angio1

In Vitro Models for Studying Angiogenesis BD Biosciences - Discovery Labware Suparna Sanyal Overview Angiogenesis Important considerations for studying angiogenesis in vitro BD in vitro angiogenesis models EC Migration EC Invasion EC tube formation Angiogenesis • Angiogenesis – formation of new blood vessels (sprouting, branching etc.) from existing vessels • Vasculogenesis – formation of new blood vessels from angioblasts or progenitor stem cells (i.e. de novo vessel synthesis) Diseases Associated With Angiogenesis Cancer Rheumatoid Arthritis Aids Complications Blindness (Diabetic Retinopathy) Excessive ANGIOGENESIS Psoriasis Stroke Insufficient Infertility Heart Disease Ulcers Scleroderma Tumor Angiogenesis Localized solid tumor •Necessary for tumor growth and metastasis Tumor Angiogenesis •Inhibition of angiogenesis represents a critical point of intervention for cancer treatment. •Circumvents problems of cellular toxicity and tolerance development seen with chemotherapy Progression of Angiogenesis 1 Pro-angiogenic factors released from hypoxic cells VEGF bFGF 2 Factors Bind to EC receptors MMps activated and released Endothelial Cell Basement membrane degradation 3 4 5 6 7 Proliferation Invasion Adhesion Migration 9 Tube formation 10 Highly permeable pericyte 11 • Pericyte recruitment • Vessel stabilization In vitro cell-based angiogenesis assay systems in Drug Discovery • Molecular and cellular mechanisms of angiogenesis and drug action mechanisms • Target validation • Secondary screen and lead optimization Clinical Relevance of Endothelial Cell-Based in vitro Angiogenesis Assays (Kerbel and Folkman, Nat Rev Cancer, 2002, 2:727-739) Important Considerations for Developing Angiogenesis Studies • Choice of Endothelial Cells • Establishing a good dynamic range to measure stimulation and/or inhibition of angiogenesis • ECM complex to facilitate cell growth and assay outcome • Choice of in vitro angiogenesis assay Sources of Endothelial Cells BD™ Human Umbilical Vein Endothelial Cells • Most commonly studied human EC type in angiogenesis • Source, isolation procedure and initial culturing conditions can influence response to pro-angiogenic factors (e.g. VEGF, bFGF). • BD™ HUVEC-2 (Cat. No. 354151) cells have been pre-screened for responsiveness to VEGF in migration assays. Extracellular Matrix • • • • • Cell attachment Cell morphology and spreading Survival and Proliferation EC function Examples: gelatin, fibronectin, vitronectin, laminin, collagen, BD Matrigel™ BD in vitro Angiogenesis Assays • EC cell migration • EC cell invasion • EC cell tube formation Using BDTM FluoroBlokTM Insert Systems BD Falcon™ FluoroBlok™ Insert Systems • Quantitative and High-Throughput Analysis of Tumor Cell Invasion Using Fluorescence-Blocking Insert Systems Webinar, September 2005 http://www.bdbiosciences.com/discovery_labware/ webinar/archive/tumor_cells.html BD Falcon™ FluoroBlok™ Inserts and BD BioCoat Angiogenesis Systems BD Falcon FluoroBlok Inserts: for Migration • 24-multiwell (3 & 8 micron pores) • 96-well (3 & 8 micron pores) BD BioCoat Angiogenesis Systems •hFN coated 3 micron pores •24- and 96-well format BD BioCoat Angiogenesis Systems: for Invasion • Patented Matrigel Coating Process • 24- and 96-well format • 3 micron pores (EC) • 8 micron pores (tumor cells) Shipped on dry ice, Store at -20ºC Do not store in frost free or -70ºC freezer BD BioCoatTM FluoroBlokTM Migration System No serum + 10% serum Endpoint or Real-time kinetic Assay POST-LABELING PRE-LABELING Cell migration Calcein AM Real-Time Analysis of Calcein AM-Labeled HUVEC Migration through 3 mm BD Falcon™ FluoroBlok™ Inserts 