Cancer Immunoprevention: Methods and Protocols

Description

This volume provides methods and techniques to further the study of cancer immunoprevention. Chapters describe tumor-associated antigens, cancer immune-preventive vaccines, generation of TILs, development of monoclonal antibodies, immunoprofiling technologies, tissue multispectral imaging techniques, mass cytometry on suspensions, mutiparametric flow cytometry, genomic expression analysis, and proteomic profiling of tumor microenvironment cell populations and metabolic assessment through novel imaging technologies.  Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Cancer Immunoprevention: Methods and Protocol aims to further understanding, development of interventional active strategies, and immune-interception of cancer. Preface Contents Contributors Chapter 1: Mechanisms of Antitumor Immunity and Immunosurveillance 1 Introduction 2 Components of Immune System Involved in Antitumor Immunity and Immune Surveillance 2.1 Innate Immunity 2.2 Adaptive Immunity 3 The Cancer Immunity Cycle 4 Clinical Implications References Chapter 2: Targets and Strategies for Cancer Immunoprevention 1 Introduction 2 Inflammatory Factors 2.1 Proinflammatory Cytokines (IL-6, IL-1, and IL-8) 2.2 Cytotoxic Lymphocyte-Associated Antigen-4 (CTLA-4) 2.3 Programmed Cell Death-1 (PD-1) 2.4 Thelper17 (T17) Cells and IL-17 2.5 STAT3 2.6 Tregs 2.7 Myeloid-Derived Suppressor Cells (MDSC) 2.8 Protumoral Neutrophils (N2) and Macrophages (M2) 3 Antitumorigenic Immune Activators 3.1 NKT Cells 3.2 Dendritic Cells 3.3 Tumor-Infiltrating Lymphocytes (TILs) 4 Vaccines References Chapter 3: Discovery of Targets for Cancer Immunoprevention 1 Introduction 2 Materials 2.1 Reagents 2.2 Reagents for Two-Dimensional Gel Electrophoresis (2-DE) 2.3 Consumables 2.4 Equipment 3 Methods 3.1 Serological Proteome Analysis (SERPA) 3.1.1 Sample Preparation 3.1.2 Bidimensional Electrophoresis (2-DE) (See Note 2) 3.1.3 Western Blotting 3.1.4 Mass Spectrometry Analysis 3.2 Immunological Characterization of Candidate Targets for Cancer Vaccines 3.2.1 Generation and Pulsing of DC 3.2.2 Co-culture of Pulsed DC and Autologous T Cells 3.2.3 Proliferation Assay 3.2.4 ELISpot Assay 3.2.5 Cr51 Release Assay 3.2.6 Win Assay (In Vivo Co-culture of Tumor and Effector T Cells) 4 Notes References Chapter 4: Exosome-related Methods and Potential Use as Vaccines 1 Introduction 2 Materials 3 Methods 3.1 Isolation of Exosomes 3.2 Characterization of Exosomes 3.3 Quantification of Exosomes 3.3.1 Loading Samples into the Nanosight Chamber 3.3.2 Visualization and Analysis of the Particles 3.4 Antigen Pulsing 3.5 In Vivo Vaccination 4 Notes References Chapter 5: Isolation and Maintenance of Tumor-Infiltrating Lymphocytes for Translational and Clinical Applications: Establishe... 1 Introduction 2 Materials 3 Methods 3.1 Pre-REP 3.1.1 Traditional Pre-REP Procedure Tumor Preparation TIL Culture TIL Cryopreservation 3.1.2 TIL 3.0 (Pre-REP): The Three Signal Approach Tumor Preparation TIL Culture TIL Cryopreservation 3.2 REP 3.2.1 Day -3, -2, -1, or 0 3.2.2 Day 0 3.2.3 Day 4 or 5 3.2.4 Day 7 3.2.5 Day 9 or 10 3.2.6 Day 11 or 12 3.2.7 Day 14 4 Notes References Chapter 6: Monoclonal Antibodies Generation: Updates and Protocols on Hybridoma Technology 1 Introduction 2 Materials 2.1 Animals 2.2 Cells 2.3 Antigens 2.4 Media/Reagents 2.5 Sterile Tissue Culture Plastics, Surgical Instruments, PAGE 2.6 Equipment 2.7 IACUC Protocols Approved for 3 Methods 3.1 Antigen Preparation 3.2 Immunization, Route, and Schedule 3.3 Euthanasia and Harvesting the Popliteal Lymph Nodes or Spleen 3.4 Hybridoma Generation 3.5 Hybridomas Colony Pickup 3.6 ELISA Screening 3.7 Selection and Confirmation of Hybridoma Clones 3.8 Hybridoma Subcloning 3.9 Purification and Concentration of the Monoclonal Antibodies 3.10 Antibody Isotyping 3.11 Antibody Characterization by SDS PAGE 4 Notes References Chapter 7: Transcriptomic-Assisted Immune and Neoantigen Profiling in Premalignancy Abbreviations 1 Introduction 2 Materials 3 Methods 3.1 Sequence Alignment and Preprocessing 3.2 Whole Transcriptomic Analysis 3.3 Mutation Analysis 3.4 Mutational Burden 3.5 Mutational Signature 3.6 Neoantigen Discovery 4 Notes 5 Conclusions References Chapter 8: Assessment of the Murine Tumor Microenvironment by Multiplex Immunofluorescence 1 Introduction 2 Materials 2.1 FFPE Tissue Slide Preparation 2.2 Opal Multiplexed Staining and Imaging 3 Methods 3.1 Tissue-Slide Fixation and Processing 3.2 Optimization of Antibodies 3.3 Immune