Inflammation and Cancer: Methods and Protocols (Methods in Molecular Biology, 2691)

Description

This second edition volume expands on the previous edition with an update on the broad spectrum of research models, techniques, and protocols used in laboratories by basic and clinical researchers. The chapters in this book are divided into two parts. Part One discusses the latest findings on the development and characterization of representative research models for chronic immune-based diseases and inflammation-associated cancers. Part Two covers biochemical, molecular, and cellular biological techniques that are commonly used to dissect the molecular mechanisms and cellular processes that drive the pathogenesis of certain disease states. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Inflammation and Cancer: Methods and Protocols, Second Edition is a valuable resource for those with a diverse range of laboratory-based experience, ranging from novice undergraduate students to established basic or clinical researchers who wish to diversify their existing portfolio of practical knowledge in the field.  Preface Contents Contributors Part I: Experimental Model Systems Chapter 1: Identifying Adult Stomach Tissue Stem/Progenitor Cells Using the Iqgap3-2A-CreERT2 Mouse 1 Introduction 2 Materials 2.1 Mouse Model 2.2 Animal Treatment 2.3 Formalin-Fixed Paraffin Embedded (FFPE) Mouse Tissue 2.4 Antibodies 2.5 Immunofluorescence Staining 2.6 Hematoxylin-Eosin Staining 2.7 Microscope 3 Methods 3.1 Mice and Treatment 3.2 Formalin-Fixed Paraffin Embedded (FFPE) Stomach at Room Temperature 3.3 Immunofluorescence (IF) Staining and Visualization 3.4 Hematoxylin-Eosin (HE) Staining and Visualization 4 Notes References Chapter 2: In Vitro and In Vivo Models for Metastatic Intestinal Tumors Using Genotype-Defined Organoids 1 Introduction 2 Materials 2.1 Mouse Intestinal Tumor-Derived Organoids 2.2 Standard Culture (2D) 2.3 Collagen Gel Culture (2D or 3D) 2.4 Culture Medium 2.5 Spleen Transplantation for Liver Metastasis Model 2.6 Bioimaging of Tumor Growth by Luciferase Activity 3 Methods 3.1 AKTP Organoid Standard Culture on 2D Culture Plates (Fig. 1) 3.2 Organoid Growth Analyses in 3D collagen Gel (See Note 6) 3.3 Competition Co-culture Analysis on 2D Collagen Gel (See Notes 9 and 10) 3.4 Liver Metastasis Model (See Note 12) 3.5 In Vivo Imaging Analyses 4 Notes References Chapter 3: On Target: An Intrapulmonary Transplantation Method for Modelling Lung Tumor Development in its Native Microenviron... 1 Introduction 2 Materials 2.1 Intrapulmonary Injection 2.2 PET/CT Imaging of Lung Tumors 3 Methods 3.1 Intrapulmonary Injection 3.2 PET/CT Imaging of Lung Tumor Development 4 Notes References Chapter 4: Endoscopic Ultrasound-Guided Fine-Needle Biopsies to Generate Preclinical Disease Models to Study Inflammation in P... 1 Introduction 2 Materials 2.1 Human Specimen Collection 2.2 Preparing EUS-FNB for Xenograft 2.3 Subcutaneous Implantation of Tumor into Immunodeficient Mice 2.4 Peripheral Blood Mononuclear Cell Engraftment 2.5 Passage of Xenografts 2.6 Freezing Down Tumors 3 Methods 3.1 Human Tumor Sample Collection 