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- Materials and Methods
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Lymphatic spread is an important clinical determinant in the prognosis of many human cancers. The lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) is implicated in the promotion of lymphatic metastasis through the development of lymphatic vessels in some human cancers. In this study, we developed an anti-VEGF-D monoclonal antibody, cVE199, and investigated its in vitro properties, in vivo effects against tumors and possible target indications to evaluate its potential as a therapeutic antibody. The cVE199 molecule was revealed to have a specific binding reactivity against human VEGF-D, as well as a specific inhibitory activity against the binding of human VEGF-D to VEGFR-3. In addition, cVE199 was found to inhibit the biological activity of VEGF-D against lymphatic cells in vitro. Because we determined that a neuroblastoma cell line, SK-N-DZ, abundantly expressed VEGF-D, an in vivo efficacy study was performed using a xenograft model of SK-N-DZ. We found that cVE199 significantly decreased lymphatic metastasis of SK-N-DZ as well as lymphangiogenesis in primary lesions. Finally, we investigated VEGF-D expression in human neuroblastoma, finding that the molecule was expressed in 11 of 29 human neuroblastoma specimens (37.9%). In conclusion, we found that a novel anti-VEGF-D monoclonal antibody, cVE199, with specific reactivity against human VEGF-D, prevents lymphatic metastasis of neuroblastoma through the inhibition of lymphangiogenesis in an animal model. In addition, our results show that VEGF-D is expressed in some cases of human neuroblastomas, which suggests that cVE199 is a potential anti-metastasis therapeutic antibody in neuroblastoma treatment.
Lethality in cancer is primarily associated with metastasis. In one type of this phenomenon, known as lymphatic metastasis, cancer cells spread to the lymph nodes. This type of metastasis serves as an important prognostic indicator for disease state and, indeed, lymphatic metastasis is correlated with poor prognosis in many tumor types.[2-7]
Multiple steps are required for lymphatic metastasis to occur, including: detachment from the primary tumor mass; invasion into lymphatic vessels; transport through draining lymphatic vessels; and, finally, arrest, survival, and growth in the lymph nodes.[8, 9] The first step is lymphangiogenesis, the formation in tumors of new lymphatic vasculature from preexisting vessels. Reliable markers for lymph vessels (e.g. CD44-related hyaluronan receptor LYVE-1) have revealed that tumor-associated upregulation of lymphangiogenesis is linked to increased lymphatic spread and poor prognosis in human cancers. Hence, lymphangiogenesis has become a focus of interest in lymphatic metastasis.
A novel member of the vascular endothelial growth factor (VEGF) family, VEGF-D, has been implicated in the promotion of lymphangiogenesis. This molecule is synthesized as a proprotein. Following synthesis, the VEGF homology domain region is proteolytically cleaved and secreted into the extracellular environment. This processing progressively increases the affinity of the molecule for its receptors. In general, VEGF-D binds to VEGF receptor (VEGFR)-2, found on both blood and lymphatic vessels, and VEGFR-3, found predominantly on lymphatic vessels and in some angiogenic tumor blood vessels.[12, 13] VEGFR-3 signaling is the primary factor responsible for the lymphangiogenic response to VEGF-D stimulation and leads to lymphangiogenesis in mouse models. Several experimental and clinical studies have shown positive correlations among VEGF-D expression, tumor lymphatic vessel density and lymphatic metastasis. Moreover, a number of reports have shown that higher VEGF-D expression correlates with poor prognosis in human tumors.[16-21] Therefore, the blockade of VEGF-D seems to be a promising method of preventing the lymphatic metastasis of tumors.
To explore this possibility, we generated a novel anti-VEGF-D monoclonal antibody, cVE199, and investigated its in vitro binding and neutralizing activity against human VEGF-D, its in vivo anti-lymph node metastasis efficacy against VEGF-D-expressing tumors and its possible indications in human cancer treatment.
