Medulloblastoma: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Childhood Medulloblastoma: An Overview
	1 Introduction
	2 History of MB Classification
		2.1 Histopathological Subtypes
		2.2 Molecular Subgroups
		2.3 Second Generation Molecular Subtypes
		2.4 Tissue of Origin
	3 Cancer Stem Cell Hypothesis
		3.1 Defining ``Stem Cell´´
		3.2 Cancer Stem Cells in Brain Cancer
	4 Therapeutic Avenues
		4.1 Targeted Therapy
		4.2 MB Immunotherapy and Challenges
		4.3 Personalized Medicine in  MB
	References
Part I: Molecular Assay-Based Methods
	Chapter 2: Real-Time PCR Assays on Formalin-Fixed, Paraffin-Embedded Medulloblastomas
		1 Introduction
		2 Materials
			2.1 Total RNA Extraction from FFPE Tumor Tissues
			2.2 cDNA Synthesis
			2.3 Gene Expression Analysis by Real-Time PCR
			2.4 MiRNA Expression Analysis by Real-Time PCR
		3 Method
			3.1 Total RNA Extraction from FFPE Tumor Tissues
			3.2 cDNA Synthesis
			3.3 Gene Expression Analysis by Real-Time PCR
				3.3.1 Primer Designing
				3.3.2 Real-Time PCR
			3.4 MiRNA Expression Analysis by Real-Time PCR
				3.4.1 Preparation of Custom RT  Pool
				3.4.2 Preparation of the RT Reaction  Mix
				3.4.3 Preparation of Custom Preamplification (PreAmp) primer  Pool
				3.4.4 PreAmp Reaction Mix (See Note 7)
				3.4.5 Real-Time PCR
			3.5 Molecular Subgrouping of Medulloblastomas Using Prediction Analysis of Microarrays (PAM)
		4 Notes
		References
	Chapter 3: Validation of microRNA Target Genes Using Luciferase Reporter assay and Western Blot Analysis
		1 Introduction
		2 Luciferase Reporter Assay
			2.1 Materials
			2.2 Method
		3 Western Blot Analysis
			3.1 Extraction of Total Proteins from Cultured Cells
				3.1.1 Materials
				3.1.2 Method
		4 Estimation of Protein Concentration
			4.1 Materials
			4.2 Method
		5 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
			5.1 Materials
			5.2 Method
		6 Western Blot Analysis and Immunodetection
			6.1 Materials
			6.2 Method
		7 Notes
		References
	Chapter 4: Chromatin Immunoprecipitation Assays on Medulloblastoma Cell Line DAOY
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 5: Quantification of microRNAs in Cells and Tissues Using Stem-Loop RT PCR and qPCR
		1 Introduction
		2 Extraction of RNA from Human or Mouse Tissues
			2.1 Materials
			2.2 Method
		3 Stem-Loop Reverse Transcription for miRNA
			3.1 Materials
			3.2 Method
		4 Real-Time PCR
			4.1 Materials
			4.2 Method
		5 Notes
		References
	Chapter 6: Analysis of Microarray Data from Medulloblastoma Tissue Samples
		1 Introduction
		2 Materials
		3 Methods
			3.1 Data Processing, Screening for DEGs, and Functional Enrichment Analyses
				3.1.1 Raw Data Quality Control
				3.1.2 Preprocessing
				3.1.3 Estimation of Differentially Expressed Genes (DEGs)
				3.1.4 Functional Enrichment Analyses
		4 Notes
		References
	Chapter 7: Advancement of Single-Cell Sequencing in Medulloblastoma
		Abbreviations
		1 Introduction
		2 Methods
			2.1 Single-Cell Sequencing
			2.2 Isolation of Single  Cell
			2.3 Amplification
			2.4 Single-Cell Genome Sequencing
			2.5 Single-Cell Transcriptome Sequencing
			2.6 Application and Types of Single-Cell Analysis Techniques
			2.7 Computational Analysis
			2.8 Experimental Analysis
		3 Ongoing Research in Brain Cancer
		4 Conclusion
		References
Part II: Cellular Assays and Inhibitors for Medulloblastoma
	Chapter 8: Flow Cytometry Approaches to Obtain Medulloblastoma Stem Cells from Bulk Cultures
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents
		3 Methods
			3.1 Harvesting Adherent Cells for Staining
			3.2 Harvesting Suspension Cells for Staining
			3.3 Staining Protocol for Surface Markers
