Cancer Cell Biology: Methods and Protocols (Methods in Molecular Biology, 2508)

Preface
Contents
Contributors
Chapter 1: Overview of the Maintenance of Authentic Cancer Cell Lines
	1 Introduction
	2 Biosafety
	3 Cell Line Authentication
	4 Avoiding Cross-Contamination
	5 Genetic Drift
	6 Contamination with Mycoplasmas
	References
Chapter 2: Culturing Suspension Cancer Cell Lines
	1 Introduction
	2 Materials
		2.1 Thawing and Reviving Frozen Suspension Cancer Cell Lines
		2.2 Subculturing of Suspension Cell Lines
		2.3 Cryopreservation of Suspension Cell Lines
	3 Methods
		3.1 Reviving Frozen B Cell Lines from Liquid Nitrogen Storage
		3.2 Subculturing of Suspension Cell Lines Grown in 100-mm Culture Plates
		3.3 Cryopreservation of Suspension Cell Lines
	4 Notes
	References
Chapter 3: Culture of Adherent Cancer Cell Lines
	1 Introduction
	2 Materials
		2.1 Preparing Cell Culture Medium from Powder
		2.2 Thawing and Reviving Frozen Adherent Cancer Cell Lines
		2.3 Passaging Adherent Cancer Cell Lines
		2.4 Counting Adherent Cancer Cells
		2.5 Freezing Adherent Cancer Cell Lines
	3 Methods
		3.1 Preparation of 1 L of Powdered Cell Culture Medium
		3.2 Thawing and Reviving Frozen Adherent Cancer Cell Lines from Liquid Nitrogen Storage
		3.3 Passaging of Adherent Cancer Cell Lines
		3.4 Counting Adherent Cancer Cells
		3.5 Freezing of Adherent Cancer Cell Lines
	4 Notes
	Reference
Chapter 4: Breast Cancer Xenograft Murine Models
	1 Introduction
		1.1 Grafting of Breast Cancer Cell Lines
		1.2 Xenografting Patient-Derived Tumors
	2 Materials
	3 Methods
		3.1 Personnel Preparation for Procedures
		3.2 Protocol 1: Passage of Patient-Derived Xenografts as Tumors Pieces and Freezing
		3.3 Protocol 2: Xenograft of Patient-Derived Xenografts into  Mice
		3.4 Protocol 3: Generation of Single-Cell Suspensions from Patient-Derived Xenografts
		3.5 Protocol 4: Xenografting of Cells Derived from Cell Lines and the Single-Cellular Suspensions Prepared from  PDXs
	4 Notes
	References
Chapter 5: Processing and Cryopreservation of Blood, Cancer Tissues, and Cancer Cells for Viable Biobanking
	1 Introduction
		1.1 Peripheral Blood and Bone Marrow Aspirates
		1.2 Solid Tumor Tissue
	2 Materials
		2.1 General Materials and Equipment
		2.2 Additional Materials and Equipment for Bone Marrow Aspirate or Peripheral Blood
		2.3 Additional Materials and Equipment for Tissue
		2.4 Additional Materials and Equipment for Dissociating Tissue to Individual Cells
	3 Methods
		3.1 Peripheral Blood Processing for Plasma and Viable PBMCS
		3.2 Bone Marrow Processing
		3.3 Tissue Processing for Frozen Viable Tissue Fragments
		3.4 Tissue Processing for Dissociated Cells
	4 Notes
	References
Chapter 6: Adipocyte-Breast Cancer Cell Co-Culture in Transwells
	1 Introduction
	2 Materials
	3 Methods
		3.1 Pre-differentiated SGBS Cell Retrieval
		3.2 Pre-differentiated SGBS Cell Subculture
		3.3 SGBS Cell Differentiation
		3.4 MCF-7 Cell Retrieval
		3.5 MCF-7 Cell Subculture
		3.6 Cell Co-Culture
		3.7 Oil Red O Staining of Adipocytes
	4 Notes
	References
Chapter 7: Three-Dimensional Co-Culture Method for Studying Interactions Between Adipocytes, Extracellular Matrix, and Cancer ...
