vtt high throughput screening
TRANSCRIPT
High-throughput screening at VTT
Merja Perälä, PhD, Principal ScientistVTT Medical Biotechnology, Turku
224/08/2012
Research ServicesFocus on cell-based assays
• Functional drug assays (MoA, EC50)• Toxicity and safety (chemical / material)• In vitro cell-based metabolomics• Target identification and validation• Bio-informatics• In-depth knowledge of cell signalling pathways• Immunotechnologies for cell research
Bio-Informatics• Data analyses (from genes to proteins and pathways)• Development of solutions and software • Data visualisation and navigation
Enabling Technologies• HT-robotics platforms• In vitro biochips • Protein arrays• 3-D organotypic models• Immunotechnologies• Imaging platforms• In-silico tools and software
Instrumentation• HT-robotics core• Versatile imaging facility• Modern laboratories • Biacore T-200
Reagent Libraries• 70K LMW compounds• +140 human cell lines • Genome-wide RNAi• Genome-wide miRNA (anti-miR and pre-miR)
In Vitro Cell Biology• Technology development (next-gen biochips, 3-D models)• Identification of new therapeutics + drug repurposing• Target validation at genetic and protein levels• Cell signalling research + metabolomics
In vitro Cell Biology for H&W
324/08/2012
High-throughput and high-content cell based assaying capabilities at VTT Turku
Areas of expertiseCancer systems biologyBreast and prostate cancer biologyHigh-throughput and high-content screening BioinformaticsSolutions for data analysis and graphics
Mechanistic insights,Clinically and functionally relevant drug targets,Development of therapeutic,Diagnostic / predictive biomarkers
Proteomics, Epigenetics, Metabolomics, Protein interactions
BioinfoBioinfointegrationintegration
Gene copyGene copynumbernumber
aCGHaCGH
Gene / Gene / miRNAmiRNAExpressionExpression
arraysarrays
Clinical Clinical screening / screening / translationtranslation
TMAs, Lysate arrays
Other Other platformsplatforms
RNAiRNAi & & miRNAmiRNAscreensscreens
Compound Compound screensscreens
LysateLysate arraysarrays
TransfectedTransfectedcell arrayscell arrays
Functional Functional data on cell data on cell line modelsline models
Molecular Molecular profiling data profiling data on cell lines on cell lines and tumorsand tumors
Genome-scale research
Mechanistic insights,Clinically and functionally relevant drug targets,Development of therapeutic,Diagnostic / predictive biomarkers
Proteomics, Epigenetics, Metabolomics, Protein interactions
BioinfoBioinfointegrationintegration
Gene copyGene copynumbernumber
aCGHaCGH
Gene / Gene / miRNAmiRNAExpressionExpression
arraysarrays
Clinical Clinical screening / screening / translationtranslation
TMAs, Lysate arrays
Other Other platformsplatforms
RNAiRNAi & & miRNAmiRNAscreensscreens
Compound Compound screensscreens
LysateLysate arraysarrays
TransfectedTransfectedcell arrayscell arrays
Functional Functional data on cell data on cell line modelsline models
Molecular Molecular profiling data profiling data on cell lines on cell lines and tumorsand tumors
BioinfoBioinfointegrationintegration
Gene copyGene copynumbernumber
aCGHaCGH
Gene / Gene / miRNAmiRNAExpressionExpression
arraysarrays
Clinical Clinical screening / screening / translationtranslation
TMAs, Lysate arrays
Other Other platformsplatforms
RNAiRNAi & & miRNAmiRNAscreensscreens
Compound Compound screensscreens
LysateLysate arraysarrays
TransfectedTransfectedcell arrayscell arrays
Functional Functional data on cell data on cell line modelsline models
Molecular Molecular profiling data profiling data on cell lines on cell lines and tumorsand tumors
Genome-scale research
424/08/2012
High-throughput screening technologies
• analysis of gene functions by HT-RNA interference
• drug target identification and validation in disease
• identification of genes with a functional role in drug response (resistance /sensitivity)
• functional exploration of cell signaling pathways
• primary and secondary compound screening in understanding of mechanism of action
524/08/2012
• analysis of gene functions• drug target identification and validation in disease• functional exploration of cell signaling pathways• identification of genes with a functional role in drug response
1 drug 1000s RNAis
Which genes functionally modulate resistance/sensitivity to existing
therapeutic agents?
