign tissue microenvironments. Followingtransport within the vasculature, tumor cells arrest tothe endothelium and then assisted by blood cells

transmigrate into the surrounding tissue. The escapefrom circulation (extravasation) is one of the rate-limiting steps in metastasis cascade and its probabilityis determined both by intrinsic characteristics of tumorcell and by specific features of endothelium in second-ary tissue. We identified transcription factor c-Myb as acritical player controlling the extravasation capacity ofbreast tumor cells. By means of Ccl2 suppression c-Myb prevents monocyte-assisted transmigration of tumorcells across lung endothelium without compromising thepassage across liver endothelial cells. c-Myb-inducedblock in transendothelial migration is dependent on en-dothelial Ccl2-Ccr2 signaling that has tissue-specificfeatures. This unique extravasation guiding force of c-Myb is projected in the absence of lung foci inorthotopic mouse model and in the site-specific relapsefree survival of breast carcinoma patients. In conclusion,c-Myb is a clinically relevant regulator of the metastaticspread of breast carcinomas.This work was funded by grant NT 13441-4/2012 of the In-ternal Grant Agency of Ministry of Health of theCzech Republic, by the European Regional DevelopmentFund Project FNUSA-ICRC (No. CZ.1.05/1.1.00/02.0123),and by grant SCOPES/SNF (IZ73Z0-152361).

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Trop-2 in Breast Cancer Adaptation to SpecificConditions of Tumor Microenvironment

Petr Benes1,2, Tereza Nehybova1,2, Lucia Knopfova1,2, JanRemsik1,2,3, Karel Soucek1,2,3, Jan Smarda21Center of Biomolecular and Cellular Engineering, Interna-tional Clinical Research Center, St. Anne's University Hospi-tal, Brno, Czech Republic2Department of Experimental Biology, Faculty of Science,Masaryk University, Brno, Czech Republic3Department of Cytokinetics, Institute of Biophysics, Academyof Sciences of the Czech Republic, Brno, Czech Republic

The ability of tumor cells to adapt to dynamic microenviron-ment is considered a key requirement for their growth, surviv-al and dissemination. One of the molecules that may interlinkprocesses of plasticity of cancer cells, their dissemination ca-pability and adaptation to microenvironmental factors is thetransmembrane glycoprotein Trop-2. This protein is often as-sociated with regulation of cell proliferation, survival, tumor-igenicity and cancer progression, however, the specific func-tion of Trop-2 during tumorigenesis and metastasis has notbeen yet fully elucidated. Here we analyzed the molecularbasis of Trop-2 expression and function in the context of dy-namic tumor/metastatic microenvironment. We observed a

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dramatic decrease in the frequency of Trop2 positive cells inbreast cancer cells exposed to repeated cycling of hypoxia/starvation. Furthermore, we found that cells with low or noexpression of Trop2 are significantly more resistant to thesetumor microenvironment-like conditions and that Trop2 ex-pression is regulated by both environmental factors. More-over, we identified JNK/AP-1 signaling as an important reg-ulator of Trop2 expression/function in breast cancer cells.These results demonstrate that Trop2-mediated signaling af-fect breast cancer cell adaptation to specific conditions oftumor/metastatic microenvironment.This work was funded by grant NT 13441-4/2012 of the In-ternal Grant Agency of Ministry of Health of theCzech Republic, by the European Regional DevelopmentFund Project FNUSA-ICRC (No. CZ.1.05/1.1.00/02.0123)and by FP7 REGPOT project ICRC-ERA-HumanBridge(no. 316345).

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Re-Modulation Innate Immune Activityin the Tumor Microenvironment by a Combinationof Inducer on Micro-Particles

Ehud Shahar1, Raphael Gorodetsky2, Elina Aizenshtein1,Lior Lalush1,3, Jacob Pitcovski1,31Lab of Virology and Vaccine Development, MIGAL – GalileeResearch Institute, Kiryat Shmona, Israel2Lab of Biotechnology and Radiobiology, Sharett Institute ofOncology, Hadassah – Hebrew University Medical Center,Jerusalem, Israel3Biotechnology, Tel Hai Academic College, Upper Galilee,Israel

Targeted cancer immunotherapy is a challenge due to the cel-lular diversity and imposed immune tolerance in the tumormicroenvironment (TME). A possible promising route toovercome those drawbacks is by activation of the innate im-mune cells (IIC) in the TME, toward tumor destruction. Stud-ies have shown the ability to “re-educate” pro-tumor-activatedIIC toward antitumor responses. The current research wasaimed to stimulate such activation with a combination of in-nate activators loaded onto micro-particles (MP). Inducers ofToll-like receptors 4 (TLR-4), the complement C5a receptor(C5aR) and their combinations were loaded on MP, and theirinfluence on immune cell activation was evaluated. Stimula-tion of immune cell activation by MP was tested in vivo usinga subcutaneous B16-F10 melanoma model induced inC57BL6 mice. Exposure to the TLR4 ligand lipopolysaccha-ride (LPS) bound toMP induced acute inflammatory cytokineand chemokine activity in the TME. Elevation of CD45+ leu-kocytes, in particular the GR-1+ neutrophils, and F4/80

macrophages in the TME was evident. Nevertheless, LPSalone bound to the MP was insufficient to significantly delaytumor progression. LPS combined with the C5aR ligand C5a-pep on the same MP resulted in a similar pattern of inflamma-tion activation. However, interleukin-10 and TGF-β3 levelswere lowered, and tumor growth was significantly delayed.Mixtures of these two ligands on separateMP did not yield thesame cytokine activation pattern, indicating the importance ofthe need for cells’ dual activation to yield the adequate re-sponse. The results suggest that delivery of inducers of distinctinnate immune activation pathways holds promise for suc-cessful redirection of TME-residing IIC toward antitumoralactivation.

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Nano-Ghosts for Selective Drug Deliveryto and Across the Blood Brain Barrier as a PotentialTherapy for Glioblastoma Multiforme

Noa Cohen Anavy, Marcelle MachlufBiotechnology and Food Engineering, Technion-Israel Insti-tute of Technology, Haifa, Israel

The central nervous system (CNS) is protected by the blood–brain barrier (BBB). The BBB, which is formed by imperme-able tight junctions (TJs) between Brain Capillary Endothelialcells (BCEC), serves as an active and selective barrier regu-lating the homeostasis of the brain and protecting it from toxicsubstances. Unfortunately, the high selectivity of the BBBalso hampers the passage of drugs. Despite the rapid develop-ment of drugs with well-established activity in the brain, manydisorders such as Alzheimer`s disease (AD), Parkinson`s dis-ease (PD), brain tumors and Multiple Sclerosis (MS) remainseverely undertreated.In order to overcome these limitations, we aim to modify ourmesenchymal stem cell (MSC) nano-ghosts (NGs) drug deliv-ery system to allow transport across the BBB. The NGs arenano-vesicles produced from the plasma membrane of humanmesenchymal stem cells (hMSCs) which possess membrane-associated targeting and migratory abilities to and through theBBB, and towards sites of inflammation. The NGs are expect-ed to retain the cells` surface moieties and encompass theirunique targeting capabilities, and therefore may serve as aneffective drug delivery system for targeting neurological dis-orders. Our primary hypothesis is therefore that brain targetingof a healthy or diseased BBB can be accomplished by NGs.The hypothesis will be tested both in vitro, using a 3D modelof the BBB, which is composed of a co-culture of rat glial cellsand bovine Brain Capillary Endothelial Cells (BCEC), and invivo, using mice with glioblastoma multiforme (GBM)—anaggressive brain tumor.

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