Tumor Associated Macrophages Mitigate Oncolytic Herpes Simplex Virus Anti-Tumor Efficacy in Ewings Sarcoma

Abstract

Introduction: Ewing sarcoma is a highly aggressive bone tumor that is often lethal following recurrence or metastasis. Oncolytic viruses (OVs), such as the rRp450 herpes simplex virus, are promising anticancer therapeutics designed to selectively replicate in cancer cells. While OV anti-tumor efficacy is partially caused by direct infection and lysis of cancer cells, stimulation of an anti-cancer immune response also contributes to virus-mediated efficacy. Immunologic responses to infections are known to be modulated by macrophages via various cytokines and chemokines and it is now appreciated that tumors are replete with tumor associated macrophages (TAMs). M2 alternatively activated macrophages in particular express pro-tumor immunosuppressive cytokines, such as IL-10 and TGF-β. We hypothesize that TAMs reduce therapeutic efficacy by producing an immunosuppressive tumor microenvironment via IL-10 and TGF-β signaling. Research methods: Human Ewing sarcoma xenografts were implanted into athymic nude mice and macrophages were depleted using liposomal clodronate prior to intratumoral injection of rRp450 oncolytic HSV. Tumors were allowed to grow for tumor progression. In vitro cytotoxicity was determined using MTS assay. In vitro and in vivo virus replication was determined through plaque assay. Bone marrow derived macrophages were cocultured with Ewing sarcoma cell lines and harvested for flow cytometry and PCR analysis of tumor inflammatory signaling and M1/M2 macrophage gene profiles. F4/80+ tumor associated macrophages were extracted from tumors using magnetic bead separation. TGF-β cytokine superfamily receptor signaling was inhibited with A83-01 (Sigma-Aldrich) small molecule treatment prior to intratumoral injection of rRp450. Results: Macrophage depletion significantly inhibited tumorigenesis and enhanced rRp450 anti-tumor efficacy in A673, but not 5838 Ewing sarcoma xenografts. No change in virus titer was observed in the macrophage depleted tumors, suggesting the effect isn’t due to enhanced virus replication. Macrophages cocultured with A673 cells had higher expression of M2 pro-tumor macrophage genes than macrophages cocultured with 5838 cells. Macrophages in A673 tumors also demonstrated higher expression of IL-10 and TGF-β than 5838 tumor associated macrophages. Inhibition of TGF-β signaling enhanced rRp450 oncolytic virus anti-tumor efficacy in A673 tumors. Conclusions: Macrophages play a significant role in mitigating OV anti-tumor efficacy. Specifically, our tumor models that promote M2 macrophage polarization are significantly more resistant to oncolytic virus therapy, in part due to TGF-β signaling. Our results suggest that the combination of oncolytic virus therapy with a macrophage modulatory therapy will improve OV anti-tumor efficacy in patients with highly immunosuppressive tumors.