Cancer Cell Signaling: Methods and Protocols

Preface
Contents
Contributors
Part I: Cancer Resistance
	Chapter 1: Fighting Cancer Resistance: An Overview
		1 Cancer Resistance: Main Obstacle to Overcome in Cancer Therapy
		2 Challenges to Defeat Resistance Using Targeted Anticancer Therapies
		3 The Role of HIFs in Cancer Resistance: Challenges to Overcome Resistance Targeting Autophagy and Metabolism
		4 Cancer Stem Cells as a Hub in Cancer Resistance
		5 Challenges to Beat Resistance with Immunotherapy
		References
	Chapter 2: Prostate Cancer Spheroids: A Three-Dimensional Model for Studying Tumor Heterogeneity
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Cell Lines
			2.3 Cell Culture Equipment
		3 Methods
		4 Notes
		References
	Chapter 3: Enrichment and Transcriptional Characterization of Stem Cells Isolated from Human Glioblastoma Cell Lines
		1 Introduction
		2 Materials
			2.1 Cell Culture (See Note 1)
			2.2 Immunostaining for Flow Cytometry
			2.3 Immunofluorescence
			2.4 RT-qPCR
		3 Methods
			3.1 Glioma Stem Cells Enrichment and Culture
			3.2 Determination of CD133+ and CD15+ Cells by Flow Cytometry Analysis
			3.3 Glioma Stem Cells Characterization
			3.4 Analysis of the Expression of Genes Associated With Stemness in  GSCs
		4 Notes
		References
	Chapter 4: Reverse Docking for the Identification of Molecular Targets of Anticancer Compounds
		1 Introduction
		2 Materials
			2.1 Computational Workstation
			2.2 Python Environments Manager (See Note 4)
			2.3 Molecular Docking Software
			2.4 Bioinformatics and Cheminformatic Tools
			2.5 Analysis Tools Working on Python 3.6
		3 Methods
			3.1 Generation of Protein Structures Database
			3.2 Setting Up the Software and Conda Environments
			3.3 Protein Dataset Preparation for Reverse Docking Assays (See Note 7)
			3.4 Ligand Building and Preparation for Reverse Docking (See Note 8)
			3.5 Protein Pocket Search for Reverse Docking
			3.6 Reverse Docking
			3.7 Analysis of Docking Results
		4 Notes
		References
	Chapter 5: Mouse Model for Efficient Simultaneous Targeting of Glycolysis, Glutaminolysis, and De Novo Synthesis of Fatty Acid...
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Drug Treatments
			2.3 Seahorse Assays
			2.4 Mice
			2.5 Glucose Tolerance Tests
			2.6 Magnetic Resonance Imaging
			2.7 Indirect Calorimetry
		3 Methods
			3.1 Cell Culture and Viability Measurement
			3.2 Viability Curves
			3.3 Pharmacological Interaction
			3.4 Seed and Treatment of Cells for Seahorse Assays
			3.5 Measurement of Oxidative Phosphorylation Parameters With the Seahorse XF Cell Mito Stress  Kit
				3.5.1 The Day Prior of the Assay
				3.5.2 Day of Assay
			3.6 Measurement of Glycolytic Parameters With the Seahorse XF Glycolysis Stress  Kit
				3.6.1 Day of Assay
			3.7 Tumor Growth in  Mice
			3.8 In Vivo Evaluation of the Tumor Growth
			3.9 Glucose Tolerance Tests
			3.10 Magnetic Resonance Imaging
			3.11 Indirect Calorimetry
		4 Notes
		References
Part II: Epigenetic Control of Cancer
	Chapter 6: Developing a Portable Device for the Identification of miRNAs in Fluids
		1 Introduction
		2 Materials
			2.1 Expression of has-mir-141-5p in HEK293 Cells
			2.2 Extraction of microRNAs from HEK293 Cells
			2.3 Preparation of Silk Gels
