Cancer Stem Cells: Methods and Protocols (Methods in Molecular Biology, 2777)

Preface
Contents
Contributors
Chapter 1: Cancer Stem Cells: Current Challenges and Future Perspectives
	1 Introduction
	2 Challenges in CSC Research
		2.1 The CSC Definition Is a Functional Trait Rather than a Specific  Cell
		2.2 Molecular Markers Used to Identify the CSC Do Not Specifically Nor Uniformly Mark  CSCs
		2.3 The Origins of  CSCs
		2.4 Drug Resistance
		2.5 Drug Development
	3 Future Perspectives
		3.1 Single-Cell RNA Sequencing
		3.2 Genome-Wide CRISPR Screens
		3.3 Precision Medicine
		3.4 Clinical Trial Design
	References
Chapter 2: Immunohistochemistry for Cancer Stem Cell Detection: Principles and Methods
	1 Introduction
	2 Materials
		2.1 Buffer, Diluents, and Antigen Retrieval Solutions
		2.2 Substrates, Chromogens, and Counterstain Solutions
		2.3 Endogenous Activity Blocking Solutions
	3 Methods
		3.1 Preanalytical Stage: Tissue Fixation and Processing of FFPE Samples
		3.2 Antigen Retrieval Methods
		3.3 Endogenous Activity Block
		3.4 Signal Revelation Systems
		3.5 Automated Immunohistochemical Systems
		3.6 Advantages of the Automated Immunohistochemical Approach
		3.7 Automated Immunohistochemistry Staining Protocol
		3.8 Immunohistochemical Detection of Multiple Antigens
		3.9 Main Markers of Cancer Stem Cells
			3.9.1 Aldehyde Dehydrogenase 1A1 (ALDH1A1)
			3.9.2 CD44
			3.9.3 Prominin-1/CD133
		3.10 Troubleshooting
			3.10.1 Excessive Background Staining
			3.10.2 Inadequate or No Staining of the Test Slide and Adequate Staining of the Positive Control Slide
			3.10.3 Weak or No Staining of Positive Control and Weak or No Staining of Test Slides
			3.10.4 No Staining of the Positive Control Slide and Adequate Staining of the Test Slide
	4 Notes
	5 Pitfalls in TMA Use to Detect CSCs by IHC
	References
Chapter 3: Isolating Cancer Stem Cells from Solid Tumors
	1 Introduction
	2 Materials
		2.1 Reagents
			2.1.1 Cell Culture Reagents and Materials
			2.1.2 Antibodies
			2.1.3 Side Population Reagents
			2.1.4 ALDH Activity Reagents
			2.1.5 Equipment and Plates
	3 Methods
		3.1 Primary Cell Culture
			3.1.1 Glioblastoma Cell Culture
			3.1.2 Breast Cancer Cell Culture
			3.1.3 Lung Cancer Cell Culture
			3.1.4 Sarcoma Cell Culture
		3.2 Spheres
			3.2.1 Neurospheres from Glioblastoma
			3.2.2 Mammospheres
			3.2.3 Lung Tumorspheres
			3.2.4 Sarcospheres
		3.3 Detection and Isolation of CSCs by Flow Cytometry
			3.3.1 Stemness Marker Expression
			3.3.2 Side Population Detection
			3.3.3 ALDH Activity
		3.4 Flow Cytometer Setup and Data Acquisition
			3.4.1 Cytometric Detection of Stemness Markers
			3.4.2 Cytometric Analyses of Side Population
			3.4.3 Cell Sorting
	4 Notes
	References
Chapter 4: Surface Markers for the Identification of Cancer Stem Cells
	1 Introduction
		1.1 CSC Surface Markers
		1.2 Isolation and Detection
	2 Materials
		2.1 Cell Culture
		2.2 Single-Cell Suspension
		2.3 Isolation and Characterization of CSCs
			2.3.1 Flow Cytometry
			2.3.2 Aldefluor Assay Conjugated Flow Cytometry
			2.3.3 Magnetic Separation
		2.4 Detecting CSC Functions
			2.4.1 Sphere-Forming Assay
			2.4.2 Western Blotting
			2.4.3 Immunostaining
			2.4.4 Polymerase Chain Reaction (PCR)
	3 Methods
		3.1 Preparation of Single-Cell Suspension
		3.2 Isolation and Detection
			3.2.1 Flow Cytometry-Based Method
			3.2.2 Aldefluor Assay Conjugated Flow Cytometry Method
			3.2.3 Magnetic Separation-Based Method
		3.3 In Vitro Growth Properties of  CSCs
		3.4 Molecular Expression of CSC Markers
