Phenotypic Characterization by Mass Cytometry of the Microenvironment in Ovarian Cancer and Impact of Tumor Dissociation Methods

Abstract

Improved molecular dissection of the tumor microenvironment (TME) holds promise for treating high-grade serous ovarian cancer (HGSOC), a gynecological malignancy with high mortality. Reliable disease-related biomarkers are scarce, but single-cell mapping of the TME could identify patient-specific prognostic differences. To avoid technical variation effects, however, tissue dissociation effects on single cells must be considered. We present a novel Cytometry by Time-of-Flight antibody panel for single-cell suspensions to identify individual TME profiles of HGSOC patients and evaluate the effects of dissociation methods on results. The panel was developed utilizing cell lines, healthy donor blood, and stem cells and was applied to HGSOC tissues dissociated by six methods. Data were analyzed using Cytobank and X-shift and illustrated by t-distributed stochastic neighbor embedding plots, heatmaps, and stacked bar and error plots. The panel distinguishes the main cellular subsets and subpopulations, enabling characterization of individual TME profiles. The dissociation method affected some immune (n = 1), stromal (n = 2), and tumor (n = 3) subsets, while functional marker expressions remained comparable. In conclusion, the panel can identify subsets of the HGSOC TME and can be used for in-depth profiling. This panel represents a promising profiling tool for HGSOC when tissue handling is considered.

Analysis of the Single-Cell Heterogeneity of Adenocarcinoma Cell Lines and the Investigation of Intratumor Heterogeneity Reveals the Expression of Transmembrane Protein 45A (TMEM45A) in Lung Adenocarcinoma Cancer Patients

“…Mistry et al reviewed the advantages of CyTOF in studying solid tumors, namely for the quantitative analysis of certain proteins in single cells [ 61 ]. Using CyTOF, CD24 was previously used to grade ovarian cancer [ 62 ], cytokeratin was applied in labeling melanoma cells [ 63 ], and EpCAM was recently selected for labeling of ovarian carcinoma cells [ 64 ]. However, our study is the first to show the single-cell CyTOF profile of the non-immune compartment of human primary lung adenocarcinoma for the expression of the 14 investigated markers.…”

Mass Cytometry for the Characterization of Individual Cell Types in Ovarian Solid Tumors

“…A prerequisite for the study of solid tissues is to optimize their disaggregation into viable single cells [1,9,10]. Despite the difficulties and inevitable significant depletion of some cell populations (e.g., stromal cells), we have acquired valuable and clinically relevant data [2,3,11].…”

CyTOF® for the Masses

“…It is important to optimize tissue digestion protocols to minimize cell debris (reduces staining quality) and maximize viability. Multiple published protocols exist for a variety of human and mouse tissues, including tumors ( 43 , 57 , 61 , 122 – 130 ). In general, when attempting to enrich cells, protocols should leave the cells of interest unmanipulated (e.g., negative selection procedures for magnetic bead separation or fluorescence-activated cell sorting/FACS).…”