The Tumor Microenvironment: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Imaging Mass Cytometry in Immuno-Oncology
	1 Introduction
	2 Materials
	3 Methods
		3.1 Histology and Sample Preparation
		3.2 Panel Design Considerations
		3.3 Sample Staining
		3.4 Data Acquisition
		3.5 Data Visualization, Pre-processing, and Analysis
	4 Notes
	References
Chapter 2: Analyzing the Tumor-Immune Microenvironment by Flow Cytometry
	1 Introduction
		1.1 General Principles of Flow Cytometry
		1.2 Instrumentation
		1.3 Panel Design
		1.4 Flow Cytometry Controls
		1.5 Instrument Controls
		1.6 Sensitivity Controls
		1.7 Biological Controls
	2 Materials
		2.1 Sample Preparation
		2.2 External/Internal Staining
	3 Methods
		3.1 Sample Preparation (Solid Tumors)
		3.2 Sample Preparation (Spleen/Lymphoid Tissues)
		3.3 External Epitope Staining Protocol
		3.4 Internal Epitope Staining Protocol
		3.5 Sample Analysis and Gating Strategies
	4 Notes
	References
Chapter 3: Blood Sample Processing and Banking for Functional and Molecular Analyses
	1 Introduction
	2 Materials
	3 Methods
		3.1 Plasma Isolation
		3.2 PBMC Isolation
		3.3 PBMC Thawing
	4 Notes
	References
Chapter 4: Quantitatively Assessing the Respiratory Burst in Innate Immune Cells
	1 Introduction
	2 Materials
		2.1 Preparation of Mouse Neutrophils or Macrophages
		2.2 Preparation of Human Neutrophils or Macrophages
		2.3 ROS Detection
	3 Methods
		3.1 Ex Vivo Culture-Derived Common Myeloid Progenitors (CMPs)
		3.2 Generation of Ex Vivo Culture-Derived Mouse Neutrophils
		3.3 Generation of Ex Vivo Culture-Derived Mouse Macrophages
		3.4 In Vitro Generation of Human Neutrophils from Myeloblastic HL-60 Cells
		3.5 In Vitro Generation of Human Macrophages from Monocytic U937 Cells
		3.6 Quantitation of Stimulated Neutrophil or Macrophage Respiratory Burst
	4 Notes
	References
Chapter 5: Visualizing NETosis Using a Novel Neutrophil Extracellular Trap-Specific Marker
	1 Introduction
	2 Materials
		2.1 Neutrophil Isolation and Preparation
		2.2 Immunofluorescent Staining
	3 Methods
		3.1 Neutrophil Isolation and Preparation
		3.2 Immunofluorescent Staining
	4 Notes
	References
Chapter 6: In Vitro Methods to Evaluate Macrophage Polarization and Function in Cancer
	1 Introduction
	2 Materials
		2.1 Cell Culture Media
		2.2 Isolation of PBMCs from Whole Blood
		2.3 Differentiation of PBMCs and Macrophage Polarization
		2.4 RNA Extraction and Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)
		2.5 ELISA Assays
		2.6 Flow Cytometry
		2.7 Analysis of Human Monocyte-Derived Macrophages for In Vitro Tumor Cytotoxicity Assays
	3 Methods
		3.1 Isolation of Human Peripheral Blood Mononuclear Cells
		3.2 Isolation of Monocytes
		3.3 Monocyte Differentiation into Macrophages
		3.4 RT-qPCR to Assess Macrophage Polarization
		3.5 Flow Cytometry to Assess Macrophage Polarization
		3.6 ELISA Assays to Assess Macrophage Polarization
		3.7 Cytotoxic Activity of Macrophages Toward Cancer Cells In Vitro
	4 Notes
	References
Chapter 7: In Vivo Analysis of Tumor-Associated Macrophages in the Tumor Microenvironment
	1 Introduction
	2 Materials
		2.1 Establishing In Vivo Models to Evaluate Tumor Growth Using Immunocompetent Mice
		2.2 RT-qPCR Analysis
