Cancer Immunoprevention: Methods and Protocols

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