Mammary Stem Cells: Methods and Protocols (Methods in Molecular Biology, 2471)

Preface
Contents
Contributors
Chapter 1: Protocol for Studying Embryonic Mammary Gland Branching Morphogenesis Ex Vivo
	1 Introduction
	2 Materials
		2.1 Dissection of E13.5 Embryo Flank with Mammary Gland
		2.2 Separation of E13.5 Skin Epithelium from the Mammary Gland Explant
		2.3 Ex Vivo Culture of E13.5 Embryonic Mammary Gland Explants
		2.4 Live Imaging of Cultured E13.5 Mammary Gland Explants
		2.5 3D Stroma-Free Organoid Culture of E16 Mammary Rudiments
	3 Methods
		3.1 Dissection of E13.5 Embryo Flank with Mammary Gland
		3.2 Separation of E13.5 Skin Epithelium from the Mammary Bud Explant
		3.3 Explant Culture of E13.5 Embryonic Mammary Gland Explants
		3.4 Live Imaging of Cultured E13.5 Mammary Gland Explants
		3.5 3D Stroma-Free Organoid Culture of E16 Mammary Rudiments
			3.5.1 Dissection of the Mammary Tissue
			3.5.2 Epithelial-Mesenchymal Separation
	4 Notes
	References
Chapter 2: Multidimensional Fluorescence Imaging of Embryonic and Postnatal Mammary Gland Development
	1 Introduction
	2 Materials
		2.1 Imaging Window (MIW) Preparation
		2.2 Imaging Window Implantation
		2.3 Intravital Microscopy (IVM)
		2.4 Embryonic Mammary Gland Dissection
		2.5 Ex Vivo Embryonic Mammary Gland Culture and 4D Live-Cell Imaging
		2.6 Enzymatic Digestion and Wholemount Immunostaining of Mammary Tissues
		2.7 CUBIC-Based Optical Tissue Clearing
		2.8 SeeDB-Based Optical Tissue Clearing
	3 Methods
		3.1 Mammary Imaging Window Implantation for 4D-IVM
			3.1.1 Mammary Imaging Window (MIW) Preparation
			3.1.2 Surgery Preparation
			3.1.3 Surgical Implantation of the MIW
		3.2 Longitudinal 4D-IVM by Multiphoton Microscopy
		3.3 Establishing Mammary Embryonic Buds in Culture for 4D Ex Vivo Imaging
			3.3.1 Dissection of the Embryonic Mammary Gland
			3.3.2 Separating the Embryonic Skin Epithelium and Mesenchyme
			3.3.3 Establishing Mammary Buds in Ex Vivo Culture
		3.4 4D Time-Lapse and Longitudinal Imaging of Ex Vivo Embryonic Mammary Cultures
		3.5 3D Fluorescence Imaging of Fixed Mammary Gland Tissues
			3.5.1 Proteolytic Digestion-Based Immunostaining of Mammary Gland Tissues
			3.5.2 Modified CUBIC Tissue Clearing and Immunostaining of Mammary Gland Tissues
			3.5.3 SeeDB Tissue Clearing and Immunostaining of Mammary Gland Tissues
	4 Notes
	References
Chapter 3: Single-Cell Transcriptomic and Epigenetic Analyses of Mouse Mammary Development Starting with the Embryo
	1 Introduction
	2 Materials
	3 Methods
		3.1 Production of Timed Pregnant Females
		3.2 Embryo Collection and Identification of Females
		3.3 Isolation of Late (E15.5 to E18.5) Mammary Rudiments
		3.4 Isolation of Early (E12.5 to E14.5) Mammary Rudiments
		3.5 Mammary Rudiment Dissociation into Single-Cell Suspension
		3.6 Performing RNA/ATAC-seq Using a Preferred Method
		3.7 Processing of scRNA-seq  Data
			3.7.1 FastQC
			3.7.2 Running 10x Cell Ranger Pipeline
			3.7.3 Quality Control in Seurat
			3.7.4 Marker Gene Identification and Differential Gene Analysis
			3.7.5 Pseudotime Trajectory Inference Using monocle2 Package
		3.8 Analysis of snATAC-seq  Data
			3.8.1 Import Data into ArchR Object (See Note 25)
			3.8.2 Quality Control
			3.8.3 Dimensionality Reduction with ArchR
			3.8.4 Explore the Clusters Identify Using ArchR Gene Score
			3.8.5 Generate Cluster Aggregated ATAC Signal Tracks
			3.8.6 Pseudo-Bulk Replicates and Peak Calling
			3.8.7 Call Peaks in Each Cluster
			3.8.8 Explore Transcription Motif Deviations Using chromVar TF Activity Score
			3.8.9 Enrichment Analysis of Custom Genome Annotations
			3.8.10 Peak-Peak Co-accessibility
		3.9 Integration of scRNA-seq and snATAC-seq  Data
	4 Notes
	References
Chapter 4: Computational Methods to Identify Cell-Fate Determinants, Identity Transcription Factors, and Niche-Induced Signali...
