The Tumor Microenvironment: Methods and Protocols (Methods in Molecular Biology, 1458)

✍ Scribed by Josie Ursini-Siegel; Nicole Beauchemin

• Tongue: English
• Leaves: 355
• Category: Library

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Methods of Immunohistochemistry and Immunofluorescence: Converting Invisible to Visible
1 Introduction
2 Materials
2.1 Reagents for IHC and IF
2.2 Other Materials
3 Methods
3.1 Tissue Preparation
3.1.1 Tissue Preparation for Paraffin Section
3.1.2 Tissue Preparation for Frozen Section
3.2 Preparation of Tissue Sections from Tissue Blocks
3.2.1 Preparation of Tissue Sections from Paraffin-
3.2.2 Preparation of Tissue Sections from Frozen OCT Tissue Blocks
3.3 Reducing Autofluorescence from Tissue (Only for IF)
3.4 Reducing Nonspecific Antibody Binding in IF and IHC
3.5 From Antibody Reactions to Visualizing Target Molecule
3.5.1 IHC Reactions
3.5.2 IF Reactions
4 Notes
References
Chapter 2: Laser Capture Microdissection as a Tool to Study Tumor Stroma
1 Introduction
2 Materials
2.1 Total RNA Extraction for Quality Control
2.2 H&E Staining for LCM
2.3 HistoGene Staining
2.4 Laser Capture Microdissection
3 Methods
3.1 Total RNA Extraction for Quality Control
3.2 H&E Staining for LCM
3.3 HistoGene Staining
3.4 Laser Capture Microdissection
4 Notes
References
Chapter 3: Quantitative Analysis of Human Cancer Cell Extravasation Using Intravital Imaging
1 Introduction
2 Materials
2.1 Preparation of the Shell-Less Ex Ovo Chicken Embryos
2.2 Cancer Cell Line Preparation
2.3 Intravenous Injection of Cancer Cells or Agents to Visualize Chicken Embryo Vasculature
3 Methods
3.1 Cancer Cell Preparation for Injection
3.2 Intravenous Injection of Cancer Cells for Extravasation Assay
3.3 Intravenous Injection of Lectin into Chorioallantoic Membrane
3.4 Imaging of Cell Extravasation In Vivo
3.5 Quantification of Cancer Cell Extravasation
4 Notes
References
Chapter 4: Studies on the Tumor Vasculature and Coagulant Microenvironment
1 Introduction
2 Materials
3 Methods
3.1 Fluorescent Multicolor Tissue Immunostaining (IF)
3.2 Tissue Factor Procoagulant Activity Assay (TF-PCA)
4 Notes
References
Chapter 5: A Microfluidic Method to Mimic Luminal Structures in the Tumor Microenvironment
1 Introduction
2 Materials
2.1 Device Fabrication Components
2.2 Collagen Preparation
2.3 Sample Fixation and Staining
3 Methods
3.1 SU-8 Mold Preparation
3.2 PDMS Device Fabrication
3.3 PDMS Rod Fabrication
3.4 Microfluidic Device Setup
3.5 Collagen Gel Preparation
3.6 Creating Lumens
3.7 Cell Seeding (Blood Vessel Next to Tumor Example)
3.8 Cell Fixation and Immunofluorescent Staining
4 Notes
References
Chapter 6: Measuring Vascular Permeability In Vivo
1 Introduction
2 Materials
2.1 Microscope Setup Measuring Vascular Permeability in Mice Using 2D Method
2.2 Microscope Setup Measuring Vascular Permeability in Mice Using 3D Method
3 Methods
3.1 Measuring Vascular Permeability in Mice Using 2D Method
3.2 Measuring Vascular Permeability in Mice Using 3D Method
4 Notes
References
Chapter 7: Hydroxylation-Dependent Interaction of Substrates to the Von Hippel-Lindau Tumor Suppressor Protein (VHL)
1 Introduction
2 Materials
2.1 In Vitro Binding Assay
2.2 In Vitro Hydroxylation Reagents
3 Methods
3.1 In Vitro Binding Assay
3.2 In Vitro Hydroxylation Reaction
4 Notes
References
Chapter 8: Analyzing the Tumor Microenvironment by Flow Cytometry
1 Introduction to Flow Cytometry
1.1 General Information on Flow Cytometry
1.2 Instrumentation
1.3 Panel Design
1.4 Flow Cytometry Controls
1.4.1 Instrument Controls
Unstained Control
Compensation Control
1.4.2 Sensitivity Controls
Isotype Control
Fluorescence Minus One Control
Antibody Competition Control
1.4.3 Biological Controls
2 Materials
2.1 Sample Preparation
