Mouse Models of Cancer: Methods and Protocols (Methods in Molecular Biology, 2773)

Preface
Contents
Contributors
Chapter 1: In Vivo Modeling of Leukemia by Murine Stem Cell Transplantation
	1 Introduction
	2 Materials
		2.1 Stimulation of HSC Proliferation in Donor  Mice
		2.2 Production of Retroviral Particles
		2.3 Isolation of Bone Marrow Cells
		2.4 Transduction of Bone Marrow Cells
		2.5 Stem Cell Transplantation
		2.6 Analysis
	3 Methods
		3.1 Stimulation of HSC Proliferation in Donor  Mice
		3.2 Production of Retroviral Particles
		3.3 Isolation of Bone Marrow Cells
		3.4 Transduction of Bone Marrow Cells
		3.5 Stem Cell Transplantation
		3.6 Analysis
	4 Notes
	References
Chapter 2: NOTCH1-Induced T-Cell Acute Lymphoblastic Leukemia In Vivo Models
	1 Introduction
	2 Materials
		2.1 Retroviral Supernatant Preparation
		2.2 Hematopoietic Precursor Isolation
		2.3 Hematopoietic Precursor Infection
		2.4 Recipient Mouse Transplant and Follow-Up
	3 Methods
		3.1 Retroviral Supernatant Preparation
		3.2 Hematopoietic Precursor Isolation
		3.3 Hematopoietic Precursor Infection
		3.4 Recipient Mouse Transplant and Follow-Up
	4 Notes
	References
Chapter 3: Urinary Bladder Cancer Induced by N-Butyl-N-(4-Hydroxybutyl)-Nitrosamine
	1 Introduction
	2 Materials
	3 Methods
		3.1 BBN Solution
		3.2 Experimental Animals
			3.2.1 Animals Treated with 0.05% BBN
			3.2.2 Control Animals
	4 Notes
	References
Chapter 4: Establishment of Mouse Orthotopic Urinary Bladder Tumor Model and Its Analysis by Light and Electron Microscopy
	1 Introduction
	2 Materials
		2.1 Materials for Cell Cultivation, Lentiviral Transduction, Generation of a Stable Fluorescent MB49 Cell Line, and Labeling o...
		2.2 Materials for Animal Model
	3 Methods
		3.1 Preparation of Fluorescent Monoclonal Stable Cancer Cell Line MB49-GFP Using Lentiviral Transduction (See Note 4)
		3.2 In Vitro Labeling of MB49-GFP Cancer Cells with Metal Nanoparticles
		3.3 Establishment of Orthotopic Bladder Tumor Model
		3.4 Euthanasia of  Animals
		3.5 Preparation of Whole-Mount Bladder Tissue Samples
		3.6 Observation of Bladder Tumor Growth with the Stereomicroscope
		3.7 Preparation of Paraffin Sections of Tumor Tissue (for Long-Term Experiments)
		3.8 Preparation of Cryosections of Bladder Tissue for Fluorescence Microscopy Analysis (for Short- and Long-Term Experiments)
		3.9 Preparation of Semithin and Ultrathin Sections of Bladder Tissue for Transmission Electron Microscopy (TEM) Analysis
		3.10 Preparation of Bladder Tissue Specimens for Scanning Electron Microscopy (SEM) Analysis
	4 Notes
	References
Chapter 5: Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) Model of Colorectal Cancer
	1 Introduction
	2 Materials
		2.1 Mice
		2.2 AOM Solution
		2.3 DSS Solution
		2.4 Drinking Water
		2.5 Additional Materials and Equipment
	3 Methods
		3.1 Chemical Induction of Tumor Development
		3.2 Macroscopic Inspection and Tissue Sampling for the Evaluation of Tumor and Precancerous Lesions
	4 Notes
	References
Chapter 6: Isolating Primary Tumor Cells from the MMTV-PyMT Mouse Model and Their Use in Developing an Orthotopic Mouse Model ...
