The recent introduction of imaging mass cytometry has considerably advanced the potential to simultaneously obtain information on phenotypes, their localization within a tissue, and to map cellular interactions. Mass cytometry makes use of metal isotopes conjugated to antibodies of interest, in contrast to flow cytometry and immunofluorescence techniques that rely on fluorescent dyes. 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. Keywords: imaging mass cytometry, cancer microenvironment, immunophenotyping, CyTOF, cancer immunity, immunotherapy Introduction Technologies that support the high dimensional analysis of biological systems are essential in scientific research and have become increasingly relevant in clinical contexts. For instance, the advent of T cell checkpoint blockade therapies for cancer treatment has revitalized the field of cancer immunotherapy but also introduced an urgent need for the discovery of biomarkers that guide patient selection for therapies (1, 2). Furthermore, recent works making use of single-cell platforms based on RNA sequencing and mass cytometry have delivered a wealth of data revealing previously unappreciated cell subsets and novel functionalities (3C5). Nevertheless, most immunophenotyping techniques are held back by the lack of spatial resolution, limitations in the number of targets that can be visualized simultaneously, or cumbersome protocols. Methodologies such as flow cytometry can be employed to analyze multiple markers but are insufficient to chart the vast spectrum of immune cells in an unbiased manner (6). Single-cell mass cytometry overcomes this limitation by currently allowing the simultaneous analysis of ~40 cellular markers. However, it also lacks spatial information, failing to reveal tissue context and cellular interactions which are extremely relevant in physiological and disease states (7C9). Conversely, multispectral fluorescence imaging provides spatial context but is limited to few markers and is thus EFNA1 best suited to investigate specific research questions in large cohorts (10, 11). The recent introduction of imaging mass cytometry has considerably CYP17-IN-1 advanced the potential to simultaneously obtain information on phenotypes, their localization within a tissue, and to map cellular interactions. Mass cytometry makes use of metal isotopes conjugated to antibodies of interest, in contrast to flow cytometry and immunofluorescence techniques that rely on fluorescent dyes. The metal isotopes are distinguished by mass in a time-of-flight mass spectrometer and, thus, the number of markers that can be detected simultaneously is not limited by spectral overlap. Since its discovery in ’09 2009 (12), mass cytometry continues to be requested the immunophenotyping of cancers microenvironments successfully. It has accelerated the breakthrough of new immune system cell subsets, the evaluation of potential relationship and biomarkers of immune-phenotypical adjustments to healing final results (5, 13C15). Imaging mass cytometry employs a higher resolution laser that’s coupled towards the mass cytometer (16). Successive ablations of little portions of tissues (~1 m2) are examined by CyTOF (Cytometry Time-Of-Flight) thus quantifying the current presence of CYP17-IN-1 steel isotopes per section of tissues. This data is normally reconstructed into an artificial multilayer picture producing a wide and comprehensive summary of proteins appearance in situ. Imaging mass cytometry may be employed for imaging up to 40 markers in various tissues CYP17-IN-1 resources (e.g., snap-frozen, FFPE), however the combination of a lot of antibodies in the same test.
