Melanoma: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Model Systems for the Study of Malignant Melanoma
1 Introduction
2 Mouse Models of Melanoma
2.1 Transplantation of Murine Melanoma Cell Lines in Syngeneic Mice
2.2 Xenograft Transplantation
2.3 Genetically Engineered Mice (GEM)
2.4 Carcinogen- and UV-Induced Melanoma
3 Other Animal Models of Melanoma
3.1 Dog Model
3.2 Horse Model
3.3 Swine Model
3.4 Zebrafish Model
3.4.1 Genetically Engineered Zebrafish (GEZ)
3.4.2 Xenograft Transplantation in Zebrafish
4 Tissue-Engineered 3D Models of Melanoma
5 Conclusion
References
Part I: 2D Cell Culture-Based Approaches for Studying Melanoma Biology
Chapter 2: Generation of Functional Gene Knockout Melanoma Cell Lines by CRISPR-Cas9 Gene Editing
1 Introduction
2 Materials
2.1 Cell Culture Media and Supplies
2.2 Transfection Reagents
2.3 DNA Analysis
3 Methods
3.1 Designing a gRNA for CRISPR-Cas9 Gene Editing
3.2 Transfection of Melanoma Cells with CRISPR All-In-One Plasmid Vector
3.3 Antibiotic Selection of Transfected Cells
3.4 Limiting Dilution Cloning of Transfected Melanoma Cells
3.5 Isolation of DNA from Melanoma Clones
3.6 PCR Amplification of DNA of Interest and Purification by Gel Extraction
3.7 DNA Sequencing and Analysis
4 Notes
References
Chapter 3: A Fluorescent Gelatin Degradation Assay to Study Melanoma Breakdown of Extracellular Matrix
1 Introduction
2 Materials
2.1 Preparation of Fluorescein-Labeled Gelatin-Coated Coverslips
2.2 Cell Seeding
2.3 Cell Staining
3 Methods
3.1 Preparation of Fluorescein-Labeled Gelatin-Coated Coverslips (See Note 5)
3.2 Cell Seeding (See Note 5)
3.3 Cell Staining (See Notes 12 and 13) and Fluorescence Imaging
3.4 Quantification of Invadopodia Formation
3.4.1 Calculating the Percentage of Cells Able to Form Invadopodia
3.4.2 Calculating the Number of Invadopodia per Cell
3.5 Quantification of Invadopodia Activity
3.5.1 Calculating the Percentage of Cells Associated with Gelatin Digestion
3.5.2 Calculating the Mean Gelatin-Digested Area per Cell
3.5.3 Calculating the Gelatin-Digested Area per Area of a Single Cell
3.5.4 Gelatin Degradation in Third Dimension
4 Notes
References
Chapter 4: Wound Healing Assay for Melanoma Cell Migration
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 5: A Fluorescence-Based Assay for Measuring Glucose Uptake in Living Melanoma Cells
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture and Treatment
3.2 Flow Cytometric Analysis of Glucose Uptake
3.2.1 Flow Cytometry Setup
3.2.2 Comparison of Glucose Uptake Between the Cell Lines
3.2.3 Measurement of the Effects of Drugs on Glucose Uptake
4 Notes
References
Chapter 6: Analyzing Melanoma Cell Oxygen Consumption and Extracellular Acidification Rates Using Seahorse Technology
1 Introduction
2 Materials
2.1 Tumor Processing and Culture
2.2 Seahorse Mito Stress Test
3 Methods
3.1 Tumor Processing and Cell Preparation
