© FIMM - Institiute for Molecular Medicine Finland www.fimm.fi

High Throughput BiomedicineUnit (HTB) at FIMM Technology Centre

www.fimm.fi

FIMM Technology Centre

High-throughput Biomedicine

Biomarker validation

Metabolomics

Genomics & Bioinformatics

IT

Imaging & clinicalinformatics

State-of-the-art equipment

~ 50 Technology experts-PIs-Senior scientists-Post Docs-Masters-Bioinformaticians-Lab technicians

National and international research core unit providing an extensive spectrum of biomedical research services.

High-throughput Biomedicine

www.fimm.fi

High density format

96 well plateVol/well= 100ul 1536 well plate

Vol/well= 4 ul

Microarray format

384 well plateVol/well= 25ul

What is needed for a HTS?What is needed for a HTS?

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High density format

High throughput reader

What is needed for a HTS?What is needed for a HTS?

www.fimm.fi

What is needed for a HTS?What is needed for a HTS?

High density format

High throughput reader

Data analysis

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HTB Equipment

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BeckmanCoulter integrated robotic system-used for splitting libraries on Labcyte ECHO source plates, by pipetting-running fully automated screens with cell or biochemistry based assays

Motoman robotic armBiomek FXp pipetting robotMultidrop Combi (x2) and Combi nl (x1)Cytomat 6001-plate incubator for cellsPlatelock-plate sealerV-spin-centrifugeDelidding stations and plate hotels on robot deckParadigm plate readerCytomat 24 plate hotel

Labcyte Access robotic system-used for creating assay plates with chemicals and/or siRNAs-miniaturized cell-based and biochemical screening

Labcyte robotic armLabcyte 550 Omics2 Screening2 (2.5 nl droplet)Echo 525 (25 nl droplet)Labcyte LX bulk filler (4 source liquids)Xpeel-plate peelerPlatelock-plate sealerV-spin-centrifugeDelidding stations and plate hotels on robot deck

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Collaborations

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550 525 550

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RNAi screening Chemical screening Microarray printing

High Throughput Biomedicine UnitHigh Throughput Biomedicine Unit

Sharing of chemicals with

academic groups

•siRNA screens

•miRNA screens

•Combination screens•of siRNA, miRNA, chemicals

•Drug Sensistivity and Resistance Testing

(DSRT)

•Chemical Screens

•Biochemical Assays

•Reverse Phase Protein Lysate Array (RPPA)

•Serum printing

•Oligonucleotide printing

Personalized medicine

ex vivo testing of patient Cells with oncology drugs:

LeukemiaOvarian cancerGlioblastoma

Assay miniaturisation

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1. RNAi screening1. RNAi screening

Screening is mainly performed on 384-well plates (96-well possible)Any adherent cell-line is applicableScreening volume/ sample 25 ul1 plate: 320 samples + 64 controls

Readout: high content microscopy, live cell microscopy or cell viability

Analysis: image analysis, statistical analysis, pathway analysis

siRNA library: Ambion Silencer Select full genomeCustom made library: Qiagen or other vendors

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Ambion SilencerAmbion Silencer® ® SelectSelect Human siRNA Human siRNA LibraryLibrary V4V4

Nuclear hormones (47)

Ion channels (338)

Proteases (494)GPCR (380)

Kinases (710)Phosphatases

(298)

Druggable genome (9032)

Extended Druggable genome (10 415)

Human Genome Collection (21 585)

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Incubation 72 h-7 days at 37oCDispensing of detection reagent on cells

Luminescence / intensity readout

Cell culture 384 well assay plates

1) Dispensing of transfection reagent on assay plates 2) Dispensing of cells on the plates

siRNA Screen WorkflowsiRNA Screen Workflow

siRNA libraries on 384 well ECHO plates

siRNA administration (nl)

A

B

Fixing and staining of cellsMicroscopy readout

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High Content ScreeningHigh Content Screening

Image analysisNumbers

cells

Picture

Microscope

Microtiter plate

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2. Chemical Screening2. Chemical Screening

• Drug sensitivity and resistance testing (DSRT)

• Custom screens (mammalian, yeast, prokaryotes)

• Biochemical screens

• Chemical library combined with siRNA KO

Screening is mainly performed on 384-well plates (1536 well possible)Cell-lines, patient cells, suspension cells...Readout: can be chosen among several plate readers, microscopy

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HTB chemical LibrariesHTB chemical Libraries

› HTB Unit has access to several chemical & genomic libraries:

306 FIMM and NIH National Cancer Institute oncology drug collection

446 NIH Clinical collection 2000+640 Microsource and ENZO, including FDA approved drug

collection and natural products 1120 Tocris Tocriscreen mini 2500 NIH NCI (National Cancer Institute) collections 6000 Tripos Structures collection 15 000 ChemDiv Peptidomimetics 25 000 ChemDiv TP02 and TP03 collections 30 000 ChemBridge DivSet and CNS Set 30 000 Specs Consortium collection

1410.10.2013

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Cell culture 384 well assay plates

Dispensing of cells on the plates-patient cells-cell lines

Drug administration (nl)

Incubation 72 h at 37oCDispensing of detection reagent on cells-cell viability mesurement

Luminescence readout

Drug sensitivity and resistance testing (DSRT)Drug sensitivity and resistance testing (DSRT)

Oncology collection 306 drugs5 conc. 10,000-fold conc. range

300 dose response curves

Effective drugs

Resistant drugs

Microscopy readout under development

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What is in the FIMM oncology collection? What is in the FIMM oncology collection?

