Single-cell identification of an endothelial cell proximal SPP1+ macrophage population defines the metastatic vascular niche in lymph nodes.

Head and neck squamous cell carcinoma (HNSCC) is a highly prevalent malignancy with poor prognosis, largely driven by lymph node metastasis (LNM). Despite its clinical significance, the underlying mechanisms of LNM remain elusive. In this study, we used single-cell transcriptomic data to dissect the cellular and molecular interactions within metastatic lymph nodes (MET). Specifically, we analyzed Single-cell RNA sequencing (scRNA-seq) data from GSE195655, GSE140042, GSE227156, and GSE159929 (n = 41) to delineate cellular heterogeneity, intercellular communication networks, and functional enrichment in primary tumors (PT), MET, and non-metastatic lymph nodes (LN). Our analysis identified a subpopulation of tumor-associated macrophages (TAMs) distinctly enriched in MET, characterized by high expression of SPP1. Functional analysis revealed that this TAM subpopulation promotes angiogenesis through specific ligand-receptor interactions with endothelial cells (ECs), involving the SPP1-ITGa9b1 and FN1-ITGa2b1 signaling axes. Furthermore, we leveraged bulk RNA-seq for prognostic research. Immunohistochemistry (IHC) confirmed the increased density and number of blood and lymphatic vessels in MET. Spatial analysis via multiplex immunohistochemistry (mIHC) confirmed the preferential localization of SPP1+ TAMs near ECs in MET. These results suggest a potential communication between TAMs and ECs that contributes to LNM in HNSCC, providing critical insights for HNSCC prognosis and precision treatment.