	1 Introduction
	2 Materials
		2.1 SeesawPred: Cell-Fate Determinants
		2.2 TransSyn: Cell Identity  TFs
		2.3 SigHotSpotter: Niche-Induced Signaling Pathways
	3 Methods
		3.1 SeesawPred: Cell-Fate Determinants
		3.2 TransSyn: Cell Identity  TFs
		3.3 SigHotSpotter: Niche-Induced Signaling Pathways
	4 Notes
	References
Chapter 5: Differential Proteomic Analysis of Complex Mixtures by Label-Free nLC MS/MS
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 MS Analysis
	3 Methods
		3.1 Protein Extraction
		3.2 Protein Quantification
		3.3 Protein Digestion by FASP (Filter Aided Sample Preparation)
		3.4 Peptide Concentration and Desalting by Reverse-Phase Microcolumns
		3.5 Sample Load onto the Chromatographic System
		3.6 MS Acquisition
		3.7 MS Data Analysis
		3.8 Label-Free Quantification
		3.9 Bioinformatic Analysis
	4 Notes
	References
Chapter 6: Orthotopic Transplantation of Mouse Mammary Epithelial Cells
	1 Introduction
	2 Materials
		2.1 Preparation of Mammary Epithelial Cells and Fragments for Transplantation
		2.2 Adenovirus Infection and Mammosphere Culture
		2.3 Animal Surgery
		2.4 Outgrowth Analysis by Carmine-Stained Whole Mounts
	3 Methods
		3.1 Preparing Material for Surgery
		3.2 Clearing the Fat  Pad
		3.3 Preparation and Transplantation of Mouse Mammary Epithelial Fragments
		3.4 Preparation of Single Mouse Mammary Cells
		3.5 Injection of Mammary Epithelial Cells into Cleared Fat  Pads
		3.6 Injection of Mammary Cells After Adeno-Cre Infection
		3.7 Injection of Cultured Mammospheres
		3.8 Closing/Revival of Mouse
		3.9 Outgrowth Analysis by Carmine-Stained Whole Mounts
	4 Notes
	References
Chapter 7: Lineage Tracing Methods to Study Mammary Epithelial Hierarchies In Vivo
	1 Introduction
	2 Materials
		2.1 Genetically Modified Mouse Models
			2.1.1 Tamoxifen-Inducible Cre/Lox System
			2.1.2 Tetracycline-Inducible Tet/Lox System
			2.1.3 Dre/Rox System
		2.2 Reporter Lines
		2.3 Inducing Tamoxifen-Mediated Recombination
		2.4 Inducing Tetracycline-Mediated Recombination
		2.5 Harvesting the Mammary Glands
		2.6 Immunostaining
		2.7 Mammary Gland Cell Dissociation
	3 Methods
		3.1 Generate the Mouse Colony
		3.2 Cre Recombinase Activation
		3.3 Analysis of the Cells of Interest and Their Progeny
		3.4 Other Lineage Tracing-Related Applications
	4 Notes
	References
Chapter 8: Lentiviral Transduction of Mammary Epithelial Cells
	1 Introduction
		1.1 Lentiviral Vector Production
		1.2 Lentiviral Vector Production
		1.3 Packaging Vectors
		1.4 Lentiviral Expression Vectors
	2 Materials
		2.1 Components for Lentiviral Production
			2.1.1 For Calcium Phosphate Transfection
			2.1.2 For PEI Transfection
			2.1.3 Plasmids to Make a Simple GFP Expression Vector
		2.2 Components for Titration of Viruses
		2.3 Components for RCL  Test
			2.3.1 Reagents for Routine Molecular Biology
	3 Methods
		3.1 Production of Lentiviral Particles
			3.1.1 Calcium Phosphate Protocol
			3.1.2 PEI Protocol
			3.1.3 Continue Both Protocols
		3.2 Infection of Target Cells and Titration of Lentiviral Vectors
			3.2.1 Titration by Antibiotic Selection
				Calculate Viral Titre
			3.2.2 Titration by Flow Cytometry
				Calculate Viral Titre
			3.2.3 Titration by p24 ELISA
		3.3 Replication Competent Lentivirus (RCL)  Test
			3.3.1 Infect 293T Cells
			3.3.2 Extract  RNA