	1 Introduction
	2 Materials
		2.1 Adipogenesis
		2.2 Three-Dimensional Culture
		2.3 Fixation and Immunofluorescence (IF) Staining
	3 Method
		3.1 Adipogenic Differentiation of Pre-Adipocytes
		3.2 Three-Dimensional Co-Culture
		3.3 Fixation
		3.4 Immunofluorescence Staining and Slide Mounting
	4 Notes
	References
Chapter 8: Modeling Collective Invasion and Single-Cell Mesenchymal Invasion in Three-Dimensional Matrigel-Collagen I Cultures
	1 Introduction
	2 Materials
		2.1 Aliquoting and Storing Matrigel
		2.2 Coating Coverslips with Matrigel
		2.3 Plating Cells
		2.4 Generating Fibrillar Collagen I Overlay
		2.5 Imaging and Quantifying Spheroids
		2.6 Permeabilizing and Staining Spheroids
	3 Methods
		3.1 Aliquoting and Storing Matrigel
		3.2 Preparing Sterilized Coverslips
		3.3 Coating Sterilized Coverslips with Matrigel
		3.4 Plating Single Cells on Top of Pre-polymerized Matrigel
		3.5 Plating Single Cells Within Diluted Matrigel
		3.6 Plating Pre-clustered Spheroids Within Diluted Matrigel
		3.7 Generating Fibrillar Collagen I Overlay on Matrigel (Sandwich Assay)
		3.8 Imaging Live Spheroids
		3.9 Quantifying Spheroid Invasion: Elongation Index in Collectively Invasive Spheroids
		3.10 Quantifying Spheroid Invasion: Protrusion Length in Mesenchymal-Type Invasive Spheroids
		3.11 Visual Representation of Spheroid Invasion over  Time
		3.12 Permeabilizing and Staining Spheroids
	4 Notes
	References
Chapter 9: CellTrace Violet Flow Cytometric Assay to Assess Cell Proliferation
	1 Introduction
	2 Materials
		2.1 CellTrace Violet
		2.2 Viability Dyes Compatible with CellTrace Violet
		2.3 Cell Suspension Preparation
		2.4 Cell Acquisition on the Flow Cytometer
		2.5 Others
	3 Methods
		3.1 Prior to Day 0
		3.2 Day 0
			3.2.1 Harvest and Preparation of Single-Cell Suspension
			3.2.2 Cell Staining with CellTrace Violet
			3.2.3 Cell Staining with Viability Dye for Same-Day Flow Cytometric Acquisition of Fresh Cells
			3.2.4 Cell Staining with Viability Dye for Flow Cytometric Acquisition of Fixed Cells
		3.3 Day 3: Cell Suspensions and Staining of Cells Labeled with CellTrace Violet
		3.4 Day 6: Cell Suspension and Staining of Cells Labeled with CellTrace Violet
	4 Notes
	References
Chapter 10: Enzyme-Linked Immunosorbent Assay (ELISA)
	1 Introduction
		1.1 Primary Antibodies
			1.1.1 IgG
			1.1.2 IgM
			1.1.3 IgA
			1.1.4 IgE
		1.2 Antigens
		1.3 Secondary Antibodies
		1.4 Enzyme-Conjugated Antibodies
			1.4.1 Horseradish Peroxidase (HRP)
			1.4.2 Alkaline Phosphatase (ALP)
		1.5 Substrates
			1.5.1 3,3′,5,5′-Tetramethylbenzidine (TMB)
			1.5.2 2,2′-Azinobis (3-Ethylbenzothiazoline-6-Sulfonic Acid) Diammonium Salt (ABTS)
			1.5.3 p-Nitrophenylphosphate, Disodium Salt (pNPP)
			1.5.4 Luminescent and Fluorescent Probes
		1.6 Stop Buffer
		1.7 Coating Buffer
		1.8 Blocking Buffer
		1.9 Washing Buffer
		1.10 Types of ELISA
			1.10.1 Direct ELISA
			1.10.2 Indirect ELISA
			1.10.3 Sandwich  EISA
			1.10.4 Competitive ELISA
			1.10.5 Nanoparticle-Based ELISA
	2 Materials
	3 Methods
		3.1 Direct ELISA
		3.2 Indirect ELISA
		3.3 Sandwich ELISA
		3.4 Competitive ELISA
		3.5 Gold Nanoparticle-Based ELISA
		3.6 Analysis
	4 Notes
	References
Chapter 11: Flow Cytometry Analysis to Detect Lapatinib-Induced Modulation of Constitutive and IFN-γ-Induced HLA Class I Expre...
