PURPOSE: Cytokine therapies such as interleukin-2 and -12 suffer from impractically small therapeutic windows driven by their on-target, off-tumor activity, limiting their clinical potential despite potent antitumor effects. We previously engineered cytokines that bind and anchor to tumor collagen following intratumoral injection, and sought to test their safety and biomarker activity in spontaneous canine soft tissue sarcomas (STS). METHODS: Collagen-binding cytokines were canine-ized to minimize immunogenicity and were used in a rapid dose-escalation study in healthy beagles to identify a maximum tolerated dose. Ten client-owned pet dogs with STS were then enrolled into trial, receiving cytokines at different intervals prior to surgical tumor excision. Tumor tissue was analyzed through immunohistochemistry (IHC) and Nanostring RNA profiling for dynamic changes within treated tumors. Archived, untreated STS samples were analyzed in parallel as controls. RESULTS: Intratumorally administered collagen-binding IL-2 and IL-12 were well-tolerated by STS-bearing dogs, with only Grade 1/2 adverse events observed (mild fever, thrombocytopenia, neutropenia). IHC revealed enhanced T cell infiltrates, corroborated by an enhancement in gene expression associated with cytotoxic immune function. We found concordant increases in expression of counterregulatory genes that we hypothesize would contribute to a transient antitumor effect, and confirmed in mouse models that combination therapy to inhibit this counterregulation can improve responses to cytokine therapy. CONCLUSIONS: These results support the safety and activity of intratumorally-delivered, collagen-anchoring cytokines for inflammatory polarization of the canine soft tissue sarcoma tumor microenvironment. We are further evaluating the efficacy of this approach in additional canine cancers, including oral malignant melanoma.