| OBJECTIVEThere are two important characteristics in tumor microenvironment:hypoxia and accumulation of extracellular adenosine,which are important factors leading to tumor immunosuppression,proliferation deterioration,development and metastasis,and are closely related to patients'poor prognosis.An important factor in the hypoxia pathway is hypoxia inducible factor 2alpha(HIF-2alpha),the adenosine pathway contains two important targets,5'extracellular nucleotidase(CD73),adenosine receptor A2a(A2aR).Microvessel density(MVD)is an important indicator for assessing tumor neovascularization,and we label it with CD34 protein.In non-small cell lung cancer(NSCLC),the expression of HIF-2?,CD73,A2aR and their relationship are still unclear,and the relationship with tumor neovascularization has not yet clarified.In this study,we used immunohistochemistry to detect the expression of HIF-2?,CD73 and A2aR in NSCLC,determined the MVD value,analyzed the expression and correlation of them.METHODSWe collected a total of 72 resected NSCLC tumor specimens from the patients who underwent curative surgical treatment between June 1,2008 and June 1,2014 at Affiliated Hospital of Shandong Academy of Medical Sciences.Immunohistochemistry was used to evaluate the expression of CD73 protein and MVD has been assessed by using CD34 protein.We collected 12 specimens of primary lung adenocarcinoma resected surgically in Shandong Cancer Hospital And Institute from November 1,2017 to January 1,2018 for western blot analysis.RESULTS1.HIF-2alpha was mainly expressed in the cytoplasm of tumors and partly in the nucleus.The positive expression was 66.7%(48/72),which was associated with high TNM stage(?~2=11.708,P=0.003),lymph node metastasis(?~2=16.457,P=0.000),and high histological grade(?~2=6.613,P=0.037).2.CD73 protein is mainly expressed on the membrane of tumor cells,and a small part in the cytoplasm.Positive CD73 expression was 70.8%(51/72),The CD73 expression level was significantly higher in TNM III stage than in I&II stage(Mean Rank 46.00>31.45)(z=-2.931,P=0.003),in Node-Positive than in Node-Negative(Mean Rank 44.57>30.74)(z=-2.884,P=0.004).3.A2aR protein is mainly expressed on the membrane of tumor cells,and a small part in the cytoplasm.Positive A2aR expression was 54.2%(39/72).In women(?~2=3.981,P=0.046)and adenocarcinoma(?~2=5.469,P=0.019),the A2aR expression is increased.4.Western blot analysis of lung adenocarcinoma:HIF-2 alpha protein expression in lymph node metastasis group was higher than that in non-lymph node metastasis group(P=0.001),the CD73 protein expression in lymph node metastasis group was higher than that in non-lymph node metastasis group(P=0.0049),and there was no significant difference in the expression of A2aR between lymph node metastasis group and non-metastasis group(P=0.6504).5.There was a positive correlation between positive expression of HIF-2alpha and positive expression of CD73(r=0.389,P=0.001),between positive expression of CD73 and positive expression of A2aR(r=0.372,P=0.001).But there was no correlation between positive expression of HIF-2alpha and positive expression of A2aR(r=0.157,P=0.187).6.Kaplan-Meier survival curves were modeled in a univariate analysis:Patients with HIF-2alpha positive expression experienced significantly reduced overall survival(Log-Rank,?~2=5.563,P=0.018).Patients with CD73 positive expression experienced significantly reduced overall survival(Log-Rank,?~2=4.09,P=0.043).There was no significant difference in survival rates between groups with different expression of A2aR(Log-Rank,?~2=0.849,P=0.357).Among NSCLC patients,high CD73 expression was an independent indicator of poor prognosis in multivariate Cox regression analyses for overall survival[HR=2.524,95%CI:1.167~5.458].7.The MVD value was significantly higher in TNM III stage(49.72±10.44)than in I&II stage(31.55±8.75),(t=-7.836,P=0.000);in tumor size>3cm(39.82±12.41)than in tumor size?3cm(31.53±12.02),(t=-2.423,P=0.018);in Node-Positive(47.13±10.76)than in Node-negative(31.24±9.56),(t=-6.600,P=0.000).8.The value of MVD with HIF-2?positive expression(41.81±12.491)was significantly higher than with HIF-2?negative expression(29.96±9.196),(t=-4.119,P=0.000).The expression level of HIF-2?was correlated significantly with MVD(r=0.442,P=0.000).The value of MVD with CD73 positive expression(41.71±11.592)was significantly higher than with CD73 negative expression(28.52±10.543),(t=-4.498,P=0.000).The expression level of CD73 was correlated significantly with MVD(r=0.474,P=0.000).There was no significant difference in MVD count among different groups of A2aR(t=-0.208,P=0.836).CONCLUSIONIn NSCLC patients,the expression of HIF-2alpha,CD73 and A2aR protein increased,between them HIF-2alpha and CD73 were closely related to the tumor development,malignancy,poor prognosis and neovascularization,while the expression of A2aR was only related to women and adenocarcinoma.The expression of HIF-2alpha is closely related to the expression of CD73,but they are not related to the expression of A2aR.This suggests that there is a close relationship between hypoxia and adenosine pathways in NSCLC,and both of HIF-2?and CD73 are related to neovascularization,which may become a new potential targets for NSCLC therapy.However,because of the limited number of samples,the expression of protein and its specific mechanism need to be further studied. |
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