Monoplex Staining for Multispectral Library Development 3.4 Immune Monoplex Staining for Validation 3.5 Immune Multiplexed Staining for Multispectral Imaging 3.6 Image Collection and Analysis 4 Notes References Chapter 9: Cytometry of Mass for Murine Immunoprofiling Abbreviations 1 Introduction 2 Materials 2.1 General Supplies 2.2 CyTOF Supplies 2.3 Antibody Panel Design and Considerations 3 Experimental Protocol 3.1 Preparation of Single-Cell Suspension 3.2 Viability Staining (Optional) 3.3 Surface Marker Staining 3.4 Fixation 3.5 Barcoding (Optional) 3.6 Intracellular Marker Staining 3.7 DNA Intercalation Staining 3.8 Sample Acquisition 3.9 Data Analysis 4 Notes References Chapter 10: Interrogating the Tumor Microenvironment by Deep Tissue Immunoprofiling 1 Introduction 2 Materials 2.1 Immunohistochemistry 2.1.1 Reagents 2.1.2 Equipment 2.2 Laser Capture Microdissection and RNA Isolation 2.2.1 Reagents 2.2.2 Consumables 2.2.3 Equipment 2.3 Real-Time PCR 2.3.1 Reagents 2.3.2 Consumables 2.3.3 Equipment 2.4 Tumor Tissue Processing for Lymphocytes 2.4.1 Reagents 2.4.2 Consumables 2.4.3 Equipment 2.5 Intracellular and Extracellular Staining for Multi-parameter Flow Cytometry Analysis 2.5.1 Reagents 2.5.2 Consumables 2.5.3 Equipment 3 Methods 3.1 Immunohistochemistry 3.1.1 Deparaffinization 3.1.2 Antigen Retrieval 3.2 Laser Capture Microdissection and RNA Isolation 3.2.1 Preparing Slides 3.2.2 RNA Isolation and RT-PCR: Adapted from Applied Biosystems Protocols Preamplification of cDNA PCR Amplification with TaqMan Array Plate 3.3 Tissue Collection and Storage 3.4 Weigh Tissue for Immune Cell Density 3.5 Tissue Processing 3.6 Percoll Gradient 3.7 Freezing Cells 3.8 Thawing for Frozen Cells 3.9 Counting Cells and Making Single-Cell Suspension 3.10 Surface Stain with Viability Dye 3.11 Intracellular Stimulation 3.12 Fixation and Permeabilization 3.12.1 Intracellular Stain 4 Notes References Chapter 11: Proteomic Profiling of the Tumor Microenvironment 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Collection of Total Cell Extract 2.3 Collection of Conditioned Media 2.4 Collection of Cell Surface Protein Extract 2.5 Collection of Nuclear Protein Extract 2.6 Exosome Protein Extract 2.7 Protein Fractionation and LC-MS/MS Analysis 3 Methods 3.1 Protein Extraction from Different Cell Compartments 3.1.1 Cell Culture 3.1.2 Collection of Total Cell Extract 3.1.3 Collection of Conditioned Media 3.1.4 Collection of Cell Surface Protein Extract 3.1.5 Collection of Nuclear Protein Extract 3.1.6 Exosome Protein Extract Exosome Total Protein Extract Exosome Surface Protein Extract 3.1.7 Protein Fractionation and LC-MS/MS Analysis Protein Fractionation and Trypsin Digestion Nano-LC-Coupled Ion Mobility HDMSe LC-MS/MS System 4 Notes References Chapter 12: Metabolic Imaging Using Hyperpolarization for Assessment of Premalignancy 1 Introduction 2 Materials 2.1 Genetically Engineered Mice 2.2 Hyperpolarizing [1-13C] Pyruvate 2.3 Magnetic Resonance Spectroscopy 3 Methods 3.1 Hyperpolarization Methodology 3.2 MRI and MRS 4 Notes References Chapter 13: Murine Models for Lineage Tracing Cancer Initiating Cells 1 Introduction 2 Materials 2.1 Transgenic Mice 2.2 Genotyping 2.3 Tamoxifen Administration to Achieve Cre-Mediated Recombination 2.4 Sacrificing Mouse to Collect Pancreas 2.5 Whole Mount Analysis 2.6 Flow Cytometry 2.7 IHC/IF Analysis 3 Methods 3.1 Transgenic Mice Breeding 3.2 Genotyping 3.3 Induction of Cre-Mediated Recombination with Tamoxifen 3.4 Mouse Dissection 3.5 Flow Cytometry 4 Notes References Chapter 14: Murine Model of Obesity-Induced Cancer 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 15: Lung Cancer Murine Models and Methodology for Immunopreventive Study 1 Introduction 2 Models 2.1 COPD Models 2.1.1 Cigarette Smoke Exposure 2.1.2 Bacterial Lysate Exposure 2.2 Immunomodulation 2.2.1 Neoadjuvant Immunomodulation 2.2.2 Adjuvant Immunomodulation 3 Materials 3.1 Lung Tissue and Bronchioalveolar Lavage Fluid (BALF) Collection 3.2 Lung Tissue Harvesting for Histology 3.3 Lung Tissue Harvesting for Flow Cytometry 4 Methods 4.1 Lung Tissue and Bronchioalveolar Lavage Fluid (BALF) Collection 4.2 Lung Tissue Collection for Histology 4.3 Lung Tissue Harvesting for Flow Cytometry 5 Notes References Chapter 16: Mouse Models to Study Secondary Cancer Prevention 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Surgical Procedure 3 Methods 3.1 Cell Manipulation 3.2 Pancreatic Orthotopic Tumor Implantation 3.3 Pancreatic Engrafted Tumor Removement 4 Notes References Index