			3.4.1 PCR Technique
				Extraction of Total  RNA
				cDNA Synthesis
				Real-Time Polymerase Chain Reaction
			3.4.2 Western  Blot
			3.4.3 Immunological Analysis
	4 Notes
	References
Chapter 5: CD44-Based Detection of CSCs: CD44 Immunodetection by Flow Cytometry
	1 Introduction
	2 Materials
		2.1 Cell Dissociation from Tissue Using gentleMACS Dissociator
		2.2 CD44 Staining for Flow Cytometry
	3 Methods
		3.1 Sample Preparation
		3.2 CD44 Staining for Flow Cytometry
		3.3 Sample Reading and Analysis
	4 Notes
	References
Chapter 6: ALDH Activity Assay: A Method for Cancer Stem Cell (CSC) Identification and Isolation
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Sample Preparation
	3 Methods
		3.1 Aldefluor Assay
		3.2 Flow Cytometer Setup and Data Acquisition
			3.2.1 Prepare an Acquisition Template
			3.2.2 To Set up Analyzer and Acquire   Data
			3.2.3 Cell Sorting
	4 Notes
	References
Chapter 7: In Vitro Tumorigenic Assay: A Tumor Sphere Assay for Cancer Stem Cells
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Isolation of Cell Suspensions from Tumor Tissues
		3.2 Preparation of Cells from Prostate Cancer Cell Lines
		3.3 Seeding Cells for Sphere Formation Assay
		3.4 Propagating Spheres
	4 Notes
	References
Chapter 8: Multicellular Tumoroids for Investigating Cancer Stem-Like Cells in the Heterogeneous Tumor Microenvironment
	1 Introduction
	2 Materials
		2.1 Cells and Culture Medium
		2.2 General Tissue Culture
		2.3 Lentiviral Transduction
		2.4 Cell Labeling with CellTracker Dye
		2.5 3D Hanging Drop Platform
		2.6 Fluorescence-Activated Cell Sorting
		2.7 RT-qPCR
		2.8 Transwell Migration Assays
	3 Methods
		3.1 Lentiviral Transduction of Adherent Cells
		3.2 Lentiviral Transduction of Nonadherent Cells
		3.3 Labeling Adherent or Nonadherent Cells with Fluorescent Cell Tracker
		3.4 Preparation of Hanging Drop Plates
		3.5 Differentiation of U937 Monocytes to Macrophages
		3.6 Generation of 3D Spheroids or Tumoroids with Single or Multiple Cell Types, Respectively
		3.7 Maintenance of 3D Spheroids and Tumoroids
		3.8 FACS to Separate Cell Types from Tumoroids
		3.9 Gene Expression Analysis of Individual Cell Types
		3.10 Flow Cytometry to Analyze Stem Markers on Cancer Cells
		3.11 Flow Cytometry to Analyze Drug Response of Cancer Cells
		3.12 Transwell Assays to Investigate Cancer Cell Migration
	4 Notes
	References
Chapter 9: Generation, Expansion, and Biobanking of Gastrointestinal Patient-Derived Organoids from Tumor and Normal Tissues
	1 Introduction
	2 Materials
		2.1 Sample Collection and Preparation (or Handling)
		2.2 Tissue Processing
		2.3 Organoid Culture in  BME
		2.4 Organoid Passaging and Cryopreservation
		2.5 Drug Sensitivity Assay of Tumor Organoids
	3 Methods
		3.1 Primary Tissue Processing
		3.2 Organoid Establishment Protocol
		3.3 Passaging and Expansion of Organoids
		3.4 Cryopreservation and Thawing of Organoids
		3.5 Drug Sensitivity Assay of Tumor Organoids
	4 Notes
	References
Chapter 10: Prostate Cancer Organoids for Tumor Modeling and Drug Screening
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Isolation of Single-Cell Suspensions from Prostate Tumor Tissues (See Fig. 1)
		3.2 Preparation of Cells from Prostate Cancer Cell Lines
		3.3 Seeding Cells for Organoid Formation Assay (See Fig. 1)
		3.4 Propagation of Prostate Tumor Organoids (See Fig. 1)
		3.5 Treatment of Prostate Tumor Organoids
	4 Notes
	References
Chapter 11: Mimicking the Tumor Niche: Methods for Isolation, Culture, and Characterization of Cancer Stem Cells and Multicell...
