Liver Carcinogenesis: Methods and Protocols (Methods in Molecular Biology, 2769)

Preface
Contents
Contributors
Chapter 1: Orthotopic Model of Hepatocellular Carcinoma in Mice
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 Surgical Supplies
	3 Methods
		3.1 HCC Cell Preparation for Injection_ Time: 40-60 min
		3.2 Implantation of Syngeneic HCC Cells in Liver_ Time: 20-30 min/Mouse
			3.2.1 Pre-surgical Setup
			3.2.2 Surgical Procedure
			3.2.3 Postoperative Care
		3.3 Tumor Growth Follow-Up_ Time: 6-8 Weeks
	4 Notes
	References
Chapter 2: Diethylnitrosamine Induction of Hepatocarcinogenesis in Mice
	1 Introduction
		1.1 Generalities
		1.2 Molecular Mechanisms of DEN-Induced Hepatocarcinogenesis
		1.3 Characteristics of DEN-Induced HCC
			1.3.1 Histomorphology
			1.3.2 Mutational/Transcriptomic Landscape
			1.3.3 Immune Infiltration
		1.4 General Recommendations
		1.5 Advantages and Limitations of the Model
		1.6 Materials
	2 Methods
	3 Notes
	References
Chapter 3: Diethylnitrosamine-Induced Liver Tumorigenesis in Mice Under High-Hat High-Sucrose Diet: Stepwise High-Resolution U...
	1 Introduction
	2 Materials
		2.1 DEN Injection
		2.2 High-Fat High-Sucrose  Diet
		2.3 Ultrasonographic Equipment
		2.4 Necropsy and Liver Sampling for Histology
	3 Methods
		3.1 DEN Preparation and Administration to Animals
		3.2 High-Fat High-Sucrose Feeding
		3.3 Ultrasonographic Assessment of the Liver
			3.3.1 Work Station Preparation
			3.3.2 Mouse Preparation
			3.3.3 Transducer Positioning and Ultrasound Settings
			3.3.4 B-Mode Imaging and Ultrasonographic Description
			3.3.5 Ultrasound Examination
				Cranial Abdomen Imaging and Anatomical Landmarks
				Normal Liver Ultrasonography
				Pathologic Ultrasonographic Liver Findings
			3.3.6 Post-Imaging Mouse  Care
		3.4 Necropsy and Histological Sample Collection
			3.4.1 Sampling
			3.4.2 Interpretation of Histopathological Liver Findings
		3.5 Tumor Development Modifiers
	4 Notes
	References
Chapter 4: A Mouse Model of Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma Induced by Western Diet and Carbon Tetr...
	1 Introduction
	2 Materials
		2.1 Animals
		2.2 Diet and Treatment
		2.3 CCl4 Injection
		2.4 Equipment and Other Materials
	3 Methods
		3.1 Western Diet (WD) and High-Sugar Water Feeding
		3.2 CCl4 Preparation and Administration to Mice
		3.3 Ultrasonographic Assessment of HCC
	4 Notes
	References
Chapter 5: A Mouse Model of Hepatocellular Carcinoma Induced by Streptozotocin and High-Fat Diet
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Prepare Animals: Time 4-6 h
		3.2 Fresh Streptozotocin Buffer Preparation: Time 30 min
		3.3 Diabetes Mellitus Induced by Streptozotocin: Time 4 Weeks
		3.4 NAS, NASH, Fibrosis, and HCC Accelerated by HFD: Time 20 Weeks
	4 Notes
	References
Chapter 6: Hydrodynamic Transfection of Hepatocytes for the Study of Hepatocellular Carcinogenesis
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Disposables
		2.3 Mice and Plasmids
	3 Methods
		3.1 Preparation of Plasmid Mix Solution
		3.2 Hydrodynamic Tail Vein Injection
		3.3 Monitoring and Tumor Collection
	4 Notes
	References
