Characterization of a monoclonal antibody that antagonizes the function of human endostatin

Endostatin, a 20-kDa proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. The anti-angiogenic effects of endostatin include inhibition of endothelial cell migration and proliferation, and inhibition of the activity of MMP2. Structure-function analysis of endostatin that implies this contravention function buried in separate fragments of endostatin introduces new issues into the understanding of the structure-function relationship of endostatin. We developed and characterized a novel murine MAb, 4E7, to human endostatin, which antagonizes the function of endostatin. As we show here, MAb 4E7 blocks the anti-migration/adhesion effects of endostatin in vitro and the anti-angiogenesis effect of endostatin in vivo, but the inhibition effect of endostatin on endothelial cell proliferation is not affected by MAb4E7. These results suggest that the anti-migration and anti-proliferation functions of endostatin may have distinct structural foundations.     

Enhanced antiangiogenic therapy of squamous cell carcinoma by combined endostatin and epidermal growth factor receptor-antisense therapy.

PURPOSE: We tested the combined effects of antiangiogenic endostatin and epidermal growth factor receptor (EGFR) antisense gene therapy on squamous cell carcinoma (SCC). EXPERIMENTAL DESIGN and Results: The 1483 cell line of human head and neck SCC (HNSCC) and SCC-VII/SF murine SCC cells was used to establish tumors in nude mice and immunocompetent C3H mice, respectively. Tumor-bearing mice were treated with endostatin (20 mg/kg/day, s.c.), liposomal EGFR-antisense expression plasmid (25 microg/mouse, three times/week, intratumoral), a combination of both agents, or liposomal EGFR-sense plasmid as a control. Endostatin or EGFR-antisense alone significantly, yet partially, inhibited the growth of 1483 and SCC-VII/SF tumors, and a combination of both treatments completely blocked tumor growth. Immunohistochemistry analysis demonstrated that a complete suppression of tumor angiogenesis was achieved by the combination treatment. Down-regulation of vascular endothelial growth factor was shown in EGFR-antisense-treated tumors. These results suggest that the EGFR-antisense treatment, in addition to its inhibitory activity on tumor cell proliferation, might have a synergistic effect with endostatin on SCC-induced angiogenesis. In vitro studies demonstrated that EGFR inhibition by antisense oligonucleotides or EGFR-specific tyrosine kinase inhibitor down-regulated the production of VEGF in HNSCC cells. Additional experiments demonstrated that these EGFR inhibition approaches also directly suppressed the growth of endothelial cells. CONCLUSION: A combination of endostatin and EGFR targeting strategies profoundly inhibited the angiogenesis and growth of SCC in vivo. EGFR-antisense therapy might have multiple inhibitory effects against both tumor cells and endothelial cells, leading to enhanced antitumor efficacy. Such a combination strategy might represent a novel and promising approach for HNSCC therapy.     

Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.     

Endostatin regulates endothelial cell adhesion and cytoskeletal organization

Endostatin, an endogenous angiogenesis inhibitor, attenuates endothelial cell migration through an unknown mechanism. We show that endostatin induced tyrosine phosphorylation of focal adhesion kinase and paxillin, and promoted formation of focal adhesions and actin stress fibers, similar to fibroblast growth factor-2 (FGF-2). In cells cotreated with endostatin and FGF-2, focal adhesions and actin stress fibers were decreased, indicating that endostatin disturbs cell-matrix adhesion. Reduced tyrosine phosphorylation and cytoplasmic relocalization of beta-catenin in cells treated with FGF-2 and endostatin indicates that loosening of cell-cell adhesion is also disturbed by endostatin. These data provide a molecular basis both for the lack of effect of endostatin on the normal, quiescent vasculature, and its antagonistic effects on stimulated tumor vessels.     

Tumor escape from endogenous, extracellular matrix-associated angiogenesis inhibitors by up-regulation of multiple proangiogenic factors.