3.1.1 Murine Tumors
3.1.2 Human Tumors
3.2 Preparation of Cells from Culture
3.3 Seahorse Mito Stress Test
3.4 Data Analysis
4 Notes
References
Chapter 7: Analysis of Melanoma Cell Glutamine Metabolism by Stable Isotope Tracing and Gas Chromatography-Mass Spectrometry
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Sample Collection
2.3 Standards
2.4 Derivatization
2.5 GC-MS
2.6 Data Analysis
3 Methods
3.1 Cell Culture and Labeling
3.2 Sample Collection and Extraction
3.3 Derivatization (Polar Samples and Standards)
3.4 GC-MS (Polar Samples and Standards)
3.5 Derivatization (Nonpolar Samples, for Fatty Acids)
3.6 GC-MS (Fatty Acid Samples and Standards)
3.7 13C Data Analysis (Polar Metabolites)
3.8 Metabolite Quantification (Polar Metabolites)
3.9 13C Data Analysis (Fatty Acids)
3.10 Metabolite Quantification (Fatty Acids)
4 Notes
References
Chapter 8: Determination of Cytotoxic Activities Against Melanoma Cells Using Flow Cytometry
1 Introduction
2 Materials
2.1 Reagents and Equipment
2.2 Cell Lines and Animals
3 Methods
3.1 Prepare Target Cells
3.1.1 Thawing, Culturing, and Harvesting of Target Cells
3.1.2 CFSE Labeling of Target Cells (See Note 4)
3.2 Isolation of Single Cells from Mouse Spleen
3.3 Co-culture of Target Cells with Effector Cells
4 Notes
References
Chapter 9: In Vitro Differentiation of Tumor-Associated Macrophages from Monocyte Precursors with Modified Melanoma-Conditione...
1 Introduction
2 Materials
2.1 For the Preparation of Melanoma Cell Culture Medium and Modified Melanoma-Conditioned Medium
2.2 For the Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Fresh Leukopak
2.3 For Monocyte Enrichment from PBMCs and Differentiation into MCMI-Mphi
3 Methods
3.1 Culture Melanoma Cells and Harvest Cell Culture Medium
3.1.1 Thaw Frozen Cells
3.1.2 Determine Cell Viability with Trypan Blue Exclusion
3.1.3 Culture and Subculture Melanoma Cells
3.1.4 Generation of Modified Melanoma-Conditioned Medium
3.2 Isolation of Monocytes
3.2.1 Isolation of PBMCs from Leukopak Buffy Coats
3.2.2 Isolate Human Monocytes from PBMCs
3.3 Differentiating Monocytes into MCMI-Mphi
4 Notes
References
Chapter 10: A Flow Cytometric Assay for Investigating Melanoma Cell Adhesion to Lymphatic Endothelial Cells
1 Introduction
2 Materials
2.1 Cell Culture Media and Supplies
2.2 Supplies and Reagents for Melanoma Cell/Lymphatic Endothelial Cell Adhesion Assay
3 Methods
3.1 Routine Maintenance and Passaging of Cell Cultures
3.2 Preparation of Lymphatic Endothelial Cell Monolayers
3.3 Preparation and CFSE Labeling of Melanoma Cells
3.4 Melanoma Cell/Lymphatic Endothelial Cell Adhesion Assay
3.5 Flow Cytometry Sample Acquisition and Analysis
4 Notes
References
Part II: 3D Cell Culture Systems for Studying Melanoma
Chapter 11: An Approach to Study Melanoma Invasion and Crosstalk with Lymphatic Endothelial Cell Spheroids in 3D Using Immunof...
