Within research exploring the mechanisms of tumor metastasis, there are more and more studies examining the relationship between tumor lymphangiogenesis and tumor metastasis. The important role of lymphatic vessels in tumor metastasis, and especially solid tumor metastasis, has been recognized gradually. Clinicopathological research has demonstrated that the earliest path for solid tumor metastasis is regional spreading to the lymph nodes through lymphatic vessels. The regulatory mechanism for lymphangiogenesis in tumors is complicated and there are multiple factors involved. Among them, VEGF-C, VEGF-D, and Flt-4 were thought to be the major regulatory factors for tumor lymphangiogenesis, and likely play an important role in the lymphatic tumor metastasis. Animal tumor models with overexpression of VEGF-C or VEGF-D have shown that the lymphatic endothelial cells proliferated quickly in tumor tissues, LVD increased significantly, and lymph node metastasis was also enhanced. Jeltsch et al.  reported that high-expression of VEGF-C in a K14-VEGF-C transgenic mice model promoted lymphatic endothelial cell proliferation and enlargement of lymph vessel cavity in the dermis of mice. Von Marschall et al.  transplanted human pancreatic duct cancer cells that overexpressed VEGF-D into nude mice and detected LYVE-1 positive lymphatic vessels. They found that VEGF-D significantly induced the lymphangiogenesis, increased LVD in the tumor tissues, and was closely related to the significantly increased lymphatic vessel invasion and lymph node metastasis. In our study, we showed that VEGF-C, VEGF-D, and Flt-4 were significantly correlated with lymph node metastasis and lymphatic vessel invasion. Meanwhile, we used a polyclonal LYVE-1 antibody to label lymphatic vessels. Since the measurement of lymphatic vessel density can be quite subjective, a strategy was applied in which the lymphatic vessel density was measured by two expert pathologists, who were blinded for clinical data. LVD counting demonstrated that LVD was associated with lymph node metastasis and lymphatic vessel invasion and was closely related to levels of VEGF-C and VEGF-D, which is consistent with previous clinical studies [7–9]. The underlying mechanism may be secretion of VEGF-C and VEGF-D by tumor cells, which then function through the receptor tyrosine kinase Flt-4 in lymphatic endothelial cells in a paracrine manner and promote endothelial proliferation, differentiation and cavity formation. These newly generated lymphatic vessels in tumor tissues are structurally similar to the physiological lymphatic vessels, but occur in large numbers and in thin walls. These features provide more paths for tumor cell infiltration and facilitate tumor metastasis.
The mechanism of tumor lymphangiogenesis is complicated and involves an interaction between tumor cells and lymphatic endothelial cells. It has been shown that increased lymphatic vessels in tumor not only provide paths for tumor cell metastasis, but also release proteases under the stimulation of VEGF-C and promote basal membrane infiltration of tumor cells . On the other hand, VEGF-C also changed the adherent features and expression of surface chemo-attractants and receptors, affected the process by which tumor cells enter lymphatic vessels and therefore actively promote the tumor lymphatic metastasis .
Although increased LVD provides more metastatic pathways and plays an important role in tumor lymphatic metastasis, the process of tumor lymphatic metastasis is complicated and has multiple steps, including tumor cell migration, degradation of extracellular matrix, and relocation. Migration and invasion of tumor cells are prerequisites for tumor metastasis and infiltration. As the receptor for VEGF-C and VEGF-D, Flt-4 is expressed in not only the lymphatic endothelial cells, but also in the liver and spleen blood sinus, during injury repair, and in newly generated tumor blood vessel endothelium. Recent studies have shown that Flt-4 was also expressed in many types of tumor cells [12, 13] and played an important role in tumor lymphatic metastasis and tumor progression by promoting tumor cell proliferation, growth, and migration .
Su et al.  used in vitro migration and invasion methods and found that some tumor cells with a strong invasion ability, such as cervical carcinoma cell SiHa, had not only a high expression level of VEGF-C, but also a high level of Flt-4. Human recombinant VEGF-C (Cys 156 Ser) protein could promote the migration and invasion of tumor cells. Application of recombinant Flt-4/Fc blocked signaling of VEGF-C and also significantly decreased tumor migration and invasion. This suggested that Flt-4/Fc enhances lymphangiogenesis by affecting paracrine signaling, and that VEGF-C, VEGF-D and Flt-4 might also have an autocrine function in promoting tumor cell migration and invasion, which could eventually lead to tumor lymphatic metastasis. Van et al.  found that in the transition from localized cervical epithelial neoplasia to metastatic cervical carcinoma, the expression of VEGF-C, VEGF-D, and Flt-4 increased gradually. Therefore, it was speculated that VEGF-C, VEGF-D and Flt-4 could be involved in the process of phenotypic transition to lymphangiogenesis and could facilitate lymphatic metastasis in the early stages of cervical cancer. In addition, Masood et al.  found that VEGF-C and VEGFR-3 activation promoted the growth of malignant pleural endotheliomas. Consistently, the application of antisense oligos against VEGF-C, recombinant VEGFR-3/Fc, or VEGFR-3 antibody to inhibit VEGF-C/VEGFR-3 signaling led to a significantly lower survival of malignant pleural endotheliomas cells. In the current study, we found that in cervical carcinoma, Flt-4 was expressed not only in blood vessel and lymphatic vessel endothelial cells, but also in tumor cells, and that the level of Flt-4 was positively correlated with lymph node metastasis and lymphatic vessel infiltration. This is inconsistent with the results from a previous study by Jüttner et al. . One possible reason for this inconsistency is that there are multiple receptors of VEGF-C and VEGF-D and their expression is heterogeneous in different tumor cells. Except for Flt-4, VEGFR-2, NRP-1 and NRP-2 can all function as receptors for VEGF-C and VEGF-D . Therefore, the roles of VEGF-C, VEGF-D, and Flt-4 in the progress of tumors are omnifarious and the underlying mechanisms of these growth factors need to be further studied.
Our research showed that the specificity of Flt-4 as a lymphatic vessel marker was not high. Some of the Flt-4 positive vessels were morphological blood vessels and other vessels were lymphatic vessels. We found that FVD was positively associated with the FIGO stage of cervical cancer, but was not related to the other clinicopathological features including histological grade, lymph node metastasis, or lymphatic vessel infiltration. In addition, we found that FVD was correlated with the expression of VEGF-C and VEGF-D. This is inconsistent with Yasuoka et al. . The VEGF receptor tyrosine kinase family includes VEGFR-1, VEGFR-2, and VEGFR-3. VEGF-1 and VEGF-2 are primarily expressed in blood vessel endothelial cells and are involved in tumor angiogenesis. Since Flt-4 is expressed in the endothelial cells of blood vessels and lymphatic vessels, VEGF-C, VEGF-D, and Flt-4 may also play important roles in tumor angiogenesis .
In summary, our results indicated that VEGF-C, VEGF-D, and Flt-4 may promote tumor lymphangiogenesis and may provide a spreading route for tumor metastasis through a paracrine mechanism. On the other hand, they may function in an autocrine manner to enhance tumor cell migration and invasion and may therefore play an important role in the lymphatic vessel metastasis of early-stage cervical carcinoma.