VTT High Throughput Screening

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

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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



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TMAs, Lysate arrays










aCGHaCGH


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TMAs, Lysate arrays








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

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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

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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

Documents

VTT High Throughput Screening