Single‐cell profiling reveals peripheral blood immune landscape remodelling in breast cancer lymph node metastasis

BACKGROUND: Axillary lymph node (LN) metastasis significantly impacts breast cancer (BC) prognosis. The role of the systemic immune environment in promoting metastasis remains unclear. OBJECTIVES: This study describes peripheral blood immune alterations associated with LN metastasis in BC patients. METHODS: Peripheral blood mononuclear cells (PBMCs) from six BC patients with LN metastasis-positive (Pos) and negative (Neg) underwent single-cell RNA/T-cell receptor sequencing (scRNA-seq/scTCR-seq). Findings were validated by in vitro functional experiments and single-cell data from additional 14 BC tissues. RESULTS: scRNA-seq identified pro-metastatic immune subpopulations enriched in Pos group: neutrophilRSAD2 with expressing interferon-stimulated genes (ISG) and neutrophilMMP9 with expressing pro-angiogenic factors, and immunosuppressive CD4TregFOXP3 and cytotoxic-exhausted TandNK cells CD8TeffGZMH and NKTGNLY; and anti-metastatic CD8TeffCCL5 in Neg group. Pos group showed expanded large clonal T cells expressing cytotoxicity markers GZMH/GNLY. LGALS9‒HAVCR2 checkpoint interactions were elevated in Pos group through the neutrophil/mononuclear phagocyte (MP) ‒MP/TandNK axis, which was associated with an immunosuppressive microenvironment and metastatic status. These pro- and anti-metastatic immune signatures and LGALS9‒HAVCR2 axis were confirmed by in vitro experiments and consistently observed in BC tissues. Furthermore, functional co-culture assays established a causal relationship: interferon-stimulated neutrophils secreted LGALS9 and impaired their cytotoxic function on T cells. Conversely, neutralising LGALS9 or disrupting this axis restored T-cell-mediated tumour cell killing. CONCLUSION: LN metastasis is associated with a remodelled systemic immunity, enriches immunosuppressive neutrophils and TandNK cells and activates LGALS9‒HAVCR2 signalling. These findings reveal immune signatures associated with LN metastasis status and identify candidate biomarkers and therapeutic targets warranting further validation.