	1 Introduction
		1.1 Properties and Characterization of CSCs
		1.2 Interplay Between TME and CSCs
	2 Materials
		2.1 CSC Enrichment from Cell Lines: Primary and Secondary Tumorsphere Generation
		2.2 Soft Agar Colony Formation Assay
		2.3 CSC Characterization by Flow Cytometry
			2.3.1 Hoechst Exclusion Assay
			2.3.2 CSC Marker Evaluation
		2.4 In Vivo Tumorigenic Properties of CSCs (Orthotopic Limiting Dilution Assay)
		2.5 Multicellular Spheroid Production
	3 Methods
		3.1 CSC Enrichment from Cell Lines: Primary and Secondary Tumorsphere Generation
		3.2 Soft Agar Colony Formation Assay
		3.3 Cancer Stem Cell Characterization by Flow Cytometry
			3.3.1 Hoechst Exclusion Assay
			3.3.2 Cancer Stem Cell Marker Evaluation
		3.4 In Vivo Tumorigenic Properties of CSCs (Orthotopic Limiting Dilution Assay)
		3.5 Multicellular Spheroids
			3.5.1 Cell Culture
			3.5.2 Formation of Multicellular Spheroids Following the Hanging-Drop Protocol
	4 Notes
	References
Chapter 12: Detection of Cancer Stem Cells in Normal and Dysplastic/Leukemic Human Blood
	1 Introduction
	2 Materials
	3 Methods
		3.1 RBC Lysis and Sample Preparation
		3.2 Direct Staining Procedure
		3.3 Flow Cytometric Acquisition and Parameter Setting
		3.4 Blast Phenotyping
	4 Notes
	References
Chapter 13: Methods to Study the Role of Mechanical Signals in the Induction of Cancer Stem Cells
	1 Introduction
	2 Materials
		2.1 Hydrogel Preparations
		2.2 Isolation and Seeding of Primary Mammary Luminal Cells
	3 Methods
		3.1 Hydrogel Preparations
		3.2 Mammary Gland Dissociation and Isolation of Luminal Differentiated Cells
		3.3 Seeding and Infection of Mammary LD Cells
		3.4 Clonogenic Assay
	4 Notes
	References
Chapter 14: Co-Delivery Polymeric Poly(Lactic-Co-Glycolic Acid) (PLGA) Nanoparticles to Target Cancer Stem-Like Cells
	1 Introduction
		1.1 Cancer Stem Cells Show Increased Chemoresistance, Metastasis, and ALDH Activity due to PDGF Signaling with Mesenchymal Ste...
		1.2 Nanoparticles for Co-Delivery of Sunitinib and Paclitaxel
	2 Materials
		2.1 Nanoparticle Synthesis and Collection
			2.1.1 Materials and Solvents
			2.1.2 Supplies
		2.2 2D and 3D Cell Culture
			2.2.1 Cells and Culture Medium
			2.2.2 General Tissue Culture
	3 Methods
		3.1 Nanoparticle Synthesis and Collection
			3.1.1 Electrospray Setup
			3.1.2 Electrospray Solution Formulation
			3.1.3 Nanoparticle Production
			3.1.4 Nanoparticle Collection
		3.2 Nanoparticle Characterization
			3.2.1 Scanning Electron Microscopy (SEM): Nanoparticle Size and Morphology
			3.2.2 Nanoparticle Tracking Analysis (NTA): Nanoparticle Size, Polydispersity, and Quantity
			3.2.3 Liquid Chromatography-Mass Spectrometry (LCMS): Drug Loading of Nanoparticles
		3.3 Nanoparticle Drug Delivery and Cell Assays
			3.3.1 Hetero-Spheroid Generation and Co-Delivery of Paclitaxel and Sunitinib
	4 Notes
	References
Chapter 15: Isolating Circulating Cancer Stem Cells (CCSCs) from Human Whole Blood
	1 Introduction
	2 Materials
		2.1 Antibodies, Cells, and Kits
		2.2 Equipment and Disposable Plastic/Glassware
	3 Methods
		3.1 Procurement of Human Whole Blood Samples
		3.2 Depletion of Red Blood Cells and White Blood Cells from Human Whole Blood Samples
		3.3 Analyzing Residual Cells in the Final Elution from the Human Blood Samples
		3.4 Verifying Efficient Depletion Process from Human Blood Samples
		3.5 Confirming Presence of CCSCs in the Final Elution
	4 Notes
	References
Chapter 16: Generation of Cancer Stem Cells by Co-Culture Methods
	1 Introduction
	2 Materials
		2.1 Co-Culture of Cancer Cells with TAMs
		2.2 Co-Culture of Cancer Cells with CAFs
	3 Methods
		3.1 Co-Culture of Cancer Cells with TAMs
			3.1.1 Cell Culture
			3.1.2 Differentiation of THP-1 to M1 TAMs Using External Stimuli in a Co-Culture of THP-1 and Cancer Cells
			3.1.3 In Vitro Generation of Monocyte-Derived Macrophages (MDMs) Using THP-1 Cells
			3.1.4 Enrichment of the CSCs by Incubating Cancer Cells with the CM
		3.2 Co-Culture of Cancer Cells with CAFs
			3.2.1 Cell Culture
			3.2.2 Activation of CAFs in HFB Cells Using External Stimuli
			3.2.3 Preparation of Chemokine-Enriched Conditioned Medium (CM)
			3.2.4 Generation of CSCs by Co-Culturing CAFs and Cancer Cells
	4 Notes
	References
Chapter 17: Deep Learning of Cancer Stem Cell Morphology
	1 Introduction
	2 Materials
		2.1 Typical PC Hardware, OS, and Software Framework
		2.2 Microscopy and CSC Culture
	3 Methods
		3.1 Cell Image Acquisition from Cell Culture
		3.2 Image Processing for pix2pix
		3.3 Construction of an AI Model Predicting CSC in Phase Contrast Cell Image
			3.3.1 Basic Concepts of AI Models
			3.3.2 GAN, Conditional GAN, and pix2pix
		3.4 Evaluation of CSC Image Created Using an AI Model
		3.5 Cancer Stem Cell Image Classification and Visualization
			3.5.1 Overview
			3.5.2 Installation
			3.5.3 Sample  Data
			3.5.4 Usage
	4 Notes
	References
Index