			2.4 Preparation of Molecular Beacons and Surface Gel
			2.5 Agarose Preparation for Surface Gel
			2.6 Printing Microarrays on Conventional Coverslips
			2.7 Production of Low-Cost Equipment for the Identification of microRNAs in Samples and Fluids
		3 Methods
			3.1 Expression of has-mir-141-5p in HEK293 Cells
			3.2 Extraction of microRNAs from HEK293 Cells
			3.3 Preparation of Silk Gels
			3.4 Preparation of Molecular Beacons and Surface Gel
			3.5 Agarose Preparation for Surface Gel
			3.6 Printing Microarrays on Conventional Coverslips
			3.7 Recording Microarray Fluorescence Increments in Real-Time
			3.8 Low-Cost Equipment for the Identification of microRNAs in Samples and Fluids
			3.9 Traditional Versus 3D-Enhanced TIRF Arrays
			3.10 Identification of hsa-miR-141-5p, a microRNA Marker for Cancer
			3.11 Microarray Analysis
		4 Notes
		References
	Chapter 7: Methods for the Study of Long Noncoding RNA in Cancer Cell Signaling
		1 Introduction
			1.1 The Human Genome Contains Many Thousands of Unexplored lncRNAs
			1.2 lncRNAs Regulate Gene Expression and Protein Functions Via Various Mechanisms
			1.3 lncRNA Expression Is Deregulated in Human Cancer
			1.4 lncRNAs Serve as Tumor Suppressor Genes or Oncogenes
			1.5 lncRNAs Represent Promising Biomarker and Therapeutic Candidates for Cancer Diagnosis and Treatment
			1.6 Methods in lncRNAs Research
		2 Materials
			2.1 lncRNA-Immunoprecipitation
			2.2 lncRNA Pull-Down
			2.3 lncRNA Northern Blot Analysis
				2.3.1 DIG labeled RNA Probe Synthesis
				2.3.2 Separating RNA by Electrophoresis
				2.3.3 Transferring RNA to the Membrane
				2.3.4 Probe-RNA Hybridization
				2.3.5 Detection of Probe-RNA Hybrids
			2.4 lncRNA In Situ Hybridization
			2.5 lncRNA Knockdown
				2.5.1 lncRNA Knockdown Using siRNAs
				2.5.2 lncRNA Knockdown Using shRNAs
		3 Methods
			3.1 lncRNA-Immunoprecipitation
				3.1.1 Whole Cell Lysate Preparation (See Note 6)
				3.1.2 Cell Harvest and Nuclei Lysate Preparation (See Note 6)
				3.1.3 RNA Immune-Precipitation and Purification
			3.2 lncRNA Pull-Down
				3.2.1 Biotinylated RNA Synthesis by In Vitro Transcription
				3.2.2 Whole Cell Lysate Preparation (See Note 6)
				3.2.3 Nuclear Lysate Preparation (See Note 6)
				3.2.4 RNA Pull-Down (See Note 14)
			3.3 lncRNA Northern blot Analysis (See Note 17)
				3.3.1 DIG Labeled RNA Probe Synthesis by In Vitro Transcription
				3.3.2 Separating RNA Samples by Electrophoresis
				3.3.3 Transfer RNA from Agarose Gel to the Membrane
				3.3.4 Hybridization of DIG-Labeled Probes to the Membrane (See Note 28)
				3.3.5 Detection of DIG-Probe-Target RNA Hybrids
			3.4 lncRNA In Situ Hybridization
				3.4.1 DIG Labeled RNA Probe Synthesis by In Vitro Transcription
				3.4.2 Cell Preparation and Pretreatment
				3.4.3 In Situ Hybridization and Detection of Probe-Target Hybrid
			3.5 Method for shRNA Knockdown
				3.5.1 lncRNA Knockdown Using siRNAs
				3.5.2 lncRNA Knockdown Using shRNAs
		4 Notes
		References
	Chapter 8: RNA-Sequencing Analysis Pipeline for Prognostic Marker Identification in Cancer
		1 Introduction
		2 Materials
			2.1 System Requirements
			2.2 Data Availability
			2.3 Clinical Data Availability
			2.4 Data Download
			2.5 Data Analysis
			2.6 Quality Control
			2.7 Alignment to a Reference Genome and Sorting