1 Introduction
2 Materials
2.1 Separation of the Two Cell Types from 2D Melanoma-LEC Co-cultures
2.2 Preparation of Fibrin Droplets
2.3 Preparation of LEC Spheroids
2.4 Indirect Immunofluorescence (IF) Staining and Imaging of Fibrin Droplets
3 Methods
3.1 Separation of the Two Cell Types in Melanoma-LEC 2D Co-cultures
3.2 Embedding of Single Melanoma Cells in Fibrin (See Note 4)
3.3 LEC Spheroid Preparation and Embedding into Fibrin
3.4 Preparation of 3D Melanoma-LEC Co-cultures
3.5 Staining of Fibrin Droplets
4 Notes
References
Chapter 12: Evaluating Melanoma Viability and Proliferation in 3D Microenvironments
1 Introduction
2 Materials
2.1 Reagents
3 Methods
3.1 Preparation of Melanoma Cells in 3D Collagen Matrices
3.2 Drug Treatment of Melanoma Cells in 3D Collagen Matrices
3.3 Viability Assay in 3D Culture
3.4 Proliferation Assay in 3D Culture Systems
3.5 3D Image Analysis Using Cell Profiler
4 Notes
References
Chapter 13: Preparation, Drug Treatment, and Immunohistological Analysis of Tri-Culture Spheroid 3D Melanoma-Like Models
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Reagent Setup
2.4 Cell Lines
3 Methods
3.1 Preparation of Tri-Culture Spheroids
3.2 Drug Treatment
3.3 Agarose Mold Culture to Avoid Loss of Cells Upon Drug Treatment
3.4 Immunohistological Analysis
3.4.1 Sample Preparation for Whole Mount Staining and Imaging
3.4.2 Sample Preparation for Section Staining and Imaging
4 Notes
References
Chapter 14: Enrichment of Melanoma Stem-Like Cells via Sphere Assays
Abbreviations
1 Introduction
2 Materials
2.1 Standard Materials Necessary Throughout the Procedure
2.2 Preparing Non-adherent Plates for Sphere Assays
2.3 Primary and Secondary Sphere Assays
3 Methods
3.1 Preparing PolyHEMA-Coated Plates
3.2 Seeding Primary Sphere Assay
3.3 Maintenance of Primary Sphere Cultures
3.4 Seeding Secondary Sphere Assay
3.5 Quantification of Spheres
3.6 Evaluation of Sphere Viability
4 Notes
References
Part III: Techniques for Isolating and Studying Circulating Melanoma Cells
Chapter 15: Capture and Isolation of Circulating Melanoma Cells Using Photoacoustic Flowmetry
1 Introduction
2 Materials
2.1 Blood Separation
2.2 Photoacoustic Flowmetry
2.3 Capture and Isolation
2.4 Immunohistochemistry
2.5 RT-PCR and cDNA Library Preparation
3 Methods
3.1 Blood Separation
3.2 Photoacoustic Flowmetry
3.3 Analysis of CTC
3.3.1 Immunocytochemistry
3.3.2 Isolation of CTC RNA and Generation of cDNA Library
4 Notes
References
Chapter 16: Multi-Marker Immunomagnetic Enrichment of Circulating Melanoma Cells
1 Introduction
2 Materials
2.1 Blood Collection and PBMC Isolation
2.2 Immunomagnetic Beads
2.3 Immunomagnetic Enrichment and Immunostaining
2.4 Instruments
3 Methods
3.1 PBMC Isolation | Timing: 45 min
3.2 CTC Isolation | Timing: 1 h 15 min
3.3 Immunostaining | Timing: 2 h
4 Notes
References
Chapter 17: PD-L1 Detection on Circulating Melanoma Cells
1 Introduction
2 Materials
2.1 Immunomagnetic Bead Preparation
2.2 Peripheral Blood Mononuclear Cell (PBMC) Separation
2.3 Melanoma Patient Circulating Tumor Cell (CTC) Enrichment
2.4 Immunodetection of PD-L1 Expression in Melanoma Patient CTCs
3 Methods
3.1 Immunomagnetic Bead Preparation
3.2 PBMC Separation
3.3 Melanoma CTC Enrichment
3.4 Immunodetection of PD-L1 Expression in Melanoma Patient CTCs
4 Notes
References
Chapter 18: Transcript-Based Detection of Circulating Melanoma Cells
1 Introduction
2 Materials
2.1 RNA and DNA Extraction
2.2 Reverse Transcription and Targeted Preamplification
2.3 Real-Time PCR Transcript-Based CTC Detection Assay
2.4 ddPCR Transcript-Based CTC Detection Assay
3 Methods
3.1 RNA/DNA Extraction
3.1.1 RNA Extraction
3.1.2 DNA Extraction
3.2 Reverse Transcription
