Expression level of vascular endothelial growth factor-C and -A in cultured human oral squamous cell carcinoma correlates respectively with lymphatic metastasis and angiogenesis when transplanted into nude mouse oral cavity

Angiogenesis is induced by various angiogenic factors including vascular endothelial growth factors (VEGFs), such as VEGF-A, -B, -C and -D, and is involved in tumor progression and metastasis. In an effort to define the expression pattern of VEGFs in oral squamous cell carcinoma (OSCC) and its correlation with clinicopathological factors, we determined the expression levels of VEGFs in OSCC cell lines (HSC-2, HSC-3, HSC-4 and OSC-19) by quantitative RT-PCR and examined their relationship with regional lymph node (LN) and distant metastasis, intratumoral microvessel density (MVD) in tumor transplanted nude mice. We found that HSC-2 and OSC-19 expressed significantly higher levels of VEGF-A and VEGF-C, and caused frequent regional LN metastasis and higher MVD than did the other cell lines. Since VEGF-C is a lymphangiogenic factor, these results suggest that expression of VEGF-C is a useful predictor for LN metastasis.     

VEGF-A and VEGFR-3 correlate with nodal status in operable non-small cell lung cancer: inverse correlation between expression in tumor and stromal cells

BACKGROUND: Lymph node metastasis is an essential determinant for stage and clinical management of non-small cell lung cancer (NSCLC). The vascular endothelial growth factors (VEGFs) and receptors (VEGFRs) are fundamental molecules in angiogenesis and lymphangiogenesis. We aimed to explore the correlations between nodal metastasis and the expression of VEGFs and VEGFRs in tumor cells and in tumor-related stroma. PATIENTS AND METHODS: Tumor tissue samples from 335 resected patients with stage I-IIIA NSCLC were obtained and tissue microarrays were constructed from duplicate cores of tumor cells and surrounding stromal tissue from each resected specimen. Immunohistochemistry was used to evaluate the expression of VEGF-A, VEGF-C, and VEGF-D and VEGFR-1, VEGFR-2 and VEGFR-3. RESULTS: There were 232 N0 and 103 N+ patients (76 N1, 27 N2). In multivariate analyses, low stromal VEGF-A expression (P=0.018) is associated with N+ status. In tumor cells, strong correlations exist between high VEGF-A expression (P=0.032) and N+ status, and high VEGFR-3 expression (P<0.001) and N2-status. CONCLUSION: The converse impact by stromal VEGF-A versus tumor cell VEGF-A expression on nodal metastasis may allude the importance of the tumor-stroma interaction when trying to understand lymphatic metastasis in NSCLC.     

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.     

ErbB2 overexpression correlates with increased expression of vascular endothelial growth factors A,C, and D in human breast carcinoma

BACKGROUND: The angiogenic factor vascular endothelial growth factor (VEGF)-A plays an important role in breast cancer progression. However, the involvement of VEGF-C and VEGF-D, two newer members of the VEGF family, in breast carcinoma and their relationship with clinicopathologic parameters have not been clearly demonstrated. METHODS: In this study, the expression levels of VEGF-A, VEGF-C, and VEGF-D protein in 107 breast carcinoma cases and 22 nonmalignant breast tissue samples were examined by immunohistochemistry and quantitated by image analysis. RESULTS: Higher expression of VEGF-C and VEGF-D was found in breast carcinomas than in nonmalignant breast tissue samples. Moreover, expression of VEGF-A, VEGF-C, and VEGF-D was significantly and positively correlated with ErbB2 expression. High levels of VEGF-A expression were associated with shorter disease-free survival (DFS). Patients with tumors expressing high levels of VEGF-C or VEGF-D showed a notable trend for worse DFS, however, it was not statistically significant. The combination of VEGF-A and VEGF-C status predicted survival better than either marker alone. CONCLUSIONS: Our study suggests that expression of the angiogenic and lymphangiogenic factors (i.e., VEGFs) might be regulated at least in part by ErbB2. In addition, the combination of VEGF-A and VEGF-C status may better predict prognosis of patients with breast carcinoma than VEGF-A alone.     