			3.4 Staining Protocol for Intracellular Markers of Medulloblastoma
			3.5 Flow Cytometer Analysis
			3.6 Data Analysis of Flow Data
			3.7 Sorting of Stained Cells
		References
	Chapter 9: Drug Screening Assays on Medulloblastoma Stem Cells Using Compound Libraries
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation and Plating of MB Stem Cells
			3.2 Compound Library Treatment
			3.3 Tumorsphere Formation Assay
			3.4 Cell Cytotoxicity Assay
		4 Notes
		References
	Chapter 10: 3D-on-2D: A Physiologically Relevant and Gel-Free In Vitro Coculture Method to Assay Antimetastatic Agents
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 11: In Vitro Screening Assay for Activators of T-Cell Migration to Solid Tumors
		1 Introduction
		2 Methods
		3 Notes
		References
	Chapter 12: Impact of Nano Preparation of Phytoconstituents in Medulloblastoma
		1 Introduction
		Box 1 Ideal Property of a Nanopreparation
		2 Conclusion
		References
	Chapter 13: Challenges and Recent Advances of Novel Chemical Inhibitors in Medulloblastoma Therapy
		Abbreviation
		1 Introduction
		2 Materials
			2.1 A Novel Peptide Doxorubicin Conjugates and Its Effect on Medulloblastoma Cells
			2.2 CAT3, a Novel Agent, Reduces Tumor Growth by Distracting the Hedgehog Signaling Pathway
			2.3 Novel Non-peptide Small Molecular STAT3 Inhibitor
			2.4 ApoE Mimetic Peptide Conjugated PEG-PBC Polymer
		3 Methods
			3.1 Artemisinin Derivatives as Novel Compounds
			3.2 MK-4101 Drug Molecule
			3.3 Novel Chemical Compound TAK441
			3.4 NVP-LEQ506-Derived Drug Molecules
			3.5 Cystine-Rich Domain with Smoothened Receptor Modulators
			3.6 DYRK1B Inhibitors
			3.7 GLl Expression Suppressors
			3.8 Biflavone Ginkgetin Novel Compounds Act as Wnt Inhibitors
			3.9 Newly Synthesized Compounds as Hedgehog Signaling Pathway Inhibitors
			3.10 Compounds Derived from Marketed Taladegib Drug
		4 Conclusion
		References
	Chapter 14: Role of Ayurvedic Plants as Anticancer Agents
		1 Introduction
			1.1 Pathogenesis in Medulloblastoma
		2 Reported Phytoconstituents for the Management of Medulloblastoma
			2.1 Curcumin
			2.2 Genistein
			2.3 Tetrahydrocannabinol (THC) and Cannabidiol (CBD)
		3 Conclusion
		References
Part III: Animal Models in Medulloblastoma
	Chapter 15: In Vivo Tumorigenicity Assays Using Subcutaneous and Orthotopic Xenografts in Immunodeficient Mice
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Equipment
			2.3 Animals
			2.4 Cell Lines
		3 Methods
			3.1 Generation of Subcutaneous Xenografts
			3.2 Generation of Orthotopic (Intracerebellar) Xenograft
				3.2.1 Preparation of Cells
				3.2.2 Equipment Setup and Preoperative Animal Preparation
				3.2.3 Surgical Procedure
				3.2.4 Bioluminescence Imaging
		4 Notes
		References
	Chapter 16: Protocols to Manufacture an Oncolytic Measles Virus-Sensitive Immunocompetent Mouse Model of Medulloblastoma
		1 Introduction
		2 Materials
			2.1 Mouse Strain, Surgery, and Bioluminescence Imaging
			2.2 Cells, Plasmids, and Culture Media
			2.3 Isolation of Cerebellar Stem Cells
			2.4 Measles Virus Preparation
		3 Methods
			3.1 Preparation of Cerebellar Stem Cell Neurospheres
			3.2 Preparation of Murine MV-Sensitive Group 3 Medulloblastoma Cell Line
				3.2.1 Packaging of Retroviruses
				3.2.2 Harvesting Virus
				3.2.3 Preparation of Lentivirus
				3.2.4 Harvesting Virus
			3.3 Transduction of Cerebellar Stem Cells
			3.4 Preparation of Murine Tumor Cell Line
		4 Notes
		References
Part IV: Patient Data Based Approaches for Biomedical Research in Medulloblastoma
	Chapter 17: Preclinical Research Strategy Development for RNAi-Based Therapies in Oncology Using Patient-Centered Information ...