800 Mean Fluorescence 600 400 200 0 0 1 2 3 Time (hours) 4 5 Chemoattractant Control Optimization of chemotaxis incubation time 2.50 2.00 1.50 1.00 0.50 5 10 15 20 25 30 35 Time (mins) 45 60 Fold increase over control 3.00 Fold increase over control THP-1 2.50 2.00 1.50 1.00 0.50 0.00 5 10 Monocytes 90 15 20 25 30 45 Time of incubation (mins) Importance of optimizing cell seeding density THP-1 2.400 Fold increase over control (mean + SD) 2.200 2.000 1.800 1.600 1.400 1.200 1.000 0.800 1 5 10 15 20 25 30 100,000 50,000 25,000 10,000 Monocyte 3.500 CV % < 10% Fold increase over background 3.000 2.500 2.000 1.500 1.000 0.500 0.000 20 25 Time of incubation 30 50,000 Cells/well 100,000 cells/well Time of incubation (mins) BD BioCoat™ Endothelial Cell Migration System: 24- and 96-well Comparison HUVEC-2 Migration 14 Fold Stimulation 12 10 8 6 4 2 0 FBS VEGF 24-well FBS VEGF 96-well BD BioCoat™ Endothelial Cell Migration System: Importance of hFN coating Comparison of hFN coated and uncoated 24-well Inserts 6000 Fibronectin coated insert 5000 Uncoated Insert Fluorescent Units 4000 3000 2000 1000 0 Control VEGF FBS Chemotaxis Response of Different EC Types HAEC Cells 12 2.5 HUVEC-2 Cells Fold increase over control Fold increase over control 10 mean + se (n=4) 2.0 8 6 4 2 0 mean + se (n=4) 1.5 1.0 0.5 0.0 0.000 1.000 3.125 6.250 12.500 25.000 0.000 1.000 3.100 6.200 12.500 25.000 VEGF (ng/ml) 8-10 fold stimulation VEGF (ng/ml) 2-3 fold stimulation Chemotaxis stimulation and inhibition using HMEC-1 Cells Fold increase in migration over control (mean + SE) 12 Cell migration (% inhibition) 10 8 6 4 2 0 -13 HMEC-1 100 80 60 40 20 EC50 = 10 nM IC50 = 50 nM 0 -5 -12 -11 -10 -9 -8 -7 -6 -9 -8 -7 -6 -5 log CHEMOATTRACTANT (M) log [Inhibitor] (M) This assay is well suited for medium throughput drug screening BD BioCoat™ Endothelial Cell Invasion System Cross section of one of 24 inserts per Multiwell Insert plate Pore Occluding BD Matrigel™ Matrix Layer BD FluoroBlok™ membrane, 3 micron pores Human Primary Vascular Endothelial Cell Invasion through BD Matrigel™ Matrix Control Chemoattractant BD BioCoat™ Endothelial Cell Invasion System Effe ct of M M P inhibitor 1'10' P he na nthroline on HM V EC Inva sion 1800 1600 1400 1200 1000 800 600 400 200 0 Fluorescent Units VE G F( 4n g/ m l) 0. 1u g/ m l V EGF(4ng/ml)+ 1'10' Phenathroline 20 ug /m l 1u g/ m l tro l 10 ug /m C on l BD BioCoat™ Endothelial Cell Invasion System Endothelial Cell Invasion: Inter-assay Reproducibility 100 90 80 Assay 1 Assay 2 Assay 3 70 Percent Invasion 60 50 40 30 20 10 0 -10 3T3 HMEC-1 HMVEC BD BioCoat™ Endothelial Cell Invasion System Endothelial Cell Invasion: lot-to-lot Reproducibility 100 90 80 Lot 1 Lot 2 Lot 3 Percent Invasion 70 60 50 40 30 20 10 0 3T3 HMEC-1 HMVEC Successful Assay Development Tips • A practical guide for running a successful assay on the BDTM FluoroBlokTM Insert Systems • • • • • • Detection instrument Cell labeling methodology Choice of pore sizes ECM coating Assay conditions Criteria of a robust assay Detection Instrument • A fluorescent plate reader with bottom reading capability, and an inverted fluorescent microscope for confirmation and troubleshooting • A fluorescent Imager • Set Up Guidelines and Dimensional Templates for Fluorescence Plate Readers Used With BD Falcon™ HTS FluoroBlok™ Insert Systems and BD BioCoat™ Multiwell Insert Cell-Based Assays, Technical Bulletin # 436 • http://www.bdbiosciences.com/discovery_labware/ technical_resources/pdf/tb436_fluoroblok.pdf Choice of membrane pore sizes • 8 µm • • • • • 3 µm • Epithelial cells Fibroblasts Most of tumor cells Smooth muscle cells • Leukocytes • Endothelial cells In addition to pore size, pore density also matters. A side-by-side comparison of 3 and 8 µm for your application is always a good idea. Assay Conditions • Cell Seeding density • 24-well: >= 25,000 cells/well • 96-well: >= 10,000 cells/well Chemoattractant • A titration of chemoattractant concentration is recommended ECM coating • e.g. fibronectin, collagen, laminin etc. Assay Buffer Serum Starvation Incubation time • Migration: • Leukocytes:10 minutes to two hours • Others: A few hours to one day • Invasion: Overnight to days • • • • • Criteria for a Robust Migration/Invasion Assay on the BD™ FluoroBlok™ Insert System • Definition: • Positive control: Cells with chemoattractant • Negative control: Cells without chemoattractant • Background fluorescence: • Pre-labeled cells: zero time point • Post labeled cells: Unlabeled cell or no cells • Assay criteria: • %CV • Dynamic range (signal-to-noise ratio) • Z’=1-(3SD of POS +3SD of NEG/(Mean of POS-Mean of NEG)* • >=0.5 is preferred • *Zhang, etc. 1999 Angiogenesis Tube Formation Assay steps in angiogenesis process Angiogenesis Tube Formation Assay - Simplified Angiogenic Stimulators VEGF, FGF 1. EC lysis of basement 2. EC invasion/migration 4. Capillary tube formation & 3. EC proliferation differentiation membrane and extracellular matrix BD BioCoat™ Angiogenesis Assay: Endothelial Cell Tube Formation • 96-well Black/Clear Plate • Surface modified for elimination of meniscus • Coated with BD MatrigelTM Matrix • Manufacturing Technique to product flat surface • Screened for ability to promote tube formation • Concentration and volume optimized • Mat Cover and Lid • Ensures product stability Shipped on dry ice, Store at -20ºC Do not store in frost free or -70ºC freezer Assay Flow Chart Day 1 Thaw, 6-24 hours Gel, 30 min. Frozen 96-well Tube Formation product Trypsinize & resuspend EC in assay medium 37 C/5% CO2 incubation Day 2 Fluorescent dye labeling BD Matrigel™ Matrix Endothelial cells Angiogenesis stimulating factors Automated Image Acquisition & Data Processing Endothelial Cell Tube Formation Collagen I 2D BD Matrigel Bright Field Human Microvascular Endothelial Cells BD Matrigel Fluorescence Angiogenesis – Confocal vs. Non-confocal Confocal Non-confocal HUVEC-2 cells, stained with Calcien AM, 4X Confocal images show entire network Improved Segmentation Effect of Cell Number Endothelial Cell Tube Formation Total tubule length (pixels) HMEC-1 Cell Number Titration 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 HUVEC Cell Number Titration 10 14 20 24 28 Number of cells per well (x 1000) 40 10 14 20 24 28 Number of cells per well (x 1000) 40 Endothelial Cell Tube Formation • • • • Assay setup to data within 24 hours Validated Protocols High-throughput: 96-well format Automated image acquisition and data processing • Labeling with fluorescent dye • Morphometric analysis • Can use Fluorescent Microscope • Reproducible • Consistent endothelial cell tube formation: • Well to well, lot to lot , assay to assay • Z' value= 0.6, Good for screening Angiogenesis - Dose Response to Suramin 0 160 uM Different Analysis Parameters - Similar Results 8000 350 Tube Complexity (total # of segments) Total Tube Length (pixels) 7000 6000 5000 4000 3000 2000 1000 0 1.0×10 -7 1.0×10 -6 1.0×10 -5 1.0×10 -4 1.0×10 -3 300 250 200 150 100 50 0 1.0×10 -7 1.0×10 -6 1.0×10 -5 1.0×10 -4 1.0×10 -3 IC50 = 2.93E-05 IC50 = 2.97E-05 Log [M] Suramin 70000 Log [M] Suramin Total Tube Area (pixels) 60000 50000 40000 30000 20000 10000 0 1.0×10 -7 1.0×10 -6 1.0×10 -5 1.0×10 -4 1.0×10 -3 IC50 = 2.94E-05 Log [M] Suramin Tube Formation using various Human