3.3 Targeted Preamplification
3.4 Real-Time PCR Assay
3.5 ddPCR Assay
4 Notes
References
Chapter 19: Isolation and Quantification of Plasma Circulating Tumor DNA from Melanoma Patients
1 Introduction
2 Materials
2.1 cfDNA Extraction
2.2 ctDNA Quantification
3 Methods
3.1 General Recommendations
3.2 cfDNA Extraction
3.3 ctDNA Quantification
3.3.1 PCR Set Up
3.3.2 Droplet Reader Setup
3.3.3 Data Analysis
4 Notes
References
Chapter 20: Simultaneous BRAFV600E Protein and DNA Aberration Detection in Circulating Melanoma Cells Using an Integrated Mult...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 IMMS Device Fabrication
2.3 IMMS Cell Capture, Release, and Lysis
2.4 IMMS BRAFV600E DNA Detection
2.5 IMMS BRAFV600E Protein Detection
3 Methods
3.1 IMMS Device Fabrication
3.1.1 Preparation of Electrode Pattern on Glass Wafer
3.1.2 Preparation of Microfluidic Structures
3.2 IMMS Cell Capture, Release, and Lysis
3.3 IMMS BRAFV600E DNA Detection
3.4 IMMS BRAFV600E Protein Detection
4 Notes
References
Chapter 21: Single-Cell Analysis of BRAFV600E and NRASQ61R Mutation Status in Melanoma Cell Lines as Method Generation for Cir...
1 Introduction
2 Materials
2.1 Culture and Preparation
2.2 Single Cell Micromanipulation
2.3 Whole-Genome Amplification of DNA Derived from Single Cells
2.4 Mutation Detection Using Droplet Digital PCR (ddPCR)
3 Methods
3.1 Culture and Preparation of Melanoma Cell Lines
3.2 Single Cell Micromanipulation
3.3 Whole Genome Amplification of DNA Derived from Single Cells
3.4 Mutation Detection Using Droplet Digital PCR
4 Notes
References
Part IV: Methods to Study Melanoma-Derived and Melanoma-Associated Exosomes
Chapter 22: A Rapid Exosome Isolation Using Ultrafiltration and Size Exclusion Chromatography (REIUS) Method for Exosome Isola...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Harvesting of Supernatants and Exosome Isolation
2.3 Counting of Exosomes by NTA
2.4 Western Blot
2.5 Transmission Electron Microscopy (TEM)
3 Methods
3.1 Cell Culture for Exosome Production
3.2 REIUS Method for Isolating Exosomes
3.3 NTA by ZetaView
3.4 Western Blot Analysis of Exosome Samples
3.5 Preparation of Exosomes for TEM
4 Notes
References
Chapter 23: Immunoaffinity-Based Isolation of Melanoma Cell-Derived and T Cell-Derived Exosomes from Plasma of Melanoma Patien...
1 Introduction
2 Materials
2.1 Isolation of Exosomes from Plasma Using Mini-SEC
2.2 Characterization of Exosomes Recovered from Plasma
2.3 Immunoaffinity-Based MTEX or CD3+ Exosome Capture
2.4 Detection of MTEX or CD3+ Exosomes
3 Methods
3.1 Isolation of Exosomes from Plasma
3.1.1 Preclearing of Blood Samples
3.1.2 Mini-Size Exclusion Chromatography (Mini-SEC)
3.2 Exosome Characterization
3.2.1 Protein Measurements
3.2.2 Transmission Electron Microscopy
3.2.3 Measurements of Exosome Size and Concentration by Tunable Resistive Pulse Sensing (TRPS)
3.2.4 Western blot Analysis
3.3 Separation of MTEX from NMTEX
3.3.1 Immunoaffinity-Based Capture of MTEX on Magnetic Beads
3.3.2 Immunoaffinity-Based Capture of CD3+ Exosomes from NMTEX
3.3.3 Immunoaffinity-Based Capture of CD63+Exosomes from the Noncaptured Fractions
3.4 Detection of Antigens on the Immunocaptured MTEX or CD3+ Exosomes
4 Notes
References
Chapter 24: An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Consumable Material and Instruments
2.3 Cell Lines and Cell Culture Reagents
2.4 Immunocapture Reagents and Antibodies
3 Methods
3.1 Sample Preparation for EV Analysis by Flow Cytometry
3.1.1 Conditioned Medium Pretreatment for Direct EV Immunocapture
3.1.2 Plasma or Serum Pretreatment for Direct EV Immunocapture
3.2 EV Enrichment from Conditioned Media or from Plasma by Sequential Centrifugation
