Lung Cancer: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Automated TTF-1 Immunohistochemistry Assay for the Differentiation of Lung Adenocarcinoma Versus Lung Squamous Cell...
	1 Introduction
	2 Materials
		2.1 Ready to Use Reagents (see Note 1)
		2.2 Immunohistochemistry and Histology Reagents
		2.3 Additional Equipment and Materials
	3 Methods
		3.1 Tissue Fixation and Processing
		3.2 Tissue Sectioning
		3.3 Immunohistochemistry Staining in the Dako Omnis IHC Staining System
		3.4 Counterstaining
		3.5 Cover Slipping
		3.6 Staining Evaluation and Interpretation
	4 Notes
	References
Chapter 2: Immunohistochemical Detection of p40 Expression in Lung Cancer Clinical Samples
	1 Introduction
	2 Materials
		2.1 IHC Buffer Solutions and Histology Reagents
		2.2 Antibodies and Detection  Kits
		2.3 Laboratory Equipment
	3 Methods
	4 Notes
	References
Chapter 3: Automated Immunohistochemistry Assay for the Detection of Napsin-A in Formalin-Fixed Paraffin-Embedded Tissues from...
	1 Introduction
	2 Materials
		2.1 Standard Histology Reagents
		2.2 Immunohistochemistry Kits, Antibodies and Reagents
		2.3 Laboratory Equipment and Consumables
	3 Methods
		3.1 Reagent Preparation
		3.2 Tissue Fixation and Processing
		3.3 Tissue Sectioning
		3.4 Immunohistochemistry Staining in the Dako Omnis IHC Staining System
		3.5 Cover Slipping
		3.6 Staining Evaluation and Interpretation
	4 Notes
	References
Chapter 4: Detection of Programmed Cell Death Ligand 1 Expression in Lung Cancer Clinical Samples by an Automated Immunohistoc...
	1 Introduction
	2 Materials
		2.1 IHC Reagents and Antibody Clones
		2.2 IHC Equipment
		2.3 Standard Reagents and Additional Equipment
	3 Methods
		3.1 Automated PD-L1 Immunohistochemistry Staining
		3.2 Image Acquisition, Digital Analysis and Staining Analysis
	4 Notes
	References
Chapter 5: Western Blot as a Support Technique for Immunohistochemistry to Detect Programmed Cell Death Ligand 1 Expression
	1 Introduction
	2 Materials
		2.1 Gel Electrophoresis Separation
		2.2 Protein Transfer
		2.3 Membrane Blocking, Immunolabeling, Washing and Development
		2.4 Cell Lines and Tissues
		2.5 Additional Materials
	3 Methods
		3.1 Sample Preparation, Electrophoresis, and Transfer
		3.2 Immunolabeling and Signal Development
	4 Notes
	References
Chapter 6: Creation of Formalin-Fixed, Paraffin-Embedded 3D Lung Cancer Cellular Spheroids for the Optimization of Immunohisto...
	1 Introduction
	2 Materials
		2.1 Cell Lines and Cell Culture Reagents
		2.2 Histology Reagents and Supplies
		2.3 Immunohistochemistry Reagents
		2.4 General Laboratory Equipment
	3 Methods
		3.1 Spheroid Preparation from Cell Cultures
		3.2 Preparation of Spheroid Paraffin Blocks
		3.3 Microtome Sectioning of Spheroid Paraffin Blocks
		3.4 Hematoxylin and Eosin (H&E) Staining
		3.5 Immunohistochemistry (IHC) of Spheroid Sections
	4 Notes
	References
Chapter 7: Immunoblot Validation of Phospho-Specific Antibodies Using Lung Cancer Cell Lines
	1 Introduction
	2 Materials
		2.1 Cell Lysis and Protein Extraction and Quantification
		2.2 Alkaline Bovine Intestinal Phosphatase (BIP) Treatment of Protein Lysates
		2.3 SDS Polyacrylamide Gel Electrophoresis
		2.4 Transfer
		2.5 Immunoblotting Reagents
		2.6 Additional Laboratory Equipment and Plasticware
	3 Methods
		3.1 Cell Lysis and Preparation of Protein Extracts
		3.2 Protein Lysate Dephosphorylation with Alkaline Bovine Intestinal Phosphatase (BIP)
		3.3 SDS Polyacrylamide Gel Electrophoresis and Transfer
		3.4 Transfer of Proteins to Nitrocellulose Membranes
		3.5 Immunoblotting, Image Development, and Capture
		3.6 Interpretation of Results
	4 Notes
	References
Chapter 8: Detection of Non-Small Lung Cell Carcinoma-Associated Genetic Alterations Using a NanoString Gene Expression Platfo...
