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

Abstract

Background Single-cell RNA sequencing (scRNAseq) is a powerful tool for studying complex biological systems, such as tumour heterogeneity and tissue microenvironments.However, the sources of technical and biological variation in primary solid tumour tissues and patient-derived mouse xenografts for scRNAseq, are not well understood. Here, we used low temperature (6°C) protease and collagenase (37°C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNAseq dataset comprising 128,481 cells from patient cancer tissues, patient-derived breast cancer xenografts and cancer cell lines. ResultsWe observe substantial variation in standard quality control (QC) metrics of cell viability across conditions and tissues. From FACS sorted populations gated for cell viability, we identify a sub-population of dead cells that would pass standard data filtering practices, and quantify the extent to which their transcriptomes differ from live cells. We identify a further subpopulation of transcriptomically "dying" cells that exhibit up-regulation of MHC class I transcripts, in contrast with live and fully dead cells. 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. We observe that the yield of cancer and non-cancer cell types varies between tissues and dissociation methods. ConclusionsThe method and conditions of tumour 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 sur-2 face immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with identification of such effects in dissociated scRNA-seq experiments.

Mapping hormone-regulated cell-cell interaction networks in the human breast at single-cell resolution

“…Programs in module 7 primarily consisted of ribosomal transcripts and genes involved in cellular respiration, whereas programs in module 8 consisted of stress response genes such as heat shock and chaperone proteins (Figures 2C and S7C). We speculate that module 8 represents an artifact of tissue processing rather than biologically meaningful transcriptional variation, since prior studies have identified a similar signature in dissociated solid tissues (O'Flanagan et al, 2019). However, one advantage of DECIPHER-seq is that it describes cells as a combination of activity programs rather than forcing cells into distinct clusters.…”

Somatostatin-expressing parafacial neurons are CO2/H+ sensitive and regulate baseline breathing

“…Genes related to cell cycle, stress response, the Y-chromosome, hemoglobin as well as ribosomal, mitochondrial, and the gene Xist were excluded from this list of highly variable genes prior to the computation of PCs. Briefly, the list of cell cycle genes was adapted from ( 9), using all annotated genes except those with labels "Other", "Function known but", and "Uncharacterized"), whereas the stress response genes were adapted from (49). The first 25 PCs To further analyze the neuronal populations, all cells were first classified as neuronal or nonneuronal using a simple two-state Gaussian mixture model (39), and neuronal cells were further classified as inhibitory or excitatory using a three-state Gaussian mixture model.…”

Shades of white: new insights into tissue‐resident leukocyte heterogeneity

“…O'Flanagan et al . [42] highlighted the potentially confounding effect of 37 °C tissue digestion, promoting cold active proteases for primary tissue gene expression analyses. Typically, the confounding variable of tissue manipulation is controlled by treating all samples equally; however, it is possible that some leukocytes are more vulnerable and reactive to manipulation and handling than others.…”

“…This is often achieved by subjecting the tissue sample to enzymatic digestion at 37 °C for periods of 1-2 h. At 37 °C, cells are transcriptionally and metabolically active, and will alter their gene expression profile, often inducing a stress response. O'Flanagan et al [42] highlighted the potentially confounding effect of 37 °C tissue digestion, promoting cold active proteases for primary tissue gene expression analyses. Typically, the confounding variable of tissue manipulation is controlled by treating all samples equally; however, it is possible that some leukocytes are more vulnerable and reactive to manipulation and handling than others.…”