COX‐2‐Mediated Regulation of VEGF‐C in Association With Lymphangiogenesis and Lymph Node Metastasis in Lung Cancer

The mechanisms underlying the effects of COX‐2 on tumor lymphangiogenesis remain largely undefined. Here, the human lung cancer cell lines A549, 95D, Anip973, and AGZY83‐a with different metastatic capacities were investigated by immunostaining, western blotting, and real‐time RT‐PCR. We observed increased expressions of COX‐2 and VEGF‐C in the three highly metastatic cell lines compared with the less metastatic AGZY83‐a cell line. The COX‐2‐specific inhibitor Celecoxib suppressed VEGF‐C expression whereas the main COX‐2 metabolite PGE₂ elevated VEGF‐C expression in Anip973 and AGZY83‐a cells in positive and negative experiments. To determine the functional link to COX‐2 more specifically and elucidate the mechanistic pathway, we used a siRNA to knock down the high COX‐2 expression in Anip973 cells and transfected a COX‐2 cDNA to enhance the low COX‐2 expression in AGZY83‐a cells, and then treated the cells with EP1/EP4 agonists or antagonists, respectively. The results revealed that the EP1/EP4 agonists significantly increased VEGF‐C production in the COX‐2‐knockdown Anip973 cells. In contrast, the EP1/EP4 antagonists diminished VEGF‐C production in the COX‐2‐overexpressing AGZY83‐a cells. Furthermore, animal models provided evidence that Celecoxib decreased VEGF‐C expression, lymphangiogenesis, and lymph node metastases in Anip973 cells, whereas PGE₂ treatment increased the same factors in the parental AGZY83‐a cells. A positive correlation between COX‐2 and VEGF‐C was also confirmed in vivo. The present data suggest that COX‐2 regulates VEGF‐C expression via the PGE₂ pathway, and that EP1/EP4 receptors are involved in PGE₂‐mediated VEGF‐C production. Thus, COX‐2 may represent a candidate gene for blocking tumor lymphangiogenesis and lymph node metastasis. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of VEGF‐C and VEGF‐D as Significant Markers for Assessment of Lymphangiogenesis and Lymph Node Metastasis in Non‐Small Cell Lung Cancer

Vascular endothelial growth factor (VEGF)‐C and VEGF‐D induce lymphangiogenesis through activation of VEGF receptor 3 (VEGFR‐3) and have been implicated in tumor spread to the lymphatic system. Lymph node dissemination critically determines clinical outcome and therapeutic options of patients with non‐small cell lung cancer (NSCLC). However, the relationship of VEGF‐C, VEGF‐D, and lymph node metastasis in cancers, including NSCLC, is still controversial. To evaluate the relationship between lymphangiogenesis and lymph node metastasis, the expression of VEGF‐C and VEGF‐D in NSCLC tumors were detected by immunohistochemistry and quantitative real‐time polymerase chain reaction (QRT‐PCR). QRT‐PCR revealed that in marginal region VEGF‐C and VEGF‐D mRNA was significantly higher than in tumor center, and VEGF‐D mRNA was also higher than that in peritumoral lung tissue. Immunohistochemically, we observed the same heterogeneous expression of VEGF‐C and VEGF‐D proteins. The group with high expression of VEGF‐C and VEGF‐D in marginal region had a higher incidence of lymph node metastasis compared with the group with low expression. Furthermore, the group with high expression of VEGF‐D in marginal region had a higher incidence of lymphatic invasion. The group with high peritumoral lymphatic vessel density (LVD) had higher expression of VEGF‐C and VEGF‐D mRNA compared with the group with low peritumoral LVD. Our studies suggested that the expression of VEGF‐C and VEGF‐D at invasive edge was significantly associated with lymph node metastasis or lymphatic invasion in patients with NSCLC and may be involved in regulation of lymphangiogenesis and lymph node metastasis in NSCLC. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Lentivirus‐Mediated Small Interfering RNA Targeting VEGF‐C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma

