Stromal Expression Profiling Reveals Immune‐Driven Adaption to Malignancy in Canine Melanoma Subtypes

ABSTRACTCanine mucosal melanoma (CMM) is the most common oral malignancy in dogs and is significantly more aggressive than its cutaneous counterpart (CCM), yet the reasons for this disparity remain unclear. Cancer‐associated stroma (CAS) plays a crucial role in tumour progression, but a detailed understanding of CAS in canine melanoma is missing. To assess stromal reprogramming, 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 and RNA in situ hybridisation. Stromal reprogramming was evident in both subtypes but significantly more pronounced in CMM. Immune‐excluded tumours exhibited higher MC than desert/cold ones. MC strongly correlated with genes associated with B‐cells, T‐helper cells and CTLA4 in CCM, suggesting CAS reprogramming to depend on tumour malignancy. Finally, we identify an immune‐suppressive stromal signature in a subset of CMM characterised by the downregulation of key immune checkpoints and pathways. Together, these findings provide a solid foundation for understanding the role of CAS in canine melanoma, specific to cutaneous and mucosal subtypes.