Metastasis: Methods and Protocols (Methods in Molecular Biology, 2294)

✍ Scribed by Ulrike S. Stein (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 346
• Category: Library

✦ Synopsis

This volume provides the most recent developments and methodologies on metastatic process, formation, and detection. Chapters guide readers through functional metastasis in vitro assays, non-mouse and mice metastasis models, methods for imaging metastasis, analyzing the tumor microenvironment, senescence and inflammation with respect to metastasis, methods to investgate the premetastatic/ metastatic, detecting biomarkers in patient, and bioinformatics to simulate the metastatic process. 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.

Authoritative and cutting-edge, Metastasis: Methods and Protocols aims to be a useful practical guide to researches to help further their study in this field.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Metastasis Models
Chapter 1: Cancer Cell Invasion and Metastasis in Zebrafish Models (Danio rerio)
1 Introduction
2 Materials
2.1 Injection Station
2.2 Manipulating Fish
2.3 Visualization
3 Methods
3.1 Set up Embryos for Injection and Make Stock Solutions
3.2 Labeling Cells with Red Lipophilic Fluorescent Dye
3.3 Injecting Cells into the Precardiac Sinus of Zebrafish Embryos
3.4 Scoring Extravasation of Cancer Cells in Zebrafish Embryos
3.5 Mounting Zebrafish onto Slides and Subsequent Fluorescence Imaging
3.6 Alternative Application: Injecting Cancer Cells into the Yolk Sac
4 Notes
References
Chapter 2: The Chicken Egg Chorioallantoic Membrane (CAM) Model as an In Vivo Method for the Investigation of the Invasion and...
1 Introduction
2 Materials
2.1 Equipment
2.2 Consumables
2.3 Reagents
3 Experimental Procedure
3.1 Incubation and Preparation of Eggs
3.2 Isolation of Samples
3.3 Isolation of Genomic DNA
3.4 TaqMan-Based Quantitative PCR of Samples and Standard Curve
3.5 TaqMan-Based Absolute Quantification of Invasive Cells
4 Notes
References
Chapter 3: In Vitro 3D Models of Tunable Stiffness
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Method
3.1 Reagent Preparation
3.2 Alginate Hydrogel Preparation
3.3 Monitoring Spheroid Formation
4 Notes
References
Chapter 4: Patient-Derived Xenografts from Solid Tumors (PDX) for Models of Metastasis
1 Introduction
2 Materials
2.1 Materials for Collection of Primary Tumor Material and PDX Establishment
2.1.1 Media and Instruments
2.1.2 Material for Xenotransplantation of Tumor Samples
2.1.3 Materials for Cryoconservation
2.2 Mouse Strains for PDX Establishment and Formation of Metastasis
2.3 Materials for Generation of PDX-Derived Metastasis
2.3.1 Media and Supplements
2.3.2 Instruments for Generation of Single Cells, Inoculation of Cells and Collection of Samples
2.4 Materials for Molecular Analyses of Metastasis
2.4.1 DNA Isolation
2.4.2 PCR, Primer and Probes
3 Methods
3.1 Establishment of PDX Models
3.1.1 Collection, Shipment and Processing of Primary Tumor Material
3.1.2 Xenotransplantation
3.1.3 Harvesting of PDX Tumor Tissue
3.1.4 Cryoconservation and Biobanking of PDX Specimen
3.2 Generation of PDX-Derived Metastasis Models
3.2.1 Generation of Single-Cell Suspensions from PDX Tumor Tissue
3.2.2 Inoculation of Tumor Cells for Metastasis Generation
3.2.3 Collection of Liver and Lung Samples for Metastasis Analysis
3.2.4 Determination of Macroscopic Metastasis by Metastatic Counts and Scoring
3.3 Molecular Analyses for Metastasis Detection and Quantification by Human-Specific PCR
3.3.1 Isolation of DNA from Metastatic Sites (E.g., Liver and Lung)
3.3.2 qPCR for Detection of Human Satellite DNA in Mouse Tissue
4 Notes
References
Chapter 5: Preparation and Culture of Organotypic Hippocampal Slices for the Analysis of Brain Metastasis and Primary Brain Tu...
