Cancer Systems and Integrative Biology (Methods in Molecular Biology, 2660)

Preface
Contents
Contributors
Chapter 1: Introduction: Cancer Systems and Integrative Biology
	References
Chapter 2: Genome-Wide Analysis of Palindrome Formation with Next-Generation Sequencing (GAPF-Seq) and a Bioinformatics Pipeli...
	1 Introduction
	2 Materials
		2.1 GAPF
		2.2 Library Construction
	3 Methods
		3.1 DNA Fragmentation (Restriction Enzyme Digestion)
		3.2 Snap-Back
		3.3 S1 Digestion
		3.4 Purify DNA (Monarch PCR and DNA Cleanup Kit)
		3.5 Library Construction (NEBNext Ultra II FS DNA Library Prep Kit for Illumina)
		3.6 Data Analysis
	4 Notes
	References
Chapter 3: Sample Preparation and Differential Gene Expression Analysis of Human Cancer Cell Lines by RNA Sequencing
	1 Introduction
	2 Materials
		2.1 RNA Preparation
		2.2 RNA-Seq Library Preparation
		2.3 Bioinformatic Software and Tools
	3 Methods
		3.1 RNA Isolation (see Note 6)
		3.2 rRNA Hybridization
		3.3 First-Strand cDNA Synthesis
		3.4 Second-Strand cDNA Synthesis
		3.5 cDNA Cleanup
		3.6 cDNA End  Prep
		3.7 cDNA Adapter Ligation
		3.8 Ligation Cleanup
		3.9 Library  PCR
		3.10 PCR Cleanup
		3.11 Library Quantification and Assessment
		3.12 Assessing Sequencing Quality
		3.13 Understanding Pre-Alignment Data and Preparation
		3.14 Building the Reference Transcriptome
		3.15 Read Alignment
		3.16 Differential Expression Analysis
		3.17 Gene Set Enrichment Analysis
	4 Notes
	References
Chapter 4: Generating Mitochondrial-Nuclear Exchange (MNX) Mice to Identify Mitochondrial Determinants of Cancer Metastasis
	1 Introduction
		1.1 Challenges to Studying Mitochondrial Genetics
		1.2 Models for Studying Mitochondrial Genetics
		1.3 Mitochondrial Genetics in Cancer Metastasis
	2 Materials
	3 Methods
		3.1 Collection of Presumptive Zygotes
		3.2 Zona Drilling
		3.3 Pronuclear Exchange
		3.4 Genotyping
	4 Notes
	References
Chapter 5: 3D-Autologous Culture Method for Precision Oncology
	1 Introduction
	2 Materials
		2.1 Liquid Samples (Ascites or Pleural Effusions)
		2.2 Solid Tissue Samples
	3 Methods
		3.1 Cell Culture for Liquid Samples
		3.2 Cell Culture for Solid Tissue Samples
	4 Notes
	References
Chapter 6: High-Plex Spatial Profiling of RNA and Protein Using Digital Spatial Profiler
	1 Introduction
	2 Materials
		2.1 Equipment and Hardware
		2.2 Reagents
		2.3 Consumables and Accessories
	3 Methods
		3.1 Morphological Marker Validation on Tissues
		3.2 Tissue Processing
		3.3 RNA-Based Profiling (WTA and CTA)
			3.3.1 Working Solution Preparation
			3.3.2 Tissue Baking and Rehydration
			3.3.3 Antigen Retrieval
			3.3.4 RNA Target Exposure
			3.3.5 Tissue Fixation and in Situ WTA Probe Hybridization
			3.3.6 Removal of Extra Probes, Blocking, and Morphological Staining
			3.3.7 Post-collection Process and Library Preparation
			3.3.8 Library Purification and Quality Control (QC)
		3.4 Protein-Based Profiling
			3.4.1 Working Solution Preparation
			3.4.2 Tissue Baking and Rehydration
			3.4.3 Antigen Retrieval
			3.4.4 Tissue Blocking and Antibody Incubation
			3.4.5 Tissue Fixation and Nuclear Staining
			3.4.6 Post-collection Processes
	4 Notes from Our Experience
	References
Chapter 7: Integration of Single-Cell RNA-Sequencing and Network Analysis to Investigate Mechanisms of Drug Resistance
	1 Introduction
	2 Materials
		2.1 Software (see Note 1)
		2.2 Datasets
	3 Methods
		3.1 Constructing PANDA Regulatory Networks
		3.2 Compare PANDA Networks
		3.3 Finding Enriched Edges
		3.4 Finding Enriched TF Nodes
		3.5 Finding Enriched Gene Nodes
		3.6 Finding Key TF and Gene  Node
		3.7 Creating a Generalized Network
		3.8 GSEA of TF Specific-Targeted Genes
		3.9 Connectivity Map (CMAP) Analysis
	4 Notes
	References
Chapter 8: Combination of Tissue Microarray Profiling and Multiplexed IHC Approaches to Investigate Transport Mechanism of Nuc...
