Immuno-oncology and immunotherapy Part C

1st Edition, Volume 191 - January 16, 2025
Editors: Lorenzo Galluzzi, Norma Bloy, Maud Charpentier
Language: English
Hardback ISBN:
9 7 8 - 0 - 4 4 3 - 2 9 6 2 0 - 8
eBook ISBN:
9 7 8 - 0 - 4 4 3 - 2 9 6 2 1 - 5

Immuno-oncology and immunotherapy, Part C, Volume 191 in the Methods in Cell Biology series, highlights new advances in the field, with this new volume presenting interesting c… Read more

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Immuno-oncology and immunotherapy, Part C, Volume 191 in the Methods in Cell Biology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variety of timely topics, including Extraction and quantification of histones from human cells, Expression and characterization of Phosphatidylserine-targeting antibodies for biochemical and therapeutic applications, ILC differentiation from HSCs in vitro, Methods to expand human Treg cells and assay their function, Monitoring rapid activation of human gamma/delta T cells by multicolor flow cytometry, Methods to induce T cell exhaustion in vitro, Ex vivo assessment of human neutrophil motility and migration, and much more.

Additional chapters focus on Flow cytometry-based monitoring of myeloid-derived suppressor cells in the peripheral blood of patients with solid tumors, Deciphering human blood and tumor neutrophil heterogeneity: Methods for isolation and assessing suppression of T-cell proliferation, Splenocyte anticancer citotoxicity assessment after prophylactic vaccination or drug treatment of tumor-bearing mice, Therapeutic treatment of tumor-bearing mice with drug-killed cancer cells: a method to confirm immunogenic cell death and assess its therapeutic effectiveness, and much more.

