The TriStar Cancer Stem Cell Platform

Figure 13.8 The Biology of Cancer (© Garland Science 2007)

“CLASSICAL” CELL LINES FAIL TO FAITHFULLY REPRODUCE PRIMARY TUMOR HISTOLOGY

“The inadequacies of currently available human tumor models and the resulting inability to accurately predict how clinically effective anticancer drugs will be, cost the pharmaceutical industry hundreds of millions of dollars annually and therefore represent one of the major impediments to the development of new anti-cancer drugs.” Robert A. Weinberg

The Biology of Cancer (© Garland Science 2007)

Are we using the wrong preclinical models ?

Cancer is rarely cured and relapse is common

Increasing body of evidence exists for the role of Cancer Stem Cells in tumor establishment, metastasis, chemoresistance and relapse

Failure to target appropriate cell subpopulations?

Are we targeting the wrong cells ?

CANCER STEM CELLS (CSC)

THE TRISTAR CANCER STEM CELL (CSC) PLATFORM

Largest collection of Cancer Stem Cells available for drug and target discovery

Robust and well characterized cells that reproducibly recapitulate cellular hierarchies in vitro and patient tumor histology in vivo

Validated high throughput in vitro screening assays and in vivo models (subcutaneous and orthotopic)

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

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

AVAILABLE RESOURCES

Diagnosis and Tissue

Banking

Tumor Dissociation

Stem Cell Culture

And Expansion

Cancer Stem Cell

Banking

Tumor Type Histotype Total availableColon Colon 8Glioblastoma - 11Lung Squamous 2

Adenocarcinoma 1Large Cells 1Small Cells 1

Tumor Type Histotype Total availableMelanoma - 9Breast Infiltrating ductal 5

Infiltrating lobular 1

ThyroidAnaplastic 4Papillary 8Follicular 6

H&E

Tumor database

CD31CEA CD45

Weeks

Co

ntr

ol

CD

13

3

2.97%

0

0.1%

82.3%

6

0.25%

17.43%

3

0.01%

CULTURE CONDITIONS ENRICH FOR CSCS

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

Tu

mo

r s

ize

(cm

3)

Highly tumorigenic in vivo

Reproduce patient tumor histology

Small cell lung cancer Lung adenocarcinoma

Reproduce phosphoproteomic profile

CHARACTERISTICS OF CSCS (2)

Stable propagation in serum free medium over many passages

CHARACTERISTICS OF CSCS (3)

Generate differentiated nontumorigenic progeny in the presence of serum

Colon cancer

CHARACTERISTICS OF CSCS (4)

Chemoresistance

Glioblastoma

Colon

Lung

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 IDENTIFICATION Screening of antibody or shRNA libraries

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

Donor Samples Cancer Type

5 Colon

4 Lung

3 Breast

8 Melanoma

11 Glioblastoma

5 Thyroid

Glioblastoma Colon Lung Melanoma Thyroid

Cores taken from formalin fixed cytospin cell blocks to create a microarray

CD133

CD44

CD44v6c-Met

CD133

Msi-1

Immunofluorescence analysis of stem cell markers

GENE EXPRESSION MICROARRAYS ON CSCS

Gene expression data available on CSCs from• Glioblastoma• Colon• Lung• Breast

FURTHER CHARACTERIZATION OF CSC LINES

miR expression data are available for colon, glioblastoma and melanoma 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

IN VITRO DRUG SCREENING

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 OR using BrdU incorporation

Liquid handling station interconnected to a multimode plate reader

Stem Cell CultureAnd Expansion

Single Cell Suspension Distributed in 96-well

Plates

Incubation

Readout

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 hrs and viability was measured by cell titer

glo:

Cell Killing

IN VITRO DRUG SCREENING: EXAMPLE 1

IN VITRO DRUG SCREENING: EXAMPLE 2

Focused library screen on colon CSCs

In vitro testing In vivo efficacy

IN VITRO DRUG SCREENING: EXAMPLE 3

Secondary assays for hit characterization

ALD

H1

CD

29

treated untreated

Stem cell markers

treated

untreated

Differentiation markers

Clonogenicity assays

untreated treated

Cell Cycle Analysis Apoptosis

IN VIVO MODELS (COLON CSCS)

S.C. xenograft Orthotopic xenograft

Lung metastasis

Spleen metastasisBrain metastasis

Liver metastasis

IMAGING OF CSC-BASED TUMORS

The CSC samples are genetically labeled with markers that allow 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

IDENTIFICATION OF NOVEL CSC-DIRECTED ANTIBODIES (1)

Generation of novel mouse monoclonals by direct immunization with CSCs

IDENTIFICATION OF NOVEL CSC-DIRECTED ANTIBODIES (2)

Possible flowchart for a screen using an antibody library

Antibody library

CSCs vsdifferentiated cells

cell-based ELISA

Unique CSCbinding clones

ILLUSTRATIVE SCREENING PROJECTS USING CSCS

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 COMPOUNDS4 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 & POST TREATED CSC XENOGRAFTS

ILLUSTRATIVE SCREENING PROJECTS USING CSCS

IN-VIVO EFFICACY STUDY ON 50 SCID MICE

Subcutaneously implanted with CSC Xenografts.

Drug treatment and twice weekly caliper measurement of tumor volume and body weight (4 weeks).

Tumor harvest at end of experiment, formalin fixation and RNA extraction for microarray analysis.

CSC derived Xenograft TMA consisting of untreated Xenografts plus samples that have undergone one cycle of treatment

TriStar Technology Group, LLC9700 Great Seneca Highway

Rockville, MD 20850U.S.A

www.tristargroup.us

THANK YOU

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