1000sisogenic
cell variants
1 RNAi1000s compounds
Which compounds are effective in cells lacking specific gene functions?
RNA interference is a powerful tool for specific gene knockdowns in mammalian cells
624/08/2012
• Qiagen Genome wide library v1 (22 000 x 2 = 44 000 siRNAs) -separate sets for 2x 509 GPCR siRNAs,
2x 212 phosphatase siRNAs, 2x 709 kinase siRNAs
• Qiagen Druggable genome v3 (4 x 6 941 = 27 764 siRNAs) • Qiagen Human Cancer siRNA Set (1 183 x ~2 = 2 375)
-Apoptosis, Angiogenesis, Cell Cycle, DNA Repair, Growth Factors, Metastasis, Tumor Suppressors
• Ambion kinase siRNA library (747 x 3 = 2 241 siRNAs)• Specific custom siRNA libraries from Qiagen
-secondary screening -targeted screens: epigenetic players, prostate and breast cancer related genes…..
• 321 Ambion Anti-miRs against mature miRNAs• 319 Ambion Pre-miR Precursors
• 810 Dharmacon miRIDIAN® mimics• 896 Dharmacon miRIDIAN® inhibitors
Reagent libraries: siRNAs and miRNAs
724/08/2012
Reagent libraries: compounds• Sigma LOPAC 1280 compounds • Microsource Spectrum 2000 compounds• Microsource cancer plate 80 compounds• Tocris Tocriscreen 1120 compounds• FDA approved drugs (Enzo) 640 compounds • FDA approved drugs (Selleck) 426 compounds • IBS 1500 natural compounds
• BioMol, kinase and phosphatase inhibitors 84 compounds• Enzo BioMol Bioactive lipid library 202 compounds• Enzo BioMol Epigenetics library 43 compounds• Enzo BioMol Protease inhibitor library 47 compounds• Enzo BioMol Autophagy Library 96 compounds• >200 individually ordered compounds
•ChemDiv 25000 structurally diverse compounds•ChemBridge compounds (9998 CNS-set + 19989 Diverse set)•Tripos 6000 structurally diverse compounds ~70 000 compounds
824/08/2012
Integrated high-throughput robotic system enables up to 40,000 RNAi molecules or chemical compounds to be screened at one time in a 384 well format
Platform and method for high-throughput RNAi screening of living cancer cells in 384 well plate format
924/08/2012
High-throughput screening of 4910 known drugs and drug-like LMW molecules for their tumor cell growth suppressing activity in prostate cancer cell lines
Comparison of cell viability results:
Iljin et al., 2009, Clin. Cancer Res.
