Cancer Immunotherapy: Methods and Protocols (Methods in Molecular Biology, 2748)

Preface: Cancer Immunotherapy
Contents
Contributors
Chapter 1: Isolation of Live Immune Cells from the Tumor Microenvironment by FACS
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Reagents
		2.3 Solutions
		2.4 Equipment
	3 Experimental Procedure
		3.1 Metastatic Colonization in the Lung Using an In Vivo Mouse Model
		3.2 Mouse Lung Tissue Processing to Obtain a Single Cell Suspension
		3.3 Staining Procedure to Identify and Isolate Neutrophils from the TME
		3.4 Downstream Applications for Isolated Neutrophils (Ex Vivo)
			3.4.1 Giemsa Staining
			3.4.2 ROS Production Assay
			3.4.3 Phagocytosis Assay
	4 Notes
		4.1 Metastatic Colonization in the Lung Using an In Vivo Mouse Model
		4.2 Mouse Lung Tissue Processing to Obtain a Single-Cell Suspension
		4.3 Staining Procedure to Identify and Isolate Neutrophils from the TME
		4.4 Downstream Applications for Isolated Neutrophils (Ex Vivo)
	References
Chapter 2: In Vitro Evaluation of Cancer Cell Immunogenicity and Antigen-Specific T-Cell Cytotoxicity by Flow Cytometry
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Disposable
		2.2 Equipment
		2.3 Reagents
	3 Methods
		3.1 Cell Culture Routine
		3.2 Bone Marrow (BM) Processing and DC Differentiation (See Note 10)
		3.3 Spleen Processing and CD8 OT-1 T Cell Isolation
		3.4 CD8 OT-1 T Cell Cross-Priming and Activation.
		3.5 CD8 OT-1 T Cell Proliferation Analysis by Flow Cytometry
		3.6 Cancer Cell Killing Analysis by Flow Cytometry
	4 Remarks
	5 Notes
	References
Chapter 3: Retroviral Transduction of Human Primary T Cells Followed by Real-Time T-Cell-Mediated Cancer Cell Cytolysis Analys...
	1 Introduction
	2 Materials
		2.1 Culturing of Packaging Cells
		2.2 Transfection
		2.3 Isolation and Activation of T Cells
		2.4 Preparing Plates for Transfected Cells
		2.5 Transduction of T Cells
		2.6 Test Transduction Efficiency by Flow Cytometry
		2.7 xCELLigence Assay
	3 Methods
		3.1 Seeding of Packaging Cells: Day 1
		3.2 Transfection of Packaging Cells: Day 2
		3.3 Isolation and Activation of T Cells: Day 2
		3.4 Coat Plates with RetroNectin: Day 3
		3.5 Give New Media to Packaging Cells: Day 3
		3.6 Transduction of Human Primary T Cells (Hit 1): Day 4
		3.7 Transduction of Human Primary T Cells (Hit 2): Day 5
		3.8 Transfer Cells to TCT Plate
		3.9 Test Transduction Efficiency by Flow Cytometry: Day 8
		3.10 Sort Cells
		3.11 Culturing of Cells
		3.12 Titration of Target Cells in xCELLigence RTCA SP System (See Note 9)
		3.13 Cytolysis Assay in xCELLigence
	4 Notes
	References
Chapter 4: Expansion and Retroviral Transduction of Primary Murine T Cells for CAR T-Cell Therapy
	1 Introduction
	2 Materials
		2.1 293T Cell Culture
		2.2 Transfection
		2.3 Splenocyte Collection
		2.4 T-Cell Activation and Culture
		2.5 Transduction
			2.5.1 Validation of Transduction
	3 Methods
		3.1 Retrovirus Production
			3.1.1 Day -1: Plate 293 T Cells for Transfection
			3.1.2 Day 0: 293T Cell Transfection
			3.1.3 Day 0: Splenocyte Collection
			3.1.4 Day 1: Replace Media on 293T Cells
		3.2 Transduction
			3.2.1 Method A: Spinoculation Using Transduction Enhancer RetroNectin
				Day 1: RetroNectin Coating
				Day 2: Harvest Retrovirus Supernatant
				Day 2: T-Cell Transduction
				Day 3: Expand T Cells
			3.2.2 Method B: Spinoculation Using Transduction Enhancer Vectofusin-1
				Day 2: Harvest Retrovirus Supernatant
				Day 2: T-Cell Transduction
				Day 3: Expand T Cells
			3.2.3 Method C: Static Transduction Using Vectofusin-1
				Day 2: Harvest Retrovirus Supernatant
				Day 2: T-Cell Transduction
				Day 3: Maintain T-Cell Activation (Only for Method C1)
		3.3 Evaluation of CAR Transduction
			3.3.1 Day 4 or 5: Harvest CAR T Cells
	4 Notes
	References
Chapter 5: In Situ Decellularization of Tissues Applied to the Topographical Analysis of Tumor-Associated Extracellular Matrix
	1 Introduction
	2 Materials
		2.1 Surgery for Decellularization (Fig. 2)
		2.2 For Tissue Preparation and Immunostaining
		2.3 For Imaging
		2.4 Reagents (See Note 2)
		2.5 Immunostaining
		2.6 Secondary Antibodies
	3 Methods
		3.1 Setup
			3.1.1 Two-Photon Microscopy Setup
		3.2 Procedure
			3.2.1 Preparation
			3.2.2 Surgery
			3.2.3 Surgery to Decellularize Tissues in the Territory of the Left Subclavian Artery and the Cardiopulmonary Complex (i.e., P...
