• Maxillofacial Plastic and Reconstructive Surgery
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• v.36 no.3
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• pp.85-93
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• 2014

Purpose: Nodal metastasis is the main prognostic factor in the patients with oral squamous cell carcinoma (OSCC). We investigated the association between tumor-associated lymphatics and OSCC characteristics. Methods: Thirty-four specimens were used for the immunohistochemical staining with the antibody for vascular endothelial growth factor (VEGF)-C, VEGF-D, VEGF receptor (VEGFR)-3, phosphorylated VEGFR-3, D2-40, and matrix metallproteinases (MMPs). We observed the distribution of the lymphangiogenic factors and quantified the degree of expression. We determined lymphatic vessel density (LVD) and lymphatic vessel dilatation with D2-40 immunostaining. We assessed the association of LVD or lymphatic vessel dilatation with tumor progression or tumor differentiation. Results: OSCC cells expressed lymphangiogenic ligands. Lymphangiogenic receptor, VEGFR-3, was expressed and activated in some tumor cells as well as in tumor-associated endothelial cells. LVD was not associated with tumor size or nodal status, but lymphatic vessel dilatation was higher in tumors with nodal metastasis, and also higher in poorly differentiated tumors. In stromal area of OSCC, MMP-1 and MMP-10 were up-regulated and the basement membrane of tumor-associated endothelial cells was destroyed by these collagenases. Conclusion: In the primary tumors with nodal metastasis, especially in poorly differentiated OSCC, tumor cells invaded the dilated lymphatic vessels via ruptured sites. MMP-1 and MMP-10 are important in the lysis of the glycocalyx inside the tumor-associated lymphatic endothelial cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.35 no.1
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• pp.1-12
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• 2013

Purpose: Vascular endothelial growth factor (VEGF)-C and its tyrosine kinase receptor, VEGF receptor (VEGFR)-3 are recently known to have lymphangiogenic activities in various tumor types. In this study, we determined whether the expression of lymphangiogenic factors correlate with nodal metastasis or survival in a nude mouse model of oral squamous cell carcinoma (OSCC). Methods: Three OSCC cells (KB, SCC4, SCC9) were xenografted into the right mandibular gland of athymic nude mice. The mice were followed for tumor development and growth, and the mice were sacrificed when they had lost more than 20% of their initial body weight, or the diameter of the induced tumor exceeds 20 mm. After necropsy, the murine tumors were examined histologically and radiologically (micro-positron emission tomography computed tomography) for regional or distant metastasis. We performed immunohistochemical assays with anti-VEGF-C, VEGFR-3, CD105, and D2-40 antibodies. Immunofluorescence double staining for LYVE-1/CD31 was also performed. To quantify the VEGF-C and VEGFR-3 level in the cancer tissue, Western blotting was performed. Finally, we determined the correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time. Results: OSCC tumor cells into the mandibular gland of the nude mice successfully resulted in the formation of recapitulating orthotopic tumor. Tumor cells of the induced tumor did not express VEGF-C. VEGF-C/VEGFR-3 expression was mainly distributed in the endothelial cells of the stromal area. There were no correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time of mice injected with different OSCC cell lines. Conclusion: An recapitulating orthotopic model of OSCC in nude mice was established, which copies the cervical nodal metastasis of human OSCC. Overexpression of lymphangiogenic factors seems to have no effect on survival of hosts in this in vivo experiment.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.1
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• pp.1-8
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• 2010

