Abstract

Background: Approximately 30% of patients with stage T1 non-small cell lung cancer (NSCLC) have mediastinal (N2) lymph node metastasis; however, the underlying mechanism remains unclear.

Methods: The cells likely mediating N2 lymph node metastasis in T1 NSCLC were identified by single-cell sequencing. The expression and function of the main functional gene high fibrinogen-like protein 1 (FGL1) in this cell subgroup were analyzed by single-cell analysis. Transcriptome sequencing, metabolome sequencing, and mass spectrometry combined with in vitro and in vivo experiments were conducted, and therapeutic validation was performed using shFGL1_AAV9 and shFGL1_AAV6.

Results: A novel cell subgroup characterized by FGL1 expression was identified (CCNE1(+) cells). FGL1 expression coincided with the appearance of this cell subgroup, suggesting that FGL1?+?cells mediate T1 NSCLC N2 lymph node metastasis. Mass spectrometry combined with transcription sequencing and metabonomics revealed that FGL1 may affect glycolysis regulators and participate in epithelial-to-mesenchymal transition in NSCLC via the PI3K/AKT/HIF-1α pathway. Further analyses suggested that FGL1 promotes tumor proliferation, metastasis, and lymph tube formation, ultimately inducing lymph node metastasis. This was verified in vivo and in vitro. FGL1 knockdown inhibited these processes. Finally, shFGL1_AAV9 and shFGL1_AAV6 were verified as novel targeted therapies to knock down FGL1 in vivo, supporting the identification of new therapeutic targets to inhibit NSCLC metastasis.

Conclusion: We elucidated the role of FGL1 in NSCLC, proposing that FGL1 acts like a "shield machine cutter" in mediating T1 NSCLC N2 lymph node tube formation, creating metastasis channels. This provides the basis for novel FGL1-targeting treatment strategies.