Immuno-oncology and immunotherapy Part A

1st Edition, Volume 189 - October 9, 2024
Editors: Lorenzo Galluzzi, Norma Bloy, Maud Charpentier
Language: English
Hardback ISBN:
9 7 8 - 0 - 4 4 3 - 2 9 6 2 2 - 2
eBook ISBN:
9 7 8 - 0 - 4 4 3 - 2 9 6 2 3 - 9

Immuno-Oncology And Immunotherapy – Part A Volume 189, in the Methods in Cell Biology series, continues the legacy of this premier serial with quality chapters authored by leade… Read more

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Immuno-Oncology And Immunotherapy – Part A Volume 189, in the Methods in Cell Biology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Specific chapters to this release include, Generation of transmitochondrial cybrids in cancer cells, Methods for flow cytometry analysis for tumor infiltrating cells functions and phenotype, Innovative 2D and 3D methods of tumor cells culture, and more.

Immuno-oncology and immunotherapy - Part A
Cover image
Title page
Table of Contents
Series Page
Copyright
Contributors
Chapter One Assessment of translation rate in leukemic cells and immune cells of the microenvironment by OPP protein synthesis assay
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Cells and reagents
2.3 Equipment
2.4 Software
3 Methods
3.1 Human PBMC isolation (see Note 20)
3.2 Preparation of treatment and staining stock solution (see Note 35)
3.3 Cell culture and treatments (see Note 35)
3.4 OPP incorporation
3.5 Zombie NIR live/dead staining
3.6 Antibody staining
3.7 OPP staining (see Note 51)
3.8 Sample acquisition by flow cytometry
3.9 Data analysis
4 Notes
5 Concluding remarks
Competing interests
References
Chapter Two Generation of transmitochondrial cybrids in cancer cells
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables (see Note 1)
2.2 Cells and reagents (see Note 1)
2.3 Equipment (see Note 1)
2.4 Software (see Note 1)
3 Methods
3.1 rho0 cell generation (see Notes 3 and 4)
3.2 Quantification of mtDNA by qPCR
3.3 Isolation of rho0 cell clones by limiting dilution
3.4 Expansion of donor cells, mitochondria isolation and purification
3.5 Mitochondrial Transfer (see Note 22)
3.6 Analysis of mitochondrial purity
4 Notes
5 Concluding remarks
Competing interests
References
Chapter Three Enzyme-linked ImmunoSpot (ELISpot) assay to quantify peptide-specific IFN-γ production by splenocytes in a mouse tumor model after radiation therapy
Abstract
Keywords
1 Introduction
2 Materials
2.1 Disposables
2.2 Mice, cell line
2.3 Reagents
2.4 Equipments
3 Experiment steps
3.1 Cell preparation
3.2 Engraftment
3.3 Treatment
3.4 Splenocyte isolation
3.5 ELISpot
3.6 ELISpot data analysis
4 Notes
5 Conclusion
Declaration of interests
Ethical statement
References
Chapter Four Tumor slice culture system for ex vivo immunotherapy studies
Abstract
Keywords
1 Introduction
1.1 Ex vivo systems to personalized cancer immunotherapy
1.2 Tumor slice culture system
2 Materials and methods
2.1 Cell culture and reagents
2.2 Animals
2.3 Preparation of tumor slices
2.4 Hematoxylin and eosin staining
2.5 AlamarBlue HS Viability Assay
2.6 Tumor slice dissociation
2.7 Flow cytometry
2.8 CFSE-labeled
3 Procedure and results
3.1 Tumor slice preparation
3.2 Assessment of viability and tumor slice structural integrity
3.3 Tumor slices depict the bulk tumor immune microenvironment
3.4 Tumor slices with immunostimulatory agents
3.5 Specific anti-tumor CD8 response in tumor slice cultures
4 Concluding remarks
Acknowledgments
References
Chapter Five Identification and analysis of alloreactive T lymphocytes from peripheral blood mononuclear cells
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Cells and reagents
2.3 Equipment
2.4 Software
3 Methods
4 Notes
5 Concluding remarks
Conflicts of interest statement
References
Chapter Six Assessment of pDCs functional capacity upon exposure to tumor-derived soluble factors
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Reagents
2.3 Equipment
2.4 Software
3 Methods
3.1 Preparation of tumor-derived cell culture supernatants
3.2 pDC Isolation
3.3 pDC seeding and treatment for ELISA
3.4 pDC seeding and treatment for flow cytometry
4 Notes
5 Concluding remarks
References
Chapter Seven Degranulation assay to evaluate NK cell natural and antibody-dependent cell-mediated cytotoxicity against A549 tumor spheroids
Abstract
Keywords
1 Introduction
2 Materials and equipment
2.1 Common disposables
2.2 Equipment
2.3 Reagents
3 Methodology
3.1 A549 cell culture
3.2 Quality pre-assessment of A549 spheroids
3.3 A549 spheroid formation
3.4 NK cell isolation
3.5 Co-culture of spheroids and NK cells: Degranulation assay
3.6 Flow cytometry
3.7 Analysis of NK cell degranulation
4 Concluding remarks
5 Notes
References
Chapter Eight Characterization of Natural Killer cells infiltrating irradiated murine tumors through flow cytometry
Abstract
Keywords
1 Introduction
2 Material
2.1 Equipment
2.2 Common disposables (see Note 1)
2.3 Reagents
2.4 Cells and animals
2.5 Software
3 Methods
3.1 Cells preparation
3.2 Establishment of CT26 tumors
3.3 Radiotherapy
3.4 Flow-cytometry assay
3.5 FACS analysis
4 NK cell characterization
5 Notes
6 Conclusion
Declaration of interests
References
Chapter Nine Flow cytometry detection and quantification of circulating leukocyte subpopulations in mice after brain irradiation
Abstract
Keywords
1 Introduction
2 Material
2.1 Disposables
2.2 Mice and reagents
2.3 Reagent preparation
2.4 Equipment
2.5 Software
3 Method
3.1 Radiation treatment plan preparation
3.2 Radiation treatment procedure
3.3 Sample collection
3.4 FACS staining procedure
3.5 Data acquisition by flow cytometry and gating
3.6 Data analysis and quantification
4 Concluding remarks/notes
Conflict of interest statement
References
Chapter Ten Flow cytometry-assisted analysis of phenotypic maturation markers on an immortalized dendritic cell line
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables (see Note 1)
2.2 Cells and reagents (see Note 1)
2.3 Equipment (see Note 1)
2.4 Software
3 Methods
3.1 In vitro de-immortalization and maturation of iniDCs
3.2 Staining
3.3 Acquisition and gating
4 Notes
5 Concluding remarks
Competing interests
References
Chapter Eleven Optimization and intra-assay validation of a multiparametric flow cytometric test for monitoring circulating TREGs
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Reagents
2.3 Equipment
2.4 Software
3 Methods
3.1 Safety warning
3.2 Preliminary procedures
3.3 Panel design
3.4 Conjugated antibody titration
3.5 Whole blood sample collection
3.6 Panel compensation set-up
3.7 Panel first trial
3.8 Panel validation
4 Concluding remarks
5 Notes
Competing interests
References
Chapter Twelve Cytofluorometric assessment of calreticulin exposure on CD38+ plasma cells from the human bone marrow
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables (see Note 1)
2.2 Cells and reagents (see Note 1)
2.3 Equipment (see Note 1)
2.4 Software (see Note 1)
3 Methods
3.1 Culture and isolation of mononuclear cells from human BM samples
3.2 Surface-expose CALR quantification
3.3 Acquisition and gating
3.4 Surface-exposed PS quantification
3.5 Acquisition and gating
4 Notes
5 Concluding remarks
References

