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Mammary Stem Cells: Methods and Protocols

Tác giả: Vivanco, Maria del Mar

Chủ đề:

Chủ đề: Medicine; Cancer; Molecular Biology

Nhà xuất bản: Humana Press

Năm xuất bản: 2015

Loại tài liệu: Book

Mô tả vật lý: 279 p.

Định danh: 978-1-4939-2519-3

Ngôn ngữ: en

Liên kết: Methods in Molecular Biology;Book 1293

Đọc trực tuyến

The identification of normal and breast cancer stem cells has offered a new vision of this heterogeneous disease and new hopes for its prognosis and treatment. This volume provides an overview of recent developments in mammary stem cell research and discusses the many varieties of approaches used by researchers to investigate the properties and functions of mammary stem cells. The beginning chapters provide readers with an introduction to mammary stem cells, and the processes used to characterize stem cells and isolate them via fluorescent activated cell sorting. The next few chapters discuss DNA and mRNA sequencing, proteomic techniques to help profile cells, lentiviral cell transduction for gene expression, and in vivo lineage tracing. The final few chapters are dedicated to following stem cells from their initial niche to the new microenvironment at their metastasis site, and to studying these cells using physical and mathematical approaches

Cover
Methods in Molecular Biology
Title pages
Preface
Contents
Contributors
1 Breast Cancer Stem Cells: Current Advances and Clinical Implications
- 1 Introduction
- 2 Stemness as a Concept in Cancer Biology
  - 2.1 CSC Model as a Cellular Mechanism of Carcinogenesis and Tumor Heterogeneity
  - 2.2 CSCs Are Not Necessarily Rare
- 3 Isolation and Identification of BCSCs
- 4 Cellular Origins and Phenotypic Plasticity of BCSCs
  - 4.1 BCSCs Transition Between EMT- and METưLike States
  - 4.2 Cell Origins of BCSCs and Breast Tumorigenesis
- 5 BCSCs and Treatment Resistance
  - 5.1 BCSCs Are Associated with Treatment Resistance
  - 5.2 Mechanisms Involving Treatment Resistance in BCSCs
    - 5.2.1 BCSCs with Enhanced Drug Efflux Activity
    - 5.2.2 ALDH Enzymatic Activity Expressing in BCSCs Directly Involves in Drug Resistance
    - 5.2.3 BCSCs Exhibit Low Reactive Oxygen Species Levels and Radioresistance
    - 5.2.4 BCSCs Have Enhanced DNA-Damage Repair Responses
- 6 BCSCs and Tumor Metastasis
- 7 Circulating Tumor Cells (CTCs)
  - 7.1 Clinical Importance of Circulating BCSCs
  - 7.2 Isolation Methods for Characterization of CTCs
- 8 Patient Derived Xenograft Models for Cancer Stem Cell Research
- 9 Key Signaling Pathways Regulating the Self-Renewal of CSCs
  - 9.1 Hedgehog Signaling Pathway
  - 9.2 Notch Signaling
  - 9.3 HER2 Signaling
  - 9.4 Wnt Pathway
  - 9.5 Regulation of BCSCs by Tumor Microenvironment
  - 9.6 Regulation of BCSCs by MicroRNAs
- 10 Therapy Implications of CSC-Targeted Approaches
  - 10.1 Cancer Treatment and Prognosis: Why a New Paradigm Is Necessary
  - 10.2 Clinical Implications of CSCs
- 11 Future Perspectives
- References
2 A Protocol for Studying Embryonic Mammary Progenitor Cells During Mouse Mammary Primordial Development in Explant Culture
- 1 Introduction
- 2 Equipment and Materials
  - 2.1 Preparation of Culture Medium
  - 2.2 Dissection of Embryo Flank
  - 2.3 Embryonic Explant Culture
- 3 Methods
  - 3.1 Preparation of Materials and Equipment for Explant Culture
  - 3.2 Dissection of Embryo Flanks
  - 3.3 Embryonic Explant Culture
- 4 Notes
- References