			3.3.3 Treat with DNase  I
			3.3.4 Reverse Transcribe
			3.3.5 Perform Quantitative  PCR
	4 Notes
	References
Chapter 9: Modification of Single Cells Within Mouse Mammary Gland Derived Acini via Viral Transduction
	1 Introduction
	2 Materials
	3 Methods
		3.1 Organotypic Culture of Primary Mammary Epithelial Cells
		3.2 Lentiviral Transduction
	4 Notes
	References
Chapter 10: Surgical Procedure for Implantation of Human Tumor Tissue into the Epithelium-Free Mammary Fat Pad of Immunocompro...
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Supplies
		2.3 Autoclaved Surgical Pack #1
		2.4 Autoclaved Surgical Pack #2
	3 Methods
		3.1 Preparation of the Non-sterile Surgical Area (Can Be Done Concurrently by the Prepper and Surgeon in Regular Gloves)
		3.2 Preparation of the Sterile Surgical Area (Prepper and Surgeon Will Work Together While Maintaining Sterility)
		3.3 Preparing the Mouse for Surgery (Prepper)
		3.4 Surgical Procedure to Implant the Tissue Fragment (Surgeon)
		3.5 Post-Surgical Procedures (Prepper and Surgeon Will Work Together While Maintaining Sterility)
	4 Notes
	References
Chapter 11: The Chicken Embryo Chorioallantoic Membrane (CAM): A Versatile Tool for the Study of Patient-Derived Xenografts
	1 Introduction
	2 Materials
		2.1 Egg Housing and Preparation
		2.2 PDX Engraftment
		2.3 Tumor Harvest
	3 Methods
		3.1 Housing of Fertilized  Eggs
		3.2 Egg Preparation
		3.3 PDX Engraftment
		3.4 Tumor Harvest
	4 Notes
	References
Chapter 12: Intraductal Injections into the Mouse Mammary Gland
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Mice/Removal of Fur Surrounding Nipples (See Note 2)
		3.2 Preparation of Materials and Equipment
		3.3 Preparation of Mice (Pre-surgical)
		3.4 Intraductal Injection
		3.5 Postoperative Care
		3.6 Monitoring Cell or Tumor Growth
	4 Notes
	References
Chapter 13: Modeling Breast Cancer in Organoid and Intraductal Models
	1 Introduction
		1.1 Hormone Receptor Positive Breast Cancer Models
		1.2 Culture Systems for Breast Tumors and Normal Mammary Epithelial Cells
		1.3 Mammary Intraductal Injection (MIND)
	2 Materials
		2.1 Components for Isolation of Mammary Epithelial Cells
		2.2 Components for Lentiviral Transduction of Mammary Epithelial Cells
		2.3 Components for Clevers Breast Organoid Medium (CBOM)
		2.4 Components for Injection of Mammary Ducts (MIND Protocol)
			2.4.1 General Animal House Equipment
	3 Methods
		3.1 Prepare Mammary Epithelial Cells from Tumors and Mammoplasties
		3.2 Purify EPCAM+ Mammoplasty Cells on Magnetic Beads
		3.3 Infect Mammary Epithelial Cells
		3.4 Inject the Mammary Ducts
	4 Notes
	References
Chapter 14: Single Organoids Droplet-Based Staining Method for High-End 3D Imaging of Mammary Organoids
	1 Introduction
	2 Materials
		2.1 Biological Materials
		2.2 Reagents
		2.3 Equipment
	3 Methods
		3.1 Organoid Culture Remarks
		3.2 Organoid Collection
			3.2.1 Preparation of a Staining Chamber  Dish
			3.2.2 Organoid Collection from Living Cultures, Followed by Fixation
			3.2.3 Organoid Collection from Fixed 3D Cultures
		3.3 Blocking and Permeabilization
		3.4 Staining
		3.5 Clearing
		3.6 Mounting and Microscopy
	4 Notes
	References
Chapter 15: Divide and Conquer: Isolating Cell Populations to Investigate How Breast Cancer Risk Factors Alter the Breast Micr...
