N1-like neutrophils polarization as a new approach for cancer treatment

INTRODUCTION: Cancer, a complex disease with uncontrolled cell growth, can invade tissues and spread, being a leading global cause of death. Hanahan and Weinberg (2011) proposed cancer Hallmarks, where inflammation became a key marker. Neutrophils, observed in tumours since 1863 by Virchow, can be recruited by tumours, promoting growth and progression. Tumor-associated neutrophils (TANs) can be N1 (antitumor) or N2 (pro-tumor). N1s are cytotoxic to tumour cells, producing CD95, TNF-α, and ROS, while N2s support tumour growth and metastasis with arginase-1, CCL2, VEGF, IL-8, and MMP-9. Our group pioneered in vitro polarization of human neutrophils to the N2-like phenotype, studying their role in tumour growth. We have identified strategies to reduce N2-like neutrophils. Understanding N1-like polarization could reveal how these neutrophils kill tumour cells. Thus, our main objective was to polarize, in vitro, neutrophils from healthy patients to the N1-like phenotype and investigate how these immune system cells will interact separately with two different tumour cell lines. METHODS: Neutrophils were isolated from the peripheral blood of healthy donors (CAAE 65372522.4.0000.5257) and treated with LPS and IFNy for three h. Flow cytometry showed increased CD95, indicating N1-like polarization. We used MDA-MB-231 (breast cancer) and U87-MG (glioblastoma) cell lines for viability assays (MTT) and ROS production (DCF probe, one h kinetics). RESULTS: LPS and IFNy-polarized neutrophils showed higher CD95, confirming N1-like polarization. Tumour cells treated with these neutrophils showed about 50% reduced viability, and N1-like neutrophils increased total ROS production in contact with MDA-MB-231 cells. CONCLUSION: Our findings suggest a novel N1-like polarization capable of combating MDA-MB-231 and U87-MG tumour cells in vitro, offering a promising model for cancer therapy. REFERENCES: Cell (2011) 144 (5). Nature (2008) 436. Cancer Cell (2009) 183, (v.3). Cells (2022) v.12.