Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses

Abstract

Background Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood. Results We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. Conclusions The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.

ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics

“…ACME is a cell dissociation approach that fixes cells while they are being dissociated. This has enormous advantages over enzymatic and mechanical approaches, as disrupting the cellular environment has effects on the cellular transcriptomic profiles that are only beginning to be realised [36][37][38]. Unlike nuclei extraction approaches, ACME preserves the cell cytoplasm, where most cellular mRNAs reside.…”

“…trypsin, papain or similar) or mechanical (e.g. dounce homogenisation) approaches [35], which introduce dissociation artefacts and cellular stress on the samples [36][37][38].…”

“…Dissociated cells are stripped from their extracellular context and washed, incubated, centrifuged, stained and often sorted by FACS while still alive, which changes their gene expression patterns [36][37][38].…”

Targeted single-cell RNA-seq identifies minority cell types of kidney distal nephron that regulate blood pressure and calcium balance

“…Cells sometimes suffer from stress responses during single-cell dissociation characterized by overexpression of immediate early genes, confounding the biological interpretation of scRNAseq data 42 . As seen in Supplementary Fig.…”

Dimension reduction and denoising of single-cell RNA sequencing data in the presence of observed confounding variables

“…As scRNA-seq is now ubiquitously applied to human patient samples [11][12][13] and large cohorts in population-wide expression quantitative trait locus (eQTL) studies [14][15][16], subject-level variables such as age, sex, and ancestry may confound the question of interest. On top of these subject level covariates are the technical differences between samples in their preparation and processing time, to which batch effects are usually attributed [17][18][19]. In addition, cell-level covariates such as library size, cell cycle, and mitochondrial RNA content, the latter often used as a proxy for cell quality [20], may need to be accounted for in a proper analysis [21][22][23].…”