Introduction Raised blood neutrophil-to-lymphocyte ratio (NLR) correlates with worse outcomes in NSCLC. Increasing evidence shows neutrophils are not the short-lived bystander they were once considered to be, but instead a persistent active player in cancer pathophysiology. By studying neutrophils from pleural fluid in NSCLC, we sought to establish the role they play in dictating immune responses to cancer at the metastatic site, and investigate whether features of the pleural environment itself impact upon their behaviour.

Methods Pleural fluid and blood was obtained from patients with NSCLC (n=33). Cells were extracted using magnetic negative selection kits (blood) and flow sorting (pleural fluid). Neutrophils (CD66b⁺CD11b⁺CD15⁺CD14^-CD49d^-) were examined for PD-L1 expression. NSCLC neutrophil apoptosis (% Annexin⁺) was measured, and compared to that of healthy donors. To model the metastatic environment, healthy donor neutrophils and T cells were co-cultured ± NSCLC pleural fluid supernatant (cell-free pleural fluid) ± PD-L1 inhibitor, and CD8⁺ proliferation observed (CFSE).

Results NSCLC blood neutrophils were functionally different, living longer than those from healthy donors (Apoptosis 34.5% vs. 51.6%, p<0.05). NSCLC pleural neutrophils had even lower rates of apoptosis (19.1%), and were phenotypically different, expressing higher levels of PD-L1 (PD-L1⁺91.7% vs. 0.9%, p<0.05). Healthy donor blood neutrophils exposed to NSCLC pleural fluid supernatant also expressed higher levels of PD-L1 and suppressed CD8+ T cell proliferation (CD8⁺ T cells divided 8.6% vs. 52.4%, p<0.05), an effect partially reversed by PD-L1 inhibition (CD8⁺ T cells divided 16.6%).

Conclusions Neutrophils in NSCLC are intrinsically different, living longer and expressing higher levels of PD-L1: features augmented by the pleural environment. We have shown evidence to support the hypothesis that neutrophils are detrimental at the pleural metastatic site, with an immunosuppressive role that is partially mediated via PD-L1. This inappropriate innate immune response may indirectly aid tumour progression.