Background and Purpose: Vascular endothelial growth factor (VEGF)-C and VEGF receptor (VEGFR)-3 are involved in tumor lymphangiogenesis. Oral mucosal squamous cell carcinoma (OMSCC) preferentially metastasizes to cervical lymph nodes, so we investigated the expression and distribution of VEGFR-3 signaling proteins in OMSCC. Materials and Methods: Tissue samples of 18 OMSCC, 10 oral mucosal leukoplakia, and 3 normal oral mucosa were evaluated for expression of VEGF-C, VEGF-D, and VEGFR-3 by immunohistochemical staining. The presence of lymphatic vessels was determined using D2-40 staining, by which we also measured lymphatic vessel density (LVD). Results: 72% (13/18) and 56% (10/18) of tissue samples showed VEGF-C and VEGF-D immunopositivity in tumor cells and tumor-associated endothelial cells. VEGFR-3 was also expressed in most of OMSCC, which was up-regulated when compared with normal mucosa or with leukoplakia. Furthermore, LVD was higher in OMSCC than in leukoplakia. Conclusion: Taken together, our results suggest that autocrine activation of lymphatic endothelial cell via VEGFR-3 by VEGF-C and/or VEGF-D could be involved in progression of OMSCC. Therefore, VEGF-C/VEGFR-3 signaling pathway can be a molecular target for anti-metastatic therapy in OMSCC.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.6
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• pp.453-460
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• 2009

Background and Purpose: Vascular endothelial growth factor (VEGF)-C, VEGF-D and their tyrosine kinase receptor, VEGF receptor (VEGFR)-3 are recently known to have lymphangiogenic activities in various tumor types. Oral mucosal squamous cell carcinoma (OMSCC) easily metastasizes to cervical lymph nodes, so we determined the expression levels of VEGF-C, VEGF-D and VEGFR-3 in oral squamous cell carcinoma. Materials and Methods: We performed Western blot analyses with 4 OMSCC cultured tumor cell lines (SCC9, KB, YD-10B, YD-38), and with 7 surgical specimens of OMSCC for the detection of VEGF-C, VEGF-D and VEGFR-3 proteins. Expression of VEGF-C mRNA as well as mRNA for VEGFR-3 in 4 OMSCC cell lines (KB, SCC-4, SCC-9, YD-10B) was investigated by RT-PCR. We also measured VEGFC/VEGF-D protein concentrations in the media and protein concentration of VEGFR-3 in cell lysates of 4 OMSCC cell lines (SCC9, KB, YD-10B, YD-38) using commerical ELISA kits. Finally, we performed immunoprecipitation for the detection of VEGF-C in cell lysates of 4 OMSCC cells (KB, SCC-4, SCC-9, YD-10B) and real-time RT-PCR for the quantification of VEGF-C mRNA. Results: In the result of Western blotting with cell lysates of 4 OMSCC cells, we could not detect the protein expression of VEGF-C, VEGF-D, and VEGFR-3. But, all tumor tissues demonstrated VEGF-C and VEGFR-3. VEGF-C mRNA was detected at various levels in 4 OMSCC cell lines. Moreover, OMSCC cells secreted VEGF-C, not VEGF-D and VEGFR-3 was also detected in cell lysates of OMSCC by ELISA. Immunoprecipitation and real-time RT-PCR revealed VEGF-C was also expressed in 4 OMSCC cell lines. Conclusion: Taken together, tumor cells of OMSCC secrete VEGF-C, not VEGF-D. And VEGFR-3 is expressed tumor cells as well as OMSCC tumor tissues, needs further study.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.3
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• pp.991-994
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• 2012

Objective: VEGF-C has recently been identified as a key molecule which is involved in tumor lymphangiogenesis. The aim of this research was to investigate the role of PARP-1 inhibition in the regulation of VEGF-C expression in CT26 cells. Methods: CT26 cells were treated with or without the PARP-1 inhibitor 5-aminoisoquinolinone (5-AIQ). The expression of PARP-1, NF-kB, and VEGF-C proteins in CT26 cells was measured by Western blot analysis and the VEGF-C mRNA level was determined by reverse transcription polymerase chain reaction (RT-PCR). CT26-secreted VEGF-C was detected by enzyme-linked immunosorbent assay (ELISA). Results: The results of Western blot analysis showed that the expression levels of PARP-1, NF-kB, and VEGF-C were reduced in 5-AIQ treated CT26 cells and the levels of VEGF-C mRNA in 5-AIQ treated CT26 were significantly lower than t in 5-AIQ-untreated cells (P<0.05). The concentrations of CT26-secreted VEGF-C were also dramatically decreased (P<0.05). Conclusion: Here, we provide evidence for the first time that PARP-1 inhibition dramatically reduces VEGF-C expression via the nuclear factor NF-kB signaling pathway. We therefore propose that PARP-1 inhibition has an anti-lymphangiogenic effect and may contribute to the prevention of metastatic dissemination via the lymphatic system.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2823-2828
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• 2012

