tristar csc platform 2010
TRANSCRIPT
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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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CHARACTERISTICS OF CSCS (2)
Stable propagation in serum free medium over many passages
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CHARACTERISTICS OF CSCS (3)
Generate differentiated nontumorigenic progeny in the presence of serum
Colon cancer
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CHARACTERISTICS OF CSCS (4)
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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IN VITRO DRUG SCREENING: EXAMPLE 2
Focused library screen on colon CSCs
In vitro testing In vivo efficacy
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IN VITRO DRUG SCREENING: EXAMPLE 3
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