Immuno-Oncology and Immunotherapy - Part C
Cover image
Title page
Table of Contents
Series Page
Copyright
Contributors
Chapter One Simplified acid extraction and quantification of histones in human tumor cells
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Cells and reagents
2.3 Solutions
2.4 Equipment
2.5 Software
3 Methods
3.1 Cell culture (see Note 1)
3.2 Acid extraction for histones
3.3 Bradford protein quantification assay
3.4 Western blot
3.5 Protein level quantification
3.6 Normalizing protein quantification
4 Notes
5 Conclusions
Conflicts of interest
References
Chapter Two Expression, purification and characterization of phosphatidylserine-targeting antibodies for biochemical and therapeutic applications
Abstract
Keywords
1 Introduction
2 Materials
2.1 Disposable materials
2.2 Cell lines and reagents
2.3 Equipment
2.4 Software
3 Methods
3.1 Cloning of PS antibodies Bavituximab, 11.31, and 1N11
3.2 Expression of PS antibodies using Expi293 system
3.3 Supernatant harvest
3.4 Antibody purification
3.5 PS antibody characterization
4 Concluding remarks
Competing interests
References
Chapter Three In vitro ILC differentiation from human HSCs
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 Isolation of CD34+ HSCs from mobilized HD PB
3.2 CD34+ HSCs culture and differentiation toward ILCPs
3.3 Sorting of ILCPs
3.4 ILCPs culture and differentiation toward ILCs
4 Notes
5 Concluding remarks
Acknowledgments
References
Chapter Four Optimizing protocols for human regulatory T isolation, expansion, and characterization
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Cells and reagents
2.3 Equipment
2.4 Software
3 Methods (see Note 10)
3.1 Large scale generation of human Tregs
3.2 Phenotypic and functional characterization of human Treg
3.3 Data analysis
4 Notes
5 Concluding remarks
Conflicts of interest
References
Chapter Five Specific selection of stimulation-responsive γδ T-cells utilizing a short-term activation assay
Abstract
Keywords
1 Introduction
2 Material
2.1 Common disposables
2.2 Reagents
2.3 Buffers and cell culture media
2.4 Equipment
2.5 Software
3 Methods
3.1 Cell culture and treatment
3.2 Sample collection and preparation
3.3 Immunolabeling for flow cytometry
3.4 Acquisition
3.5 Gating
3.6 Cell sorting
3.7 Expansion of sorted activated γδ T cells
4 Notes
5 Concluding remarks
Acknowledgments
References
Chapter Six Generation of human and murine exhausted CD8+ T cells in vitro
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 Generation of exhausted human CD8+ T cells
3.2 Generation of exhausted mouse CD8+ T cells
3.3 Staining of human and mouse CD8+ T cells
3.4 Flow cytometry acquisition
3.5 Data analysis
4 Notes
5 Concluding remarks
References
Chapter Seven Ex vivo assessment of human neutrophil motility and migration
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 Neutrophil isolation and staining
3.2 Neutrophil motility assay
3.3 Neutrophil migration assay
4 Notes
5 Concluding remarks
Conflicts of interest
References
Chapter Eight Flow cytometry-based monitoring of myeloid-derived suppressor cells in the peripheral blood of patients with solid tumors
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 buffers and antibody mixes
3.2 Staining procedure and sample preparation
3.3 Data acquisition
3.4 Data analysis: Evaluation of the gate borders on the unstained control sample
3.5 Data analysis: Determination of the m-MDSCs and g-MDSCs
4 Notes
5 Concluding remarks
Conflict of interest
References
Chapter Nine Deciphering neutrophil heterogeneity in human blood and tumors: Methods for isolating neutrophils and assessing their effect on T-cell proliferation
Abstract
Keywords
1 Introduction
2 T-cell isolation and mature DC generation
2.1 PBMC isolation from whole blood
2.2 Separation of CD2+ and CD2− cells by rosetting with sheep erythrocytes
2.3 Isolation of T-cells from CD2+ cells by negative selection
2.4 Isolation of CD14+ cells from CD2− cells by positive selection
2.5 Control of cell purity by flow cytometry
2.6 Generation of mature DC
2.7 Freezing and thawing of cells
3 Neutrophil isolation
3.1 Neutrophil preparation from blood
3.2 Neutrophil preparation from solid tissue
3.3 Neutrophil isolation by fluorescence-activated cell sorting
4 Labeling of T-cells with fluorescent tracker dye
5 Preparation of proliferation assay
5.1 Preliminary notes
5.2 T-cell stimulation
5.3 Cells plating
6 Neutrophil-T-cell co-culture and analysis of T-cell proliferation
6.1 Proliferation analysis by flow cytometry
6.2 Data analysis
7 Concluding remarks
Acknowledgments
References
Chapter Ten Splenocytes antitumor cytotoxicity assessment after prophylactic vaccination or drug treatment of tumor-bearing mice
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 Prophylactic vaccination with cells treated with an ICD-inducing agent
3.2 In vivo antitumor treatment with ICD inductor
3.3 Splenocytes obtention
3.4 Cancer cell staining and co-culture
3.5 Flow cytometry acquisition
3.6 Data analysis
4 Notes
5 Concluding remarks
References
Chapter Eleven Use of drug-killed cancer cells: A method to assess the therapeutic effectiveness of immunogenic cell death
Abstract
Keywords
1 Introduction
2 Material
2.1 Common disposables
2.2 Cells and reagents
2.3 Equipment
3 Methods
3.1 Tumor establishment
3.2 Therapeutic treatment with drug-killed cancer cells
4 Notes
5 Concluding remarks
Competing interests
References
Chapter Twelve Single-cell RNA flow cytometry to assess intratumoral production of cytokines/chemokines
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Tissue and cell processing reagents
2.3 Flow cytometry reagents
2.4 Equipment
2.5 Software
3 Methods
3.1 Reagent preparation
3.2 Tumor collection and processing
3.3 Ex vivo stimulation for detection of cytokines/chemokines
3.4 Flow cytometry staining
3.5 Sample acquisition
3.6 Data analysis
4 Notes
5 Concluding remarks
Acknowledgments
References
Chapter Thirteen Multidimensional profiling of cancer microenvironments in FFPE tissues by imaging mass cytometry
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables (note 1)
2.2 Reagents (see note 1)
2.3 Equipment (note 1)
2.4 Software
3 Methods
3.1 FFPE tissue preservation and preparation (note 12)
3.2 Antibody selection and validation by immunohistochemistry (note 18)
3.3 Antibody conjugation (notes 23 and 24)
3.4 Evaluation of antibody conjugation
3.5 Standard IMC immunodetection protocol (note 30)
3.6 Titration of IMC antibodies (note 34)
3.7 Analysis
4 Notes
5 Concluding remarks
Acknowledgments
References
Chapter Fourteen Evaluation of lymphocyte infiltration into cancer spheroids by immunofluorescent staining and 3D imaging
Abstract
Keywords
1 Introduction
2 Materials
2.1 General material
2.2 Buffers for staining
2.3 Instruments
2.4 Software
3 Methods
3.1 Spheroid formation (day 0–3)
3.2 Co-culture with immune cells (day 3–5)
3.3 Immunofluorescence (day 5–8)
3.4 Imaging
3.5 Analysis
4 Notes
5 Concluding remarks
Competing interests
References
Chapter Fifteen Quantitative pre-clinical imaging of hypoxia and vascularity using MRI and PET
Abstract
Keywords
1 Introduction
2 Preliminary considerations
3 PET imaging of hypoxia
3.1 PET acquisition
3.2 PET images reconstruction and quantification
4 MRI imaging of oxygenation and tumor perfusion
4.1 BOLD/TOLD-MRI
4.2 DCE-MRI
5 Reproducibility and repeatability
6 Discussion and conclusions
Acknowledgments
References
Chapter Sixteen Retrovirus-based manufacturing of chimeric antigen receptor-modified T cells for cancer therapy research
Abstract
Keywords
1 Introduction
2 Material
2.1 Common disposables
2.2 Cells and reagents
2.3 Equipment
2.4 Software
3 Methods
3.1 Peripheral blood mononuclear cell isolation
3.2 Magnetic bead-based T cell sorting
3.3 Preparation of RetroNectin-coated wells
3.4 Retroviral-based T cell transduction
3.5 T cell expansion
3.6 Evaluation of transduction efficiency
4 Notes
5 Concluding remarks
Conflict of interest
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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