The tumor microenvironment (TME) is a relevant target for novel biological therapies. MV-m-uPA and MV-h-uPA are fully retargeted, species-specific, oncolytic measles viruses (MVs) directed against murine or human urokinase receptor (PLAUR/uPAR), expressed in tumor and stromal cells. The effects of stromal selective targeting by uPAR retargeted MVs were investigated. In vitro infection, virus-induced GFP expression and cytotoxicity by MV-h-uPA and MV-m-uPA were demonstrated in human and murine cancer cells and cancer associated fibroblasts (CAFs) in a species-specific manner. In a murine fibroblast/human breast cancer 3D co-culture model, selective fibroblast targeting by MV-m-uPA inhibited breast cancer cell growth. Systemic administration of murine specific MV-m-uPA in mice bearing human MDA-MB 231 xenografts was associated with a significant delay in tumor progression and improved survival compared to controls. Experiments comparing tumor (MV-h-uPA) vs. stromal (MV-m-uPA) vs. combined virus targeting showed that tumor and stromal targeting was associated with improved tumor control over the other groups. Correlative studies confirmed in vivo viral targeting of tumor stroma by MV-m-uPA, increased apoptosis, and virus induced differential regulation of murine stromal genes associated with inflammatory, angiogenesis and survival pathways, as well as indirect regulation of human cancer pathways, indicating viral induced modulation of tumor-stromal interactions. These data demonstrate the feasibility of stromal selective targeting by an oncolytic MV, virus-induced modulation of tumor-stromal pathways, and subsequent tumor growth delay. These findings further validate the critical role of stromal uPAR in cancer progression and the potential of oncolytic viruses as anti-stromal agents.

The tumor stroma acts as a barrier that limits the efficacy of systemically administered oncolytic viruses (OV). We previously demonstrated that stromal-selective, retargeted oncolytic measles viruses (MVs) delay in vivo tumor progression. To further characterize the contribution of stromal targeting to MV's overall in vivo efficacy in an experimental cancer model, a dual targeted oncolytic measles virus (MV-CD46-muPA) able to simultaneously infect murine stromal (via murine uPAR) and human cancer (via CD46) cells was developed. MV-CD46-muPA infected, replicated, and induced cytotoxicity in both murine and human cancer cells. Viral infection was successfully transferred from stromal to tumor cells in vitro, leading to tumor cell oncolysis. Systemic administration of MV-CD46-muPA led to improved antitumor effects in colon (HT-29) cancer xenografts compared to vehicle or CD46 only targeted MVs. These effects were associated with improved tumor viral deposition, increased apoptosis, and decreases in murine stromal endothelial cells and fibroblasts. MV-CD46-muPA modulated cell cycle, survival, proliferation, and metabolic pathways, as determined by functional proteomic analysis of treated tumors. The above findings further validate the concept that dual stromal and tumor cell viral targeting enhances the therapeutic effects of systemically administered OVs and support further preclinical and clinical development of stromal directed virotherapies.

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