The Clinical Pathological Characteristics And Mechanisms Of Lymphatic Endothelial Cells Abnormal Hyperplasia In NSCLC

Objective:To investigate the clinical significance of abnormal proliferation of lymphatic endothelial cells in non-small cell lung cancer(NSCLC),and further explore the prossble mechanisms.Methods:1.Clinical sections:NSCLC patients staged TxNOMO～TxN1MO, who underwent surgical treatment in Fuzhou General hospital of Nanjing Military Command from January 1,2012 through December 31,2012, were screened and included according to the related creteria. Patient’s informations and pathological datum were collected. Lymphatic endothelial cell markers VEGFR2, VEGFR3, PROX1, LYVE1 were detected by immunohistochemical assay. Recurrence and survial atate,calculate disease free survial time(DFS) and overall surial time(OS) were followuped and analyzed by Kaplan-Meier assay, varied factors potentially affecting prognosis were analyzed by Cox regression model. Log-Rank test were used to test the difference, P values were setted at less than 0.05 indicated signifcant difference.2.Experiment sections:(1) Lung cancer stem-like cells Enrichment,screening and identification:selected A549 lung cancer cell line were cultured in serum-free circumstance through 2 weeks, combined with toxic drug Cisplatin 48h. The screened out cells (lung cancer stem-like cells) were identified and analyzed primarily by Flow Cytometry Method. Proliferative capacity was detected by CCK-8 assay. Tumor formation trial were applied in nude mice. (2) Lymphatic endothelial cell differentiation procedures:lung cancer stem-like cells(CSLC) were cultured in the specific culture medium with lymphatic endothelial cell growth factors, and classified into seveh groups:A549 group, A549-CSLC group, A549-CSLC blank group 1, A549-CSLC blank group 2, A549-CSLC TNF-a Group, A549-CSLC VEGF-C group, A549-CSLC TNF-a and VEGF-C group. Proliferative capacity was detected by CCK-8 assay and analyzed to compare the difference among groups. VEGFR2, VEGFR3, PROX1, LYVE1 were detected by Western Blot. Differences between groups were tested by T test, P less than was set as an indication of significant difference.Results:1.According to the screening process, altogether 60 patients with stage TxNOMO TxNIMO NSCLC were included. The relationship analyzed among VEGFR2, VEGFR3, PROX1, LYVE1 and clinical pathological characters showed:VEGFR2, VEGFR3, PROX1, LYVE1 expression was significantly associated with TNM stage differentely (P=0.000); compared with lymph node metastasis status, P value was 0.017,0.000,0.017,0.001 differentely.2.As showed on Kaplan-Meier survial curve, the status of VEGFR2, VEGFR3, LYVE1 and TNM potentially affected the disease-free survival time of the seclected patients.However, little significance was found at PROX1, though a few indicated a shorter disease free survival time.3.The result of cell proliferation detection show that the proliferation ability of A549 cloned ball group cells was stronger which compared to the A549 cell group, there were statistically significant difference in two groups (P＜0.05).4.FCM analysis results show that after enrichment of A549 cloned ball cells group stem cell surface marker CD44 positive rate was 64.16%, CD 133 positive rate was 70.88%; the A549 cells group stem cell surface marker CD44 positive rate was 0.51%, CD133 was 0.78%; there was significant difference between two groups (P<0.05).5.Nude mice tumor model experiment results showed that subcutaneous tumor appear significantly in the group transplanted with A549-CSLC three weekss; later, and little significantly was found in the control transplanted with A549 (P<0.05).6.Western blot results show that compared with A549 cells, the expression of VEGFR2, VEGFR3, PROX1, LYVE1 in the five groups with A549-CSLC were significantly increased. (P<0.05).Conclusion:1.Overexpression of lymphatic endothelia cell markers VEGFR2, PROX1, VEGFR3, LYVE1 are frequently detected in the selected NSCLC patients, which indicated poor prognosis.2.The novel paradigm of serum-free suspension culture combined with Cisplatin can effectively enrich lung cancer stem-like cells.3.Lung cancer stem-like cells can be differentiated into lymphatic endothelial cells by lymphatic endothelial cell culture environment.