Lymph node metastasis is a major prognostic factor for patients with breast cancer. The activation of vascular endothelial growth factor (VEGF)‐C plays a key role in lymph node metastasis through promoting lymphangiogenesis. Thus, we attempted to elucidate whether small interfering RNAs (siRNA) targeting VEGF‐C could suppress lymphangiogenesis and lymph node metastasis in vivo. A lentivirus‐based VEGF‐C siRNA vector was infected into breast cancer cells and a xenograft model. The expression of VEGF‐C mRNA and protein were quantified by quantitative real‐time polymerase chain reaction (QRT‐PCR), immunohistochemistry, and western blot analysis. The effect of VEGF‐C siRNA on breast cancer cells was investigated by an invasion assay. Lymphangiogenesis was analyzed with anti‐LYVE‐1 and anti‐D2‐40 by immunohistochemical analysis. Lentivirus‐mediated VEGF‐C siRNA stably reduced VEGF‐C mRNA and protein expression. VEGF‐C siRNA inhibited the invasive ability of breast cancer cells in vitro. Five weeks after intratumoral injection, the tumor volume was significantly smaller in the VEGF‐C siRNA group than in the control scramble siRNA group in the MDA‐MB‐231 cell xenograft model. The numbers of LYVE‐1 and D2‐40 positive vessels per microscopic field were significantly decreased in the VEGF‐C siRNA group, which indicates that VEGF‐C siRNA inhibited lymphangiogenesis. Moreover, lymph node metastasis was significantly suppressed by VEGF‐C siRNA in vivo. In conclusion, these results indicate that lentivirus‐mediated VEGF‐C siRNA offers a new approach for therapeutic intervention to prevent tumor growth and lymphatic metastasis of breast cancer. Anat Rec, 292:633–639, 2009. © 2009 Wiley‐Liss, Inc.     

高低转移肺腺癌细胞系中肿瘤抑制基因p16、p21和p53表达研究

目的 探讨肿瘤抑制基因对肺腺癌细胞生长的抑制作用。方法 利用FuGene转染方式分别将p21和p16基因的表达质粒转入－对肺腺癌细胞系Anip973和AGZY83－a中,同时用含野生型p53 基因的腺病毒感染p16基因转染前后的这一对细胞系。对P16和P21蛋白过表达的细胞系进行了细胞生长曲线、克隆形成率、原位末端标记分析和流式细胞仪分析。结果 p16基因的过表达只能使细胞系的G1期细胞比例提高,但细胞生长曲线,克隆形成率均未出现改变,未检测到凋亡信号。P21蛋白过表达的一对细胞系细胞生长曲线斜率降低,克隆形成能力下降,并出现明显的G1期阻滞,但未检测到凋亡信号。p53基因感染AGZY83－a,Anip973及经过p16基因转染的细胞AGZY83－ap16和Anip973p16后呈现时间依赖性表达,细胞生长曲线和四唑盐比色法分析提高,野生型p53基因的大量表达明显抑制以上4种细胞的生长,Anip973和Anip973p16的生长抑制率高于AGZY83－a和AGZY83－ap16;Anip973p16和AGZY83-ap16的生长抑制率高于Anip973和AAGZY83－a。这4种细胞在感染p53后出现典型的凋亡信号。结论p16基因的过表达并不能抑制细胞系的生长,而p21基因的过表达通过G1期阻滞抑制这1对肺腺癌细胞的生长;野生型p53基因在AGZY83－a和Anip973中高效表达可产生明显的细胞生长抑制效应;野生型p53基因对肺腺癌高转移细胞系Anip973抑制作用更为明显。     

Integrin α4 Induces Lymphangiogenesis and Metastasis via Upregulation of VEGF‐C in Human Colon Cancer

