Ovarian Cancer: Methods and Protocols (Methods in Molecular Biology, 2424)

Description

This detailed volume provides a robust set of methods to understand variation between patients with ovarian cancers, in vitro models to better study different stages of the disease, and in vivo models to test therapies. Beginning with clinical perspectives to orient basic scientists, the book continues with sections exploring methods to characterize features of the tumor microenvironment (TME), techniques to isolate cells for in vitro study, and in vitro and in vivo models to study the disease. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Ovarian Cancer: Methods and Protocols serves as an ideal guide for researchers seeking to improve their study of ovarian cancer and find new therapeutic approaches. Preface Clinical Perspectives Characterizing the TME Cell Isolation In Vitro and In Vivo Models Concluding Thoughts References Contents Contributors Part I: Clinical Perspectives Chapter 1: Clinical Staging of Ovarian Cancer 1 Introduction 2 FIGO Staging 3 Surgical Staging of Ovarian Cancer 4 Ovarian Cancer Grade 5 Treatment of Ovarian Cancer 6 Conclusion References Chapter 2: Pathologic Classification of Ovarian Cancer 1 Introduction 2 Materials 3 Methods 3.1 Specimen and Tissue Sampling 3.2 Slide Preparation 3.3 Approach to Histologic Assessment 3.4 Approach to Epithelial Tumors 3.5 High-Grade Serous Carcinoma 3.6 Low-Grade Serous Neoplasia 3.7 Approach to Mucinous Tumors 3.8 Approach to Endometrioid and Clear Cell Tumors 3.9 Other Tumors 3.10 Tumor Staging 4 Notes References Part II: Tumor Microenvironment Chapter 3: Multiparameter Flow Cytometry for Detailed Characterization of Peritoneal Immune Cells from Patients with Ovarian C... 1 Introduction 2 Materials 3 Methods 3.1 Staining Cocktail Preparation and Single-Stain Control Staining 3.2 Immune Cells from Ascites 3.3 Cell Surface Staining 3.4 Intracellular Staining 3.5 Instrument Quality Control and Configuration 3.6 Standardizing Flow Cytometer 3.7 Data Acquisition 3.8 Computational Data Analysis 4 Notes References Chapter 4: Mass Cytometry for the Characterization of Individual Cell Types in Ovarian Solid Tumors 1 Introduction 2 Materials 2.1 Sample Collection 2.2 Tumor Dissociation into Single Cells 2.3 Cell Counting and Viability After Tumor Disaggregation 2.4 Viability and Live-Dead Staining 2.5 Fixation and Preparation for Freezing 2.6 Reagents for 20-Plex Palladium Bar-Coding 2.7 Sample Thawing and Barcoding 2.8 Antibody Conjugation with Metal-Chelated Polymers 2.9 Antibody Titrations 2.10 Antibody Staining of Individual or Bar-Coded Samples 2.11 Sample Preparation for Loading into Mass Cytometer 3 Methods 3.1 Sample Collection 3.2 Tumor Dissociation into Single Cells 3.3 Cell Counts and Viability After Tumor Disaggregation 3.4 Live-Dead Cell Staining with Cisplatin or Rhodium 3.5 Fixation and Preparation for Freezing 3.6 Preparing Palladium Barcoding Plates 3.7 Sample Thawing and Barcoding 3.8 Antibody Conjugation with Metal-Chelated Polymers 3.9 Antibody Titrations 3.10 Antibody Staining Individual Samples 3.11 Staining Combined Barcoded Samples 3.12 Preparation for Sample Loading into Mass Cytometer After Antibody Staining 3.13 Initial Analysis steps 4 Notes References Chapter 5: Processing and Analysis of Ascites 1 Introduction 2 Materials 2.1 Ascites Processing and Analysis 2.2 Cellular Processing, Storage, and Analysis 3 Methods 3.1 Ascites Collection and Storage 3.2 Initial Cell Suspension 3.3 Cryopreservation of Ascites Cells 3.4 Separation of Aggregates and Single Cells 3.5 Analysis of Aggregate Size Distribution 3.6 Embedding Aggregates for Histological Analysis 4 Notes