Abstract Background Multiple primary lung cancer (MPLC) exhibits distinct clinicopathological features and poor response to EGFR-TKIs, yet its underlying biology remains unclear. Methods We performed single-cell RNA sequencing (scRNA-seq) on 29 samples (14 tumors, 9 normal tissues) from 13 individuals (7 MPLC, 6 solitary primary lung cancer SPLC patients). Integrated analyses included copy number variation (CNV) inference, tumor cell annotation, immune cell annotation, and cell-cell communication mapping. Results MPLC demonstrated significant CNVs in histologically normal lung tissues (P0.001), supporting the field cancerization hypothesis. Tumor cells in MPLC exhibited lower malignancy, evidenced by enrichment of alveolar differentiation genes (*SFTPA1/SFTPA2*; favorable prognosis in TCGA-LUAD, P0.05); preferential oxidative phosphorylation (OXPHOS) over glycolysis (P0.01), linked to DNMT1high/NNMTlow expression; reduced immunosuppressive Tregs and expanded cytotoxic GZMB+ CD8+ T cells and CD16+ NK cells (P 0.001); MPLC-specific TNFA-NFKB pathway activation was observed in T cells and macrophages (CCL3+/AZU1+ subsets). In contrast, SPLC showed fibroblast-dominated stroma and glycolytic metabolism. Conclusion MPLC is characterized by field cancerization-driven tumorigenesis, OXPHOS dependency, and a unique immunocompetent microenvironment. Therefore, this study reveals the difference of tumorigenesis between MPLC and SPLC and provides a potential immunotherapy strategy for MPLC. This abstract is funded by: None
Wang et al. (2026) studied this question.