Combination therapy with short interfering RNA vectors against VEGF-C and VEGF-A suppresses lymph node and lung metastasis in a mouse immunocompetent mammary cancer model

Cancer metastasis contributes significantly to cancer mortality and is facilitated by lymphangiogenesis and angiogenesis. Vascular endothelial growth factor-C (VEGF-C) and VEGF-A are involved in lymphangiogenesis and angiogenesis. To inhibit metastasis, combination therapy with vector-based small interfering RNA (siRNA) against VEGF-C and/or VEGF-A was conducted on murine metastatic mammary cancer. Syngeneic, inoculated, metastatic mammary cancers received direct intratumoral injection of plasmid siRNA vector targeting VEGF-C (psiRNA-VEGF-C), VEGF-A (psiRNA-VEGF-A), both VEGF-C and VEGF-A (both psiRNA-VEGF-C and psiRNA-VEGF-A vectors injected, referred to as the psiRNA-VEGF-C+A group) or a scrambled sequence (psiRNA-SCR) as control, once a week for 8 weeks. Gene electrotransfer was performed on the tumors after each injection. Tumor volume was significantly lower in the psiRNA-VEGF-A and the psiRNA-VEGF-C+A groups throughout the study. Lymph node metastasis was significantly less frequent in all therapeutic groups, whereas the multiplicity of lung metastases was significantly lower in the psiRNA-VEGF-C+A group only. All siRNA therapeutic groups showed a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells and microvessel density. Our data suggest that specific silencing of the VEGF-C or VEGF-A gene alone can inhibit lymph node metastasis. However, combination siRNA therapy targeting both VEGF-C and VEGF-A inhibits both lymph node and lung metastasis, rendering this combined therapy more beneficial than either alone. The observed anti-metastatic activity of siRNA-expressing vectors targeting VEGF-C or VEGF-A may be of high clinical significance in the treatment of metastatic breast cancer.     

Tumor-secreted vascular endothelial growth factor-C is necessary for prostate cancer lymphangiogenesis, but lymphangiogenesis is unnecessary for lymph node metastasis

Dissemination to draining lymph nodes is a frequent first step in prostate cancer metastasis. Although tumors metastasize to lymph nodes via the lymphatics, the importance of lymphangiogenesis in mediating the process remains controversial. Here, we inhibit intratumoral lymphangiogenesis in s.c. and surgical orthotopic implantation mouse models of human prostate cancer using several strategies. Stable expression of small interfering RNAs (siRNA) targeted against human vascular endothelial growth factor-C (VEGF-C) in PC-3 cells reduced intratumoral lymphatics by 99% in s.c. tumors, indicating that tumor-secreted VEGF-C is necessary for lymphangiogenesis. Expression of siRNAs against human VEGF-A somewhat reduced tumor lymphangiogenesis. Secretion of a soluble VEGF receptor-3/Flt4 fusion protein by PC-3 cells reduced intratumoral lymphatics by 100% in s.c. tumors. Combination of soluble Flt4 and VEGF-C siRNA yielded >92% reduction of intratumoral lymphatics in orthotopic prostate tumors. However, metastasis to lymph nodes was not significantly affected regardless of intratumoral lymphatic vessel density. The abundance of marginal lymphatics at the tumor-stromal interface was unchanged in orthotopic tumors whose intratumoral lymphatics were inhibited, suggesting that these marginal vessels could be sufficient for lymph node metastasis. Hematogenous metastasis (blood tumor burden, lung metastasis) correlated with degree of lymph node invasion. We also analyzed the lymphatics in spontaneous transgenic adenocarcinomas of the mouse prostate which metastasize to lymph nodes. Progression from well-differentiated prostate intraepithelial neoplasia to metastatic, undifferentiated adenocarcinoma was accompanied by loss of lymphatics. These results suggest that tumor-secreted VEGF-C and, to a lesser extent, VEGF-A, are important for inducing prostate cancer intratumoral lymphangiogenesis but are unnecessary for lymph node metastasis.     

VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors

The spread of cancer cells to regional lymph nodes through the lymphatic system is the first step in the dissemination of breast cancer. In several human cancers including those of the breast and prostate, the expression of vascular endothelial growth factor C (VEGF-C) is associated with lymph node metastasis. Our study was undertaken to evaluate the effect of VEGF-C on metastasis of poorly invasive, estrogen dependent human MCF-7 breast cancer cells. MCF-7 breast cancer cells transfected with VEGF-C (MCF-7-VEGF-C) were grown as tumors in the mammary fat pads of nude mice implanted with subcutaneous estrogen pellets. Tumor lymphangiogenesis and lymph node metastasis were studied immunohistochemically using antibodies against lymphatic vessel hyaluronan receptor -1 (LYVE-1), VEGF receptor-3 (VEGFR-3), PECAM-1, pan-cytokeratin and estrogen dependent pS2 protein. Overexpression of VEGF-C in transfected MCF-7 cells stimulated in vivo tumor growth in xenotransplanted mice without affecting estrogen responsiveness. The resulting tumors metastasized to the regional lymph nodes in 75% (in 6 mice out of 8, Experiment I) and in 62% (in 5 mice out of 8, Experiment II) of mice bearing orthotopic tumors formed by MCF-7-VEGF-C cells whereas no metastases were observed in mice bearing tumors of control vector-transfected MCF-7 cells (MCF-7-Mock). The density of intratumoral and peritumoral lymphatic vessels was increased in tumors derived from MCF-7-VEGF-C cells but not MCF-7-Mock cells. Taken together, our results show that VEGF-C overexpression stimulates tumor lymphangiogenesis and induces normally poorly metastatic estrogen-dependent MCF-7 tumors to disseminate to local lymph nodes. These data suggest that VEGF-C has an important role in lymph node metastasis of breast cancer even at its hormone-dependent early stage.     

姜黄素对宫颈癌裸鼠移植瘤淋巴管生成及淋巴转移的影响

目的:探讨姜黄素对人宫颈癌裸鼠皮下移植瘤淋巴管生成及淋巴道转移的影响。方法:取对数生长期CaSki细胞接种于裸鼠右腋窝皮下,建立人宫颈癌裸鼠皮下移植瘤模型,免疫组化方法检测移植瘤组织中VEGF-C和VEGFR-3蛋白表达情况。结果:免疫组化显示姜黄素处理组的肿瘤组织VEGF-C、VEGFR-3蛋白表达水平较阴性对照组降低(P<0.05)。可见姜黄素能下调肿瘤细胞中VEGF-C和VEGFR-3蛋白的表达。结论:姜黄素具有抗宫颈癌淋巴管生成的作用,对宫颈癌淋巴道转移具有抑制作用,其主要机制与抑制VEGF-C和VEGFR-3蛋白的表达有关。     

Multi-kinase inhibitor E7080 suppresses lymph node and lung metastases of human mammary breast tumor MDA-MB-231 via inhibition of vascular endothelial growth factor-receptor (VEGF-R) 2 and VEGF-R3 kinase

PURPOSE: Vascular endothelial growth factor (VEGF)-C/VEGF-receptor 3 (VEGF-R3) signal plays a significant role in lymphangiogenesis and tumor metastasis based on its effects on lymphatic vessels. However, little is known about the effect of inhibiting VEGF-R3 on lymphangiogenesis and lymph node metastases using a small-molecule kinase inhibitor. EXPERIMENTAL DESIGN: We evaluated the effect of E7080, a potent inhibitor of both VEGF-R2 and VEGF-R3 kinase, and bevacizumab on lymphangiogenesis and angiogenesis in a mammary fat pad xenograft model of human breast cancer using MDA-MB-231 cells that express excessive amounts of VEGF-C. Lymphangiogenesis was determined by lymphatic vessel density (LVD) and angiogenesis by microvessel density (MVD). RESULTS: In contrast to MDA-MB-435 cells, which expressed a similar amount of VEGF to MDA-MB-231 cells with an undetectable amount of VEGF-C, only MDA-MB-231 exhibited lymphangiogenesis in the primary tumor. E7080 but not bevacizumab significantly decreased LVD within the MDA-MB-231 tumor. E7080 and bevacizumab decreased MVD in both the MDA-MB-231 and MDA-MB-435 models. E7080 significantly suppressed regional lymph nodes and distant lung metastases of MDA-MB-231, whereas bevacizumab significantly inhibited only lung metastases. E7080 also decreased both MVD and LVD within the metastatic nodules at lymph nodes after resection of the primary tumor. CONCLUSIONS: Inhibition of VEGF-R3 kinase with E7080 effectively decreased LVD within MDA-MB-231 tumors, which express VEGF-C. Simultaneous inhibition of both VEGF-R2 and VEGF-R3 kinases by E7080 may be a promising new strategy to control regional lymph node and distant lung metastases.     

Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling

BACKGROUND: Vascular endothelial growth factor C (VEGF-C) stimulates tumor lymphangiogenesis (i.e., formation of lymphatic vessels) and metastasis to regional lymph nodes by interacting with VEGF receptor 3 (VEGFR-3). We sought to determine whether inhibiting VEGFR-3 signaling, and thus tumor lymphangiogenesis, would inhibit tumor metastasis. METHODS: We used the highly metastatic human lung cancer cell line NCI-H460-LNM35 (LNM35) and its parental line NCI-H460-N15 (N15) with low metastatic capacity. We inserted genes by transfection and established a stable N15 cell line secreting VEGF-C and a LNM35 cell line secreting the soluble fusion protein VEGF receptor 3-immunoglobulin (VEGFR-3-Ig, which binds VEGF-C and inhibits VEGFR-3 signaling). Control lines were transfected with mock vectors. Tumor cells were implanted subcutaneously into severe combined immunodeficient mice (n = 6 in each group), and tumors and metastases were examined 6 weeks later. In another approach, recombinant adenoviruses expressing VEGFR-3-Ig (AdR3-Ig) or beta-galactosidase (AdLacZ) were injected intravenously into LNM35 tumor-bearing mice (n = 14 and 7, respectively). RESULTS: LNM35 cells expressed higher levels of VEGF-C RNA and protein than did N15 cells. Xenograft mock vector-transfected LNM35 tumors showed more intratumoral lymphatic vessels (15.3 vessels per grid; 95% confidence interval [CI] = 13.3 to 17.4) and more metastases in draining lymph nodes (12 of 12) than VEGFR-3-Ig-transfected LNM35 tumors (4.1 vessels per grid; 95% CI = 3.4 to 4.7; P<.001, two-sided t test; and four lymph nodes with metastases of 12 lymph nodes examined). Lymph node metastasis was also inhibited in AdR3-Ig-treated mice (AdR3-Ig = 0 of 28 lymph nodes; AdLacZ = 11 of 14 lymph nodes). However, metastasis to the lungs occurred in all mice, suggesting that LNM35 cells can also spread via other mechanisms. N15 tumors overexpressing VEGF-C contained more lymphatic vessels than vector-transfected tumors but did not have increased metastatic ability. CONCLUSIONS: Lymph node metastasis appears to be regulated by additional factors besides VEGF-C. Inhibition of VEGFR-3 signaling can suppress tumor lymphangiogenesis and metastasis to regional lymph nodes but not to lungs.     

VEGF-C-VEGFR3/Flt4 axis regulates mammary tumor growth and metastasis in an autocrine manner

Purpose: Lymphangiogenic factors, such as vascular endothelial growth factor-C (VEGF-C) and VEGFC-D, and their receptor, VEGF receptor-3 (VEGFR3), play a pivotal role in the promotion of metastasis to regional lymph nodes. In the present study we explored the role of VEGF-C as an autocrine growth factor for breast cancer cells. Methods: We examined the expression of VEGF-C and VEGFR3 in mammary tumor cells lines and examined whether blocking the VEGF-C-VEGFR3/Flt4 pathway using a VEGFR3 antagonist would inhibit proliferation of mammary tumor cells resulting in a decrease in tumor growth and metastasis. Results: We report expression of VEGF-C and its receptor VEGFR3 by mammary tumor cells, and their association with aggressiveness. Inhibition of VEGF-C-VEGFR3/Flt4 in mammary tumor cells decreased their proliferation and survival. Mammary tumor bearing mice treated with a VEGFR3 antagonist showed a significant decrease in tumor growth and the extent of spontaneous and experimental lung metastases. Conclusion: These findings demonstrate the VEGF-C-VEGFR3/Flt4 autocrine signaling pathway regulates mammary tumor cell survival and proliferation and that neutralization of VEGFR3 signaling might lead to development of a novel therapeutic approach for malignant breast cancer.     