	1 Introduction
	2 Materials
		2.1 Tumor Cell Content Assessment
		2.2 DNA/RNA Extraction
		2.3 NanoString Fusion Gene Detection
		2.4 NanoString Gene Expression
		2.5 General Laboratory Equipment, Supplies and Reagents
	3 Methods
		3.1 RNA/DNA Extraction
		3.2 Fusion Gene Detection Using the NanoString Technology
			3.2.1 Hybridization Using the nCounter Elements Chemistry (Combined Gene Expression and Fusion Gene Detection)
			3.2.2 Hybridization Using the nCounter XT Chemistry (Gene Expression Only)
			3.2.3 Operating the NanoString SPRINT Instrument
			3.2.4 NanoString Data Processing
		3.3 Final Report of NanoString Fusion Gene Detection and Gene Expression Profiling
	4 Notes
	References
Chapter 9: A PCR-Based Approach for Driver Mutation Analysis of EGFR, KRAS, and BRAF Genes in Lung Cancer Tissue Sections
	1 Introduction
		1.1 Driver Genes in Lung Cancer: Focus on Personalized Medicine
		1.2 The Polymerase Chain Reaction Technique: Basis and Principles
		1.3 PCR-Based Approach for Mutational Analysis of EGFR, KRAS, and BRAF Genes
	2 Materials
		2.1 Histology Reagents and Materials
		2.2 Reagents and Kits for DNA Extraction, PCR, and Sequencing
		2.3 General Laboratory Equipment
		2.4 General Use Laboratory Consumables
	3 Methods
		3.1 DNA Isolation from Lung Tumor Tissue Sections
			3.1.1 Deparaffinization of Tissues
			3.1.2 Tissue Digestion and DNA Extraction from Tumor Tissue Sections
		3.2 PCR Amplification of EGFR, KRAS, and BRAF Hotspot Regions
		3.3 Direct Sanger Sequencing
			3.3.1 Purification of the PCR Product for Sequencing
			3.3.2 Assembly of the Sequencing Reaction
			3.3.3 Sequencing Reaction Steps
			3.3.4 Purification of Sequencing Yield
			3.3.5 Genetic Analysis
	4 Notes
	References
Chapter 10: 6-Color Crystal Digital PCRTM for the High-Plex Detection of EGFR Mutations in Non-Small Cell Lung Cancer
	1 Introduction
		1.1 Genetic Mutations Commonly Associated with Non-small Cell Lung Cancer
		1.2 The Naica system Enables Robust Tumor-Derived DNA Analysis
		1.3 The Naica system 6-Color Digital PCR EGFR Assay
	2 Materials
		2.1 General Plasticware and Laboratory Equipment
		2.2 PCR Equipment and Software
		2.3 DNA Extraction and Quantification
		2.4 PCR Reagents and Solutions
	3 Methods
		3.1 DNA Template Preparation and PCR Setup
		3.2 Setting up the Fluorescence Spillover Compensation Matrix Using Control Samples
		3.3 Data Acquisition and Analysis of Test Samples
		3.4 Limit of Blank (LOB) Determination and Data Interpretation
	4 Notes
	References
Chapter 11: Detection of MET Exon 14 Skipping Alterations in Lung Cancer Clinical Samples Using a PCR-Based Approach
	1 Introduction
	2 Materials
		2.1 Isolation of Total RNA from Fresh Tissue
		2.2 Isolation of Total RNA from Formalin Fixed Paraffin Embedded (FFPE) Sections
		2.3 PCR Detection of METex14 Skipped Samples