		3.3 Decellularization
			3.3.1 Preparing Decellularized Tissues for Immunostaining
		3.4 Preparation for Imaging of the Decellularized Tissues with a Multiphoton/Confocal Fluorescence Microscope
		3.5 Image Acquisition (Fig. 3)
	4 Notes
	References
Chapter 6: Monitoring Cell Cytoskeleton Variations upon Piezoelectric Stimulation: Implications for the Immune System
	1 Introduction
	2 Materials
		2.1 Preparation of the Particle Dispersion
		2.2 Cell Culture
		2.3 Scratch/Migration Assay
		2.4 f-/g-Actin Ratio
		2.5 Trans-Well Invasion Assay (Abcam, ab235887)
		2.6 Equipment
		2.7 Image and Statistical Analysis
	3 Methods
		3.1 Preparation of the Particle Dispersion
		3.2 Cell Seeding
		3.3 Migration Assay
		3.4 Actin Cytoskeleton Organization
		3.5 Invasion Assay
	4 Notes
	5 Summary
	References
Chapter 7: Preparation Method and In Vitro Characterization of Nanoparticles Sensitive to Tumor Microenvironment
	1 Introduction
	2 Materials
		2.1 Copolymers and Conjugates
		2.2 Cell Culture Reagents
		2.3 MMP2 Cleavage Assay
		2.4 Nanoparticle Cytotoxicity
			2.4.1 Alamar Blue Assay
			2.4.2 Confocal Microscope Imaging
			2.4.3 Quantification of NP Cellular Uptake Kinetics
	3 Methods
		3.1 NP Preparation
		3.2 PELGA NP Physical-Chemical Characterization
			3.2.1 DLS
			3.2.2 SEM
			3.2.3 Cryo-TEM
		3.3 Biocompatibility of PELGA NPs
			3.3.1 Alamar Blue Assay
			3.3.2 Confocal Microscopy
			3.3.3 Uptake Quantification Analysis
		3.4 MMP2-Mediated Drug Release Assay
		3.5 Spheroid Formation
		3.6 Cytotoxicity Test
	4 Notes
	References
Chapter 8: A New Microfluidic Device to Facilitate Functional Precision Medicine Assays
	1 Introduction
	2 Materials
		2.1 SU8 Mold Fabrication
		2.2 PDMS Replica Molding
		2.3 Microfluidic-Based Dynamic BH3 Profiling
	3 Methods
		3.1 SU8 Mold Fabrication to Create PDMS Microfluidic Chip
		3.2 PDMS Microfluidic Chip Production
		3.3 Functional Assays Using the Microfluidic Platform
	4 Notes
	References
Chapter 9: Kinetic Detection of Apoptosis Events Via Caspase 3/7 Activation in a Tumor-Immune Microenvironment on a Chip
	1 Introduction
	2 Materials
		2.1 Cell Culture and Collagen Preparation
		2.2 Equipment
	3 Methods
		3.1 Microfluidic Chip Design and Process
		3.2 MC-38 Cell Culture
		3.3 Nuclear Staining
		3.4 Collagen Preparation
		3.5 Caspase 3/7 Assay
		3.6 Splenocytes Cell Culture and Coculture
		3.7 Image Acquisition
		3.8 Image Analysis
	4 Notes
	References
Chapter 10: Hypoxic 3D Tumor Model for Evaluating of CAR-T Cell Therapy In Vitro
	1 Introduction
	2 Materials
		2.1 Hypoxia Microdevice Design and Fabrication
		2.2 Establishing 3D Hypoxic Tumor Model
		2.3 Combination CAR-T and PD-1/PD-L1 Inhibition Therapy
		2.4 Cytotoxicity Assay
		2.5 Immunostaining
		2.6 Microscopy
	3 Methods
		3.1 Hypoxia Microdevice Design and Fabrication
		3.2 Establishing 3D Hypoxic Tumor Model
		3.3 Combinatorial CAR-T and PD-1/PD-L1 Inhibition Therapy
		3.4 Evaluating the Therapeutic Efficacy of PD-L1 Blockade and CAR-T Cell Combination Treatment