Endostar Combined with Cisplatin Inhibits Tumor Growth and Lymphatic Metastasis of Lewis Lung Carcinoma Xenografts in Mice

Objective: To investigate the effects of endostar, a recombined humanized endostatin, plus cisplatin on thegrowth, lymphangiogenesis and lymphatic metastasis of the Lewis lung carcinoma (LLC) in mice. Methods: Atumor model were established in C57BL/6 mice by intravenious transplantation of LLC cells. Then the mice wererandomized to receive administration with NS, endostar, cisplatin, or endostar plus cisplatin. After the mice weresacrificed, tumor multiplicity, tumor size and lymph node metastasis were assessed. Then the expression of vascularendothelial growth factor-c (VEGF-C) and podoplanin were determined by immunohistochemical staining.Results: Endostar plus cisplatin significantly suppressed tumor growth. lymphatic metastasis and prolongedsurvival time of the mice without obvious toxicity. The inhibition of lymphatic metastasis was associated withdecreased microlymphatic vessel density (MLVD) and expression of VEGF-C. Conclusions: Endostar combinedwith cisplatin was more effective to suppress tumor growth and lymphatic metastasis than either agent alone.Thus this may provide a rational alternative for lung carcinoma treatment.     

Cyclooxygenase-2 promotes tumor lymphangiogenesis and lymph node metastasis in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer and frequently metastasizes to the cervical lymph nodes, leading to poor survival of patients with OSCC. However, the mechanism of lymph node metastasis is not fully understood. To clarify the molecular mechanism underlying OSCC metastasis to regional lymph nodes, the highly metastatic fluorescent labeled OSCC cell line SAS-LM3 was successfully established allowing us to monitor the progression of lymph node metastases in a non-invasive manner. SAS-LM3 tumors showed increased lymphangiogenesis and elevated expression of VEGF-C, a potent stimulator of lymphangiogenesis, compared to parental SAS tumors. SAS-LM3 showed high expression of cyclooxygenase-2 (COX-2) compared to parental SAS cells and immunohistochemical analysis demonstrated intense COX-2 expression at the primary site. Inactivation of COX-2 by knockdown or the COX-2 inhibitor NS-398 decreased VEGF-C expression. Administration of COX-2 inhibitor NS-398 in SAS-LM3 tumor-bearing mice suppressed tumor lymphangiogenesis and lymphatic metastases. Collectively, our results indicate that COX-2 promotes tumor lymphangiogenesis and lymph node metastasis of OSCC. COX-2 ablation holds promise as a potential therapeutic approach for lymph node metastasis in OSCC.     

Development of a mouse model for lymph node metastasis with endometrial cancer

Controlling lymph node metastasis is currently a key issue in cancer therapy. Lymph node metastasis is one of the most important prognostic factors in various types of cancers, including endometrial cancer. Vascular endothelial growth factor-C (VEGF-C) plays a crucial role in lymphangiogenesis, and is implicated to play an important role in lymph node metastasis. To evaluate the role of VEGF-C in lymph node metastasis, we developed an animal model by using an endometrial cancer cell line, HEC1A. This cell line is not invasive by nature and secretes moderate amounts of VEGF-C; intrauterine injection of HEC1A cells into Balb/c nude mice resulted in uterine cancer with lymph node metastasis after 8 weeks. To analyze the contribution of VEGF-C to lymph node metastasis, its corresponding gene was stably introduced into HEC1A cells (HEC1A/VEGF-C), which then produced more than 10 times the amount of VEGF-C. The number of lymph node metastases was significantly higher in HEC1A/VEGF-C cells than in HEC1A cells (3.2 vs 1.1 nodes/animal, respectively). Augmented lymphangiogenesis was observed within tumors when HEC1A/VEGF-C cells were inoculated. These results indicate that VEGF-C plays a critical role in lymph node metastasis, in addition to serving as a platform to test the efficacy of various therapeutic modalities against lymph node metastasis.     

Inhibition of cyclooxygenase-2 suppresses lymph node metastasis via reduction of lymphangiogenesis

Cyclooxygenase-2 (COX-2) inhibitor has been reported to suppress tumor progression. However, it is unclear whether this inhibitor can also prevent lymphatic metastasis. To determine the effects of COX-2 inhibitor on lymphatic metastasis, etodolac, a COX-2 inhibitor, was given p.o. to mice bearing orthotopic xenografts or with carcinomatous peritonitis induced with a highly metastatic human diffuse-type gastric carcinoma cell line, OCUM-2MLN. Tumor lymphangiogenesis was significantly decreased in etodolac-treated mice compared with control mice. Consistent with this decrease in lymphangiogenesis, the total weight of metastatic lymph nodes was less in etodolac-treated mice than in control mice. Immunohistochemical analysis revealed that the major source of vascular endothelial growth factor-C (VEGF-C) and VEGF-D was F4/80-positive macrophages in our models. The mRNA levels of VEGF-C in mouse macrophage-like RAW264.7 cells, as well as those in tumor tissues, were suppressed by etodolac. The growth of human dermal lymphatic microvascular endothelial cells was also suppressed by etodolac. Supporting these findings, etodolac also inhibited lymphangiogenesis in a model of chronic aseptic peritonitis, suggesting that COX-2 can enhance lymphangiogenesis in the absence of cancer cells. Our findings suggest that COX-2 inhibitor may be useful for prophylaxis of lymph node metastasis by reducing macrophage-mediated tumor lymphangiogenesis.     

