VEGF-D was stably expressed in transfected cells
To detect the quality of the transfection in SKOV-3 cells, QT-PCR analysis was implemented to check he expression of VEGF-D. As a result, the expression of VEGF-D was stably expressed in cells transfected with pcDNA3.1 (+)/VEGF-D, while it was undetectable in the cells transfected with pcDNA3.1 (+) (Fig 1. P<0.05). The data indicated that the transfected cells were stable and VEFG-D was stably expressed.
The tumor growth in each group
To explore the tumor growth, the tumor volume was measured at the 0, 20 th, 40 th and 60 th day after cell inoculation in each group. The results was shown in Fig 2, the tumor growth began to accelerate from the 15 th day after tumor cell implantation in SR group. Moreover, the tumor growth rate was quicker than that in SV and SC group at every subsequent point of observation since the 20 th day after inoculation (P<0.05, Fig. 2). What’s more, the tumor growth was obviously expedited since the 25 th day. Nevertheless, there was no significant difference between SV group and SC group. These data demonstrated that the over-expression of VEGF-D promoted the tumor growth of ovarian carcinoma.
The hyperplasia of tumor lymphatic
To investigate the hyperplasia of tumor lymphatic, Evan’s Blue assay was implemented. The result manifested that the most lymphatic in draining areas and popliteal lymph nodes from mice in SR group were perfused with blue at the 20-minute time point, while infused functional lymphatic were hard to be found in the SV and SC groups (Fig. 3A-C). Furthermore, we checked the proliferation of tumor lymphatic. Consequently, the lymphatic was distinctly expanded in SR group, while the lumen of lymphatic vessels was much narrow even not visible in SC and SV groups (Fig. 3D-F). The data demonstrated that the expansion and proliferation of lymphatic could be enhanced by the over-expression of VEGF-D.
The dynamic metastasis of lymph nodes
To research the metastasis of lymph nodes after tumor inoculation, consecutive observation for gradient lymph nodes of the mice in three groups was conducted for two months, including inspection and palpation of the popliteal and inguinal lymph node (sentinel lymph node). As consequence, the inguinal lymph nodes of two mice in SR group were palpable in two weeks after inoculation, while the popliteal lymph node of one mouse was touched after four weeks of inoculation in SC group. Moreover, HE assay manifested that the nodal involvement occurred from the lower grading lymph nodes to the upper ones step by step (Fig. 4A-E). What’s more, the difference of the metastasis rates of gradient lymph nodes between SC and SV group wasn’t significant. However, the metastasis of lymph nodes was dramatically quicker in SR group compared to that in SC or SV group after being inoculated for 45 day (Fig. 4F-I).
The distinction of nodal metastasis morph
Although it is easy to confirm the typical metastasis focus using H&E staining, the atypical nodal involvement is hard to estimate. Herein, we analyzed the atypical nodal involvement via checking the expression of CA-125 and CD40 with immunohistochemical (IHC) staining. The data displayed that there were various types of atypical nodal involvement according to detect the expression of CA-125 (Fig. 5A-C) and CD40 (Fig. 5D-F) in comparison to the primary tumor sections.
VEGF-D increases the intratumoral lymphatic vessel density
To know whether VEGF-D played a role in blood vessel sprouting, IHC analysis was performed to detect the expression of LYVE-1 and CD31. Immunostaining with an antibody against LYVE-1 indicated that the lymph vessels density was obviously elevated while the immunostaining of CD31 showed that the density of microvessels was distinctly increased in SR group compared to that in SC and SV group (Fig. 6). These results suggested that the over-expression of VEGF-D could promote the proliferation of lymph vessels and the formation of tumor blood vessels.
MMP-2 was up-regulated by the over-expression of VEGF-D
Furthermore, we explored that whether the promoting effects of VEGF-D on the tumor lymph nodes metastasis was correlated with the enhanced expression of MMP-2. IHC analysis exhibited that MMP-2 were all positive expressed in three groups. Moreover, MMP-2 in SC and SV groups was weakly positive expressed (+) in tumor stroma instead of tumor cells (Fig. 7A, B), while more than 90% tumor cells and stroma of SR group showed much stronger staining for MMP-2 (+++) (Fig. 7C), which was properly corresponded with the over-expression of VEGF-D (Fig. 7D-F). All above, the tumor lymph metastasis was intensified by the over-expression of MMP-2 induced by VEGF-D.