Efeito de vesículas extracelulares liberadas por células de melanoma MV3 na polarização de macrófagos humanos

Abstract

To acknowledge the mechanisms involved on tumor growth, it is necessary to understand its microenvironment, composed by several extracelular matrix componentes, like growth factors, cytokines and different cell types. Immune cells present in the microenvironment may have their functions modified in order to support tumor growth. The macrophage polarization into M1 and M2 phenotypes, as well as their implications in the microenvironment have been extensively described. Evidences also show that in the tumor microenvironment there is a huge presence of extracelular vesicles (EV), producted abundantly by the tumor cells. EV not only transport molecular information from their origin cells, but are also capable of modulating the activity of target cells. It has already been shown that tumor-derived EV are able to interact with different cell types present in the microenvironment and recently our group has shown that human melanoma-derived MV3 EV were able to skew neutrophils into a pro-tumoral phenotype in vitro. However, their effects on human macrophages have not been elucidated yet. The aim of this study was to investigate if MV3-EV would modulate macrophages activity, shifting these cells towards a M2-like phenotype. EV were released from human melanoma MV3 cells and quantified through real-time microscopy ZetaView™ and flow cytometry. We have observed that EV induced monocytes chemotaxis in a concentration-dependent manner, and that such effect was dependent on the phosphatidylserine exposed on their membranes, since the blockage of phosphatidylserine with annexin V reverted this effect. EV were also capable of inducing the expression of CD206, a M2 marker, on the membrane of macrophages, as well as increased the secretion of IL-10 and TGF-β and the mRNA expression of CCR5 and CCL3. Moreover, EV-stimulated macrophages exhibited increased ROS production, although their NO levels remained at basal levels. Corroborating this, EV-stimulated macrophages presented increased arginase activity, and did not demonstrate cytotoxicity properties against melanoma cells in coculture. Furthermore, EV were biodistribucted to several organs, such as liver, intestine, kidneys and lungs, what could contribute to primary tumor metastasis to secondary sites such as the lungs in nude mice. Our data reveal a possible mechanism in which EV may modulate TAM activity, suggesting them as a potential therapeutic target, aiming the reversal of macrophages polarization and tumor inhibition