Esophageal Squamous Cell Carcinoma: Methods and Protocols (Methods in Molecular Biology, 2129)

Preface
Contents
Contributors
Chapter 1: Introduction: Esophageal Squamous Cell Carcinoma-Current Status and Future Advances
	1 Global Burden of Esophageal Squamous Cell Carcinoma
	2 Risk Factors
	3 Predisposing Lesions
	4 Clinicopathological Features
	5 Prognosis
	6 Future Perspectives
	References
Chapter 2: Histopathological Assessment for Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 3: Application of Pathological Staging in Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 4: Macroscopic Examination of Surgical Specimen of Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 5: Endoscopic Diagnosis and Treatment of Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
		2.1 Diagnostic Endoscopy
		2.2 Endoscopic Ultrasound (EUS)
		2.3 Endoscopic Resection
	3 Methods
		3.1 Diagnostic Endoscopy
			3.1.1 White-Light Endoscopy
			3.1.2 Narrowband Imaging (NBI) (Fig. 2b, c)
			3.1.3 Lugol Chromoendoscopy (Fig. 2d)
			3.1.4 Endoscopic Ultrasound (EUS)
			3.1.5 Endoscopic Resection
			3.1.6 Endoscopic Mucosal Resection (EMR)
			3.1.7 Endoscopic Submucosal Dissection (ESD) (Fig. 3)
	4 Notes
	References
Chapter 6: Macroscopic Assessment and Sampling of Endoscopic Resection Specimens for Squamous Epithelial Malignancies with Sup...
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 7: Roles of Pathological Assessments of Frozen Sections in Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Frozen Blocks
		3.2 Cutting of Frozen Blocks
		3.3 Staining of Frozen Sections
		3.4 Interpret the Sections with Microscope by Pathologist (See Note 7)
		3.5 Tissues for Clinical Diagnosis Should Transfer Back to formlin fixation and then embedded in paraffin After Frozen Section...
	4 Notes
	References
Chapter 8: Biobanking for Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
		2.1 Raw Materials
		2.2 Reagents and Consumables
		2.3 Equipment
	3 Method
		3.1 Collection Procedures
		3.2 Tissue Handing Protocol
		3.3 Blood Handling Protocol
		3.4 Data Management Protocol
	4 Notes
	References
Chapter 9: Whole-Slide Imaging of Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
	3 Methods
		3.1 Prepare the Slides Before Scanning
		3.2 System Start-Up
		3.3 Loading the Slides for Scanning (Fig. 2)
		3.4 Set Up the Scanning Field
		3.5 Scanning
		3.6 Completion
		3.7 Work with the Images (See Note 9)
	4 Notes
	References
Chapter 10: Use of Tissue Microarray in Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
		2.1 ``Recipient´´ Paraffin Block
		2.2 ``Donor´´ Paraffin Blocks
		2.3 TMA Layout Design
		2.4 Tools and Punchers
	3 Methods
		3.1 Manual Method by Alignment Mold
		3.2 Automatic Method by Coaxial Puncher
		3.3 Preparation for Sectioning
		3.4 Image Processing
	4 Notes
	References
Chapter 11: Patient-Derived Xenograft and Mice Models in Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
		2.1 Implantation of ESCC into Mice
		2.2 Experimental Phase
		2.3 Dissection of Mice
	3 Method
		3.1 Implantation of ESCC into Mice
		3.2 Experimental Phase
		3.3 Dissection of Mice
	4 Notes
	References
Chapter 12: Orthotopic Xenograft Mouse Model in Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
		2.1 Reagents and Consumables
		2.2 Equipment
	3 Methods
		3.1 Preparation of ESCC Cells
		3.2 Orthotopic Inoculation
		3.3 Live Animal Imaging
	4 Notes
	References
Chapter 13: In Vitro Assays of Biological Aggressiveness of Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Cell Proliferation
		2.3 Cytotoxicity Assay
		2.4 Colony Formation Assay
		2.5 Wound Healing Assay
		2.6 Invasion and Migration Assays
	3 Methods
		3.1 Cell Culture
		3.2 Cell Proliferation Assay
		3.3 Cytotoxicity Assay
		3.4 Colony Formation Assay
		3.5 Wound Healing Assay
		3.6 Invasion and Migration Assays
