Frozen but Faithful: Single-Cell Study Shows Cryopreserved Insulinoma Cells Keep Their Transcriptomic Identity

Insulinoma is a rare but formidable adversary. It is the most common pancreatic neuroendocrine tumour in both dogs and humans, yet its rarity, an estimated 30 cases per million dogs and just 1 to 3 cases per million people each year, has left researchers with a limited understanding of the genes that drive its growth and survival. In dogs, more than 95 percent of insulinomas are considered malignant because they almost invariably spread to abdominal lymph nodes and the liver, while only 5 to 16 percent of human insulinomas metastasise. Even with aggressive multimodal treatment combining surgery, glucocorticosteroids, diazoxide, somatostatin receptor ligands and cytotoxic chemotherapy, outcomes remain sobering: surgically treated dogs survive a median of only 14 months, and five-year survival for human patients with malignant disease ranges from 24 to 67 percent. Against this backdrop, a new study published in Veterinary Oncology offers both a sharper picture of insulinoma biology and a practical breakthrough for how future samples can be collected and stored.

A research team led by Floryne O. Buishand, Phoebe Y. K. Chan, Dong Xia and Lucy J. Davison at the Royal Veterinary College turned to single-cell RNA sequencing, a technique that reads the gene expression of individual cells rather than averaging signals across a bulk tumour sample. This matters because tumours are not uniform populations; they contain rare subpopulations that may underlie treatment resistance or drive metastasis. Bulk RNA sequencing of canine insulinomas had previously shown that early-stage primary tumours resemble normal pancreas, while late-stage tumours resemble metastatic lymph nodes, but such bulk approaches cannot resolve the internal diversity of a tumour. By profiling cells one by one, the team could detect distinct clusters within each sample and identify marker genes that would be diluted into invisibility in a bulk measurement.

The researchers applied this technology to four insulinoma cell lines spanning three species: canINS, a canine line established from a spontaneously occurring insulinoma; CM, a human insulinoma line; INS-1, a rat line derived from a radiation-induced tumour; and MIN6, a murine line from a transgenic mouse expressing the large T antigen under the rat insulin promoter. Each line carries its own history and its own limitations. CM has large chromosomal rearrangements involving the insulin gene that abolished insulin secretion from early passages. MIN6 and INS-1 both secrete insulin, but their artificial genetic backgrounds do not resemble spontaneous insulinoma. Canine insulinoma, sharing clinical and molecular features with the human malignant disease, has been proposed as a translational model, and earlier work showed that Notch pathway inhibition could target chemoresistant cancer stem cells in both canINS and CM cells.

Between 5,427 and 9,423 cells were analysed per cell line, with a median of 3,606 to 5,964 genes detected per cell and 43,042 to 68,959 mean reads per cell. Computational clustering in R and Seurat revealed five or six distinct cell clusters in each line. All four lines expressed neuroendocrine markers, including ENO2, MAP2 and NCAM1, confirming their neuroendocrine identity, but a striking split emerged in their differentiation status. INS-1 and MIN6 expressed the full panel of islet hormones and mature beta-cell markers tested, including insulin genes Ins1 and Ins2, glucagon, Mafa and Pdx1. In contrast, canINS and CM expressed only ACVR1C, and in the case of CM also MAFA, among the mature beta-cell markers, and neither expressed insulin. Instead, canINS and CM expressed genes normally activated during pancreatic development or in early endocrine progenitors, such as GATA4 and ISL1, which were the only lineage-specific pancreatic progenitor markers detected in canINS.

This differentiation landscape has direct practical value. Dedifferentiation of pancreatic beta cells in adherent monolayer culture, with loss of insulin-secretory capacity, is one of the main obstacles to building representative preclinical insulinoma models. Notably, canINS cells have previously regained insulin expression and secretion when grown in non-adherent conditions, suggesting that their epigenetic memory remains intact and that the absence of insulin expression in monolayer culture does not disqualify a cell line as an insulinoma model. The study’s single-cell maps now allow researchers to make informed choices: INS-1 and MIN6 for questions requiring mature, insulin-producing beta-cell features, and canINS and CM, with their spontaneous-tumour origins, for studies closer to the clinical disease.