1 Introduction
1.1 Brain Metastases and Other Brain Tumors
1.2 Use of Cell Culture 3D Models and Animal Experiments
1.3 Organotypic Hippocampal Slice Cultures (OHSCs)
2 Materials
2.1 OHSC Preparation
2.2 Seeding of Cells
2.3 Life-Dead Staining
2.4 Fixing and Staining of OHSCs with Seeded Cells
2.5 Cryosection
2.6 Immunofluorescence Staining
3 Methods
3.1 Preparation of OHSCs
3.2 Seeding of Cells
3.3 Life-Dead Staining
3.4 Fixing and Staining of OHSCs with Seeded Cells
3.5 Cryosection
3.6 Immunofluorescence Staining
4 Notes
References
Part II: Functional Mechanisms of Metastasis
Chapter 6: Measuring Mechanical and Adhesive Properties of Single Cells Using an Atomic Force Microscope
1 Introduction
2 Materials
2.1 Mechanical Measurements
2.2 Adhesive Measurements
3 Methods
3.1 Cell Seeding
3.2 Cantilever Coating
3.3 Cantilever Mounting and Calibration
3.4 Mechanical Measurements
3.4.1 Weakly Adherent Cells
3.4.2 Fully Adherent Cells
3.5 Adhesive Measurement
3.5.1 Weakly Adherent Cells
3.5.2 Fully Adherent Cells
3.6 Data Analysis
3.6.1 Mechanical Measurements: Calculating the Young´s Modulus
3.6.2 Adhesive Measurement: Calculating Total Adhesion Energies
4 Notes
References
Chapter 7: Analyzing the Roles of Rho GTPases in Cancer Cell Adhesion to Endothelial Cells Under Flow Conditions
1 Introduction
2 Materials
2.1 Cell Culture
2.2 siRNA Nucleofection
2.3 Cancer Cell Fluorescent Labeling
2.4 Flow Adhesion Assay
2.5 Quantification
3 Methods
3.1 PC3 Cell Culture
3.2 siRNA Transfection of PC3 Cells
3.3 HUVEC Cell Culture
3.4 HUVEC Seeding in Ibidi μ-Slide VI0.4 48 H Pre-Assay
3.5 Microscope Setup for Live Imaging
3.6 ``Flow System´´ Setup
3.7 Cancer Cell Preparation for the Flow Assay
3.8 Endothelial Cell Preparation for the Flow Assay
3.9 Flow-Based Adhesion Assay Coupled to Live Cell Imaging
3.10 Quantification of Images
4 Notes
References
Chapter 8: Probing Intravascular Adhesion and Extravasation of Tumor Cells with Microfluidics
1 Introduction
2 Materials
2.1 Cell
2.2 Cell Culture
2.3 Microfluidic Equipment (Fig. 2a-d)
2.4 Microchannels (Fig. 2e)
2.5 Live Microscopy (Fig. 2f)
2.6 Confocal Microscopy
2.7 Fluorescent Beads for Flow Calibration
2.8 Scanning Electron Microscopy Reagents
2.9 Scanning Electron Microscopy Reagents
3 Methods
3.1 Endothelial Cell Seeding in Microchannels
3.2 Tubing Setup (Fig. 2a, c)
3.3 Calibrating the Microfluidic Perfusion and Probing Flow Profiles
3.4 Microfluidic Shear Stress Resistance Assay
3.5 Live Cell Adhesion Assay
3.6 Microfluidic Extravasation Assay
3.7 Live Imaging
3.8 Immunostaining and 3D Confocal Imaging
3.9 Scanning Electron Microscopy (Fig. 4)
4 Notes
References
Chapter 9: Real-Time Cell Migration Monitoring to Analyze Drug Synergism in the Scratch Assay Using the IncuCyte System
1 Introduction
2 Materials
2.1 Cell Cultivation
2.2 Materials for Preparation of Cells for Scratch Assay
2.3 Software for Qualitative and Quantitative Migration Analysis
3 Methods
3.1 Preparation of Cell Cultures, Seeding of Assay Plates, Scratch Assay, Drug Treatment
3.2 Data Analysis
3.3 Anticipated Results
4 Notes
References
Chapter 10: Measurement of Metabolites from Migrating Cells
1 Introduction
2 Materials
2.1 Materials for Migration Assays
2.2 Materials for Metabolic Assays
3 Methods
3.1 Migration Assay and Sample Collection
3.2 Metabolism Assays
4 Notes
References
Chapter 11: Matrix Degradation Assay to Measure the Ability of Tumor Cells to Degrade Extracellular Matrix
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Acid Wash Coverslips Cleaning and Gelatin Coating
2.3 Gelatin Degradation Assay, Fluorescence/Immunocytochemistry Assay
3 Methods
3.1 Glass Coverslips Cleaning (Acid Wash)
3.2 Fluorescent Gelatin Coating
3.3 Fluorescent Gelatin Degradation Assay
3.4 Imaging and Quantification of Invadopodia Formation and Function
4 Notes
References
Chapter 12: Evaluation of Real-Time In Vitro Invasive Phenotypes
1 Introduction
2 Materials
3 Methods
3.1 Standardized Conditions for Real-Time Assessment of Migration and Invasion Phenotypes
3.2 Visualization of Invading Cells
3.3 Real-Time Migration and Invasion Phenotypes
3.4 Informatic Analysis: Connecting the Invasion Phenotypes with Genes and Pathways
3.5 Informatic Analysis: Connecting the Invasive Phenotypes with the NCI Drug Screening Database
3.6 Discussion
4 Notes
References
Chapter 13: Detection of Tumor Cell-Induced Platelet Aggregation and Granule Secretion
1 Introduction
2 Materials
2.1 Platelet Isolation
2.2 Investigation of a TCIPA Using Light Transmission Aggregometry
2.3 Quantification of a Tumor Cell-Induced Platelet Dense Granule Release
3 Methods
3.1 Platelet Isolation
3.2 Investigation of a TCIPA Using Light Transmission Aggregometry
3.2.1 Preparation of Platelets and Tumor Cells
3.2.2 Induction of a TCIPA and Detection in an LTA Approach
3.3 Quantification of a Tumor Cell-Induced Platelet Dense Granule Release
4 Notes
References
Chapter 14: Fatty Acid-Driven Polarization of Suppressive Bone Marrow-Derived Macrophages Including Metabolic and Functional A...
