TriStar CSC Platform 2010

8/9/2019 TriStar CSC Platform 2010

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The TriStar Cancer Stem Cell Platform


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Figure 13.8 The Biology of Cancer( Garland Science 2007)

CLASSICAL CELL LINES FAIL TO FAITHFULLY REPRODUCE PRIMARY TUMOR HISTOLOG

The inadequacies of currently availablehuman tumor models and the resultinginability to accurately predict howclinically effective anticancer drugs willbe, cost the pharmaceutical industryhundreds of millions of dollars annually

and therefore represent one of the majorimpediments to the development of newanti-cancer drugs.

Robert A. WeinbergThe Biology of Cancer( Garland Science 2007)

Are we using thewrong preclinical models ?


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lCancer is rarely cured and relapse is common

lIncreasing body of evidence exists for the role of Cancer Stem Cells intumor establishment, metastasis, chemoresistance and relapse

lFailure to target appropriate cell subpopulations?

Are we targeting thewrong cells ?

CANCER STEM CELLS (CSC)


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THE TRISTAR CANCER STEM CELL (CSC) PLATFORM

lLargest collection ofCancer Stem Cells available for drug andtarget discovery

l

lRobust and well characterized cells that reproducibly recapitulatecellular hierarchies in vitro and patient tumor histology in vivo

l

lValidated high throughput in vitro screening assays and in vivomodels (subcutaneous and orthotopic)

l

l

Clinical annotation of all CSC samples and growing preclinicaldatabase (work in progress: mRNA, miR, phospho-proteomics,sequence data)


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THE TRISTAR CANCER STEM CELL (CSC) PLATFORM: CUTTING-EDGE SCIENCE

Nature 445, 111, 2007

Cell Stem Cell 1, 389, 2007

Proc. Natl. Acad. Sci. U.S.A. 105, 13427, 2008

Cell Death and Differentiation 15, 5004, 2008


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AVAILABLE RESOURCES

Diagnosis andTissue Banking

TumorDissociation

Stem Cell CultureAnd Expansion

Cancer Stem CellBanking

umor Type Histotype otal availableolon Colon 8lioblastoma - 11ung Squamous 2Adenocarcinoma 1Large Cells 1Small Cells 1 umor Type Histotype otal availableelanoma - 9reast Infiltratingductal

5Infiltratinglobular

1Thyroid Anaplastic 4Papillary 8Follicular 6

H&E

Tumordatabase


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CD31CEA CD45

Weeks

Control

CD133

2.97%

0

0.1%

82.3%

6

0.25%

17.43%

3

0.01%

CULTURE CONDITIONS ENRICH FOR CSCS


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CHARACTERISTICS OF CSCS (1)

Time (weeks)

3,000 AC133+

105 AC133-

106 total

0

0.5

1.0

1.5

2.0

2.5

3.0

0 2 4 6 8 10 12

Tumorsi z

e(c

m3

)

Highly tumorigenic in vivo

Reproduce patient tumor histology

Small cell lung cancer Lung adenocarcinoma

Reproduce phosphoproteomic profile


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Stable propagation in serum free medium over many passages


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Generate differentiated nontumorigenic progeny in the presence of serum

Colon cancer


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Chemoresistance

Glioblastoma

Colon

Lung


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New wnt-reporter colon cancer stem cells


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q Mutational activation of the wnt pathway occurs in virtually allcolon cancers

q Yet, in most cancers -catenin does not show homogeneousnuclear localization in all tumor cells

q Using a wnt-reporter (TOP GFP) stably transfected into coloncancer stem cells the selective activation of wnt signaling inthe CSC population was shown

Tumor-associated myofibroblasts secrete HGF that stimulates wnt signaling in CSCs


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TOP-GFP high cells express highlevels of nuclear -catenin andstem cell markers, whereas TOPGFP low cells express markers of

differentiated cells

TOP-GFP high cells are highly tumorigenic

TOP-GFP intensity correlates with CD133

expression


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Tumor-associated myofibroblasts (MF) secrete HGF that activateswnt signaling in adjacent

tumor cells. MF or condition media from MF (MFCM) are able to

restore tumorigenicityof differentiated colon cancer cells


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q Marker-selected pure stem cell populations

q Allow to study wnt signaling in relevant cells

q Study effect of stroma-targeting agents(angiogenesis) on CSCs

q Preclinical biomarker for development of newtargeted agents and combinations

Advantages of TOP-GFP colon cancer stem cells


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TRISTAR OFFERSTHE FOLLOWING CONTRACT RESEARCH CAPABILITIES(CSCS)

TARGET EXPRESSION AND PATHWAY ANALYSIS

IHC Screening: Formalin Fixed Cancer Stem Cell Array

mRNA and protein analysis: Western Blot, RTPCR, Flow Cytometry,

Immunoflourescence, Confocal Analysis,

IN-VITRO COMPOUND SCREENING

IN-VIVO EFFICACY MODELS CSC DERIVED XENOGRAFTS

TARGET IDENTIFICATIONScreening of antibody or shRNA libraries


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CYTOINCLUSION MICROARRAYS OF CANCER STEM CELLS AS A TOOL FOR CANCERRESEARCH

