Correlation Between Lymphatic Metastasis And The Expression Of VEGF-C, VEGF-D And VEGFR-3 In Non-small Cell Lung Cancer

Infiltrating and metastasis are significant biological characteristics of cancer.The blood circulation and lymphangitic spread arc two important ways for neoplasm metastasis.The lymphatic vessel is one of the earliest accesses for entity neoplasm metastasis.However,compared with tumor vessel,the research for lymphatic vessel is too little.In recent years,with the identification of vascular endothelial growth factor-C(VEGF-C),VEGF-D and lymphatic endothelial markers including lymphatic vessel endothelial hyaluronan receptor-1(LYVE-1),vascular endothelial growth factor receptor-3(VEGFR-3),glomerular podocyte mcmbrance mucoprotein (podoplanin) and the homeobox transcription factor(Prox-1),lymphangiogenesis has become one of the highlights of in the field of tumor metastasis study.It has been demonstrated that lymphangiogenesis induced by VEGF-C/VEGF-D plays a crucial role in promoting tumor cell dissemination to regional lymph nodes in animal models.Anti-lymphangiogenesis will become a new method for cancer.In human,many solid tumors,including breast cancer,melanoma and head and neck carcinoma,the mechanism and clinical significance of lymphangiogenesis have been identified.A positive association between VEGF-C,VEGF-D,VEGFR-3 expressions and lymph node metastasis has been reported in several cancers.However,the relationship of VEGF-C,VEGF-D and VEGFR-3 expressions and lymph node metastasis in some cancers,including non-small cell lung cancer(NSCLC),is controversial.The main focuses are followed:one is whether functional lymphatic vessels exist within tumors or not;the other is which one on earth plays a dominant role in tumor metastasis, intratumoral or periturnoral lymphangiogenesis? Presently,the relationships among the expression of VEGF-C,VEGF-D,VEGFR-3,lymph node metastasis and prognosis have been studied in the field of lymphangiogenesis in NSCLC,while few data exists so far about impact of lymphangiogenesis on the prognosis of NSCLC patients.Further study on the mechanism and significance of lymphangiogenesis in NSCLC would be of benefit to providing experimental evidence for anti-lymphangiogenic therapy.Objective To study the expressions of lymphangiogenetic growth factors as follow,proprotein convertase(PC)5,PC7,vascular endothelial growth factor (VEGF)-C,VEGF-D and their receptor-3(VEGFR-3) in patients with NSCLC,the relationship with microlymphatic vessel density(MLVD),lymph node metastasis and their clinicopathological value.To investigate the expressions of lymphatic vessel markers,such as lymphatic vessel endothelial hyaluronan receptor-1(LYVE-1) and the homeobox transcription factor(Prox-1) in patients with NSCLC,the relationship with MLVD,lymph node metastasis and their clinicopathological value.Methods We evaluated the quantitative expressions of VEGF-C,VEGF-D, VEGFR-3,PC5 and PC7 mRNA in NSCLC tissue samples from patients who had undergone surgery between the October of 2007 and the June of 2008 using real-time quantitative reverse transcriptase polymerase chain reaction(real-time quantitative RT-PCR).At the same time,we evaluated the qualitative expressions of VEGF-C, VEGF-D,VEGFR-3,LYVE-1 and Prox-1 protein in NSCLC tissue samples using immunohistochemistry(IHC) and microlymphatic vessel density(MLVD) was counted.Results1.The real-time quantitative RT-PCR study demonstrated that the expressions of PC5,PC7,VEGF-C,VEGF-D and VEGFR-3 mRNA in specimens of NSCLC were significantly higher than those of the peritumoral and the pulmonary benign diseases tissues and the genes above were correlated significantly with lymph node metastasis and PTNM stage of NSCLC.Significantly positive correlations between PC5,PC7 and VEGF-C,VEGF-D,VEGFR-3 mRNA were observed in NSCLC. 2.The immunohistological study indicated that VEGF-C and VEGF-D were expressed as buffy granules not only in the cytoplasm of cancer cells,but also in macrophages in NSCLC,and that VEGFR-3 immunoreactivity was present in the cytoplasm of cancer cells,macrophages and endothelial cells of lymphatic vessels in the stroma surrounding the cancer cells.LYVE-1 immunoreactivity was present in the cytoplasm of endothelial cells of lymphatic vessels in the stroma,while Prox-1 immunoreactivity was present in the nuclear of endothelial cells of lymphatic vessels.The positive rates of VEGF-C,VEGF-D and VEGFR-3 protein in specimens of NSCLC were significantly higher than those of the peritumoral and pulmonary benign diseases tissues and the proteins aforesaid were correlated remarkably with lymph node metastasis and PTNM stage of NSCLC.The immunohistological study of 146 lymph nodes manifested that the positive rates of VEGF-C,VEGF-D and VEGFR-3 protein in the positive lymph node metastasis tissue were significantly higher than those in the negative lymph node metastasis tissue.According to the correlation analysis,the expressions of VEGF-C,VEGF-D and VEGFR-3 protein in NSCLC were correlated with lymph node metastasis.The MLVDs marked by LYVE-1 and Prox-1 in the cancerous invasive edge of NSCLC were significantly higher than those in the center of cancerous tissues and those in the pulmonary benign diseases tissues.The MLVDs signed by LYVE-1 and Prox-1 in the cancerous invasive edge of NSCLC with the protein expressions of VEGF-C,VEGF-D and VEGFR-3 were significantly higher than those without,which shows the remarkable correlations between MLVD and lymph node metastasis and PTNM stage.3.Comprehensive analysis with the results of real-time quantitative RT-PCR and immunohistochemistry showed that in the level of gene,significant positive correlations between VEGF-C and VEGF-D,VEGF-C and VEGFR-3,VEGF-D and VEGFR-3 mRNA expressions were observed in NSCLC;in the level of protein, significant positive correlations between VEGF-C and VEGFR-3,VEGF-D and VEGFR-3 protein expressions were observed in NSCLC,while,no correlation between VEGF-C and VEGF-D was observed;the immunoreactivities of VEGF-C, VEGF-D and VEGFR-3 were roughly correlated to the mRNA levels of VEGF-C, VEGF-D and VEGFR-3 in real-time RT-PCR.Condusions1.All these suggest that the expressions of VEGF-C,VEGF-D,VEGFR-3 mRNA and protein in specimens of NSCLC are significantly higher.NSCLC cells may secrete lymphangiogenetic growth factors VEGF-C,VEGF-D and their receptor VEGFR-3,which induce the growth of endothelial cells of lymphatic vessels and lymphangiogenesis by VEGF-C,VEGF-D/VEGFR-3 signaling pathway,further accelerate lymphatic metastasis and the growth of NSCLC.2.As specific markers of lymphatic vessel endotheliocyte,LYVE-1 and Prox-1 could identify blood vessels and lymphatic vessels strictly and evaluate vascular system of tumor relatively exactly.The functional microlymphatic vessels correlated with lymphatic metastasis are mainly located in the cancerous invasive edge rather than the center of cancerous tissues.3.Lymphangiogenetic growth factors VEGF-C,VEGF-D,VEGFR-3 and specific markers of lymphatic vessel LYVE-1,Prox-1 might be acted as important markers for evaluating lymphatic metastasis and prognosis in patients with NSCLC.