血小板反應蛋白-4募集淋巴管內皮細胞誘導人類膀胱腫瘤的淋巴管新生

仇光宇¹、黃一勝^1,3,4、蔡德甫¹、何肇晏¹、張安辰²

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

Recruitment of lymphatic endothelial cells by thrombospondin-4 induces lymphangiogenesis in human bladder tumors

Kuang-Yu Chou¹, Thomas I-Sheng Hwang^1,3,4,Te-Fu Tsai¹, Chao-Yen Ho¹and, An-Chen Chang²

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.

Purpose:

Bladder cancer (BC) ranks second among the most prevalent urinary tract malignancies. Thrombospondin-4 (TSP4), an extracellular calcium-binding protein, has been shown to regulate the extracellular matrix in the tumor microenvironment (TME). Although TSP4 has been reported to have the ability to promote cancer cell invasiveness, its role in regulating lymphatic metastasis is not well understood. The aim of this study was to investigate whether the secretion of TSP4 by BC cells leads to the recruitment of lymphatic endothelial cells (LEC) to the TME and subsequently 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.

Results:

In our in vitro investigation, we observed that high-grade BC cells (5637 and T24) had significantly higher levels of TSP4 mRNA and protein expression than low-grade BC cells (RT4). The conditioned medium (CM) from 5637 and T24 cells induced recruitment of LECs, and this effect was disrupted when T24 CM was pretreated with TSP4 monoclonal antibody (mAb). Interestingly, 5637 and T24 CM also promoted LEC tube formation, but TSP4 mAb had no effect on this. To confirm the lymphangiogenic function of TSP4, we stimulated LECs with recombinant human TSP4 protein (rhTSP4), which directly increased LEC recruitment through the integrin αvβ3/FAK/ERK signaling pathway. Furthermore, rhTSP4 promoted the expression of vascular endothelial growth factor-C (VEGF-C) in BC cells, indicating that BC-derived TSP4 may induce its own production of VEGF-C to regulate lymphangiogenesis. Finally, we found a positive correlation between TSP4 and VEGF-C in human BC tissues.

Conclusions: 

Our findings revealed for the first time the underlying mechanism of TSP4-mediated lymphangiogenesis, which involves the recruitment of LECs to the TME and upregulation of VEGF-C expression in BC. These findings highlight the potential of TSP4 as a therapeutic target to inhibit lymphatic metastasis in BC.