Ovarian carcinoma is the fifth leading cause of death among all gynecological malignancies in development countries [16]. Due to the lack of typical clinical symptoms and available precise biomarkers, 70% patients have widespread metastasis at advanced stages when diagnosed [17]. Although the overall survival of patients with ovarian carcinoma has greatly improved along with the advances in clinical diagnosis and treatment, the effective treatment and control method still lack for patients at advanced stage, especially for those with lymph metastasis. Therefore, it is central to uncover the underlying mechanism of tumor metastasis so as to develop effective therapeutic strategy. Abundant investigations have been done on angiogenesis and vascular relevant molecules, and much progress have been made, while with respect to the lymphatic spread, much smaller number of researches has been done.
The observation and definition of nodal invasion is the basement for estimate the lymphatic spread, which is the first step of tumor dissemination. To our knowledge, there are no previous reports about the determination of metastasis. In our study, we established the lymphogeneous high metastasis animal model with SKOV-3 cells transfected with VEGF-D [6], and explored the underlying mechanisms of the lymph metastasis.
VEGF-D, an angiogenic and lymphangiogenic glycoprotein, can facilitate tumor growth and distant organ metastasis [11]. Accumulated evidences have proved the expression of VEGF-D is usually connected with tumor metastasis and poor patient outcome. For instance, Wei et al., found VEGF-D was involved and played an important role in gallbladder cancer progression, which indicted that VEGF-D was a potential molecular target in the treatment of gallbladder cancer [18]. VEGF-D was highly expressed and could predict the lymph node metastasis in patients with urothelial carcinoma of the bladder at the time of radical cystectomy, which combined with the low MMP-2 serum levels [14]. Moreover, the up-regulation of VEGF-D was significantly correlated with CXCR4, CCR7 and VEGF-C in the lymph node metastasis of patients with cervical cancer [19]. Besides, we had verified that VEGF-D could promote the lymph metastasis [6]. Our study revealed that the over-expression of VEGF-D was positive to the tumor growth, indicating that VEGF-D could raise the proliferative capability of tumor cells. It was reported that the lower the degree of differentiation of tumor cells, the stronger the ability of cell proliferation and invasion, the stronger the sex, the more malignant it is [20]. However, whether VEGF-D could increase the malignancy of the tumor via strengthening the tumor cell proliferation needs further studies.
Sentinel lymph node (SLN) has been demonstrated to become functional blood vessel-enriched and lymph vessel/sinus-enriched before metastasis formation, and there are reorganizations of the lymphatic channels and the vasculature before the establishment of metastasis in the node [21]. The dilation of the lymph sinuses is correlated with the primary tumor weight and the proliferation rate of the endothelial cells is significantly increased [22]. In our study, we found that there were a few isolated tumor cells in enlarged marginal sinus of SLN in SR group before apparent metastasis focus was established, which might be related to the dilation of lymph sinuses. In addition, we discovered that it is not a very short interval from formation of primary tumor mass to nodal involvement, comprising four steps. Firstly, presence of increased lymphatic sinus in size. Secondly, invasion of single or scattered cancer cells via sinus. Thirdly, formation of cancer cell clusters in the sinus or into the parenchyma. Finally, cancer cells defeated the lymphocytes and proliferated in the cortex. What we have found suggests and emphasizes it is vital to diagnose at earlier stages and to seek routinely realistic effective screening program.
Moreover, we investigated the morphology of the invaded nodes which displayed that some types of atypical morph of metastatic tumor cells were occurred in the involved lymph nodes. Herein, we speculated this phenomenon might occur in clinical cases. Many papers have revealed that the poor patient survival of unsuitable treatment was caused by missing microinvasion. Consistent with this, we assumed missing atypical metastases also resulted in the worse outcome of patients. It should be argued that immunostaining for CA125 or CD31 might be a promising approach to tell the atypical lymphatic metastasis, which deserved further investigations.
Furthermore, the role of VEGF-D and the underlying mechanisms of ovarian tumor metastasis was investigated. A high tendency of earlier and dilated lymph node appeared in SR group, and enlargement, fixation and harder feature (osseous metaplasia) of lymph nodes seemed to be indicators of metastases.
Finding that microscopic blood vessels were increased in VEGF-D overexpression tumors was not anticipated, which may be another reason for tumorigenesis and tumor growth caused by VEGF-D. It may be due to the activation between VEGF-D and up-regulated VEGFR-3 on blood vessels within tumors, and to the increased microscopic vessel density caused by strong MMP-2 expression. It could not be excluded the possibility that VEGF-D bind to the VEGFR-3-VEGFR-2 heterodimers [23, 24]. Currently, we used Evan’s blue dye to explore tumor-draining lymph flux. More rapid dye perfusion into the SR lymph nodes was observed, both in the popliteal and iliac nodes (more central lymph nodes), which indicated an accelerated lymph flow both into and out of lymph nodes. We deduced that VEGF-D-induced lymphangiogenesis and enlarged lymphatic sinus might facilitate tumor cell transport via accelerate lymph flow and finally act as a stimulator of lymphatic spread.