Background: Background: In this multicentric retrospective study, we evaluated the relationship between pre- treatment blood neutrophils and neutrophil extracellular traps (NET) in biopsy samples and their predictive value for progression progression-free survival (PFS) in patients with non-small-cell lung cancer (NSCLC) receiving immunotherapy alone or in combination with chemotherapy as a first-line treatment. Methods: Patients with metastatic NSCLC (n = 100) were retrospectively analyzed between Apr 2018 and Sep 2021; 77.5% of the patients received platinum-containing chemotherapy with Pembrolizumab, and 22.5% – Pembrolizumab only as a first-line treatment. Pre-treatment blood neutrophils were counted. Tissue sections were stained immunohistochemically for Neutrophil elastase (NE) and Histone H3. Both NE and Histone H3 stained tissue areas were calculated manually and determined by Image-J software. We considered the extracellular components that were double-positive for NE and H3 to be NET. Results: In correspondence analysis, patients with low levels (< 33^rd percentile) of pre-treatment neutrophils were grouped with low amounts (< 33^rd percentile) of NET-positive tumor areas (p = 0.02). The amounts of NET-positive tumor areas were not related to other clinicopathological characteristics of the patients. Patients with an intermediate/high amount of NET-positive areas had significantly shorter mean PFS, 10.7 (95% CI: 9.6–11.8) than those with low NET-positive areas, 14.8 (95% CI: 11.5–18.1) (log-rank test p = 0.003). Moreover, in a multivariate Cox regression model, the presence of an intermediate/high amount of NET-positive areas was an independent predictive factor for shorter PFS, HR 2.1 (95% CI: 1.2–3.7; p = 0.005). Conclusions: Our study suggests that tumor formation of NETs is related to the numbers of pre-treatment neutrophils and may mediate neutrophil-induced antitumor resistance. Excessive NET formation in tumor tissue is a potential negative predictive marker for shorter PFS.