Tumor Angiogenesis Assays: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Discovery and Development of Tumor Angiogenesis Assays
1 Introduction
2 Historical Notes
3 In Vivo Assays
3.1 Chamber Assays
3.2 Chick Chorioallantoic Membrane Assay
3.3 Zebrafish and Xenopus laevis Tadpole
3.4 Corneal Assay
3.5 Mesentery-Window Assay
3.6 Sponge/Matrix Implant
3.7 Disc Angiogenesis System
3.8 Tumor Angiogenesis Models
4 In Vitro Angiogenesis Assays
4.1 Endothelial Cell Proliferation Assays
4.2 Endothelial Cell Migration Assays
4.3 Tubule Formation Assays
4.4 Ex Vivo Vessel Outgrowth Assays
4.5 In Vitro Tumor Angiogenesis Assays
5 Conclusion
References
Chapter 2: The Role of the Stroma in Tumor Angiogenesis and Progression: The Fundamental Contribution of Pietro M. Gullino
1 Biographic Profile
2 Interstitial Pressure and Blood Dissemination
3 Angiogenesis as a Marker for Neoplastic Transformation
4 Tumor Cells Release Molecules Able to Stimulate Angiogenesis: The Role of Prostaglandin E-1
5 Microenvironment and Tumor Angiogenesis
6 Concluding Remarks
References
Chapter 3: Methods for Isolation of Tumor-Associated Endothelial Cells for Surface Protein Analysis and Sorting by Flowcytomet...
1 Introduction
2 Materials
2.1 Collagenase Digestion of B16-F10 Melanoma Tumors
2.2 Magnetic Enrichment of CD31 Positive Cells
2.3 Staining CD31+ Fraction for Simultaneously Analyzing Expression of Surface Proteins and Sorting Endothelial Cells
3 Methods
3.1 Collagenase Digestion of B16-F10 Melanoma Tumors
3.2 Magnetic Enrichment of CD31 Positive Cells
3.3 Staining CD31+ Fraction for Simultaneously Analyzing Expression of Surface Proteins and Sorting TECs
4 Notes
References
Chapter 4: Perfusion-Based Fluorescent Dye Labeling to Sort Cancer Cells Based on Their Distance from Blood Vessels
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Tumor Inoculation
2.3 PFDLC
2.4 Fluorescence Activate Cell Sorting (FACS)
3 Methods
3.1 Fluorescently-Tagged Cancer Cell Preparation
3.2 Tumor Inoculation
3.3 Perfusion-Based Fluorescent Dye Labeling of Cancer Cells
3.4 Dissociation of Tumor Tissue into Single Cells
3.5 Sorting Cancer Cells as a Function of Distance from BVs
4 Notes
References
Chapter 5: Visualizing the Interactions Shaping the Imaging of the Microenvironment in Human Cancers
1 Introduction
2 Materials
2.1 Flash Freezing Tissue
2.2 Cryosections of the OCT Tissue Block
2.3 Tissue Fixation
2.4 Tissue Staining
2.5 Gene Expression
2.6 Library Construction
3 Methods
3.1 Sample Preparation: Fresh Frozen Solution Highlights
3.2 Tissue Fixation
3.3 Tissue Staining and Brightfield Imaging
3.4 Library Preparation
3.5 Data Analysis and Visualization
References
Chapter 6: Quantification of Tumor and Angiogenesis-Related Markers in Ovarian Cancer Models by a Digital Pathology Approach
1 Introduction
2 Materials
2.1 Patient-Derived Ovarian Cancer Xenografts
2.2 Mice
3 Methods
3.1 Generation of Subcutaneous Tumor Xenografts
3.2 Antiangiogenic Treatment
3.3 Immunohistochemistry
3.4 Digital Pathology
3.4.1 Image Acquisition
3.4.2 Classification of Tissue Components (Tumor, Stroma, and Necrosis)
3.4.3 Microvessel Analysis
3.4.4 Quantification of MCT4
4 Conclusions
5 Notes
References
Chapter 7: Monitoring Angiotropic Extravascular Migratory Metastasis In Vitro
1 Introduction
2 Materials
2.1 Reagents and Cells
2.2 Reagent Set Up
2.3 Equipment
3 Methods
3.1 Endothelial Cell Culture
3.2 Tubule Formation
3.3 Cancer and Endothelial Cell Coculture
3.4 Time Required for Each Step
3.5 Quantitative Fluorescence Real-Time Imaging
3.6 Notes: Tips, Troubleshooting, and Applications
References
Chapter 8: Double Immunohistochemical Staining on Formalin-Fixed Paraffin-Embedded Tissue Samples to Study Vascular Co-option
Abbreviations
1 Introduction
1.1 Vascular Co-option
2 Materials
2.1 Basic Laboratory Supplies
2.2 Histology Laboratory Supplies
2.3 Image Acquisition and Analysis Supplies
3 Methods
3.1 Tissue Specimens Preparation
3.2 Double Immunohistochemistry Protocol
3.3 Image Acquisition
3.4 Image Analysis
4 Note
References
Chapter 9: Development of Experimental Three-Dimensional Tumor Models to Study Glioblastoma Cancer Stem Cells and Tumor Microe...