3.2 Preparing EUS-FNB for Xenograft 3.3 Subcutaneous Implantation of Tumor into Immunodeficient Mice 3.4 Engrafting PMBCs into Immunodeficient Mice 3.5 Passage of Xenografts 3.6 Freezing of Tumor Pieces for PDXs 4 Notes References Chapter 5: Modeling Intestinal Carcinogenesis Using In Vitro Organoid Cultures 1 Introduction 2 Materials 2.1 Isolation of Crypts from Tissue and Organoid Culture 2.2 Isolation of Single Cells from Intestinal Tissue and Organoids, FACS, and Single-Cell Seeding 2.3 Tamoxifen-Mediated Induction of Cre Recombinase 2.4 Induction of Recombination Using the Cell Permeant TAT-Cre 2.5 Analysis of Cell Viability 2.6 Whole-Mount Immunofluorescence Staining of Organoids 2.7 Phenotypic Analysis of Organoids 3 Methods 3.1 Crypt Isolation and Organoid Culture 3.2 Isolation of Single Cells from Tissue 3.3 Induction of Tamoxifen-Mediated Cre Recombination 3.4 Induction of Recombination Using the Cell Permeant TAT-Cre 3.5 Analysis of Cell Viability 3.6 Whole-Mount Immunofluorescence Staining of Organoids 3.7 Phenotypic Analysis of Organoids 4 Notes References Chapter 6: An In Vitro Model for Assessing Acute Lung Injury During Pancreatitis Development Using Primary Mouse Cell Co-cultu... 1 Introduction 2 Materials 2.1 Isolation and Culturing of Mouse Lung Endothelial Cells (MECs) 2.2 Isolation and Culturing of Mouse Lung Progenitor Cells 2.3 Isolation and Culturing of Mouse Pancreatic Acinar Cells 3 Methods 3.1 Isolation and Culturing of Mouse Lung Endothelial Cells (MECs) 3.2 Isolation and Culturing of Mouse Lung Progenitor/Stem Cells 3.3 Isolation and Culturing of Mouse Pancreatic Acinar Cells 3.4 Co-culturing 3D Lung Organoids with Acinar Cells 4 Notes References Chapter 7: Tracking the Host Response to Infection in Peritoneal Models of Acute Resolving Inflammation 1 Introduction 2 Materials 2.1 Preparation of Staphylococcus epidermidis Cell-Free Supernatant 2.2 SES Bioassay 2.3 Acute Resolving Inflammatory Challenge 2.4 Tracking Innate Immune Responses 2.5 Tracking Stromal Tissue Responses in Mice 3 Methods 3.1 Isolation and Culture of S. epidermidis Single Colonies 3.2 Preparation of SES 3.3 Test Validation: SES Bioassay 3.4 In Vivo Administration of SES and Assessment of Acute Resolving Inflammation 3.5 Repeated Administration of SES and Profiling of Recurrent Inflammatory Challenge 3.6 Live Model of S. epidermidis Infection 3.7 Tracking Innate Responses to Infection 3.8 Biochemical and Genomic Analysis of Stromal Tissue 4 Notes References Chapter 8: Assessing Lung Inflammation and Pathology in Preclinical Models of Chronic Obstructive Pulmonary Disease 1 Introduction 2 Materials 2.1 Cigarette Smoke Exposure 2.2 Characterization of Cigarette Smoke Exposure: Total Suspended Particulate Mass and Particle Number Concentration 2.3 Bronchoalveolar Lavage and Lung Collection 2.4 Total and Differential Cell Counts 2.5 Carboxyhemoglobin Measurements 2.6 RNA Extraction, cDNA Synthesis, and qPCR 2.7 Histology 2.8 Acute Exacerbations of COPD 2.9 Flow Cytometry 3 Methods 3.1 Mice 3.2 Cigarette Smoke Exposure and Euthanasia 3.3 Total Suspended Particulate Mass and Particle Number