Inhibition of VEGFR-3 activation in tumor-draining lymph nodes suppresses the outgrowth of lymph node metastases in the MT-450 syngeneic rat breast cancer model

For many types of human cancer, the expression of vascular endothelial growth factor-C (VEGF-C) correlates with enhanced tumor-associated lymphatic vessel density, metastasis formation and poor prognosis. In experimental animals, VEGF-C produced by primary tumors can induce lymphangiogenesis within and/or at the periphery of the tumor, and promotes metastasis formation. Tumor-induced lymphangiogenesis is therefore thought to expedite entry of tumor cells into the lymphatic vasculature and their trafficking to regional lymph nodes, thereby fostering metastatic dissemination. Tumour-produced VEGF-C can also drain to the regional lymph nodes and induce lymphangiogenesis there. Whether this activity promotes metastasis formation remains unclear. To address this issue we manipulated VEGF-C activity and VEGFR-3 activation in the lymph nodes draining syngeneic rat breast cancers using intra-dermal delivery of either recombinant VEGF-C or VEGFR-3 blocking antibodies to induce or suppress lymph node lymphangiogenesis, respectively. Recombinant VEGF-C induced lymph node lymphangiogenesis, but was not sufficient to promote metastasis formation by poorly metastatic NM-081 breast tumours. Conversely, inhibition of lymph node lymphangiogeneis induced by highly metastatic MT-450 breast tumours suppressed the outgrowth of lymph node metastases, but not the initial colonization of the lymph nodes. Lung metastasis was also not affected. We conclude that tumor-derived VEGF-C draining to regional lymph nodes promotes the outgrowth of lymph node metastases. VEGF-C may induce lung metastasis independently of its effects on lymph node metastasis.     

Angiogenic and lymphangiogenic microvessel density in breast carcinoma: correlation with clinicopathologic parameters and VEGF-family gene expression.

Angiogenesis and lymphangiogenesis are essential for breast cancer progression and are regulated by vascular endothelial growth factors (VEGF). To determine clinical and molecular correlates of these processes, we measured blood and lymphatic vascular microvessel density in 29 invasive carcinomas (22 ductal, six lobular, one papillary), using the vascular marker CD31 and the novel lymphatic marker D2-40. Microvessel density was assessed microscopically and by image cytometry, and was compared with tumor histology, grade, stage, lymph node metastasis, hormone receptors, HER2/neu status, and expression of VEGF, VEGF-C and VEGF-D by immunohistochemistry or quantitative RT-PCR. Strong correlation was observed between visual and image cytometric microvessel density using D2-40 but not CD31 (P=0.016 and 0.1521, respectively). Image cytometric CD31 microvessel density correlated with tumor size, grade, stage and lymph node metastasis (P=0.0001, 0.0107, 0.0035 and 0.0395, respectively). D2-40 microvessel density correlated with tumor stage (P=0.0123 by image cytometry) and lymph node metastasis (P=0.0558 by microscopy). Immunohistochemical VEGF signal in peritumoral blood vessels correlated with image cytometric CD31 and D2-40 microvessel density (P=0.022 and 0.0012, respectively), consistent with the role of VEGF in blood and lymphatic vascular growth. Intratumoral VEGF-C and VEGF-D expression by quantitative RT-PCR correlated with D2-40 (P=0.0291 by image cytometry) but not with CD31 microvessel density, which could suggest a selective role of VEGF-C and VEGF-D in lymphangiogenesis. CD31 and D2-40 microvessel density correlated significantly with several prognostic factors, including lymph node metastasis. Thus, measurements of angiogenesis and lymphangiogenesis may have utility for breast cancer pathology, particularly for estimation of metastatic risk.     

Signaling for lymphangiogenesis via VEGFR-3 is required for the early events of metastasis

Metastasis to regional lymph nodes is an important and early event in many tumors. Vascular endothelial growth factor-C (VEGF-C), VEGF-D and their receptor VEGFR-3, play a role in tumor spread via the lymphatics, although the timing of their involvement is not understood. In contrast, VEGFR-2, activated by VEGF-A, VEGF-C and VEGF-D, is a mediator of angiogenesis and drives primary tumor growth. We demonstrate the critical role for VEGFR-3, but not VEGFR-2, in the early events of metastasis. In a tumor model exhibiting both VEGF-D-dependent angiogenesis and lymphangiogenesis, an antibody to VEGFR-2 (DC101) was capable of inhibiting angiogenesis (79 % reduction in PECAM + blood vessels) and growth (93 % reduction in tumor volume). However, unlike an anti-VEGFR-3 Mab (mF4-31C1), DC101 was not capable of eliminating either tumor lymphangiogenesis or lymphogenous metastasis (60 % reduction of lymph node metastasis by DC101 vs 95 % by mF4-31C1). Early excision of the primary tumors demonstrated that VEGF-D-mediated tumor spread precedes angiogenesis-induced growth. Small but highly metastatic primary human breast cancers had significantly higher lymphatic vessel density (23.1 vessels/mm²) than size-matched (11.7) or larger non-metastatic tumors (12.4) thus supporting the importance of lymphatic vessels, as opposed to angiogenesis-mediated primary tumor growth, for nodal metastasis. These results suggest that lymphangiogenesis via VEGF-D is more critical than angiogenesis for nodal metastasis.     