Vascular endothelial growth factor‐C (VEGF‐C) is a key regulator in lymphangiogenesis, and is overexpressed in various malignancies. Integrin α4β1, a new member of the VEGF‐C/VEGF receptor pathway, was found to be overexpressed in melanoma tumors. However, little is known regarding the potential role of integrin α4β1 in lymphangiogenesis and other solid tumors. The aim of this study was to investigate the expression patterns of integrin α4 and VEGF‐C in relation to lymphangiogenesis and clinicopathological parameters in human colon cancer. The expression of integrin α4, VEGF‐C, and VEGFR‐3 was assessed in 71 human colon cancer tissues and 30 paracancerous normal tissues by immunohistochemical staining. Lymphatic microvessel density (LMVD) was measured after D2‐40‐labeling, and the correlations among different factors were statistically analyzed. The expression of integrin α4, VEGF‐C, VEGFR‐3, and LMVD was higher in colon cancer tissues compared with the normal paracancerous colon tissues. There was a positive correlation between the expression of integrin α4 and VEGF‐C. Integrin α4 and VEGF‐C were significantly associated with the clinicopathological parameters (LMVD, Duke's stage, and lymph node metastasis). Kaplan–Meier analyses indicated that patients with high integrin α4 or VEGF‐C expression had significantly shorter overall survival and tumor‐free survival time. Multivariate analyses suggested that integrin α4 and VEGF‐C may serve as independent prognostic factors for human colon cancer. Both integrin α4 and VEGF‐C are involved in lymphangiogenesis and lymphatic metastasis. Our results demonstrated that integrin α4 is a novel prognostic indicator for human colon cancer. Anat Rec, 299:741–747, 2016. © 2016 Wiley Periodicals, Inc.     

应用mRNA差异显示技术克隆人肺腺癌转移相关基因

肿瘤转移是恶性肿瘤最基本的生物学特征。为了探讨恶性肿瘤的侵袭和转移的机理，选择两株具有相同的细胞来源、但具有不同转移能力的人肺腺癌细胞系ＡＧＺＹ８３ａ和Ａｎｉｐ９７３作为研究材料，采用ｍＲＮＡ差异显示（ｍＲＮＡｄｉｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙ）技术，对这两个细胞系的基因表达差异情况进行了分析。结果显示，在这两个细胞系之间存在明显的基因表达差异，说明在低转移的ＡＧＺＹ８３ａ向高转移的Ａｎｉｐ９７３演变过程中涉及多个基因的激活与失活。研究结果还提示，在高转移的Ａｎｉｐ９７３细胞系中，某些基因的表达或高表达与人类逆转录病毒基因ＬＴＲ的整合有关。另一个克隆与人类线粒体基因ＮＤ４具有高度同源性，其高度表达与Ａｎｉｐ９７３的转移表型有关。表明肿瘤的转移是一个多基因参与的过程，由于这些基因的相互作用及细胞内外环境因素的影响，最终决定了肿瘤细胞的转移表型。     

Intratumoral Lymphatics and Lymphatic Vessel Invasion Detected by D2‐40 Are Essential for Lymph Node Metastasis in Bladder Transitional Cell Carcinoma

Bladder cancer is frequently associated with regional lymph node metastasis at the time of diagnosis or after initial treatment, and lymph node metastasis is crucial for clinical therapeutic strategies. Lymphangiogenesis, detected by antibodies specific for lymphatic endothelial cells, is correlated with cancer spread, but the mechanisms that underlie lymphatic spread and the role of lymphangiogenesis in cancer metastasis has been less clear. The aim of this study was to investigate the association of vascular endothelial growth factor (VEGF)‐D expression, intratumoral lymphatics, and lymphatic invasion associated with lymph node metastasis as well as the prognostic analysis in patients with bladder transitional cell carcinoma (TCC). The VEGF‐D expression was evaluated by immunohistochemistry in 72 specimens, and tumoral lymphatic vessels were measured by D2‐40. Counts of lymph vessels were taken in intratumoral and peritumoral areas. Survival analyses and their independent roles were investigated using univariate and multivariate analysis models. The high expression of VEGF‐D was closely associated with the intratumoral lymphatic vessels, tumoral lymphatic invasion, and lymph node metastasis as well as a shorter overall survival. Higher lymphatic vessel density, intratumoral lymphatics, and lymphatic invasion showed a significant association with lymph node metastasis. Univariate analysis indicated that VEGF‐D, intratumoral lymphatics, and lymphatic invasion were associated with overall survival, but they were not independent prognostic factors for bladder TCC in multivariate analysis. We conclude that VEGF‐D plays an essential role in tumoral lymphangiogenesis. Intratumoral lymphatics and lymphatic invasion are important predictive factors of pelvic lymph node metastasis in patients with bladder cancer. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Lymphangiogenesis and Prognostic Significance of Vascular Endothelial Growth Factor C in Gastro‐oesophageal Junction Adenocarcinoma

