N2-like neutrophil-derived extracellular vesicles enhance malignancy of MDA-MB-231 breast tumor cells

INTRODUCTION: Breast cancer is the first most common among women. The relationship between cancer and inflammation is well established, and neutrophil infiltration has been described in tumors since 1863¹. Neutrophils are recruited by the tumor microenvironment and begin to favor tumor growth, thus they are called tumor-associated neutrophils (TAN) and can present two phenotypes: N1 (anti-tumor) or N2 (pro-tumor)^2. Our research group observed that neutrophils treated with extracellular vesicles (EVs) derived from melanoma (MV3) or breast tumor cells (MDA-MB-231) were able to adopt an N2-like phenotype³. As an in vitro polarization for TAN N2, we used EVs derived from breast tumor cells. We aimed to investigate the role of N2 TAN-derived EVs (N2-EVs) in MDA-MB-231 cells. METHODS: Neutrophils were isolated using ficoll gradient from the peripheral blood of healthy donors (CAAE 65372522.4.0000.5257) and treated with MDA-EVs for 3h. After polarization, we observed some specific markers of neutrophil polarization to a pro-tumor or N2-like phenotype³. N2-EVs were collected after 2h of polarization and characterized by cytometry. The effect of N2-EVs on AKT activation, mitochondrial ROS, invasion and migratory capacity, MMP-9, and NF-κB activity of MDA-MB-231 was observed by treating the cells with the EVs by 1h. RESULTS: We observed that N2-EVs present L-selectin on their surface and that its blocker fucoidan is capable of interacting and blocking this selectin. N2-EVs were able to increase AKT activation and mitochondrial ROS production in tumor cells. N2-EVs were also able to regulate the migratory and invasive capacity of MDA-MB-231 cells through the activity and transfer of MMP-9. CONCLUSION: Taken together, our results show that N2-EVs are capable of inducing tumor cells, in vitro, to a greater migratory and invasive capacity of MDA-MB-231 cells in an L-selectin-dependent manner.

 1- Nature, (2008) 3: 436

2- Cancer Cell, (2009) 3: 183

3- Cells, (2022) 12: 1