Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis

Abstract

Mass cytometry is a single cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >104 relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies where investigators used mass cytometry to measure protein and phospho-protein expression in millions of cells, characterize rare cell types in healthy and diseased tissues, and reveal novel, unexpected cells. However, these advances largely occurred in studies of blood, lymphoid tissues, and bone marrow, since the cells in these tissues are readily obtained in single cell suspensions. This protocol establishes a primer for single cell analysis of solid tumors and tissues and has been tested with mass cytometry. The cells obtained from this protocol can be fixed for study, cryopreserved for long term storage, or perturbed ex vivo to dissect responses to stimuli and inhibitors.

High risk glioblastoma cells revealed by machine learning and single cell signaling profiles

“…As of February 2019, the median progression free survival (PFS) and overall survival (OS) after diagnosis were 6.3 and 13 months, respectively, similar to those observed in larger populations of patients undergoing standard therapy 27 . Resected tissues were immediately dissociated into single-cell suspensions as previously reported 38 . Cells were stained with a customized antibody panel for mass cytometry designed to capture the expression of known cell surface proteins, intracellular proteins, and phospho-signaling events (Figure 1, 2, Supplementary Table 2, Supplemental Information) that are critical for gliomagenesis and pathogenesis 32,39,40,30,34,35 .…”

“…The second round of data analysis used an equal number of each patient's glioblastoma cells to create a single, common t-SNE map of glioblastoma cell phenotypes across all patients (N = 131,880 cells; 4,710 cells x 28 patients). Prior to creating this common map, mass cytometry standardization beads were used to remove batch effects and to set the variance stabilizing arcsinh scale transformation for each channel following field-standard protocols 11,38,41 . This common t-SNE map was generated using 24 of 34 measured markers (Supplementary Table 2) and was used for automated analysis of risk stratifying cell subsets.…”

“…Surgical resection specimens of 28 IDH-wildtype glioblastomas collected at Vanderbilt University Medical Center between 2014 and 2016 were processed into single cell suspensions following an established protocol 38 . Only samples that were confirmed to be IDH-wildtype glioblastomas by standard pathological diagnosis were used.…”

“…Cells derived from patient samples were prepared as previously described 38 . A multi-step staining protocol was used, which included 1) live surface stain, 2) 0.02% saponin permeabilization intracellular stain, and 3) intracellular stain after permeabilization with ice-cold methanol ( Supplementary Table 2).…”

OMIP‐045: Characterizing human head and neck tumors and cancer cell lines with mass cytometry

“…As this panel contains markers expressed in 3 distinct cellular compartments (cell surface, cytoplasm and nucleus), multiple permeabilization protocols were tested to identify one that allowed for optimal detection of all parameters in all compartments. We tested the panel on 3 head and neck solid tumors disaggregated with a standardized protocol for obtaining single cells for mass cytometry analysis of human tissue and tumor biopsies . The protocol as well as information on staining patient samples can be found in the Supporting Information Materials in Section “Panel optimization on tumor biopsies.”…”