- Top of page
- Materials and Methods
- Disclosure Statement
- Supporting Information
Lymphatic spread is an important clinical determinant in the prognosis of many tumors. Therefore, therapeutic modalities capable of inhibiting lymphatic spread would be predicted to convey a clinical benefit in the treatment of many tumors. VEGF-D is implicated in lymphatic spread through the promotion of lymphangiogenesis in human cancers. In addition, high VEGF-D expression has been shown to correlate well with poor prognosis in many cancers. These findings suggest that anti-VEGF-D would be a promising agent in cancer treatment.
In this study, we generated a novel anti-VEGF-D chimeric monoclonal antibody, cVE199, which has a specific binding reactivity and a neutralizing activity against human VEGF-D. The result of an assay using HMVEC-Lly cells suggested that cVE199 preferentially inhibited VEGF-D/VEGFR-3 as compared to the VEGF-D/VEGFR-2 signal (Fig. 2b). A recent study showed that N-terminal residues of VEGF-D are important for VEGF-D binding and activation of VEGFR-3, but not of VEGFR-2. This finding suggests that cVE199 binds to N-terminal residues of VEGF-D, and so cVE199 preferentially inhibits the VEGF-D/VEGFR-3 signal.
In a xenograft study to evaluate the in vivo effect of cVE199, we used SK-N-DZ that natively expresses VEGF-D, although previous studies have used VEGF-D-transfected cell lines. This approach is important because it has a higher clinical relevance compared to overexpression experiments. Our result showing that cVE199 significantly decreased lymph node metastasis of SK-N-DZ with reduction of lymphangiogenesis strongly supports previously published conclusions drawn from VEGF-D overexpressing in vivo studies. These reports largely attribute the metastatic action of VEGF-D to an induction of lymphangiogenesis. For instance, VEGF-D stimulated lymphangiogenesis when the molecule was overexpressed in skin keratinocytes; it promoted the development of lymphatics and lymph node metastasis when overexpressed in tumors.[24, 26]
In this study, cVE199 did not alter blood vessels in the primary tumors of SK-N-DZ-luc xenografts. Although the VEGF-D/VEGFR-3 pathway is known to play a role not only in tumor lymphangiogenesis but also in pathological angiogenesis the VEGF-A/VEGFR-2 signal is known as the predominant regulator of physiological and tumor angiogenesis. We found that SK-N-DZ-luc cells expressed VEGF-A (Fig. S6). In addition, VEGFR-3 kinase inhibitor or VEGFR3-Ig, a potent inhibitor of VEGF-C/VEGF-D signaling, did not always affect tumor angiogenesis.[28, 29] These data suggest that the contribution of VEGF-D/VEGFR-3 signaling to tumor angiogenesis is affected by in vivo circumstances, such as VEGF-A expression. Furthermore, these findings might explain the reason for the ineffectiveness of cVE199 on tumor angiogenesis in the SK-N-DZ-luc xenograft.
Because cVE199 did not affect tumor growth (Fig. 5a,b) or angiogenesis (Fig. S5), it seems that cVE199 inhibits lymph node metastasis of SK-N-DZ-luc by inhibiting lymphangiogenesis. Thus, our finding also supports previously published data showing that formation of a dense lymphatic network facilitates metastatic tumor spread by increasing the likelihood that tumor cells will leave the primary tumor site and invade the lymphatics.
One of the most important findings of the present study was that cVE199 inhibits the lymphatic metastasis in an experimental neuroblastoma model (Fig. 5) and that VEGF-D was expressed in some human neuroblastomas (Fig. 6), implicating VEGF-D in the lymphatic metastasis of neuroblastoma; these findings further suggest cVE199 as a possible therapeutic antibody for neuroblastoma. Although many lower-stage neuroblastomas regress completely or differentiate into benign ganglioneuroblastoma without treatment, metastatic neuroblastoma (stage IV) in children over 1 year of age is lethal for most patients despite aggressive multimodality therapy.[31-34] Therefore, new therapeutic agents for neuroblastoma are still necessary, and our finding that cVE199 has potential as a therapeutic antibody for neuroblastoma is of significance.
In conclusion, the results of the present study implicate VEGF-D in the promotion of lymphatic metastasis in neuroblastoma and identify cVE199 as a potential therapeutic antibody against neuroblastoma in the prevention of this process.