Vascular endothelial growth factor C (VEGF‐C) is a crucial regulator of the development of lymphatic vessels and is involved in the lymph node metastasis of cancer. The levels of VEGF‐C expression and lymphatic vessel density (LVD) in 128 gastro‐oesophageal junction adenocarcinoma (GEJA) tissues were examined by immunohistochemistry and analysed for their association with clinicopathological features and disease‐free survival. We found that 75.0% of tumour samples displayed strong immunoreactivity to VEGF‐C. The levels of VEGF‐C expression in the tumour tissues were associated with the stages of the clinical tumours and the lymph node metastasis status, but not with the age, gender and the size and type of tumours in the cohort. Similarly, LVD, as evaluated by anti‐D2‐40 staining, was also associated with the clinical stages of GEJA. The values of LVD were positively correlated with the levels of VEGF‐C expression in these samples (r = 0.3760, P = 0.0001). High levels of VEGF‐C expression and high values of LVD were associated with shorter periods of disease‐free survival (DFS) in patients with GEJA (P < 0.001). In addition, GEJA at N1 and N2 stages, at T4 stage, chemotherapy after surgery, high levels of VEGF‐C expression and lower marginal resection were independent factors for the prognosis of DFS in patients with GEJA. Our data indicate that VEGF‐C may promote the lymphangiogenesis and lymphatic metastasis of GEJA and that VEGF‐C may be a valuable biomarker for the diagnosis of lymphatic metastasis and a prognostic factor of the survival of patients with GEJA.     

血管内皮生长因子C及其受体3在非小细胞肺癌组织中的表达及意义

目的　探讨血管内皮生长因子C(VEGF C)和受体 (VEGFR) 3在人非小细胞肺癌(NSCLC)组织中的表达及其与微血管、微淋巴管形成、淋巴转移、预后之间的关系。方法　对 76例人NSCLC及相应癌旁组织行VEGF C、VEGFR 3及CD34免疫组织化学染色链霉素抗生物素蛋白 过氧化物酶 (SP)法检测 ,进行淋巴管密度计数、微血管密度 (MVD)计数 ,并结合临床和病理资料进行分析。结果　NSCLC中 ,VEGF C的表达与肺癌分化程度负相关 (P =0 0 0 9)。VEGF C和VEGFR 3的表达水平与淋巴结转移呈正相关 (分别P =0 0 0 8,P =0 0 13) ,与淋巴浸润呈正相关 (分别P =0 0 2 7,P =0 0 2 0 )。VEGF C的表达与VEGFR 3在肺癌细胞中的表达呈正相关 (P =0 0 0 9)。VEGF C与淋巴管密度 (P =0 0 0 6 )、MVD(P =0 0 4 6 )呈正相关。淋巴管密度与淋巴结转移 (P =0 0 10 )、淋巴浸润 (P =0 0 19)、TNM分期 (P =0 0 15 )呈正相关 ,MVD与血行转移 (P <0 0 0 1)、TNM分期 (P <0 0 0 1)呈正相关。VEGF C阳性表达与生存时间、5年生存率呈负相关 (P <0 0 0 1)。结论　NSCLC中 ,VEGF C通过自分泌方式作用于受体VEGFR 3,促进肺癌组织生长 ,抑制分化。VEGF C促使肺癌内淋巴管形成 ,促进肺癌淋巴结转移。VEGF C和VEGFR 3表达增高、淋巴管密度增加     

VEGF‐D deficiency in mice does not affect embryonic or postnatal lymphangiogenesis but reduces lymphatic metastasis

