Abstract
Background: In non-small cell lung cancer (NSCLC), lymph node (LN) metastasis is a crucial prognostic factor. Asparagine synthetase (ASNS) plays a crucial role in cellular aspartate metabolism and promotes LN metastasis. However, the mechanisms by which LN metastasis affects immune microenvironment remodeling in situ and tumor-draining LNs (TdLNs), as well as the role of ASNS in this process remains unclear. Methods: LN metastatic lung cancer cell lines were established through in vivo selection in a murine model and subsequently analyzed via metabolomic profiling. ASNS expression and its role in modulating immunogenicity were assessed using transcriptomic analysis, western blotting, and immunohistochemistry. Metabolomic profiling, combined with in vitro stimulation assays, identified key metabolic regulators involved in the axis. Furthermore, T-cell kinetics were monitored via flow cytometry, multiplex immunofluorescence and patient datasets. Tissue samples from NSCLC patients with LN metastases following neoadjuvant immunotherapy were employed to validate findings. Results: Elevated aspartate metabolism and ASNS expression were observed in LN metastasis based on metabolomic analyses of LN metastatic lung cancer cell lines and immunohistochemistry of tissue samples from LN metastasis, intrapulmonary implantation, LN injection models and NSCLC patients-derived samples. Higher ASNS expression in LN metastases correlated with enhanced immunogenicity. Mechanically, ASNS promoted the expression of major histocompatibility complex through α-aminobutyric acid auto-secretion in lung cancer cells. Moreover, in vivo and clinical studies revealed that metastatic tumor areas with high ASNS expression facilitated the formation of lymphocyte niches conducive to CD8+T cell activation, memory, and stemness within metastatic TdLNs, particularly in the vicinity of metastatic foci, thus reshaping the immune landscape in both tumors in situ and metastatic LNs. Clinical research confirmed that high ASNS expression in LN metastases correlated with improved efficacy of neoadjuvant immunotherapy in NSCLC patients. Conclusions: ASNS promotes anti-tumor immunity in NSCLC via regulating immunogenicity of cancer cells and immune microenvironment remodeling in metastatic TdLNs. Lung cancer cell-intrinsic ASNS appears to be a promising marker for anti-PD-1-based neoadjuvant immunotherapy.