Concentration 3.4 Bronchoalveolar Lavage and Lung Collection 3.5 BALF Cell Counts 3.6 Carboxyhemoglobin Measurements 3.7 RNA Extraction, cDNA Synthesis, and qPCR 3.8 Histology 3.9 Acute Exacerbations of COPD 3.10 Preparation of Lung Cells for Flow Cytometry 3.11 Analyzing Innate Immune Cells Using Flow Cytometry 4 Notes References Chapter 9: Preclinical Mouse Model of Silicosis 1 Introduction 2 Materials 2.1 Intranasal Delivery of Silica 2.2 Euthanasia of Mice 2.3 Harvesting Lung Tissue for Histology 2.4 Histological Staining of Lung Tissue 3 Methods 3.1 Intranasal Delivery of Silica 3.2 Euthanasia of Mice 3.3 Harvesting of Lung Tissue 3.4 Histological Staining of Lung Tissues 3.4.1 H & E Staining of Lung Tissue Sections 3.4.2 Masson´s Trichrome Staining of Lung Tissue Sections (see Note 7) 3.5 Visualizing Histological Changes in Lung Tissue Sections Following Silica Exposure 3.6 Visualizing Silica Particles in Lung Tissue Sections Following Silica Exposure 4 Notes References Part II: Experimental Techniques Chapter 10: Tracking Cardiovascular Comorbidity in Models of Chronic Inflammatory Disease 1 Introduction 2 Materials 2.1 Animal Ethics 2.2 Recovery of the Thoracic Aorta and Isometric Tension Myography 2.3 Immunohistochemistry 3 Methods 3.1 Recovering the Thoracic Aorta from In Vivo Models of Chronic Inflammatory Disease 3.2 Preparing the Aorta for Isometric Tension Myography 3.3 Measuring Vascular Constriction Responses by Isometric Tension Myography 3.4 Histological Processing of the Aorta 3.5 Immunohistochemistry (IHC) 4 Notes References Chapter 11: Live Imaging of Pyroptosis in Primary Murine Macrophages 1 Introduction 2 Materials 2.1 Preparation of Murine Bone Marrow-Derived Macrophages (BMDMs) and Their Retroviral Transduction to Express Fluorescent Pro... 2.2 Activation of Canonical Inflammasome in Murine Macrophages 2.3 Activation of Noncanonical Inflammasome in Murine Macrophages 2.4 Live Imaging Pyroptosis in Murine Macrophages 3 Methods 3.1 Retroviral Transduction of Fluorescent Probe into Murine Macrophages (see Note 12) 3.2 Activation of the NLRP3 Inflammasome in Murine Macrophages 3.3 Activation of Noncanonical Inflammasome in Murine Macrophages 3.4 Live Imaging Pyroptosis in Murine Macrophages 4 Notes References Chapter 12: A Streamlined Method for Detecting Inflammasome-Induced ASC Oligomerization Using Chemical Crosslinking 1 Introduction 2 Materials 2.1 Reagents for Assays 3 Methods 3.1 Cell Treatments and ASC Crosslinking 3.2 Western Blotting to Detect ASC Oligomerization 4 Notes References Chapter 13: DNA Methylation Analysis 1 Introduction 2 Materials 2.1 Conventional Protocol for Bisulfite-Mediated Conversion (See Note 1) 2.2 Bisulfite-Mediated Conversion Using a Kit 2.3 Infinium BeadArray Analysis (Fig. 2a; See Note 4) 2.3.1 Quantification, Bisulfite-Mediated Conversion, and Amplification of DNA 2.3.2 Hybridization of DNA to the BeadChip 2.3.3 Wash, Single-Base Extension, and Stain BeadChips 2.3.4 Imaging of BeadChips 2.3.5 Data Processing, Quality Check, and Annotation 2.3.6 Data Visualization 2.4 Quantitative MSP (See Note 6) 2.4.1 Preparation of Fully Unmethylated DNA (See Note 7) 2.4.2 Preparation of Fully Methylated DNA (See Note 7) 2.4.3 Quantitative MSP 2.5 Conventional Bisulfite Sequencing (See Note 11) 2.6 Amplicon Bisulfite Sequencing Using a Next-Generation Sequencer (See Note 11) 3 Methods 3.1 Conventional Protocol for Bisulfite-Mediated DNA Conversion 3.2 Bisulfite-Mediated Conversion Using a Kit 3.3 Infinium BeadArray Analysis 3.3.1 Quantification, Bisulfite-Mediated Conversion, and Amplification of DNA 3.3.2 Hybridization of DNA to the BeadChip 3.3.3 Wash, Single-Base Extension, and Stain BeadChips 3.3.4 Imaging of BeadChips 3.3.5 Data Processing 3.3.6 Data Visualization 3.4 Quantitative MSP 3.4.1 Preparation of Fully Unmethylated DNA 3.4.2 Preparation of Fully Methylated DNA 3.4.3 Primer Design for MSP 3.4.4 Optimization of Real-Time MSP 3.4.5 Quantitative MSP Using Test Samples 3.5 Bisulfite Sequencing 3.5.1 Primer Design for Bisulfite Sequencing 3.5.2 Optimization of PCR for Bisulfite Sequencing (See Note 15) 3.5.3 Conventional Protocol for Bisulfite Sequencing 3.5.4 Protocol for Amplicon Bisulfite Sequencing Using a Next-Generation Sequencer 4 Notes References Chapter 14: Flow Cytometry Identification of Cell Compartments in the Murine Brain 1 Introduction 2 Materials 2.1 Dissociation of Brain Tissue 2.2 Flow Cytometry 2.3 Quantitative RT-PCR 3 Methods 3.1 Dissociation of Murine Brain Tissue 3.2 Preparing Cells for Fluorescence Activated Cell Sorting 3.3 Cell Sorting 3.4 Quantitative RT-PCR of Isolated Cells 4 Notes References Chapter 15: Expression and Purification of Inflammasome Sensor NOD-Like Receptor Protein-1 Using the Baculovirus-Insect Cell E... 1 Introduction 2 Materials 2.1 Expressing Recombinant Protein 2.2 Purification of hNLRP1 2.3 Denaturing Polyacrylamide Gel Electrophoresis (SDS-PAGE) 2.4 Immunoblotting 3 Methods 3.1 Expression and Purification of hNLRP1 (See Note 3) 3.2 SDS-PAGE and Western Blot Transfer 3.3 Western Blot Membrane Washing and Imaging 4 Notes References Chapter 16: Dissecting Interleukin-6 Classic and Trans-signaling in Inflammation and Cancer 1 Introduction 2 Materials 2.1 Culture of Ba/F3-gp130 and Ba/F3-gp130-IL-6R Cell Lines 2.2 Serum Starvation 2.3 Stimulation with IL-6 and Hyper-IL-6 2.4 Measurement of STAT3 Phosphorylation 2.5 Measurement of Ba/F3-gp130(-IL-6R) Cell Viability 2.6 Analysis of Antibodies or Designer Proteins That Interfere with IL-6 Classic Signaling 2.7 Analysis of Small Molecules That Interfere with IL-6 Classic Signaling 2.8 Analysis of Antibody Activity from In Vivo Experiments 3 Methods 3.1 Permanent Culture of Ba/F3-gp130 and Ba/F3-gp130-IL-6R Cell Lines 3.2 Serum Starvation and Stimulation with IL-6 or Hyper-IL-6 3.3 Measurement of STAT3 Phosphorylation 3.4 Measurement of Ba/F3-gp130(-IL-6R) Cell Viability 3.5 Analysis of Antibodies or Designer Proteins That Interfere with IL-6 Classic Signaling 3.6 Analysis of Small Molecules That Interfere with IL-6 Classic Signaling 3.7 Analysis of Antibody Activity from In Vivo Experiments 4 Notes References Chapter 17: Selecting Therapeutic Antisense Oligonucleotides with Gene Targeting and TLR8 Potentiating Bifunctionality 