3.2.1 Preparation of Conditioned Media and EV Isolation from Cell Culture Supernatants
3.2.2 EV Isolation from Plasma
3.3 EVs Characterization
3.4 Extracellular Vesicle Immunocapture
3.5 Staining for Flow Cytometry
3.6 Flow Cytometry Acquisition
3.7 Flow Cytometry Analysis
4 Notes
References
Chapter 25: Postlymphadenectomy Analysis of Exosomes from Lymphatic Exudate/Exudative Seroma of Melanoma Patients
1 Introduction
2 Materials
3 Methods
3.1 Purification of Exosomes from Human Exudative Seroma/Lymphatic Exudate by Differential Ultracentrifugation
3.2 Preparing Highly Purified Exosomes by Density Gradient-Based Ultracentrifugation
3.3 Exosome Protein Measurement
3.4 Exosome Particles Measurement
3.5 Analysis of Purified Exosomes by Electron Microscopy
3.6 Proteomic Analysis
3.7 DNA Extraction
3.8 Analysis of DNA Mutations (e.g., BRAF V600E/K)
4 Notes
References
Part V: Assessing Melanoma Metastasis in Pre-Clinical and Clinical Settings
Chapter 26: Fate Mapping of Cancer Cells in Metastatic Lymph Nodes Using Photoconvertible Proteins
1 Introduction
2 Materials
2.1 Animal Models and Cell Lines
2.2 General Purpose and Animal Surgery Materials
2.3 Equipment and Materials for Photoconversion and Intravital Imaging
2.4 Tissue Staining and Clearing
3 Methods
3.1 In Vitro Measurement of Photoconversion Efficiency
3.2 Tumor Inoculation, Growth, and Spontaneous Lymph Node Formation
3.3 Chronic Lymph Node Window Implantation
3.4 Photoconversion of Cancer Cells in the Lymph Node
3.5 Tissue Staining and Clearing
4 Notes
References
Chapter 27: Detection of Melanoma Cells in Lymphatic Drainage (LD) After Lymph Nodes Dissection Via Nested RT-PCR Analysis of ...
1 Introduction
2 Materials
3 Methods
3.1 Lymph Fluid Collection
3.2 RNA Isolation
3.3 RT-PCR
4 Notes
References
Chapter 28: A Clonogenic Assay to Quantify Melanoma Micrometastases in Pulmonary Tissue
1 Introduction
2 Materials
2.1 Materials and Reagents for the Generation and Monitoring of Melanoma Mouse Model
2.2 Solutions for Lung Tissue Dissection, Ex Vivo B16 Plating, Cell Staining, and Image Acquisition
2.3 Other Supplies and Equipment
3 Methods
3.1 Melanoma Cell Culture
3.2 Mice Handling and In Vivo Treatments
3.3 Mice Sacrifice and Pulmonary Tissue Dissection
3.4 Clonogenic Assay Plating Setup
3.5 Micrometastasis Determination and Quantification of the Stained Micrographs
3.5.1 Manual Analysis of the Acquired Images
3.5.2 Automated Analysis of the Acquired Images
3.6 Preliminary Evaluation of the ImageJ Datasets by Generation of Heatmap Layouts
3.7 Statistical Analysis and Plotting of Data
3.7.1 Statistical Analysis
3.7.2 Plotting Data
4 Notes
References
Chapter 29: PET Imaging of Melanoma Using Melanin-Targeted Probe
1 Introduction
2 Materials
3 Methods
3.1 Synthesis of the Precursor (Br-P3BZA) and Standard (19F-P3BZA)
3.2 Radiochemistry of the 18F-P3BZA Probe
3.3 PET/CT Imaging of Melanoma in Mouse Models
3.4 PET/CT Imaging of Melanoma in Human Patients
4 Notes
References
Chapter 30: Imaging and Isolation of Extravasation-Participating Endothelial and Melanoma Cells During Angiopellosis
1 Introduction
2 Materials
2.1 Zebrafish Tumor Injections
2.2 Melanoma Cell Culture and Preparation
2.3 Isolation of Injected Melanoma Cells from Zebrafish
3 Methods
3.1 Tumor Cell Injection into Zebrafish Embryo
3.2 Light Sheet Imaging of Injected Zebrafish Embryos
3.3 Isolation of Injected Tumor Cells from Zebrafish Embryos for Culturing
3.4 Isolation of Extravasating Tumor Cells and Extravasation-Participating Endothelial Cells for Direct Analysis
4 Notes
References
Part VI: Strategies for Diagnostic and Prognostic Biomarker Identification for Melanoma and Computational/Bioinformatic Approa...
