Vasculogenic Mimicry: Methods and Protocols

Preface
Contents
Contributors
Part I: Vasculogenic Mimicry Background
	Chapter 1: Vasculogenic Mimicry-An Overview
		1 Introduction
		2 The Vasculogenic Mimicry
			2.1 VM Debates
		3 VM Identification
		References
	Chapter 2: In Vitro Models to Study Angiogenesis and Vasculature
		1 Introduction
		2 Classical Angiogenesis Assays
			2.1 ECM Degradation/Remodeling
			2.2 Cell Proliferation
			2.3 Cell Migration/Chemotaxis
			2.4 Cell Adhesion
			2.5 Vascular Morphogenesis: The Tube Formation Assay
		3 Modern Angiogenesis Models: Complex Multicellular 3D Systems
			3.1 3D Bioprinting
			3.2 3D ``Vasculature on a Chip´´: Microfluidic Devices
		4 Measuring Vasculogenic Mimicry In Vitro
		5 Conclusion
		References
Part II: Protocols for VM In Vitro
	Chapter 3: In Vitro Tube Formation Assays in Matrigel
		1 Introduction
		2 Materials
		3 Methods
			3.1 Coating Plates with Matrigel
			3.2 Cell Preparation
			3.3 Plating Cells on Matrigel
			3.4 Imaging
			3.5 Image Analysis
		4 Notes
		References
	Chapter 4: Vasculogenic Mimicry in a 3D Model In Vitro
		1 Introduction
		2 Materials
		3 Methods
			3.1 BNC-IKVAV Hydrogel Preparation
			3.2 Cell Preparation
			3.3 Seeding Cells on Model
			3.4 Imaging
		4 Notes
		References
	Chapter 5: Cancer Cell Spheroids as a 3D Model for Exploring the Pathobiology of Vasculogenic Mimicry
		1 Introduction
		2 Materials
		3 Methods
			3.1 Production of Cancer Spheroids
			3.2 Removing Spheroids from the  Mold
		4 Notes
		References
	Chapter 6: A Three-Dimensional Culture-Based Assay to Detect Early Stages of Vasculogenic Mimicry in Ovarian Cancer Cells
		1 Introduction
		2 Materials
			2.1 Cell Cultures and Buffer Solutions
		3 Methods
			3.1 Maintenance of Cell Cultures
			3.2 Transfection of Pre-miR-765 in SKOV3 Cancer Cells
			3.3 Cell Culture under Hypoxic Conditions
			3.4 Preparation of Plates Coated with Matrigel
			3.5 Vasculogenic Mimicry Assay
			3.6 Immunostaining of Three-Dimensional (3D) Channel-like Networks-Preparing Cell Cultures for Immunostaining
			3.7 Immunostaining of Three-Dimensional (3D) Channel-like Networks-Cell Fixation for Immunostaining
			3.8 Immunostaining of 3D Channel-like Networks
			3.9 Periodic Acid Staining
		4 Notes
		References
	Chapter 7: Co-immunoprecipitation of Protein Complexes from Different Subcellular Compartments in Vasculogenic Mimicry Studies
		1 Introduction
		2 Materials
			2.1 Buffers
			2.2 Immunoprecipitation (IP)
			2.3 Western Blotting
		3 Methods
			3.1 Cell Subfractionation
			3.2 Immunoprecipitation (IP) Using Protein A Sepharose
			3.3 Immunoprecipitation (IP) Using Magnetic Beads (Dynabeads)
			3.4 Protein Quantification
			3.5 Western Blotting
		4 Notes
		References
Part III: Protocols for VM In Vivo
	Chapter 8: In Vivo Models to Evaluate Antitumor Drugs Effect on Vasculogenic Mimicry
		1 Introduction
		2 Materials
			2.1 Cancer Cell Lines
			2.2 Xenograft Model
			2.3 Drug Test
			2.4 Tissue Processing and Histology
			2.5 Periodic Acid-Schiff Staining/CD31 Double Staining
		3 Methods
			3.1 Cancer Cell Lines
			3.2 Xenograft Model
			3.3 Drug Test
			3.4 Tissue Processing and Histology
			3.5 Periodic Acid-Schiff Staining/CD31 Double Staining
		4 Notes
		References
	Chapter 9: Patient-Derived Xenograft Models for Studying Vascular Mimicry in Melanoma In Vivo
		1 Introduction
		2 Materials
			2.1 Tissue Collection and Transport
			2.2 Monitoring Tumor Growth
			2.3 Tumor Harvest and Reimplantation
		3 Methods
			3.1 Patient-Derived Xenograft Melanoma Model
