Abstract
Tissue-resident and recruited immune cells are essential mediators of natural and therapy-induced immunosurveillance of liver neoplasia. This idea has been recently reinforced by the clinical approval of immune checkpoint inhibitors for the immunotherapy of hepatocellular carcinoma and cholangiocarcinoma. Such research progress relies on the in-depth characterization of the immune populations that are present in pre-neoplastic and neoplastic hepatic lesions. A convenient technology for advancing along this path is high-dimensional cytometry.
In this chapter, we present a protocol to assess the subtype and differentiation state of hepatic lymphocyte populations by multicolor immunofluorescence staining and flow cytometry. We detail the steps required for viability assessment and immune cell phenotyping of single-cell suspensions of liver cells by means of surface and intracellular staining of more than a dozen markers of interest. This protocol does not require prior removal of debris and dead cells and allows to process multiple samples in parallel. The procedure includes the use of a fixative-resistant viability dye that allows cell fixation and permeabilization after cell surface staining and before intracellular staining and data acquisition on a flow cytometer. Moreover, we provide a panel of fluorochrome-labeled antibodies designed for the characterization of lymphocytic subsets that can be adapted to distinct experimental settings. Finally, we present an overview of the post-staining pipeline, including data acquisition on a flow cytometer and tools for post-acquisition analyses.
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Acknowledgments
J.G.P. is supported by the SIRIC Cancer Research and Personalized Medicine (CARPEM); Multi-Organism Institute (ITMO) Aviesan Cancer (National Alliance for Life Sciences and Health), Institut National du Cancer (INCa), and Fondation pour la Recherche Médicale (FRM). M.C.M. is supported by the SIRIC Cancer Research and Personalized Medicine (CARPEM). G.K. is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) – Projets blancs; AMMICa US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; European Research Council Advanced Investigator Grant “ICD-Cancer,” FRM; a donation by Elior; Equipex Onco-Pheno-Screen; European Joint Programme on Rare Diseases (EJPRD); European Research Council (ICD-Cancer), European Union Horizon 2020 Projects Oncobiome and Crimson; Fondation Carrefour; Institut National du Cancer (INCa); Institut Universitaire de France; LabEx Immuno-Oncology (ANR-18-IDEX-0001); a Cancer Research ASPIRE Award from the Mark Foundation; the RHU Immunolife; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and SIRIC Cancer Research and Personalized Medicine (CARPEM).
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Pérez-Lanzón, M. et al. (2024). Flow Cytometry Assessment of Lymphocyte Populations Infiltrating Liver Tumors. In: Kroemer, G., Pol, J., Martins, I. (eds) Liver Carcinogenesis. Methods in Molecular Biology, vol 2769. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3694-7_10
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DOI: https://doi.org/10.1007/978-1-0716-3694-7_10
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