Transcript FIMM - Biocenter

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= 100ul
384 well plate
Vol/well= 25ul
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
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 arm
Biomek FXp pipetting robot
Multidrop Combi (x2) and Combi nl (x1)
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 siRNAs
-miniaturized cell-based and biochemical screening
Labcyte robotic arm
Labcyte 550 Omics2 Screening2 (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
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Collaborations
550
525
550
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High Throughput Biomedicine Unit
RNAi screening
Chemical screening
•siRNA screens
•Drug Sensistivity and
Resistance Testing
(DSRT)
•miRNA screens
•Combination screens
•of siRNA, miRNA, 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
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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
siRNA library: Ambion Silencer Select full genome
Custom made library: Qiagen or other vendors
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Ambion Silencer® Select Human siRNA
Library V4
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
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siRNA Screen Workflow
Cell culture 384 well
assay plates
siRNA administration (nl)
1) Dispensing of transfection reagent on assay plates
2) Dispensing of cells on the plates
siRNA libraries on
384 well ECHO
plates
Luminescence / intensity readout
Dispensing of detection reagent on cells
Incubation 72 h-7 days at 37oC
A
Microscopy readout
26.3.2016
Fixing and staining of cells
B
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High Content Screening
cells
Microscope
Picture
Microtiter plate
Numbers
Image analysis
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2. 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 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
10.10.2013
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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 37oC
Microscopy readout
under development
Resistant drugs
Effective drugs
26.3.2016
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What is in the FIMM oncology collection?
Currently 306 active substances:
• Conventional chemotherapeutics
• Rapamycin analogs
•Epigenetic modulators
• Immunosuppressants
• mTOR/PI3K inhibitors
•PARP inhibitors
• Tyrosine kinase-type inhibitors
• PI3K inhibitors
•Hh inhibitors
• Bcl-2 inhibitors
•γ-secretase inhibitors
• HSP90 inhibitors
•Farnesyltransferase inhibitor
• Survivin inhibitor
•p53 activators
• HDACi:s
•Metabolic inhibitors…
• Abl, Src, EGFR, FGFR, VEGFR, JAK,
• IGF1R, PDGFR, Met, ALK Kit, Flt3….
• S/T-type inhibitors
Aurora, 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
10.10.2013
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75 mm
3. Microarray Printing
Reverse Phase Protein Array (RPPA)
Serum samples
Oligonucleotide arrays
25 mm
Aushon 2470 contact printer
Serum
Oligonucleotide
microarray
microarray
RPPA
We hope to use the
ECHO in the future to
print arrays (Echo Array
Maker software)
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Reverse Phase Protein Array (under development)
Cells on plates,
Incubation 72h
Analysing the scans
using Array-Pro Analyzer
Cell lysis
Scanning the slides
with Li-Cor Odyssey
Heating of the
plates at +95C
Transfer of the lysates on the
arrayer compatible plates
Staining the slides
with antibodies
Scanning the slides
for tot. protein
Spotting with
Aushon 2470
SyproRuby
staining of the
slides
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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
10.10.2013
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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
6. Screening
-pilot screen
-primary screen
-(counter screen)
7. Data analysis
-identification of hits, pathway analysis, statistical analysis
8. Validation
-required for publications
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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
26/03/2016
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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.
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
26/03/2016
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FIMM High Throughput Biomedicine Unit
Evgeny Kulesskiy (Graduate stud.)
Carina von Schantz-Fant (PhD)
Karoliina Laamanen (MSc)
Laura Turunen (Lic.Sc.)
Anna Lehto (BSc)
Heidi Virtanen (PhD)
Vilja Pietiäinen (PhD)
Krister Wennerberg (PhD)
Swapnil Potdar (MSc)
Päivi Östling (PhD)
Jani Saarela (PhD)
Contact Details:
Chemical screening, DSRT:
[email protected]
siRNA screening, imaging:
[email protected]
Unit Head:
[email protected]
www.fimm.fi
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