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

Abstract

Mass cytometry aka Cytometry by Time-Of-Flight (CyTOF) is one of several recently developed multiparametric single-cell technologies designed to address cellular heterogeneity within healthy and diseased tissue. Mass cytometry is an adaptation of flow cytometry in which antibodies are labeled with stable heavy metal isotopes and the readout is by time-of-flight mass spectrometry. With minimal spillover between channels, mass cytometry enables readouts of up to 60 parameters per single cell. Critically, mass cytometry can identify minority cell populations that are lost in bulk tissue analysis. Mass cytometry has been used to great effect for the study of immune cells. We have extended its use to examine single cells within disaggregated solid tissues, specifically freshly resected tubo-ovarian high-grade serous tumors. Here we detail our protocols designed to ensure the production of high-quality single-cell datasets. The methodology can be modified to accommodate the study of other solid tissues.

Single-cell technologies in multiple myeloma: new insights into disease pathogenesis and translational implications

“…By improving protein preparation and isolation procedures, researchers have been able to decrease protein loss and perform quantitative proteomics sequences at a single-cell resolution. By combining the principles of MS with flow cytometry, mass cytometry by time of flight (CyTOF) uses metal isotope-labeled antibodies conjugated with specific molecules on the cell surface or inside cells, allowing for the examination of 100 specific proteins in single cells [ 121 ]. Based on immunohistochemistry with metal-labeled antibodies and CyTOF, imaging mass cytometry (IMC) has been developed [ 122 ].…”

Multiparameter single-cell proteomic technologies give new insights into the biology of ovarian tumors

“…However, not all heavy metals within this mass range can be used due to the challenging chemistries required for antibody tagging [ 35 ]. In a typical experiment, the majority of metal isotopes are linked to antibodies, but others serve as reagents to assay a variety of key cellular readouts such as DNA intercalation (to register a single cell), cell viability, cell cycle, cellular metabolism, enzymatic activity, hypoxia, and bar coding of samples [ 36 – 38 ]. Critically, the generation of large multi-parameter single-cell datasets necessitated the development of new computational tools which identify cell populations, e.g., clustering approaches to identify cell populations [ 39 ] or uniform manifold approximation and projection (UMAP) for single-cell resolution [ 40 ].…”