Combining Mass Cytometry Data by CyTOFmerge Reveals Additional Cell Phenotypes in the Heterogeneous Ovarian Cancer Tumor Microenvironment: A Pilot Study

SIMPLE SUMMARY: High-grade serous ovarian cancer (HGSOC) has a dismal prognosis, but its tumor microenvironment (TME), which impacts disease progression and prognosis, is inadequately mapped. A better understanding of the complexity of the TME requires its characterization with multidimensional approaches that allow for simultaneous identification and categorization of the various cell populations. Here, we have performed in-depth profiling of the cellular TME by merging two high-dimensional single-cell datasets generated by mass cytometry of dissociated tumors, to determine whether combining datasets by the CyTOFmerge algorithm can provide more information on the HGSOC TME than separate dataset analyses. The results confirmed high interpatient heterogeneity and that such merging had the potential for identifying novel combinations of markers expressed on cells of the TME. These hypothesis-generating findings could help identify new combinations of expressed antigens and lead to improved mapping of the HGSOC TME and enhanced response to therapy. ABSTRACT: The prognosis of high-grade serous ovarian carcinoma (HGSOC) is poor, and treatment selection is challenging. A heterogeneous tumor microenvironment (TME) characterizes HGSOC and influences tumor growth, progression, and therapy response. Better characterization with multidimensional approaches for simultaneous identification and categorization of the various cell populations is needed to map the TME complexity. While mass cytometry allows the simultaneous detection of around 40 proteins, the CyTOFmerge MATLAB algorithm integrates data sets and extends the phenotyping. This pilot study explored the potential of combining two datasets for improved TME phenotyping by profiling single-cell suspensions from ten chemo-naïve HGSOC tumors by mass cytometry. A 35-marker pan-tumor dataset and a 34-marker pan-immune dataset were analyzed separately and combined with the CyTOFmerge, merging 18 shared markers. While the merged analysis confirmed heterogeneity across patients, it also identified a main tumor cell subset, additionally to the nine identified by the pan-tumor panel. Furthermore, the expression of traditional immune cell markers on tumor and stromal cells was revealed, as were marker combinations that have rarely been examined on individual cells. This study demonstrates the potential of merging mass cytometry data to generate new hypotheses on tumor biology and predictive biomarker research in HGSOC that could improve treatment effectiveness.