Donor Samples Cancer Type

5 Colon4 Lung

3 Breast

8 Melanoma

11 Glioblastoma

5 Thyroid

Glioblastoma Colon Lung Melanoma Thyroid

Cores taken from formalin fixedcytospin cell blocks to create amicroarray


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CD133

CD44

CD44v6c-Met

CD133

Msi-1

Immunofluorescence analysis of stem cell markers


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GENE EXPRESSION MICROARRAYS ON CSCS

Gene expression data available on CSCs from

GlioblastomaColonLungBreast


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FURTHER CHARACTERIZATION OF CSC LINES

miR expression data are available for colon, glioblastoma andmelanoma CSCs

Gene mutation data is available for the following CSCs:

Breast:ER, PR, Her2 and Ki67

NSCLC:KRAS (cod12 and 13), EGFR ex18 (2155 GtoA), ex 19 (del1,2,3,4,5),

ex 21 (2573 TtoG) and ex20 ( 2361 A to G), p53

Colon:KRAS G12V, BRAF V600E, PTEN, PI3K, p53,Smad4, MSH6, MLH1


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IN VITRO DRUG SCREENING

Resistant Sensitive

Drug screening is performed by an establishedhigh-throughput system that allows monitoring the

viability of CSCs using a luminescence-based assaythat reliably measures cellular ATP levels OR usingBrdU incorporation

Liquid handling station

interconnected to amultimode plate reader

Stem Cell CultureAnd Expansion

Single Cell SuspensionDistributed in 96-well Plates

Incubation

Readout


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Results are rainbow coded, with more active compounds in the violetarea(red: 0%, dark violet: 40%)

CSCs derived from 4 colon cancer patients (horizontal strips)were treated with 80 anti-cancer drugs (vertical bars) for 72 hrs

and viability was measured by cell titer glo:

Cell Killing

IN VITRO DRUG SCREENING: EXAMPLE 1


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Focused library screen on colon CSCs

In vitro testing In vivo efficacy


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Secondary assays for hit characterization

ALDH1

CD29

treateduntreated

Stem cell markers

treated

untreated

Differentiation markers

Clonogenicity assays

untreated treated

Cell Cycle Analysis Apoptosis


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IN VIVO MODELS (COLON CSCS)

S.C. xenograft Orthotopic xenograft

Lung metastasis

Spleen metastasis

Brain metastasis

Liver metastasis


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IMAGING OF CSC-BASED TUMORS

The CSC samples are genetically labeled with markers that allow thenon-invasive monitoring of tumor growth and response to treatment

Luciferase

ac

tivity

Control Tw-GFP-Luc

108

106

104

102

100

presorting postsorting

GFP+

LTR LTR

EGFPPGKCMV LUCIFERASE


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IDENTIFICATION OF NOVEL CSC-DIRECTED ANTIBODIES (1)

Generation of novel mouse monoclonals by direct immunization with CSCs


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IDENTIFICATION OF NOVEL CSC-DIRECTED ANTIBODIES (2)

Possible flowchart for a screen using an antibody library

Antibodylibrary

CSCs vsdifferentiated cells

cell-basedELISA

Unique CSCbindingclones


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ILLUSTRATIVE SCREENING PROJECTS USINGCSCS

CANCER STEM CELL CYTO INCLUSION ARRAY(TEST FOR CSC SPECIFIC MARKERS)

ANALYSIS OF ANTIGEN EXPRESSION BY FACS(TEST APPROXIMATELY 6 CSC TYPES, 5 DONORS EACH)

IN VITRO ANALYSIS OF COMPOUNDS 4 GROUPS TESTED IN TRIPLICATE FOR EACH

COMPOUND:CONTROL, COMPOUND, CHEMOTHERAPEUTIC AGENT,COMBINATION 5 TIME POINTS ARE STUDIED: 24, 48, 72,96, 120 HRS

TISSUE MICRO ARRAY ANALYSIS OF PRE & POSTTREATED CSC XENOGRAFTS


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ILLUSTRATIVE SCREENING PROJECTS USINGCSCS

IN-VIVO EFFICACY STUDY ON 50 SCID MICEq Subcutaneously implanted with CSC Xenografts.

q Drug treatment and twice weekly caliper measurementof tumor volume and body weight (4 weeks).

q Tumor harvest at end of experiment, formalin fixationand RNA extraction for microarray analysis.

q CSC derived Xenograft TMA consisting of untreatedXenografts plus samples that have undergone onecycle of treatment


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THANK YOU

TriStar Technology Group,LLC

9700 Great Seneca HighwayRockville, MD 20850U.S.A

www.tristargroup.us

Rockville, MDHeadquarters

Catania, ItalyTissue Repository

Cancer Stem Cell Research

Hamburg, GermanyMain RepositoryArray ManufacturingContract Research

Documents

TriStar CSC Platform 2010