GP73 Regulates Macrophage Polarity And Inflammation In The Tumor Microenvironment

ObjectiveOur findings provided first insight into the pathological function of exocellular GP73,delineating the molecular mechanism of GP73 in affecting the density of TAMs in the microenvironment of HCC tumors,and providing a potential therapeutic target for combating HCC progression.Method1.TCGA database was used to obtain clinical data of 129 Hepatocellular carcinoma(HCC)patients;the correlation between GP73 and various inflammatory cytokines on m RNA expression was analyzed by Pearson product moment coefficient method.2.Recombinant human GP73,and C-GP73 were produced and purified in 293 T cells transfected with Flag-GP73 or Flag-C-GP73 expression plasmid;Ficoll gradient method was used to isolate monocytes from whole blood of anonymous donors,M-CSF was used to induce the differentiation of monocytes into macrophages;purified GP73 and C-GP73 proteins were added into the culture of isolated macrophages,flow cytometry was used to analysis the density of macrophages and TAMs indicated by CD163 and CD206 markers,respectively.Supernatants from above experiments were collected and protein array was used to screen the the secretion of cytokines affected by GP73 protein.3.Multiplexed immunofluorescent staining based on tyramine signal amplification(TSA)technology was used to immunostaining tissue microarray(TMA)of HCC with antibodies against GP73,CD68 and CD206.Automatic quantitative pathology imaging system(Vectra? Polaris?,Perkin Elmer)was used to analyze the correlation of GP73 with CD68 and CD206 expression in 129 tissue microarrays containing liver cancer and adjacent normal tissues.4.COX multivariate regression analysis and Kaplan-Meier parallel log-rank test were used to analyze the prognostic value of GP73 and CD206 expression in HCC tissues with complete follow-up data.5.A syngeneic tumor model and DEN-induced HCC mice model were used.After successful model construction,GP73 blocking antibody treatment was given for a certain period of time and concentration.The Serum GP73 level of the mice were regularly detected,the size and number of the tumors were monitored.After a certain period of treatment,the mice were sacrificed,a part of the livers were used for flow cytometry to detect the expression levels of macrophage surface molecular markers F4/80 and CD206,another part for immunohistochemical staining.ResultsPart I: GP73 induces TAMs polarizion and cytokines secretions1.The results of TCGA database analysis showed that the expressions of chemokine CXCL1(r=0.41,P<0.0001),CXCL20(r=0.36,P<0.0001),IL-18BPa(r=0.29,P<0.0001)and TIM-3(r=0.30,P<0.0001)were positively correlated with the expression level of GP73.2.Flow cytometry data showed that the expression of CD163 was significantly increased after stimulation with GP73 protein or C-GP73 protein(P<0.01;P<0.001)relative to that of PBS-treated cells.3.Multiple cytokines and chemokines associated with immunomodulation,immune cell recruitment,tumor growth and metastasis was induced by the addition of GP73;Notably,the expression levels of TIM-3,CXCL1,CXCL9 and CXCL10 induced by GP73 were increased at about 3,5,9 and 32 folds as compared to the control group;ELISA was used to confirm the above cytokines.Part II: GP73 and CD206 was significantly correlated with reduced overall survival of HCC1.The density of TAMs in HCC tissues was positively related with the expression level of GP73;a positive relationship between the level of GP73 and CD206 in liver cancer tissues(P<0.001)was identified,with no significant relationship between CD68 and GP73.2.COX proportional hazard regression analysis showed that vascular invasion(P=0.017),tumor stage(P<0.001)and CD206(P=0.027)were independent factors influencing the survival rate of patients with HCC;patients with high expression of GP73 had no association with the survival outcome(P = 0.8078);patients with high expression of CD206 had moderate association with the survival outcome(P = 0.0445),patients with high expression of both GP73 and CD206 was significantly correlated with reduced overall survival(P = 0.0243).Part ?: Blockage of GP73 by using GP73 specific antibody inhibits tumor growth1.An orthotropic HCC mice model with Hepa1-6 cells was first established and treated with Ig G control or GP73 antibody.As compared to the Ig G control,GP73 antibody therapy significantly improved survival and decreased tumor burden;a significant decrease in F4/80+ and CD206 +TAMs were found in GP73 antibody-treated tumors,however,the proportion of CD11c+ M1 macrophages was not affected by GP73 antibody treatment.2.DEN-induced HCC mice model was used and treated with Ig G control or GP73 antibody.As compared to the Ig G control,GP73 antibody therapy presented with a reduced number of liver tumors and smaller tumor area;GP73 antibody-treated tumors had lower expression of F4/80+ and CD206 +TAMs than Ig G-treated tumors.Conclusion1.Full-length GP73 and C-GP73 impacts macrophage polarity to modulate cytokine associated with tumor growth and metastasis release in tumor microenvironment.2.The density of TAMs in liver cancer tissues is positively correlated with the expression level of GP73;patients with high expression of both GP73 and CD206 was significantly correlated with reduced overall survival.3.Blocking GP73 leads to reduced density of TAMs in the tumor microenvironment with smaller tumor size and prolonged survival.Here we identified a novel potential therapy and diagnosis marker-GP73,which regulates HCC progression by impacting macrophages polarize and cytokines release,finally,we provided evidence that blockage of GP73 can inhibit HCC growth and prolong tumor-induced survival.