Tumor Associated Macrophages' Function And Mechanism Research In Non-small Cell Lung Cancer Epithelial Mesenchymal

Background: Tumor microenvironment composed of mesenchymal cells,and active medium constitute to tumor cells,and has played a key role in tumor progression and metastasis.Among them,the tumor associated macrophage(tumor associated macrophages,TAMs)refers to the infiltration in the macrophages of tumor stroma,is also the largest number of inflammatory cells in tumor microenvironment.Divided into classical TAMs according to its microenvironment activation(M1)and alternative activation(M2).A large number of studies have shown that TAMs in M2 phenotypes,the high expression of a variety of growth factors,proteolytic enzyme,promote angiogenesis factor and immunosuppressive cytokines,etc.,can promote tumor growth and metastasis,and the induced immunosuppression.Studies have found that the number of TAMs M2 phenotypes in the process of tumor invasion to raise a variety of solid tumor and lung cancer progression and poor prognosis.TAMs,however,affect NSCLC progression of the exact mechanism is unclear.Glycosylation is a kind of eukaryotic gene expression in the process of common translation regulation after the event.More and more studies have shown that tumor malignant transformation(oncogenic transformation)anomaly of the initial phase of glycosylation further promoted the tumor invasion and metastasis.Abnormal glycosylation is the symbol of the tumor characteristics,and many kinds of enzymes especially is related to the abnormal expression of glycosyl transferase.Enzyme catalytic fucus Protein glycosylation for fucose transferase(fucosyltransferases,FUTs),divided into Protein O-fucose base transferase(Protein O-fucosyltransferases POFUTs)and N-sugar chain end fucus saccharifying enzyme(FUT1-FUT11).Fucose base transferase(FUT)family through catalytic fucose transfer to the glycoprotein of n-acetyl glucosamine residues to participate in the synthesis of cell surface antigen.According to the different types of catalytic glycosidic bond is divided into 1,2-FUTs,1,3-FUTs and 1,6-FUTs.Lewis(Lewis antigen Y,Le Y)is combined with the oligosaccharide in the end of the glycoprotein and glycolipid,60-90% of epithelial origin of tumor are the expression of the protein,belonging to A,B,H blood group antigen family.FUT4 as 1,3-FUT,is the key enzyme of Le Y in Lewis oligosaccharides synthesis.Studies have proven through regulating fucose transferase to indirectly regulate the expression of cell surface antigen of oligosaccharides.Recent studies have shown that FUT4 / Le Y,in a variety of solid tumors with high expression,and is related to invasion,metastasis and poor prognosis.However,in view of the type M2 macrophages in lung cancer microenvironment interactions with FUT4 / Le Y,research is still little,the immune cells in the tumor microenvironment and tumor secrete factors and fucus glycosylation relationship has not yet been reported.EMT conversion epithelial mesenchymal,refers to the epithelial cells lose polarity,sample into mesenchymal cells form,obtain the ability of migration,is the key step in the tumor invasion and metastasis for higher capacity,closely related with inflammatory cells in the tumor microenvironment.Research shows that in micro environment under the regulation of inflammatory medium,epithelial cell polarity to lose adhesion between the cell decreases,cytoskeleton remodeling and migration of mesenchymal cells and plasticity and other characteristics,and involves the epithelial markers E cadherin-reduce,mesenchymal markers N-cadherin and EMT related transcription factor q increases.A large number of studies have shown that EMT in the growth of a variety of malignant tumor cells and play an important role in tumor invasion and metastasis.Using immunohistochemical SP method,this study to count of CD68 mark TAMs,according to its average divided into high and low expression group.59 cases of patients with NSCLC in lung tissue adjacent to carcinoma tissues and TAMs count analysis,and study the expression and localization of the TAMs,counting analysis with NSCLC patients' gender,age,smoking,T stage,clinical stage,pathological type,differentiation degree and lymph node metastasis and its influence on prognosis.And further by western blot,immunofluorescence and transwell cell into a tumor experiment,experiment and nude mice tumor weight,it was found that TAMs,such as volume curve expression and FUT4 / Le Y,protein,and the correlation between EMT.Methods:1.59 cases of NSCLC patients had operated in first affiliated hospital of dalian medical universityl,from August 2005 to August 2009,detect the protein expression of TAMs counts in tumor stroma,nests,and normal tissues by SP immunohistochemistry.To investigate the correlation of TAMs counts and the clinical features of patients with non-small-cell lung cancer.analysis TAMs with gender,age,smoking,pathological type,differentiation degree,T stage,clinical stages,lymph node metastasis and 5 years of survival rate,as well as to the Diease-free survival(DFS)and Overall survival(OS)such