Corning Incorporated · NYSE:GLW
Corning — Technical Presentation (February 2015)
50 slides from Corning's technical presentation, Feb 10, 2015
Slide 1: Novel Cell Culture Tools for Oncology Assays and High Throughput Screening
Novel Cell Culture Tools for Oncology Assays and High Throughput Screening David Randle, Ph.D. and Hannah Gitschier, M.Sc. Corning Life Sciences Fang Tian, Ph.D. ATCC SLAS 2015 Tutorial, Washington, D.C. February 10, 2015
Slide 2: Agenda
Agenda • Overview of Corning Life Sciences • Microplates for screening three dimensional multicellular tumor spheroids • Overview of ATCC • Permeable supports for assays at the air liquid interface • Phenotypic screening assays using tumor cell panels and Corning® Epic® technology • Q&A © 2015 Corni
Slide 3: Corning Life Sciences: An innovator in laboratory products for discovery research
Corning Life Sciences: An innovator in laboratory products for discovery research • Business Overview: − Fortune 500 company (#328), market leader for 100 years − Significant R&D investment for continuous innovation − Global manufacturing and distribution − 230+ sales representatives including Techn
Slide 4: Corning offers a comprehensive portfolio of highly respected global brands in life sciences research
Corning offers a comprehensive portfolio of highly respected global brands in life sciences research © 2015 Corning Incorporated 4
Slide 5: Microplates for screening three dimensional multicellular tumor spheroids
Microplates for screening three dimensional multicellular tumor spheroids Hannah Gitschier, M.Sc. Corning Life Sciences
Slide 6: Multicellular tumor spheroid (MCTS) model
Multicellular tumor spheroid (MCTS) model • Three dimensional MCTS provide a more physiologically relevant model as they more accurately reflect the intricate microenvironment of tumors in vivo.1 MCTS generated from single-cell Various methods used for MCTS formation suspension culture and culture S
Slide 7: Corning spheroid microplate
Corning® spheroid microplate • Corning Ultra-Low Attachment surface and unique round well-bottom design enable the formation and growth of a single, uniform spheroid per well with reproducible size. Standard ANSI/SBS footprint dimensions for 96-well and 384-well formats Clear bottom for Black sidewa
Slide 8: Multicellular tumor spheroid culture using Corning Ultra-Low Attachment surface
Multicellular tumor spheroid culture using Corning® Ultra-Low Attachment surface Tissue culture-treated Ultra-Low Attachment Ultra-Low Attachment 96-well microplate 96-well microplate 96-well spheroid microplate The combination of Corning Ultra-Low Attachment surface and the spheroid microplate un
Slide 9: MCTS formation and growth in Corning spheroid microplates
MCTS formation and growth in Corning® spheroid microplates MCTS formation is straightforward with cell aggregation occurring within the first several hours of seeding a single-cell suspension in the spheroid microplates. Duration of MCTS culture is cell-line dependent. After 48 hours of culture,
Slide 10: HT-29 Spheroids
MCTS formation and optimization is straightforward 40 cells/well 200 cells/well 1,000 cells/well 5,000 cells/well 10,000 cells/well 24 hr HT-29 Spheroids 72 hr 48 hr Scale bar = 1,000 μm © 2015 Corning Incorporated 10
Slide 11: ATCC cell lines used to generate MCTS in the Corning spheroid microplate
ATCC cell lines used to generate MCTS in the Corning® spheroid microplate Spheroid Cell Line ATCC Cat. No. Tumor Type Medium Morphology BT-474 HTB-20 Human breast/duct carcinoma RPMI/10% FBS Tight A549 CCL-185 Human lung carcinoma F-12K/10% FBS Tight HEK-293 CRL-1573 Human embryonic kidney DMEM/10%
Slide 12: Simple plug and play protocol
Simple “plug and play” protocol 1. Plate cells 2. Generate and culture spheroids in microplate 3. Assay spheroid directly in well 24 hr 48 hr 72 hr 4. Process/read microplate to generate data © 2015 Corning Incorporated 12
Slide 13: Corning spheroid microplates: evaluation with LOPAC1280
Corning® spheroid microplates: evaluation with LOPAC1280 Screening with Sigma’s Library of Pharmacologically Active Compounds (LOPAC®) (Sigma Cat. No. 1280) in BT-474 cells, a human mammary gland ductal carcinoma tumor cell line available from ATCC, resulted in the identification of several toxic