Differential in vivo and in vitro expression of vascular endothelial growth factor (VEGF)-C and VEGF-D in tumors and its relationship to lymphatic metastasis in immunocompetent rats

The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival. A number of clinical and experimental studies suggest that tumor-induced lymphangiogenesis driven by vascular endothelial growth factor (VEGF)-C- and/or VEGF-D-induced activation of VEGF receptor (VEGFR)-3 may promote metastasis to regional lymph nodes. Here we show that constitutive VEGF-C and VEGF-D expression by tumor cells of diverse origin grown in tissue culture does not correlate with metastatic potential in vivo. However, tumors derived from cell lines that do not constitutively express VEGF-C or VEGF-D in tissue culture can nevertheless express one or both of these factors. We demonstrate that both tumor and stromal cells can contribute to this expression, suggesting that tumor cell-host interactions determine tumor expression of VEGF-C and VEGF-D. Using immunocompetent rat mammary tumor models, we show in two ways that this expression can promote metastasis via the lymphatics. Firstly, ectopic expression of a soluble VEGFR-3 receptor globulin protein in MT-450 tumor cells that are highly metastatic via the lymphatics blocked VEGF-C and VEGF-D activity and suppressed metastasis formation in both the regional lymph nodes and the lungs. Secondly, ectopic expression in the weakly metastatic NM-081 cell line of a mutant form of VEGF-C that is only able to activate VEGFR-3 strongly promoted metastasis of these cells to the regional lymph nodes and lung. These data show that expression of VEGF-C and VEGF-D in tissue culture does not reflect expression in vivo and that activation of VEGFR-3 in the absence of VEGFR-2 activation is sufficient to promote tumor-induced lymphangiogenesis and metastasis, and they support the notion that blockade of VEGFR-3 activation will be useful as a novel form of cancer therapy.     

JAM-C promotes lymphangiogenesis and nodal metastasis in non-small cell lung cancer

This study aims to investigate lymphatic metastasis-related genes in non-small cell lung carcinomas (NSCLC). NSCLC tissue was analyzed for expression of junctional adhesion molecule-C (JAM-C) protein. Our data revealed novel associations between JAM-C overexpression in primary tumors and lymphatic microvessel density (LMVD), lymph node metastasis, and poorer overall survival and recurrence-free survival. We used the highly metastatic human lung adenocarcinoma cell line Anip973 and its parental line AGZY83-a, which has a low metastatic capacity, in vivo and vitro. We found that JAM-C played an important role in different metastasis capacity of lymph node. JAM-C affected tumor growth, LNM, JAM-C, VEGF-C, vasculature, and ERK1/2 phosphorylation (p-ERK1/2). β1 integrin was involved in lymph node metastasis. Moreover, JAM-C knockdown in highly metastatic Anip973 decreased cell migration in scratch-wound assays. The JAM-C knockdown in Anip973 cells and JAM-C cDNA in AGZY83-a cells regulated the vascular endothelial growth factor C (VEGF-C) expression. Immunofluorescence showed that blocked VEGF-C expression in JAM-C shRNA Anip973 cells were restored after JAM-C treatment. JAM-C-induced VEGF-C in JAM-C cDNA AGZY83-a cells was also effectively inhibited by treatment with an antibody specifically against JAM-C. Use of media from Anip973 cells, AGZY83-a, and A549cells lung cancer cells that overexpressed or downregulated JAM-C was demonstrated to affect activity of VEGF-C-induced β1 integrin subunit or ERK activity in human dermal lymphatic endothelial cells (HDLEC) treated with VEGF-C or inhibitory antibody to JAM-C. Overall, these results indicate that JAM-C could mediate metastasis as it contributes to VEGF-C expression in cancer cells. JAM-C affects β1and ERK activation in HDLEC, thus promoting lymphangiogenesis and nodal metastasis. Our findings indicate that JAM-C may be a therapeutic target for preventing and treating lymphatic metastases.     