1 Introduction
2 Materials
2.1 Miscellaneous
2.2 Buffers
2.3 Bone Marrow-Derived Macrophages
2.4 Magnetic Sort
2.5 Metabolic Analysis
2.6 Flow Cytometry
2.7 Devices
3 Methods
3.1 Bone Marrow Isolation
3.2 Macrophage Differentiation and Polarization
3.3 Gr1- Macrophages Purification
3.4 Metabolic Analysis (Seahorse XFe96 Analyzer, Agilent)
3.5 T-Cell Suppression Assay
3.5.1 CD4 T Cells Isolation
3.5.2 T-Cell Suppression Assay
4 Notes
References
Chapter 15: From Whole Blood to Isolated Pro-Metastasis Immune Cells: An Ex Vivo Approach to Isolate and Manipulate Immune Cel...
1 Introduction
2 Materials
2.1 PBMCs Isolation and Counting
2.2 PBMCs Thawing
2.3 Isolation of Macrophages or Cytotoxic T Cells
2.4 Differentiation of Monocytes into M2 Macrophages
2.5 Seeding and Transfection of Isolated Cells
2.6 RNA Extraction
2.7 RT-PCR for cDNA Synthesis
2.8 qRT-PCR for Quantification of Genes
3 Methods
3.1 PBMCs Isolation and Counting
3.2 PBMCs Thawing
3.3 Isolation of Macrophages or Cytotoxic T-Cells
3.4 Differentiation of Monocytes into M2 Macrophages
3.5 Seeding and Transfection of Isolated Cells
3.6 RNA Extraction
3.7 RT-PCR for cDNA Synthesis
3.8 qRT-PCR for Quantification of Genes
4 Notes
References
Part III: Prognostication of Metastasis
Chapter 16: A Bioinformatic Pipeline to Identify Biomarkers for Metastasis Formation from RNA Sequencing Data
1 Introduction
2 Materials
2.1 Tools Required for this Pipeline
2.2 Current FASTA and Annotation Files of the Human Genome
3 Methods
3.1 Quality Control and Preprocessing
3.2 Quantification of Gene Expression
3.3 Mutational Analysis of RNAseq Data
3.3.1 Alignment of Fragments to the Reference Genome
3.3.2 Addition of Read Groups and Sorting by Coordinates
3.3.3 Deduplication and Splitting of Reads Containing Undefined Base Calls (N)
3.3.4 Base Quality Score Recalibration (BQSR) and Haplotype Calling
3.4 Identification of Differentially Expressed Genes with DESeq2
4 Notes
References
Chapter 17: Barcoding Technology for Multiplexed Analysis of Metastatic Ability In Vivo
1 Introduction
2 Materials
2.1 Barcode Cell Line Generation
2.2 Screening Platform
2.3 Barcode Amplification
2.4 Next-Generation Sequencing
2.5 Laboratory Equipment
3 Methods
3.1 Identification of a Suitable Metastatic Cell Line
3.2 Production of Barcode Virus
3.3 Generation of Barcoded Cell Lines
3.4 Elimination Unbarcoded Cells
3.5 Compound Treatment and Cell Pool Injection
3.6 Isolation of Barcoded Cells
3.7 DNA Isolation
3.8 Barcode Amplification
3.9 Library Preparation and NGS
3.10 Bioinformatic Analysis/Calculation of Metastatic Ability
4 Notes
References
Chapter 18: In Vivo Assessment of Metastatic Cell Potential in Prostate Cancer
1 Introduction
2 Material
2.1 Cell Culture
2.2 Retrovirus Production
2.3 Fluorescent-Activated Cell Sorting (FACS)
2.4 Cell Inoculation In Vivo
2.5 In Vivo Imaging System
3 Methods
3.1 Transduce Cells with a Luciferase-Expressing Vector
3.1.1 Produce Retroviral Particles
3.1.2 Transduce PC3, DU145, and 22RV1 Cells with GFP-LUC-Expressing Vector
3.2 Sort GFP+ Cells
3.3 In Vivo Metastatic Potential
3.3.1 Intra-Tibiae Injection
3.3.2 Left Ventricle Injection
3.3.3 Intra-Iliac Injection
3.4 Bioluminescent Imaging and Data Analysis
4 Notes
References
Chapter 19: Isolation of Nucleoli for Characterization of Nucleolar Contents to Uncover Clues to Metastatic Progression
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 20: Analyzing the Role of Proteases in Breast Cancer Progression and Metastasis Using Primary Cells from Transgenic On...