References Chapter 6: Multispectral Staining and Analysis of Extracellular Matrix 1 Introduction 2 Materials 2.1 Tissue Sections 2.2 Deparaffinization 2.3 Wash and Slide Preparation 2.4 Antigen Retrieval 2.5 Staining 2.6 Coverslip Mounting 2.7 Microscopy and Analysis 3 Methods 3.1 Generation of the Spectral Library 3.2 Imaging the Spectral Library 3.3 Computing the Spectral Library 3.4 Multispectral Detection of ECM Proteins 3.5 Acquisition and Processing of Multispectral ECM Images 3.6 Image Quantification 4 Notes References Chapter 7: Quantitative Analysis of the Extracellular Matrix by Immunoblot 1 Introduction 2 Materials 2.1 FFPE Blocks 2.2 Collagen Type I Standard 2.3 Protein Extraction 2.4 Quantitative Dot Blot 2.5 Fluorescent Immunostaining 3 Methods 3.1 Preparing a Standard 3.2 Protein Extraction 3.3 Dot Blot 3.4 Deparaffinization 3.5 Antigen Retrieval 3.6 Immunostaining 3.7 Imaging 3.8 Analysis 4 Notes References Part III: Cell Isolation Chapter 8: Multiparameter Single-Cell Characterization of Ovarian Intratumor Heterogeneity 1 Introduction 2 Materials 2.1 Generation of Single-Cell Suspension 2.2 Beacon Chip Preparation and Cell Loading 2.3 Single-Cell DNA Sequencing Preparation 2.4 Single-Cell DNA Sequencing 3 Methods 3.1 Generation of Single-Cell Suspension 3.2 Beacon Chip Preparation and Cell Loading 3.3 Phenotypic Characterization of Cells 3.4 Single-Cell DNA Sequencing Preparation 3.5 Single-Cell DNA Sequencing 4 Notes References Chapter 9: Culturing Primary Human Mesothelial Cells 1 Introduction 2 Materials 3 Methods 3.1 Tissue Procurement 3.2 HPMC Isolation 3.3 Second Wash (HPMC) 3.4 Maintenance of Growing HPMCs 4 Notes References Chapter 10: Isolation of Normal and Cancer-Associated Fibroblasts 1 Introduction 2 Materials 2.1 Isolation of CAFs and Normal Fibroblasts 2.2 Cryopreservation of Primary Cells 2.3 Cryorecovery of Primary Cells 3 Methods 3.1 Isolation of CAFs and Normal Fibroblasts 3.2 Cryopreservation of Primary Cells 3.3 Cryorecovery of Primary Cells 4 Notes References Chapter 11: Isolation of Primary Normal and Cancer-Associated Adipocytes from the Omentum 1 Introduction 2 Materials 2.1 Tissue Procurement 2.2 General Materials 2.3 Solutions 2.4 Nylon Mesh-Lined Funnel 3 Methods 3.1 Isolation of Adipocytes from Benign Tissue 3.2 Isolating Cancer-Associated Adipocytes 4 Notes References Part IV: Model Systems Chapter 12: Isolation of Fallopian Tube Epithelium for Assessment of Cilia Beating Frequency (CBF) 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Media 2.3 Equipment and Plates 3 Method 3.1 Isolation of Fallopian Tube Epithelium 3.2 Isolation of Murine Ovaries and Oviducts 3.3 Capturing Cilia Beating 3.4 Measuring CBF 4 Notes References Chapter 13: Ex Vivo Ovarian Culture to Model the Initial Metastasis in Ovarian Cancer 1 Introduction 2 Materials 2.1 Mouse Ovaries 2.2 Cell Culture 2.3 Labeling Cells with CellTracker Dye 2.4 Cell Attachment to Ovaries 2.5 Counting and Imaging Cells Attached to Ovaries 3 Methods 3.1 Isolation of Mouse Ovaries 3.2 Removal of Ovaries from the Bursa 3.3 Collecting Cells Stably Expressing a Fluorescent Protein 3.4 Collecting and Labeling Cells with Fluorescent CellTracker Dye 3.5 Attachment of Fluorescent Cells to Ovaries 3.6 Counting Cells Attached to Each Ovary 3.7 Generating Images of Cells Attached to the Ovary 4 Notes References Chapter 14: In Vivo and Ex Vivo Analysis of Omental Adhesion in Ovarian Cancer 1 Introduction 1.1 Murine In Vivo Models 1.2 Microscopy: Second Harmonic Generation Microscopy 1.3 Microscopy: Scanning Electron Microscopy 1.4 Histology 1.5 Ex Vivo Adhesion 2 Materials 2.1 In Vivo Adhesion and Tumor Study 2.2 Second Harmonic Generation Microscopy 