Canine malignant insulinoma is a functional neuroendocrine tumour of pancreatic beta cells: a cancer that not only grows and spreads but keeps manufacturing insulin as though it were still serving the body. Middle-aged and older dogs are typically affected, with Boxers, German shepherds, Labrador retrievers and West Highland white terriers among the predisposed breeds. Affected animals may show increased appetite and weight gain, weakness between meals or after exercise, and, as the disease advances, collapse and hypoglycaemic seizures. Diagnosis rests on documenting clinical signs alongside inappropriately high serum insulin concentrations in the face of low blood glucose, often supported by imaging of a pancreatic nodule. Surgical excision is the mainstay of treatment but is rarely curative, because functional metastases frequently seed new insulin-producing lesions and bring the clinical signs roaring back.

The prognosis is sobering. The largest study of canine insulinoma to date, covering 93 dogs, reported a median survival of eight months with medical management and twenty months with surgery. Adjunctive drugs such as prednisolone, octreotide and diazoxide aim to blunt insulin’s effects rather than attack the tumour itself, while small studies of beta-cell-toxic streptozotocin and the tyrosine kinase inhibitor toceranib have hinted at benefit without establishing clear efficacy. This therapeutic vacuum is precisely what makes a high-resolution map of the tumour’s cellular landscape so valuable: it offers both candidate drug targets and potential biomarkers to identify which patients are at risk of metastasis before it happens.

Single-cell RNA sequencing overcomes the fundamental limitation of bulk RNA-sequencing, which averages gene expression across thousands of heterogeneous cells and thereby blurs the very distinctions that matter most. By profiling individual cells, researchers can identify malignant sub-populations, reconstruct the tumour microenvironment, and infer the communication channels between cancer and immune cells. Until now, only human and mouse pancreatic neuroendocrine tumours had been examined this way, and the mouse work relied on an experimentally induced model rather than spontaneous disease. The new study is the first to apply the technique to naturally occurring insulinoma in any species, using tissue surplus to diagnostic requirements after planned therapeutic surgery, with owner consent and no influence on clinical management.

The technical execution was meticulous. Fresh tumour tissue was transported in cold buffer within two hours of excision, minced, enzymatically digested with Liberase TL, and processed into single-cell suspensions before library preparation on the 10X Chromium system and sequencing on an Illumina HiSeq4000. Reads were aligned to the canine reference genome CanFam3.1, and clustering and differential expression analyses were performed in Seurat, with cell types assigned through canonical marker genes and automatic classifiers. Quality control demanded more than fifty detected genes per cell and less than ten percent mitochondrial read content, yielding an average of 123,536 reads per cell and transcripts from a median of 1,010 genes per cell.

The headline discovery is that all three tumour samples contained two transcriptionally distinct populations of insulin-expressing cancer cells, separated by roughly 8,000 differentially expressed genes. One population, dubbed INS+ FOS+ EGR1+ TP53+ and abbreviated INS+, maintains expression of the tumour suppressor TP53, the transcription factor EGR1, and the AP-1 component FOS. The second, INS+ FOSlow, shows strong downregulation of these genes and of around sixty other tumour suppressors, including NF1, ARID1A and CDKN1A, together with a 23.5-fold reduction in DUSP1, a regulator of MAPK signalling. Pathway analysis of the genes separating the two populations flagged the MAPK, mTOR, PI3K-AKT, Notch, Wnt and p53 signalling pathways, along with cellular senescence and apoptosis, all of them famous for their deregulation in cancer.

Despite their deep differences, both malignant populations retained the molecular identity of their beta-cell origin, expressing insulin-related genes such as IAPP, PCSK2, NKX2-2 and SLC30A8, and the chromogranin and secretogranin family markers CHGA, CHGB, SCG2, SCG3, SCG5 and SCGN that define neuroendocrine tumours. Strikingly, one of the very few genes ubiquitously expressed and significantly upregulated in both insulin-expressing populations was COX7A2L, elevated more than twenty-fold, with a mean of twenty-seven-fold, over other captured cells. The gene encodes a subunit of cytochrome c oxidase involved in mitochondrial respiration, has been linked to poor prognosis in pancreatic, ovarian and breast cancers, and its knockdown reduces tumour growth in mice, making it an obvious candidate for further investigation in insulinoma.

