Abstract
Objectives: In vitro and in vivo analyses were conducted to examine the correlations between the production, expression, and secretion of the VEGF-C/D-VEGFR-3/NRP-2 axis and various levels of lymphatic metastatic potential in murine hepatocellular carcinoma cells. Methods: Quantitative real-time PCR, western blotting, cytoimmunofluorescence, immunohistochemistry, and enzyme-linked immunosorbent assay were employed to assess the expression and secretion of the VEGF-C/D-VEGFR-3/NRP-2 axis at the gene, protein, cytological, and histological levels. Results: In both in vitro and in vivo experiments, the ligands VEGF-C/D and receptors VEGFR-3/NRP-2 were primarily localized in the cytoplasm or cell membrane of hepatocarcinoma F/P cells with high/low lymphatic metastatic potentials, and in normal hepatocytes, respectively. The production and expression levels of the VEGF-C/D-VEGFR-3/NRP-2 axis in F/P cells were significantly higher than those in normal liver cells. Additionally, the production and expression of this axis were higher in F cells compared to P cells (P < 0.01). Notably, within both F/P cells, the production and expression levels of VEGF-C and VEGFR-3 were lower than those of VEGF-D and NRP-2 (P < 0.05). However, the secretion of VEGF-C exceeded that of VEGF-D in the supernatant of cultured cells and in the serum of tumor-bearing mice (P < 0.01). Interestingly, the ratio of VEGF-C/D in F cells was significantly higher than that in P cells and normal hepatocytes. Moreover, the ratio of VEGF-C/D was notably elevated in the supernatant of tumor cells and serum from tumor-bearing mice, as determined by ELISA, compared to that in tumor cells and tissues assessed using other technologies (P < 0.01). Conclusion: The significant co-production, co-expression, and co-secretion of the VEGF-C/D-VEGFR-3/NRP-2 axis are distinctive features of hepatocarcinoma cells, particularly those with a heightened potential for lymphatic metastasis.