Tumor-associated Macrophage-derived Exosomes Mediate Chemoresistance In Glioblastoma

Glioblastoma(GBM)is the tumor with the highest incidence in the central nervous system.Even with postoperative temozolomide(TMZ)-based concurrent and adjuvant chemoradiotherapy(Stupp Protocol.),the tumor recurrence rate is still high.Patients have poor overall survival and prognosis(overall survival time is only 14.6 months).In 2005,the US FDA approved temozolomide as a first-line chemotherapy drug for GBM,but the effective rate was less than 50%;even if the initial treatment was effective,the vast majority of patients developed resistance within 1 year of treatment,which greatly affected the treatment effect.Therefore,strengthening the study of temozolomide resistance mechanism and finding reliable new targets for inhibiting drug resistance are of great significance for improving the sensitivity of glioma comprehensive treatment and improving the prognosis.It has been reported that microRNAs of exosomes secreted by tumor-associated macrophages(TAM)in the tumor microenvironment of gastric,pancreatic and ovarian cancer can mediate the development of chemotherapy resistance of tumor cells.Studies have confirmed that there are also a large number of TAMs in the GBM microenvironment(mainly M2 type,which can account for 30%to 50%of the GBM cell population),but whether it can also participate in the occurrence of resistance to GBM chemotherapy through exosomes is currently unknown.Long non-coding RNA(LncRNA)is a non-coding RNA with a length of more than 200 nucleotides.It is usually abnormally expressed in cancer cells and is closely related to the malignant progression of tumors.LncRNA is one of the many contents(Cargo)of exosomes,and can exert biological effects in target cells.However,the role of lncRNA of ExosomesTAM in GBM progression,especially the effect on GBM chemotherapy resistance,is not clear.Therefore,the research purpose of this subject is to explore the biological role and molecular mechanism of ExosomesTAM-lncRNA in GBM chemotherapy resistance.From this perspective,it provides an important therapeutic reference for solving the problem of TMZ chemotherapy resistance in GBM.Chapter1:Induced differentiation and phenotypic identification of TAMObjective:To construct an in vitro model of human macrophages.Methods:1)Induced differentiation:polarized THP-1 cells into TAM cells by adding PMA and IL-4I/IL-13 to construct an in vitro model of human-derived macrophages;at the same time,TMZ stimulation of THP-1 was performed to exclude the possibility that chemotherapeutic drugs promote THP-1 phenotypic transformation.2)Phenotypic identification:Compare the cell morphology before and after induction by microscopy,and phenotypic identification of TAM by cell immunofluorescence and qRT-PCR.Results:Microscopically,adherent cells were round,oval,or spindle-shaped,in line with the typical growth morphology of macrophages;TMZ did not induce phenotypic transformation of THP-1.The qRT-PCR results showed that the mRNA levels of the M2-TAM phenotype markers CD 163,Fizzl,Arg1,and CD206 were significantly up-regulated after THP-1 cells were induced;meanwhile,the mRNA levels of the phenotypic markers iNOS and MHC-Ⅱ of M1-TAM cells were significantly down-regulated;Laser confocal results showed that M2-TAM cell phenotypic markers CD68,CD 163 and CD206 were positively expressed.Conclusion:In this study,THP-1 was polarized to TAM by chemical inducers PMA and IL-4I/IL-13.At the same time,the identification results showed that TAM was M2 phenotype,indicating that we have successfully constructed Human-derived macrophages in vitro model.Chapter2:Extraction,identification and tracing of ExosomesTHP-1 and ExosomesTAMObjective:Extraction,identification and tracking of THP-1 and TAM-derived exosomes.Methods:1)Extraction:Use the kit to enrich exosomes in THP-1 and M2-TAM culture supernatants.2)Identification:Characterize exosomes by TEM,SEM,NTA and WB.3)Tracer:Mark exosomes with PKH67 and incubate in GBM cell line,observe the distribution and intensity of fluorescence with laser confocal microscope.Results:TEM results showed that membrane-like structures with sizes between 50 and 150 nm were visible in the supernatants of THP-1 cells and TAM cells;NTA results showed that the peaks of these enrichment curves were concentrated between 100 and 150 nm;WB results of CD9,CD63 and CD86 protein expressions were shown;laser confocal observations showed that PKH67-labeled exosomes could enter GBM cells and were mainly distributed in the cytoplasm of the cells,especially around the nucleus.Conclusion:TEM,NTA and WB showed that this study successfully extracted and enriched exosomes.PKH67 tracer experiments showed that THP-1 and TAM-derived exosomes could be internalized by GBM cells.Chapter3:Effects of Exosomes THP-1 and ExosomesTAM on GBM chemotherapy Objective:To investigate the effects of ExosomesTHP-1 and ExosomesTAM on GBM chemotherapy.Methods:1)The intervention group of GBM cell lines was set to THP-1-CM,TAM-CM,TAM-Exos and TAM-CM depleted of Exos groups,and CCK8 proliferation experiments,clone formation experiment,apoptosis flow cytometry experiments,and subcutaneous tumor formation experiments were performed.2)The intervention group of GBM cell line was set to THP-1-Exos group and TAM-Exos group,the cell morphological changes were observed under the microscope,and the changes of EMT marker protein were detected by WB.Results:Compared with THP-1-CM and TAM-CM depleted of Exos groups,TAM-CM and TAM-Exos inhibited the cytotoxic killing effect of TMZ on GBMs.Compared with the THP-1-Exos group,the EMT level increased in the TAM-Exos group.Conclusion:ExosomesTAM may mediate GBM’s TMZ chemotherapy resistance through EMT.Chapter4:Differential lncRNA screening for ExosomesTAM mediated GBM chemotherapy resistanceObjective:To screen out differential lncRNAs that mediate GBM chemotherapy resistance in ExosomesTAM.Methods:1)The TMZ chemosensitized cell lines and drug-resistant cell lines isolated from continuous intracranial generation of GL261 cells were analyzed by lncRNA microarray chip,and the chip data was combined with the data shared by GEO DataSets chip GSE115040 for bioinformatics analysis to obtain the differential lncRNA.2)qRT-PCR was performed on the selected differentially expressed lncRNAs that were highly expressed in TAM,TAM-Exos and resistant strains.Results:Bioinformatics analysis screened differentially expressed lncRNAs that were highly expressed in TAM,TAM-Exos and drug-resistant cell lines as FAM83A-AS1 and KCNK4-TEX40.qRT-PCR results showed that only FAM83A-AS1 was highly expressed in TAM-Exos and drug-resistant GBM cell lines.Conclusion:FAM83A-AS1 may be a key molecule that is transferred to GBM through exosomesTAM and mediates chemotherapy resistance of GBM.