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Molecular and Immunohistochemical Characterization of the Immune Landscape of Spontaneous, Translationally‐Relevant Canine Cancers

Recent developments in human oncology have dramatically increased the relevance of cancer immunotherapy as a new treatment modality, with therapies such as checkpoint inhibitors providing significant clinical benefit to a subset of patients with certain tumor types. However, further advances in the field of immuno‐oncology will require continued identification of predictors of patient response to, and pre‐clinical investigations of, rational therapeutic combinations of these drugs. Current mouse tumor models have intrinsic deficiencies in predicting microenvironmental and molecular determinants of response to immunotherapy. In contrast, naturally occurring tumors in dogs have significant potential to inform human immunotherapy studies due to their spontaneous development under the selective pressure of an intact immune system, allowing for a natural evolution of the processes of immune evasion, therapy resistance, and metastasis. Yet at present, our understanding of the basic immune landscape of common canine tumors is significantly lacking. Thus, we sought to quantify the density of tumor‐infiltrating leukocytes (TILs) in four translationally relevant canine tumors (n = 115), including melanoma (CML), soft tissue sarcoma (STS), osteosarcoma (OSA) and bladder carcinoma (TCC), via immunohistochemical labeling and quantitative image analysis of CD3+ T cells, MAC387+ myeloid cells, and FoxP3+ Tregs. Immune gene expression profiling by microarray analysis was performed on a subset of canine melanoma and bladder carcinomas (n = 16) to characterize the functional orientation of TILs and investigate potential immune gene signatures and immunotherapeutic targets associated with tumors of high‐ and low‐density immune infiltrates. The range of tumor‐infiltrating CD3+ T cells varied significantly in all tumor types (CML=20–1,302; STS=0–288, OSA=1–793, and TCC=1–695 cells/mm2), suggesting the presence of immunologically “hot” and “cold” tumors across all histo‐types. Overall, the mean density of both tumor‐infiltrating CD3+ T cells and MAC387+ myeloid cells was significantly higher in CML and metastatic OSA as compared to all other evaluated histo‐types. For all tumors, a significant positive correlation existed between CD3+ T cell and MAC387+ myeloid cell density. Gene expression analysis demonstrated enrichment of gene sets associated with type I interferon response, plasmacytoid dendritic cells, and molecules associated with T cell and antigen‐presenting cell co‐stimulation in high T cell infiltrated vs. low T cell infiltrated bladder tumors. In conclusion, these data provide a preliminary foundation for determining the suitability of, as well as potential rationale for, the design of immunotherapeutic investigations in the spontaneous canine cancer model.