Tumor-infiltrating natural killer cell profiling for therapeutic stratification in smokers with resectable non-small cell lung cancer

Objective: Neoadjuvant chemoimmunotherapy has improved outcomes in resectable non-small cell lung cancer (NSCLC), yet its real-world implementation is often challenged by surgical delays, cancellations, immune-related fibrosis, and increased postoperative complications. Smokers with NSCLC, despite being at higher risk for postoperative complications, have enhanced responses to neoadjuvant chemoimmunotherapy. This study aimed to identify smoking-associated immune cell determinants that could inform treatment strategies. Methods: Single-cell RNA sequencing was performed on 61 lung samples from non-smokers, smokers, and patients with chronic obstructive pulmonary disease (COPD), as well as 8 invasive lung adenocarcinomas, to define smoking-related immune signatures. Using RNA sequencing data from 102 resected NSCLC and 24 NSCLC patients treated with neoadjuvant chemoimmunotherapy, we conducted in silico deconvolution to evaluate associations between cellular composition and clinical outcomes. Results: Among 140 lung cellular phenotypes, two natural killer (NK) cell subsets were strongly associated with smoking and COPD severity. “Stress-responsive NK (NKSR)” cells exhibited immature features and cytokine-responsive signatures. “Exhausted NK (NKExh)” cells showed mature features and elevated multiple immune checkpoint expression (PDCD1, TIGIT, and LAG3). Abundant intratumoral NKSR cells were associated with significantly improved survival after surgery, particularly in current smokers. Conversely, tumors with low NKSR and high NKExh cell profiles were associated with better responses to neoadjuvant chemoimmunotherapy. Conclusions: Tumor-infiltrating NK cell phenotyping may aid in therapeutic stratification in smokers with NSCLC. NKSR cell preservation predicts favorable outcomes with upfront surgery, while NKExh cell enrichment indicates greater benefit from neoadjuvant chemoimmunotherapy. These profiles may help optimize treatment by balancing therapeutic benefit and risk. Overall design: This study was conducted under protocols approved by the Institutional Review Board at Baylor College of Medicine (BCM) (H-35782) and the University of Arizona (UA) (1811124026). Written informed consent was obtained from all participants for the collection of clinical data and biospecimens. At BCM, a prospectively maintained, single-institution database was reviewed retrospectively. The BCM cohort included 102 patients with histologically confirmed NSCLC who underwent complete pulmonary resection and mediastinal lymph node dissection. Staging was determined according to the 8th edition of the TNM classification. At UA (by Dr. Polverino), lung tissue samples were obtained from patients undergoing lung volume reduction surgery or lung transplantation for severe emphysema, or from patients undergoing lung resection for a solitary peripheral nodule, with sampled tissue taken from a region at least 10 cm away from the nodule. The UA cohort included 13 non-smokers, 21 ever-smoker control subjects, 15 patients with mild chronic obstructive pulmonary disease (COPD)(GOLD stage 1-2), and 12 patients with severe COPD (GOLD stage 3-4). COPD was defined as pre-bronchodilator forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) ratio of less than 0.7 (70%). Disease severity was classified according to the GOLD criteria as follows: GOLD 1 (FEV1 = 80%), GOLD 2 (50% = FEV1 < 80%), GOLD 3 (30% = FEV1 < 50%), and GOLD 4 (FEV1 < 30%).