	1 Introduction
		1.1 Tissue Microarray (TMA) and IHC Multiplexing
		1.2 ENT1 as a Candidate for Evaluating Drug Resistance in Pancreatic Cancer
	2 Materials
		2.1 Equipment Needed for IHC Processing of the TMA Slides
		2.2 Chemicals, Reagents, and Antibodies
		2.3 Stock and Working Solutions
	3 Methods
		3.1 TMA Construction
		3.2 Deparaffinization, Rehydration, and Fixation of the TMA Slides
		3.3 Antigen Retrieval (AR) of the  TMA
		3.4 Blocking, Probing with Primary and Secondary Antibodies, and Fluorescent Labeling with OPAL System
		3.5 Staining for Additional Markers and Final Processing of the TMA Slides
		3.6 Imaging of the Multiplexed TMA Slides
		3.7 Quantification and Analysis of the Acquired Multiplexed IHC Images
		3.8 Statistical Analysis of the Data from Multiplexed  TMA
	4 Notes
	5 Loss of ENT1 During EMT Process Opal Panel: A Working Example
	6 Investigating Drug Resistance by Exploiting Multiplexed  TMA
	7 Future of TMA Multiplexing in Studying Anticancer Drug Resistance
	References
Chapter 9: In Situ Proximity Ligation Assay to Visualize Protein-Protein Interactions in Tumor Specimens
	1 Introduction
	2 Principle of PLA
	3 Materials and Methods
		3.1 Specimens
			3.1.1 Tissue Specimens
			3.1.2 Cultured Cells
		3.2 Primary Antibodies
		3.3 PLA Protocol
			3.3.1 Specimen Preparation
			3.3.2 Blocking
			3.3.3 Reaction Using Primary Antibodies
			3.3.4 Reaction Using Secondary Antibodies (PLA Probes)
			3.3.5 Ligation Reaction
			3.3.6 Amplification
			3.3.7 Nuclear Staining and Mounting
	4 Results
	5 Conclusion
	6 Notes
	References
Chapter 10: Integration of Metabolomic and Proteomic Data to Uncover Actionable Metabolic Pathways
	1 Introduction
		1.1 Omics Notebook
	2 Installation
	3 Prepare Input Files
		3.1 Search Result Input Formatting
		3.2 Annotation File
			3.2.1 Sheet One
			3.2.2 Sheet Two
	4 Running the Omics Notebook
	5 Interpreting Results
	6 Notes
	References
Chapter 11: Linking Expression of Cell-Surface Receptors with Transcription Factors by Computational Analysis of Paired Single...