Chapter 31: Salivary Proteomic Analysis of Canine Oral Melanoma by MALDI-TOF Mass Spectrometry and LC-Mass Spectrometry/Mass S...
1 Introduction
2 Materials
2.1 Saliva Preparation
2.2 First and Second MALDI-TOF MS
2.3 Sample Concentration and Purification by ZipTipC18 Pipette Tips
2.4 LC-MS/MS
2.5 Western Blotting
2.6 LC-MS to Verify Sequences of Expressed Protein from Western Blot
3 Methods
3.1 Saliva Preparation
3.2 First MALDI-TOF MS
3.3 Sample Concentration and Purification by ZipTipC18 Pipette Tips
3.4 Second MALDI-TOF MS
3.5 LC-MS/MS
3.6 Western Blotting
3.7 Verification of Expressed Protein Sequences by LC-MS/MS
4 Notes
References
Chapter 32: Detection of Uveal Melanoma by Multiplex Immunoassays of Serum Biomarkers
Abbreviations
1 Introduction
2 Materials
2.1 Antibodies and Reagents
2.2 Equipment and Accessories
2.3 Lab Consumables
3 Methods
3.1 Antibody Compatibility Test
3.2 Coupling Beads with Capture Antibodies
3.3 Development of Monoplex Immunoassays
3.4 Development of Multiplex Immunoassays
3.5 Validation and Application of Multiplex Immunoassays
4 Notes
References
Chapter 33: Unbiased Microbiome and Metabolomic Profiling of Fecal Samples from Patients with Melanoma
1 Introduction
2 Materials
2.1 Fecal Sample Collection
2.1.1 Inpatient Collection
2.1.2 Outpatient Collection
2.2 Fecal Sample Aliquot and Storage
2.3 Genomic DNA (gDNA) Extraction
2.4 gDNA Quantification
2.5 Sample Preparation for LC-MS Analysis
3 Methods
3.1 Fecal Sample Collection
3.1.1 Inpatient Collection
3.1.2 Outpatient Collection
3.2 Fecal Sample Aliquot and Storage
3.3 gDNA Extraction
3.4 gDNA Sequencing
3.5 Bioinformatic Analysis of MSS Data
3.6 Sample Preparation for Gut Metabolomic Profiling
3.7 Quantification of Sample Amount
3.8 Fecal Lyophilization
3.9 Extraction
3.10 Unbiased Gut Metabolomic Profiling with Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS)
3.11 Statistical Analysis of Metabolome
4 Notes
References
Chapter 34: Assessment of Cell-Free microRNA by NGS Whole-Transcriptome Analysis in Cutaneous Melanoma Patients´ Blood
1 Introduction
2 Materials
2.1 Serum Isolation
2.2 Plasma Isolation
2.3 Library Preparation and Cleanup
2.4 Library Quality Check
2.5 Next-Generation Sequencing
3 Methods
3.1 Serum Isolation
3.2 Plasma Isolation
3.3 Library Preparation and Cleanup
3.4 Library Quality Check
3.5 Library Normalization, Pooling, and Next-Generation Sequencing
3.6 Data Analysis
4 Notes
References
Chapter 35: High-Throughput Identification of miRNA-Target Interactions in Melanoma Using miR-CATCHv2.0
1 Introduction
2 Materials
2.1 Materials for miR-CATCHv2.0 Protocol
2.2 Materials for Small-RNA seq (Library Construction and Sequencing)
2.3 Hardware and Software for Bioinformatic Analysis
2.4 Materials for miRNA Validation Assays
3 Methods
3.1 miR-CATCHv2.0
