Because RLN metastasis is an important factor in determining the prognosis of EC [2–4], the relationship between VEGF-c mRNA expression and RLN metastasis was evaluated in an in vivo model in the present study. The establishment of EC with RLN metastasis was confirmed at day 21 post-injection with VX2 cells. In addition, VEGF-c mRNA expression increased significantly over time in the tumor site, RLN tissue, and peripheral white blood cells. Metastatic RLNs expressed higher VEGF-c mRNA expression as compared to those without metastasis. Furthermore, significantly higher VEGF-c mRNA expression was observed in peripheral white blood cells of rabbits with RLN metastasis, indicating that VEGF-c levels may have predicative value for metastasis in EC.
The VX2 cell line is composed of squamous cell carcinoma cells derived from Shope virus-induced papilloma in rabbit . Their high survival rates make them a suitable candidate for in vivo inoculation , which has been carried out in liver, lung, uterus, and breast tissues to establish the corresponding animal models [14–16]. In the endometrium, inoculation of VX2 cells induced EC with lymph node metastasis after 14-21 days post-injection [14, 15], which is similar to the results of the present study. These previous studies employed blocks of VX2 cells in the myometrium using microsurgical instruments while the present study introduced the VX2 cells via an injection, which removes the dependence on microsurgical instruments, is less difficult, and may increase the success rate of establishing the model as was evident in the 100% success rate of establishing EC and RLN metastasis observed in this study. The high success rate of this study using this method is consistent with results reported by Chen et al. .
In addition to the advantage of a high success rate, the in vivo model of EC employed in the present study induced EC with lymph node metastasis by day 21, which was faster than that reported for other models. For example, establishment of a mouse model of EC with lymph node metastasis via 5 million HEC1A cells required eight weeks . Of note, 100% of the animals in the present study developed EC with RLN metastasis, which was greater than that observed using the HEC1A mouse model of 86.5% .
In the present study, an in vivo model of EC with RLN metastasis was established by injecting 500 million VX2 cells into the uterine muscularis mucosae of rabbits. Previous studies in our laboratory indicated that this number of cells was necessary for successful RLN metastasis (data not shown). Analysis of the peritoneal cavity revealed that primary tumors were restricted to within the uterus as peritoneal dissemination of the VX2 cells was not observed.
Tumor angiogenesis is related to its invasion and metastasis , and VEGF is an important factor regulating angiogenesis in cancers . The VEGF-c gene is mapped to chromosome 4q34 , and in vitro studies have confirmed that it induces lymphangiogenesis but not angiogenesis . Straume et al.  also reported that lymphatic vessel density in nodular melanoma was positively related to the VEGF-c level. Furthermore, previous studies have shown that the VEGF-c expression in solid cancers, including endometrial adenocarcinoma, was significantly higher than that observed in adjacent normal tissues [22–24], which is consistent with the data presented in this study. In patients with gastro-oesophageal junction adenocarcinoma, VEGF-c levels were associated with tumor stage, lymph node metastasis, and shorter periods of disease-free survival . Further studies will assess if the increase in VEGF-c expression is directly related to EC growth and metastasis through lymphangiogenesis. In addition, a possible synergism between VEGF-c and FGF-2 as reported by Cao et al.  will be explored.
In the present study, differences in VEGF-c mRNA expression were observed among rabbits without and with RLN metastasis; differences were also noted between rabbits with full and partial metastasis with the highest expression found in rabbits with metastasis to all RLNs. These results suggest that the increased VEGF-c may arise from the EC during its development and progression, which may subsequently promote lymph node metastasis . This data is consistent with that reported by Kimura et al.  in which VEGF-c levels correlated with lymph node metastasis and lymphatic involvement in esophageal squamous cell carcinoma. Although VEGF-c expression increased with tumor growth and RLN metastasis, further studies will evaluate whether its level is reflective of the cancer load and metastatic state of EC.
The current study was limited in that it did not confirm whether VEGF-c protein levels were correlated with those obtained for mRNA. Further studies will evaluate the VEGF-c protein levels, as well as markers of epithelial-mesenchymal transition (EMT), in tissues as well as serum samples using immunohistochemistry and enzyme-linked immunosorbent assays (ELISAs). In addition, although previous studies have reported a relationship between VEGF-c and lymphangiogenesis with metastasis [6–8], no mechanistic data were presented in this study. Finally, VEGF receptor expression was not determined.