Single-cell transcriptome conservation in a multispecies comparative analysis of fresh and cryopreserved insulinoma cell lines

Buishand, Floryne O; Chan, Phoebe Y K; Xia, Dong; Davison, Lucy J

Abstract

Background Insulinoma is the most common pancreatic neuroendocrine tumour in dogs and humans. The understanding of driving factors and critical survival genes in insulinomas is limited and overall survival is poor for canine and human malignant insulinoma. This study aimed to use single-cell RNA-sequencing to conduct a multispe-cies analysis of insulinoma cell lines to understand their single-cell transcriptomic landscape. Secondly, the impact of freeze-thawing on the pancreatic beta single-cell transcriptome was investigated, to determine whether cryo-archiving of primary insulinoma samples may be feasible in future studies. Methods Single-cell transcriptomic analysis was performed using fresh and cryopreserved multispecies insulinoma cell lines (canINS, CM, INS-1 and MIN6). R and Seurat were used to perform cell clustering and specific cluster marker genes were identified by the FindMarkers function. Metascape was used to identify statistically enriched pathways for specific cell clusters. Differentially expressed genes between fresh and cryopreserved single-cell transcriptome profiles, were defined as genes with a log2 fold change > 0.25 and a Bonferroni-adjusted P < 0.05, based on the Wil-coxon rank sum test. Results Based on the specific cell line single-cell transcriptome profiles, five or six cell clusters were constructed per cell line. All cell lines expressed neuroendocrine markers and additionally INS-1 and MIN6 displayed a gene signature indicative of mature/functional pancreatic islet/beta-cells. DEPTOR, BICC1, GHR, CCNB2, CENPA, LMO4, VANGL1, and L1CAM were identified as cross-species conserved insulinoma cluster marker genes. Little effect was found of cryopreservation and thawing on overall gene expression at the single-cell level in insulinoma cell lines: only 6 and 29 genes had a log2 fold change > 1 in cryopreserved versus fresh canINS and CM, respectively. Conclusions canINS, CM, INS-1 and MIN6 are all principally relevant as insulinoma models and the demonstrated differences in their single-cell transcriptomic profiles could aid researchers in selecting the appropriate cell lines for specific study objectives. Cross-species conserved insulinoma cluster marker genes were identified that harbour oncogenes and their involvement in insulinoma tumourigenesis should be investigated in future studies. The good comparability between cryopreserved and fresh insulinoma cells allows for inclusion of cryopreserved insulinoma patient samples in future studies, which allows for reduced assay-based variability.

Citation

Buishand, F. O., Chan, P. Y. K., Xia, D., & Davison, L. J. (2025). Single-cell transcriptome conservation in a multispecies comparative analysis of fresh and cryopreserved insulinoma cell lines. Veterinary Oncology, 2(1), https://doi.org/10.1186/s44356-025-00025-4

• Journal Article Type: Article
• Acceptance Date: Apr 7, 2025
• Online Publication Date: May 27, 2025
• Publication Date: May 27, 2025
• Deposit Date: May 27, 2025
• Publicly Available Date: Jun 5, 2025
• Journal: Veterinary Oncology
• Print ISSN: 3004-9814
• Electronic ISSN: 3004-9814
• Publisher: BioMed Central
• Peer Reviewed: Peer Reviewed
• Volume: 2
• Issue: 1
• DOI: https://doi.org/10.1186/s44356-025-00025-4
• Keywords: Canine; Cryopreservation; Pancreas; Pancreatic β-cells; Pancreatic neuroendocrine neoplasm; Neuroendocrine tumour model; Single-cell RNA-sequencing
• Additional Information: Received: 19 December 2024; Accepted: 7 April 2025; First Online: 27 May 2025; : ; : Not applicable.; : Not applicable.; : The authors declare no competing interests.