			2.8 Assembly and Quantification
				2.8.1 STAR Pipeline
			2.9 Differential Expression Analysis
				2.9.1 DESeq
			2.10 Survival Analysis
		3 Differential Expression and Survival Analysis Using RNA-Seq Data from Glioblastoma (GBM) Patients
			3.1 Differential Expression Analysis
			3.2 Cox-Regression and Kaplan-Meier Analysis
		4 Notes
		References
Part III: Metastasis Promotion
	Chapter 9: Correlation of Circulating Tumor Cell Measurements with 3D Quantitative Tumor Characterization to Predict Clinical ...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Protocol Overview
			3.2 MR Imaging
			3.3 Blood Samples (see Note 2)
			3.4 Circulating Tumor Cell Analysis Using the CellSearch System
				3.4.1 Processing with the CELLTRACKS AUTOPREP System
				3.4.2 Analysis Using the CELLTRACKS ANALYZER II
			3.5 Quality Control
			3.6 Quantification of Circulating Tumor Cells
		4 Notes
		References
	Chapter 10: Extracellular Vesicles and Their Roles in Cancer Progression
		1 EVs: A Brief Introduction and History
		2 EV Isolation and Classification
		3 Mechanisms of EV Formation and Release
			3.1 MV Biogenesis
			3.2 Exosome Biogenesis
		4 EVs and Cancer Progression
			4.1 How EVs Mediate Cancer Cell Phenotypes
			4.2 EVs and Tumor Angiogenesis
		5 EVs and Metastasis
		6 EVs and Cancer-Mediated Immunosuppression
		7 EVs and Their Potential Uses in the Clinics
			7.1 EVs and Liquid Biopsies
			7.2 EVs as a Drug Delivery System
		8 Concluding Remarks
		References
	Chapter 11: Analysis of Tumor-Derived Exosomes by Nanoscale Flow Cytometry
		1 Introduction
			1.1 Exosomes
		2 Biological Relevance of Exosomes in Cancer
			2.1 Mechanisms of Tumorigenesis and Cancer Progression via Exosomes
			2.2 Tumor Exosomes as Prognostic and Diagnostic Tools in Cancer
		3 Current Methods for Isolation and Characterization of Tumor Exosomes
			3.1 Ultracentrifugat-ion
			3.2 Density Gradients
			3.3 Size Exclusion Chromatography
		4 Biochemical and Physical Analysis of Exosomes
			4.1 Biochemical Characterization
			4.2 Physical Characterization of Exosomes
		5 Flow Cytometry as an Approach for Exosome Analysis
		6 Nanoscale Flow Cytometry
			6.1 Considerations for the use of Nanoscale Flow Cytometry for Tumor Exosome Detection and Analysis
				6.1.1 Instrument Calibration
					Fluidic System Settings
					Acquisition Settings
					Sample Preparation
					Data Analysis
		References
	Chapter 12: In Vitro Models for Studying Tumor Progression
		1 Introduction
		2 Materials
			2.1 Scratch-Wound Assay to Evaluate Cancer Cell Migration in 2D Cell Cultures
			2.2 Transwell Invasion Assay to Evaluate Cancer Cell Invasion in 2D Cell Cultures
			2.3 Cell Migration Assay in 3D Cell Culture Conditions
			2.4 Transwell Invasion Assay to Evaluate Cancer Cell Invasion in 3D Cell Culture Conditions
		3 Methods
			3.1 Scratch-Wound Assay to Evaluate Cancer Cell Migration in 2D Cell Cultures
			3.2 Transwell Invasion Assay to Evaluate Cancer Cell Invasion in 2D Cell Cultures
			3.3 Cell Migration Assay in 3D Cell Culture Conditions
			3.4 Transwell Invasion Assay to Evaluate Cancer Cell Invasion in 3D Cell Culture Conditions
		4 Notes
		References
	Chapter 13: Assessment of Cell Cycle in Primitive Chronic Myeloid Leukemia Cells by Flow Cytometry After Coculture with Endoth...