2.3 Histology 2.4 Ex Vivo Adhesion Assay 2.5 Scanning Electron Microscopy 3 Methods 3.1 In Vivo Adhesion and Tumor Study Analysis 3.2 Second Harmonic Generation Microscopy: In Vivo Assays 3.3 Histology: Hematoxylin and Eosin Assay 3.4 Histology: Trichrome Staining 3.5 Histology: Immunohistochemistry 3.6 Ex Vivo Adhesion Fluorescence Assay 3.7 Scanning Electron Microscopy: In Vivo or Ex Vivo Adhesion Assays 4 Notes References Chapter 15: The Role of the Tumor Microenvironment in CSC Enrichment and Chemoresistance: 3D Co-culture Methods 1 Introduction 2 Materials 2.1 Cells and Culture Medium 2.2 General Tissue Culture 2.3 3D Hanging Drop Platform 2.4 Fluorescence-Activated Cell Sorting 2.5 Lentiviral Transduction 2.6 MTS Assay 2.7 Transwell Assay 2.8 ELISA Assay 2.9 RT-qPCR 3 Methods 3.1 Isolation of CSC Using FACS 3.2 Preparation of Hanging Drop Plate 3.3 Generation of 3D Monoculture Tumoroids 3.4 Generation of 3D CSC-MSC Co-culture Tumoroids 3.5 Generation of 3D CSC-U937 Monocyte Co-culture Tumoroids 3.6 Differentiation of Monocytes Prior to Co-culture 3.7 Maintenance of 3D Hanging Drop Cultures 3.8 Transduction of Adherent Cells 3.9 Transduction for Nonadherent Cells 3.10 FACS Analysis of Cell Populations and Viability 3.11 FACS to Collect Cells for Downstream Analysis 3.12 MTS Assay 3.13 Transwell Assay 3.14 ELISA 3.15 Evaluating Transcriptional Changes in Co-culture Tumoroids 4 Notes References Chapter 16: Establishment of In Vivo Ovarian Cancer Mouse Models Using Intraperitoneal Tumor Cell Injection 1 Introduction 2 Materials 2.1 Cell Culture and Collection 2.2 Animal Injection 2.3 In Vivo Imaging and Quantification of Tumor Burden 3 Methods 3.1 Preparation of Cells for Injection 3.2 Intraperitoneal (I.P.) Injection 3.3 In Vivo Imaging and Quantification of Tumor Burden 4 Notes References Chapter 17: Humanized Patient-Derived Xenograft Models of Ovarian Cancer 1 Introduction 2 Materials 2.1 Primary Tumor Digestion 2.2 Feeder Cells from PBMCs 2.3 Artificial Antigen-Presenting Cells (aAPCs) 2.4 TIL Rapid Expansion 2.5 Characterization of Expanded T Cells 2.6 Development of Orthotopic PDX Models 2.7 Tumor Monitoring 2.8 Tumor Collection 2.9 Preclinical Studies with TILs 3 Methods 3.1 Primary Tumor Digestion 3.2 Feeder Cells from PBMCs 3.3 Artificial Antigen Presenting Cells (aAPCs) 3.4 TIL Rapid Expansion 3.5 Characterization of Expanded T Cells 3.6 Development of Orthotopic PDX Models 3.7 Tumor Monitoring 3.8 Tumor Collection for Expansion or Banking 3.9 Preclinical Studies with Administration of Autologous TILs 4 Notes References Chapter 18: Xenograft Models of Ovarian Cancer for Therapy Evaluation 1 Introduction 2 Materials 2.1 Preparation of Ovarian Cancer Cell Lines for Injection 2.2 General, for In Vivo Experiments 2.3 Subcutaneous Model 2.4 Orthotopic Model 2.5 Optical Imaging for Tumor Burden 2.6 Treatment Protocols: Drug Treatments 2.7 Treatment Protocols: Surgical 3 Methods 3.1 Preparation of Ovarian Cancer Cell Lines for Injection 3.2 Subcutaneous Model 3.3 Orthotopic Model 3.4 Optical Imaging for Tumor Burden 3.5 Treatment Protocols: Drug Treatments 3.6 Treatment Protocols: Surgical 4 Notes References Chapter 19: Confocal Imaging of Single-Cell Signaling in Orthotopic Models of Ovarian Cancer 1 Introduction 2 Materials 2.1 Cell Lines and Lentiviral Transduction 2.2 Orthotopic Tumor Inoculation and Treatment 2.3 Confocal Microscopy 3 Methods 3.1 Plasmid Preparation 3.2 Lentivirus Preparation 3.3 Reporter Cell Line Generation 3.4 In Vitro Reporter Cell Line Characterization 3.5 Orthotopic Tumor Inoculation and Treatment 3.6 Fluorescent Probe Injection 3.7 Terminal Dissection and Confocal Microscopy 4 Notes References Index