		2.3 Fluorescence-Activated Cell Sorting (FACS) Analysis
		2.4 Multiplex Immunophenotyping (Akoya Biosciences)
	3 Methods
		3.1 Subcutaneous Injection of Tumor Cells
		3.2 Tumor Measurement and Tissue Harvesting
		3.3 RT-qPCR Analysis
		3.4 FACS Analysis of Tumor-Infiltrating Immune Cells
		3.5 Multiplex Immunophenotyping
	4 Notes
	References
Chapter 8: Assessing the Immune Regulatory Role of Metabolites In Vitro
	1 Introduction
	2 Materials
		2.1 Activate and Assess T Cell Proliferation in the Presence of MNA
		2.2 Assess T Cell Function by Flow Cytometry
	3 Methods
		3.1 Prepare Anti-CD3 Bound Plates
		3.2 T Cell Isolation
		3.3 Activate and Assess T Cell Proliferation in the Presence of MNA
		3.4 Assess T Cell Function by Flow Cytometry
	4 Notes
	References
Chapter 9: Mouse Models of Obesity to Study the Tumor-Immune Microenvironment
	1 Introduction
	2 Materials
		2.1 Mouse Models: Mice and Diets Required
		2.2 Monitoring Obesity
		2.3 Assessing Inflammation by Flow Cytometry
	3 Methods
		3.1 C57BL/6 Mouse Model
		3.2 Balb/c Mouse Model
		3.3 Ob/Ob Mouse Model
		3.4 Monitoring Obesity (Weight Curves)
		3.5 Monitoring Obesity (Glucose Tolerance Test)
		3.6 Monitoring Obesity (Body Mass Composition)
		3.7 Blood Processing to Assess Inflammation
		3.8 Tissue Processing to Assess Inflammation
		3.9 Assessing Inflammation with Flow Cytometry
		3.10 Syngeneic Tumor Cell Transplantation
	4 Notes
	References
Chapter 10: Generation and Quantification of Cytotoxic Lymphocytes Following Oncolytic Virus Infection of Multi-cellular Tumor...
	1 Introduction
	2 Materials
		2.1 Generation of Ultra-Low Binding Plates
		2.2 Cell Culture
		2.3 Buffers and Reagents
	3 Methods
		3.1 Coating Ultra-Low Binding Plates
		3.2 Generation and OV Infection of Multi-cellular Tumor Spheroids (MCTS)
		3.3 Isolation and Culture of Antigen-Presenting Cells (CD14+ Cells) and T Cells (CD14- Cells)
		3.4 Stimulation of T Cells with Antigen-Loaded iDCs
		3.5 Restimulating Cytotoxic T Cell Cultures
		3.6 Measuring CTL Responses by Peptide Recall
	4 Notes
	References
Chapter 11: Assessing the Efficacy of Immune Checkpoint Inhibitors in Preclinical Tumor Models
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Subcutaneous Tumor Cell Injection
		2.3 Experimental Lung Metastasis (Tail Vein Injection)
		2.4 4 T1.2 Mammary Fat Pad Injection
	3 Methods
		3.1 Preparation of Tumor Cells for Injection
		3.2 Subcutaneous Injection of Cancer Cells
		3.3 Monitoring Tumor Growth and Immunotherapy Treatment
		3.4 Tail Vein Injection of Cancer Cells
		3.5 Immunotherapy Treatment of Established Lung Metastases
		3.6 Immunotherapy Treatment of 4T1.2 Mammary Tumors and Spontaneous Metastases
	4 Notes
	References
Chapter 12: Fibroblast Isolation from Mammary Gland Tissue and Syngeneic Murine Breast Cancer Models
	1 Introduction
	2 Materials
		2.1 Mammary Fibroblast Isolation from Naïve Murine Mammary Glands
		2.2 Cancer-Associated Fibroblast Isolation from Syngeneic Murine Breast Cancer Models
	3 Methods
		3.1 Mammary Fibroblast Isolation from Naïve Murine Mammary Glands
		3.2 Cancer-Associated Fibroblast Isolation from Syngeneic Murine Breast Cancer Models
	4 Notes
	References
Chapter 13: Multiscale Label-Free Imaging of Fibrillar Collagen in the Tumor Microenvironment