	1 Introduction
	2 Materials
		2.1 Isolation of Primary Tumor Cells
		2.2 Orthotopic Transplantation of Primary Tumor Cells
	3 Methods
		3.1 Isolation of Primary Tumor Cells from MMTV-PyMT-Induced Mammary Carcinomas
		3.2 Orthotopic Transplantation of Primary Tumor Cells into the Mammary Gland of Congenic Mice
	4 Notes
	References
Chapter 7: Transplantable Subcutaneous Tumor Models
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents, Cells, and Animals
	3 Methods
	4 Notes
	References
Chapter 8: Preclinical Mouse Metastatic Model Established Through Induced Lung Metastases
	1 Introduction
	2 Materials
		2.1 Cell Suspension
		2.2 Injection of Tumor Cells
		2.3 Histopathological Examination of Metastases
	3 Methods
		3.1 Cell Suspension
		3.2 Induction of Metastases
		3.3 Histopathological Examination of Metastases
	4 Notes
	References
Chapter 9: In Vivo Wound Healing Model for Characterization of Gene Electrotransfer Effects in Mouse Skin
	1 Introduction
	2 Materials
		2.1 Experimental Animals
		2.2 Splints
		2.3 Surgical Materials and Equipment
		2.4 Electroporation Devices
		2.5 Reagents
		2.6 Monitoring of the Wound Healing Process
	3 Methods
		3.1 Anesthesia and Operative Preparation
		3.2 Excision of Wounds
		3.3 Treatment
		3.4 Splinting of Wounds
		3.5 Postoperative Management
		3.6 Monitoring of the Wound Healing Process
		3.7 Analysis of the Results
	4 Notes
	References
Chapter 10: Partial-Volume Irradiation of Murine Tumors
	1 Introduction
	2 Materials
		2.1 Tumor Models and Cell Culture
		2.2 Anesthesia
		2.3 Irradiation Device
	3 Methods
		3.1 Heterotopic Tumor Induction
		3.2 Preparation of the Irradiation Device and Anesthesia
		3.3 Partial-Volume Tumor Irradiation
	4 Notes
	References
Chapter 11: In Vivo Bioluminescence and Fluorescence Imaging: Optical Tool for Cancer Research
	1 Introduction
	2 Materials
		2.1 Cell-Associated Work
			2.1.1 Equipment
			2.1.2 Reagents
		2.2 In Vivo Imaging
			2.2.1 Equipment
			2.2.2 Reagents
	3 Methods
		3.1 General Maintenance of Cell Lines
			3.1.1 Adherent Cell Line (HEK293T, Bioware Brite B16F10-Red-FLuc)
			3.1.2 Suspension Cell Line (Raji-FLuc and Jurkat)
		3.2 Lentivirus Production
		3.3 Determination of Viral Titer by Flow Cytometry
		3.4 Generation and Expansion of CAR-T Cells
			3.4.1 Isolation of Human T Cells
			3.4.2 Transduction of Human T Cells
			3.4.3 Expansion of Human T Cells
			3.4.4 Validation of CAR Expression
		3.5 Establishment of a Preclinical Cancer Model
			3.5.1 CD19 CAR-T Cell Treatment for Burkitt´s Lymphoma Animal Model
			3.5.2 Melanoma Solid Cancer Model
			3.5.3 iRFP-Labeled CD3 Fluorescence Detection
		3.6 In Vivo Imaging
			3.6.1 Bioluminescence Imaging
			3.6.2 Fluorescence Imaging
	4 Notes
	References
Chapter 12: Cancer Imaging by Intravital Microscopy: The Dorsal Window Chamber Model
	1 Introduction
	2 Materials
		2.1 Mice, Analgesia, and Anesthesia
		2.2 Reagents and Equipment for Mice Surgery
			2.2.1 Reagents for Mice Preparation and Surgical Area Preparation
			2.2.2 Equipment for Mice Preparation
		2.3 Equipment Specific for Dorsal Window Chamber Surgery
		2.4 Equipment for Tumor Implantation
		2.5 Reagents and Equipment for Mice Imaging
		2.6 Imaging-Specific Equipment
	3 Methods
		3.1 Instruments Preparation and Sterilization
		3.2 Preparation of the Solutions and Cells
		3.3 Surgical and Dissection Areas Preparation
		3.4 Surgery Procedure
		3.5 Animal Housing and Recovery
		3.6 Imaging
			3.6.1 Wide-Field Microscopy
			3.6.2 Multiphoton Microscopy
	4 Notes
	References
Chapter 13: Imaging of Extravasation of Splenocytes in the Dorsal Skinfold Window Chamber
	1 Introduction
	2 Materials
		2.1 Implantation of the Dorsal Skinfold Window Chamber (DSWC)
		2.2 Tumor Induction in the DSWC
		2.3 Splenocyte Isolation and Staining
		2.4 Intravenous Splenocyte Injection
		2.5 Intravital Imaging of Splenocytes in the DSWC
	3 Methods
		3.1 Implantation of the Dorsal Skinfold Window Chamber (DSWC)
		3.2 Tumor Induction in the DSWC
		3.3 Splenocyte Isolation and Staining
		3.4 Intravenous Splenocyte Injection
		3.5 Intravital Imaging of Splenocytes in the DSWC
	4 Notes
	References
Chapter 14: Mouse Melanoma Model in Tumor Vaccines and Immunotherapy Research
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Preparation
		3.2 Tumor Induction by Cell Injection
		3.3 Tumor Growth Measurements and Animal Survival Curve
		3.4 Infiltration of LPS as a Positive Control for the Infiltration of Immune Cells
		3.5 Immunohistochemistry Staining
	4 Notes
	References
Chapter 15: Immunospot Assessment of T-Cell Responses in Preclinical Tumor Models with Undefined Target Antigens
	1 Introduction
	2 Materials
		2.1 Lymphocyte Isolation
		2.2 Sample Thawing and Cell Recovery
		2.3 FluoroSpot and Data Analysis
	3 Methods
		3.1 Lymphocyte Isolation
		3.2 Sample Thawing
		3.3 FluoroSpot
		3.4 Data Analysis
	4 Notes
	References
Chapter 16: Necropsy in Mouse Research
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Solutions
	3 Methods
		3.1 External Examination
		3.2 Integumentary System and Mammary Glands
		3.3 Opening of the Abdominal Cavity and Sampling of the Abdominal Organs
			3.3.1 Gastrointestinal Tract
			3.3.2 Genitourinary Organs
		3.4 Sampling of the Neck Organs
		3.5 Opening of the Thoracic Cavity and Sampling of Thoracic Organs
		3.6 Head and Cranial Cavity
		3.7 Skeletal Muscle, Bones, and Peripheral Nerve
		3.8 Sampling of the Organs for Histopathological Analysis
	4 Notes
	References
Index