Abstract 2498: Melanoma cell -derived extracellular vesicles - immunomodulators of the tumor microenvironment

Abstract The role of extracellular vesicles (EVs) in tumor-stroma communication has been under extensive investigation, and their contribution to inflammation and immunosuppression, angiogenesis and metastasis has been demonstrated. However, the exact molecular mechanisms and the EV cargos responsible for mediating these effects are still partly unknown. In tumor microenvironment, macrophages are one of the most abundant cell types and they are composed of several distinct populations that share features of both pro-inflammatory (M1) and immunosuppressive (M2) macrophages. In this study, we investigated the effects of melanoma cell secreted EVs on macrophage polarization. In several cancers hyaluronan synthesis is elevated and overexpression of its producing enzyme, hyaluronan synthase 3 (HAS3), induces EV shedding from the plasma membrane. These HAS3 induced EVs contain a hyaluronan coat around the vesicles. To elucidate the effects of HAS3-induced EVs on target cells, THP1 monocytes were treated with EVs derived from EGFP-HAS3 overexpression MV3 melanoma cells, or from parental MV3 cells, and the expression of cytokines and other factors related in cancer -induced immune modulation was studied. EVs were isolated with ultracentrifugation and characterized with Nanoparticle tracking analysis (NanoSight NS300) and transmission electron microscopy. THP1 cells were activated with PMA for 48 h and thereafter cells were treated with either control EVs from MV3 cells or hyaluronan coated EVs from HAS3 overexpressing cells for 4-24 h. The expression of pro-inflammatory and immunosuppressive markers was studied with qPCR, and the effects on cell morphology with confocal microscopy. The size distribution of EVs derived from MV3 and HAS3-MV3 cells was relatively similar, being approximately 100-200 nm in range, while the particle concentration (/ml) was higher in samples derived from HAS3-MV3 cells. The expression of proinflammatory cytokines and chemokines, like IL-1β and IL-8, and immunosuppressive factors like TSG-6 and PD-L1 was elevated in macrophages treated with MV3 melanoma cell secreted EVs. The effect on expression of the studied factors was more obvious when cells were treated with hyaluronan coated EVs (HAS3-MV3 EVs). This indicates that either the hyaluronan coat around the EVs or their cargo is different compared to control EVs, which causes the effects. Inhibiting TLR4 receptor decreased the cytokine expression induced by HAS3-MV3 EVs, suggesting TLR4-mediated activation after HAS3-MV3 EV exposure. Our results suggest that melanoma cell derived EVs induce mixed M1/M2 polarization of THP1 monocytes. Citation Format: Sanna Marika Pasonen-Seppänen, Piia Takabe, Kirsi Kainulainen, Kaisa Mäki-Mantila, Riku Korkiamäki, Silvia Lopez Borrego, Taija Hukkanen, Niina Aaltonen, Kirsi Rilla. Melanoma cell -derived extracellular vesicles - immunomodulators of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2498.