1 Introduction
2 Materials
2.1 Neurospheres
2.2 Glioblastoma Organoids
2.3 Organotypic Brain Culture
3 Methods
3.1 Neurospheres
3.1.1 Prepare Neurosphere Media
3.1.2 Cancer Stem Cell Isolation from Glioblastoma Tumor
3.1.3 Cell Culture
3.1.4 Fixation
3.2 Glioblastoma Organoids
3.2.1 Small Sphere Formation from Single Glioblastoma Stem Cells
3.2.2 Generation of Matrigel Droplet
3.2.3 GBM Organoid Growth in Matrigel with Shaking Agitation
3.3 Organotypic Brain Culture: Mouse or Human
3.3.1 Prepare the Agarose Solution
3.3.2 Prepare Organotypic Media
3.3.3 Prepare Culture Plates
3.3.4 Mouse Brain Dissection
3.3.5 Mouse Brain Slices
4 Notes
References
Chapter 10: IKOSA CAM Assay Application to Quantify Blood Vessels on Chick Chorioallantoic Membrane (CAM)
Abbreviations
1 Introduction
2 Materials
2.1 Fertilized Eggs
2.2 Equipment
2.3 Reagents
2.4 Image Acquisition
2.5 Analysis Systems
3 Methods
3.1 Egg Preparation
3.1.1 Day 0-2
3.1.2 Day 3
3.1.3 Day 8-Day 12
3.1.4 Day 12
3.2 Image Acquisition
3.3 Image Analysis
4 Representative Results
5 Notes
References
Chapter 11: Application of Laser Speckle Contrast Imaging (LSCI) for the Angiogenesis Measurement of Tumors in the Chorioallan...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Primary Tumors
2.3 CAM Model
2.4 PeriCam PSI HR Model
3 Methods
3.1 Cell Culture
3.2 CAM Model
3.3 Angiogenesis Measurement with PSI HR Model on the Basis of LSCI
3.4 Evaluation of the Acquired Measurement Data with the Pimsoft Software
4 Notes
References
Chapter 12: The Chick CAM as an In Vivo System to Study Stem Cell Activity
1 Introduction
2 Materials (and Equipment)
2.1 Cell Culture
2.2 Egg Incubation and Windowing
2.3 CAM Implantation
2.4 Collection and Processing of CAM-Xenografted Tumor Samples
2.5 Immunohistochemistry
3 Methods
3.1 Incubation of Chicken Eggs
3.2 Windowing of Chicken Eggs
3.3 Cell Suspension Preparation for CAM Implantation
3.4 CAM Implantation
3.5 Collection of CAM-Xenografted Tumors and CAM Imaging
3.6 Tissue Processing
3.7 Determination of CSCs Frequency in CAM
3.8 Haematoxylin & Eosin (H&E) Staining and CSCs Marker Validation by Immunohistochemistry (IHC)
4 Notes
References
Chapter 13: Detection of Possible Symmetries in Vascular Networks by Computer-Assisted Image Analysis
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Software Tools
3 Methods
3.1 Imaging Chick Embryo CAM
3.2 Computer-Assisted Image Pre-processing
3.3 Morphometric Characterization of a Symmetry Index
3.4 Detection of Symmetry Axes Using Orientation Histograms
3.5 Evaluation of Branching Asymmetry
4 Notes
References
Chapter 14: Alternative In Vivo Models to Study Teratoma
1 Introduction
2 Base Labware Equipment and Materials
3 Chick Embryo CAM Model of Teratoma Formation
3.1 Eggs Preparation
3.2 ESCs Implant on CAM
4 Murine Model of Teratoma Formation
4.1 ESCs Preparation
4.2 ESCs Injection
5 Conclusions
References
Chapter 15: How to Generate a Vascular-Labelled Transgenic Zebrafish Model to Study Tumor Angiogenesis and Extravasation
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Generation of Vascular-Labelled Tg Zebrafish Animals
3.2 Performing Tumor Angiogenesis Assays in Tg Zebrafish
3.3 Tumor Cell Extravasation in the Zebrafish Embryo
3.4 Imaging and Analyses of the Zebrafish Embryos
4 Notes
References
Chapter 16: 3D Human Tumor Tissues Cultured in Dynamic Conditions as Alternative In Vitro Disease Models
1 Introduction
2 Materials
3 Methods
3.1 Alginate Embedding
3.2 Cell Recovery
4 Notes
References
Index