PURPOSE: Thrombospondin-1 (Tsp1), endostatin, and tumstatin are extracellular matrix-associated proteins that inhibit angiogenesis. We examined the mechanisms by which tumor cells may bypass the antiangiogenic effects of these endogenous regulators. EXPERIMENTAL DESIGN: CT26 colon and RenCa renal carcinoma cells were stably transfected with Tsp1, endostatin, or tumstatin cDNA. Subcutaneous and metastatic tumor growth in syngeneic mice was analyzed. Expression of proangiogenic factors in resulting tumors was measured by quantitative real-time PCR. The combination of Tsp1 and vascular endothelial growth factor (VEGF) receptor-2 inhibition was also examined. RESULTS: There was significant suppression of angiogenesis in flank tumors and liver metastases formed from cells overexpressing Tsp1, endostatin, or tumstatin. However, all tumors ultimately escaped angiogenesis inhibition. The combination of all three angiogenesis inhibitors had no additive effect beyond overexpression of a single inhibitor. Using quantitative real-time PCR, we found that VEGF and platelet-derived growth factor (PDGF)-A levels were routinely up-regulated at least 5-fold in all CT26 tumors overexpressing any antiangiogenic protein, and there were variable increases in angiopoietin 2 (Ang2), basic fibroblast growth factor, and PDGF-B. In contrast, RenCa tumors, which have high baseline levels of VEGF and PDGF-B, relied on basic fibroblast growth factor, Ang1, and PDGF-A up-regulation to counteract Tsp1 overexpression. Growth of CT26 cells with Tsp1 overexpression was suppressed when anti-VEGFR-2 treatment was added. CONCLUSIONS: Cancer cells with overexpression of three different endogenous angiogenesis inhibitor eventually escape angiogenesis inhibition by up-regulation of various proangiogenic factors. Tsp1, endostatin, and tumstatin may be functionally redundant in this system. These endogenous angiogenesis inhibitors are likely best used in combination with the blockade of proangiogenic pathways or with traditional chemotherapy or radiation therapy.     

Prolactin antagonist-endostatin fusion protein as a targeted dual-functional therapeutic agent for breast cancer

In previous studies (Chen, W. Y. et al., Clin. Cancer Res., 5:3583-3593, 1999; Chen, N Y. et al., Int. J. Oncol., 20:813-818, 2002), we have demonstrated the ability of the human prolactin (hPRL) antagonist, G129R, to inhibit human breast cancer cell proliferation in vitro and to slow the growth rate of tumors in mice. We further revealed that the possible mechanisms of G129R antitumor effects act through the induction of apoptosis via the regulation of bcl-2 gene expression. It has been established that to sustain tumor growth, it is necessary for the development of a network of blood vessels to bring in nutrients, a process called angiogenesis. The disruption of angiogenesis has been proven to be an effective strategy to cause regression of certain tumors. One of the best-studied angiogenesis inhibitors is endostatin, which acts through the inhibition of endothelial cells. In this study, we combine the anti-breast tumor effects of G129R and the antiangiogenic effects of endostatin by creating a novel fusion protein (G129R-endostatin) specifically for breast cancer therapy. The data presented here demonstrated that this novel fusion protein was able to bind to the PRL receptor (PRLR) on T-47D human breast cancer cells and inhibit the signal transduction induced by PRL. At the same time, G129R-endostatin inhibited human umbilical vein endothelial cell (HUVEC) proliferation and disrupted the formation of endothelial tube structures with potency similar to that of endostatin. More importantly, the therapeutic efficacy of G129R-endostatin was confirmed using a mouse breast cancer cell line 4T1 in vivo. G129R-endostatin has a significantly prolonged serum half-life as compared with that of G129R or endostatin alone, and exhibited greater tumor inhibitory effects than G129R and endostatin individually or in combination. Taken together, these data demonstrate the dual therapeutic effects of G129R-endostatin, and suggests that this fusion protein has great promise as a novel anti-breast cancer agent.     

Angiopoietin‐1 alters tumor growth by stabilizing blood vessels or by promoting angiogenesis