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Flow Cytometry Assay
	3 Method
		3.1 Cell Preparation: Day 1
		3.2 Treatment of Cells with Lapatinib and IFN-γ: Day 2
		3.3 Preparation of Treated Cells for Flow Cytometry: Day 3
		3.4 Flow Cytometry to Determine HLA Class I Expression
		3.5 Interpretation of Data and Statistical Analysis
	4 Notes
	References
Chapter 12: An End-to-End Workflow for Interrogating Tumor-Infiltrating Myeloid Cells Using Mass Cytometry
	1 Introduction
	2 Materials
		2.1 Blood Collection and Fixation/RBC Lysis
		2.2 NOP12 Tumor Cell Culture
		2.3 Tumor Processing
		2.4 CyTOF Staining
		2.5 CyTOF Data Analysis Software
		2.6 Myeloid Cell Deep Phenotyping R Packages
	3 Methods
		3.1 Growing NOP12 Tumor Cells
		3.2 Transplantable Tumor Model
		3.3 Blood and Tumor Processing at Endpoint for CyTOF Profiling
		3.4 CyTOF Staining
		3.5 CyTOF Data Pre-processing
		3.6 Major Immune Cell Type Annotation
		3.7 Myeloid Lineage Cell Deep Phenotyping
	4 Notes
	References
Chapter 13: Manual Immunofluorescence of Formalin-Fixed Paraffin-Embedded Human Tumor Tissues
	1 Introduction
	2 Materials
		2.1 Tissue and Equipment
		2.2 Buffers and Solutions
	3 Methods
		3.1 Tissue Preparation
		3.2 Deparaffinization and Rehydration (Fig. 2) (from this step forward, ensure that slides are not left without liquid atop th...
		3.3 Tissue Fixation
		3.4 Antigen Retrieval
		3.5 Blocking
		3.6 Antibody Incubations (all the following steps should be protected from direct light exposure when possible)
		3.7 Quenching
		3.8 Counterstaining
		3.9 Mounting
		3.10 Imaging
	4 Notes
	References
Chapter 14: Western Blot Analysis of Lapatinib-Mediated Inhibition of the Epidermal Growth Factor Receptor 2 (HER2) Pathway in...
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 SDS PAGE
		2.3 Western Blotting
		2.4 Immunodetection of Proteins
	3 Method
		3.1 Preparation of Whole Cell Lysates
		3.2 Protein Concentration Using BCA Assay
		3.3 Preparation of Sample for SDS-PAGE
		3.4 SDS-PAGE
		3.5 Western Blotting - Electrophoretic Protein Transfer
		3.6 Western Blotting - Immunodetection
	4 Notes
	References
Chapter 15: Evaluating Cell Membrane Localization and Intracellular Transport of Proteins by Biotinylation
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Stock Solutions
		2.3 Biotinylating Solutions
		2.4 Cell Harvesting and Sample Preparation Solutions
	3 Methods
		3.1 Experimental Design
		3.2 Cell Preparation
		3.3 Surface Biotinylation
		3.4 Internalization
		3.5 Recycling
		3.6 Sample Processing and Immunoprecipitation of Biotin-Labeled Proteins
	4 Notes
	References
Chapter 16: Multiplexed Quantitative Proteomic Profiling of Cancer Cells and Tissues Using Isobaric Labeling-Based Tags
	1 Introduction
	2 Materials
		2.1 Lysate Preparation
		2.2 Sample Preparation for Mass Spectrometry
		2.3 High pH Reversed Phase HPLC
		2.4 Equipment
	3 Methods
		3.1 Tissue/Cell Lysis
		3.2 Total Protein Quantification (see Note 7)
		3.3 Reduction and Alkylation of Proteins
		3.4 SP3 Cleanup of Protein Samples and Tryptic Digestion (see Note 11)
		3.5 TMT Labeling of Peptides
		3.6 Cleanup of Sample Using SepPak Cartridge
		3.7 Fractionation of Peptides
		3.8 Purification of Peptides Using Stage  Tips
		3.9 Mass Spectrometry Analysis
		3.10 Data Analysis
	4 Notes