Slide 14: Summary of hits from LOPAC1280 screen
Summary of hits from LOPAC1280 screen Compound Name Class Hit Type 2D or 3D Compound Name Class Hit Type 2D or 3D Y-27632 dihydrochloride Phosphorylation Proliferative 2D & 3D H-8 dihydrochloride Phosphorylation Toxic 2D Chlorpromazine hydrochloride Dopamine Proliferative 3D Podophyllotoxin Cytoskel
Slide 15: Evaluation with Tocriscreen Kinase Inhibitor Toolbox (TKIT)
Corning® spheroid microplates: evaluation with Tocriscreen Kinase Inhibitor Toolbox (TKIT) BT-474 cells were screened with the 80 kinase inhibitors comprising the Tocriscreen Kinase Inhibitor Toolbox (46% hit rate for 2D, 42% hit rate for 3D) Iressa, used as a control (red), as well as a compoun
Slide 16: Compound potency and differences in magnitude of cytotoxic responses
Compound potency and differences in the magnitude of cytotoxic responses revealed in hit confirmation Table of EC50 Values Compound 2D (M) 3D (M) Ouabain 1.3x10-7 1.1x10-7 MTX 9.3x10-9 8.7x10-9 Idarubicin 1.2x10-5 5.2x10-7 Iressa 2.3x10-6 1.4x10-6 Iressa and methotrexate (MTX) display comparable s
Slide 17: Summary
Summary Corning® spheroid microplates are an optimal tool for generating, culturing, and assaying multicellular tumor spheroids (MCTS): • Uniform, single spheroid formation in every well aids in maintaining reproducibility and accuracy of assay. • The design of the spheroid microplates allows for in
Slide 18: Air-Liquid Interface (ALI) 3D Culture Model
AIR-LIQUID INTERFACE (ALI) 3D CULTURE MODEL Fang Tian, Ph.D. Lead Scientist, ATCC February, 2015
Slide 19: About ATCC
About ATCC • Founded in 1925, ATCC is a non-profit organization with headquarters in Manassas, VA • World’s premiere biological materials resource and standards development organization • ATCC collaborates with and supports the scientific community with industry-standard products and innovative solu
Slide 20: ATCC Normal Human Primary Cells
ATCC Normal Human Primary Cells • ATCC Primary Cells provide complete culture reagents formulated for optimal cell growth, morphology, and functionality • ATCC Primary Cells are provided at very low passage Epithelial Endothelial Smooth muscle Dermal Bronchial/Tracheal Aortic Aortic Keratinocytes Sm
Slide 21: Comparison of 2D and 3D culturing
Comparison of 2D and 3D culturing Culture Strengths Limitations • Simplistic model • Monolayer structure • Easy to culture • Tight junctions 2D • Time to develop models • Non-optimal physiologic response • Complex - closer to in vivo tissue • Complexity of design • Reduces need for animal models • T
Slide 22: In vitro respiratory model
In vitro respiratory model Bronchial and small airway epithelial cell differentiation in air-liquid interface (ALI) respiratory model Areas of application: • Development of airway epithelium • Disease models • Pre-clinical drug development • Inhalation and airborne toxicology studies • Physio-pharma
Slide 23: Corning Permeable Supports
Corning® Permeable Supports • Corning • Snapwells™ Inserts HTS Plates Custom Fluoroblok™ • Transwell inserts • Netwells® Products • Transwell® • Falcon® • Falcon • HTS Plates • Made-to-order • Snapwell 12 mm • 6, 12, and • Netwell 15 mm, • 24 and 96- 24 mm 24-well well plates inserts • 24 and 96- we
Slide 24: Corning Permeable Supports
Corning® Permeable Supports Permeable supports are convenient, easy-to-use, cell culture devices for the study and analysis of anchorage-dependent and -independent cell lines. insert Upper (apical) compartment Microporous Lower (basolateral) membrane compartment • A two-chamber well separated by a m
Slide 25: ALI cultures
ALI cultures Normal Human Small Airway Epithelial Cells (ATCC® PCS-301-010) Normal Human Bronchial/Tracheal Epithelial Cells (ATCC® PCS-300-010) 3-4 days 2-5 days 21-28 days Expansion Differentiation Pre-expansion (Submerged) (ALI) ATCC Primary Epithelial Cells (used at less than passage 4) Airway E
Slide 26: Human airway epithelium
