High Throughput Biomedicine Unit HTB at FIMM Technology

High Throughput Biomedicine Unit (HTB) at FIMM Technology Centre © FIMM - Institiute for Molecular Medicine Finland www. fimm. fi

FIMM Technology Centre Genomics & Bioinformatics Metabolomics IT High-throughput Biomedicine Imaging & clinical informatics Biomarker validation National and international research core unit providing an extensive spectrum of biomedical research services. State-of-the-art equipment ~ 50 Technology experts -PIs -Senior scientists -Post Docs -Masters -Bioinformaticians -Lab technicians www. fimm. fi

What is needed for a HTS? High density format Microarray format 96 well plate Vol/well= 100 ul 384 well plate Vol/well= 25 ul 1536 well plate Vol/well= 4 ul www. fimm. fi

What is needed for a HTS? High throughput reader High density format www. fimm. fi

What is needed for a HTS? High throughput reader High density format Data analysis www. fimm. fi

HTB Equipment Beckman. Coulter 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 arm Biomek FXp pipetting robot Multidrop Combi (x 2) and Combi nl (x 1) Cytomat 6001 -plate incubator for cells Platelock-plate sealer V-spin-centrifuge Delidding stations and plate hotels on robot deck Paradigm plate reader Cytomat 24 plate hotel Labcyte Access robotic system -used for creating assay plates with chemicals and/or si. RNAs -miniaturized cell-based and biochemical screening Labcyte robotic arm Labcyte 550 Omics 2 Screening 2 (2. 5 nl droplet) Echo 525 (25 nl droplet) Labcyte LX bulk filler (4 source liquids) Xpeel-plate peeler Platelock-plate sealer V-spin-centrifuge Delidding stations and plate hotels on robot deck www. fimm. fi 6

Collaborations 550 525 550 www. fimm. fi 7

High Throughput Biomedicine Unit RNAi screening Chemical screening • si. RNA screens • Drug Sensistivity and Resistance Testing (DSRT) • mi. RNA screens • Combination screens • of si. RNA, mi. RNA, chemicals • Chemical Screens Microarray printing • Reverse Phase Protein Lysate Array (RPPA) • Serum printing • Oligonucleotide printing • Biochemical Assays Personalized medicine ex vivo testing of patient Cells with oncology drugs: Leukemia Ovarian cancer Glioblastoma Sharing of chemicals with academic groups Assay miniaturisation www. fimm. fi 8

1. RNAi screening Screening is mainly performed on 384 -well plates (96 -well possible) Any adherent cell-line is applicable Screening volume/ sample 25 ul 1 plate: 320 samples + 64 controls Readout: high content microscopy, live cell microscopy or cell viability Analysis: image analysis, statistical analysis, pathway analysis si. RNA library: Ambion Silencer Select full genome Custom made library: Qiagen or other vendors www. fimm. fi 9

Ambion Silencer® Select Human si. RNA Library V 4 Kinases (710) Phosphatases (298) Nuclear hormones (47) GPCR (380) Proteases (494) Ion channels (338) Druggable genome (9032) Extended Druggable genome (10 415) Human Genome Collection (21 585) dd. mm. yyyy Presentation name or name of the guest www. fimm. fi 10

si. RNA Screen Workflow Cell culture 384 well assay plates si. RNA administration (nl) 1) Dispensing of transfection reagent on assay plates 2) Dispensing of cells on the plates si. RNA libraries on 384 well ECHO plates Luminescence / intensity readout Dispensing of detection reagent on cells Incubation 72 h-7 days at 37 o. C A Microscopy readout 3/15/2018 Fixing and staining of cells B www. fimm. fi 11

High Content Screening cells Microscope Picture Microtiter plate Numbers Image analysis www. fimm. fi

2. Chemical Screening • Drug sensitivity and resistance testing (DSRT) • Custom screens (mammalian, yeast, prokaryotes) • Biochemical screens • Chemical library combined with si. RNA 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 www. fimm. fi 13