Copy number analysis added an evolutionary dimension. The INS+ FOSlow population carried more chromosomal alterations than the INS+ population, and in the patient with more advanced disease, these cells showed frequent deletion of chromosomes 13, 20 and 29 and amplification of 11, 14 and 15, alterations that were rare in the other population and largely absent in the less-affected patient. This pattern suggests that whole-chromosome copy number changes are not the initiating event in these tumours but accumulate as the disease progresses, potentially serving as a marker of advanced disease state. Meanwhile, comparisons between the two primary tumours revealed far fewer differences, roughly 600 differentially expressed genes, than existed between the two cancer cell populations within each patient, including genes such as TMSB4X, proposed as a cancer prognostic marker and reportedly overexpressed in toceranib-resistant liver cancer cells, and CLTRN, a stimulator of beta cell replication that was upregulated in the patient with more advanced disease.

Two findings from the metastatic lesion were genuinely unexpected. First, the metastasis contained all the cell types represented in the primary tumour, including immune populations, rather than presenting as a pure mass of neoplastic endocrine cells, a pattern consistent with locally invasive spread rather than lymph node metastasis. Second, and more startling, both insulin-expressing populations in the metastasis showed more than twenty- to seventy-fold upregulation of exocrine pancreatic genes, including CLPS, PRSS2, PRSS and CTRC, markers normally associated with digestive enzyme production and pancreatitis. For a tumour of endocrine origin, this is deeply atypical, and the authors suggest these genes may mark metastatic transformation, invasion or de-differentiation, though their significance remains to be determined.

The immune analysis completed the picture. The tumours harboured effector and memory T cells, naive CD4-positive T cells, B lymphocytes, and two macrophage populations distinguished by MS4A7 and S100A12 expression, with the greatest inter-patient differences appearing in the macrophage cluster. Cell communication analysis using CellChat identified significant interactions between cancer and immune cells, most notably CD40LG expressed by both insulin-expressing tumour populations engaging CD40 on infiltrating B cells, an axis implicated in angiogenesis and described as a prognostic indicator in breast cancer, and an APP interaction with the TREM2-TYROBP complex on macrophages. Insulin-expressing cells also showed upregulation of inflammatory response genes including C15orf48 and TRAF3IP2, while insulin-expressing tumour cells had the highest proportion of cells in S phase of any cell type examined, with the peak in the metastasis, underscoring why DNA replication remains a therapeutic target. The authors are candid about the study’s limits: two dogs, one breed, and no matched blood or healthy beta-cell reference data. Yet the demonstration that single-cell data can be recovered from veterinary surgical material surplus to diagnostic needs opens a translational window, since canine insulinoma shares histopathological features with human pancreatic neuroendocrine tumours, and any future therapy would need to hit both malignant sub-populations simultaneously, in dogs and potentially in people.

Subject of Research: Single-cell transcriptomic profiling of canine insulinoma and its tumour microenvironment

Article Title: Single-cell transcriptomic analysis of canine insulinoma reveals distinct sub-populations of insulin-expressing cancer cells

Article References: Wallace, M. D., Herrtage, M. E., Gostelow, R., Owen, L., Rutherford, L., Hughes, K., Denyer, A., Catchpole, B., O’Callaghan, C. A., & Davison, L. J. (2025). Single-cell transcriptomic analysis of canine insulinoma reveals distinct sub-populations of insulin-expressing cancer cells. Veterinary Oncology, 2(1), Article 13. https://doi.org/10.1186/s44356-025-00026-3

Image Credits: AI Generated

DOI: 10.1186/s44356-025-00026-3

Keywords: single-cell RNA sequencing, canine insulinoma, pancreatic neuroendocrine tumour, beta cells, tumour microenvironment, TP53, EGR1, COX7A2L, metastasis, copy number alteration, tumour immunology, veterinary oncology

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