	4 Notes
	References
Chapter 14: Detention and Identification of Cancer Stem Cells in Esophageal Squamous Cell Carcinoma
	1 Introduction
		1.1 Implications of CSC Markers in ESCC Prognosis
		1.2 Therapeutic Applications of CSCs in ESCC
	2 Materials
		2.1 Cell Culture
		2.2 Cell Staining and Sorting
		2.3 In Vitro Functional Assay
		2.4 In Vivo Xenotransplantation
	3 Methods
		3.1 Cell Culture
		3.2 Cell Staining and Sorting
		3.3 In Vitro Tumor Spheres Formation Assay
		3.4 Xenotransplantation
	4 Notes
	References
Chapter 15: Liquid Biopsy: Detection of Circulating Tumor Cells in Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
	3 Method
		3.1 Extraction of CTC
		3.2 Detection Protocol
	4 Notes
	References
Chapter 16: Liquid Biopsy for Investigation of Cancer DNA in Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
	3 Method
		3.1 Isolation of Cell-Free DNA from Plasma
			3.1.1 Isolation of Plasma
			3.1.2 Protein Digestion
			3.1.3 Purification of DNA
		3.2 Isolation of DNA from Exosomes
			3.2.1 Plasma Separation
			3.2.2 Filtration and Separations of Exosomes
			3.2.3 Digestion of Proteins and Lysis of Exosomes
			3.2.4 Purification of DNA
	4 Notes
	References
Chapter 17: Genome Sequencing in Esophageal Squamous Cell Carcinoma
	1 Introduction
		1.1 Influence of Next-Generation Sequencing on ESCC Research
		1.2 Whole-Genome Sequencing (WGS)
		1.3 Whole-Exome Sequencing
		1.4 Whole-Transcriptome Sequencing/RNA Sequencing
		1.5 Targeted Next-Generation Sequencing
	2 Materials
		2.1 Library Preparation Reagents and Materials
		2.2 Ion Chef Reagents and Materials
		2.3 Ion S5 Sequencing Reagents and Materials
	3 Methods
		3.1 Overview of Next-Generation Sequencing
		3.2 Construction of Library
			3.2.1 Library Purification
			3.2.2 Library Quantification
		3.3 Template Preparation and Chip Loading
		3.4 Loading the Diluted Libraries to the Ion Chef
		3.5 Initialize the Sequencer
			3.5.1 Before Initializing the Sequencer
			3.5.2 Initialize the Sequencer
			3.5.3 Start Sequencing Run
		3.6 Data Analysis
	4 Notes
	References
Chapter 18: Roles of MicroRNAs in Esophageal Squamous Cell Carcinoma Pathogenesis
	1 Introduction
		1.1 Aberrant miRNAs Expression and Pathogenesis of ESCC
		1.2 miRNAs in Prognosis of Patients with ESCC
	2 Materials
		2.1 Isolation of Total RNA
		2.2 Microarray
			2.2.1 Post-processing
			2.2.2 Labelling of RNA
			2.2.3 Hybridization
			2.2.4 Washing and Scanning
		2.3 qRT-PCR
	3 Methods
		3.1 Extraction of Total RNA Including miRNAs from Tissue, Blood, or Cells
		3.2 miRNAs Microarray: A microarray Used for miRNAs Expression has 5 Steps
		3.3 Post-processing
		3.4 Labelling
		3.5 Hybridization
		3.6 Washing and Scanning
		3.7 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
			3.7.1 Performing the Reverse Transcription Reaction
			3.7.2 Performing the Polymerase Chain Reaction (PCR)
	4 Notes
	References
Chapter 19: Mass Spectrometry for Biomarkers Discovery in Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Cell Lysis
		2.3 Protein Digestion and Mass Spectrometry
		2.4 Data Analysis
	3 Methods
		3.1 Cell Culture
		3.2 Cell Lysis
		3.3 Protein Digestion and Mass Spectrometry
		3.4 Data Analysis
	4 Notes
	References
Chapter 20: Immunoblotting in Detection of Tumor-Associated Antigens in Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
		2.1 Western Blotting
	3 Methods
	4 Notes
	References
Chapter 21: Immunohistochemistry for Protein Detection in Esophageal Squamous Cell Carcinoma
	1 Introduction
		1.1 Concept of Use of Immunohistochemistry
		1.2 Application of Immunohistochemistry in Esophageal Squamous Cell Carcinoma
		1.3 Principles of Immunohistochemistry for Formalin-Fixed Paraffin-Embedded Tissue
			1.3.1 Tissue Fixation
			1.3.2 Paraffin Embedding
			1.3.3 Sectioning
			1.3.4 Dewaxing
			1.3.5 Antigen Retrieval