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

Norma Bloy

Norma received her master’s degree in health biology from Paris Saclay. In 2013, she joined the laboratory of Dr. Guido Kroemer at the Centre de Recherche des Cordeliers (Paris, France) and at Gustave Roussy (Villejuif, France), the largest center for oncological patients in Europe. She worked on several projects linked to immunosurveillance, culminating with her obtaining her PhD in 2017 with an original work on “Immunogenic stress and death of cancer cells: Contribution of antigenicity vs adjuvanticity to immunosurveillance”. She then moved to Weill Cornell Medicine to join the program in radiation and immunity under the mentorship of Dr. Galluzzi. Her current research is focused on investigating resistance to immunotherapy in a mouse model of HR+ breast cancer.

Maud Charpentier

Maud Charpentier received her M.Sc. in Cellular Biology and her Ph.D. in Immunology from the University of Nantes, France. She chose to pursue an academic career and continued her postdoctoral training in the United States. She joined the Department of Radiation Oncology at Weill Cornell Medicine under the mentorship of Dr. Sandra Demaria. Maud has a long-standing interest in the anti-tumor immune response and its role in controlling cancer progression and treatment outcomes. Her research focuses on understanding the synergy between radiation therapy and immunotherapy in solid tumors, with the aim of overcoming resistance to treatment and developing innovative therapeutic approaches in preclinical models.

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