1. Vast majority of anti-cancer drugs are equally effective in cancer and control cells
2. Disulfiram, a relatively safe FDA approved drug used as a alcohol-abuse deterrent, was identified among the few selective inhibitors of prostate cancer cell growth
3. Disulfiram reduced prostate cancer xenograft growth in vivo but was not able to block it combinatorial approaches needed
Drug Repositioning: A New Indication for Disulfiram (antabuse)
Research Example on VTT’s Compound Screening Capabilities
Cancer cell viability
Cont
rol c
ell v
iabi
lity
1024/08/2012
Identification of novel prostate cancer drug targets usingin silico gene expression profiling and high-throughput
siRNA screeningStudy outline:1. Selection of prostate cancer specific genes
2. Functional studies in cultured prostate cancer cells
3. Validation in vitro and in vivo
• Vainio P et al., Arachidonic acid pathway members PLA2G7, HPGD, EPHX2, and CYP4F8 identified as putative novel therapeutic targets in prostate cancer. Am J Pathol. Feb;178(2):525-36, 2011
• Vainio P et al., Integrative genomic, transcriptomic, and RNAi analysis indicates a potential oncogenic role for FAM110B in castration-resistant prostate cancer. Prostate 72(7):789-802, 2012
• Vainio P, et al., High-throughput transcriptomic and RNAi analysis identifies AIM1, ERGIC1, TMED3 and TPX2 as potential drug targets in prostate cancer. PLoS ONE 2012;7(6):e39801, 2012
1124/08/2012
VTT’s offering: Tissue/Cell lysate microarrays for rapid systematic profiling of multiple proteins
Multiple slides enable many readouts from a single screen
1224/08/2012
3-channel staining of LMA slides
Sypro(total protein)
• Sypro Ruby protein stain (Invitrogen) for totalprotein followed by double immunostainingwith two primary antibodies and labelledsecondary antibodies with IR-labels AlexaFluor680 (Invitrogen) & IRDye800 (Rockland)
• NC has very low background fluorescence at near IR-wavelenghts
• Up to 3 individual signals from same sample(single spot)- signal normalization within spot- reduces slide-to-slide variation
Nitrocellulose-coated microscope slideArrayed protein lysate spot
Primary antibody
Fluorescent IR dye-taggedsecondary antibody
Fluorescent total protein stain
Antibody staining 700 & 800 nm
1324/08/2012
Prediction of targets
The role of microRNA in breast and prostate cancer: VTT project overview
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MicroRNA expression (Agilent) Functional screens on 384-well plates
Pre-miR
Validation
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Target search by lysate microarrays
1424/08/2012
Research example: Protein lysate microarray analysis to identify microRNAs regulating Estrogen Receptor (ERa) signaling in breast cancer cell lines
Pre-miRNAlibrary
Validation by reduction of ER proteinOverexpression of microRNAs results in downregulation of ERalpha mRNA and protein, and this leads in suppression of estrogen-responsive genes and in inhibition of estrogen-stimulated growth of estrogen-dependent breast cancer cells.
Clinical validation: miRNAs having inverse correlation with ER in breast tumor samples: low expression in ER-positive and higher expression in ER-negative tumors
miR-18a miR-18b
Leivonen et al., Oncogene 28, 3926-3936, 2009
Breast cancer cells transfected with pre-miR miRNA libraryLysate microarray screen to identify miRNAs regulating ER
1524/08/2012
miRNA libraries:overexpression of 1129 miRNA molecules
Five AR positive cell lines:LNCaP, LAPC-4, MDA-PCa-2b, 22Rv1, CWR-1R
Protein lysates
AR ARResults:- A total of 71 unique miRNAs influence the protein levels of AR
in all five cell lines (52 decreasing, 19 increasing)- Top 20 were validated in LNCaP and 22Rv1 cells by Western Blotting- 13 miRNAs directly target the 6.8 kb AR 3’UTR - 11 of these decrease androgen-induced proliferation in vitro
Protein lysate microarraymeasure AR protein
1. miR-135b2. miR-185 3. miR-299-3p4. miR-34a5. miR-34c6. miR-371-3p 7. miR-449a8. miR-449b9. miR-63410. miR-654-5p11. miR-9
Results demonstrate that miRNAs are important regulators of AR protein levels.
This could be applied for developing novel therapeutic strategies to inhibit AR function and androgen-dependent cell growth.