The maturation of blood vessels requires mural cell adhesion to endothelial cells. Angiopoietin‐1 (Ang1), a ligand for Tie2 receptor expressed on endothelial cells, plays a critical role in cell adhesion between mural cells and endothelial cells and in endothelial cell sprouting from preexisting vessels in the absence of mural cells. Much information has been amassed on the Tie2–Ang1 system in physiological blood vessel formation during embryogenesis; however, the role of Ang1 in the tumor environment and its interaction with mural cells has not been well documented. Here we studied how Ang1 regulates maturation of blood vessels using the human colon cancer cell line HT29 and the human prostate cancer cell line PC3, and studied how Ang1 affects tumor growth. In a xenograft tumor model using female nude mice, we found that Ang1 enhanced angiogenesis and resulted in tumor growth in the case of PC3 tumors but suppressed tumor growth in the case of HT29 tumors. In PC3 tumors, the number of mural cells adhering to endothelial cells was less than that in HT29 tumors. Ang1 induced sprouting angiogenesis in PC3 tumors although there was little maturation of blood vessels. On the other hand, there was abundant mural cell adhesion to endothelial cells in HT29 tumors and Ang1 did not induce angiogenesis. These results suggest that Ang1 alters tumor growth in a manner that is dependent on the adhesion of mural cells and their localization in the tumor environment. (Cancer Sci 2008; 99: 2373–2379)     

Inhibition of orthotopic human bladder tumor growth by lentiviral gene transfer of endostatin.

PURPOSE: Inhibitors of endothelial cell proliferation, such as endostatin, result in suppression of tumor-associated angiogenesis and can achieve growth-inhibitory effects depending on the type of tumor treated. The purpose of this study was to investigate whether local overexpression of endostatin could serve to diminish tumor growth of bladder cancer in vivo. EXPERIMENTAL DESIGN: We examined the capability of lentiviral-mediated gene transfer in vitro and therapeutic effects of lentivirus-based vectors expressing endostatin on tumor growth using an orthotopic human bladder tumor model. RESULTS: We found that self-inactivating lentivirus vectors containing green fluorescent protein, alone or in combination with endostatin, were capable of efficient and stable gene transfer to a variety of human bladder tumor cell lines. The production and secretion of endostatin from lentivirus-transduced KU-7 human bladder cancer cells was confirmed by Western blot and competitive enzyme immunoassay. Intravesical instillation of untransduced, green fluorescent protein control lentivirus-transduced, and endostatin-transduced KU-7 cells was performed in murine models to establish orthotopic tumors. Sustained long-term expression of endostatin was achieved in lentivirus-transduced orthotopic bladder tumors, and it was associated with decreased vascularization and inhibition of tumor growth. Lentivirus vector-mediated overexpression of endostatin did not affect the intrinsic production of basic fibroblast growth factor and vascular endothelial growth factor. CONCLUSIONS: These findings suggest that lentivirus-mediated gene transfer might represent an effective strategy for expression of angioinhibitory peptides to achieve inhibition of human bladder cancer proliferation and tumor progression.     

Deletion of the endothelial Bmx tyrosine kinase decreases tumor angiogenesis and growth

Bmx (Bone marrow kinase in chromosome X), also known as Etk, is a member of the Tec family of nonreceptor tyrosine kinases. Bmx is expressed mainly in arterial endothelia and in myeloid hematopoietic cells. Bmx regulates ischemia-mediated arteriogenesis and lymphangiogenesis, but its role in tumor angiogenesis is not known. In this study, we characterized the function of Bmx in tumor growth using both Bmx knockout and transgenic mice. Isogenic colon, lung, and melanoma tumor xenotransplants showed reductions in growth and tumor angiogenesis in Bmx gene-deleted ((-/-)) mice, whereas developmental angiogenesis was not affected. In addition, growth of transgenic pancreatic islet carcinomas and intestinal adenomas was also slower in Bmx(-/-) mice. Knockout mice showed high levels of Bmx expression in endothelial cells of tumor-associated and peritumoral arteries. Moreover, endothelial cells lacking Bmx showed impaired phosphorylation of extracellular signal-regulated kinase (Erk) upon VEGF stimulation, indicating that Bmx contributes to the transduction of vascular endothelial growth factor signals. In transgenic mice overexpressing Bmx in epidermal keratinocytes, tumors induced by a two-stage chemical skin carcinogenesis treatment showed increased growth and angiogenesis. Our findings therefore indicate that Bmx activity contributes to tumor angiogenesis and growth.     