血管内皮生长因子D的表达与乳腺癌淋巴管生成及淋巴结转移的相关性

目的 探讨乳腺癌患者不同区域淋巴管生成、淋巴管浸润特点以及与血管内皮生长因子D(VEGF-D)的表达关系,并结合腋淋巴结转移状态进行分析. 方法 选取乳腺癌根治术石蜡标本79例,分4个区域(肿瘤区、癌周区、近癌区、远癌区)取材.切片行免疫组织化学染色,采用D2-40对淋巴管进行标记,检测各区域淋巴管密度(LVD)、淋巴管浸润(LVI)及VEGF-D表达情况. 结果 癌周区LVD最高(20.25±2.03),肿瘤区VEGF-D和LVI阳性率最高,分别为87.34%和63.29%.肿瘤区VEGF-D表达与LVD之间、LVD与LVI之间、VEGF-D表达与LVI之间差异无统计学意义(P>0.05),但在其他各区域它们之间差异有统计学意义(P<0.05),且呈正相关.癌周区LVD与腋淋巴结转移状态有关(P<0.05);癌周区和近癌区的VEGF-D表达及LVI与腋淋巴结转移之间差异有统计学意义(P<0.05),但在其他区域差异无统计学意义(P>0.05). 结论 VEGF-D可能促进乳腺癌淋巴管生成,增加淋巴管浸润机会.LVD的增高易致淋巴管浸润,促进腋淋巴结转移.癌周区和近癌区在乳腺癌淋巴道转移以及评估腋淋巴结转移状态的研究中可能更具有意义.     

VEGF-D in association with VEGFR-3 promotes nodal metastasis in human invasive lobular breast cancer

We assessed the expression of vascular endothelial growth factors (VEGF-C and VEGF-D) in breast cancer cells and the density of lymph vessels and VEGF receptor-3 (VEGFR-3)-positive vessels in and around the tumor in invasive lobular breast cancer. We found significant correlation between peritumoral lymph vessel density and presence of lymph node metastases (P=.001) and the number of metastatic lymph nodes (P<.001). A significant correlation was detected between tumor cell VEGF-D expression and lymph node status (P=.001) and density of lymphatic vessel endothelial receptor (LYVE)-1-positive vessels (P=.035). VEGFR-3+/VEGF-D+ and VEGFR-3+/VEGF-C+ tumors had a significantly higher number of metastatic lymph nodes than tumors with other staining patterns (P<.001). Tumors positive for neither VEGF-D nor VEGFR-3 had a lower density of LYVE-1+ vessels than tumors with other staining patterns (P=.033). Our results indicate that peritumoral lymph vessel density is associated with lymph node metastases in invasive lobular breast cancer and that invasive lobular cancer producing VEGF-D, surrounded by VEGFR-3+ vessels, has a significantly higher peritumoral lymph vessel density and a higher number of metastatic lymph nodes.     

Mammary cancer gene therapy targeting lymphangiogenesis: VEGF-C siRNA and soluble VEGF receptor-2, a splicing variant

Metastasis contributes significantly to cancer mortality, and the most common pathway of initial dissemination is via the afferent ducts of the lymphatics. Overexpression of vascular endothelial growth factor (VEGF)-C has been associated with lymphangiogenesis and lymph node metastasis in a multitude of human neoplasms, including breast cancers. We recently reported that both VEGF-C siRNA and endogenous soluble vascular endothelial growth factor receptor-2 (esVEGFR-2, a new splicing variant) inhibit VEGF-C function and metastasis in a mouse model of metastatic mammary cancer. Here we briefly review our previous experimental work, specifically targeting tumor lymphangiogenesis, in which metastatic mouse mammary cancers received direct intratumoral injections of either expression vectors VEGF-C siRNA or esVEGFR-2, or the empty plasmid vector, once a week for 6 or 8 weeks, followed by in vivo gene electrotransfer of the injected tumors. Throughout our study, both tumor lymphangiogenesis and the multiplicity of lymph node metastasis were significantly inhibited, with an overall reduction in tumor growth, by both VEGF-C siRNA and esVEGFR-2; further, a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells was observed with both treatments. Thus, therapeutic strategies targeting lymphangiogenesis may have great clinical significance for the treatment of metastatic human breast cancer.     

