Dogs’ Insulin-Secreting Tumors Reveal Two Hidden Cancer Cell Types in First-of-Its-Kind Study

In a quiet operating theatre at a British veterinary hospital, tissue that would normally have been discarded after surgery instead became the basis of a scientific first. Researchers have now produced the single-cell RNA sequencing analysis of naturally occurring insulinoma in any species, dissecting, cell by cell, the rare and dangerous pancreatic tumours that cause dogs to collapse from dangerously low blood sugar. The study, published in Veterinary Oncology, captured the transcriptomic profiles of 5,532 individual cells drawn from two primary insulinomas and one metastatic lesion in two unrelated Boxer dogs, and in doing so revealed a hidden architecture that no bulk sequencing experiment could have exposed.

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