Chapter 7: Experimental Model of Biliary Tract Cancers: Subcutaneous Xenograft of Human Cell Lines in Immunodeficient Nude Mice
	1 Introduction
	2 Materials
		2.1 In Vitro  Step
			2.1.1 Human BTC Cell Lines
			2.1.2 Cell Culture Reagents and Equipment
		2.2 In Vivo  Step
			2.2.1 Mouse Strains and Housing
			2.2.2 Equipment Required at the Animal Facilities
			2.2.3 Equipment Required from the Laboratory
	3 Methods
		3.1 In Vitro  Step
			3.1.1 Preparation of BTC Cell Lines
			3.1.2 Preparation of Matrigel
		3.2 In Vivo  Step
			3.2.1 Subcutaneous Injection in  Mice
			3.2.2 Staining and Immunostainings
			3.2.3 Representative Results from the BTC Xenograft Model
	4 Notes
	References
Chapter 8: Oncogene-Driven Induction of Orthotopic Cholangiocarcinoma in Mice
	1 Introduction
	2 Materials
		2.1 Mice
		2.2 Preparation of the Plasmid Solution
		2.3 Hydrodynamic Injection
		2.4 Liver Collection
		2.5 MRI Imaging
	3 Methods
		3.1 In the Animal Facility
			3.1.1 Mouse Weighing
		3.2 In the Laboratory
			3.2.1 Preparation of the Plasmid Solution
		3.3 In the Animal Facility
			3.3.1 Induction of Cholangiocarcinoma-Hydrodynamic Injection
			3.3.2 Liver Collection
		3.4 In the Laboratory
		3.5 In the MRI Core Facility
			3.5.1 MRI Imaging
				Setting the Parameters of the MRI Scanner
				Liver Immobilization in the MRI Scanner
	4 Notes
	References
Chapter 9: Isolation of Primary Mouse Hepatocytes and Non-Parenchymal Cells from a Liver with Precancerous Lesions
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Disposables and Equipment
	3 Methods
		3.1 Isolation of Primary Mouse Hepatocytes and NPCs by Liver Perfusion
			3.1.1 Preparation of Buffers_ Time: 30 min
			3.1.2 Isolation of Cell Populations from Liver
				Before Perfusion_ Time: 20 min
				Perfusion_ Time: 30 min
				After Perfusion: Hepatocyte Isolation_ Time: 90 min
				After Perfusion: Non-Parenchymal Cells Isolation_ Time: 40 min
		3.2 Generation of Leukocyte-Rich Single Cell Suspensions from Non-Perfused Whole Livers
			3.2.1 Reagent and Material Preparation_ Time: 15 min
			3.2.2 Liver Collection_ Time: 2-5 min per Sample
			3.2.3 Liver Processing and Dissociation into a Single Cell Suspension_ Time: 1 h 30 min per Sample (But Simultaneous Sample Is...
	4 Notes
	References
Chapter 10: Flow Cytometry Assessment of Lymphocyte Populations Infiltrating Liver Tumors
	1 Introduction
	2 Materials
		2.1 Materials and Equipment
		2.2 Reagents
	3 Methods
		3.1 Preparation of Buffers_Time: 10 min
		3.2 Preparation of the Single Cell Suspension for Immunostaining_Time: 1 min per Sample (See Notes 1-4)
		3.3 Cell Surface Immunostaining_Time: 150 min per Sample (but Simultaneous Sample Staining Is Possible) (See Notes 8-11)
		3.4 Intracellular/nuclear Flow Cytometry Staining_Time: 60 min per Sample (but Simultaneous Sample Staining Is Possible)
		3.5 Quick Overview of Sample Acquisition Through a Flow Cytometer, Analysis, and Cell Count Normalization
			3.5.1 Sample Acquisition Through Flow Cytometer_Time: 120 min for Compensations and 5 min per Sample Acquired (See Note 16)
			3.5.2 Analyses and Cell Count Normalization_Time: 120 min per Sample (but Simultaneous Sample Analysis Is Possible) (See Note ...