	3 Methods
		3.1 RNA Isolation from Fresh Tissues
		3.2 RNA Isolation from FFPE Tissues
		3.3 Detection of METex14 Skipped Samples Using  PCR
		3.4 Agarose Gel Electrophoresis
	4 Notes
	References
Chapter 12: Immunocytochemical Detection of ALK and ROS1 Rearrangements in Lung Cancer Cytological Samples
	1 Introduction
	2 Materials
		2.1 Laboratory Equipment
		2.2 General Reagents and Solutions and Immunohistochemistry Reagents
	3 Methods
		3.1 Slide Preparation
		3.2 Immunocytochemistry
		3.3 Data Analysis
	4 Notes
	References
Chapter 13: A Method for the Establishment of Human Lung Adenocarcinoma Patient-Derived Xenografts in Mice
	1 Introduction
	2 Materials
		2.1 Materials, Laboratory Equipment, and Solutions for Tumor Implantation
		2.2 Materials for Tumor Freeze  Down
		2.3 Materials for Peripheral Blood Mononuclear Cell (PBMC) Engraftment
		2.4 Materials for Generating and Freezing Tumor Cell Suspension
	3 Methods
		3.1 Subcutaneous Implantation of Tumor Pieces
		3.2 Freezing of Tumor Pieces for  PDXs
		3.3 Engrafting PBMCs into Immunodeficient  Mice
		3.4 Subcutaneous Implantation of Tumor Cells into Immunodeficient  Mice
		3.5 Freezing Tumor Cells for  PDXs
	4 Notes
	References
Chapter 14: A Method for the Establishment and Characterization of Mouse Lung Adenocarcinoma Cell Lines that Mimic Traits of H...
	1 Introduction
	2 Materials
		2.1 Solutions and Reagents
		2.2 Mice and Surgical Procedure Equipment
		2.3 Laboratory Equipment
		2.4 Plasticware and Other Consumables
	3 Methods
		3.1 Injection of Carcinogen to  Mice
		3.2 Isolation of Lung Tumors
		3.3 Histology and Lung Adenocarcinoma Diagnosis
			3.3.1 Tissue Preparation for Histology
			3.3.2 H&E Staining
			3.3.3 Periodic Acid-Schiff Staining with (α-Amylase) Diastase (PAS-D)
		3.4 Immunohistochemical Localization of Lung Adenocarcinoma Markers
		3.5 Derivation of Mouse Lung Adenocarcinoma Cell Lines
		3.6 In Vitro Validation of Proliferative Capacity by MTT Assay
		3.7 In Vitro Validation of Stemness by Tumorsphere Formation Assay
		3.8 In Vivo Validation of Malignancy and Spontaneous Metastatic Potential by Flank Assay
	4 Notes
	References
Chapter 15: Whole Transcriptome Sequencing Analysis of Cancer Stem/Progenitor Cells Obtained from Mouse Lung Adenocarcinomas
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 RNA Extraction
		2.3 RNA-Seq Workflow and Analysis, Kits and Software
		2.4 Reverse Transcription of RNA to  cDNA
		2.5 General Laboratory Equipment, Supplies, and Solutions
	3 Methods
		3.1 Culturing Cells for Sphere-Formation Assay
		3.2 Preparation of Cell Suspension in Matrigel Mixture at a Defined Plating Density
		3.3 Plating the Cell Suspension in Matrigel for Sphere-Formation Assay
		3.4 Calculation of Sphere-Formation Efficiency
		3.5 Sphere Propagation
		3.6 RNA Isolation from Lung Cancer Spheres