VEGF-C在卵巢癌局部淋巴结内淋巴管生成中的作用

目的观察血管内皮生长因子-C(VEGF-C)在卵巢癌组织内的表达,分析其与卵巢癌局部淋巴结内淋巴管生成之间的关系。方法取卵巢癌64例,其中,有淋巴结转移40例,无淋巴结转移24例。应用免疫组化法和Western blot技术观察VEGF-C在卵巢癌组织内的表达。以D2-40作为淋巴管内皮特异性标记物,检测卵巢癌局部淋巴结内淋巴管生成情况。结果 VEGF-C主要表达于卵巢癌细胞浆和胞膜以及癌组织周围浸润的炎性细胞,在有淋巴结转移组的表达率明显高于其在无淋巴结转移组的表达率。Western blot检测结果表明,VEGF-C蛋白在有淋巴结转移的卵巢癌组织中的表达量高于其在无淋巴结转移的卵巢癌组织内的表达量。D2-40表达于卵巢癌局部淋巴结内的淋巴管内皮细胞,在有转移的淋巴结内可见大量新生的淋巴管,淋巴管腔内存在入侵的肿瘤细胞,在无转移的淋巴结内观察到新生的淋巴管。在无淋巴结转移组病例中,卵巢癌组织VEGF-C阳性者局部淋巴结内淋巴管密度明显高于VEGF-C阴性者淋巴结内的淋巴管密度。结论 VEGF-C的表达与卵巢癌淋巴结转移密切相关,卵巢癌在发生局部淋巴结转移之前存在淋巴结内淋巴管生成的现象,卵巢癌组织内VEGF-C的表达在卵巢癌局部淋巴结内的淋巴管生成中可能发挥重要作用。     

Smad4和VEGF-C在喉癌中的表达及其与淋巴管生成之间的关系

目的观察Smad4与VEGF-C在喉癌组织内的表达情况,分析Smad4和VEGF-C的表达与喉癌组织内淋巴管生成及淋巴结转移之间的关系。方法取喉癌病例58例,其中淋巴结转移组34例,无淋巴结转移组24例。应用免疫组化法和Western blot技术观察Smad4和VEGF-C在喉癌组织内的表达。以D2-40特异性标检测喉癌组织内淋巴管生成情况。结果 Smad4在无淋巴结转移的喉癌组织内的表达率明显高于其在有淋巴结转移组的表达率。Smad4表达阳性组的淋巴管数密度(LVD)明显低于Smad4表达阴性组的LVD。VEGF-C在淋巴结转移组的表达率明显高于其在无淋巴结转移组的表达率。Smad4的表达与VEGF-C的表达呈显著的负相关性(r=-0.391)。结论 VEGF-C在喉癌淋巴管的发生及淋巴结转移中发挥重要作用。Smad4与VEGF-C的表达呈负相关,Smad4可能有抑制喉癌淋巴管生成和淋巴道转移的作用。     

Lymphangiogenesis and lymphatic metastasis in breast cancer

Lymphatic metastasis is the main prognostic factor for survival of patients with breast cancer and other epithelial malignancies. Mounting clinical and experimental data suggest that migration of tumor cells into the lymph nodes is greatly facilitated by lymphangiogenesis, a process that generates new lymphatic vessels from pre-existing lymphatics with the aid of circulating lymphatic endothelial progenitor cells. The key protein that induces lymphangiogenesis is vascular endothelial growth factor receptor-3 (VEGFR-3), which is activated by vascular endothelial growth factor-C and -D (VEGF-C and VEGF-D). These lymphangiogenic factors are commonly expressed in malignant, tumor-infiltrating and stromal cells, creating a favorable environment for generation of new lymphatic vessels. Clinical evidence demonstrates that increased lymphatic vessel density in and around tumors is associated with lymphatic metastasis and reduced patient survival. Recent evidence shows that breast cancers induce remodeling of the local lymphatic vessels and the regional lymphatic network in the sentinel and distal lymph nodes. These changes include an increase in number and diameter of tumor-draining lymphatic vessels. Consequently, lymph flow away from the tumor is increased, which significantly increases tumor cell metastasis to draining lymph nodes and may contribute to systemic spread. Collectively, recent advances in the biology of tumor-induced lymphangiogenesis suggest that chemical inhibitors of this process may be an attractive target for inhibiting tumor metastasis and cancer-related death. Nevertheless, this is a relatively new field of study and much remains to be established before the concept of tumor-induced lymphangiogenesis is accepted as a viable anti-metastatic target. This review summarizes the current concepts related to breast cancer lymphangiogenesis and lymphatic metastasis while highlighting controversies and unanswered questions.     