Human airway epithelium Goblet cells Cilia Membrane Polarized differentiated airway epithelium has the following features: • Presence of goblet cells for mucin secretion (Periodic Acid-Schiff (PAS)- Alcian blue) • Presence of ciliated cells (ciliogenesis) • Presence of good barrier function (transep
Slide 27: ATCC Human Bronchial Epithelial Cells
ATCC Human Bronchial Epithelial Cells ATCC Airway Epithelial Cell Basal Medium (ATCC® PCS-300-030) plus Bronchial Epithelial Cell Growth kit (ATCC® PCS-300-040) or Small Airway Epithelial Cell Growth kit (ATCC® PCS-301-040) At passage 3: 21 days in PneumaCult ALI differentiation media Pseudostratifi
Slide 28: Mucin secretion and production in Primary Small Airway and Bronchial Epithelial Cells
Mucin secretion and production in Primary Small Airway and Bronchial Epithelial Cells • Excessive abnormal mucus is retained in the various human airways diseases (i.e., asthma, COPB, chronic bronchitis). • The amounts of mucins, such as the MUC5AC variant produced primarily by epithelial goblet cel
Slide 29: Effects of IL-13 and GGE on mucin secretion and production
Effects of IL-13 and GGE on mucin secretion and production Differentiation 28 days post-airlift Secretion Production 16.00 20.00 14.00 18.00 16.00 12.00 MUC5AC (ng/mL) 14.00 10.00 12.00 8.00 10.00 6.00 8.00 6.00 4.00 4.00 2.00 2.00 0.00 0.00 Control IL-13 IL-13 + Control IL-13 IL-13 + GGE GGE Interl
Slide 30: 3D organotypic skin epithelial model
3D organotypic skin epithelial model Co-cultures: • Primary Normal Human Neonatal Foreskin Epidermal Keratinocytes (ATCC® PCS-200-010), or Ker-CT (ATCC® CRL-4048™) • Primary Normal Human Neonatal Fibroblasts (ATCC® PSC-201-010) 30 Adapted from Kalabis J, et al. Nature Protocols 7:235-246, 2012.
Slide 31: Primary Keratinocytes and Ker-CT differentiation day 11
Primary Keratinocytes and Ker-CT differentiation day 11 A Primary Keratinocytes P+2 Cornified layer Keratinocytes Fibroblasts in matrix B Ker-CT P+6 C Ker-CT P+15 Immortalized Ker-CT Fibroblasts in matrix 31
Slide 32: Conclusions
Conclusions 3D culture can provide a model system which reflects the phenotypic and functional characteristics of the in vivo tissue microenvironment. Corning® permeable supports are convenient, easy-to-use cell culture devices, and encourage more in-vivo-like morphology, metabolic activities, and c
Slide 33: Genetic Alteration Cell Panels: Effective Tools for High Throughput Screening Using Corning Epic Technology
GENETIC ALTERATION CELL PANELS: EFFECTIVE TOOLS FOR HIGH THROUGHPUT SCREENING USING CORNING® EPIC® TECHNOLOGY Fang Tian, Ph.D. David H. Randle, Ph.D. February, 2015
Slide 34: Background and definitions
Background and definitions Genetic Alteration Cell Panels • ATCC has designed a number of panels to facilitate basic and translational cancer research. • Panels contain key components of cell signaling pathways which have been experimentally validated at ATCC. Molecular signature panels 34
Slide 35: EGFR introduction
EGFR introduction Ligand: EGF, TGF-α, HB-EGF, Amphiregulin, δ cellulin, etc. Family members • EGFR/ERBB1/HER1 • EGFR2/ERBB2/HER2 • EGFR3/ERBB3/HER3 • EGFR4/ERBB4/HER4 3 domains • Extracellular • Transmembrane • Intracellular Functions: • Proliferation • Invasion • Angiogenesis • Metastasis • Inhibit
Slide 36: EGFR mutation in cancer
EGFR mutation in cancer Mutations associated with drug resistance Mutations associated with drug sensitivity 36 Sharma S., et al., Nat Rev Cancer, 2007
Slide 37: EGFR Genetic Alteration Panel composition and characterization
EGFR Genetic Alteration Panel composition and characterization EGFR Genetic Alteration Cell Panel (ATCC® TCP-1027™) EGFR copy ERBB2 copy Cell line Amino acid Tumor ATCC® No. Gene cDNA change Zygosity number number name change source variation variation c.2236_2250delGAATTAA CRL-2868™ HCC827 EGFR Het
Slide 38: Panel includes relevant EGFR mutations for drug screening
Panel includes relevant EGFR mutations for drug screening ATCC® No. Cell line name Gene Amino acid change CRL-2868™ HCC827 EGFR p.ELREA746del EGFR Activating inhibitor CRL-2871™ HCC4006 EGFR p.ELR746del mutations sensitive cell lines CCL-231™ SW48 EGFR p.G719S p.T790M Resistant mutation EGFR CRL-590
Slide 39: Panel includes different levels of EGFR and ERBB2 gene amplification
