Tumor-Derived Extracellular Vesicles Induce Phenotypic and Functional Changes on Human Monocytes

Introduction: Focused on breast cancer, particularly the aggressive triple-negative subtype, this study explores the role of monocytes in the tumor microenvironment (TME). These cells, capable of differentiating into tumor-associated macrophages (TAMs), interact with extracellular vesicles (EVs) carrying biomolecules and genetic material. Investigating EVs from MDA-MB-231 (MDA-EVs), a cell lineage of triple-negative breast cancer, the study aims to characterize phenotypic and functional alterations of human monocytes stimulated with MDA-EVs. Methods: MDA-MB-231 conditioned medium (MDA-CM) was obtained for 24 hours of culture with medium supplemented with 1% of fetal bovine serum. The EVs were isolated from MDA-CM at 20.000 x g for 70 minutes (4ºC) to obtain microparticles. Monocytes were stimulated with MDA-EVs for 24 hours, and flow cytometry was performed to investigate monocytes' phenotypic changes (expression of CD14, CD16, CCR2, CCR5, CX3CR1) after stimulation. The chemotactic capacity of MDA-EVs, the ability to induce invasion in monocytes, and NF-κB activity were also accessed. Arginase-1 (ARG1) expression was obtained through western blot analysis. Monocytes were differentiated into macrophages with 7 days of culture with MDA-EVs, and flow cytometry was performed to assess phenotypic (CD14 and CD16 expression) and polarization (CD80 and CD206 expression) changes. Results: Following exposure to MDA-EVs, there was a notable rise in the classical monocyte population (CD14⁺⁺CD16^-), accompanied by a decline in markers associated with intermediate (CD14⁺CD16+) and non-classical populations (CD14^dimCD16⁺). Moreover, MDA-EV stimulation reduced CCR2 expression specifically within the classical monocyte population, while other markers did not show significant effects. The exposure to MDA-EVs prompted monocyte migration and invasion, suggesting their chemotactic ability. Additionally, MDA-EVs were observed to reduce the activity of NF-κB, possibly indicating a decrease in the pro-inflammatory profile of monocytes. There was an increase in ARG1 expression. Macrophages differentiated from monocyte cultures presented an increase in non-classical subtype markers and heightened the presence of CD14^dimCD16⁺CD206⁺ cells, indicating a shift in the polarization of these cells towards an M2-type profile. Conclusion: It is possible to observe that MDA-EVs induce phenotypic changes in monocytes after 24 hours of stimulation, enhancing the expression of classical population markers in monocytes and non-classical makers in macrophages after 7 days of stimulation, also inducing the polarization of these cells to a pro-tumoral phenotype. The chemotactic activity of MDA-EVs supports the idea of recruiting monocytes to establish TAMs in the TME.