			3.4.1 Cytotoxicity Assay
			3.4.2 CAR-T Cell Infiltration
	4 Notes
	References
Chapter 11: Rapid Screening of CAR T Cell Functional Improvement Strategies by Highly Multiplexed Single-Cell Secretomics
	1 Introduction
	2 Materials
		2.1 Cell Culture and Single-Cell Secretome Assay
		2.2 Dead Cell Removal
		2.3 Intracellular Cytokine Staining
		2.4 Equipment and Consumables
	3 Methods
		3.1 Cultivation of Target and Nontarget Cell Lines
		3.2 Thawing Cryopreserved CAR T Cells
		3.3 Antigen-Specific Stimulation of CD19-Specific CAR CD8+ T Cell
		3.4 Target Cell Depletion
		3.5 Chip Thawing
		3.6 Membrane Staining (Optional)
		3.7 CD8-AF647 Staining
		3.8 Chip Loading
		3.9 Loading Reagent Tubes on IsoLight
		3.10 Run the Assay on IsoLight
		3.11 Data Analysis
	4 Notes
	References
Chapter 12: Genome Editing in CAR-T Cells Using CRISPR/Cas9 Technology
	1 Introduction
	2 Materials
		2.1 T-Cell Isolation
		2.2 Generation of CRISPR/Cas9-Edited CAR-T Cells
		2.3 Analysis of Knockout Efficiency
	3 Methods
		3.1 Generation of CAR-T Cells
			3.1.1 T-Cell Isolation
			3.1.2 T-Cell Stimulation and Transduction
		3.2 Gene Editing by CRISPR/Cas9 Technology
			3.2.1 Debeading
			3.2.2 Single-Guide RNA (sgRNA) Design
			3.2.3 T-Cell Preparation
			3.2.4 Preparation of RNP Complexes
			3.2.5 Electroporation of RNP Complexes
			3.2.6 Postediting CAR-T Cell Expansion
		3.3 Analysis of Knockout Efficiency
			3.3.1 DNA Level
			3.3.2 Protein Level
	4 Notes
	References
Chapter 13: Genetic Modification of Tumor-Infiltrating Lymphocytes, Peripheral T Cells, and T-Cell Model Cell Lines
	1 Introduction
	2 Materials
		2.1 Preparation of TIL Cultures
			2.1.1 Establishment of TIL Cultures
			2.1.2 Expansion of TIL Cultures
		2.2 Preparation of PBMCs, T Cells, and T-Cell Lines
			2.2.1 Activation of PBMCs
			2.2.2 Preparation of T Cells from PBMCs
			2.2.3 T-Cell Substitutes
		2.3 Genetic Modification of TILs, PBMCs, T Cells, or Cell Lines
			2.3.1 Viral Transduction
				Virus Preparation
				Preparation of RetroNectin-Coated Dishes
				Binding of Retrovirus to RetroNectin-Coated Plates
				Retrovirus Transduction of T Cells and  TILs
				Maintenance of Transduced  TILs
			2.3.2 mRNA Transfection of T Cells and  TILs
		2.4 Flow Cytometry to Assess Transduction/Transfection Efficiency of  TILs
	3 Methods
		3.1 Preparation of  TILs
			3.1.1 Establishment of Unselected/Young TIL Cultures
				Cultivation of Fragments
				Cultivation of TILs and Tumor Cells Obtained by Enzymatic Digestion of Tumor Tissue Fragments
				Tissue Remnant Culture (TRC)
				Pooling of Cells Obtained from the Three Methods Described Above and Separation of TILs and Melanoma Cells
			3.1.2 Rapid Expansion Protocol (REP) of  TILs
		3.2 Preparation of PBMCs, T Cells, or T-Cell Lines
			3.2.1 Activation of PBMCs
				Cell Culture Setup and Activation
				Cell Release by Dissolution of Cloudz CD3/CD28 Microspheres and Cell Collection