Research example: Functional analyses of microRNAs targeting Androgen Receptor (AR) in prostate cancer cells
Päivi Östling et al, Cancer Res 2011, 71: 1956-1967
1624/08/2012
Reduced miR-34a/c expression is linked to increased AR immunostaining in prostate tumour samples
47 prostatic tissues obtained by transurethral resection(TURP) of the prostate (Hagman et al., Int J Cancer 2010)
1) miR-34c and miR-34a levels were measured with qRT-PCR
2) AR protein content was determined by immunostaining and scored by overall intensity
Statistically significant inverse correlation was observed for miR-34c and miR-34a expression compared to AR
weak moderate to strong intense
Intensity score 1 + 2 versus 3 (Mann-Whitney test) miR-34c: p=0.0082 miR-34a: p=0.0085
Päivi Östling et al., Cancer Res 71: 1956-1967, 2011
1724/08/2012
VTT’s offering: Cell arrays for high-throughput RNAi screening• Individual reverse transfection microarray spots on SBS assay plates• Cells are growing only on the defined arrayed spots• ~15 000 transfections can be screened on a single assay plate• 150µm spots with 375µm spacing, 150-250 cells/spot
•Novel, ultra-high-throughput miniaturised cell spot array platform for genome-scale RNAi analyses on a single microplate
•Fully compatible for microarray scanning and high-content screening with equipment widely available
•Multiparametric readout for genome-scale screen with four markers in 20 minutes
•92 adherent cell types have been tested for compatibility with the CSMA cell patterning method
Rantala J et al., A cell spot microarray method for production of high density siRNA transfection microarrays BMC Genomics . Vol. 12 (2011) No: 1:162
Rantala J, Pouwels et al., SHARPIN is an endogenous inhibitor of 1-integrin activation. Nature Cell Biology, 13:1315-24, 2011
1824/08/2012
Applications of CSMA
CSMA is a functional genomics tool for drug discovery and development with applications in
Drug target discovery and validation in high-content with multiple parameters
Testing of the gene/drug sensitization effect in ultra HT manner (miniaturization enables screening also when the amount of compound or other reagents is limited)
Modulation of drug efficacy by siRNA-mediated synthetic lethal gene-knockdowns
Mechanism-of-action studies
1924/08/2012
Benefits of Using Cell Spot Microarray
Reliable and reproducible
More information
Very fast results
Rapid and cost-effective
Less cells needed
Miniaturized: all samples with up to 28.000 siRNAs areanalyzed in a single chip -> no interassay variability
Multiparametric phenotype analysis
100-fold decrease in reagent needs
20x less cells required-> Compatible with rare cell types e.g. primary or patient-derived cells
Long term cell culture possible
Fully compatible with microarray scanners -> Readout for genome-scale screen with four markers in <20 min
2024/08/2012
Bioinformatics Solutions for Data Analysis and Presentation
2124/08/2012
• Service-based data mining across the world’s largest fully integrated and annotated human gene expression data source
• 20.000 normalized tumour samples• 9.400 genes profiled per sample• Multitude of annotation information per sample • Versatile gene comparisons across all samples• Customised bioinformatics for translational cancer biology
In-Silico Transcriptomics (IST) Human Gene Expression Database
1) High quality custom data mining and contract research based on the IST database available from VTT Medical Biotechnology
2) Open access with limited functions http://www.genesapiens.org/
3) Database and interactive web access available from VTT spin-off MediSapiens
What makes MediSapiens platform different?- Open Platform -philosophy: APIs fully and openly available- Advanced technology platform: flexible analysis development and integrations- Data unification: high quality and published array type unification methodology- Ease-of-use: online data access and distribution for research teams
2224/08/2012
Summary of the VTT-MBT Capabilities
Assays• Proliferation• Apoptosis and Cell Death• Toxicity• Growth Dynamics• Cell Differentiation &
Morphogenesis• Cancer Invasion• Biomarker expression
Applications• Compound / siRNA Screening• Toxicology • Target Validation• Basic Cell Biology• Co-Culture Models
Areas of expertiseHigh-throughput and high-content screening Cancer systems biologyBioinformaticsSolutions for data analysis and graphics
TechnologiesHTS with- 384 well plate format - protein lysate array- cell-spot microarrayHC live-cell imagingConfocal microscopyImmunofluorescencemRNA and protein expressionOrganotypic cancer cell modelsProximity ligation assay for P-P interaction detection
2324/08/2012
VTT creates business from technologywww.vtt.fi/mbt