		3.7 Whole Transcriptome Sequencing
			3.7.1 Experimental Workflow
			3.7.2 Analysis Workflow
		3.8 Interrogation of Select Differentially Expressed Genes by Quantitative Real Time PCR (qRT-PCR)
			3.8.1 Reverse Transcription of RNA to  cDNA
	4 Notes
	References
Chapter 16: In Vivo Imaging of Orthotopic Lung Cancer Models in Mice
	1 Introduction
	2 Materials
		2.1 Cell Lines and Culture Reagents
		2.2 Plasticware for Cell Cultures
		2.3 Mice and Related Materials and Solutions
		2.4 Equipment
		2.5 Software
	3 Methods
		3.1 Cell Culture
		3.2 Preparation of Cellular Suspension for Injection
		3.3 Intravenous Injection of TC1-Luc Cells
		3.4 Intercostal Injection of LLC-Luc Cells
		3.5 Bioluminescent Imaging and Data Analysis
		3.6 Preparation of Reagents and Treatments of Tumor Bearing  Mice
	4 Notes
	References
Chapter 17: An Annexin V-FITC-Propidium Iodide-Based Method for Detecting Apoptosis in a Non-Small Cell Lung Cancer Cell Line
	1 Introduction
	2 Materials
		2.1 Reagents and Solutions
		2.2 Cell Culture Reagents and Equipment
		2.3 Other Labware and Equipment
	3 Methods
		3.1 Cell Preparation and Drug Treatment
		3.2 Cell Staining
		3.3 Flow Cytometer Instrument Setup
		3.4 Cell Analysis
	4 Notes
	References
Chapter 18: Detection of DNA Adduct Formation in Rat Lungs by a Micro-HPLC/MS/MS Approach
	1 Introduction
	2 Materials
		2.1 Synthesis, Purification, and Characterization of 1,N2-propanodGuo, [15N5]-1,N2-propanodGuo and [13C2,15N5]-1,N2-propanodGuo
		2.2 DNA Extraction and Enzymatic Hydrolysis
		2.3 Quantification of 2′-Deoxyguanosine
		2.4 Enrichment and Purification of the 1,N2-propanodGuo Adducts
		2.5 Analysis by Micro-LC-ESI/MS/MS of 1,N2-propanodGuo in  Rats
	3 Methods
		3.1 Synthesis of 1,N2-propanodGuo, [15N5]-1,N2-propanodGuo and [13C4,15N5]-1,N2-propanodGuo Standards
		3.2 DNA Extraction and Enzymatic Hydrolysis
		3.3 Quantification of 2′-Deoxyguanosine
		3.4 Enrichment and Purification of the 1,N2-propanodGuo Adducts
		3.5 Analysis by Micro-LC-ESI/MS/MS of 1,N2-propanodGuo in Rat Lungs
	4 Notes
	References
Chapter 19: A Method for Liposome Co-encapsulation of Phenethyl Isothiocyanate and Cisplatin and Determining Its Toxicity Agai...
	1 Introduction
	2 Materials
		2.1 Liposome Preparation
		2.2 1,2-Benzenedithiol (BDT) Assay
		2.3 Release
		2.4 Cell Lines, Cell Culture Media, and Cytotoxicity Assay Reagents
		2.5 Additional Laboratory Equipment and Reagents
	3 Methods
		3.1 Preparation and Characterization of Liposomes
		3.2 Determination of PEITC Loading in Liposomal-PEITC and Liposomal-PEITC-CDDP Using 1,2-Benzenedithiol (BDT) Assay
		3.3 Determination of CDDP Loading in Liposomal-CDDP and Liposomal-PEITC-CDDP by Inductively Coupled Plasma-Mass Spectrometry (...
		3.4 In Vitro Drug Release Studies
		3.5 Cytotoxicity Studies
	4 Notes
	References
Index