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Currently 306 active substances:

• Conventional chemotherapeutics

• Immunosuppressants

• Tyrosine kinase-type inhibitors• Abl, Src, EGFR, FGFR, VEGFR, JAK,• IGF1R, PDGFR, Met, ALK Kit, Flt3….

• S/T-type inhibitorsAurora, PLK1, MEK, TTK, PDK1, Akt, Wee1, PKCs, Cdks, Chk1…

Scattered layout for controls:•Benzethonium chloride for low control•DMSO for high control

Algorithms and scripts are being developed to QC and analyse the data

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3. Microarray Printing3. Microarray Printing

Serum microarray

RPPA

25 mm

75

mm

Oligonucleotide microarray

We hope to use the ECHO in the future to print arrays (Echo Array Maker software)

Reverse Phase Protein Array (RPPA)Serum samplesOligonucleotide arrays

Aushon 2470 contact printer

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Cells on plates, Incubation 72h Cell lysis

Heating of the plates at +95C

Spotting with Aushon 2470

SyproRuby staining of the slides

Scanning the slides for tot. protein

Staining the slides with antibodies

Scanning the slides with Li-Cor Odyssey

Analysing the scans using Array-Pro Analyzer

Transfer of the lysates on the arrayer compatible plates

Reverse Phase Protein Array Reverse Phase Protein Array (under development)(under development)

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•Preplating of chemicals on assay plates with ECHO

•Distributing of single aliquots to different research groups

•Partner in the DDCB network, a national infrastructurefor drug discovery and chemical biology research in

Finland

Sharing of chemicals with academic groupsSharing of chemicals with academic groups

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1. You have an idea for a screen

2. First planning meeting-To estimate the feasibility of the project, to give hints about assay development.

3. Assay development

4. Assay robustness and quality testing-The assay needs to fill quality measurements (e.g. Z’-value, signal-to-noise ratio)

5. Second planning meeting-To estimate wether screening can be started, planning the scedule

6. Screening-pilot screen-primary screen-(counter screen)

7. Data analysis-identification of hits, pathway analysis, statistical analysis

8. Validation-required for publications

OverviewOverview of the screening process of the screening process

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Some statistics of last year:Some statistics of last year:

•392 DSRT sets (627,200 samples)

•150 Custom designed chemical plates (48,000 samples)

•A large chemical screen with 100,000 compounds for an international pharma company

•2 full genome siRNA screens with image analysis (64,755 siRNAs each)

•~300 plates of custom designed siRNA/miRNA + compound screens (96,000 samples)

•546 distributed compound aliquotes

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Published papersPublished papers

Antila H. et al. 2014. Utilisation of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells. J Neurosci Methods

Stylinaou M. et al. 2013. Antifungal application of non-antifungal drugs. Antimicrob Agents Chemother.

PemovskaT. et al. 2013.Individualized Systems Medicine (ISM) strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discovery

Tang J. et al. 2013. Target Inhibition Networks: Predicting Selective Combinations of Druggable Targets to Block Cancer Survival Pathways. PLOS Computational Biology

Nybond S. et al. 2013. Antimicrobial assay optimization and validation for HTS in 384-well format using a bioluminescent E. coli K-12 strain. EUFEPS

Denisova O.V. et al. 2012. Obatolax, Saliphenylhalamide, and Gemcitabine Inhibit Influenza A Virus Infection. J.Biol. Chem.

Koskela H.L.M. et al. 2012. Somatic STAT3 Mutations in Large Granular Lymphocytic Leukemia. NEJM

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FIMM High Throughput Biomedicine Unit FIMM High Throughput Biomedicine Unit

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Evgeny Kulesskiy (Graduate stud.)

Karoliina Laamanen (MSc)

Anna Lehto (BSc)

Vilja Pietiäinen (PhD)

Swapnil Potdar (MSc)

Jani Saarela (PhD)

Contact Details:Contact Details:

Chemical screening, DSRT: jani.saarela@fimm.fi

siRNA screening, imaging: carina.vonschantz-fant@fimm.fi

Unit Head:Krister.wennerberg@fimm.fi

Carina von Schantz-Fant (PhD)

Laura Turunen (Lic.Sc.)

Heidi Virtanen (PhD)

Krister Wennerberg (PhD)

Päivi Östling (PhD)