Phenotypic and Functional Alterations in Human Monocytes Induced by Tumor-Derived Extracellular Vesicles

Introduction: This study investigates the effects of extracellular vesicles derived from MDA-MB-231 cells (MDA-EVs), a breast cancer cell line, on the phenotype and functional properties from induced human monocytes (Mo). Methods: In Mo (UERJ/CAAE 36880914.0.0000.5259) stimulated with MDA-EVs for 24 hours, the phenotype and polarization status was performed by flow cytometry, investigating the expression of CD14, CD16, CCR2, CCR5, CX3CR1, CD80, and CD206. The chemotactic and invasive capacity of Mo towards MDA-EVs were assessed by Boyden chamber and gelatin-coated transwell inserts. Arginase-1 (ARG1), AMPK, and Superoxide dismutase 1 (SOD1) expression were evaluated by western blotting. In macrophages (MØ) differentiated from Mo in the presence of MDA-EVs for 7 days flow cytometry was performed to assess phenotypic and polarization changes (CD14, CD16, CD80, CD206 expression). NO e total intracellular ROS production was measured in Mo (with DAF and CellROX probes, respectively. Results: Exposure to MDA-EVs increased the CD14++CD16- Mo population while decreased the CD14⁺CD16+ and CD14^dimCD16⁺ populations. The exposure to MDA-EVs prompted Mo migration and invasion. There was an increase in ARG1 expression, a marker of pro-tumoral phenotype, and a decrease in p-AMPK, indicating a possible metabolic alteration in Mo. There was no change in NO production. Intracellular ROS production was lower after stimulation, with no change in SOD1 expression. In MØs differentiated from Mo the presence of CD14^dimCD16⁺CD206⁺ cells were heightened, indicating the polarization towards an M2-like profile. Conclusion: MDA-EVs induce phenotypic changes in Mo after 24 hours of stimulation, enhancing the expression of classical population markers in Mo and non-classical makers in MØs 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 Mo to establish TAMs in the TME.