	1 Introduction
	2 Materials
		2.1 Python Packages
		2.2 Input Files
	3 Methods
		3.1 Importing and Preprocessing CITE-seq Data
		3.2 Fitting SPaRTAN Models for Each Cell Type
		3.3 Inferring TF Activities
		3.4 Identifying TF-Surface Protein Relationships
	4 Notes
	References
Chapter 12: Mass Spectrometry-Based Tissue Imaging of the Tumor Microenvironment
	1 Introduction
	2 Materials
		2.1 Tissue Slides
		2.2 Tissue Processing
		2.3 MCA Conjugation
		2.4 Metal-Conjugated Antibody Cocktail
		2.5 Tissue Preservation
		2.6 Mass Cytometry Imaging Platforms
	3 General Methods
		3.1 Tissue Selection
			3.1.1 Tissue Quality
			3.1.2 Control Tissue Sourcing
		3.2 Panel Design
			3.2.1 Machine Sensitivity
			3.2.2 Organic Interference
			3.2.3 Organic Metal Adducts
			3.2.4 Overnight Versus Day 2 Panel
		3.3 Antibody Sourcing
			3.3.1 Commercial MCAs
			3.3.2 Unconjugated Antibodies
		3.4 Antibody Conjugation
			3.4.1 Establishing Input Material
			3.4.2 Antibody Purification
			3.4.3 Antibody Reduction
			3.4.4 Antibody: Metal Polymer Conjugation
			3.4.5 MCA Purification, Measurement, and Stabilization
		3.5 Marker Validation
	4 Methods for Multiplexed Ion Beam Imaging
		4.1 Tissue Preparation (See Notes 6.2)
		4.2 Antibody Staining
		4.3 Tissue Preservation
	5 Methods for Imaging Mass Cytometry
		5.1 Tissue Preparation (See Notes 6.3)
		5.2 Antibody Staining
		5.3 Counter Staining
		5.4 Tissue Preservation
	6 Notes
		6.1 Antibody Conjugation Notes
		6.2 MIBI Methods Notes
		6.3 IMC Methods Notes
	References
Chapter 13: Multiplexed Immunoassay Using Quantum Dots to Monitor Proteins Secreted from Single Cells at Near-Single Molecule ...
	1 Introduction
	2 Materials
		2.1 Antibody Conjugation
			2.1.1 Biotinylated Capture Antibodies
			2.1.2 TCO-Modified Detection Antibodies
		2.2 Tetrazine-Modified Quantum Dots
		2.3 QD Immunoconjugates
		2.4 Fabrication of PDMS Microwell Arrays
		2.5 Preparation of Detection Slides
			2.5.1 Calibration Detection Slides
			2.5.2 Single-Cell Assay Detection Slides
		2.6 Calibration Experiments
		2.7 U-937 Cell Culture and Single-Cell Secretion Assays
		2.8 Imaging and Analysis
		2.9 Phenotypic Analysis
	3 Methods
		3.1 Antibody Conjugations
			3.1.1 Biotinylated Capture Antibodies
			3.1.2 TCO-Modified Detection Antibodies
		3.2 Tetrazine-Modified Quantum Dots
		3.3 QD Immunoconjugates
		3.4 Fabrication of PDMS Microwell Arrays
		3.5 Preparation of Detection Slides
		3.6 Calibration Experiments
		3.7 U-937 Cell Culture and Single-Cell Secretion Assays
		3.8 Imaging and Analysis
		3.9 Phenotypic Analysis
	4 Notes
	References
Chapter 14: Isolation and Identification of Plasma Extracellular Vesicles Protein Biomarkers
	1 Introduction
	2 Materials
	3 Methods
		3.1 Enrichment of Extracellular Vesicles (EVs)
		3.2 EV Lysis
		3.3 Protein Digestion and Surfactant Removal
		3.4 Peptide Desalting
		3.5 Tandem Mass Tag (TMT) Labeling
		3.6 Alternative Approach: Mass Spec Analysis Via Data-Independent Acquisition (DIA)
	4 Additional Notes
	References
Chapter 15: Discovery of a Hidden Proinflammatory Signaling Proteome Using a Large-Scale, Targeted Antibody Microarray Platform
	1 Introduction
	2 Materials
		2.1 Radioactively Labeled Cultured Cells
		2.2 Protein Labeling
		2.3 Antibody Microarray
		2.4 Bioinformatic Analysis of Antibody Microarrays
	3 Methods
		3.1 Radioactive Labeling
			3.1.1 35[S]-Methionine Radioactive Labeling
			3.1.2 32[P]-Orthophosphate Radioactive Labeling
		3.2 Protein Labeling
		3.3 Antibody Microarray
		3.4 Bioinformatic Analysis of Antibody Microarray
	4 Notes
		4.1 Radioactive Labeling
		4.2 CyDye Labeling
		4.3 Antibody Microarray
		4.4 Bioinformatic Analysis
	References
Chapter 16: Multiplex Fluorescent Immunohistochemistry for Preservation of Tumor Microenvironment Architecture and Spatial Rel...
