Stromal expression profiling reveals immune-driven adaption to malignancy in canine melanoma subtypes

Canine mucosal melanoma (CMM) is the most common oral malignancy in dogs and exhibits significantly higher aggressiveness than its cutaneous counterpart (CCM), yet the reasons for this disparity remain unclear. The influence of cancer-associated stroma (CAS) on tumour progression is well-recognized, but a detailed understanding of CAS in canine melanoma is missing. To comprehensively assess stromal reprogramming in canine melanoma, we analysed CAS from 21 CMM, 14 CCM and normal stroma from 10 skin and 9 oral mucosa samples by laser-capture microdissection followed by RNA sequencing. Results were assessed in relation to subtypes, prognostic factors including mitotic count (MC), ulceration, necrosis, pigmentation, and immune cell infiltration (CD3, CD20 and CD68), scored using immunohistochemistry. While stromal reprogramming was evident in both subtypes, it was significantly more pronounced in CMM. Furthermore, MC strongly correlated with stromal gene expression in both subtypes, suggesting CAS reprogramming to depend on tumour malignancy. Comparison of subtypes revealed specific downregulation of key regulators of leukocyte activation and proliferation in mucosal CAS, supporting a role for impaired immune control in CMM aggressiveness. CMM presented with significantly more CD20 infiltrated (excluded or hot) cases than CCM. In CMM, CD20 infiltration was significantly associated with MC, with excluded cases displaying highest MC. Finally, we identify an immune-suppressive stromal signature in a subset of CMM that influences tumour aggressiveness and predicts overall survival in human melanoma patients. Together, these findings provide a solid foundation for understanding the role of CAS in canine melanoma, with relevance for the human disease. Clinical FFPE specimen of canine mucosal and cutaneous melanoma. Comparison of gene expression in cancer-associated stroma compared to non-matched cutaneous or mucosal normal stroma.