The Effects And Molecular Mechanism Of Nanocomplex AS1517499-USPIO-CD163 Regulating The Polarization Of Tumor Associated Macrophages In Wilms’ Tumor

Background Wilms’ Tumor(WT)is a common malignant tumor of the abdomen in children and originates from renal blastocyst cells,the incidence in the population is approximately 1/10000.Wilms’ Tumor can be combined with hypospadias,cryptorchidism,sporadic aniridia,and with high grade malignancy and recurrence rate.Currently,the standard of treatments by the National Wilms’ Tumor Study Group(NWTS)and the International Society of Pediatric Oncology(SIOP)are widely adopted,individualized treatment can be given according to the clinical stage,age,tumor weight and histologic type.Nowdays,surgical resection combined with postoperative chemotherapy and radiotherapy can significantly improve the survival rate,and the overall survival rate in developing countries has reached 80%.However,the side effects of extended surgical resection and postoperative high-dose chemotherapy have been paid more and more attention,which can seriously affect the quality of life of surviving children.At present,doctors are faced with many problems,including the prospective identification of low recurrence patients,the exploration for targeted molecular therapy,the reduction of side effects during treatment and the improvement of overall survival rate.Tumor associated macrophages(TAMs)are an important parts of tumor stromal cells,accounting for about 50%of the total number of immune cells in the tumor stroma.They derived from differentiation and proliferation of monocytes which are recruited in the tumor site.Recent studies have shown that TAMs plays an important role in tumor proliferation,invasion and metastasis,immune escape and angiogenesis.According to the influence of chemokines in the tumor microenvironment,TAMs can be differentiated into subtypes with different functions,which can be broadly divided into classical activation pathway(M1-type macrophages,the anti-tumor subtype)and alternative activation pathway(M2-type macrophages,the pro-tumor subtype).In recent years,some studies have confirmed that the aggregation of TAMs in tumor predict the poor prognosis of patients,and more and more studies support this view.For example,the increase of TAMs in epithelial ovarian cancer,pancreatic cancer,and medulloblastoma breast cancer predicts a poor prognosis,but the expression and role of TAMs in Wilms’ Tumor have not been clearly studied,which requires further study.TAMs can be polarized into different subtypes with different effects according to tumor microenvironment,the role of TAMs in tumor are ambidextrous.In the early stage of tumor,TAMs mainly polarize to M1-type macrophages in the tumor stroma,with the progression of the tumor,TAMs largely polarize to M2-type macrophages,which promote the invasion and metastasis.TAMs locate in the tumor stroma,and interacte directly with tumor cells.And CSF-1 receptors in TAMs are high expression,and the tumor cells could produce CSF-1,then stimulates TAMs polarized to pro-tumor subtype,and pro-tumor subtype of TAMs can produce peptides to promote the growth of tumor,such as CCL2,EGF,PDGF could promote the proliferation,VEGF could promote angiogenesis,MMPs can hydrolysis substrate membrane to promote migration.With development of research,cancer cells are the main targets in treatment,but targeted-TAMs treatment has also become an auxiliary therapy.There are two main approaches to targeted-TAMs therapy:one is to inhibit the accumulation of monocytes/TAMs and reduce the number of TAMs in the tumor stroma;the other is to regulate the polarization of TAMs into the anti-tumor subtype.Molecular targeting treatment inhibit the aggregation and polarization of TAMs based on the characters,to regulate the tumor microenvironment and promote the immune response of T cells with an extensive prospect in antitumor therapy.The role of subtypes of TAMs in the Wilms’ tumor has not been elucidated,so the aim of the study is to illuminate the expression and mechanism of different subtypes of TAMs in the Wilms’ tumor.According to the result,targeting different subtypes of TAMs enhance the anti-tumor therapy,and providing a foundation for molecular targeted therapy in Wilms’ tumor.Objective Firstly,explore the distribution subtypes of TAMs and analyze the correlation between TAMs and overall survival rate in Wilms’ tumor.Then,research was made to illuminate action and mechanism subtypes of TAMs in Wilms’ tumor progress and metastasis in vitro,and modificate the polarization of TAMs to suppress tumor.Lastly,Ultrasmall Superparamagntic Iron Oxide(USPIO)were used as a carrier to synthesis targeted nanocomplex to accurately regulate the polarization process of TAMs and inhibit the proliferation and invasion of Wilms’ tumor.Methods Overall,61 patients with Wilms’ tumor underwent nephrectomy without chemotherapy before operation from April 2006 to March 2014 in shandong provincial hospital,including 44 males and 17 females,and the median age was 19 months(2-106 months).According to the National Wilms Tumor Study(NWTS)-5 guidelines,the distribution of tumor stages was as follows:stage I,27 patients;stage Ⅱ,18 patients;and stage III,16 patients.Western Blot,immunohistochemistry and immunofluorescence were used to explore the distribution of M1 and M2 macrophages in tumor and adjacent tissues.Kaplan-Meier analysis with regard to the relationship between the presence of TAMs and follow-up information was performed.THP-1 cell lines differentiated into Ml and M2 macrophages with the stimulation of cytokines,and the phenotype were identified at gene and protein level by PCR,Western Blot and immunofluorescence.M1-type and M2-type macrophages were co-cultured with SK-NEP-1 cells lines respectively,and cell viability and invasion were detected by CCK8 and Transwell assay to