Panel includes different levels of EGFR and ERBB2 gene amplification EGFR copy Measured ERBB2 copy Measured ® Cell line Tumor ATCC No. Gene number CNV of number CNV of name source variation EGFR variation ERBB2 CRL-2868™ HCC827 EGFR Amplification 63.01 − − Lung HTB-132™ MDA-MB-468 EGFR Amplification
Slide 40: Additional mutations represent the genetic complexity observed in clinical patients
Additional mutations represent the genetic complexity observed in clinical patients EGFR genetic alteration cell panel (ATCC® TCP-1027™) EGFR copy Cell line Amino acid ERBB2 copy Tumor Other mutations observed (COSMIC ATCC® No. Gene number name change number variation source database) variation CRL-
Slide 41: Corning Epic technology is a label-free drug discovery platform
Corning® Epic® technology is a label-free drug discovery platform • 2 key components: − Reader (Epic Gen 1 or BT) − Microplates (96, 384, and 1536-well) • Used for cell-based, biochemical, and compound aggregation assays • Uses optical biosensor technology © 2015 Corning Incorporated 41
Slide 42: Label-free assays measure dynamic mass redistribution (DMR) within cells
Label-free assays measure dynamic mass redistribution (DMR) within cells • Following stimulation with ligands, changes in intracellular components result in subtle morphology changes that we call DMR. • The DMR response is mediated by changes in cell attachment to surface substrate via integrins*. T
Slide 43: High magnitude EGF-induced DMR correlates with EGFR gene amplification
High magnitude EGF-induced DMR correlates with EGFR gene amplification Mechanism EGFR gene copy number amplification increases EGFR protein expression on the cell membrane. The cell lines showed enhanced responses to the EGF stimuli. Supported by Characterization data © 2015 Corning Incorporated 43
Slide 44: High magnitude EGF-induced DMR correlates with ERBB2 gene amplification
High magnitude EGF-induced DMR correlates with ERBB2 gene amplification Mechanism • EGFR2/ERBB2 gene copy number amplification increases ERBB2 protein expression on the cell membrane. • Over-expression of ERBB2 enhances the stability of EGF and retention of EGFR on cell membrane. • Therefore, the ce
Slide 45: Low magnitude EGF-induced DMR when endogenous EGFR is hyperactivated
Low magnitude EGF-induced DMR was observed when endogenous EGFR is hyperactivated in cells DMR Profile: EGF Cell Line Feature Mechanism HCC827 Response EGFR activation • EGFR is already hyper-activated and recruits related kinases and adaptor 50 pm proteins to the cell membrane. EGFR p.ELREA746 dele
Slide 46: DMR profiles demonstrate selective inhibition of EGFR by Iressa
DMR profiles demonstrate selective inhibition of EGFR by Iressa based on EGFR mutations DMR Profile:EGF+Iressa Inhibition Curve Cell Line Feature HCC827 • EGFR p.ELREA746 deletion (activation mutation) • EGFR gene amplification • Verified Iressa sensitive cell line NCI-H1975 • EGFR T790M (drug resis
Slide 47: Identification of new target in Iressa-resistant cell line NCI-H1975
Identification of new target in Iressa-resistant cell line NCI- H1975 by screening Tocriscreen™ Kinase Library • Selective inhibitors of IκB kinase (IKK16) or CK2 inhibitor (TBB) showed dramatic inhibition on Iressa-resistant cell line NCI-H1975. • Selective inhibitor (NSC693868) of CDKs and GSK-3 s
Slide 48: ATCC panels and Corning Epic technology are effective tools for profiling tumor pathways
ATCC Genetic alteration panels and Corning® Epic® technology are effective tools for profiling of tumor pathways ATCC Molecular Signature Cell Panels Corning Epic technology • Extensive characterization • High throughput screening • Represent patient tumor molecular profiles • ≤ 2 hours assay • Tool
Slide 49: Corning Life Sciences offers complete workflow solutions for drug discovery
Corning Life Sciences offers complete workflow solutions for drug discovery Through acquisition and ongoing R&D investment, we now offer a more comprehensive range of high quality, innovative laboratory products and solutions for a wide spectrum of life science applications. Our trusted brands, back
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