	1 Introduction
	2 Imaging
	3 Imaging Modalities for Characterizing Fibrillar Collagen
		3.1 Magnetic Resonance Imaging (MRI)
			3.1.1 MRI Collagen Contrast
			3.1.2 MRI Resolution, Scale, and Range
			3.1.3 MRI Strengths and Weaknesses
			3.1.4 MRI Clinical Suitability and Research Advances
		3.2 Ultrasound
			3.2.1 Ultrasound Collagen Contrast
			3.2.2 Ultrasound Resolution, Scale, and Range
			3.2.3 Ultrasound Strengths and Weaknesses
			3.2.4 Ultrasound Clinical Suitability and Research Advances
		3.3 Optical Coherence Tomography (OCT)
			3.3.1 OCT Collagen Contrast
			3.3.2 OCT Resolution, Scale, and Range
			3.3.3 OCT Strengths and Weaknesses
			3.3.4 OCT Clinical Suitability and Research Advances
		3.4 Polarized Light Microscopy
			3.4.1 Polarized Light Microscopy Collagen Contrast
			3.4.2 Polarized Light Microscopy Resolution, Scale, and Range
			3.4.3 Polarized Light Microscopy Strengths and Weaknesses
			3.4.4 Clinical Suitability and Research Advances
		3.5 Second Harmonic Generation (SHG)
			3.5.1 SHG Collagen Contrast
			3.5.2 SHG Resolution, Scale, and Range
			3.5.3 SHG Strengths and Weaknesses
			3.5.4 SHG Clinical Suitability and Research Advances
	4 Computational Analysis
		4.1 Quantification of Collagen Fibers
		4.2 Rendering, Visualization, and Enhancement for Collagen Fiber Imaging
			4.2.1 Image Stitching and Rendering
			4.2.2 Multimodal Image Registration
			4.2.3 Deep Learning-Based Cross-Modality Image Synthesis
		4.3 Analysis at Runtime
	5 Complementary and Multimodal Imaging of Collagen
		5.1 Complementary Imaging Methods
		5.2 Multimodal Imaging Advances
	6 Discussion
		6.1 Importance of Multimodal Analysis of Collagen
		6.2 What Are the Challenges?
		6.3 Progress Toward Improved Imaging of Collagen Fibers
		6.4 Biological Relevance
		6.5 Where Can We Go from Here, How Do We Bridge the Gap?
	References
Chapter 14: In Situ Measurement of Intra-tumoral Tissue Rigidity
	1 Introduction
	2 Materials
	3 Methods
		3.1 Oxygen-Free Microgel Fabrication
		3.2 Polyacrylamide Phantom Tissue Fabrication For μTAM Calibration
		3.3 Embedding Sensors in Microwell-Generated Spheroids
		3.4 Imaging Under Temperature Control
		3.5 Image Analysis
		3.6 Sensor Calibration
	4 Notes
	References
Chapter 15: Extracellular Matrix Glycation and Crosslinking in Mammary Tumor Progression
	1 Introduction
	2 Materials
		2.1 Preparation of Ribose-Crosslinked Collagen-I/BME Hydrogels
		2.2 Mouse Surgical Procedure Preparation
		2.3 Patient-Derived Xenograft Preparation
		2.4 Orthotopic Mammary Fat Pad Injection of ECM Hydrogels and Implantation of Patient-Derived Xenograft Tissues
	3 Methods
		3.1 Preparation of Ribose-Crosslinked Collagen I/BME Hydrogels
		3.2 Preparing for Mouse Surgical Procedures
		3.3 Patient-Derived Xenograft Preparation
		3.4 Orthotopic Mammary Fat Pad Injection of ECM Hydrogels and Implantation of Patient-Derived Xenograft Tissues
	4 Notes
	References
Chapter 16: Proteomic Assessment of the Murine Mammary Gland Extracellular Matrix
	1 Introduction
	2 Materials
		2.1 Enrichment of Extracellular Proteins (ECM) from Murine Mammary Glands
		2.2 Protein Extraction, Reduction, and Alkylation
		2.3 Protein Concentration Determination
		2.4 Proteolytic Digestion and Peptide Cleanup
		2.5 Nano-LC-MS/MS Analysis
		2.6 Data Analysis
	3 Methods
		3.1 Enrichment of Extracellular Proteins (ECM) from Murine Mammary Glands
		3.2 Protein Extraction, Reduction, and Alkylation
		3.3 Proteolytic Digestion and Peptide Cleanup
		3.4 Nano-LC-MS/MS Analysis
		3.5 Data Analysis
	4 Notes
	References
Chapter 17: Spheroids as a 3D Model of the Hypoxic Tumor Microenvironment
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Forming Spheroids
			2.2.1 Scaffold-Free Protocol
			2.2.2 Scaffold Protocol
		2.3 Spheroid Harvesting and Analysis
	3 Methods
		3.1 Cell Culture
		3.2 Scaffold Free Spheroid Formation: Hanging Drop Method
		3.3 Scaffold-Free Spheroid Formation: Low Adhesion Plate Method