Vascular endothelial growth factor‐D (VEGF‐D) is one of the two ligands of the VEGFR‐3 receptor on lymphatic endothelial cells. Gene‐silencing studies in mice and Xenopus tadpoles recently showed that the role of endogenous VEGF‐D in lymphatic development is moderate. By contrast, exogenous VEGF‐D is capable of stimulating lymphangiogenesis. Nonetheless, its endogenous role in pathological conditions remains largely unknown. Hence, we reassessed its role in disease, using Vegf‐d^null mice. Vegf‐d^null mice were generated that, under physiological conditions, displayed normal embryonic and postnatal lymphangiogenesis and lymphatic remodelling, efficient lymphatic functioning and normal health. Vegf‐d^null mice also reponded normally in models of skin wound healing and healing of infarcted myocardium, despite enhanced expression of VEGF‐D in these models in wild‐type mice. In contrast, Vegf‐d^null mice displayed reduced peritumoral lymphangiogenesis and lymph node metastasis in an orthotopic pancreatic tumour model. Together, our data indicate that endogenous VEGF‐D in mice is dispensible for lymphangiogenesis during development, in postnatal and adult physiology and in several pathological conditions, but significantly contributes to lymphatic metastasis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Vascular Endothelial Growth Factor D and Intratumoral Lymphatics as Independent Prognostic Factors in Epithelial Ovarian Carcinoma

Lymph node metastasis is an important prognostic indicator for disease progression and is crucial for therapeutic strategies of epithelial ovarian carcinoma. Vascular endothelial growth factor (VEGF)‐D has been confirmed to have potent lymphangiogenic function in experimental models, but the role in the progression of human ovarian carcinoma remains presently controversial. The purpose of this study was to investigate the prognostic significance of VEGF‐D and the presence of intratumoral lymphatics in patients with epithelial ovarian carcinoma. The VEGF‐D expression was evaluated by immunohistochemistry in 78 specimens of epithelial ovarian carcinoma and tumoral lymphatic vessels were measured by D2‐40. The expression of VEGF‐D protein was detected in the cytoplasm of the tumor cells and in stroma occasionally. The high expression of VEGF‐D was closely associated with the FIGO stage, intratumoral lymphatic vessels, tumoral lymphatic invasion, and lymph node metastasis as well as a shorter overall survival. Univariate and multivariate analysis indicated that VEGF‐D, intratumoral lymphatics, and lymphatic invasion were independent prognostic factors for overall survival and disease‐free survival in patients with epithelial ovarian carcinoma. We conclude that VEGF‐D plays an essential role in tumoral lymphangiogenesis and lymphatic spread, VEGF‐D expression, and the intratumoral lymphatics may be clinically useful indicators for prognostic evaluation in patients with epithelial ovarian carcinoma. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Vascular endothelial growth factor-C expression in human gallbladder cancer and its relationship to lymph node metastasis

Lymph node metastasis is a major prognostic factor in human cancer. Vascular endothelial growth factor C (VEGF-C) is a lymphangiogenic polypeptide that has been implicated in several human solid tumors. However, the clinical significance of VEGF-C has remained unknown in gallbladder carcinoma. Paraffin-embedded tumor specimens of 52 surgically resected gallbladder cancers were immunohistochemically stained for VEGF-C, VEGF, and CD34. The correlations among VEGF-C expression, VEGF expression, microvessel density (MVD), clinicopathologic features, and clinical outcomes were statistically analyzed. Thirty-three (63%) of the 52 gallbladder cancers were highly positive for VEGF-C protein by immunohistochemistry. VEGF-C expression was significantly correlated with lymphatic vessel involvement, lymph node metastasis, and worse outcomes after operation (p<0.001, p<0.001, p<0.001, respectively), but not with MVD. By the Cox regression model, lymphatic vessel involvement emerged as an independent prognostic parameter. These results suggest that VEGF-C may play a role in tumor progression via lymphangiogenesis and lymph node metastasis in human gallbladder cancer.     

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.     

Tumor lymphangiogenesis predicts melanoma metastasis to sentinel lymph nodes.