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Cell Transfection 2.3 Luciferase Assay 2.4 ELISA 3 Methods 3.1 HEK TLR8 Cells 3.1.1 Reverse-Transfection of the NF-κB Reporter Construct 3.1.2 Oligonucleotide Treatment and Uridine Stimulation 3.1.3 Luciferase Assay 3.2 THP-1 Cells 3.2.1 Oligonucleotide Treatment and TLR8 Stimulation 3.2.2 IP-10 Production Analysis by ELISA 4 Notes References Chapter 18: Exploring Allosteric Inhibitors of Protein Tyrosine Phosphatases Through High-Throughput Screening 1 Introduction 2 Materials 2.1 Reagents and Equipment for Assays 3 Methods 3.1 Pilot Operation Using a 384-Well Microplate Format 3.2 Quality Control of the Multi-sample Assay on a Full-Plate Format 3.3 Primary Screening Using a Chemical Library 3.4 Secondary Screening Using Extracted Hit Candidates 4 Notes References Chapter 19: Intravital Imaging of Regulatory T Cells in Inflamed Skin 1 Introduction 2 Materials 2.1 Contact Hypersensitivity Model 2.2 Imaging and Surgical Materials and Instruments 3 Methods 3.1 Contact Hypersensitivity (CHS) Model 3.2 Preparation of Flank Skin for Multiphoton Imaging (See Note 5) 3.3 Multiphoton Intravital Microscopy of Skin Flank for Treg Visualization 4 Notes References Chapter 20: Confocal Endomicroscopy Monitoring of Tumor Formation 1 Introduction 2 Materials 2.1 Mice 2.2 Ethical Approval 2.3 Reagents 2.4 Equipment 3 Methods 3.1 Preparation of Equipment for Imaging 3.2 Visualization of Colonic Tumors in Live Mice 3.3 Cleaning the Endoscopy Units 4 Notes References Chapter 21: Detection of Free Bioactive IL-18 and IL-18BP in Inflammatory Disorders 1 Introduction 2 Materials 2.1 Free IL-18 ELISA 2.2 Organ Dissociation for Western Blot Analysis 2.3 Western Blot Analysis for Recombinant IL-18 in Cell or Tissue Lysates 2.4 Detection of Mouse Free IL-18 Bioassay 2.5 Detection of Human Free IL-18 Bioassay 2.6 Detection of Murine IL-18BP by Western Blot 3 Methods 3.1 Free IL-18 ELISA (See Note 3) 3.2 Organ Dissociation for Western Blot Detection of IL-18 or IL-18BP 3.3 Western Blot Analysis for IL-18 (See Note 4) 3.4 Detection of Mouse Free IL-18 Bioassay (See Note 7) 3.5 Detection of Human Free IL-18 Bioassay 3.6 Western Blot Analysis for IL-18BP 4 Notes References Chapter 22: MAC-Seq: Coupling Low-Cost, High-Throughput RNA-Seq with Image-Based Phenotypic Screening in 2D and 3D Cell Models 1 Introduction 2 Materials 2.1 Cell Culture and Recovery 2.2 Imaging and Assay End Point 2.3 Library Preparation and Next-Generation Sequencing 2.4 Oligonucleotides (See Tables 1 and 2 for Sequences) 2.5 Lysis Buffer 2.6 Reverse Transcription Master Mix 2.7 Pre-Amplification PCR and Cycling 2.8 End Preparation Mix and Cycling 2.9 Adaptor Ligation Mix 2.10 Index PCR Mix and Cycling 2.11 NextSeq 500 Sequencing (See Note 4) 2.12 Consumables and Equipment 3 Methods 3.1 Automated Cell Dispensing in 2D 3.2 Automated Cell Seeding for 3D Cells in Matrigel 3.3 Compound Library Preparation and Delivery (Optional) 3.4 Automated Dye Dispensing (Optional) 3.5 Fixing and Staining (Optional) 3.6 Automated Imaging 3.7 Cell Sample Storage for 2D Assay 3.8 Cell Recovery in 3D Assay 3.9 Cell Lysis and Reverse Transcription 