Role of Extracellular Vesicles Derived from the 4T1 Triple-Negative Breast Cancer Cell Line in the Phenotypic and Functional Modulation of Macrophages

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype in which tumor–microenvironment communication contributes to disease progression. Extracellular vesicles (EVs) are important mediators of this communication and may modulate the highly plastic functional state of macrophages. 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. Macrophage viability was assessed by MTT and flow cytometry, while phenotypic modulation was evaluated by iNOS expression, IL-6, IL-10 and TNF-α secretion, and NO production. 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. EVs showed no significant cytotoxicity toward macrophages. 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.