	1 Introduction
	2 Materials
		2.1 Wash Buffers
		2.2 Antigen Retrieval
		2.3 Staining Reagents
		2.4 Special Equipment
	3 Methods
		3.1 Slide Preparation and Antibody Optimization
		3.2 Staining Preparation
		3.3 Slide Staining
		3.4 Multiplex Workflow
	4 Notes
	References
Chapter 17: Circle Damage Sequencing for Whole-Genome Analysis of DNA Damage
	1 Introduction
	2 Materials
		2.1 Genomic DNA Preparation and Formation of DNA Double-Strand Breaks
		2.2 CD-seq Library Preparation
		2.3 Data Analysis
	3 Methods
		3.1 Preparation of Circularized Genomic DNA
			3.1.1 Introduction of DNA CPD Adducts
			3.1.2 Genomic DNA (gDNA) Isolation and Fragmentation
			3.1.3 Intramolecular Circularization of DNA Fragments
		3.2 Introduction of Double-Strand Breaks at CPD Sites
			3.2.1 Cleavage of DNA at CPD Sites
			3.2.2 Reversion of the Dimerized Pyrimidines
			3.2.3 Generation of Double-Strand Breaks
		3.3 Preparation of the CD-seq Sequencing Library
			3.3.1 End-Preparation and Adaptor Ligation
			3.3.2 CD-seq Library Amplification by PCR
			3.3.3 Quantification of the CD-seq Library
		3.4 Data Analysis
	4 Notes
	References
Chapter 18: CRISPR-Directed Gene Editing as a Method to Reduce Chemoresistance in Lung Cancer Cells
	1 Introduction
		1.1 Significance and Therapeutic Methodological Goals
		1.2 The NRF2 Gene as the Target
	2 Methods and Protocols
		2.1 Cell Line and Culture Conditions
		2.2 NFR2 R34G Cell Line Engineering
		2.3 NRF2 R34G Targeting with LNP
		2.4 Gene Editing Analysis
		2.5 Chemosensitivity Testing
	3 Results
	4 Discussion and Conclusion
	References
Chapter 19: Integrated In Silico Analysis of Proteogenomic and Drug Targets for Pancreatic Cancer Survival
	1 Introduction
	2 Clinical Data from cBioPortal
	3 Methods
		3.1 Copy Number Alterations
		3.2 Survival Curves
		3.3 Protein Drug Analysis
		3.4 Pancreatic Cancer Categories
		3.5 Data Analysis
			3.5.1 Copy Number Alterations Based on Race Categories
			3.5.2 Association of CNA Amplification with Survival Outcome
			3.5.3 Protein Expression and Protein-Drug Interaction
	4 Notes
	References
Chapter 20: Advanced Computational Methods to Evaluate Vascular Heterogeneity in Tumor Tissue Based on Single Plane Illuminati...