		1 Introduction
		2 Materials
			2.1 Primary Human Cells
			2.2 General
			2.3 Isolating Mononuclear Cells from Human Bone Marrow Samples by Density Gradient
			2.4 Enrichment of CD34+ Hematopoietic Primitive Cells
			2.5 Endothelial Cells Culture
			2.6 Primitive Hematopoietic Cells (PHC) in Coculture with Endothelial Cells
			2.7 Cell Cycle
		3 Methods
			3.1 Isolating Mononuclear Cells from Human Bone Marrow Samples by Density Gradient
			3.2 CD34+ PHC Enrichment
			3.3 Obtaining Endothelial Cells
			3.4 Contact Co-culture
			3.5 Separation of Cell Populations
			3.6 Cellular Identification by Sorting
			3.7 Cell Cycle Assessment in CD34+ Cells
			3.8 Analysis of Cell Cycle by Flow Cytometry
		4 Notes
		References
Part IV: New Technologies in the Study of Cancer
	Chapter 14: Chimeric Antigen Receptor (CAR) T Cell Therapy for Cancer. Challenges and Opportunities: An Overview
		1 Introduction
		2 Chimeric Antigen Receptor (CAR) T Cells
			2.1 Structure of Chimeric Antigen Receptor (CAR)
			2.2 Clinical Manufacturing of CAR T Cells
		3 Clinical Applications of CAR T Cells
			3.1 CAR T Cell for Hematologic Malignancies
				3.1.1 Leukemias
				3.1.2 Lymphomas
			3.2 Advances in CAR T Cell Therapies for Solid Tumors
		4 Current Challenges and Perspectives
			4.1 CAR T Therapy Derived-Toxicities
				4.1.1 On-Target On-Tumor Toxicity
				4.1.2 On-Target Off-Tumor Toxicity
				4.1.3 Off-Target Toxicity
				4.1.4 Genotoxicity
				4.1.5 Immunogenicity
				4.1.6 Neurotoxicity
			4.2 Strategies to Overcome Toxicity
				4.2.1 Suicide Gene Switch
				4.2.2 Antibody-Mediated Suicide Switch
				4.2.3 Combinatorial Target Antigen Recognition
				4.2.4 Synthetic Notch Receptors
				4.2.5 Inhibitory Chimeric Antigen Receptor
				4.2.6 Bispecific T Cell Engager
				4.2.7 On-Switch CAR
			4.3 Current Challenges in CAR T Cell Therapy
				4.3.1 Exhaustion of CAR T Cells
				4.3.2 Resistance to CAR T Cell Therapy
					Antigen Escape and Down Modulation of Target Antigen
		References
	Chapter 15: Cell-Internalization SELEX of RNA Aptamers as a Starting Point for Prostate Cancer Research
		1 Introduction
		2 Materials
			2.1 Aptamer Library
			2.2 Oligonucleotide for Retrotranscription (RV)
			2.3 Oligonucleotides for PCR
		3 Methods
			3.1 Cell Culture
			3.2 Library Preparation
			3.3 Cell-Selex
			3.4 RNA Isolation
			3.5 Retrotranscription
			3.6 PCR and Purification
		4 Notes
		References
	Chapter 16: Generation of Functional Genetic Study Models in Zebrafish Using CRISPR-Cas9
		1 Introduction
		2 Materials
			2.1 sgRNA Annealing and In Vitro Transcription
			2.2 Cas9 In Vitro Transcription
			2.3 CRISPR Injection
		3 Methods
			3.1 Single-Guide RNA In Vitro Transcription
			3.2 Cas9 In Vitro Transcription
			3.3 CRISPR System Injection into Zebrafish Embryos
			3.4 DNA Extraction and Genotyping of Injected Embryos
		4 Notes
		References
	Chapter 17: Developing a Model for a siRNA Delivery System by Cancer Implantation into Zebrafish Circulation
		1 Introduction
		2 Materials
			2.1 Zebrafish Breeding Resources
			2.2 Cell Culture and Stable Expression of Fluorescent Protein Components
			2.3 Preparation of Zebrafish for Cancer Cell Implantation
			2.4 Cancer Cell Implantation Components
			2.5 Components for LPEI-Coated siRNA-PLGA Hybrid Micelles
			2.6 Components and Software for Imaging
		3 Methods
			3.1 Fluorescent mCherry Labeling of A375, Human Malignant Melanoma Cells
			3.2 Preparation of LPEI-Coated siRNA-PLGA Hybrid Micelles
			3.3 Preparation for Cancer Cell Implantation
			3.4 Cancer Cell Implantation
			3.5 Injection of LPEI-Coated siRNA-PLGA Hybrid Micelles
			3.6 Evaluation of Anticancer Effects by Image Analysis
		4 Notes
		References
	Chapter 18: Fabrication of Adhesive Substrate for Incorporating Hydrogels to Investigate the Influence of Stiffness on Cancer ...
		1 Introduction
			1.1 Wound Healing, Chronic Inflammation and Cancer
			1.2 The Tumor Microenvironment
			1.3 Extracellular Matrix
			1.4 Signalling Pathways
			1.5 Cancer Cell Behaviors and Phenotypic Plasticity
			1.6 Hydrogels for Mimicking Microenvironments to Study Cancer Cells
		2 Materials
			2.1 Preparation of Samples
			2.2 Polyacrylamide (PAA) Hydrogels
			2.3 Cell Culture
			2.4 Immunofluorescence
			2.5 Imaging and Statistics
		3 Methods
			3.1 Hydrogel on Loctite
			3.2 Cell Culture and Immunofluorescence
		4 Notes
		References
Index