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
		2.3 Media
	3 Methods
		3.1 Digestion of Reduction Mammoplasty Tissue
		3.2 Isolation of Adipocytes
		3.3 Separation of Stromal Cells and Epithelial Cells
		3.4 Dissociation of Breast Epithelial Cells
		3.5 Macrophage Isolation
	4 Notes
	References
Chapter 16: An Organotypic Assay to Study Epithelial-Fibroblast Interactions in Human Breast
	1 Introduction
	2 Materials
		2.1 Isolation of Fibroblasts from Human Breast
		2.2 Primary Fibroblast Culture and Freezing
		2.3 Culture and Freezing of D492 Cells
		2.4 D492 Spheroid Formation and 3D Coculture Assay
		2.5 General Materials and Equipment
	3 Methods
		3.1 Isolation of Fibroblasts from Human Breast
		3.2 Culture of Primary Human Breast Fibroblasts
			3.2.1 Passaging of Human Breast Fibroblasts
			3.2.2 Freezing of Primary Human Breast Fibroblasts
		3.3 Culture of D492 Cells
			3.3.1 Coating of Culture Vessels with Collagen  I
			3.3.2 Culture and Passaging of D492 Cells
			3.3.3 Freezing of D492 Cells
		3.4 3D Coculture of D492 Spheroids with Human Breast Fibroblasts
			3.4.1 D492 Spheroid Formation
			3.4.2 3D Coculture Assay
	4 Notes
	References
Chapter 17: Patient-Derived Explant Cultures of Normal and Tumor Human Breast Tissue
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 18: Negative-Selection Enrichment of Circulating Tumor Cells from Peripheral Blood Using the Microfluidic CTC-iChip
	1 Introduction
	2 Materials
		2.1 Blood Collection and Incubation
		2.2 Reagents and Buffers
		2.3 IFD Tower (See Note 5)
	3 Methods
		3.1 Preparing the Blood Sample
		3.2 Setting Up CTC-iChip Tower
		3.3 Running the iChip
		3.4 Cleanup
	4 Notes
	References
Chapter 19: Measuring Mechanical Properties of Breast Cancer Cells with Atomic Force Microscopy
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Sample Preparation for Atomic Force Microscopy
		2.3 Force Spectroscopy (See Note 3)
		2.4 Chemicals
		2.5 Software
	3 Methods
		3.1 Cell Culture
		3.2 Sample Preparation
			3.2.1 Slide Preparation
			3.2.2 Cell Sample Preparation
		3.3 Force Spectroscopy
			3.3.1 Assembling the System (See Note 3)
			3.3.2 Calibration of the System
			3.3.3 Calibration of System in  Air
			3.3.4 Calibration of System in Liquid
			3.3.5 Force Spectroscopy Measurements
			3.3.6 Disassembling the System and Cleaning
		3.4 Data Evaluation
			3.4.1 Elasticity
			3.4.2 Viscoelastic Measurements: Stress Relaxation and Creep
	4 Notes
	References
Chapter 20: Breast Cancer Stem Cells: A Clinician´s View
	References
Index