	1 Introduction
	2 Materials
		2.1 Experimental Agents
			2.1.1 Selective Plane Illumination Microscopy on Healthy Mice Brain and Mice Injected with Glioblastoma/Glioma Tumor Cells
		2.2 Image Processing and Analysis Software
	3 Methods
		3.1 Experimental Methods
			3.1.1 Tumor Cell Injection (Example for Glioblastoma and Glioma Cells)
			3.1.2 Lectin Injection, Mouse Sacrifice, and Clearing Protocol for Selective Plane Illumination Microscopy
		3.2 Vascular Segmentation and Image Post-Processing
			3.2.1 Tumor Volume Segmentation
			3.2.2 Vessel Segmentation Using Ilastik
			3.2.3 Vascular Segmentation Post-Processing
			3.2.4 Vascular Skeletonization
			3.2.5 Vascular Thinning Due to Lectin Fluorescence Overexposure
		3.3 Vascular Geometry Analysis
		3.4 Vascular Network Topology
			3.4.1 Scale-Free and Basic Topological Characteristics of the Vascular Network
			3.4.2 Community Measures on the Vascular Network
			3.4.3 Community Structure and Connectivity
	4 Notes
	5 Conclusion
	References
Chapter 21: Illuminating DEPDC1B in Multi-pronged Regulation of Tumor Progression
	1 Introduction
	2 High DEPDC1B Expression During the G2/M Phase Promotes Disassembly of Focal Adhesion, De-adhesion, and Cell Entry into Mitos...
	3 DEPDC1B Is a Positive Upstream Effector of pERK
	4 DEPDC1B Is a Novel Binding Partner of the p85 Subunit of PI3K, and DEPDC1B Expression Correlates with Downregulation of Insu...
	5 DEPDC1B Is Implicated in Other Protein-Protein Interactions and Tumor Progression
	6 Conclusions and Future Perspectives
	References
Chapter 22: Single-Cell and Spatial Analysis of Emergent Organoid Platforms
	1 Introduction
	2 Emergence and Construction of Organoids
		2.1 Emergence of Organoids
		2.2 2D and 3D Culture Models
	3 Structural and Molecular Analysis of Organoids
		3.1 Structural Heterogeneity of Organoids
		3.2 Single-Cell and Spatial Analysis of Organoids
	4 Applications of Organoid Platforms
	5 Challenges and Practical Issues of Organoids
		5.1 Technical Challenges
		5.2 Practical Challenges
	6 Conclusion
	References
Chapter 23: Analysis of Phase-Separated Biomolecular Condensates in Cancer
	1 Introduction
	2 Does it Happen? Analysis of Phase Separation for the Protein of Interest
		2.1 In Silico
		2.2 In Vitro
			2.2.1 Why In Vitro?
			2.2.2 Reconstituted Systems with Purified Molecules
			2.2.3 How to Demonstrate that the POI Forms Condensates Instead of Just ``Aggregates´´?
		2.3 In Cells
	3 Is it Important? Functional Analysis of LLPS in Cancer
		3.1 LLPS Perturbation by Protein-Extrinsic Factors and Conditions
		3.2 LLPS Perturbation by Changing Protein Sequences
		3.3 Analysis of Cancer-Associated Mutations and Variations
	4 How Does it Work? Mechanistic Studies on how Phase-Separated Biomolecular Condensates Regulate Cancer
	5 Concluding Remarks
	References
Chapter 24: Gain-of-Function Variomics and Multi-omics Network Biology for Precision Medicine
	1 Gain-of-Function Mutations in Cancer
	2 Epigenetic Regulation
		2.1 Histone Acetylation and Methylation
	3 Transcription Factors
	4 Noncoding Elements
		4.1 Enhancers and Promoters
	5 Protein-Protein Interactions
	6 Post-translational Regulation
	7 Gain-of-Function Mutations in Other Diseases
		7.1 Neurological Diseases
		7.2 Inflammatory Diseases
	8 Protein Aggregation and Liquid-Liquid Phase Separation
	9 Computational Resources for Predicting GOF Mutations (See Table 1)
	10 Conclusion
	References
Index