The team also examined epithelial-mesenchymal status, a critical axis in cancer metastasis. When cultured in monolayers, pancreatic beta cells undergo epithelial-mesenchymal transition, becoming highly proliferative cells that can re-differentiate into insulin producers. All four lines expressed the epithelial or ductal markers ANXA4, CDH1 and SLC4A4, but vimentin, a mesenchymal marker, was detected in all lines except MIN6 and was significantly higher in canINS and CM than in INS-1. This means canINS and CM contain substantial populations of hybrid epithelial/mesenchymal cells, a state previously observed in prostate, lung and colorectal cancer lines. Hybrid E/M cells are thought to migrate collectively as clusters of circulating tumour cells, enhancing metastatic potential compared with individually migrating cells, making canINS and CM the preferred models for studying how insulinoma cells acquire metastatic behaviour.

Perhaps the most tantalising discovery came from the cross-species comparison. Because single-cell analysis exposes genes robustly expressed in subpopulations that bulk methods would miss, the researchers catalogued the top ten marker genes for each of the 23 clusters identified across the four lines. Nineteen of these clusters were characterised by genes consistently expressed across all cell lines. Filtering for unique cluster markers with plausible cancer-related functions that were conserved across species and expressed in all four lines yielded eight candidate genes: DEPTOR, BICC1, GHR, CCNB2, CENPA, LMO4, VANGL1 and L1CAM. Several of these have documented pro-tumour roles in other cancers. BICC1 drives pancreatic cancer stemness and chemoresistance, L1CAM promotes perineural invasion in pancreatic cancer, CENPA has been implicated in chromosomal instability of pancreatic neuroendocrine tumours, and LMO4 is overexpressed in late-stage pancreatic cancer.

Among these candidates, the growth hormone receptor gene GHR stands out for its translational potential. GHR expression has previously been demonstrated by immunohistochemistry in canine primary insulinomas and their metastases, and growth hormone and insulin-like growth factor 1 expression were increased in metastases compared with primary tumours. This has led to the hypothesis that targeting the GH/IGF-1 axis might inhibit insulinoma proliferation and prevent micrometastatic outgrowth after surgery. Pegvisomant, currently the only clinically available GHR antagonist, is FDA approved for acromegaly, and promising preclinical results have been obtained with pegvisomant and a variant called compound G against pancreatic cancer xenografts, particularly in combination with gemcitabine. Because GHR emerged as a cross-species conserved cluster marker in all four insulinoma lines, the study provides a direct rationale for testing GHR inhibition in these models.

The second major achievement of the study addresses a logistical bottleneck that has long hampered single-cell research on rare tumours. Ideally, fresh patient samples are dissociated into single-cell suspensions immediately upon retrieval to prevent ischaemia-related gene expression changes and RNA degradation, then loaded onto droplet-based platforms within minutes. For rare diseases, where samples arrive sporadically and often far from sequencing facilities, this workflow is difficult to sustain. Cryopreservation offers an obvious solution, but only if freezing does not distort the transcriptomic picture. To test this, the team froze canINS and CM cells in 90 percent fetal calf serum with 10 percent dimethyl sulfoxide, stored them at minus 80 degrees Celsius for four weeks, thawed them, and sequenced them alongside fresh counterparts.

The verdict was emphatically reassuring. Cryopreserved samples yielded five canINS clusters and seven CM clusters, and most clusters matched their fresh counterparts closely, sharing five to nine of the top ten marker genes. Merged datasets showed near-equal distribution of fresh and frozen cells across all clusters, and expression of neuroendocrine, epithelial-mesenchymal, islet hormone and progenitor markers was fully maintained. Differential expression analysis identified 1,395 genes changed in cryopreserved canINS, but only six exceeded a log2 fold change of one, with BTF3, MEI4, NUPR1, FOS and SLC25A6 upregulated; in CM, 1,484 genes were differentially expressed and 29 exceeded the threshold, with TNFRSF12A, CKS1B and PTTG1 upregulated. Only FOS upregulation had previously been linked to DMSO cryopreservation in other cell types. These modest perturbations, the authors note, will need consideration but do not compromise data quality. The message for the field is twofold: insulinoma researchers now have a validated, species-spanning toolkit of cell lines matched to specific experimental questions, and banks of cryopreserved patient samples can finally enter single-cell studies without sacrificing fidelity, reducing assay-based variability and opening the door to larger, multi-centre studies of one of veterinary and human medicine’s most stubborn tumours.

Subject of Research: Single-cell transcriptomic analysis of fresh and cryopreserved multispecies insulinoma cell lines

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

Article References: 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), Article 14. https://doi.org/10.1186/s44356-025-00025-4

Image Credits: AI Generated

DOI: 10.1186/s44356-025-00025-4

Keywords: insulinoma, single-cell RNA sequencing, cryopreservation, canine cancer, pancreatic neuroendocrine tumour, beta cells, transcriptomics, comparative oncology, GHR, oncogenes, cell lines, veterinary oncology