Papel das vesículas extracelulares derivadas da linhagem de câncer de mama triplo-negativo 4T1 na modulação fenotípica e funcional de macrófagos

Abstract

Triple-negative breast cancer (TNBC) represents one of the most aggressive subtypes of breast cancer, characterized by a high proliferative rate, great invasive potential, and increased propensity for metastatic dissemination. In this context, the communication between tumor cells and components of the tumor microenvironment plays a major role in disease progression, a process in which extracellular vesicles (EVs) stand out as important mediators. In this scenario, macrophages are highly plastic cells whose functional activity directly influences tumor dynamics. Thus, this study aimed to evaluate the effects of EVs derived from the murine TNBC 4T1 cell line on the phenotype and viability of RAW 264.7 macrophages. EVs were isolated by differential centrifugation and ultracentrifugation and characterized by size, protein concentration, and morphology through dynamic light scattering (DLS), BCA assay, and transmission electron microscopy. Cellular viability was assessed by both MTT and flow cytometry techniques. Phenotypic modulation was evaluated through iNOS expression by flow cytometry, while the cytokines IL-6, IL-10 and TNF-α were measured by ELISA, and NO production by the Griess method, respectively. EVs exhibited features compatible with the literature, evidenced by spherical morphology and average sizes of 203.2 nm and 151.7 nm for MVs and Exos. In addition, they did not exert a significant cytotoxic effect on macrophages under the evaluated experimental conditions. Regarding phenotypic modulation, EV-treated M0 macrophages exhibited increased iNOs expression and IL-6 secretion, although TNF-a levels decreased. Concerning the M1 macrophages, EV-treatment reduced IL-10 production, without additional changes in iNOS expression; whereas in M2 macrophages, an increase in iNOS expression associated with reduced IL-10 was observed. Despite the increased iNOS expression in M0 and M2 macrophages, no significant changes in NO production were detected. Overall, these findings indicate that EVs derived from the 4T1 cell line modulate the functional state of macrophages on the dependency of their basal activation phenotype. These results reinforce the current concept that macrophage activation occurs along a continuous spectrum of functional states, in which intermediate phenotypes may coexist performing distinct functions within the tumor microenvironment.