3.1.1 Probe Design and Preparation
3.1.2 Cross-Linking
3.1.3 Lysis and Sonication
3.1.4 Probes Hybridization (Capture)
3.1.5 Target Affinity Purification and Elution
3.2 Small-RNA Seq
3.3 Bionformatic Analysis
3.3.1 Target Abundance Scaling Correction
3.3.2 miR-CATCHv2.0 miRNA Selection
3.3.3 Classification of miR-CATCHv2.0 Enriched miRNAs by Comparison with the miRNome
3.4 Validation of miRNA-Target Interactions Identified by miR-CATCHv2.0
3.4.1 MS2-Trap
3.4.2 Luciferase Reporter Assay
3.4.3 Relative Translational Efficiency Assay
3.4.4 Analysis of Target Expression After miRNA Overexpression
3.4.5 Analysis of mRNA Target Stability After miRNA Overexpression by qRT-PCR
4 Notes
References
Part VII: Characterization of Melanoma-Associated Tumor-Infiltrating Lymphocytes and Tertiary Lymphoid Structures
Chapter 36: Immunotyping and Quantification of Melanoma Tumor-Infiltrating Lymphocytes
1 Introduction
2 Tumor Sample Preparation and Tissue Microarray
3 TIL Analysis and Quantification
3.1 Multiplex IHC Analysis of TIL
3.2 Flow Cytometric Analysis of TIL from Tumor Cell Suspensions
3.3 Time-of-Flight Mass Cytometry (CyTOF) Analysis of TIL
4 TIL Immunotyping
5 Future Directions
References
Chapter 37: Single-Cell Gene Expression, Clonality, and Feature Barcoding of Melanoma Tumor-Infiltrating Lymphocytes
1 Introduction
2 Materials
3 Methods
3.1 Single-Cell Suspension from Melanoma Tumors
3.2 Thawing Protocol
3.3 Staining of the Cells with Feature Barcode-Conjugated Antibodies for Fluorescent Activated Cell Sorting
3.4 Preparation of Cells for Loading onto the 10x Chromium Controller
3.5 Library Preparation on 10x Chromium Controller
3.6 Sequencing and Analysis of the Data
4 Notes
References
Chapter 38: Using Mass Cytometry to Analyze the Tumor-Infiltrating Lymphocytes in Human Melanoma
1 Introduction
2 Materials
2.1 TIL Preparation and Freezing Media
2.2 CyTOF Staining and Analysis
3 Methods
3.1 Generation of Single-Cell Suspension from Melanoma Tumor Sample
3.2 Thawing Cryopreserved Melanoma Samples
3.3 Preparing the Sample for Stimulation with Phorbol 12-Myristate 13 Acetate (PMA) and Ionomycin
3.4 CyTOF Staining Workflow
3.5 Sample Acquisition on Fluidigm Helios CyTOF Instrument
4 Notes
Reference
Chapter 39: Multiplex Immunohistochemistry Analysis of Melanoma Tumor-Infiltrating Lymphocytes
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Patient Samples and Optimization Process
3.2 Staining on the Leica BOND RX System
3.3 Imaging on the Vectra Microscope
3.4 Deconvoluting Raw Files Using inForm Tissue Finder Software
3.5 Analyzing Deconvoluted Files Using HALO Software
3.6 Generate Cell Densities and Spatial Relationships Using R
4 Notes
Reference
Chapter 40: Multiplex Immunofluorescence Histology for Immune Cell Infiltrates in Melanoma-Associated Tertiary Lymphoid Struct...