Expression analysis of vascular endothelial growth factors and their relationships to lymph node metastasis in human colorectal cancer

This study was undertaken to determine whether expressions of the vascular endothelial growth factor (VEGF) family (VEGF-A, VEGF-B, VEGF-C, and VEGF-D) are correlated with clinicopathological parameters, with particular reference to lymph node metastasis in colorectal cancer. Total RNA was isolated from 82 surgical specimens of colorectal cancer and matched to normal mucosa with (n = 41) or without (n = 41) lymph node metastasis. The mRNA expression of each VEGF family member was quantified by real-time quantitative (RTQ) RT-PCR assay. VEGF-B and VEGF-C mRNA were significantly higher both in the tumors with lymph node metastasis (p = 0.027 and p = 0.024, respectively) and in tumors with lymphatic invasion (p = 0.042 and p = 0.005, respectively). In contrast, VEGF-D mRNA was down-regulated in tumors with lymphatic involvement (p = 0.047). Among the other clinicopathological factors, we noted that VEGF-A mRNA was higher in tumors with liver metastasis than in those without (p = 0.018) and was higher in tumors with venous invasion than in those without (p = 0.007). The results of this study demonstrate that high levels of VEGF-B, C and low levels of VEGF-D mRNA expression are associated with lymph node metastasis and lymphatic involvement. These results suggest that a balance among VEGF-B, VEGF-C, and VEGF-D might contribute to the lymphangiogenic process and metastasis in colorectal cancer.     

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.     

The number of residual metastatic lymph nodes following neoadjuvant chemotherapy predicts survival in patients with stage III NSCLC

The prognosis of patients with stage III non-small-cell lung cancer (NSCLC) who achieve a pathological complete response or downstaging following neoadjuvant therapies are better than the prognosis of patients with residual metastatic lymph nodes (LN). However, the prognostic significance of the number of residual metastatic LNs remains unclear. From January 2001 to January 2006, 42 consecutive patients with stage IIIAN2 (22 patients) and IIIB without pleural effusion (20 patients) were treated with neoadjuvant chemotherapy. Thirty-four (81.0%) of the 42 patients were pathologically staged by mediastinoscopy. Neoadjuvant chemotherapy consisted of 3 cycles of platinum-based doublet (21 patients with gemcitabine, 15 with paclitaxel, and 6 with docetaxel). After neoadjuvant chemotherapy, a pathological complete response was achieved in one patient and downstaging was achieved in 24 patients. Pathological LN metastasis was absent in 9 patients (21.4%) and present in 33 patients (78.6%). With a median follow-up of 23 months, the 2-year disease-free survival (DFS) rate of patients without residual LN metastasis was statistically better than that of patients with residual LN metastasis (46% vs. 18% respectively, P=0.03). Among 33 patients with residual LN metastasis, age (P=0.01), pathological downstaging (P=0.098) and the number of residual metastatic LNs (median 14 months in 1-4 LN vs. median 5 months in LN > or =5; P=0.011) were significant predictors of DFS in univariate analysis. In multivariate analysis, the number of residual metastatic LNs was an independent predictor of DFS among patients with residual LN metastasis, irrespective of pathological downstaging. The number of residual metastatic lymph nodes following neoadjuvant chemotherapy is an independent predictor of DFS in patients with stage III NSCLC.     

Expression of vascular endothelial growth factor receptor-3 by lymphatic endothelial cells is associated with lymph node metastasis in prostate cancer.

PURPOSE: The molecular mechanisms underlying lymph node metastasis are poorly understood, despite the well-established clinical importance of lymph node status in many human cancers. Recently, vascular endothelial growth factor (VEGF)-C and VEGF-D have been implicated in the regulation of tumor lymphangiogenesis and enhancement of lymphatic invasion via activation of VEGF receptor-3. The purpose of this study was to determine the expression pattern of the VEGF-C/VEGF-D/VEGF receptor-3 axis in prostate cancer and its relationship with lymph node metastasis. EXPERIMENTAL DESIGN: The expression pattern of VEGF-C, VEGF-D, and VEGF receptor-3 in localized prostate cancer specimens (n = 37) was determined using immunohistochemistry. RESULTS: Widespread, heterogeneous staining for VEGF-C and VEGF-D was observed in all cancer specimens. Intensity of VEGF-C staining was lower in benign prostate epithelium than in adjacent carcinoma, whereas no difference between benign epithelium and carcinoma was observed for VEGF-D staining. VEGF receptor-3 immunostaining was detected in endothelial cells of lymphatic vessels in 18 of 37 tissue samples. The presence of VEGF receptor-3-positive vessels was associated with lymph node metastasis (P = 0.0002), Gleason grade (P < 0.0001), extracapsular extension (P = 0.0382), and surgical margin status (P = 0.0069). In addition, VEGF receptor-3 staining highlighted lymphatic invasion by VEGF-C-positive/VEGF-D-positive carcinoma cells. CONCLUSIONS: Together, these results suggest that paracrine activation of lymphatic endothelial cell VEGF receptor-3 by VEGF-C and/or VEGF-D may be involved in lymphatic metastasis. Thus the VEGF-C/VEGF-D/VEGF receptor-3 signaling pathway may provide a target for antilymphangiogenic therapy in prostate cancer.     