Gene transfer of endostatin enhances the efficacy of doxorubicin to suppress human hepatocellular carcinomas in mice

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death, and is chemoresistant to anticancer drugs. Anti-angiogenic therapy has been shown to enhance the efficacy of chemotherapy to treat solid tumors. The aim of the present study was to determine whether endostatin, a potent antiangiogenic agent, could enhance the efficacy of doxorubicin to combat HCC. An endostatin expression plasmid was constructed and its expression in vitro and in vivo was detected after gene transfer. Recombinant endostatin inhibited angiogenesis in the chorioallantoic membrane assay, and showed synergistic effects with doxorubicin in inhibiting the in vitro proliferation of endothelial cells, but not that of tumor cells. Both endostatin gene therapy and doxorubicin suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, and tumor angiogenesis. Combination therapy with endostatin gene therapy and doxorubicin showed a stronger effect in suppressing tumor growth, and tumor angiogenesis, than the respective monotherapies. Gene transfer of endostatin down-regulated the expression of both hypoxia-inducible factor-1alpha and vascular endothelial growth factor (VEGF), whereas doxorubicin only down-regulated VEGF expression. Endostatin and doxorubicin synergized to down-regulate VEGF expression. Endostatin and doxorubicin combination therapy warrants investigation as a therapeutic strategy to combat HCC.     

Vascular endothelial growth factor (VEGF)-C differentially affects tumor vascular function and leukocyte recruitment: role of VEGF-receptor 2 and host VEGF-A

Unlike vascular endothelial growth factor (VEGF)-A, the effect of VEGF-C on tumor angiogenesis, vascular permeability, and leukocyte recruitment is not known. To this end, we quantified in vivo growth and vascular function in tumors derived from two VEGF-C-overexpressing (VC+) and mock-transfected cell lines (T241 fibrosarcoma and VEGF-A-/- embryonic stem cells) grown in murine dorsal skinfold chambers. VC+ tumors grew more rapidly than mock-transfected tumors and exhibited parallel increases in tumor angiogenesis. Furthermore, VEGF-C overexpression elevated vascular permeability in T241 tumors, but not in VEGF-A-/- tumors. Surprisingly, unlike VEGF-A, VEGF-C did not increase leukocyte rolling or adhesion in tumor vessels. Administration of VEGF receptor (VEGFR)-2 neutralizing antibody DC101 reduced vascular density and permeability of both VC+ and mock-transduced T241 tumors. These data suggest that VEGFR-2 signaling is critical for tumor angiogenesis and vascular permeability and that VEGFR-3 signaling does not compensate for VEGFR-2 blockade. An alternate VEGFR, VEGFR-1 or neuropilin-1, may modulate adhesion of leukocytes to tumor vessels.     

CT7 (MAGE-C1) antigen expression in normal and neoplastic tissues

Angiogenesis is a vital component of the development and progression of many human solid tumors. Glioblastoma multiforme is one of the most highly vascularised class of solid tumors. Thus, we have investigated the potential antitumourigenic activity of endostatin, an angiogenic inhibitor, in the rat C6 glioma model. We have engineered C6 cells that endogenously express mouse endostatin in order to assess the growth of C6 tumors in vivo when endostatin is constitutively expressed. Endostatin secreted by stably transfected C6 cells is biologically active as shown by its inhibition (26%) of bFGF-stimulated proliferation of BAECs in culture. The subcutaneous implantation of endostatin-C6 cells in athymic (nu/nu) mice resulted in a reduced tumor growth rate (90% inhibition) compared to control cell lines throughout the duration of our experiments. Tumor inhibition was associated with a 50% reduction in the number of vessels, which were also smaller in morphology. However, endostatin-C6 tumors were no more necrotic than control tumors. The implantation of endostatin-C6 cells into immunocompetent Wistar rat brains also resulted in reduced tumor volumes (71% inhibition) compared to controls. Tumor cells were sparsely localised along the injection tract but had not formed discrete tumors. Despite the inhibitory response mediated by endostatin on C6 growth, complete tumor inhibition or dormancy was not observed in either the athymic or immunocompetent tumor models. These findings demonstrate that the endogenous expression of endostatin by C6 glioma cells results in a reduced tumor growth rate in vivo that is associated with an inhibition of tumor angiogenesis. Our data suggest that endostatin should be developed as an adjuvant gene therapy for the effective treatment of gliomas.     