Rapamycin, a specific inhibitor of the mammalian target of rapamycin, suppresses lymphangiogenesis and lymphatic metastasis

Tumor lymphangiogenesis is now known to play a causal role in lymph node metastasis, and thus its inhibition would have great significance for the prevention of lymph node metastasis in cancer therapy. VEGF-C has recently been identified as a key molecule that involved in tumor lymphangiogenesis and lymphatic metastasis. However, the expressional regulation of VEGF-C is not fully understood. We investigated the role of mTOR, which is a downstream kinase of the phosphatidylinositol 3-kinase/Akt pathway, and the MAPK family (MEK1/2, p38, and JNK) in the regulation of VEGF-C and VEGF-A expression in B13LM cells, a lymphatic metastasis-prone pancreatic tumor cell line. We also investigated the antilymphangiogenic effect of rapamycin, a specific inhibitor of mTOR in vivo using male BALB/c nu/nu mice. VEGF-C expression was inhibited by the inhibitors for mTOR, p38, and JNK, but not by the inhibitor for MEK1/2, whereas VEGF-A expression was inhibited by all four of these inhibitors. The serum starvation-induced expression of VEGF-C was inhibited by rapamycin, whereas that of VEGF-A was incompletely inhibited. The metastatic experiment in vivo demonstrated that the number and the area of lymphatic vessels in the primary tumors were significantly decreased by rapamycin. Finally, the lymph node metastasis was significantly suppressed in rapamycin-treated mice. Our results suggest that mTOR, p38, and JNK play important roles in VEGF-C expression, and that rapamycin has an antilymphangiogentic effect and exerts the expected inhibition of lymphatic metastasis.     

Endostatin overexpression inhibits lymphangiogenesis and lymph node metastasis in mice

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. We studied the development of carcinogen-induced skin tumors in transgenic J4 mice overexpressing endostatin in their keratinocytes. Unexpectedly, we did not observe any differences in tumor incidence and multiplicity between these and control mice, nor in the rate of conversion of benign papillomas to malignant squamous cell carcinomas (SCC). We did find, however, that endostatin regulates the terminal differentiation of keratinocytes because the SCCs in the J4 mice were less aggressive and more often well differentiated than those in the control mice. We observed an inhibition of tumor angiogenesis by endostatin at an early stage in skin tumor development, but more strikingly, there was a significant reduction in lymphatic vessels in the papillomas and SCCs in association with elevated endostatin levels and also a significant inhibition of lymph node metastasis in the J4 mice. We showed that tumor-infiltrating mast cells strongly expressed vascular endothelial growth factor-C (VEGF-C), and that the accumulation of these cells was markedly decreased in the tumors of the J4 mice. Moreover, endostatin inhibited the adhesion and migration of murine MC/9 mast cells on fibronectin in vitro. Our data suggest that endostatin can inhibit tumor lymphangiogenesis by decreasing the VEGF-C levels in the tumors, apparently via inhibition of mast cell migration and adhesion, and support the view that the biological effects of endostatin are not restricted to endothelial cells because endostatin also regulates tumor-associated inflammation and differentiation, and the phenotype of epithelial tumors.     

肺腺癌和鳞癌中VEGF-C和新生淋巴管表达的预后价值

背景与目的血管内皮生长因子-C(vascu larendothelial growth factor-C,VEGF-C)是VEGF家族的成员之一,且已被证明是相对特异的血管内皮生成因子,其与受体VEGFR-3结合后可激活淋巴管的生成,为肿瘤的淋巴结转移创造有利的条件。本研究旨在探讨VEGF-C和新生淋巴管在肺腺癌和鳞癌中的表达特点及其预后意义。方法以跨粘膜样受体蛋白(podoplanin)标记新生淋巴管内皮细胞,采用免疫组化方法检测98例IIIa(N2)期肺腺癌和鳞癌中VEGF-C和新生淋巴管的表达。结果 VEGF-C的表达率与微淋巴管密度(lymphatic microvessel density,LMVD)之间存在正相关(r=0.783,P<0.01)。VEGF-C高表达组的LMVD大于低表达组(P<0.01)。肺腺癌VEGF-C和新生淋巴管表达率明显高于肺鳞癌(P<0.01)。VEGF-C阳性表达患者的生存率明显低于阴性患者(P<0.05),VEGF-C是影响预后的独立因素。结论 IIIa(N2)期肺腺癌新生淋巴管的表达较鳞癌明显。VEGF-C是影响IIIa(N2)期肺腺癌和鳞癌患者预后的独立因素。     