3.10 Pooling, First0Strand cDNA Synthesis, and Cleanup 3.11 Pre-Amplification PCR 3.12 cDNA Cleanup 3.13 cDNA QC and Quantification 3.14 Shearing 3.15 End Preparation 3.16 Adaptor Ligation 3.17 Size Selection of Adaptor-Ligated DNA 3.18 Index PCR 3.19 Index PCR Cleanup 3.20 Library QC and Quantification: TapeStation 3.21 Library QC and Quantification: qPCR 3.22 Sequencing 3.23 Bioinformatic Analysis 4 Notes References Chapter 23: Primary Intestinal Fibroblasts: Isolation, Cultivation, and Maintenance 1 Introduction 2 Materials 2.1 Extraction Solution (Volume per Colon) 2.2 Digestion Media (Volume per Colon) 2.3 Culture Media (Also Known as Complete Media) 2.4 Ammonium-Chloride-Potassium (ACK) Lysis Buffer 2.5 Tissue Processing 3 Methods 3.1 Extraction of Mouse Colon and Denudation of Epithelium 3.2 Isolation of Colon Fibroblasts 3.3 Passaging of Colon Fibroblasts 3.4 Immunofluorescence Staining and Immunoblotting of Colon Fibroblasts 4 Notes References Chapter 24: Lipid Nanoparticle-Mediated Delivery of miRNA Mimics to Myeloid Cells 1 Introduction 2 Materials 2.1 Encapsulation of MiRNA Mimic (miR146a) into Lipid Nanoparticles with Quantification and Validation 2.2 Polyacrylamide (PAGE) Gel Reagents 2.3 Western Blotting Reagents 2.4 NF-kB Reporter Assay Using RAW-Blue Cells 2.5 LNP (miR146aCy3) Uptake by Primary Mouse Splenocytes 2.6 The Assessment of IL-6 Splenocyte Cytokine Levels 3 Methods 3.1 Formulation of MiR146 into Lipid Nanoparticles 3.2 Characterization of LNP Formulation 3.3 Validation of MiR146 Encapsulation in LNPs Using Gel Electrophoresis 3.4 Functional Verification of LNP (miR146) in Mouse Macrophages by Western Blot 3.5 In Vitro RAW-Blue Assay to Verify on-Target Activity of LNP (miR146a) 3.6 Cell-Selective Uptake of LNP (miR146a) by Primary Mouse Immune Cells 3.7 Cytokine Assay to Verify Biological Activity of LNP (miR146a) 4 Notes References Chapter 25: Engineering Cell Lines for Specific Human Leukocyte Antigen Presentation 1 Introduction 2 Materials 2.1 Cancer Cell Line Culture 2.2 Plasmids for Generating Lentivirus 2.3 Lentivirus Production 2.4 Lentivirus-Mediated Transduction 2.5 Cell Staining and Sorting 3 Methods 3.1 Preparation and Propagation of Plasmids for Lentiviral Vector Production 3.1.1 Construction of Transfer Plasmids 3.1.2 Propagation of Plasmids 3.2 Production of Lentiviral Vectors for B2M KO and B2M-HLA-A*02:01 KI 3.2.1 Viral Production Cell Culture 3.2.2 Transfection 3.2.3 Harvesting Lentiviral Vectors 3.2.4 Concentrate Lentiviral Vectors by Sucrose Gradient Ultracentrifugation 3.3 Transduction of Lentivirus to the Cancer Cell Line of Choice to Knock out B2M 3.3.1 Cancer Cell Line Culture 3.3.2 Transduction 3.3.3 Media Change and Selection 3.4 Sorting Cells without HLA-I Expression 3.4.1 Preparation of Staining Samples 3.4.2 Staining 3.4.3 Cell Sorting and Culture 3.5 Transduction of Lentivirus to B2M-/- Cells to Knock in B2M-HLA-A*02:01 3.5.1 Cancer Cell Line Culture 3.5.2 Transduction, Media Change, and Selection 3.6 Sorting Cells Expressing HLA-A*02:01 3.6.1 Preparation of Samples and Staining 3.6.2 Cell Sorting and Culture 4 Notes References Index