The TriStar Cancer Stem Cell Platform

“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 ?

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

CANCER STEM CELLS (CSC)

 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 ?
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
Tumor
Tissue Banking database

H&E
Tumor
Dissociation
Stem Cell Culture Cancer Stem Cell
And Expansion Banking

Tumor Type Histotype Total available Tumor Type Histotype Total available
Colon Colon 8 Melanoma - 9
Glioblastoma - 11 Breast Infiltrating ductal 5
Lung Squamous 2
Adenocarcinoma 1 Infiltrating lobular 1

Large Cells 1 Anaplastic 4

Small Cells 1 Thyroid Papillary 8
Follicular 6
CULTURE CONDITIONS ENRICH FOR CSCS

0.01% 0.25% 0.1%

Control

2.97% 17.43% 82.3%

CD133

Weeks 0 3 6

CEA CD45 CD31

 CHARACTERISTICS OF CSCS (1)
Reproduce patient tumor histology
Highly tumorigenic in vivo

3.0
3,000 AC133+

2.5 105 AC133-

106 total

2.0
Tumor size (cm3)

1.5

1.0
Small cell lung cancer Lung adenocarcinoma

0.5
Reproduce phosphoproteomic profile
0
0 2 4 6 8 10 12
Time (weeks)
 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)Colon

Chemoresistance
Glioblastoma

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

Glioblastoma Colon Lung Melanoma Thyroid

 Immunofluorescence analysis of stem cell markers

CD133
Msi-1 CD133
CD44

CD44v6
c-Met
 GENE EXPRESSION MICROARRAYS ON CSCS

Gene expression data available on CSCs from

• Glioblastoma
• Colon
• Lung
• Breast
FURTHER CHARACTERIZATION OF CSC LINES

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

miR expression data are available for colon, glioblastoma and

melanoma CSCs
IN VITRO DRUG SCREENING

Drug screening is performed by an established

high-throughput system that allows monitoring the
Stem Cell Culture
viability of CSCs using a luminescence-based assay
And Expansion that reliably measures cellular ATP levels OR using
BrdU incorporation

Single Cell Suspension

Distributed in 96-well Plates
Liquid handling station
interconnected to a
multimode plate reader

Incubation

Readout

Resistant Sensitive
IN VITRO DRUG SCREENING: EXAMPLE 1

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

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

Clonogenicity assays Stem cell markers
untreated treated

ALDH1
untreated

CD29
treated

Differentiation markers
Cell Cycle Analysis Apoptosis
untreated treated
 IN VIVO MODELS (COLON CSCS)

S.C. xenograft Orthotopic xenograft

Liver metastasis Lung metastasis

Brain metastasis
Spleen 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

CMV LUCIFERASE PGK EGFP

LTR LTR

presorting postsorting

GFP+

108
Luciferase activity

106

104

102

100
Control Tw-GFP-Luc
 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
cell-based
ELISA

CSCs vs
differentiated cells Unique CSC
binding
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 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 & 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
THANK YOU TriStar Technology Group, LLC
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Washington DC, USA www.tristargroup.us
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