Neutrophil Polarization as an Anti-Tumor Approach

INTRODUCTION AND OBJECTIVES: Neutrophils are plastic immune cells that can polarize into N1-like antitumor or N2-like protumor phenotypes. N1 neutrophils exhibit increased cytotoxicity associated with reactive oxygen species (ROS), pro-inflammatory mediators, and NOX activity. We investigated N1-like polarization of human neutrophils using LPS and IFN-γ and their interaction with breast cancer (MDA-MB-231), melanoma (MV3), and glioblastoma (U87-MG) cells. METHODS: Neutrophils were isolated from the peripheral blood of donors (CAAE 65372522.4.0000.5257) by immunomagnetic negative selection and stimulated with LPS and IFN-γ for 3 h. Total ROS (DCF probe), nitric oxide (NO; DAF probe), peroxynitrite (ONOO⁻; APF probe), lipid peroxidation (TBARS), extracellular vesicle (EV) release (NanoSight), and tumor cell viability after co-culture were evaluated. RESULTS: N1-like neutrophils significantly increased total ROS production in MDA-MB-231 (+16%, p=0.0197), MV3 (+32%, p<0.0001), and U87-MG (+21%, p=0.0075); NO in MDA-MB-231 (+54%, p=0.0473) and U87-MG (+14%, p=0.0293); and ONOO⁻ in MDA-MB-231 (+9%, p=0.0164), MV3 (+27%, p=0.0026), and U87-MG (+34%, p=0.0080). Lipid peroxidation also increased in MDA-MB-231 (+18%, p=0.0306), MV3 (+15%, p=0.0390), and U87-MG (+13%, p=0.0257). These effects reduced tumor cell viability: MDA-MB-231 (−46%, p=0.0442), MV3 (−7%, p=0.0008), and U87-MG (−36%, p=0.0055). N1-EVs showed higher protein content than N0-EVs (+110%, p=0.0073) and reduced viability of MDA-MB-231 (−12%, p=0.0004), MV3 (−9%, p=0.0062), and U87-MG (−14%, p=0.0285). Fucoidan supported the importance of N1-EV-mediated communication in tumor cell death, suggesting that these effects rely, at least in part, on a L-selectin-dependent pathway. CONCLUSION: N1-like neutrophils exert potent antitumor activity in vitro through oxidative stress and lipid peroxidation, while their EVs may contribute to this cytotoxic response, supporting their potential as antitumor therapeutic tools.