1 Introduction
2 Materials
2.1 Specimen Preparation
2.2 Immunofluorescent Staining
2.3 Image Acquisition and Analysis
3 Methods
3.1 Immunofluorescent Staining of Cell Surface Markers on Formalin-Fixed Tumor Sections
3.1.1 Deparaffinization, Rehydration, Fixation, and Antigen Retrieval
3.1.2 Multiplex Stain
3.1.3 Spectral DAPI Stain, Mount, and Coverslip
3.2 Identification and Characterization of Immune Infiltrate and TLS in Melanoma by Immunofluorescence
4 Notes
References
Part VIII: Development and Evaluation of Therapeutic Strategies for Melanoma Treatment
Chapter 41: Use of Nanoparticles in Delivery of Nucleic Acids for Melanoma Treatment
1 Introduction to Melanoma and Its Main Characteristics
2 Current Therapies for Melanoma and Their Limitations
3 Nucleic Acids as Therapeutics
3.1 DNA
3.2 mRNA
3.3 Micro-RNAs (miRNAs)
3.4 Small Interfering Ribonucleic Acid (siRNA)
3.5 Antisense RNA and Oligonucleotides
3.6 Ribozymes
3.7 Aptamers
4 NPs Drug Delivery Systems
4.1 Lipid NPs
4.2 Polymeric NPs
4.3 Polymeric Micelles
4.4 Dendrimers
4.5 Liposomes
4.6 Gold NPs
5 The Application of NPs for the Delivery of Nucleic Acids into Melanoma Cells
References
Chapter 42: siRNA Delivery to Melanoma Cells with Cationic Niosomes
1 Introduction
2 Materials
2.1 Materials for Preparation and Characterization of Cationic Niosomes
2.2 Materials for Cell Culture
3 Methods
3.1 Preparation of Cationic Niosomes
3.1.1 Preparation of Cationic Niosomes by Microfluidic Mixing (See Notes 3-5)
3.1.2 Preparation of Cationic Niosomes by the Thin-Film Hydration Method
3.2 Characterization of Cationic Niosomes
3.3 Assessing Cytotoxicity of Cationic Niosome Formulations
3.4 Preparation of Lipoplexes (See Note 28)
3.5 In Vitro Cellular Uptake
4 Notes
References
Chapter 43: Controlled Delivery of Plasmid DNA to Melanoma Tumors by Gene Electrotransfer
1 Introduction
2 Materials
2.1 Reagents and Animals
2.2 Equipment
3 Methods
3.1 Tumor Induction
3.2 Determination of Correct GET Parameters
3.3 Evaluation of Response Using Optimized GET Parameters
4 Notes
References
Chapter 44: Generation of Murine Chimeric Antigen Receptor T Cells for Adoptive T Cell Therapy for Melanoma
1 Introduction
2 Materials
2.1 Retroviral Supernatant Production
2.2 T Cell Isolation and Activation
2.3 Transduction, Expansion, and Selection of T Cells
3 Methods
3.1 Retroviral Supernatant Production
3.2 T Cell Activation
3.3 Retroviral Transduction of T Cells
3.4 T Cell Expansion and Selection
4 Notes
References
Chapter 45: Generation of Phosphopeptide-Specific T Cell Lines as Tools for Melanoma Immunotherapy
1 Introduction
2 Materials
2.1 Standard Supplies
2.2 Reagents and Cell Lines for Bone Marrow-Derived Dendritic Cell (BMDC) Generation and T Cell Stimulation
2.3 In Vitro Cell Culture Reagents
3 Methods
3.1 Generation of Tg Murine T Cell Lines That Recognize Melanoma Phosphopeptides Presented in the Context of HLA-A*0201
3.1.1 Isolation, Maturation, and Activation of BMDCs
3.1.2 Immunization
3.1.3 In Vitro Culture of Phosphopeptide-Specific T Cells
3.1.4 Assessment of Antigen Specificity of the T Cell Lines
3.2 Human T Cell Lines
3.2.1 Isolation and Preparation of Human Dendritic Cells
3.2.2 In Vitro Culture of Human Phosphopeptide-Specific T Cells
4 Notes
References
Index