Abstract
BACKGROUND: Lymph node (LN) metastasis is not only a prognostic indicator but also a driver of systemic immune suppression. However, the mechanisms of immune escape within LNs remain unclear. METHODS: Our pan-cancer single-cell transcriptomic analysis integrated public and in-house datasets from 11 cancer types, comprising 859,744 cells across 132 samples. We subsequently applied unsupervised clustering and cell-type annotation to identify dominant immunosuppressive cell populations within metastatic lymph nodes. Focused transcriptomic and pathway enrichment analyses were used to characterize the molecular features of these populations. Mechanistic studies, including in vitro and in vivo functional assays, were conducted to elucidate the signaling pathways underlying their immunoregulatory activity. RESULTS: Our analysis revealed that LN metastases exhibit a profoundly immunosuppressive microenvironment compared to primary tumors, characterized by: (1) Significant enrichment of an IL-10-producing macrophage subpopulation (Galectin-9(high)) specifically in LNs; (2) Galectin-9-mediated activation of the BTK/STAT3 pathway, driving IL-10 secretion and creating an immune-tolerant niche; (3) STAT3 inhibition selectively depleted these macrophages and, combined with PD-1 blockade, reduced LN metastatic burden in vivo. CONCLUSION: Our study identifies a Galectin-9/BTK/STAT3 axis that drives macrophage-mediated immune suppression in LN metastases, offering a potential therapeutic strategy.