Cutaneous melanoma is a common melanocytic neoplasm that can quickly metastasize to regional lymph nodes. Currently, prognosis is determined by measuring tumor thickness but more reliable markers for metastatic spread are urgently needed. We investigated whether the extent of tumor lymphangiogenesis can predict melanoma metastasis to sentinel lymph nodes. We quantified the extent of tumor lymphangiogenesis, as well as other factors, in excised primary tumors and in sentinel lymph node biopsy samples from 45 patients with primary cutaneous melanoma. The results were correlated with histological and clinical outcome. Primary melanomas from patients whose tumors had metastasized to the sentinel lymph nodes contained prominent 'hot spots' of increased lymphatic vessel density, compared to nonmetastatic tumors. Multivariate risk analysis revealed that the lymphatic vascular area of primary melanomas, an index of tumor lymphangiogenesis, was the most sensitive prognostic marker for sentinel lymph node metastasis, and was even able to more accurately predict which tumors were metastatic to sentinel lymph nodes than the currently used method of measuring tumor thickness. Highly lymphangiogenic melanomas maintained their lymphangiogenic activity after metastasis to the sentinel lymph node. The extent of tumor lymphangiogenesis is a highly sensitive (83%) and specific (89%) prognostic marker of lymph node metastasis. Assessment of lymphangiogenesis in primary melanomas may be a more effective approach than the currently used technique of measuring tumor thickness in selecting patients with early metastatic disease for aggressive therapy.     

Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model

We reported that cyclo-oxygenase (COX)-2 expression in human breast cancer stimulated cancer cell migration and invasiveness, production of vascular endothelial growth factor (VEGF)-C and lymphangiogenesis in situ, largely from endogenous PGE2-mediated stimulation of prostaglandin E (EP)1 and EP4 receptors, presenting them as candidate therapeutic targets against lymphatic metastasis. As human breast cancer xenografts in immuno-compromised mice have limitations for preclinical testing, we developed a syngeneic murine breast cancer model of spontaneous lymphatic metastasis mimicking human and applied it for mechanistic and therapeutic studies. We tested the roles of COX-2 and EP receptors in VEGF-C and -D production by a highly metastatic COX-2 expressing murine breast cancer cell line C3L5. These cells expressed all EP receptors and produced VEGF-C and -D, both inhibited with COX-2 inhibitors or EP4 (but not EP1, EP2 or EP3) antagonists. C3H/HeJ mice, when implanted SC in both inguinal regions with C3L5 cells suspended in growth factor-reduced Matrigel, exhibited rapid tumor growth, tumor-associated angiogenesis and lymphangiogenesis (respectively measured with CD31 and LYVE-1 immunostaining), metastasis to the inguinal and axillary lymph nodes and the lungs. Chronic oral administration of COX-1/COX-2 inhibitor indomethacin, COX-2 inhibitor celecoxib and an EP4 antagonist ONO-AE3-208, but not an EP1 antagonist ONO-8713 at nontoxic doses markedly reduced tumor growth, lymphangiogenesis, angiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in responding mice revealed reduced VEGF-C and -D proteins, AkT phosphorylation and increased apoptotic/proliferative cell ratios consistent with blockade of EP4 signaling. We suggest that EP4 antagonists deserve clinical testing for chemo-intervention of lymphatic metastasis in human breast cancer.     

Differential expression of RAB5A in human lung adenocarcinoma cells with different metastasis potential