Endostatin-cytosine deaminase fusion protein suppresses tumor growth by targeting neovascular endothelial cells

Endostatin, an angiogenesis inhibitor tested in multiple clinical trials, selectively targets neovascular endothelial cells, suppressing tumor growth. To enhance the therapeutic efficacy of endostatin, we fused endostatin with cytosine deaminase, which converts a prodrug 5-flucytosine into a cytotoxic 5-fluorouracil. This therapeutic strategy was developed based on the observation that the endostatin-green fluorescence protein gene and endostatin-luciferase gene selectively target to endothelial cells in vitro and to the tumor site in vivo, respectively. When we used the endostatin-cytosine deaminase fusion protein to treat s.c. grafted tumors or experimental metastasis tumors, our results showed that endostatin-cytosine deaminase treatment provided stronger tumor growth suppression and increased mean survival time of the mice compared with the treatments of endostatin alone, cytosine deaminase alone, or endostatin plus cytosine deaminase. The endostatin-cytosine deaminase protein significantly inhibited the growth of endothelial cells and preferentially induced tumor cell apoptosis. This endostatin-cytosine deaminase fusion approach opens an avenue for cancer-targeting therapy.     

Therapeutic efficacy of endostatin exhibits a biphasic dose-response curve

We show here that recombinant endostatin protein has a biphasic effect on the inhibition of endothelial cell migration in vitro. In tumor-bearing animals, there is a similar biphasic effect on the inhibition of tumor growth and on circulating endothelial cells after once-daily s.c. injections. This biphasic effect is revealed as a U-shaped curve in which efficacy is optimal between very low and very high doses depending on the tumor type. This result may be applicable to other inhibitors of endothelial growth and to angiogenesis. Furthermore, these results have important implications for clinicians who administer angiogenesis inhibitors for cancer or other angiogenesis-dependent diseases. When these results are taken together with two previous reports of angiogenesis inhibitors with a U-shaped dose-response, they suggest that other regulators of endothelial growth may display a similar pattern.     

Prediction of in vivo synergistic activity of antiangiogenic compounds by gene expression profiling

Angiogenesis, an essential phenotype for tumor formation, requires the interaction of many cells within the tumor microenvironment. Therefore, successful antiangiogenic therapies must be able to block all of the different mechanisms tumors use to induce neovascularization. A major challenge for developing such protocols is determining which agents are likely to have the highest degree of synergistic activity in vivo. We treated human microvascular endothelial cells with six inhibitors of angiogenesis and used microarrays to seek divergent patterns of gene expression suggestive of potential synergies. The expression profiles of a thrombospondin-mimetic peptide (DI-TSPa) and TNP-470 (TNP) were very similar, whereas endostatin had a dramatically different profile. In vitro, endostatin was synergistically antiangiogenic with either TNP-470 or DI-TSPa. In vivo, mice bearing Lewis lung carcinoma cells treated with a combination of endostatin and either DI-TSPa or TNP-470, at doses that were ineffective when used alone, resulted in a marked inhibition of tumor growth and decreased tumor angiogenesis. Conversely, animals treated with both DI-TSPa and TNP-470 demonstrated a modest effect on both tumor growth and angiogenesis. These results suggest that even in the absence of a complete mechanistic understanding of how these inhibitors work, gene expression profiling may be used to predict synergistic antiangiogenic activity and thus maximize their antitumor efficacy.     

Estradiol and tamoxifen regulate endostatin generation via matrix metalloproteinase activity in breast cancer in vivo

Matrix metalloproteinases (MMP) are important regulators of tumor progression and angiogenesis. MMPs generate both proangiogenic and antiangiogenic fragments, such as vascular endothelial growth factor and endostatin. The in vivo activation of MMPs and endostatin generation occur mainly in the extracellular environment by interactions of different cell types. Therefore, these processes are necessary to study in the extracellular space in vivo. Sex steroids play a dominant role in breast carcinogenesis, by largely unknown mechanisms. In the present study, we used in vivo microdialysis to directly quantify MMP-2 and MMP-9 activity and sample endostatin from both stroma (murine) and tumor (human) cells in vivo in solid MCF-7 tumors in nude mice. We found that tamoxifen in combination with estradiol increased tumor MMP-2/MMP-9 in vivo activity, endostatin levels, and decreased tumor vascularization compared with estradiol treatment only. The stroma-derived endostatin was three to five times higher than cancer cell-generated endostatin. After inhibition of MMP-2/MMP-9, endostatin levels decreased, providing evidence that these proteases are highly involved in the generation of endostatin. Our results support the previously reported concept that MMPs may serve as negative regulators of angiogenesis. The regulation of endostatin generation by modulation of MMP-2/MMP-9 activities suggests a previously unrecognized mechanism of estradiol and tamoxifen, which may have implications for the pathogenesis of breast cancer.     