2.2 Staining
3 Methods
3.1 Sample Preparation
3.1.1 Solid tumor
3.1.2 Spleen or Other Lymphoid Tissues
3.2 Staining Protocol
3.2.1 External Staining
3.2.2 Internal Staining
3.3 Sample Analysis and Gating Strategies
4 Notes
References
Chapter 9: Detecting Secreted Analytes from Immune Cells: An Overview of Technologies
1 Introduction
2 Supernatant-Based Technology
2.1 ELISA
2.1.1 Key Principles
2.1.2 Overview of Methodology
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA
2.1.3 Detection Limit
2.1.4 Sample Volume
2.1.5 Multiplexing
2.1.6 Time and Cost
2.1.7 Advantages and Disadvantages
2.2 Cytokine Bead Arrays
2.2.1 Key Principles
2.2.2 Overview of Methodology
Cytometric Bead Array
Polystyrene and Superparamagnetic Bead Array
2.2.3 Detection Limit
Cytometric Bead Array
Polystyrene and Superparamagnetic Bead Array
2.2.4 Sample Volume
2.2.5 Time and Cost
2.2.6 Advantage and Disadvantages
2.3 Cytokine Membrane Arrays
2.3.1 Key Principles
2.3.2 Overview of Methodology
2.3.3 Detection Limit
2.3.4 Sample Volume
2.3.5 Time and Cost
2.3.6 Advantages and Disadvantages
2.4 Commercial Services
3 Cell-Based Technologies
3.1 ELISpot
3.1.1 Key Principles
3.1.2 Overview of Methodology
3.1.3 Detection Limit
3.1.4 Sample Volume
3.1.5 Time and Cost
3.1.6 Advantages and Disadvantages
3.2 Intracellular Cytokine Staining
3.2.1 Key Principles
3.2.2 Overview of Methodology
3.2.3 Detection Limit
3.2.4 Sample Volume
3.2.5 Time and Cost
3.2.6 Advantages and Disadvantages
4 Discussion
References
Chapter 10: Purification of Immune Cell Populations from Freshly Isolated Murine Tumors and Organs by Consecutive Magnetic Cell Sorting and Multi-parameter Flow Cytometry-Based Sorting
1 Introduction
2 Materials
2.1 Generation of Single-Cell Suspensions from Freshly Isolated Solid Tumors and Organs
2.2 Purification of Immune Cell Subsets by Consecutive Magnetic Cell Sorting and Multi-
2.3 Other Materials
3 Methods
3.1 Tissue Digestion and Single-Cell Suspension
3.2 Quantitative and/or Phenotypic Characterization of Immune Cells by Multi-­parameter Flow Cytometry
3.3 Immune Subset Purification by Consecutive Magnetic Cell Sorting (See Note 4) and Multi-­parameter Flow Cytometry-Based Cell Sorting
4 Notes
References
Chapter 11: Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes
1 Introduction
2 Materials
2.1 Tissue Culture Considerations
2.2 Common Materials and Reagents
2.3 Gamma-
2.4 Murine CAR-T Cell Preparation
2.5 Lentivirus Preparation
2.6 Human CAR-T Cell Generation
3 Methods
3.1 Murine CAR-T cells
3.1.1 Gamma-Retrovirus Generation
3.1.2 Murine CAR-T Cell Generation
3.2 Human CAR-T Cells
3.2.1 Lentivirus Preparation
3.2.2 Human CAR-T Cell Generation
4 Notes
References
Chapter 12: Methods to Evaluate the Antitumor Activity of Immune Checkpoint Inhibitors in Preclinical Studies
1 Introduction
2 Materials
2.1 Cell Culture Before the Injection of Tumor Cells
2.2 Subcutaneous Injections
2.3 4T1 Tumor Model
2.4 Experimental Metastasis Through Tail Vein Injections
2.5 Analysis of Tumor Immune Infiltrate by Flow Cytometry
3 Methods
3.1 Preparation of the Tumor Cells Before Subcutaneous or Intravenous Injections
3.2 Subcutaneous Injections of Syngeneic Tumor Cell Lines, Monitoring of Tumor Growth and Treatment with Immune Checkpoint Inhibitors
3.3 Spontaneous Lung Metastasis Model Using the 4T1 Tumor Cell Line
3.3.1 Tumor Growth and Treatment with Immune Checkpoint Blockers
3.3.2 Evaluation of Lung Metastasis Tumor Burden
3.4 Immune Checkpoint Inhibitors Testing in Experimental Lung Metastasis Models
3.4.1 Tail Vein Injections
3.4.2 Evaluation of Lung Metastasis Tumor Burden
3.5 Analysis of the Tumor Immune Infiltrate by Flow Cytometry
3.5.1 Tumor Disaggregation and Isolation of Tumor Infiltrating Leukocytes