as prognostic factors.All cases were diagnosed by histopathology and cytology clear.Clinical total installment and T stage lung cancer on the basis of international research organization for staging criteria(AJCC/UICC).2.Using Western blot detection E cadherin,N-cadherin,beta-catenin,Vimentin,Snai EMT marker protein and FUT4 / Le Y,fucus glycosylation related protein expression.3.using indirect immunofluorescence method to detect E cadherin,beta-catenin expression.4.the use of scratch experiment and transwell ability to observe and analyze the cell invasion and metastasis.5.the dalian medical university laboratory animal center offers-nu SPF BALB/c male nude mice(6-8 weeks),weight of 16 ~ 18 g,on the body subcutaneous tumor experiment.6.SPSS 17.0 software package is adopted to establish the database,the results of the related information into the database,for statistical analysis.The count data to rate(%),said comparison between measurement data set using the single factor analysis of variance;DFS and OS by Kaplan Meier-survival analysis,Log-Rank method to compare the survival rate;Many factor correlation analysis using COX regression analysis.Take a = 0.05 for inspection level,P < 0.05 for the difference was statistically significant.Results:1.NSCLC tumor stroma TAMs count in the cytoplasm is significantly higher than the nests and normal tissue adjacent to carcinoma,the positive rate and cell count was 94%(53.65-14.52),88%(9.92-3.12)and 85%(6.83 + /-2.26),statistically significant difference(P < 0.01).2.TAMs count is associated with NSCLC lymph node metastasis and TNM stages,lymph node metastasis,phase III-IV positive rate and positive count were 65.3%(74.36 + /-14.78),90.4%(59.83 + /-16.21),lymph node metastasis is significantly higher than the negative,I-II(41.2 + /-10.23),10% and 52.6%(41.69 + 15.6)(P < 0.05).TAMs expression and with gender,age,smoking history,pathological type,differentiation degree,T stage no correlation(P > 0.05).3.Kaplan-Meier single factor analysis showed that TAMs expression and NSCLC survival(P < 0.01).TAMs high expression group(month)is 26.512,the median DFS time was lower than that in group TAMs high expression of 42.938.This suggests that TAMs for NSCLC predictor of recurrence;TAMs high expression group(month)is 65.563,the median OS time was lower than that in group TAMs high expression of 91.105;Single factor survival analysis showed that smoking history,TNM staging,lymph node metastasis positive are associated with the OS of NSCLC(P < 0.05 or P < 0.01).Gender,age,pathological type,differentiation degree,T stage has nothing to do with the OS(P > 0.05).This suggests that TAMs can be used as a prognostic factor NSCLC;Multiariable COX regression analysis showed that TAMs count,a positive lymph nodes,TNM stage were independent prognostic risk factors for NSCLC.4.this study first PMA and IL 4,IL-13 type M2 macrophages was derived,and then through the type M2 macrophages with lung adenocarcinoma cells A549 and H1299 after cocultivation,observed under white light specific multilateral,epithelial cells of the lung cancer cells lost of rules and forms,are actually presents the spindle fiber cells instead kind of form,the arrangement of cells is more dispersed,mixed and disorderly,western blot,immunofluorescence,transwell and cell scratch test results also suggest the M2 macrophages can be caused by TGF-beta EMT,invasion and migration of tumor cells.5.Western blot test found that M2 macrophage conditioned medium group and the concentration gradient of the exogenous TGF-beta FUT4 / LEY protein expression significantly increased than the control group(P < 0.05);After the further application of TGF-beta blockers SB431542,FUT4 / LEY protein expression significantly decreased(P < 0.05);In exogenous TGF-beta under the same conditions,western blot show si-FUT4 group than the control group,compared to calmodulin N-cadherin,Vimentin nucleoprotein express significantly decreased(P < 0.05),the result suggests that TGF-beta mediated FUT4 / Le Y is closely related to the EMT happened.6.nude mice subcutaneously into tumor found that mixed injection group(M2 macrophages + A549)tumor size and volume were significantly higher than the pure injection group(A549);Tumors immunohistochemical detection of EMT related proteins found mixed injection group E-Cadherina reduce,Vimentin elevated(P < 0.05),suggesting the TAM by promoting the TGF-beta FUT4 / Le Y expression in NSCLC,which mediated EMT,tumor growth,invasion and metastasis.Conclusion:1,TAMs count associated with the TNM stages and lymph node metastasis of NSCLC patients,to participate in the development process of the occurrence of NSCLC.2,TAMs expression and NSCLC DFS that TAMs count can be used as NSCLC predictor of recurrence.3,TAMs expression and NSCLC OS,counting TAMs,TNM stage,lymph node positive is an independent prognostic factor in patients with NSCLC.4,FUT4 / Le Y as TAMs cause the key link of EMT,affects the invasion and metastasis of tumor growth and,as research gradually thorough,in the future is expected to become the new target for cancer immunotherapy.