Smad4的表达与卵巢癌淋巴管生成及淋巴结转移之间的关系

目的观察Smad4和血管内皮生长因子(VEGF)-C在卵巢癌组织内的表达情况,分析Smad4和VEGF-C的表达与卵巢癌淋巴管生成及淋巴结转移之间的关系。方法取卵巢癌病例60例,其中,淋巴结转移组36例,无淋巴结转移组24例。应用免疫组化法和Westernblot技术观察Smad4和VEGF-C在卵巢癌组织内的表达。以D2-40作为淋巴管内皮特异性标记物,检测卵巢癌组织内淋巴管生成情况。结果 Smad4表达于卵巢癌细胞胞浆和胞核内,其在无淋巴结转移组的表达率明显高于其在有淋巴结转移组的表达率。Smad4表达阳性组的淋巴管数密度(LVD)明显低于Smad4表达阴性组的LVD。VEGF-C主要表达于卵巢癌细胞胞浆内,其在淋巴结转移组的表达率明显高于其在无淋巴结转移组的表达率。Smad4的表达与VEGF-C的表达呈显著的负相关性。Western blot检测结果表明,VEGF-C蛋白在有淋巴结转移卵巢癌组织中的表达量高于其在无淋巴结转移卵巢癌组织内的表达量,而Smad4在有淋巴结转移卵巢癌组织内的表达量明显低于其在无淋巴结转移组的表达量。结论 Smad4与VEGF-C的表达呈负相关,Smad4可能通过调节VEGF-C蛋白的表达而抑制卵巢癌淋巴管生成和淋巴道转移。     

Lymphatic Vessel Density and Vascular Endothelial Growth Factor Expression in Squamous Cell Carcinomas of Lip and Oral Cavity: A Clinicopathological Analysis with Immunohistochemistry Using Antibodies to D2-40, VEGF-C and VEGF-D

Background

Squamous cell carcinoma (SCC) of the oral region often metastasizes to the cervical lymph nodes. To investigate whether the risk of cervical lymph node metastasis are predictable through lymphatic vessel density (LVD) and vascular endothelial growth factor (VEGF) expression, we assessed the relationship between LVD and clinicopathological parameters, and VEGF expression in SCC of the oral region.

Methods

The subjects were 109 patients with SCC of the oral region including the lip. Clinicopathological parameters examined for the association with LVD in a peritumoral hot spot were lymph node metastasis, histological grade and disease stage. The association with VEGF expression was similarly studied. LVD was detected by immunohistochemistry using D2-40.

Results

LVD was significantly higher in lip cancer than in other oral tumors (P < 0.0001), while there were no significant differences of LVD among other cancers of the oral cavity. LVD tended to decrease with disease progression, increase of tumor size and increase of metastatic lymph node size. Eighty-four of 109 tumors were positive for VEGF-C or D. VEGF-C-positive tumor lesions were also positive for VEGF-D. Significantly higher levels of VEGF-C and D expressions were associated with large size of lymph node metastases (P = 0.02).

Conclusion

SCC of the oral region including the lip that produces VEGF-C and D is significantly more likely to cause cervical lymph node metastasis. LVD in a peritumoral hot spot does not directly indicate the risk of cervical lymph node metastasis, but instead may reflect lymphangiogenesis due to VEGF together with loss of lymphatic vessels through tumor growth and progression.     