3 FACS Sorting Mammary Stem Cells
- 1 Introduction
- 2 Materials
- 3 Methods
  - 3.1 Setting Up the Experiment (Antibody Selection and Titration)
  - 3.2 Cell Preparation
  - 3.3 Cell Staining
  - 3.4 Setting Up for Sorting
  - 3.5 Analysis and Sorting
- 4 Notes
- References
4 Side Population
- 1 Introduction
- 2 Materials
- 3 Methods
  - 3.1 Hoechst 33342 Staining of Cells
  - 3.2 Analysis and Sorting
- 4 Notes
- References
5 Single-Cell Genome and Transcriptome Processing Prior to High-Throughput Sequencing
- 1 Introduction
- 2 Materials
  - 2.1 Single-Cell Whole-Genome DNA Library Preparation
  - 2.2 Single-Cell Whole-Genome mRNA Library Preparation
- 3 Methods
  - 3.1 Single-Cell Whole-Genome DNA Preparation for HighưThroughput Sequencing
  - 3.2 Total DNA Library Preparation
  - 3.3 Library Enrichment
  - 3.4 Enriched Library Cleanup
  - 3.5 Library Quality Control and Quantification
  - 3.6 Pooling Libraries
  - 3.7 mRNA Preparation from Single Cells
  - 3.8 Double Stranded cDNA Amplification and Purification
  - 3.9 cDNA Quality Control
  - 3.10 cDNA Library Preparation
- 4 Notes
- References
6 Shotgun Proteomics on Tissue Specimens Extracted with Acid Guanidinium-Thiocyanate-Phenol-Chloroform
- 1 Introduction
- 2 Materials
- 3 Methods
  - 3.1 Patients and Tumor Tissue
  - 3.2 Tissue Cryostat Sectioning
  - 3.3 AGPC Extraction
  - 3.4 Protein Isolation
  - 3.5 FASP Digestion
- 4 Notes
- References
7 Antibody-Based Capture of Target Peptides in Multiple Reaction Monitoring Experiments
- 1 Introduction
- 2 Materials
  - 2.1 Selection of Target Peptides
  - 2.2 Peptides and Antibody Buffers
  - 2.3 Heavy Peptide Standard Stock Solutions and Mixtures
  - 2.4 Anti-peptide Antibody Master-Mix Solutions
  - 2.5 Cross-Linking Solutions
- 3 Methods
  - 3.1 Antibody Cross-Linking Using KingFisher (Optional) (See Note 8)
  - 3.2 Antibody Capture Efficiency Evaluation
  - 3.3 Evaluation of Passenger Peptide
  - 3.4 Generation of Reverse Curve (Fig. 2)
- 4 Notes
- References
8 Lentiviral Transduction of Mammary Epithelial Cells
- 1 Introduction
- 2 Overall Strategy
- 3 Packaging Vectors
- 4 Lentiviral Expression Vectors
- 5 Materials
  - 5.1 Components for Lentiviral Production
  - 5.2 Components for Titration of Viruses
  - 5.3 Components for Isolation of Mammary Epithelial Cells
  - 5.4 Components for Lentiviral Transduction of Mammary Cells
  - 5.5 Components for RCL Test
- 6 Methods
  - 6.1 Production of Lentiviral Particles
  - 6.2 Titration of Lentiviral Vectors
    - 6.2.1 Titration by Antibiotic Selection
    - 6.2.2 Titration by Flow Cytometry
    - 6.2.3 Titration by p24 ELISA
  - 6.3 Isolation of Mammary Epithelial Cells
  - 6.4 Infection of Mammary Epithelial Cells
  - 6.5 Replication-Competent Lentivirus Test
- 7 Notes
- References
9 The Transplantation of Mouse Mammary Epithelial Cells into Cleared Mammary Fat Pads
- 1 Introduction
- 2 Materials
  - 2.1 Preparation of Mammary Epithelial Cells and Fragments for Transplantation
  - 2.2 Animal Surgery
  - 2.3 Outgrowth Analysis
- 3 Methods
  - 3.1 Clearing the Fat Pad
  - 3.2 Preparation and Transplantation of Mouse Mammary Epithelial Fragments
  - 3.3 Preparation of Single Mouse Mammary Cells
  - 3.4 Injection of Mammary Epithelial Cells into Cleared Fat Pads
  - 3.5 Closing/Revival of Mouse
  - 3.6 Outgrowth Analysis
- 4 Notes
- References
10 Humanization of the Mouse Mammary Gland
- 1 Introduction
- 2 Materials
  - 2.1 Isolation of Primary Mammary Fibroblasts and Epithelial Cells