			1.3.6 Blocking
			1.3.7 Incubating with Antibody
			1.3.8 Detection
			1.3.9 Mounting and Covering
	2 Materials
		2.1 Specimens
		2.2 Reagents
		2.3 Equipment
	3 Method
		3.1 Preparing Tissue Sections
		3.2 Entering Case Identification Information
		3.3 Heat-Induced Epitope Retrieval
		3.4 Setting up the Antibodies
		3.5 Starting a Run
		3.6 Completing a Run
		3.7 Counterstaining
		3.8 Staining Results
		3.9 DAB Disposal
	4 Notes
	References
Chapter 22: Radiotherapy for Cervical Esophageal Squamous Cell Carcinoma
	1 Introduction
	2 Materials
		2.1 Definitive Chemoradiotherapy
		2.2 Chemoselection
		2.3 Salvage Pharyngolaryngectomy with Total Esophagectomy Following Definitive Chemoradiotherapy
		2.4 Adjuvant Radiotherapy or Chemoradiotherapy After Pharyngo-Laryngo-Esophagectomy
	3 Methods
		3.1 Procedures of Radiotherapy
			3.1.1 Patient and Simulation
			3.1.2 Target Volumes
			3.1.3 Dose Prescription
			3.1.4 Radiotherapy Planning and Treatment
	4 Notes
	References
Chapter 23: Radiotherapy for Thoracic Esophageal Squamous Cell Carcinoma
	1 Introduction
		1.1 Landmarks of ESCC
		1.2 Local Spread and Lymphatic Spread of ESCC
		1.3 Indications for Radiotherapy
			1.3.1 Neoadjuvant Chemoradiotherapy
			1.3.2 Definitive Chemoradiotherapy
			1.3.3 Postoperative Radiotherapy
			1.3.4 Palliative Radiotherapy
				External Beam Radiotherapy
				Brachytherapy
	2 Materials
		2.1 Pre-treatment Investigations
		2.2 Planning and Treatment
	3 Methods
		3.1 Procedures of Radiotherapy
			3.1.1 Simulator
			3.1.2 CT Planning
				Immobilization
				CT Scanning
				Target Volume Definition
				Beam Arrangement (Fig. 2a, b)
				Dose Acceptance and Organs-at-Risks (OARs)
	4 Notes
	References
Chapter 24: Systemic Therapy for Esophageal Squamous Cell Carcinoma
	1 Introduction
		1.1 Chemotherapy
			1.1.1 Perioperative
			1.1.2 Palliative
		1.2 Target Therapy
			1.2.1 Epidermal Growth Factor Receptor (EGFR)
			1.2.2 Immune Checkpoint Inhibitor
			1.2.3 Other Target Therapy
	2 Materials
		2.1 Therapeutic Agents
		2.2 Investigations
			2.2.1 Pre-treatment Investigations
			2.2.2 On-Treatment Investigations
	3 Methods
		3.1 Neoadjuvant
		3.2 Palliative
			3.2.1 First Line
			3.2.2 Second Line
			3.2.3 Third Line or beyond
	4 Notes
	References
Chapter 25: Surgical Protocols for Squamous Cell Cancer of the Esophagus
	1 Introduction
		1.1 Depth of Invasion for ESCC and Relation to Incidence of Nodal Metastases
	2 Materials
		2.1 Patient Selection
			2.1.1 Disease Staging
			2.1.2 Physiological Assessment
		2.2 Preoperative Preparations
	3 Methods
		3.1 Location of the Tumor and Implications
		3.2 Lymphadenectomy
			3.2.1 Types of Lymphadenectomy
		3.3 Surgical Approaches for Resection
		3.4 Reconstruction: Organ for Esophageal Substitution and Route of Reconstruction
		3.5 Surgical Technique
			3.5.1 Open Surgical Resection Method
				Positioning and Anesthetic Technique
				Operating Technique: Abdominal Phase
				Operating Technique: Thoracic Phase
				Operating Technique: Cervical Phase
				Operating Technique: Minimally Invasive Esophagectomy
		3.6 Postoperative Management
	4 Notes
	References
Chapter 26: Anesthetic Management for Squamous Cell Carcinoma of the Esophagus
	1 Introduction
	2 Materials
		2.1 Patients
			2.1.1 Risk Assessment and Stratification
			2.1.2 Preoperative Anemia
			2.1.3 Prehabilitation
		2.2 Equipment
			2.2.1 Epidural Catheter and Thoracic Epidural Analgesia
			2.2.2 Special Endotracheal Tubes and Intraoperative Lung Isolation Techniques
		2.3 Drugs
			2.3.1 Propofol-Based Total Intravenous Anesthesia
			2.3.2 Nonsteroidal Anti-Inflammatory Drugs (NSAIDs)
			2.3.3 Perioperative Beta-Blockers
	3 Methods
		3.1 Preoperative Anesthetic Management
		3.2 Intraoperative Anesthetic Assessment
		3.3 Postoperative Anesthetic Management
	4 Notes
	References
Index