cancer stem cells presentation
DESCRIPTION
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.”TRANSCRIPT
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
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