For the sake of better understanding the molecular mechanism of neoplasia, we have used the mRNA differential display technique to analyze two human lung adenocarcinoma cell lines, AGZY83-a and Anip973. Anip973 was isolated from AGZY83-a, but manifested much higher metastatic potential than the parent line. We found that a significant differential cDNA fragment in Anip973 was over-expressed, then over-expressed cDNA fragment was cloned and sequenced. It showed that the over-expressed cDNA in Anip973 was RAB5A cDNA. And the RAB5A cDNA sequence was corresponding between the two cells. To determine whether RAB5A may be differentially expressed in the two human lung adenocarcinoma cells at protein level, we further detected RAB5A protein in the two cells by using immunofluorescent method. RAB5A protein was upregulated in highly metastatic Anip973. We also detected the difference in RAB5A gene expression at RNA level in human non-small cell lung carcinoma by RT-PCR. Using immunohistochemical staining, we also examined RAB5A change at protein level in 45 cases human non-small cell lung carcinoma paraffin sections. The results proved the evidence of upregulation of RAB5A in malignant tumor, indicated over-expression of RAB5A gene was correlated with the malignant degree and metastatic potential of lung cancer(² test, p <0.01). The RAB5A gene is a member of RAS superfamily, which can transcribe GTP-binding protein that plays an important role in signal transduction of protein trafficking at the cell surface and GDP/GTP cycle in the regulation of endocytotic membrane traffic. Thus our results indicated that over-expression of the RAB5A gene was involved in the process of transformation from AGZY83-a to the higher metastatic cell line Anip973. The result may be a powerful experimental evidence that over-expression of RAB5A gene associated with neoplasia metastasis.     

Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis

Lymphangiogenesis is associated with human and murine cancer metastasis, suggesting that lymphatic vessels are important for tumor dissemination. Lymphatic vessel alterations were examined using B16-F10 melanoma cells implanted in syngeneic C57Bl/6 mice, which form tumors metastasizing to draining lymph nodes and subsequently to the lungs. Footpad tumors showed no lymphatic or blood vessel growth; however, the tumor-draining popliteal lymph node featured greatly increased lymphatic sinuses. Lymph node lymphangiogenesis began before melanoma cells reached draining lymph nodes, indicating that primary tumors induce these alterations at a distance. Lymph flow imaging revealed that nanoparticle transit was greatly increased through tumor-draining relative to nondraining lymph nodes. Lymph node lymphatic sinuses and lymph flow were increased in mice implanted with unmarked or with foreign antigen-expressing melanomas, indicating that these effects are not due to foreign antigen expression. However, tumor-derived immune signaling could promote lymph node alterations, as macrophages infiltrated footpad tumors, whereas lymphocytes accumulated in tumor-draining lymph nodes. B lymphocytes are required for lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes, as these alterations were not observed in mice deficient for B cells. Lymph node lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes may actively promote metastasis via the lymphatics.     

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.     

Lymphangiogenesis in regional lymph nodes predicts nodal recurrence in pathological N0 squamous cell carcinoma of the tongue

Hirota K, Wakisaka N, Sawada‐Kitamura S, Kondo S, Endo K, Tsuji A, Murono S & Yoshizaki T
(2012) Histopathology
Lymphangiogenesis in regional lymph nodes predicts nodal recurrence in pathological N0 squamous cell carcinoma of the tongue Aims: Cancer cells induce de‐novo lymphatic vessel growth within draining lymph nodes before they metastasize. The aim of this study was to retrospectively evaluate lymph node lymphangiogenesis before the establishment of nodal recurrence in squamous cell carcinoma (SCC) of the tongue. Methods and results: Surgical specimens from 28 patients with pT2–T3N0M0 SCC of the tongue after local excision with supraomohyoid neck dissection were studied by immunohistochemistry. Intranodal lymphatic endothelium was highlighted by podoplanin staining to evaluate lymphatic vessel counts (LVCs). Primary tumour sections were examined for the expression of lymphangiogenic factors: vascular endothelial growth factor (VEGF)‐C and VEGF‐D. LVCs in regional lymph nodes were significantly increased in the cases with nodal recurrence (P = 0.0013). Simultaneous increases in VEGF‐C and VEGF‐D expression were significantly associated with both an increase in LVC in regional lymph nodes (P = 0.0001) and a decrease in the rate of survival without nodal recurrence (P = 0.016). Conclusions: Knowing the status of lymphangiogenesis in the regional pN0 lymph nodes in tongue cancer would help in predicting which patients will develop nodal recurrence. The use of a therapeutic approach which blocks lymphangiogenic factors, such as VEGF‐C and VEGF‐D, may be beneficial in suppressing the lymphatic spread of tongue cancer with intense intranodal lymphangiogenesis.