	References
Chapter 17: The Use of Seahorse XF Assays to Interrogate Real-Time Energy Metabolism in Cancer Cell Lines
	1 Introduction
	2 Materials
	3 Methods
		3.1 Real-Time ATP Rate Assay with Suspension Cells
		3.2 Real-Time ATP Rate Assay with Adherent Cells
	4 Notes
	References
Chapter 18: Using CRISPR-Cas9 to Dissect Cancer Mutations in Cell Lines
	1 Introduction
	2 Materials
		2.1 Buffers and Reagents
		2.2 Cell Culture
		2.3 Kits, Reagents, and Equipment
	3 Methods
		3.1 sgRNA Design to Inactivate a Specific Mutation of Interest
			3.1.1 Traffic-Light Reporter Assay
		3.2 Lentivirus Production
		3.3 Virus Particles Concentration (>100x)
		3.4 Transductions and Flow Cytometry Analysis
		3.5 Primer Design for Amplification of the Targeted Genomic Locus
		3.6 Harvesting Genomic DNA and Genotyping Using Sanger Sequencing
		3.7 CRISPR-Cas9 Dropout Screen to Identify Novel Cancer Vulnerability Mutations
		3.8 Generation of Isogenic hiPSCs with Cancer Mutations
		3.9 In Vitro mRNA Transcription
		3.10 TP53 Base Editing in Human iPS Cells
		3.11 Quantifying Base-Editing Efficiencies
	4 Notes
	References
Chapter 19: Single-Cell RNA Sequencing Analysis Using Fluidigm C1 Platform for Characterization of Heterogeneous Transcriptomes
	1 Introduction
	2 Materials
		2.1 Single-Cell Preparation
		2.2 Buoyancy  Test
		2.3 Construction of 3′-End Enriched cDNA Libraries (see Note 1)
		2.4 Bioinformatic Analyses
		2.5 DEG Validation at Gene Level
		2.6 DEG Validation at Protein Level
	3 Methods
		3.1 Single-Cell Preparation
		3.2 Buoyancy Test
		3.3 Construction of 3′-End Enriched cDNA Library Pools for  NGS
		3.4 Bioinformatic Analysis
		3.5 DEG Validation at Gene Level
		3.6 DEG Validation at Protein Level
	4 Notes
	References
Chapter 20: Sequences to Differences in Gene Expression: Analysis of RNA-Seq Data
	1 Introduction
	2 Materials
		2.1 Experimental Setups
		2.2 Single and Paired-End Sequencing
		2.3 Library Preparation Strandedness
			2.3.1 Unstranded Libraries
			2.3.2 Stranded Libraries
		2.4 Mate Orientation
		2.5 Resource Considerations and Workflows
		2.6 Implementation
	3 Methods
		3.1 Preprocessing and Data Preparation
			3.1.1 Annotation Files Checklist: Adapter Sequences
			3.1.2 Annotation Files Checklist: Annotations Required to Build Indexes and Processing
			3.1.3 Fastq Files
			3.1.4 Workflow Initialization
			3.1.5 Index Building
			3.1.6 FastQC Generation and Inspection: FastQC and MultiQC
			3.1.7 FastQC Generation and Inspection: Assessment of Base Qualities
			3.1.8 FastQC Generation and Inspection: Adapter Content
			3.1.9 FastQC Generation and Inspection: G/C Content
			3.1.10 Adapter Trimming
		3.2 Upstream Processing
			3.2.1 Alignment
			3.2.2 Bam File Filtering
			3.2.3 Visualization and Coverage File Generation
			3.2.4 Quantification
		3.3 High-Level Analysis
			3.3.1 Underlying Statistics and Principles
			3.3.2 Differential Expression Analysis: Principles
			3.3.3 Differential Expression Analysis: Gene-Level Filtering
			3.3.4 Differential Expression Analysis: Fold Change
			3.3.5 Normalization
			3.3.6 Batch Effect Removal
			3.3.7 Quality Control
			3.3.8 Pathway Analyses
			3.3.9 Integration with Further RNA-Seq Datasets
	4 Notes
	References
Chapter 21: Reverse Transcription-Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) for Gene Expression Analyses