HTB 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 Tocriscreen mini § 2500 NIH NCI (National Cancer Institute) collections § 6000 Tripos Structures collection § 15 000 Chem. Div Peptidomimetics § 25 000 Chem. Div TP 02 and TP 03 collections § 30 000 Chem. Bridge Div. Set and CNS Set § 30 000 Specs Consortium collection 10. 2013 www. fimm. fi 14

Drug sensitivity and resistance testing (DSRT) Cell culture 384 well assay plates Dispensing of cells on the plates -patient cells -cell lines Drug administration (nl) Oncology collection 306 drugs 5 conc. 10, 000 -fold conc. range Luminescence readout Dispensing of detection reagent on cells -cell viability mesurement 300 dose response curves Incubation 72 h at 37 o. C Microscopy readout under development Resistant drugs Effective drugs 3/15/2018 www. fimm. fi 15

What is in the FIMM oncology collection? Currently 306 active substances: • Conventional chemotherapeutics • Rapamycin analogs • Epigenetic modulators • Immunosuppressants • m. TOR/PI 3 K inhibitors • PARP inhibitors • Tyrosine kinase-type inhibitors • PI 3 K inhibitors • Hh inhibitors • Bcl-2 inhibitors • γ-secretase inhibitors • HSP 90 inhibitors • Farnesyltransferase inhibitor • Survivin inhibitor • p 53 activators • HDACi: s • Metabolic inhibitors… • Abl, Src, EGFR, FGFR, VEGFR, JAK, • IGF 1 R, PDGFR, Met, ALK Kit, Flt 3…. • S/T-type inhibitors Aurora, PLK 1, MEK, TTK, PDK 1, Akt, Wee 1, PKCs, Cdks, Chk 1… 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 10. 2013 www. fimm. fi 16

75 mm 3. Microarray Printing Reverse Phase Protein Array (RPPA) Serum samples Oligonucleotide arrays 25 mm Aushon 2470 contact printer Serum Oligonucleotide microarray RPPA We hope to use the ECHO in the future to print arrays (Echo Array Maker software) www. fimm. fi 17

Reverse Phase Protein Array (under development) Cells on plates, Incubation 72 h Analysing the scans using Array-Pro Analyzer Cell lysis Scanning the slides with Li-Cor Odyssey Heating of the plates at +95 C Transfer of the lysates on the arrayer compatible plates Staining the slides with antibodies Scanning the slides for tot. protein Spotting with Aushon 2470 Sypro. Ruby staining of the slides www. fimm. fi

Sharing of chemicals with academic groups • Preplating of chemicals on assay plates with ECHO • Distributing of single aliquots to different research groups • Partner in the DDCB network, a national infrastructure for drug discovery and chemical biology research in Finland 10. 2013 www. fimm. fi 19

Overview of the screening process 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, signalto-noise ratio) 5. Second planning meeting -To estimate wether screening can be started, planning the scedule 1. 6. Screening 2. 3. 4. -pilot screen -primary screen -(counter screen) 5. 7. Data analysis 6. -identification of hits, pathway analysis, statistical analysis 7. 8. Validation 8. -required for publications www. fimm. fi 20

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 si. RNA screens with image analysis (64, 755 si. RNAs each) • ~300 plates of custom designed si. RNA/mi. RNA + compound screens (96, 000 samples) • 546 distributed compound aliquotes 15/03/2018 www. fimm. fi 21

Published 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. Pemovska. T. 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 STAT 3 Mutations in Large Granular Lymphocytic Leukemia. NEJM 15/03/2018 www. fimm. fi 22

FIMM High Throughput Biomedicine Unit Evgeny Kulesskiy (Graduate stud. ) Carina von Schantz-Fant (Ph. D) Karoliina Laamanen (MSc) Laura Turunen (Lic. Sc. ) Anna Lehto (BSc) Heidi Virtanen (Ph. D) Vilja Pietiäinen (Ph. D) Krister Wennerberg (Ph. D) Swapnil Potdar (MSc) Päivi Östling (Ph. D) Jani Saarela (Ph. D) Contact Details: Chemical screening, DSRT: jani. saarela@fimm. fi si. RNA screening, imaging: carina. vonschantz-fant@fimm. fi Unit Head: Krister. wennerberg@fimm. fi www. fimm. fi 23