		1 Introduction
		2 Materials
		3 Methods
			3.1 GeneCards
			3.2 Human Protein Atlas
			3.3 R2: Genomics Analysis and Visualization Platform
				3.3.1 Clinical Trials
		4 Notes
		References
	Chapter 18: Magnetic Resonance Imaging in the Contemporary Management of Medulloblastoma: Current and Emerging Applications
		1 Introduction
		2 Diagnostic Imaging
		3 Acquisition and Interpretation of MRI Sequences
			3.1 T1W Intensity
			3.2 T2W Intensity
			3.3 Anatomic Tumor Location
			3.4 Tumor  Size
			3.5 Contrast-Enhancement
			3.6 Tumor Margin(s)
			3.7 Peritumoral Edema
			3.8 Cyst(s)
			3.9 Necrosis
			3.10 Hemorrhage
			3.11 Calcification
			3.12 Hydrocephalus
			3.13 Metastases
			3.14 Diffusion Imaging
			3.15 Magnetic Resonance Spectroscopy (MRS)
		4 Differential Diagnosis of Medulloblastoma
		5 Postsurgery Imaging, Risk Stratification, and Treatment Planning
		6 Risk Stratification and Adjuvant Therapy
		7 Surveillance Imaging
		8 Radiogenomics of Medulloblastoma
		9 Future Directions
		10 Conclusions
		References
	Chapter 19: Multidisciplinary Management of Medulloblastoma: Consensus, Challenges, and Controversies
		1 Introduction
		2 Clinical Presentation
		3 Baseline Evaluation
		4 Neuro Imaging: Diagnostic and Prognostic Role
			4.1 Computerized Tomography (CT) Scans
			4.2 Magnetic Resonance Imaging (MRI)
		5 Treatment Strategies
			5.1 Initial Stabilization
			5.2 Surgery
			5.3 Pathological Examination of Specimen and Its Prognostic Implication
			5.4 Risk Stratification on the Basis of Imaging and Conventional Histopathology
		6 Molecular Pathology and Subgrouping
			6.1 WNT MB
			6.2 SHH Activated MB
			6.3 Non-WNT Non-SHH MB
			6.4 Impact of Molecular Subgrouping on Risk Stratification and Management
		7 Radiotherapy
			7.1 Evolution of Treatment Paradigms
				7.1.1 Average Risk Medulloblastoma
				7.1.2 High Risk Medulloblastoma
				7.1.3 Modulation of Dose, Volume and Fractionation
				7.1.4 Delay or Omission of Radiotherapy in Very Young Children
					Treatment-Related Toxicities
		8 Future Directions
			8.1 Experience with Deescalation: Cautionary Tale
			8.2 Newer Risk Adaptive and Molecular Classification Based Trials
				8.2.1 Experimental Therapies and Rehabilitation: The Brave New World
		References
Index