A 40-Marker Panel for High Dimensional Characterization of Cancer Immune Microenvironments by Imaging Mass Cytometry

Multiplex immunophenotyping technologies are indispensable for a deeper understanding of biological systems. Until recently, high-dimensional cellular analyses implied the loss of tissue context as they were mostly performed in single-cell suspensions. The advent of imaging mass cytometry introduced...

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Bibliographic Details
Published in:Frontiers in immunology Vol. 10; p. 2534
Main Authors: Ijsselsteijn, Marieke E., van der Breggen, Ruud, Farina Sarasqueta, Arantza, Koning, Frits, de Miranda, Noel F. C. C.
Format: Journal Article
Language:English
Published: Switzerland Frontiers Media S.A 29.10.2019
Subjects:
cancer immunity
cancer microenvironment
CyTOF
imaging mass cytometry
Immunology
immunophenotyping
immunotherapy
imaging mass cytometry
cancer immunity
cancer microenvironment
immunophenotyping
CyTOF
immunotherapy
ISSN:1664-3224, 1664-3224
Online Access:Get full text
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Summary:Multiplex immunophenotyping technologies are indispensable for a deeper understanding of biological systems. Until recently, high-dimensional cellular analyses implied the loss of tissue context as they were mostly performed in single-cell suspensions. The advent of imaging mass cytometry introduced the possibility to simultaneously detect a multitude of cellular markers in tissue sections. This technique can be applied to various tissue sources including snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissues. However, a number of methodological challenges must be overcome when developing large antibody panels in order to preserve signal intensity and specificity of antigen detection. We report the development of a 40-marker panel for imaging mass cytometry on FFPE tissues with a particular focus on the study of cancer immune microenvironments. It comprises a variety of immune cell markers including lineage and activation markers as well as surrogates of cancer cell states and tissue-specific markers (e.g., stroma, epithelium, vessels) for cellular contextualization within the tissue. Importantly, we developed an optimized workflow for maximum antibody performance by separating antibodies into two distinct incubation steps, at different temperatures and incubation times, shown to significantly improve immunodetection. Furthermore, we provide insight into the antibody validation process and discuss why some antibodies and/or cellular markers are not compatible with the technique. This work is aimed at supporting the implementation of imaging mass cytometry in other laboratories by describing methodological procedures in detail. Furthermore, the panel described here is an excellent immune monitoring tool that can be readily applied in the context of cancer research.
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Edited by: Giovanna Schiavoni, National Institute of Health (ISS), Italy
Reviewed by: Daniel Olive, Aix Marseille Université, France; Jan Joseph Melenhorst, University of Pennsylvania, United States
This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology
ISSN:1664-3224
1664-3224
DOI:10.3389/fimmu.2019.02534