Abstract Rationale With the widespread application of low-dose computed tomography (LDCT) in lung cancer screening, the detection rate of ground-glass nodule (GGN) featured early-stage lung adenocarcinoma (LUAD) has increased significantly. Although most GGN-type LUADs have a favorable prognosis, a subset of cases exhibits malignant progression. Current early diagnosis of GGN-type LUAD primarily relies on imaging and pathological evaluation, lacking precise molecular discrimination methods. Utilizing single-cell RNA sequencing (scRNA-seq), this study aims to decipher the underlying molecular mechanisms driving the progression across different pathological stages of GGN-type LUAD, with a specific focus on the role of key driver molecules in malignant evolution. Methods The study enrolled 1800 surgical patients with GGN-type LUAD to construct a clinical-molecular profile spanning atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), microinvasive adenocarcinoma (MIA), to invasive adenocarcinoma (IAC), including mutation analysis for 473 cases. scRNA-seq was performed on 18 tissue samples from different stages (precursor lesion group: AAH/AIS vs. invasive group: MIA+IAC) to reveal transcriptomic dynamics. Functional cell experiments were conducted to validate the role of key molecular targets in tumor progression. Association analyses with overall survival (OS) and progression-free survival (PFS) were performed using data from The Cancer Genome Atlas (TCGA). Validation was further carried out via immunohistochemistry (IHC) and mouse model experiments. Results Compared to the precursor lesion, the invasive group showed a significantly higher proportion of males. EGFR/TP53 co-mutations more concentrated in invasive stage. Single-cell analysis revealed heightened activity of E2F targets, the G2/M checkpoint, and the PI3K/AKT/mTOR pathway in tumor cells during the invasive stage. NPC2 expression significantly decreased with disease progression and was involved in regulating pathways related to apoptosis, innate immune regulation, lipopeptide binding, and alveolar surfactant metabolism. TCGA analysis indicated that low NPC2 expression was significantly associated with poorer prognosis (OS: HR = 0.53, P 0.001; PFS: HR = 0.65, P = 0.044). Functional experiments demonstrated that NPC2 knockdown enhanced PI3K/AKT pathway activation and promoted tumor cell proliferation and migration. In vivo, NPC2 knockdown promoted tumor growth. Immunohistochemistry confirmed predominant cytoplasmic expression of NPC2, with weakening expression intensity and decreased staining correlating with advancing pathological stage. Conclusion This study systematically reveals the clinical and molecular characteristics associated with the pathological progression of GGN-type LUAD and identifies NPC2 as a key regulator driving malignant evolution, primarily through activating the PI3K/AKT signaling pathway. Low NPC2 expression is closely linked to poorer survival, positioning NPC2 as a potential target for early diagnosis and precise intervention in GGN-type lung adenocarcinoma. This abstract is funded by: This study was supported by National Natural Science Foundation of China (CN) (No. 82370100)
Wang et al. (Fri,) studied this question.