Related Experiment Video
Updated: Jan 5, 2026
Author Spotlight: Exploring Strategies for Successful Immune Response Against Tumors
Published on: August 16, 2024
Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved
Ciara H O'Flanagan1, Kieran R Campbell1,2,3, Allen W Zhang1,4,5
1Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada.
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.
Insights
Dissociation methods significantly impact single-cell RNA sequencing (scRNA-seq) data by inducing stress responses in cells. Using cold active protease minimizes this stress, improving data quality for cancer research.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Single-cell RNA sequencing (scRNA-seq) is crucial for analyzing complex biological systems like tumor heterogeneity.
- Understanding technical and biological variations in scRNA-seq data from solid tumors and xenografts is essential.
Purpose of the Study:
- To investigate how tissue dissociation methods affect cell viability and transcriptional profiles in scRNA-seq.
- To identify specific gene signatures associated with different dissociation conditions.
Main Methods:
- Compared scRNA-seq data from 155,165 cells across patient cancers, xenografts, and cell lines.
- Evaluated dissociation using low-temperature (6°C) protease versus standard collagenase (37°C).
- Analyzed variations in quality control metrics and identified stress-response gene expression.
Main Results:
- Tissue dissociation methods significantly alter cell viability and transcriptome states.
- Collagenase digestion at 37°C induces a conserved heat shock and stress response (512 genes, including FOS and JUN).
- Cold active protease at 6°C minimizes this stress response.
Conclusions:
- Tumor dissociation methods are tissue- and cell-type dependent, influencing scRNA-seq outcomes.
- Stress pathway interpretation in cancer scRNA-seq, including MHC class I, can be confounded.
- A core set of 512 genes is defined to help identify dissociation-induced effects in scRNA-seq experiments.