Thrombospondin-1 associated with tumor microenvironment contributes to low-dose cyclophosphamide-mediated endothelial cell apoptosis and tumor growth suppression

Low-dose cyclophosphamide (LDC) induces selective apoptosis of endothelial cells within the vascular bed of tumors. Here, we investigated a hypothesis that the effect of LDC is mediated by the pro-apoptotic action of endogenous inhibitors of angiogenesis. Tumors treated with LDC demonstrate similar expression of matrix metalloproteinases and also basement membrane-derived angiogenesis inhibitors when compared with wild-type tumors, whereas the expression of thrombospondin-1 (TSP-1) is significantly elevated in LDC-treated tumors. We used mice with an absence of type XVIII collagen (endostatin) or type IV collagen alpha3 chain (tumstatin) or TSP-1 to assess the contribution of these endogenous inhibitors of angiogenesis on LDC-mediated tumor suppression. Lack of TSP-1 in the host in addition to tumor cells leads to diminished capacity of LDC to suppress tumor growth, whereas the absence of endostatin and tumstatin did not alter the effect of LDC. LDC treatment predominantly induces selective expression of TSP-1 in tumor cells and peri-vascular cells and facilitates apoptosis of proliferating endothelial cells, with minimal direct effect on tumor cells and peri-vascular cells. These studies indicate that TSP-1 contributes to tumor growth suppression induced by LDC and suggest that tumors that express high basal level of TSP-1 may be more susceptible to tumor suppression by such a regimen. This study also makes a strong case for TSP-1 expression levels as a potential predictive marker for the successful use of LDC in cancer patients.     

Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth

Many solid tumors produce vascular endothelial growth factor C (VEGF-C), and its receptor, VEGFR-3, is expressed in tumor blood vessels. To study the role of VEGF-C in tumorigenesis, we implanted MCF-7 human breast carcinoma cells overexpressing recombinant VEGF-C orthotopically into severe combined immunodeficient mice. VEGF-C increased tumor growth, but unlike VEGF, it had little effect on tumor angiogenesis. Instead, VEGF-C strongly promoted the growth of tumor-associated lymphatic vessels, which in the tumor periphery were commonly infiltrated with the tumor cells. These effects of VEGF-C were inhibited by a soluble VEGFR-3 fusion protein. Our data suggest that VEGF-C facilitates tumor metastasis via the lymphatic vessels and that tumor spread can be inhibited by blocking the interaction between VEGF-C and its receptor.     

Down-regulation of vascular endothelial cell growth factor-C expression using small interfering RNA vectors in mammary tumors inhibits tumor lymphangiogenesis and spontaneous metastasis and enhances survival

Tumor production of vascular endothelial cell growth factor (VEGF)-C is associated with tumor lymphangiogenesis and lymph node metastasis. In this study, we examined the effects of small interfering RNA (siRNA)-mediated inhibition of VEGF-C on murine mammary tumor growth, metastasis, and survival. The mRNA and protein expression of VEGF-C in murine mammary tumor cells stably transfected with a VEGF-C siRNA vector were significantly lower compared with VEGF-C-control vector-transfected cells. Cl66-siVEGFC tumors had lower levels of lymphangiogenesis and lymph node and spontaneous lung metastasis than Cl66-control tumors. However, we did not observe significant differences in primary tumor growth and experimental lung metastasis between mice injected with Cl66-siVEGFC and Cl66-control cells. In addition, mice bearing Cl66-siVEGFC tumors lived significantly longer than mice bearing Cl66-control tumors. Furthermore, our data suggest that inhibition of VEGF-C modulates immune cell infiltration and their function, which might be critical in tumor immunity. In summary, our data show that inhibition of VEGF-C expression using siRNA-mediated gene silencing vectors reduces lymphangiogenesis and lymph node and spontaneous lung metastasis, and enhances survival.