  - 2.2 Cell Culture
  - 2.3 Animal Surgery
- 3 Methods
  - 3.1 Isolation of Primary Human Mammary Stromal Cells and Epithelial Cells
  - 3.2 Generation of Immortalized Stromal (RMF-EG) Cells
  - 3.3 Humanizing of Mammary Fat Pads
  - 3.4 Preparation of Human Mammary Epithelial Cells (HMECs)
  - 3.5 Injection of Material for Further Study
- 4 Notes
- References
11 Lineage Tracing in the Mammary Gland Using Cre/lox Technology and Fluorescent Reporter Alleles
- 1 Introduction
- 2 Materials
  - 2.1 Compound Mutant Mice
  - 2.2 Inducing Tamoxifen-Mediated Recombination
  - 2.3 Harvesting the Mammary Glands
  - 2.4 Whole-Mount Confocal Analysis of Fluorescent Reporter Alleles
- 3 Methods
  - 3.1 Inducing Cre-Mediated Recombination
  - 3.2 Tracing
  - 3.3 Analyzing the Trace Experiment by Whole-Mount Confocal Microscopy
  - 3.4 Constructing a 3D Picture of Labeled Alveoli Using Fiji
- 4 Notes
- References
12 Modeling the Breast Cancer Bone Metastatic Niche in Complex Three-Dimensional Cocultures
- 1 Introduction
- 2 Materials
  - 2.1 For Generating Collagen Biomatrix Discoids for 3D Coculture
  - 2.2 For Cell Plating
  - 2.3 For Plate Reader Readings
  - 2.4 Medium Used for 3D Cocultures
  - 2.5 Dissociation of 3D Cocultures
  - 2.6 Cell Lines
- 3 Methods
  - 3.1 Cutting Collagen Biomatrix into Discoid Pieces for Coculture
  - 3.2 Plating Supportive and Inhibitory Niche Cultures
    - 3.2.1 Setting up 3D Cocultures
    - 3.2.2 Plating Bone Marrow Cells
  - 3.3 Plating Breast Cancer Cells
  - 3.4 Total Fluorescence Reading and Microscopy
  - 3.5 Analysis
  - 3.6 Dissociation of Cocultures in Biomatrix
- 4 Advantages, Limitations, and Potential Applications of the Bone Metastatic Niche Experimental Systems
- References
13 Mammary Cancer Stem Cells Reinitiation Assessment at the Metastatic Niche: The Lung and Bone
- 1 Introduction
- 2 Materials
  - 2.1 Cell Culture Components
  - 2.2 Fluorescence Activated Cell Sorting Components
  - 2.3 Antibodies
  - 2.4 Cells Inoculation In Vivo
  - 2.5 In Vivo Imaging System
- 3 Methods
  - 3.1 Cell Culture Establishment
  - 3.2 Fluorescence Activated Cell Sorting (FACS)
  - 3.3 Reinitiation Metastatic Potential Assessment
    - 3.3.1 Intrathoracic Injections
    - 3.3.2 Intratibiae Injections
  - 3.4 Bioluminescent Imaging
- 4 Notes
- References
14 Nanomechanical Characterization of Living Mammary Tissues by Atomic Force Microscopy
- 1 Introduction
- 2 Materials
  - 2.1 Reagents
  - 2.2 Equipment
- 3 Methods
  - 3.1 Sample Preparation
  - 3.2 ARTIDIS: Force Spectroscopy Mode
  - 3.3 Post-AFM Processing of Tissue Specimens
  - 3.4 Force Curve Processing
  - 3.5 Data Analysis
- 4 Notes
- References
15 Mathematical Modelling as a Tool to Understand Cell Selfưrenewal and Differentiation
- 1 Why Do We Need Mathematical Models?
- 2 How to Model a Cell Production System?
- 3 How to Model Growth of a Population Using Differential Equations
- 4 Multi-compartment Models of Cell Differentiation
  - 4.1 Compartmental Structure
  - 4.2 Modelling of Cell Kinetics
  - 4.3 Modelling of Signalling Feedback
  - 4.4 Model Reduction: Two- and ThreeưCompartments
  - 4.5 Model Applications and Extensions
- 5 Structured Population Models
  - 5.1 Delay Differential Equation Model
  - 5.2 Specification of Delay
  - 5.3 Specification of Progenitor Population Net Growth Factor
  - 5.4 Analysis of the Model
- References
16 Mammary Stem Cells: A Clinicians View
- References
Index