Soluble Vegfr3 gene therapy suppresses multi‐organ metastasis in a mouse mammary cancer model

Accumulating evidence on the association of VEGF‐C with lymphangiogenesis and lymph node metastasis implicates lymphatic vessels as a potential target in anti‐cancer therapy. To evaluate whether blocking VEGF‐C and VEGFR‐3 signaling can inhibit multi‐organ metastases, a mouse metastatic mammary cancer model was subjected to gene therapy using a soluble VEGFR‐3 expression vector (psVEGFR‐3). We showed that psVEGFR‐3 significantly diminished cell growth in vitro with or without added VEGF‐C, and significantly reduced primary tumor growth and tumor metastases to wide‐spectrum organs in vivo. Although apoptotic cell death and angiogenesis levels did not differ between the control and psVEGFR‐3 groups, cell proliferation and lymphangiogenesis in the mammary tumors were significantly decreased in the psVEGFR‐3 group. Furthermore, lymphatic vessel invasion was significantly inhibited in this group. Real‐time RT‐PCR analysis revealed significantly high expression of the Vegfr3 gene due to gene therapy, and the transcriptional levels of Pcna and Lyve1 tended to decrease in the psVEGFR‐3 group. Immunofluorescence staining indicated that phospho‐tyrosine expression was considerably lower in tumor cells of psVEGFR‐3‐treated mammary carcinomas than those of control tumors. Double immunofluorescence staining indicated that phospho‐tyrosine(+)/LYVE‐1(+) (a lymphatic vessel marker) tended to decrease in psVEGFR‐3‐treated mammary carcinomas compared with control mice, indicating a decline in the activity of the VEGF‐C/VEGFR‐3 axis. These findings showed that a blockade of VEGF‐C/VEGFR‐3 signaling caused by sVEGFR‐3 sequestered VEGF‐C and prevented the side‐effects of anti‐angiogenesis and suppressed overall metastases, suggesting their high clinical significance.