Thrombospondin-4 drives lymphangiogenesis through cooperation with VEGF-C in human bladder cancer

<b>Introduction:</b> Bladder cancer (BLCA) is the second most common malignancy of the male urinary tract, with frequent metastasis to pelvic lymph nodes and, in advanced stages, to distant organs such as the lungs, liver, and bones. Thrombospondin-4 (TSP4), an extracellular matrix protein, has been implicated in cell adhesion, migration, proliferation, and cytoskeletal regulation. However, its role in lymphatic metastasis remains poorly understood. <b>Methods:</b> TSP4 mRNA and protein levels were assessed by RT-qPCR and Western blot analyses. Lymphangiogenesis and lymphatic endothelial cell (LEC) migration were respectively evaluated using tube-formation and transwell assays. The Cancer Genome Atlas (TCGA)-BLCA datasets were analyzed to compare expressions of TSP family members across lymph node metastasis stages. A popliteal lymph node metastasis model was employed to isolate lymph node-tropic BLCA cell lines. <b>Results:</b> TSP4 expression was positively associated with lymph node metastasis stages in BLCA tissues. In vitro, conditioned medium (CM) from 5637 and T24 cells promoted LEC recruitment and tube formation, while a TSP4-neutralizing antibody impaired LEC migration without affecting tube formation. Mechanistically, TSP4 enhanced LEC migration by upregulating intercellular cell adhesion molecule (ICAM)-1 and activating the integrin αvβ3/focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) pathway. Notably, TSP4 also induced vascular endothelial growth factor C (VEGF-C) expression in BLCA cells. Both TSP4 and VEGF-C were upregulated in lymph node-tropic BLCA cell lines, with a positive correlation observed in TCGA-BLCA datasets. VEGF-C neutralization abolished CM-induced LEC tube formation, highlighting the role of the TSP4/VEGF-C axis in lymphangiogenesis. <b>Conclusions:</b> This is the first study to demonstrate that BLCA-derived TSP4 cooperates with VEGF-C to promote lymphangiogenesis within the tumor microenvironment. These findings suggest that TSP4 could serve as a potential therapeutic target for preventing lymphatic metastasis in human BLCA.