			3.2.2 Preparation of T Cells from PBMCs (Magnetic Labeling; Fig. 3)
			3.2.3 Preparation of T-Cell Lines
		3.3 Genetic Modification of TILs, PBMCs, T Cells, or Cell Lines (Fig. 4)
			3.3.1 Viral Transduction
				Virus Preparation
				Preparation of RetroNectin-Coated Dishes
				Binding of Retrovirus to RetroNectin-Coated Plates
				Retrovirus Transduction of T Cells and  TILs
				Maintenance of Transduced T Cells
				Maintenance of Transduced  TILs
			3.3.2 mRNA Transfection of TILs and T Cells
		3.4 Flow Cytometry to Assess Transduction/Transfection Efficiency of  TILs
	4 Notes
	References
Chapter 14: Transposon-Based Manufacturing of Human CAR-T Cells
	1 Introduction
	2 Materials
		2.1 Plasmids (Fig. 1)
		2.2 PBMC Isolation from Human Peripheral Blood Sample
		2.3 Transfection
		2.4 Cell Culture and CAR-T Cell Expansion
		2.5 Cryopreservation of CAR-T Cells Post Expansion
		2.6 Generation of HEK293Tscan-CD19+ Cells for Granzyme
		2.7 HLA Null Granzyme Reporter Cells (HEK293Tscan-CD19+) Cell Culture
		2.8 Flow Cytometry Analysis
	3 Methods
		3.1 Plasmid Construction and Preparation
		3.2 PBMC Isolation
		3.3 Stable Transfection of PBMCs
		3.4 Expansion of CAR-T Cells
		3.5 Cryopreservation of Expanded CAR-T Cells
		3.6 CAR-T Cell Phenotype Analysis
		3.7 Generation of HEK293Tscan-CD19+ Cells
		3.8 Assessing the CAR-T Killing Capacity Using HEK293Tscan-CD19+ (Granzyme Attack Assay), CAR-T Cells and HEK293Tscan-CD19+ Ce...
		3.9 CAR-T Cell Functional Analysis
	4 Notes
	References
Chapter 15: Redirecting Human Conventional and Regulatory T Cells Using Chimeric Antigen Receptors
	1 Introduction
	2 Materials
		2.1 T Cell Isolation
		2.2 Lentivirus Production
		2.3 T Cell Transduction
		2.4 CAR+ T Cell Sorting
		2.5 Immune Assays
			2.5.1 Activation
			2.5.2 Cytotoxicity
			2.5.3 Suppression
			2.5.4 Expansion
			2.5.5 Exhaustion
			2.5.6 Stability
	3 Methods
		3.1 T Cell Isolation
			3.1.1 Leukopak Processing
			3.1.2 CD4+ T Cell Enrichment (Negative Selection)
			3.1.3 CD8+ T Cell Enrichment (Negative Selection)
			3.1.4 CD25+ Cell Enrichment (Positive Selection)
			3.1.5 CD4+CD25+CD127- Treg Sorting (FACS)
			3.1.6 T Cell Activation
		3.2 Lentivirus Production
		3.3 T Cell Transduction
		3.4 CAR+ T Cell Sorting
		3.5 Immune Assays
			3.5.1 Activation
			3.5.2 Cytotoxicity
			3.5.3 Suppression
			3.5.4 Expansion
			3.5.5 Exhaustion
			3.5.6 Stability
	4 Notes
	References
Chapter 16: How to Test Human CAR T Cells in Solid Tumors, the Next Frontier of CAR T Cell Therapy
	1 Introduction
	2 Materials
		2.1 T Cell Isolation
		2.2 Transduction
		2.3 K562 Subcutaneous Injection
		2.4 CAR T Cell Intravenous Injection
		2.5 Solid Tumor Measurement
		2.6 Tissue Dissection
		2.7 Tissue Processing
		2.8 Flow Cytometry
	3 Methods
		3.1 T Cell Isolation
			3.1.1 Leukopak Processing
			3.1.2 CD4+ T Cell Enrichment (Negative Selection)
			3.1.3 CD8+ T Cell Enrichment (Negative Selection)
			3.1.4 T Cell Activation and Culture