Objective: To explore a new model of in-situ xenograft lymphangiogenesis of human colonic adenocarcinomas in nude mice. Method: On the basis of establishing subcutaneous xenograft lymphangiogenesis model of human colonic adenocarcinoms, in-situ xenografts were established through the in situ growth of the HT-29 human colonic adenocarcinoma cell line in nude mice. The numbers of lymphangiogenic microvessels, the expression of lymphatic endothelial cell markers lymphatic vessel endothelial hyaloronic acid receptor-1 (LYVE-1), D2-40 and the lymphatic endothelial growth factors vascular endothelial growth factor-C (VEGF-C), -D (VEGF-D) and receptor-3 (VEGFR-3) were compared by immunohistochemical staining, Western bolt and quantitative RT-PCR in xenograft in-situ models. Results: Some microlymphatics with thin walls, large and irregular or collapsed cavities and increased LMVD, with strong positive of LYVE-1, D2-40 in immunohistochemistry, were observed, identical with the morphological characteristics of lymphatic vessels and capillaries. Expression of LYVE-1 and D2-40 proteins and mRNAs were significantly higher in xenograpfts in-situ than in the negative control group(both P<0.01). Moreover, the expression of VEGF-C, VEGF-D and VEGFR-3 proteins and mRNAs were significantly higher in xenografts in-situ (both P<0.01), in conformity with the signal regulation of the VEGF-C,-D/VEGFR-3 axis of tumor lymphangiogenesis. Conclusions: In-situ xenografts of a human colonic adenocarcinoma cell line demonstrate tumor lymphangiogenesis. This novel in-situ animal model should be useful for further studying mechanisms of lymph node metastasis, drug intervention and anti-metastasis therapy in colorectal cancer.

• Journal of Gastric Cancer
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• v.9 no.3
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• pp.96-103
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• 2009

Purpose: Lymph node metastasis is an important factor in determining prognosis and therapeutic options for early gastric cancer (EGC) patients. Vascular endothelial growth factor (VEGF)-C and D are known as lymphangiogenic factors, and cyclooxygenase (COX)-2 is thought to play a role in lymph node metastasis in gastric carcinoma. This study was designed to determine whether the expression of VEGF-C, VEGF-D, and COX-2 is associated with clinicopathologic factors, especially lymph node metastasis in EGCs invading the submucosa. Materials and Methods: Tissue samples were obtained from 85 Patients undergoing standard gastrectomy with lymph node dissection between 1991 and 2007 in the Department of Surgery of Daejeon St. Mary's Hospital in Daejeon, Korea. All patients were diagnosed with gastric cancers and submucosal invasion. We examined the expression of VEGF-C, VEGF-D, and COX-2 using immunohistochemical methods. Results: Of the 85 patients, 16 (18.8%) had lymph node metastasis. VEGF-C, VEGF-D, and COX-2 were positively expressed in 34.1% (29/85), 22.3% (19/85), and 37.6% (32/85) of the patients. VEGF-C and COX-2 expression was significantly correlated with lymph node metastasis (P<0.05). A positive correlation existed between VEGF-C and COX-2 expression (P< 0.001). Conclusion: VEGF-C and COX-2 expression is associated with lymph node metastasis in gastric cancer with submucosal invasion. VEGF-C and COX-2 may thus be predictive markers for lymph node metastasis in EGC patients with submucosal invasion.