Inhibition of lymphogenous metastasis using adeno-associated virus-mediated gene transfer of a soluble VEGFR-3 decoy receptor

The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival in many types of cancer. It has recently been shown that the lymphangiogenic growth factor, vascular endothelial growth factor-C (VEGF-C), and its receptor, VEGF receptor-3 (VEGFR3), may play a pivotal role in the promotion of metastasis to regional lymph nodes. In this study, human prostate and melanoma tumor models that preferentially metastasize to the lymph nodes following s.c. tumor cell implantation were established from lymph node metastases via in vivo selection. Melanoma tumor cell sublines established from lymph node metastasis express higher amounts of VEGF-C than the parental tumor cells. The inhibition of tumor-derived VEGF-C with a soluble VEGFR3 decoy receptor, sVEGFR3-Fc, expressed via a recombinant adeno-associated viral vector, potently blocks tumor-associated lymphangiogenesis and tumor metastasis to the lymph nodes, when the treatment was initiated before the tumor implantation. In addition, sVEGFR3-Fc serum levels required for efficient blockade of lymph node metastases are strictly dependent on the VEGF-C levels generated by the primary tumor. Recombinant adeno-associated virus-mediated gene transfer of sVEGFR3-Fc may represent a feasible therapeutic strategy for blockade of lymphogenous metastasis.     

Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels

Lymphangiogenic growth factors vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to promote lymphatic metastasis by inducing tumor-associated lymphangiogenesis. In this study, we have investigated how tumor cells gain access into lymphatic vessels and at what stage tumor cells initiate metastasis. We show that VEGF-C produced by tumor cells induced extensive lymphatic sprouting towards the tumor cells as well as dilation of the draining lymphatic vessels, suggesting an active role of lymphatic endothelial cells in lymphatic metastasis. A significant increase in lymphatic vessel growth occurred between 2 and 3 weeks after tumor xenotransplantation, and lymph node metastasis occurred at the same stage. These processes were blocked dose-dependently by inhibition of VEGF receptor 3 (VEGFR-3) signaling by systemic delivery of a soluble VEGFR-3-immunoglobulin (Ig) fusion protein via adenoviral or adeno-associated viral vectors. However, VEGFR-3-Ig did not suppress lymph node metastasis when the treatment was started at a later stage after the tumor cells had already spread out, suggesting that tumor cell entry into lymphatic vessels is a key step during tumor dissemination via the lymphatics. Whereas lymphangiogenesis and lymph node metastasis were significantly inhibited by VEGFR-3-Ig, some tumor cells were still detected in the lymph nodes in some of the treated mice. This indicates that complete blockade of lymphatic metastasis may require the targeting of both tumor lymphangiogenesis and tumor cell invasion.     

Pathways targeting tumor lymphangiogenesis.

Tumor metastasis to sentinel lymph nodes represents the first step of tumor dissemination in most human cancers and serves as a major prognostic indicator for disease progression. Recent studies have revealed that tumors can actively induce the formation of lymphatic vessels, and that tumor lymphangiogenesis is correlated with lymph node metastasis in experimental cancer models and in several types of human cancers. Metastatic tumor cells may continue to promote lymphatic vessel growth even after their metastasis to sentinel lymph nodes, likely promoting further cancer spread. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D were the first specific lymphangiogenesis factors identified, acting predominantly via VEGF receptor-3 (VEGFR-3) that is expressed by lymphatic endothelial cells, and a large number of clinical studies have shown a correlation between tumor expression of VEGF-C or VEGF-D and lymph node metastasis. VEGFR-3 activation promotes lymphatic endothelial cell proliferation, migration, and survival via the extracellular signal-regulated kinase 1/2, the phosphatidylinositol 3-kinase/AKT, and the c-Jun NH(2)-terminal kinase 1/2 pathways. Additional tumor lymphangiogenesis factors have been recently identified, including VEGF-A. Importantly, blockade of the VEGFR-3 pathway by specific antibodies, by soluble receptor constructs, and by small molecule kinase inhibitors efficiently inhibits experimental tumor lymphangiogenesis and metastasis and might also represent a novel therapeutic avenue for the treatment of human cancers.