Endothelial Cells 14000 12000 Total tube length (pixel) 10000 8000 6000 4000 2000 0 HUVEC HMVEC HMEC-1 CV 7.6% 5.1% 5.4% Reproducibility HMVEC Tubule Formation 4500 4000 3500 3000 2500 2000 1500 1000 500 0 Tube Area (Fluorescent Units) Day 1 Day 2 Day 3 HMVEC are seeded onto Matrigel coated 96-well plates on three different days and incubated at 37°C for 24 hours. Images are obtained using a 4X objective. Tube area is calculated for each plate (n=90). Criteria for a Robust Tube Formation Assay • Assay criteria: • %CV • Dynamic range (signal-to-noise ratio) • Z’=1-(3SD of POS +3SD of NEG/(Mean of POSMean of NEG)* • >=0.5 is preferred *Zhang, etc. 1999 Z' Factor Determination 16000 14000 T o tal tu b e len g th (p ixels) 12000 10000 8000 S/N=23 S/B=5 Z'=0.6 6000 4000 2000 0 0 20 40 60 80 100 120 Well number BD Products for Angiogenesis VEGF bFGF 1 Chemokines & Growth factors 3 In vitro 3D matrix for tube formation, capillary morphogenesis e.g. collagen, fibrin, BD PuraMatrix 2 Factors Bind to EC receptors Extracellular matrices/ Coated plates for assays Proliferation Adhesion Migration Invasion Tube formation Vessel sprouting MMps activated and released Endothelial Cell Basement membrane degradation pericyte • Pericyte recruitment • Vessel stabilization 5 EC Assay buffer Reagents 4 Ex-vivo outgrowth model on BD Matrigel e.g. aortic ring assay, chick aortic arch assay Contact Us Technical Service • In US: labware@bd.com • Phone: 877.232.8995 • Outside US: help.biosciences@bd.com • Phone: Contact your local distributor or nearest BD Biosciences office • Web: http://www.bdbiosciences.com/discovery_labware/webinar/ docs/angio.pdf • Email: Suparna_Sanyal@bd.com Paula_Flaherty@bd.com Supplementary Information: BD Products for Angiogenesis BD™ Human Umbilical Vein Endothelial Cells • 354151 BD HUVEC-2 Cells 1 cryovial BD BioCoat™ Angiogenesis System: Endothelial Cell Migration • • 24-Multiwell Insert System 354143, 1 plate, 354144, 5 plates 96-Multiwell Insert System 354147, 1 plate, 354148, 5 plates BD Falcon™ HTS FluoroBlok™ Insert Systems • • Individual, 24-Multiwell, 1, 3, and 8 micron 96-Multiwell, 3 and 8 micron BD BioCoat™ Angiogenesis System: Endothelial Cell Invasion • 24-Multiwell Insert System, 354141, 1plate, 354142, 5 plates BD BioCoat™ Angiogenesis System: Endothelial Cell Tube Formation • 96-well Black/Clear Optilux™ Microplate, 354149, 1 plate, 354150, 5 plates Supplementary Information: BD Products for Angiogenesis • • ExtraCellular Matrices (ECMs), Vialed BD BioCoat™ Cellware • • • • • • • BD Matrigel™(3D, thin layer , +/- growth factors, HC) Fibronectin (FN) Poly-lysine (PDL, PLL) Laminin (LM) Gelatin Collagens I, III, IV, V Vitronectin • • • Albumin (BSA, delipidized) • • 354331 354227 Bovine serum, 10 g 2.5/500 mg Linoleic Acid/Albumin Complex Scaffolds • 354250 BD™ PuraMatrix™ Peptide Hydrogel Supplementary Information: BD Products for Angiogenesis Chemokines & Growth Factors Vascular Endothelial Growth Factor 354107 VEGF, human recombinant 10 mg Fibroblast Growth Factor (FGF) 354002 Bovine natural 356037 bFGF, bovine natural 354060 bFGF, human recombinant 10 µg 10 µg 10 µg Supplementary Information: BD Products for Angiogenesis •Growth Factors •Epidermal Growth Factors •Fibroblast Growth Factors •Hepatocyte Growth Factor/ Scatter Factor •Media Additives •Insulin-like Growth Factors •Bovine Pituitary Extract Nerve Growth Factors •Endothelial Cell Growth Supplement •Platelet-Derived Growth Factor •Transforming Growth Factor •Lymphokines •Granulocyte-Macrophage Colony Stimulating Factor •Interleukin-1 Interleukin-2 Interleukin-3 Interleukin-4 •Stem Cell Factor •Tumor Necrosis Factor-a