3.5.2 Staining for Flow Cytometry Analysis
4 Notes
References
Chapter 13: Isolation and Characterization of Low- vs. High-Density Neutrophils in Cancer
1 Introduction
2 Materials
2.1 Materials and Reagents for Cancer Cell Injection
2.2 Reagents for Low- and High-Density Neutrophil Isolation
2.3 Antibodies for FACS Analysis
2.4 Neutrophil Enrichment
2.5 Cytotoxicity Assay
2.6 Modified Winn Assay
2.7 Phagocytosis Assay
2.8 Chemotaxis Assay
2.9 Oxidative Burst Assay
3 Methods
3.1 Orthotopic Injection of Mammary Tumor Cell Lines
3.2 Isolation of Circulating Mouse Neutrophils
3.3 Neutrophil Isolation of Human Blood Samples
3.4 Analysis of Neutrophil Purity in the Low- and High-Density Fractions Using Flow Cytometry
3.5 Neutrophil Enrichment
3.6 Functional Assays
3.6.1 Cytotoxicity Assay
3.6.2 Modified Winn Assay
3.6.3 Neutrophil Chemotaxis Assay
3.6.4 Neutrophil Oxidative Burst
3.6.5 Phagocytosis Assay
4 Notes
References
Chapter 14: Analysis of Extracellular Vesicles in the Tumor Microenvironment
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Isolation of ECV
2.3 Quantification of ECV Concentration
2.4 Purity Assessment of ECV Preparation
2.5 Analyzing Transport of ECV-­Associated Proteins to Recipient Cells
3 Methods
3.1 Cell Culture
3.2 Isolation of ECV
3.3 Quantification of ECV Concentration
3.4 Purity Assessment of ECV Preparation
3.5 Uptake of ECV by Recipient Cells
4 Notes
References
Chapter 15: Visualizing the Tumor Microenvironment of Liver Metastasis by Spinning Disk Confocal Microscopy
1 Introduction
2 Materials
2.1 Microscope Setup
2.2 Anesthesia and Catheter Preparation
2.3 Surgical Tools and Other Instruments
3 Methods
3.1 Preparation of Tumor Cells for Transplantation
3.1.1 Anesthesia of Mice
3.2 Catheter Placement (5 min)
3.3 Abdominal Surgery for Intravital Imaging (10–12 min)
4 Results
5 Notes
Video Captions
References
Chapter 16: Intravital Microscopy for Imaging the Tumor Microenvironment in Live Mice
1 Introduction
2 Materials
2.1 Tumor Cell Implantation
2.2 Intravital Imaging System Set-Up
2.3 Anesthesia and Tail Vein Catheter Insertion
2.4 Surgical Tools and Other Instruments
2.5 Injection of Labeling Antibodies
3 Methods
3.1 Establishing the Tumor
3.2 Setting Up the Imaging System
3.3 Anesthetizing the Mouse and Inserting Tail Vein Catheter (See Note 7)
3.4 Preparing the Tumor for Imaging
3.5 Injecting Antibodies and Imaging
4 Notes
References
Chapter 17: Development of a Patient-Derived Xenograft Model Using Brain Tumor Stem Cell Systems to Study Cancer
1 Introduction
2 Materials
2.1 Patient Brain Tumor Processing and Primary Cell Culturing
2.2 Tumorsphere Dissociation for In Vitro and In Vivo Assays
2.3 Flow Cytometry
2.4 Intracranial Injections
2.5 Mouse Brain Harvesting
2.6 Mouse Brain Immuno-
3 Methods
3.1 Patient Brain Tumor Processing and Primary Cell Culturing
3.2 Tumorsphere Dissociation for In Vitro and In Vivo Assays
3.3 Flow Cytometry: Surface Staining
3.4 Flow Cytometry: Internal Staining
3.5 Flow Cytometry: Combined Surface and Internal Staining
3.6 Flow Cytometry Acquisition and Sorting
3.7 Intracranial Injections
3.8 Preparation of Mouse Brain for Harvesting
3.9 Preparation of Mouse Brain for Immuno-­histochemistry
4 Notes
References
Chapter 18: Modeling Breast Tumor Development with a Humanized Mouse Model
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.3 Media
2.4 Surgical Supplies
3 Methods
3.1 Dissociation of Reduction Mammoplasty Tissue
3.2 Generation of Immortalized Stromal Cells
3.3 Humanizing Mammary Fat Pads
3.4 Preparation of Human Mammary Epithelial Cells
3.5 Injection into Humanized Glands
4 Notes
References
Chapter 19: CRISPR/Cas9 Genome Editing as a Strategy to Study the Tumor Microenvironment in Transgenic Mice
1 Introduction
2 Materials
2.1 sgRNA and Cas9
