The Study On The ACT001 Reversing Immunosuppression In Glioblastoma

Objective: ACT001 is a candidate anti-tumor drug which is originally developed by Chinese scientists.It comes from the structurally modification of an ancient anti-inflammatory drug and can penetrate the blood-brain barrier.STAT3 is a key molecule in the immunosuppression of glioblastoma(GBM),and the structural characteristics of ACT001 suggest that it may target STAT3.The aim of this study was to elucidate the mechanism of ACT001 reversing immunosuppression in GBM.Content: To explore whether the expression of STAT3 is related to the malignant level of glioma,the prognosis of glioma and the immunosuppression of glioma.To investigate whether ACT001 directly binds to STAT3 to inhibit its phosphorylation and PD-L1 transcription.To study whether ACT001 can improve antitumor immune response in vivo.Methods: RNA sequencing and microarray data from The Cancer Genome Atlas(TCGA),RNA sequencing data from(Chinese Glioma Genome Atlas,CGGA),GSE16011 microarray data and their corresponding clinical data were downloaded.The differences of STAT3 expression among different glioma pathological types and GBM subtypes were analyzed.The survival time of patients with different STAT3 expression levels was analyzed.Immunohistochemical staining(IHC)was used to detect the expression of p-STAT3 and PD-L1 in glioma tissue array.The correlation between STAT3 RNA expression and RNA expressions of immune related molecules was calculated on Morpheus website.The relationships between the glioma purity or the infiltration of 22 leukocytes and the expression of STAT3 RNA were calculated by algorithms of estimation of stromal and immune cells in malignant tumors using expression data(ESTIMATE)and cell-type identification by estimating relative subsets of RNA transcripts(CIBERSORT),respectively.The expression of p-STAT3,STAT3 and PD-L1 in three GBM cell lines treated with ACT001 were detected by polymerase chain reaction(PCR),Western blot(WB)and immunofluorescence(IF).The protein was pulled down with biotin probe of ACT001,and silver staining and WB were used to detect whether STAT3 protein was pulled down.The changes of STAT3-PD-L1 pathway were detected in glioma cells which were transfected with STAT3 overexpressing plasmid and treated with ACT001.Chromatin immunoprecipitation assay(Ch IP)was used to test DNA bound to STAT3.PCR amplification and agarose gel electrophoresis were performed on PD-L1 promoter after reverse transcription.Two luciferase reporter genes assay was conducted with STAT3 overexpressing plasmid and PD-L1 promoter overexpressing plasmid.The GBM in situ implantation model in GL261-C57BL/6 mouse was established.The development of the tumor and the survival time of the mice treated by ACT001 gavage were recorded.IHC was used to detect p-STAT3,PD-L1,CD206,CD163,i NOS and IFN γ in tumor samples.Results: The m RNA level of STAT3 in GBM was higher than that in nontumor tissues and lower grade glioma(grade II and III,LGG)which is defined by world health organization(WHO).The levels of STAT3 m RNA in mesenchymal GBM and classical GBM were higher than that of proneural GBM and neural GBM.The m RNA level of STAT3 in primary GBM was higher than that in secondary GBM.Glioma and GBM patients who have high expression of STAT3 m RNA have shorter survival time and are resistant to radiotherapy and chemotherapy.IHC showed that the expressions of p-STAT3 and PD-L1 in gliomas were higher than that in nontumor tissues,and the higher-grade glioma expressed more p-STAT3 and PD-L1.STAT3 m RNA level was negatively correlated with TNFSF9,and positively correlated with PD-L1,CD86,HAVCR2,LGALS9,FCGR2 B,TGFB1,tumor purity and immune cell infiltration.Leukocyte infiltration analysis showed that STAT3 m RNA level was positively correlated with M2 macrophage infiltration.PCR,WB and / or IF experiments showed that ACT001 significantly reduced the levels of p-STAT3 and PD-L1,but the expression level of STAT3 wasn’t changed significantly.Silver staining and WB analysis showed that STAT3 was pulled down by ACT001 probe.WB showed that the ability of ACT001 to reduce p-STAT3 and PD-L1 was decreased in glioma cells which were transfected by STAT3 overexpressing plasmid.Ch IP,PCR and agarose gel electrophoresis showed that PD-L1 promoter sequence bound to STAT3 protein.The double luciferase reporter gene assay showed that the STAT3 overexpressing plasmid could increase the transcription of PD-L1.In vivo experiments showed that ACT001 treatment could prolong the survival time of GL261-C57BL/6 tumor bearing mice,reduce the expression of p-STAT3,PD-L1 and M2 macrophage markers(CD163,CD206),and increase the expression of anti-tumor markers(i NOS,IFN γ).Conclusion: STAT3 expression level is an important factor for the malignant level and prognosis of glioma patients,and is closely related to the immunosuppressive infiltration to gliomas.ACT001 can inhibit the transcription of PD-L1 by competitively binding to STAT3 protein.ACT001 can prolong the survival time of GBM mice and reverse the immunosuppression of GBM.