	1 Introduction
	2 Materials
		2.1 Total RNA Sample Purity-Testing (see Note 1)
		2.2 Total RNA Sample Integrity-Testing via Agarose Gel Electrophoresis (see Notes 2 and 3)
		2.3 qPCR Primer Design
		2.4 Reagents, Consumables, and Instruments for cDNA Synthesis (Reverse Transcription; RT)
		2.5 Reagents, Consumables, and Instruments for qPCR
	3 Methods
		3.1 Total RNA Sample Purity-Testing (see Note 1)
		3.2 Total RNA Sample Integrity-Testing via Agarose Gel Electrophoresis (see Notes 2 and 3)
		3.3 qPCR Primer Design
		3.4 cDNA Synthesis (see Note 11)
		3.5 cDNA Pool Templates for Primer Quality Testing (see Note 13)
		3.6 Individual cDNA Templates for Normalizer Testing and RT-qPCR Experiments (see Note 13)
		3.7 qPCR Mixture
		3.8 qPCR Thermal Cycling Parameters
		3.9 Primer Pair Quality Testing
		3.10 Candidate Normalizer Gene Testing
		3.11 Comparative qPCR Analysis
		3.12 RT-qPCR Experiment
	4 Notes
	References
Chapter 22: Peptide-Affinity Isolation of Extracellular Vesicles and Cell-Free DNA From Human Plasma
	1 Introduction
	2 Materials
		2.1 Vn96 Peptide
		2.2 Reagents for Elution of EVs from Vn96
		2.3 Kits and Reagents for Nucleic Acid Extraction
		2.4 Lysis of EVs for Western-Blot Analyses
	3 Methods
		3.1 Isolation of EVs and cfDNA from Plasma Using the Vn96 Synthetic Peptide
		3.2 Nanoparticle Tracking Analysis
		3.3 DNA Extraction from the Vn96-EV/cfDNA Pellet
		3.4 RNA Extraction from the Vn96-EV/cfDNA Pellet
		3.5 Protein Extraction from the Vn96-EV/cfDNA Pellet for Use in Western-Blot Analyses
	4 Notes
	References
Chapter 23: Characterizing Extracellular Vesicles Using Nanoparticle-Tracking Analysis
	1 Introduction
	2 Materials
		2.1 Generating EV Conditioned Media from Suspension Cancer Cell Line
		2.2 Generating EV Conditioned Media from Adherent Cancer Cell Line
		2.3 Isolating EVs from Human Biofluid, Namely, Plasma, BM Aspirate, and CSF
		2.4 Isolation of EVs by Ultracentrifugation (UCF) (see Note 5)
		2.5 Isolation of EVs by Size Exclusion Chromatography (SEC), Namely, qEV Izon (see Note 6)
		2.6 Isolation of EVs Using Polyethylene Glycol (PEG), Namely, ExoQuick (Systems Biosciences) (see Note 8)
		2.7 Isolation of EVs by Protein-Affinity-Based Isolation Namely, Vn96-New England peptide (see Note 9)
		2.8 Preparation of Samples for Nanoparticle Tracking
		2.9 Running Samples on the Nanoparticle Tracker
	3 Methods
		3.1 Generating EV Conditioned Media from Suspension Cancer Cell Line
		3.2 Generating EV Conditioned Media from Adherent Cancer Cell Line
		3.3 Preparation of EVs from Conditioned Media for NTA Characterization
		3.4 Preparation of EVs from Biofluid for NTA Characterization
		3.5 Isolation of EVs by Ultracentrifugation (UCF) (see Note 5)
		3.6 Preparation of UCF Isolated EVs for NTA Characterization
		3.7 Isolation of EVs by Size Exclusion Chromatography (SEC) (see Note 6)
		3.8 Polyethylene Glycol (PEG)-Based EV Isolation, Namely, ExoQuick (see Note 8)
		3.9 Preparation of SEC and PEG Isolated EVs for NTA Characterization
		3.10 Isolation of EVs by Protein-Affinity-Based Isolation Method, Namely, Vn96 (see Note 9)
		3.11 Preparation of Vn96 Isolated EVs for NTA Characterization
		3.12 Nanosight System Setup and Sample Running (see Fig. 1)
	4 Notes
	References
Index