B Lymphocytes Promote Lymphogenous Metastasis of Lymphoma and Melanoma

The prognosis of patients with many types of cancers correlates with the degree of metastasis to regional lymph nodes (LNs) and vital organs. However, the mechanisms and route of cancer cell metastasis are still unclear. Previous studies determined that B-cell accumulation in tumor-draining LNs (TDLNs) induces lymphatic sinus growth (lymphangiogenesis) and increases lymph flow, which could actively promote tumor dissemination through the lymphatic system. Using young Eµ-c-Myc mice that feature LN B-cell expansion as hosts for tumor transplants, we show that subcutaneously implanted lymphomas or melanomas preferentially spread to TDLNs over non-TDLNs, thus demonstrating that these tumors initially metastasize through lymphatic rather than through hematogenous routes. In addition, the rate and amount of tumor dissemination is greater in Eµ-c-Myc mice versus wild-type hosts, which correlates with LN B-cell accumulation and lymphangiogenesis in Eµ-c-Myc hosts. The increased lymphatic dissemination in Eµ-c-Myc hosts is further associated with rapid hematogenous tumor spread of subcutaneously implanted lymphomas, suggesting that TDLN metastasis secondarily drives lymphoma spread to distant organs. In contrast, after intravenous tumor cell injection, spleen metastasis of lymphoma cells or lung metastasis of melanoma cells is similar in Eµ-c-Myc and wild-type hosts. These studies demonstrate that the effect of Eµ-c-Myc hosts to promote metastasis is limited to the lymphatic route of dissemination. TDLN B-cell accumulation, in association with lymphangiogenesis and increased lymph flow, thus significantly contributes to dissemination of lymphomas and solid tumors, providing new targets for therapeutic intervention to block metastasis.     

Combination therapy of lymphatic drug delivery and total body irradiation in a metastatic lymph node and lung mouse model

Chemotherapy using a lymphatic drug delivery system (LDDS) targeting lymph nodes (LNs) in the early stage of metastasis has a superior antitumor effect to systemic chemotherapy. An LDDS produces a higher drug retention rate and tissue selectivity in LNs. To expand the therapeutic coverage of LDDS from local treatment of metastatic LNs to prevention of distant metastases, the combination of treatment with therapies that enhance systemic tumor immune effects is an important therapeutic strategy. Recently, total body irradiation (TBI) has been shown to activate immune responses and alter the tumor microenvironment. Here we show that combination therapy with TBI and LDDS improves the antitumor effect of metastatic LNs and lung metastasis. Tumor cells were inoculated into the subiliac LN (SiLN) to induce metastasis into the proper axillary LN (PALN) and lung in a mouse model. TBI was carried out on day 4 after inoculation using a gamma irradiator. Lymphatic drug delivery into the accessory axillary LN was used to treat PALN. In vivo bioluminescence imaging, high-frequency ultrasound, and histology showed that combination therapy using TBI (total dose 1.0 Gy once) and the LDDS suppressed tumor growth in LNs and lung metastases and was more effective than using LDDS or TBI alone. Quantitative RT-PCR of spleens after combination therapy revealed increased expression of CD4, CD8, and IL-12b, indicating an activated immune response. The results show that combination therapy with TBI and LDDS is a method to improve the efficacy of LN metastases and distant metastases therapy and is a promising novel approach to treat cancer patients.     