		3.2 Transductions
			3.2.1 T Cell Transduction
			3.2.2 K562 Culture and Transduction
			3.2.3 Antigen+ K562 Sorting
		3.3 K562 Subcutaneous Injection
		3.4 CAR T Cell Intravenous Injection
		3.5 Solid Tumor Measurements
		3.6 Dissections
			3.6.1 Tumor Dissection
			3.6.2 Spleen Dissection
		3.7 Tissue Processing
			3.7.1 Tumor Tissue
			3.7.2 Spleen
		3.8 Flow Cytometry
	4 Notes
	References
Chapter 17: Nano-optogenetic CAR-T Cell Immunotherapy
	1 Introduction
	2 Materials
		2.1 Media
		2.2 Buffers
		2.3 Antibodies, Reagents, and Kits
		2.4 Chemicals
	3 Methods
		3.1 Isolation of Human Pan T-Cells from PBMC
		3.2 Transduction of CAR Constructs into T Cells
		3.3 UCNP Synthesis
		3.4 T-Cell Biotinylation and Coupling with Stv-UCNPs
		3.5 Mouse Model
	References
Chapter 18: A Nonviral piggyBac Transposon-Mediated Method to Generate Large-Scale CAR-NK Cells from Human Peripheral Blood Pr...
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Cell Isolation
		2.3 Electroporation
		2.4 Phenotyping
		2.5 Functional Assays
	3 Methods
		3.1 Peripheral Blood Mononuclear Cells and Primary Natural Killing Cell Isolation
		3.2 NK Cell Activation and Expansion
		3.3 Generation of CAR-NK Cells with piggyBac Transposon System
		3.4 Large-Scale CAR-NK Cell Enrichment and Expansion
		3.5 Phenotyping of CAR-NK Cells
		3.6 Functional Characterization of CAR-NK Cells by In Vitro Cytotoxicity Assay (See Note 9)
		3.7 Functional Characterization of CAR-NK Cells by CD107a Degranulation Assay
	4 Notes
	References
Chapter 19: Engineering Probiotic E. coli Nissle 1917 for Release of Therapeutic Nanobodies
	1 Introduction
	2 Materials
		2.1 Cell Lines (See Notes 1 and 2)
		2.2 Plasmids
		2.3 Bacterial Strains
		2.4 Mice (See Notes 3-6)
		2.5 General Reagents
		2.6 Equipment for Tissue Culture
		2.7 Equipment for Bacteria Culture and Transformation
		2.8 Equipment for Mouse Experiments
		2.9 Reagents for Ex Vivo Analysis and Flow Cytometry
		2.10 Reagents for Protein Visualization and Purification
		2.11 Imaging and Software
	3 Methods
		3.1 Plasmids and Strain Engineering
			3.1.1 Therapeutic Plasmids
			3.1.2 SLIC Strain
			3.1.3 E. coli Expression Strains for Downstream Purification
			3.1.4 Electrocompetent EcN-lux Strains (See Note 12)
		3.2 Tissue Culture
			3.2.1 Thaw Frozen Tumor Cells from a Liquid Nitrogen Stock
			3.2.2 Expanding Tumor Cell Lines
		3.3 Characterization of Nanobodies
			3.3.1 Purification of Nanobodies
			3.3.2 Collect Probiotically Produced PD-L1 and CTLA-4 Protein (See Notes 14)
			3.3.3 PD-L1 Nanobody (PD-L1nb) Binding (See Note 17)
			3.3.4 CTLA-4 Nanobody Binding
		3.4 Therapeutic Evaluation in Mouse Models
			3.4.1 Establishment of Subcutaneous Model
			3.4.2 Therapeutic Bacteria Preparation
			3.4.3 Therapeutic Bacteria Administration
			3.4.4 Monitoring In Vivo Bacteria Dynamics
		3.5 Evaluation of Safety
			3.5.1 Biodistribution
			3.5.2 Characterization of Immune Response
	4 Notes
	References
Index