Cell-wide Metabolic Alterations Associated with Malignancy

1st Edition, Volume 543 - June 7, 2014
Imprint: Academic Press
Editors: Lorenzo Galluzzi, Guido Kroemer
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 8 0 1 3 2 9 - 8
eBook ISBN:
9 7 8 - 0 - 1 2 - 8 0 1 4 8 3 - 7

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods… Read more

Cell-wide Metabolic Alterations Associated with Malignancy

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This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods providing a a theoretical overview on metabolic alterations of cancer cells and a series of protocols that can be employed to study oncometabolism, in vitro, ex vivo and in vivo. Malignant cells exhibit metabolic changes when compared to their normal counterparts, owing to both genetic and epigenetic alterations. Although such a metabolic rewiring has recently been indicated as "yet another" general hallmark of cancer, accumulating evidence suggests that the metabolic alterations of each neoplasm rather represent a molecular signature that intimately accompanies, and hence cannot be severed from, all facets of malignant transformation.

Preface
- Acknowledgments
Chapter One: Methods to Measure Cytoplasmic and Mitochondrial Ca^2 + Concentration Using Ca^2 +-Sensitive Dyes
- Abstract
- 1 Introduction
- 2 Experimental Components and Considerations
- 3 Isolation and Differentiation of Naïve CD4⁺ CD25⁻ T Cells from Spleen and Lymph Nodes of Wild-Type and Orai1^−/− Mice
- 4 Mitochondrial Ca^2 + Measurement
- 5 Data Handling and Processing
- 6 Notes
- 7 Conclusion
- Acknowledgments
Chapter Two: Methods to Measure Intracellular Ca^2 + Fluxes with Organelle-Targeted Aequorin-Based Probes
- Abstract
- 1 Introduction
- 2 Experimental Components and Considerations
- 3 Application Examples
- 4 Aequorin Improvement by Fusion with GFP
- 5 Monitoring Ca^2 + Fluxes in Living Organism by BRET
- Acknowledgments
Chapter Three: Measuring Baseline Ca^2 + Levels in Subcellular Compartments Using Genetically Engineered Fluorescent Indicators
- Abstract
- 1 Introduction
- 2 Evolution of Recombinant Ca^2 + Probes and Organelle Targeting
- 3 Enhancing the Fluorophores
- 4 Modulating Ca^2 +-Binding Properties
- 5 Developing Organelle Targeting
- 6 Quantitative Considerations of Measurements of Organelle-Targeted Probes
- 7 Experimental Protocols
- 8 Semiquantitative Imaging of Basal Mitochondrial [Ca^2 +] with a Novel Mitochondria-Targeted GCaMP6m (2mtGCaMP6m)
- 9 Semiquantitative Imaging of Agonist-Induced Mitochondrial Ca^2 + Signals in Primary Neurons Using the Low-Affinity 2mtD4cpv Probe
- 10 Semiquantitative Imaging of Basal, Steady-State [Ca^2 +] in the ER
- 11 Concluding Remarks
Chapter Four: Autophagy and Autophagic Flux in Tumor Cells
- Abstract
- 1 Introduction
- 2 Autophagy Initiation and Autophagic Flux
- 3 Autophagic Flux and Degradation
- 4 Conclusion
- Acknowledgments
Chapter Five: Methods to Assess Autophagy In Situ—Transmission Electron Microscopy Versus Immunohistochemistry
- Abstract
- 1 Introduction
- 2 Transmission Electron Microscopy
- 3 Immunohistochemistry
- 4 Concluding Remarks
- Acknowledgments
Chapter Six: Methods to Measure the Enzymatic Activity of PI3Ks
- Abstract
- 1 Introduction
- 2 Analysis of PI3K Lipid Kinase Activity in Immunoprecipitate
- 3 Analysis of the Activity of Small Molecule PI3K Inhibitors
- 4 Analysis of the Localized Synthesis of PI3K Lipid Products
- 5 Materials
- Acknowledgments
Chapter Seven: Luciferase-Based Reporter to Monitor the Transcriptional Activity of the SIRT3 Promoter
- Abstract
- 1 Introduction
- 2 Protocol for Construction of Reporter Plasmid
- 3 Assaying SIRT3 Promoter Activity with the Construct
- 4 Verifying Reporter Results with Complementary Methods
- 5 Summary
- Acknowledgments
Chapter Eight: Metabolomic Profiling of Cultured Cancer Cells
- Abstract
- 1 Introduction
- 2 Cell Culture, Treatments, and Sample Collection
- 3 Chromatography, Mass Spectrometry, and Data Analysis
- 4 Concluding Remarks
- 5 Notes
- Acknowledgments
Chapter Nine: Pulsed Stable Isotope-Resolved Metabolomic Studies of Cancer Cells
- Abstract
- 1 Introduction
- 2 Experimental Procedures
- 3 Data Analysis
- 4 Outlook
Chapter Ten: Single-Cell Imaging for the Study of Oncometabolism
- Abstract
- 1 Introduction
- 2 Methods
- 3 MAMS Arrays
Chapter Eleven: Study of Cellular Oncometabolism via Multidimensional Protein Identification Technology
- Abstract
- 1 Introduction
- 2 Sample Protocol
- 3 Data Processing
- 4 Example Applications of MuDPIT
- 5 Summary
Chapter Twelve: In Vivo Quantitative Proteomics for the Study of Oncometabolism
- Abstract
- 1 Introduction
- 2 Sample Preparation
- 3 MS Sample Preparation
- 4 Instruments
- 5 MS Data Analysis
- 6 Additional Considerations
- 7 Conclusions
Chapter Thirteen: Metabolomic Profiling of Neoplastic Lesions in Mice
- Abstract
- 1 Introduction
- 2 Metabolic Profiling and Cancer Biology
- 3 Techniques Applied in Metabolomic Profiling of Neoplastic Lesions in Mice
- 4 Conclusion
Chapter Fourteen: Metabolomic Profiling of Tumor-Bearing Mice
- Abstract
- 1 Introduction
- 2 Mouse Models
- 3 Sample Preparation and Mass Spectrometry Analysis (Fig. 14.3)
- 4 Statistics
- 5 Biomarker Validation
- 6 Summary
Chapter Fifteen: Metabolomic Studies of Patient Material by High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy
- Abstract
- 1 MAS-NMR Spectroscopy as a Tool for Metabolic Profiling
- 2 MAS-NMR Versus Solution-State NMR Spectroscopy of Tumors
- 3 MAS-NMR Tumor Profiling in Clinical Studies
- 4 Metabolic Perturbations in Tumors Detected by MAS-NMR Spectroscopy
- 5 Protocol for the Analysis of Tissue by ¹H MAS-NMR Spectroscopy
Chapter Sixteen: Analysis of Metabolomic Profiling Data Acquired on GC–MS
- Abstract
- 1 Introduction
- 2 Spectrum Deconvolution
- 3 Metabolite Identification
- 4 Metabolite Quantification
- 5 Association Network Analysis
- 6 Metabolic Pathway Analysis
- Acknowledgments
Author Index
Subject Index

