The Meaning Of B7-H4~+Macrophages Expression In Malignant Pleural Effusion Caused By Lung Cancer And Its Role In EMT

Part I The analysis of CD68+B7-H4+macrophages in malignantpleural effusion caused by lung cancer and its clinical significanceObjective: To research the clinical significance of CD68+B7-H4+macrophages inmalignant pleural effusion caused by lung cancer.Methods: Collect82patients clinical data with pleural effusion from respiratorydepartments of The First Affiliated Hospital Of Soochow University and Suzhou Fifthpeople’s hospital from December2012to December2013.55patients with malignantpleural effusion caused by lung cancer,27patients with tuberculous pleural effusions. Thepleural effusion mononuclear cells （PEMC） were separated by ficoll hypaquegradient.Using Flow cytometry to analyse the expression of B7-H4on the surface ofCD68+macrophage, and evaluate its diagnostic value for malignant pleural effusion causedby lung cancer with ROC.Results: Compared with tuberculous pleural effusion,malignant pleural effusion oflung cancer have higher mean of B7-H4+cell percentage in totalmacrophages (33.57±30.41%VS8.87±6.20%,P＜0.001); Using ROC curve to evaluate thediagnostic values of CD68+B7-H4+macrophages in malignant pleural effusion caused bylung cancer (area under the curve is0.919,95%CI0.861to0.977, P <0.001),the cut offvalue of the proportion of CD68+B7-H4+macrophages in total CD68+macrophages is12.05%,the sensitivity and specific degrees for malignant pleural effusion caused by lungcancer are94.5%and74.1%respectively. Conclusion: Compared with tuberculous pleural effusion, malignant pleural effusioncaused by lung cancer have higher mean of B7-H4+cell percentage in total macrophages,the proportion of CD68+B7-H4+macrophages in total CD68+macrophages have gooddiagnostic value for malignant pleural effusion caused by lung cancer.PartⅡCD68+B7-H4+macrophages induced lung cancer cell EMT bysecreting TGF-beta1Objective: To evaluate the malignant pleural effusion and TGF-beta1function ofinducing lung cancer cell EMT,and study the role of CD68+B7-H4+macrophages ininducing EMT.Methods:The malignant pleural effusion,TGF-beta1cultured A549cell were set to"pleural effusion group" and “TGF-β group” respectively， ordinary culture mediumcultured A549cell is set to "control group", analyzing the expression of vimentin andN-cadherin of three groups cells with Western Blot. And using Flow cytometry to analyzeE-cadherin expression on surface of three groups cells.Using transwell experiment toevaluate the invasion ability of three group cells.Collectting PEMC of lung cancer,analyzing the expression of TGF-beta1in CD68+B7-H4+and CD68+B7-H4-macrophagesby Flow cytometry of intracellular staining.Results:The malignant pleural effusion of lung cancer and TGF-beta1could induceA549cell EMT, and compared with control group the expression of EMT associatedprotein have significant changed.Compared with "control group","pleural effusion group"and “TGF-β group” A549cells Vimentin,N-cadherin are up regulated,while the expressionof E-cadherin is down regulated(P<0.05).Transwell experiment show that A549cell whohave occurred EMT have increased its invasion ability(P<0.05),but "pleural effusiongroup" and “TGF-β group” cells invasion ability have no significant difference(P＞0.05).Flow cytometry of intracellular staining showed that the expression of TGF-beta1inCD68+B7-H4+macrophage is higher than CD68+B7-H4-macrophages in PEMC of lungcancer (13.16±3.01%VS0.97±0.55%, P<0.05). Conclusion:Malignant pleural effusion caused by lung cancer and TGF-beta1, bothinduced the EMT of A549cells. The expression of TGF-beta1in CD68+B7-H4+macrophage is higher than CD68+B7-H4-macrophages,which may be have certain significanceto promote EMT of lung cancer.