1 Introduction
2 Materials
2.1 Primary PymT Cell Preparation
2.2 Transplantation into Mammary Gland
2.3 Experimental Lung Colonization
2.4 Brain-Homing Variants of PymT Tumor Cells
2.5 Experimental Brain Colonization
2.6 Experimental Bone Colonization
2.7 Metastatic Burden by Quantitative PCR
2.8 Analysis of Metastases by Histology
3 Methods
3.1 Isolation of Primary Tumor Cells from MMTV-PymT-Induced Mammary Carcinomas (See Note 2)
3.2 Orthotopic Transplantation of Primary PymT-Derived Tumor Cells into Mammary Gland of Congenic Mice (See Note 6)
3.3 Experimental Lung Colonization in Congenic Mice Using Primary PymT-Derived Tumor Cells (See Note 11)
3.4 Generation of Brain-Homing Variants from MMTV-PymT Tumor Cells (See Notes 16-18)
3.5 Experimental Brain Colonization in Congenic Mice by Intracardiac Injection
3.6 Experimental Bone Colonization in Congenic Mice by Caudal Artery Injection
3.7 Measurement of Lung, Brain, and Bone Metastatic Burden by Quantitative Real-Time PCR (See Note 15)
3.8 Analysis of Tumor Cell Dissemination to the Lungs, the Brain, and the Bone by Histology
4 Notes
References
Part IV: Clinical Metastasis
Chapter 21: Nuclear Medicine Imaging Procedures in Oncology
1 Introduction
2 Gamma Camera
2.1 SPECT Systems
2.1.1 General SPECT/CT Imaging Protocol
2.2 Technetium Chemistry and 99mTc: Compounds for Tumor and Metastasis Diagnosis
2.2.1 99mTc-Phosphonate and Phosphate Compounds
2.2.2 Mechanism of Localization
2.2.3 SPECT/CT Protocol
2.2.4 99mTc-SESTAMIBI
2.2.5 Mechanism of Localization
2.2.6 SPECT/CT Protocol
2.3 Iodine Chemistry and 123I-Labeled Compounds for Diagnosis
2.3.1 123I-Sodium Iodide (123I-NaI)
2.3.2 123I-Metaiodobenzyl Guanidine (123I-mIBG)
3 Positron Emission Tomography/Computed Tomography (PET/CT)
3.1 PET Detectors and Instrumentation
3.2 PET Radiopharmaceuticals
3.2.1 18F-Labeled PET Radiopharmaceuticals
18F-2-Fluoro-2-Deoxyglucose (FDG)
18F-Sodium Fluoride (18F-NaF)
18F-Fluorothymidine (18F-FLT)
18F-Fluromisonidazole (18F-FMISO)
O-(2-[18F]-Fluoroethyl)-l-Tyrosine (18F-FET)
3.2.2 68Ga-Based PET Radiopharmaceuticals
68Ga-Prostate-Specific Membrane Antigen (68Ga-PSMA)
68Ga-DOTATOC
68Ga-NODAGA-Exendin
68Ga-Pentixafor
3.3 Positron Emission Tomography/Magnetic Resonance Imaging (PET/MRI)
3.4 Quantification
3.4.1 Nonlinear Regression Analysis
3.4.2 Patlak Analysis
3.4.3 Standardized Uptake Value (SUV)
4 Conclusion
References
Chapter 22: From Operating Table to Laboratory Bench: The Path Toward Metastasis Research from the Clinical Setting
1 Introduction
2 Materials
3 Methods: Processing of Solid Tissue
4 Notes
References
Index