Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4 + and CD8 + T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses.

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Aim/Background: Depletion of essential (L-tryptophan) or semi-essential (L-arginine) amino acids has been shown to suppress antitumor immune responses. Arginase-1 (Arg-1) is a cytosolic enzyme catalyzing degradation of L-arginine to L-ornithine and urea. Several studies have shown the presence of abundant Arg-1 in either tumor cells or in tumor-infiltrating myeloid cells of neutrophilic or monocytic origin. This in turn, leads to downregulation of CD3-zeta levels in T cells and correlates with suppressed antitumor immunity. In patients with ovarian cancer (OvCa), exosomes released by tumor cells (TEX) are detected in body fluids including plasma or ascites and carry proteins that are expressed by OvCa cells. Here we report that OvCa cells release Arg-1 in TEX and investigate the influence of OvCa TEX-derived Arg-1 on the antitumor effector mechanisms of immune response. Methods: TEX were isolated by ultracentrifugation or exclusion chromatography and verified by Western blotting, NanoSight and electron microscopy. The presence of Arg-1 in TEX was determined by Western blotting and the Arg-1 activity was assessed using an enzymatic assay that measured conversion of arginine into urea. Immunohistochemical Arg-1 expression data in primary OvCa lesions were correlated to clinico-pathological characteristics. Effects of TEX Arg-1 on immune cells were analyzed by in vitro proliferation assay and flow cytometry. Results: OvCa ascites contained higher levels of exosomal Arg-1 as compared with the fluids obtained from benign ovarian cysts. Enzymatically active Arg-1 was detected in TEX derived from patients’ ascites as well as from ovarian cancer cell lines. High Arg-1 expression in correlated negatively with intratumoral T-cell infiltrates and CD3-zeta expression and was associated with shorter time to recurrence (TTR). In vitro, OvCa-derived Arg-1-positive TEX inhibited CD8+ and CD4+ T-cell proliferation and decreased T-cell receptor expression. We also observed that co-culture of bone-marrow-derived dendritic cells (DC) with TEX isolated from OvCa cells results in the transfer of functionally active Arg-1 that inhibits DCs-primed proliferation of OVA-antigen specific OT-I T cells. We have used TEX isolated from OvCa cells transfected with V5-tagged Arg-1 for these experiments to ensure that this is OvCa-derived, but not endogenous DC-derived arginase that suppresses T cell proliferation. All these in vitro effects were reversed by a novel Arg-1 inhibitor (OAT-1746). Conclusions: Our findings provide the first evidence for the role of Arg-1 in the formation of an immunosuppressive microenvironment in OvCa. We identify a novel mechanism of exosomal Arg-1 distribution from the tumor cells to antigen presenting cells. Inhibition of Arg-1 activity may be an attractive novel anti-cancer strategy. Funding:  NCN - OPUS 6 Program 2013/11/B/NZ6/02790, NCBIR – STRATEGMED2/265503/3/NCBIR/15 Citation Format: Malgorzata Czystowska, Marta Szajnik, Kavita Ramji, Slawomir Gruca, Artur Stefanowicz, Jakub Golab, Dominika Nowis. Ovarian cancer cells release arginase-1-containing exosomes to suppress antitumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3975. doi:10.1158/1538-7445.AM2017-3975

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