illuminate the roles of different subtypes of TAMs in the tumor progression.The differential expression of MMP9 between M1 and M2 macrophages was measured at gene and protein level by PCR,Western Blot and ELISA,the expression of MMP9 in Wilms’ tumor and adjacent tissue was analysed by Western Blot and immunohistochemistry,and assess the related signaling pathways of TAMs in pro-tumor effect mechanism.To explore the mechanism of STAT6 signaling pathway in the polarization of M2-type macrophages,and assess the treatment effect in Wilms’ tumor when blocking this signaling pathway of TAMs.USPIO with PEG modification used as drug vehicles,coupled with STAT6 signaling pathway inhibitor AS1517499,CD 163 monoclonal antibody respectively,synthesized targeted nanocomplex AS1517499-USPIO-CD163(AUC)by the carbodiimide method.Determine stability,particle size distribution,iron concentration,encapsulation efficiency of AS 1517499,CD 163 antibody content and transmission electron microscopy observation of AUC.The activity of AS1517499 and CD 163 antibody in AUC were detected by Western Blot and immunofluorescence methods.To verify high selectivity of AUC in targeting M2-type macrophages by AO/EB and flow cytometry,and perform anti-tumor effect of AUC in the co-culture system of TAMs and Wilms’ tumor cells.Result The results showed that the density of Ml and M2 macrophages in tumor tissues were significantly greater than that in adjacent normal tissues,and located in the tumor stroma,the quantity of M1-type macrophages decreased while the quantity of M2-type macrophages increased with tumor stage progression in Wilms’ tumor.Kaplan-Meier analysis suggested that patients with high densities of M1-type macrophages had no significant difference in overall survival time with low densities patients(log-rank test,p=0.074),and high densities of M2-type macrophages had shorter overall survival time than those with low densities of M2-type macrophages(log-rank test,p=0.011).THP-1 cells lines were successfully induced into Ml-type and M2-type macrophages,and phenotype was identified by Western Blot and immunofluorescence.It was found that the specific proteins CD80 and iNos were highly expressed in M1-type macrophages,while the specific proteins Arg-1,CD 163 and CD206 were more expressed in M2-type macrophages than in M1-type macrophages.After co-culture of M1-type and M2-type macrophages with SK-NEP-1 cells,cell vitality and invasion ability were measured by CCK8 and Transwell assay,M2-type macrophages could promote the proliferation and invasion.The MMP9 expression was high in M2-type macrophage at mRNA,protein and cell culture supernatant level by PCR,Western Blot and ELISA methods,and density of MMP9 in Wilms’ tumor tissues were significantly greater than that in adjacent normal tissues.Through activating the AKT/PI3K signaling pathway,MMP9 could reduce E-cadherin protein,increase the expression of N-cadherin protein,initiate the process of epithelial mesenchymal transformation,and significantly enhance the invasion and migration ability of SK-NEP-1 cell lines.The STAT6 signaling pathways act as a critical role in TAMs polarization into M2-type macrophages,with the application of STAT6 signaling pathway inhibitor AS1517499,phosphorylation of STAT6 is reduced,so as to the protein level of CD206 and CD163 of M2-type macrophages.Blocking STAT6 signaling pathways can significantly reduce the M2-type macrophage polarization,inhibit Wilms’ tumor proliferation and invasion in co-culture system witih TAMs.Targeted nanocomplex AS1517499-USPIO-CD163 were synthesized successively with favorable physical properties.The targeted nanocomplex AUC had pharmaceutical activity of each ingredient,good stability,and the particle size distribution was less than 100 nm.There were approximately 145.9±36.9μg AS1517499 and 34±8.9μg CD163 antibody in 1mg Fe of targeted nanocomplex AUC,transmission electron microscope of AUC result showed circular or elliptic particles,regular morphous,uniform in sizes,agglomerate distribution,the iron core was about 5 nm in diameter.Targeted nanocomplex AUC could inhibit the polarization of TAMs to M2-type macrophages and reduce the quantity of M2-type macrophages.In addition,compared with M1-type macrophages,targeted nanocomplex AUC could increase the phagocytosis of M2-type macrophages,ultimately leading to their death,and also in same way obviously inhibited the proliferation and invasion ability of Wilms’ tumor cell lines in the co-culture system.Conclusion TAMs(both Ml and M2 types)are gathered in the stroma of Wilms’tumor.M1-type macrophages are inversely related to clinical stage,and M2-type macrophage densities increase with the progression of clinical stage and predict poor prognosis of patients.M2-type macrophages can promote the proliferation,invasion and migration of Wilms’ tumor,and mechanism may be that by activating the AKT/PI3K signaling pathway,MMP9 could initiate epithelial mesenchymal transformation.The STAT6 signaling pathway acts as a critical role in TAMs polarization into M2-type macrophages,the application of STAT6 signaling pathway inhibitor AS 1517499 reduces the quantity of M2-type macrophages,inhibits Wilms’ tumor proliferation and invasion,which can be further studied as a new target for the treatment of Wilms’ tumor.Targeted nanocomplex AUC could be synthesized successively by the carbodiimide method with pharmaceutical activity of each ingredient,good stability,and smaller particle size distribution.The core of targeted nanocomplex AUC is magnetic iron oxide nanoparticles which can be actively phagocytic by macrophages and lead to their death.And targeted nanocomplex AUC could be utilized as a targeted anti-TAMs medicine,as the feature of the highly targeted selectivity for M2-type macrophages,and also have an obvious inhibition effect on the proliferation and invasion of Wilms’ tumor.