Endostatin inhibits tumour lymphangiogenesis and lymphatic metastasis via cell surface nucleolin on lymphangiogenic endothelial cells.

Endostatin has potent anti-endothelial and anti-angiogenic functions. Endostatin was reported to reduce lymphangiogenesis by down-regulating the level of VEGF-C in tumour tissues. However, there is little evidence for the direct function of endostatin on lymphangiogenic endothelial cells and lymphangiogenic vessels. Here, we report that cell surface nucleolin, which was reported as an endostatin receptor mediating its anti-angiogenic and anti-tumour functions, is also selectively expressed on the cell surface of lymphangiogenic endothelial cells both in vitro and in vivo. Treatment of primary mouse lymphatic endothelial cells (mLECs) by endostatin inhibits mLEC migration, tubule formation, and activation of the Erk pathway in mLECs, while neutralization of cell surface nucleolin or nucleolin knockdown results in loss of the anti-lymphatic endothelial activities of endostatin. Also, anti-nucleolin antibody or lentivirus delivered nucleolin siRNA abolishes the anti-lymphangiogenic function of endostatin in the Matrigel plug assay. Endostatin remarkably inhibits tumour-associated lymphangiogenesis, leading to reduced lymphatic metastasis. Systemic blockade of nucleolin notably abolishes the anti-lymphangiogenic and anti-lymphatic metastatic functions of endostatin. Importantly, endostatin does not affect quiescent lymphatics in normal organs, which is consistent with the lack of expression of cell surface nucleolin in quiescent lymphatics. Taken together, our results demonstrate that endostatin directly acts on lymphangiogenic endothelial cells via cell surface nucleolin, which provides a novel mechanism for the inhibition of tumour lymphangiogenesis and lymphatic metastasis by endostatin.