		3.4 Scaffold-Based Spheroid Formation: Hydrogel Method
		3.5 Scaffold-Based Spheroid Formation: Micropatterned Plates
		3.6 Scaffold-Based Spheroid Formation: Microfluidics
		3.7 Invasion Assay
		3.8 Imaging and Harvesting Spheroids
		3.9 Data Analysis
	4 Notes
	References
Chapter 18: Tumor pH Imaging Using Chemical Exchange Saturation Transfer (CEST)-MRI
	1 Introduction
	2 Materials
		2.1 Animals
		2.2 MRI Hardware
		2.3 MRI Sequences
		2.4 Sequence Parameters for CEST pH Mapping
		2.5 Software Requirements
	3 Methods
		3.1 In Vitro Acquisition
		3.2 In Vivo Acquisition
		3.3 In Vivo MRI-CEST Acquisition
		3.4 MRI-CEST Image Analysis
		3.5 Calculation of the In Vitro pH Calibration Curve
		3.6 Image Processing for In Vivo Tumor pH Mapping
		3.7 Tumor pH Denoising
		3.8 Tumor 3D pH Map Rendering
	4 Notes
	References
Chapter 19: Profiling the Epigenetic Landscape of the Tumor Microenvironment Using Chromatin Immunoprecipitation Sequencing
	1 Introduction
		1.1 Epigenetic Reprogramming in the Tumor Microenvironment
		1.2 Epigenetic Remodeling in Stromal Cells and Immune Cells
		1.3 Technologies to Study the Epigenetic Landscape of the Tumor Microenvironment
			1.3.1 Analysis of Epigenetic Heterogeneity Within the Tumor Microenvironment
			1.3.2 Isolation of Cell Populations from the Tumor Microenvironment
			1.3.3 Analysis of Epigenetic Profiles in Distinct Intratumor Regions
	2 Materials
		2.1 Regional Tumor Microdissection (RTM) Amenable to ChIP-seq
		2.2 Single-Cell Tumor Dissociation
		2.3 Chromatin Immunoprecipitation
		2.4 ChIP Library Prep and Sequencing
	3 Methods
		3.1 Regional Tumor Microdissection (RTM)
		3.2 Single-Cell Tumor Dissociation
		3.3 Chromatin Immunoprecipitation
		3.4 Sequencing of ChIP-seq Libraries
		3.5 ChIP-seq Data Processing and Analysis
	4 Notes
	References
Chapter 20: Analysis of Spatial Molecular Data
	1 Introduction
	2 Analyzing H&E Towards Inference of Spatial Transcriptomics
	3 Labeling
	4 Training
	5 Evaluation
	6 Generating Molecular Cartography Tensors and Heterogeneity Maps
	7 Spatial Analysis of Heterogeneity
	8 Summary
	References
Chapter 21: Laser-Assisted Single-Cell Labeling and Capture
	1 Introduction
	2 Materials
		2.1 PDMS Stock Plate
		2.2 Cell Culture Medium
		2.3 Microscopy and Illumination System
	3 Methods
		3.1 PDMS 6 mm Chamber Preparation
		3.2 Sample Preparation
		3.3 Laser Calibration
		3.4 Cell Selection and Laser-Assisted Membrane Biotinylation
		3.5 Single Cell Labeling
		3.6 Magnetic Capture
	4 Notes
	References
Chapter 22: 3D Hydrogel Cultures for High-Throughput Drug Discovery
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Peptide Hydrogel
		2.3 Compound Screening
		2.4 Statistical Analysis
	3 Methods
		3.1 Cell Culture
		3.2 Dispensing Hydrogel-Cell Constructs in 384-Well Plates
		3.3 Compound Screening
		3.4 Statistical Analysis
		3.5 Dose-Response Curves of Compounds of Interest
	4 Notes
	References
Chapter 23: Intravital Microscopy of the Metastatic Pulmonary Environment
	1 Introduction
	2 Materials
		2.1 Equipment for Establishment of Lung Metastasis
		2.2 Anesthesia
		2.3 Lung Ventilation via the Trachea
		2.4 Thoracic Surgery Equipment
		2.5 Microscope for Intravital Imaging
	3 Methods
		3.1 Cancer Cell Preparation for Experimental Lung Metastasis Model
		3.2 Operation of the Microscope
		3.3 Anesthesia and Central Venous Catheter Placement
		3.4 Tracheotomy
		3.5 Ventilation and Surgical Lung Preparation
		3.6 Intravital Microscopy: Images and Videos
	4 Notes
	References
Chapter 24: A CRISPR Platform for Targeted In Vivo Screens
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Line Selection, Characterization, and Generation of Cas9 Cells
		3.2 sgRNA Library and Experimental Design
		3.3 Lentiviral Production and MOI Determination
		3.4 Transduction, Injection in Mice, and Subsequent DNA Harvest
		3.5 PCR Amplification and Next-Generation Sequencing
		3.6 Computational Analysis and Candidate Compilation
	4 Notes
	References
Index