Compound Screening Using Cancer Stem CellsRuggero DeMariaGiorgio StassiMilan Bhagat

SIGNIFICANCE OF CANCER STEM CELLS

Cancer stem cells are responsible for the development, maintenance and spreading of tumor cells

Tumor relapse results from residual cancer stem cells that survive therapeutic treatment

Cancer stem cells should represent the primary target for new therapeutic strategies aimed at tumor eradication

Cancer stem cell analysis may provide considerable information for prognostic study and patient stratification

RESEARCH OPTIONS

Characterization of Cancer Stem Cells

Immunohistochemistry Screening: Formalin Fixed Cancer Stem

Cell Array

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

Immunoflourescence, Confocal Analysis

Functional assays In vitro drug screening In vivo drug validation

DRS. DE MARIA AND STASSI HAVE PIONEERED CANCER STEM CELL RESEARCH IN SOLID TUMORS

Identification and expansion of human colon-cancer-intiating cellsLucia Ricci-Vitiani1, Dario G. Lombardi2, Emanuela Pilozzi3, Mauro Biffoni1, Matilde Todaro4, Cesare Peschle1

& Rugerro De Maria1, 2

Nature. 2007; 445:111-5.

Identification and expansion of the tumorigenic lung cancer stem cell populationA Eramo1, F Lotti2, G Sette2, E Pilozzii3, M Biffoni4, A Di Virgilio4, C Conticello2, L Ruco3, Cesare Peschle1

& Rugerro De Maria*,1

Cell Death and Differentation. 2008; 15: 504-514.

Colon Cancer Stem Cells Dictate Tumor growth and Resist Cell Death by Production of Interleukin-4Matilde Todaro1, Mileidys Perez Alea1, Anna B. Di Stefano1, Patrizia Cammereri3, Louis Vermeulen5, Flora Iovino1,

Claudio Tripodo4,, Antonio Russo2, Gaspare Gulotta3, Jan Paul Medema5,*, and Giorgio Stassi1,*

Cell Stem Cell 1, 389-402, October 2007

Established several patient derived CSC lines

Stem cell phenotype maintained over long periods in culture

Technology that allows us to isolate and expand in vitro from several different tumors

CANCER STEM CELLS (CSC)

CSCs are also known as Tumorogenic Cells, Tumor Intiating Cells etc.

Minor subpopulation of tumor cells

Undifferentiated neoplastic cells with some characteristics that represent stem cells

Are able to proliferate indefinitely & produce differentiated progeny

Able to form tumor xenografts when injected into immuno-compromised mice

Based on current reviews, likely responsible for metastatis formation

Symmetricdivision

Asymmetricdivision

Non tumorigenic

differentiated cell

Non tumorigenic transient amplifying progenitors

Tumorigeniccancer stem

cells

Potentiallymetastatic

NonMetastat

ic

NonMetastat

ic

Old view New view

Potentially

Metastatic

Potentially

Metastatic

Potentiallymetastatic

HUMAN CANCER STEM CELLS

LeukemiaBreastBrain

ProstateColonLungLiver

MelanomaHead and neck

PancreasOvaryKidneyThyroid

ColonLung

Thyroid

CANCER STEM CELLS ARE A POTENTIAL TARGET FOR THE DEVELOPMENTOF EFFECTIVE ANTI-CANCER THERAPIES

CSC

CSC

CSC

Therapies effective on cancer

stem cells

Tumor degenerati

on

Tumor regressio

n

Therapies effective on

differentiated cancer cells

Tumor relapse

Xenograft

Sorting

CANCER STEM CELLS

Cancer stem cells are isolated from solid tumors using specificCulture conditions that allow CSC-containing “tumor spheres” to grow

Culture in serum free medium,Containing EGF and FGF2

CD133

Control

Colon cancer stem cell sphere, serial sections

AVAILABLE RESOURCES

Diagnosis and Tissue

Banking

Tumor Dissociation

Stem Cell Culture

And Expansion

Cancer Stem Cell

Banking

Tumor Type Histotype Total availableColon 14Rectum 5Squamous 5Adenocarcinoma 4

Lung Large Cells 2Small Cells 1Carcinoid 1To be determined 4

Tumor Type Histotype Total availableGlioblastoma - 32Melanoma - 9Ovary - 5Breast Infiltrating ductal 5

Infiltrating lobular 1

ThyroidAnaplastic 4Papillary 8Follicular 6

H&E

Tumor database

CYTOINCLUSION MICROARRAYS OF CANCER STEM CELLS AS A TOOL FOR CANCER RESEARCH

Donor Samples Cancer Type

12 Colon

12 Lung

6 Breast

5 Ovarian

9 Melanoma

6 Glioblastoma

6 Thyroid

Colon cancer stem cells

IN VITRO DRUG TESTING

Resistant Sensitive

Drug screening is performed by an established high-throughput system that allows monitoring the viability of CSCs using a luminescence-based assay that reliably measures cellular ATP levels.

Liquid handling station interconnected to a multimode plate reader

Stem Cell CultureAnd Expansion

Single Cell Suspension Distributed in 96-well

Plates

Incubation

Readout

CSCS DISPLAY BROAD RESISTANCE TO ANTI-CANCER DRUGS

Anti-Cancer Activity

Results are rainbow coded, with more active compounds in the violet area(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 and viability was measured by cell titer glo:

INHIBITION OF IL-4 SENSITIZES COLON CANCER SPHERES TO CHEMOTHERAPY AND TRIAL-INDUCED CELL DEATH

Untreated

anti-IL-4

Oxalipl. TRAIL5-FU Ox+5-FU

IgG

IN VIVO DRUG TESTING

Cancer Stem Cells are able to generate Xenografts thatHistologically and Phenotypically Reproduce the Patient Tumor

Patient Tumor CSC Xenograft

H&E

CDX2

Colon CancerNSCLC

HMW-CK

H&E

Patient TumorCSC Xenograft

IMAGING OF CSC-BASED TUMORS

The CSC samples are genetically labelled with markers that allowed the non-invasive monitoring of tumor growth and response to treatment

Lu

cif

era

se a

cti

vit

y

Control Tw-GFP-Luc

108

106

104

102

100

presorting postsorting

GFP+

LTR LTR

EGFPPGKCMV LUCIFERASE

COLON CANCER STEM CELL-BASED MODELS

Haematoxylin & Eosin staining of an orthotopic tumor, lung and liver metastasis generated from colon cancer stem cells

Orthotopic Lung metastasis Liver metastasis

BREAST CANCER STEM CELLS GROW AS SPHERES EXPRESSING ALDH

Infiltrating Ductal Breast Cancer

Primary Cancer Cells

CK14

Differentiation in presence of FBS

Breast cancerspheres

CK14 ALDH

CK14

2 days 7 days

Serum-free cultures with EGF + FGF

COLON CANCER STEM CELL-BASED MODELS

0

20

40

60

80

100

Untreated Doxo

cell v

iab

ilit

y (

%)

PBS

IL-4 DM

w/o Doxo IL4DM +DoxoIL4DM

Orange acridine/ethidium bromide staining of breast cancer spheres treated with Doxorubicin and IL-4 DM (IL-4 blocker)

COLON CANCER STEM CELL-BASED MODELS

0

0.5

1

1.5

2

2.5

0 7 14 21 28 35

days

tum

or

siz

e (

cm

3)

IL-4 DM

IL-4 DM+Doxo

Doxo

PBS

Control IL-4DMDoxo IL-4DM

+Doxo

Blocking IL-4 in combination with chemotherapy prevents tumor growth generated by breast CSC

Tunel

Azan Mallory

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