2.2 sgRNAs Synthesis
2.3 Genotyping
2.4 Mouse Embryo Culture
3 Methods
3.1 Selecting Genome Targeting Sites and Designing sgRNAs
3.2 sgRNA Synthesis
3.2.1 PCR Generation of T7 Promoter-­Attached sgRNA DNA Templates
3.2.2 In Vitro RNA Transcription for sgRNA
3.3 Designing and Validation of a Pair of Genomic PCR Primers for Genotyping
3.4 Microinjection, Embryo Culture, and Implantation
3.5 Blastocyst Genotyping [7]
3.5.1 Genomic DNA Preparation from Blastocysts
3.5.2 Genomic PCR from Single Blastocysts
3.6 T7 Endonuclease I Assay
4 Notes
References
Chapter 20: Metabolomics Analyses of Cancer Cells in Controlled Microenvironments
1 Introduction
2 Materials
2.1 Culture of Human Cancer Cells and Tumor Microenvironment Mimicry
2.2 Quenching, Extraction, and Derivatization
2.3 GC-MS Instrumentation
3 Methods
3.1 Growth of Cancer Cells and Media Collection
3.2 Incubation with Stable Isotope Tracers
3.3 Sample Collection, Quenching, Extraction, and Storage
3.4 Methoximation and Derivatization
3.5 GC-MS Method
3.6 Standard Curve Preparation
3.7 Metabolite Identification and Quantification
3.8 Stable Isotope Tracer Analysis
4 Notes
References
Chapter 21: Analysis of the Tumor Microenvironment Transcriptome via NanoString mRNA and miRNA Expression Profiling
1 Introduction
2 Materials
2.1 RNA Extraction from FFPE (Crude Lysate)
2.2 RNA Quantification (Qubit)
2.3 mRNA Profiling
2.4 miRNA Profiling
3 Methods
3.1 RNA Extraction from FFPE (Crude Lysate)
3.2 RNA Quantification (Qubit)
3.3 mRNA Profiling
3.3.1 mRNA Sample Preparation Protocol
3.3.2 Processing of the Samples on the nCounter Prep Station (See Note 18)
3.3.3 Scanning of the Cartridge on the nCounter Digital Analyzer (See Note 25)
3.4 miRNA Profiling
3.4.1 miRNA Sample Preparation Protocol
3.4.2 miRNA CodeSet Hybridization Protocol (See Note 35)
3.5 Data Analysis
3.5.1 Quality Control (QC) of the Raw and Normalized Data Must Be Performed to Determine Data Quality
3.5.2 Data Visualization
3.5.3 Data Analysis
4 Notes
References
Chapter 22: RNA-Seq as a Tool to Study the Tumor Microenvironment
1 Introduction
2 Materials
2.1 Library Generation and Quality Control
2.2 Data Processing and Analysis
3 Methods
3.1 RNA-Seq Library Preparation
3.1.1 Ribosomal RNA (rRNA) Depletion and RNA Fragmentation
3.1.2 Cleanup
3.1.3 First Strand cDNA Synthesis
3.1.4 Second Strand cDNA Synthesis
3.1.5 Purification of Double Stranded cDNA
3.1.6 Adenylation
3.1.7 Ligation of Adapters
3.1.8 Cleanup
3.1.9 cDNA Fragment Enrichment by PCR
3.1.10 PCR Cleanup
3.2 Quantification of Libraries Using qPCR
3.2.1 Preparation of the Master Mix
3.2.2 Preparation of the Dilution Plates
3.2.3 Loading the qPCR Plate and Performing the Assay
3.2.4 Analysis of qPCR Data
3.2.5 Calculating Size-Adjusted Concentration of Libraries
3.3 Sample Preparation for Clustering
3.3.1 Denature DNA Template for Libraries with a Concentration Over 2 nM
3.3.2 Denature DNA Template for Samples Below 2 nM
3.4 Data Analysis
3.4.1 Read Trimming and Clipping of Adapters
3.4.2 Read Alignment to a Reference Genome
3.4.3 Alignment Quality Control
3.4.4 Transcripts Analysis
3.4.5 Gene-Level Analysis
3.4.6 Additional Analyses Using FPKM Values
3.4.7 Gene Ontology (GO) Analysis for the Differential Gene Expression Results
4 Notes
References
Chapter 23: Sample Preparation for Mass Spectrometry Analysis of Protein–Protein Interactions in Cancer Cell Lines and Tissues
1 Introduction
2 Materials
2.1 Tissue Culture and Protein Extraction
2.2 Affinity Purification and Sample Elution
2.3 Protein Digestion on Chromatography Medium in a Spin Tip
3 Methods
3.1 Tissue Culture and Protein Extraction
3.2 Affinity Purification and Sample Elution
3.3 Protein Digestion on Chromatography Medium in a Spin Tip
4 Notes
References
Index