			3.2 Preimplantation Processing of Surgical Excision and Surgical Biopsy Samples
			3.3 Preimplantation Processing of Core Biopsy-Derived Samples
			3.4 Preimplantation Processing of FNA Tissue
			3.5 Tumor Implantation and Injections into  Mice
			3.6 Implantation of FNA or Core Biopsy Tumor Tissue
			3.7 Monitoring Tumor Growth
			3.8 Harvest the Tumor
			3.9 Tumor Tissue Banking for Further Implantation
			3.10 Trials for PDX Therapy
		4 Notes
		References
Part IV: VM Analysis
	Chapter 10: Histological Evaluation of Long-Term Collagen Type I Culture
		1 Introduction
		2 Materials
			2.1 Collagen Type I Solution and Cell Culture
			2.2 Fixation, Paraffin Embedding, H&E Staining
			2.3 PAS Staining
			2.4 CD31 immunohistochemistry staining
		3 Methods
			3.1 Preparation and Gelification of Collagen Type I Solution and Cell Seeding
			3.2 Collagen Type I Culture Fixation, Embedding, and Sectioning
			3.3 H&E Staining of Long-Term Culture Sections
			3.4 PAS Staining of Long-Term Culture Sections
			3.5 CD31 Immunohistochemistry of Long-Term Culture Sections
		4 Notes
		References
	Chapter 11: Histochemical Staining of Vasculogenic Mimicry
		1 Introduction
		2 Materials
			2.1 Solutions and Reagents
			2.2 Supplies and Equipment
		3 Methods
			3.1 Prepare Formalin-Fixed, Paraffin-Embedded Tissue Sections
			3.2 Deparaffinize/Hydrate Sections
			3.3 Antigen Retrieval Sections
			3.4 Staining Sections
		4 Notes
		References
	Chapter 12: Identification of Vasculogenic Mimicry in Histological Samples
		1 Introduction
		2 Materials
			2.1 Safe Handling of Formalin
		3 Methods
			3.1 Tissue Source
			3.2 Formalin Fixation and Paraffin Embedding
			3.3 Periodic Acid-Schiff Staining
			3.4 Histological Assessment
		4 Notes
		References
	Chapter 13: Evaluation of Pharmaceutical Inhibition of Vasculogenic Mimicry In Vitro
		1 Introduction
		2 Materials
			2.1 Growing 3D Cultures in Matrigel and In Vitro Tube Formation Assay
			2.2 Fluorescence Microscopy Imaging
		3 Methods
			3.1 Preparation of 3D Matrix
			3.2 Seeding and Growing Tumor Cells onto Three-Dimensional Matrix
			3.3 Introduction of Test Agents to Cultures
			3.4 In Vitro Tube Formation Assay
			3.5 Fluorescence Microscopy Imaging
			3.6 3D Reconstruction of Vasculogenic Mimicry Network
		4 Notes
		References
	Chapter 14: Immunohistochemistry for VM Markers
		1 Introduction
		2 Materials
			2.1 Fixation and Sample Preparation
			2.2 Antigen Retrieval
			2.3 Staining
		3 Methods
			3.1 Sample Preparation
			3.2 Section Preparation
			3.3 Deparaffinization/Rehydration
			3.4 Antigen Retrieval
			3.5 Staining
			3.6 Dehydration and Mounting
			3.7 Section Preparation
			3.8 Fixation (Optional)
			3.9 Staining
			3.10 Dehydration and Mounting
		4 Notes
		References
	Chapter 15: Confocal Laser Microscopy for VM Analysis with DAPI and Phalloidin Staining
		1 Introduction
		2 Materials
		3 Methods
			3.1 Stain Formaldehyde-Fixed Cells
			3.2 Staining the Actin Filaments and Nucleus
			3.3 Acquiring the Images
			3.4 Sequential Mode Acquisition
			3.5 Series of Images on the Z-Axis
			3.6 Exporting the Images
			3.7 3D Projection and 3D Animations
		4 Notes
		References
	Chapter 16: Building Mathematical Models for Vascular Growth and Inhibition
		1 Introduction
		2 Materials
			2.1 Vessel Formation in the Chick Yolk Sac Membrane (YSM) In Vivo Model
			2.2 Ethics
			2.3 Experiment
		3 Methods
			3.1 Mathematical Modeling Development
			3.2 Finding a Suitable Mathematical Model
			3.3 Mathematical Modeling of Blood Vessel Formation and Destruction
		4 Notes
		References
Index