Lorenzo Galluzzi

Lorenzo Galluzzi is Assistant Professor of Cell Biology in Radiation Oncology at the Department of Radiation Oncology of the Weill Cornell Medical College, Honorary Assistant Professor Adjunct with the Department of Dermatology of the Yale School of Medicine, Honorary Associate Professor with the Faculty of Medicine of the University of Paris, and Faculty Member with the Graduate School of Biomedical Sciences and Biotechnology of the University of Ferrara, the Graduate School of Pharmacological Sciences of the University of Padova, and the Graduate School of Network Oncology and Precision Medicine of the University of Rome “La Sapienza”. Moreover, he is Associate Director of the European Academy for Tumor Immunology and Founding Member of the European Research Institute for Integrated Cellular Pathology. Galluzzi is best known for major experimental and conceptual contributions to the fields of cell death, autophagy, tumor metabolism and tumor immunology. He has published over 450 articles in international peer-reviewed journals and is the Editor-in-Chief of four journals: OncoImmunology (which he co-founded in 2011), International Review of Cell and Molecular Biology, Methods in Cell biology, and Molecular and Cellular Oncology (which he co-founded in 2013). Additionally, he serves as Founding Editor for Microbial Cell and Cell Stress, and Associate Editor for Cell Death and Disease, Pharmacological Research and iScience.

Affiliations and expertise

Assistant Professor of Cell Biology in Radiation Oncology, Department of Radiation Oncology, Weill Cornell Medical College, NY, USA

Guido Kroemer

Guido Kroemer got his M.D. in 1985 from the University of Innsbruck, Austria, and his Ph.D. in molecular biology in 1992 from the Autonomous University of Madrid, Spain. He is currently Professor at the Faculty of Medicine of the University of Paris Descartes/Paris V, Director of the INSERM research team ‘Apoptosis, Cancer and Immunity’, Director of the Metabolomics and Cell Biology platforms of the Gustave Roussy Cancer Campus, and Practitioner at the Hôpital Européen George Pompidou (Paris, France). He is also the Director of the Paris Alliance of Cancer Research Institutes (PACRI) and the Labex 'Immuno-Oncology'. Dr. Kroemer is best known for the discoveries that mitochondrial membrane permeabilization constitutes a decisive step in regulated cell death; that autophagy is a cytoprotective mechanism with lifespan-extending effects; and that anticancer therapies are successful only if they stimulate tumour-targeting immune responses. He is currently the most-cited cell biologist in Europe (relative to the period 2007-2013), and he has received the Descartes Prize of the European Union, the Carus Medal of the Leopoldina, the Dautrebande Prize of the Belgian Royal Academy of Medicine, the Léopold Griffuel Prize of the French Association for Cancer Research, the Mitjavile prize of the French National Academy of Medicine and a European Research Council Advanced Investigator Award.

Affiliations and expertise

INSERM Cordeliers Research Cancer Paris; Hopital Europeen Georges Pompidou; Universite Paris Descartes, France

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