Roughly 30% of individuals with stage T1 non-small cell lung cancer (NSCLC) exhibit mediastinal (N2) lymph node metastasis, yet the specific biological processes responsible for this phenomenon are still not well understood. Single-cell sequencing was used to identify the specific cell population most likely responsible for driving N2 lymph node metastasis in T1 NSCLC. Single-cell analysis further evaluated the expression patterns and functional significance of the primary gene of interest, fibrinogen-like protein 1 (FGL1), within this cell subset. Integrated multi-omics approaches—including transcriptome sequencing, metabolome profiling, and mass spectrometry—were combined with both in vitro and in vivo functional assays. Therapeutic potential was evaluated through targeted knockdown using shFGL1_AAV9 and shFGL1_AAV6. A previously unrecognized cell population, marked by FGL1 expression (CCNE1(+) cells), was discovered. The emergence of this subgroup closely paralleled FGL1 upregulation, indicating that FGL1-positive cells play a central role in mediating N2 lymph node metastasis in T1 NSCLC. Through mass spectrometry, transcriptional profiling, and metabolomic analyses, FGL1 was shown to modulate key glycolysis-related factors and drive epithelial-to-mesenchymal transition (EMT) in NSCLC cells primarily via activation of the PI3K/AKT/HIF-1α signaling axis. Additional experiments demonstrated that FGL1 enhances tumor cell proliferation, migratory and invasive capabilities, and lymphatic vessel formation, thereby facilitating lymph node metastatic spread. These effects were consistently confirmed across in vitro and in vivo models. Conversely, FGL1 depletion effectively suppressed these malignant behaviors. In vivo validation confirmed that shFGL1_AAV9 and shFGL1_AAV6 serve as effective targeted delivery systems for FGL1 silencing, highlighting promising new therapeutic avenues to block NSCLC progression and metastasis. This study clarifies the critical contribution of FGL1 to NSCLC pathobiology, revealing its function as a “shield machine cutter” that promotes T1 NSCLC N2 lymph node metastasis by inducing lymph tube formation and establishing metastatic routes. These findings lay the groundwork for the development of innovative treatment strategies focused on targeting FGL1.