胆囊癌组织中血管内皮生长因子C和D的表达及其与淋巴管和血管生成的关系

目的 探讨胆囊癌组织中血管内皮生长因子C(VEGF-C)和VEGF-D的表达及其与淋巴管和血管生成的关系.方法 采用免疫组化法检测50例胆囊癌组织中VEGF-C、VEGF-D、D2-40和CD31的表达,以10例癌旁正常胆囊组织和19例慢性胆囊炎作为对照,并分析其与临床病理诸因素的关系.结果 50例胆囊癌组织中,32例VEGF-C表达阳性,阳性率为64.0%,31例VEGF-D表达阳性,阳性率为62.0%,均高于癌旁正常组织(P<0.05),但与慢性胆囊炎差异无统计学意义(P>0.05).VEGF-C的表达与胆囊癌患者的年龄和淋巴结转移有关,VEGF-D的表达仅与胆囊癌患者的淋巴结转移有关.50例胆囊癌组织的微淋巴管密度(MLVD)和微血管密度(MVD)分别为6.94±3.6和36.1±12.8.VEGF-C阳性组和VEGF-D阳性组的MLVD和MVD均高于阴性组.MLVD与淋巴结转移有关,MVD与淋巴结转移、分化程度有关.VEGF-C与VEGF-D的表达呈正相关(r=0.498,P<0.01).结论 在胆囊癌中,VEGF-C和VEGF-D参与胆囊癌的淋巴生成和血管生成的调节,通过增加瘤周淋巴管的密度促进肿瘤细胞的淋巴结转移.     

VEGF-C,VEGF-D在乳腺癌中的表达及其与淋巴结转移及预后的关系

目的探讨VEGF-C和VEGF-D在乳腺癌组织中的表达以及与淋巴结转移、预后的相关性。方法采用SP免疫组化法检测78例乳腺癌癌组织和30例癌旁组织中VEGF-C,VEGF-D的表达水平,并完成所有患者的5年的随访资料。结果 78例乳腺癌癌组织中VEGF-C/D表达与癌旁组织相比差异有统计学意义(P<0.05);乳腺癌中VEGF-C表达与VEGF-D表达无相关性。VEGF-C,VEGF-D的表达水平与乳腺癌脉管内侵犯、淋巴结转移密切相关(P<0.05),但与年龄、肿瘤大小、TNM临床分期、ER、PR及Her-2的表达无关(P>0.05)。VEGF-C和VEGF-D的表达水平与乳腺癌的总生存期及无病生存期密切相关(P<0.05)。结论 VEGF-C,VEGF-D在乳腺癌组织中呈高表达;VEGF-C/D的表达水平与乳腺癌淋巴结转移能力和预后密切相关。     

VEGF121 promotes lymphangiogenesis in the sentinel lymph nodes of non-small cell lung carcinoma patients

BACKGROUND: The sentinel lymph node (SLN) concept is that lymphatic flux from a primary tumor initially flows into a SLN. The mechanism mediating tumor metastasis within SLNs remains largely unknown; however, primary tumors overexpressing vascular endothelial growth factor (VEGF)-A appear to induce SLN lymphangiogenesis prior to metastasis in animal model. Our aim was to further investigate the capacity of VEGFs to induce lymphangiogenesis within SLNs and to assess their role in SLN metastasis in non-small cell lung carcinoma (NSCLC). METHODS: Real-time quantitative RT-PCR was used to assess expression of mRNAs encoding several VEGFs (VEGF121, VEGF165, VEGFR1, VEGFR2, VEGFR3, VEGF-C and VEGF-D) in resected lymph node specimens from 35 NSCLC patients, after which we compared their expression SLNs and non-SLNs. In addition, expression of the lymphatic endothelium-specific hyaluronan receptor (LYVE)-1 was used to assess lymphangiogenesis in SLNs and non-SLNs. RESULTS: Immunohistochemical staining revealed substantial expression of LYVE-1 in SLNs. Moreover, levels LYVE-1 mRNA were significantly higher in SLNs than non-SLNs (P<0.05), as were levels of VEGF121 and VEGFR2 mRNA (P<0.01 and P=0.02, respectively). In addition metastasis-positive SLNs showed significantly higher levels of VEGF121, VEGF-C and VEGF-D mRNA than metastasis-negative SLNs (P<0.001, P=0.01 and P=0.01, respectively), and VEGF121 induced the proliferation of lymphatic endothelial cells (P<0.01). CONCLUSIONS: Our findings suggest that active lymphangiogenesis is ongoing within SLNs from NSCLC patients, even before metastasis. This lymphangiogenesis may be promoted by upregulation of VEGF121, which may in turn act in part via induction of VEGF-C.     

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.