1新光醫院 泌尿科，2轉譯醫學中心；3台北醫學大學 醫學院；4輔仁大學 醫學院
Thrombospondin-4 Induces Lymphangiogenesis by Lymphatic Endothelial Cell Recruitment in Bladder Tumor
Kuang-Yu Chou1, An-Chen Chang2, Te-Fu Tsai1, Hung-En Chen1, Chao-Yen Ho1Peng-Hui Chang1 and Thomas I-Sheng Hwang1,3,4
Department of Urology1 and Translational Medicine Center2, Shin Kong Wu Ho-Su Memorial Hospital; Department of Urology, Taipei Medical University3; Division of Urology, School of Medicine, Fu-Jen Catholic University4, Taipei, Taiwan.
Bladder cancer (BC) is the second most common malignancy of the urinary tract among men. Thrombospondin‐4 (TSP4), a member of the extracellular calcium‐binding protein family, is reported to modulate the extracellular matrix in the tumour microenvironment (TME). Although TSP4 has the capacity to promote cancer cell invasiveness, its roles in the regulation of lymphatic metastasis have not been adequately explored. Here, we aim to investigate whether TSP4 secretion from BC cells recruits lymphatic endothelial cells (LEC) to the TME and thus promotes lymphangiogenesis.
Materials and Methods:
The human BC cells (RT4, 5637 and T24) were obtained from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan). The qPCR assay and Western blot were performed to measure TSP4 mRNA and protein expression, respectively. Tube formation assay is an in vitro model for detecting lymphangiogenesis. Recruitment of LEC was analyzed by migration transwell assay.
From in vitro investigation, we found that high-grade BC cells (5637 and T24) exhibited significantly higher TSP4 mRNA and protein expression than low-grade BC cells (RT4). The conditioned medium (CM) from 5637 and T24 cells induced LEC recruitment, while pretreatment of T24 CM with TSP4 monoclonal antibody (mAb) interrupted this phenomenon. Interestingly, 5637 and T24 CM also promoted LEC tube formation; however, TSP4 mAb had no such effect. To confirm the lymphangiogenic function of TSP4, LEC were stimulated with recombinant human TSP4 protein (rhTSP4). Resulting data revealed that rhTSP4 directly elevated LEC recruitment but not LEC tube formation. Moreover, rhTSP4 promoted vascular endothelial growth factor-C (VEGF-C) expression in BC cells, indicating BC-derived TSP4 may induce its own production of VEGF-C to regulate lymphangiogenesis.
Our study is the first to describe the mechanism of TSP4-promoted lymphangiogenesis by facilitating LEC recruitment into TME and by upregulating VEGF-C expression in BC. Thus, TSP4 could serve as a therapeutic target in inhibiting lymphatic metastasis of BC.