Simple SummaryThis study aimed to characterize the molecular profiles of immune checkpoints RANK/RANK-L and IFN-γ in high-grade mast cell tumors and lymph node metastases to understand the complex activities occurring in the tumor microenvironment. All tumors showed moderate or intense immunolabeling of PD-L1, CTLA-4, RANK, RANK-L, and IFN-γ, and the lymph node metastases presented moderate or intense immunolabeling of checkpoint proteins. In conclusion, the high-grade MCTs were characterized as immunosuppressive microenvironments, showing an increase in the RANK/RANK-L signaling pathway and intensified immune checkpoint immunoreactivity, which may explain an intratumoral escape mechanism and indicating high sensitivity to immunotherapy. Therefore, PD-L1, RANK/RANK-L and IFN-γ may be useful in the clinical management of dogs with high-grade MCT.AbstractMast cell tumors (MCTs) are the most common malignant cutaneous tumors in dogs, and they present extremely variable biological behavior. The interaction between RANK, RANK-L, and immune checkpoints is frequently detected in the tumor microenvironment, and, together, they participate in every stage of cancer development. Thus, the aim of this study was to characterize the molecular profiles of PD-L1, CTLA-4, RANK/RANK-L signaling pathway, and IFN-γ in primary tumors and lymph node metastases. Formalin-fixed, paraffin-embedded slides of MCTs and metastatic lymph nodes of ten dogs were submitted to immunohistochemical investigations. The results demonstrated that the tumor microenvironment of the high-grade mast cell tumors showed moderate or intense immunolabeling of all proteins, and the lymph node metastases also showed moderate or intense immunolabeling of checkpoint proteins. In addition, MCTs larger than 3 cm were associated with intensified PD-L1 (p= 0.03) in metastatic lymph nodes and RANK-L (p= 0.049) immunoreactivity in the tumor. Furthermore, dogs with a survival time of less than 6 months showed higher PD-L1 immunoreactivity (p= 0.042). In conclusion, high-grade MCT is associated with an immunosuppressive microenvironment that exhibits elevated RANK/RANK-L signaling and enhanced immune checkpoint immunoreactivity, potentially facilitating intratumorally immune escape. These biomarkers show promise as clinical indicators of disease progression and might response to immunotherapy in dogs with high-grade MCTs, thus emphasizing their importance for guiding treatment decisions and improving outcomes.