血管内皮生长因子C及其受体3在人恶性黑色素瘤组织内的表达及意义

目的 观察人恶性黑色素瘤组织内血管内皮生长因子C(VEGF-C)及其受体3(VEGFR-3)的表达,探讨VEGF-C和VEGFR-3在恶性黑色素瘤淋巴管生成及淋巴道转移中的作用.方法 取人恶性黑色素瘤组织48例(石蜡标本30例,术后新鲜组织18例),应用免疫组织化学和RT-PCR技术,观察VEGF-C和VEGFR-3蛋白及mRNA在恶性黑色素瘤组织内的表达情况.以淋巴管内皮透明质酸受体(LYVE-1)标记淋巴管,计数恶性黑色素瘤组织淋巴管数密度.结果 VEGF-C和VEGFR-3蛋白主要表达于恶性黑色素瘤细胞胞浆内,在肿瘤周围的血管和淋巴管内皮上也可见VEGFR-3蛋白表达,VEGF-C和VEGFR-3蛋白在淋巴结转移组恶性黑色素瘤组织内的表达水平明显高于无淋巴结转移组(P<0.05).在18例新鲜恶性黑色素瘤中,淋巴结转移组VEGF-C和VEGFR-3mRNA的表达明显高于无淋巴结转移组(P<0.01).LYVE-1表达于肿瘤间质内的淋巴管内皮细胞,淋巴结转移组恶性黑色素瘤组织中的淋巴管数密度(LMVD)为9.845±2.454,无淋巴结转移组恶性黑色素瘤组织中的淋巴管数密度为6.534±2.193,淋巴结转移组恶性黑色素瘤组织内的淋巴管数密度明显高于无淋巴结转移组(P<0.01).结论恶性黑色素瘤组织内VEGF-C表达明显增高,并通过上调其受体VEGFR-3的表达促进恶性黑色素瘤组织内淋巴管的生成,从而促进恶性黑色素瘤的淋巴道转移.     

VEGF-C表达与胰腺癌淋巴结转移及预后的关系

目的 观察血管内皮生长因子(VEGF)-C在胰腺癌组织内的表达情况,分析VEGF-C的表达与胰腺癌淋巴结转移和预后之间的关系。方法 取胰腺癌病例52例,其中,伴淋巴结转移组36例,无淋巴结转移组16例。应用免疫组化法和Western blot技术观察VEGF-C在胰腺癌组织内的表达。以D2-40作为淋巴管内皮特异性标记物,观察胰腺癌组织内淋巴管生成的情况。采用Kaplan-Meier法绘制生存曲线判断VEGF-C的表达对胰腺癌预后的影响。结果 Western blot和免疫组化法检测结果表明,VEGF-C主要表达于胰腺癌细胞浆内,淋巴结转移组阳性表达量明显高于无淋巴结转移组(p<0.05)。D2-40表达于胰腺癌组织内淋巴管内皮细胞,VEGF-C阳性组淋巴管数密度明显高于VEGF-C阴性组(p<0.05),表明VEGF-C的表达与胰腺癌淋巴管生成密切相关。Kaplan-Meier生存分析表明VEGF-C表达阴性患者的生存率均高于VEGF-C表达阳性患者,VEGF-C的表达影响患者的预后。结论 VEGF-C在胰腺癌的淋巴管生成和淋巴结转移过程中发挥重要作用,VEGF-C的表达是影响胰腺癌患者预后的主要因素之一。     

Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis

Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-alpha, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.     

VEGF-C及VEGFR-3在乳腺癌中的表达

目的探讨乳腺癌中血管内皮生长因子-C(VEGF-C)、血管内皮生长因子受体-3(VEGFR-3)的表达及与淋巴管生成的关系。方法采用免疫组化SP法检测57例乳腺癌组织中VEGF-C及VEGFR-3的表达,并在显微镜下记数VEGFR-3标记的脉管。结果淋巴结转移组VEGF-C的阳性率(90.48%)显著高于无淋巴结转移组(47.22%)(P<0.05);淋巴结转移组VEGFR-3阳性脉管数(7.62±1.18)显著高于无淋巴结转移组(5.27±0.96)(P<0.05);VEGF-C的阳性表达与VEGFR-3阳性脉管数呈正相关,相关系数为r为0.882(P<0.05)。结论 VEGF-C及VEGFR-3与乳腺癌的生长,淋巴管的生长及淋巴结的转移有关。     

Lymphatic vessel density in pulmonary adenocarcinoma immunohistochemically evaluated with anti-podoplanin or anti-D2-40 antibody is correlated with lymphatic invasion or lymph node metastases

In lung cancers, lymph node metastasis of cancer cells is one of the most important prognostic factors, and lymphatic vessel invasion (LVI) is very important in the stage preceding lymph node metastases. Recently, it has been reported that lymphatic vessel density (LVD) is associated with lymph node metastasis. The aim of the present study was to evaluate the relationship between LVD and LVI based on the immunohistochemical expression of podoplanin or D2-40, which are new specific markers for lymphatic endothelium. Using 76 cases of pulmonary adenocarcinoma, the relationship between LVD and LVI, lymph node metastases, vascular endothelial growth factor C (VEGF-C), VEGF-D or hepatocyte growth factor (HGF) expression was investigated. LVD was significantly associated with LVI, lymph node metastases and VEGF-D expression. LVI was also associated with lymph node metastases, histological subtype, VEGF-C or VEGF-D expression. High LVD, induced by VEGF-C or VEGF-D expression of cancer cells, is a good indicator of lymphatic metastases and LVI in pulmonary adenocarcinoma.