Spatiotemporal Remodeling of the Tumor-Draining Lymph Node Microenvironment During Head and Neck Cancer Metastasis

Objectives.: The spatiotemporal evolution of the tumor-draining lymph node microenvironment remains incompletely characterized in head and neck squamous cell carcinoma (HNSCC). Using a reproducible buccal xenograft model with serial time-point and spatial analyses, we aimed to define stage-and region-resolved microenvironmental remodeling during cervical lymph node metastasis. Methods.: A buccal xenograft model of cervical lymph node metastasis was used, and tumor-draining lymph nodes were analyzed at serial time points using immunofluorescence staining and confocal microscopy. The lymphovascular architecture, stromal remodeling, hypoxia, and immune cell distribution were evaluated during metastatic progression. Results.: The model reproducibly induced cervical lymph node metastasis with a gradual temporal progression. Before overt tumor colonization, tumor-draining lymph nodes showed features of a pre-metastatic niche, including expansion of LYVE-1⁺ lymphatic vessels, remodeling of high endothelial venules, and increased recruitment of CD11b⁺ myeloid cells and F4/80⁺ macrophages. Confocal imaging revealed that the tumor cells were initially localized within the subcapsular sinus lymphatic vessels and subsequently expanded into the lymph node parenchyma. During invasive progression, metastatic regions showed progressive loss of CD31⁺ blood vessels and LYVE-1⁺ lymphatic vessels, accompanied by GLUT1 upregulation, fibrinogen deposition, disruption of PDGFRβ⁺ fibroreticular networks, and spatially organized myeloid cell accumulation at the invasive front. Conclusion.: This study delineated a stage- and region-resolved sequence of nodal microenvironmental remodeling during HNSCC metastasis, linking premetastatic lymphovascular changes to intralymphatic tumor colonization and subsequent invasive regional remodeling. These findings provide an integrated spatiotemporal framework for understanding the progression of the metastatic lymph node microenvironment.