	4 Notes
	References
Chapter 11: Immunofluorescent Staining of Human Hepatic Multicellular Spheroids: A Model for Studying Liver Diseases
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Disposables
		2.3 Samples and Reagents
			2.3.1 Samples
			2.3.2 Reagents Used for Spheroid Cultures and Immunofluorescent Staining
	3 Methods
		3.1 Spheroid Culture from Primary Cells
		3.2 Spheroid Culture from Cell Lines
		3.3 Immunofluorescence Staining
			3.3.1 Fixation
			3.3.2 Permeabilization and Blocking
			3.3.3 Immunostaining
			3.3.4 Mounting
			3.3.5 Data Analysis
		3.4 Expected Outcomes
	4 Notes
	References
Chapter 12: Single-Cell Characterization of the Tumor Ecosystem in Liver Cancer
	1 Introduction
	2 Materials
		2.1 Devices for Tumor Biopsy Collection
		2.2 Reagents and Disposables
		2.3 Equipment
	3 Methods
		3.1 Preparation of Tumor Dissociation  Kit
		3.2 Tumor Biopsy Collection
		3.3 Dissociation of Tumor Biopsy into Single Cells
		3.4 Single Cell Capture
		3.5 Data Analysis
			3.5.1 Convert FASTQ Files to Single-Cell Feature Counts
			3.5.2 Data Preprocessing
			3.5.3 Data Visualization in a Low-Dimensional Space
			3.5.4 Separate Malignant and Non-Malignant Cells
			3.5.5 Determine Non-Malignant Cell Types
			3.5.6 Further Analysis
	4 Notes
	References
Chapter 13: Chromatin and DNA Dynamics in Mouse Models of Liver Cancers
	1 Introduction
	2 Materials
		2.1 Materials for ATAC-seq
			2.1.1 Equipment
			2.1.2 Disposables
			2.1.3 Reagents
		2.2 Materials for ChIP
			2.2.1 Equipment
			2.2.2 Disposables
			2.2.3 Reagents
		2.3 Materials for 3C
			2.3.1 Equipment
			2.3.2 Disposables
			2.3.3 Reagents
	3 Methods
		3.1 ATAC-seq
			3.1.1 Transposition Reaction for Primary Hepatocytes (See Note 1)
			3.1.2 Transposition Reaction for Non-Tumor and Tumor Samples
			3.1.3 Amplification (See Note 2)
			3.1.4 Purification
			3.1.5 ATAC-Sequencing and Analyses
		3.2 ChIP
			3.2.1 Crosslinking (See Note 3)
			3.2.2 Lysis and Sonication (See Note 4)
			3.2.3 Determination of DNA Concentration and Fragment  Size
			3.2.4 Chromatin Immunoprecipitation (See Note 5)
			3.2.5 DNA Isolation
				For ChIP Samples
			3.2.6 For Inputs
			3.2.7 Analysis in  qPCR
		3.3 3C
			3.3.1 Preparation of Nuclei
			3.3.2 Cross-Linking
			3.3.3 Permeabilization and Restriction Digestion
			3.3.4 Ligation
			3.3.5 DNA Purification
				For Non-Digested and Digested Controls
				For 3C Samples
			3.3.6 Second Digestion and DNA Purification
			3.3.7 Primer Design
			3.3.8 Primer Efficiency Control
			3.3.9 3C Analysis in  qPCR
	4 Notes
	References
Chapter 14: Targeted Analysis of Glycerophospholipids and Mono-, Di-, or Tri-Acylglycerides in Liver Cancer
	1 Introduction
	2 Materials
		2.1 Disposables
		2.2 Equipment
		2.3 2.3. Software
		2.4 Reagents
	3 Methods
		3.1 Sample Preparations
		3.2 Targeted Analysis of Glycerophospholipids by Flow Injection Analysis (FIA) High-Performance Liquid Chromatography (HPLC) C...
		3.3 The Targeted Analysis of Mono-, Di-, and tri-Acylglycerides by Flow Injection Analysis (FIA) High-Performance Liquid Chrom...
		3.4 Quality Control  Pool
		3.5 LC/MS System Rinsing
		3.6 Data Processing with LipidView 1.2
	4 Notes
	References
Chapter 15: Biomarker Identification in Liver Cancers Using Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) Ima...
	1 Introduction
	2 Materials
		2.1 Disposables
		2.2 Equipment
		2.3 Software
		2.4 Reagents
	3 Methods
		3.1 Sample Preparations (Fig. 1)
			3.1.1 Tissue in PFA (Paraformaldehyde) (See Note 1)
			3.1.2 HES Staining
			3.1.3 Tissue in OCT (Optimal Cutting Temperature) Compound
		3.2 MSI Acquisition (Figs. 2 and 3)
		3.3 Post-Acquisition Data Treatment
	4 Notes
	References
Chapter 16: Kinetic Modeling of Hepatic Metabolism and Simulation of Treatment Effects
	1 Introduction
	2 Methods
		2.1 Model Description
		2.2 Calibration and Validation
		2.3 Application to Proteomic  Data
		2.4 Scope of Application
		2.5 Reference Tissue (Normalization) and Generation of Individual Model Instantiations
		2.6 Individual Assessment of Metabolic States under Various Conditions
			2.6.1 Maximal Capacities
			2.6.2 Assessment of Energetic Capacities and Substrate Utilization Rates under Different Dietary Conditions
			2.6.3 Diurnal Metabolic Changes
			2.6.4 Simulation of Treatment Effects
			2.6.5 Importance of Specific Enzymes for Metabolic Functions
			2.6.6 Minimal Hardware and Software Requirements
	3 Notes
	References
Index