Mechanism Of Temozolomide Resistance Induced By NKCC1 Activated By Tumor Associated Astrocyte Derived Exosomes

BackgroundGlioblastoma(GBM)is a malignant tumor with the highest incidence rate and mortality in the central nervous system.At present,the clinical treatment of GBM mainly adopts the combined treatment of surgical resection and postoperative radiotherapy and chemotherapy.Temozolomide(TMZ)is the standard first-line chemotherapy drug for GBM clinical treatment,but more than 50%of patients will develop drug resistance,resulting in poor overall prognosis,and the 5-year survival rate is only 4.7%.Therefore,how to reduce the TMZ resistance of GBM patients and improve the chemosensitivity of GBM patients is an urgent problem for GBM clinical treatment.The heterogeneity of tumor leads to the challenge of GBM treatment,which is not only reflected in the different subtypes of tumor,but also reflected in the unique tumor microenvironment(TME).Astrocytes are the most abundant and widely distributed glial cells in the central nervous system.They are involved in maintaining the blood-brain barrier,secreting/absorbing neurotransmitters,and regulating the balance of extracellular ions.In TME,astrocytes are the most abundant stromal cells.Under the action of TME,astrocytes proliferate and become activated astrocytes,also known as tumor-associated astrocytes(TAAs).The transformation of astrocytes to TAAs phenotype is accompanied by changes in molecular,cellular and functional changes.Recent studies have shown that TAAs is an important factor in promoting tumor drug resistance.However,the key molecules that cause the transformation of astrocytes to TAAs phenotype and the mechanism of TAAs inducing temozolomide resistance have not been revealed.Na+-K+-Cl-co-transporter 1(NKCC1)is an important ion transporter in the human body,mainly expressed in brain tissue,and plays an important role in maintaining cell volume homeostasis and intracellular Cl-homeostasis.Our previous study found that NKCC1 was highly expressed on TAAs,therefore inhibited the expression of glutamate transporter,disrupting the function of astrocytes.Here,we assume that NKCC1 may be involved in the phenotypic transformation of astrocytes,but a detailed study of the molecular,morphological and functional changes in astrocytes in which NKCC1 is involved is needed.In addition,it has been reported that high intercellular Cl-concentration in tumor cells is important in promoting TMZ resistance in GBM,and the concentration of intercellular Clis more significantly reduced in sensitive cell lines than in resistant cell lines,which suggested that NKCC1 in GBM tumor cells may be involved in TMZ resistance.However,the way through which NKCC1 high expressed TAAs affect GBM tumoral NKCC1 and then induce drug resistance needs to be further investigated.Exosomes(EXOs)play an important role in intercellular communication and can transfer complex contents from primitive cells,including proteins,lipids,mRNA,miRNA and DNA,to target cells to play a role.STE20-related proline/alanine-rich protein kinase(SPAK)and oxidative stress-responsive protein kinase 1(OSR1)are upstream regulatory proteins of NKCC1.It has been found that the SPAK/OSR1 protein can combine with the cell membrane NKCC1 and enter the EXOs through endocytosis to transfer to the adjacent cells,suggesting that the SPAK/OSR1 protein can be transferred between cells through EXOs.Therefore,this study speculates that the TAAs with high expression of NKCC1 can carry SPAK/OSR1 through the EXOs to GBM tumor cells and activate NKCC1 to promote TMZ resistance.AimsAiming at the problem of TMZ resistance in clinical GBM patients,taking TAAs in the tumor microenvironment as the starting point,this paper reveals the key molecules of astrocyte transforming into TAAs phenotype and the mechanism of TAAs inducing TMZ resistance,providing new theoretical and experimental basis for clinical intervention of TMZ resistance in glioblastoma.Metholds1.Involvement of NKCC1 in astrocyte phenotypic transformation:TCGA,CPTAC and other databases were used to detect the expression of TAAs markers in normal tissues and GBM patients’ tissues;To establish cell co-culture and mouse glioma model,WB and immunofluorescence were used to detect the optimal co-culture conditions for GBM tumor cells to promote astrocytes to transform into TAAs and the expression of NKCC1;The NKCC1 knockdown astrocytes were established by lentivirus infection technology,and the effect of GBM tumor cells on the phenotypic transformation of NKCC1 knockdown astrocytes was detected by WB and QPCR;Construct adenoassociated virus,use GFAP-Cre mice,specifically knock down astrocyte NKCC1,and detect the effect of knockdown NKCC1 on astrocyte phenotype transformation in vivo by immunofluorescence and QPCR.2.Astrocyte phenotypic transformation for TMZ resistance:The co-culture of cells in vitro and the mouse glioma model were established.The astrocyte NKCC1 was specifically knocked down in vivo.The effects of NKCC1 mediated astrocyte phenotype transformation on TMZ sensitivity were detected by CCK8,flow cytometry,WB,immunofluorescence,animal imaging in vivo and animal survival.3.TAAs promote TMZ resistance by secreting EXOs:TAA-EXOs were extracted by ultrahigh speed centrifugation and identified by electron microscopy,particle size analysis and WB;TAA-EXOs were labeled with membrane dye PKH67,and the uptake of TAA-EXOs by GBM tumor cells was detected by immunofluorescence;The effect of NKCC1-mediated TAA-EXOs on the sensitivity of GBM tumor cells to TMZ was detected by CCK8 and immunofluorescence after the co-culture model was treated with exogenesis inhibitor GW4869.4.Activation of NKCC1 in GBM tumor cells by TAA-EXOs regulates ATRX:The coculture model of GBM tumor cells was treated with exogenesis inhibitor GW4869,and the effect of TAA-EXOs on the phosphorylation level of NKCC1 was detected by WB and immunofluorescence;The GBM tumor cell line with NKCC1 knockdown was established by lentivirus infection technology.The effect of TAA-EXOs on the sensitivity of GBM tumor cells to TMZ after NKCC1 knockdown was detected by CCK8,EDU and WB;The expression of NKCC1,pNKCC1 and ATRX in GBM tumor cells in vitro and in vivo was analyzed by TCGA and CGGA databases and WB;WB and immunofluorescence were used to detect the effect of NKCC1-mediated TAAEXOs on GBM tumor cells NKCC1,pNKCC1 and ATRX.5.TAA-EXOs deliver SPAK/OSR1 to activate NKCC1 in GBM tumor cells:U251 tumor cells with SPAK/OSR1 and astrocytes with knockdown and overexpression of SPAK/OSR1 constructed by lentivirus infection technology.WB was used to detect the transmission of SPAK/OSR1 delivery by EXOs and the phosphorylation state of NKCC1;WB and immunofluorescence were used to detect the effect of NKCC1mediated TAA-EXOs on GBM tumor cell SPAK/OSR1.Results1.NKCC1 is involved in astrocyte phenotypic transformation:1)Astrocyte astrocytes undergo phenotypic transformation into TAAs under the action of GBM tumor,and the molecular changes of TAAs are more inclined to A2 type,accompanied by high expression of GFAP,VIM,CHI3L1 protein and hypertrophic cell morphology;2)The phenotypic transformation of astrocytes in astrocytes was accompanied by a significant increase in NKCC1.Inhibition of NKCC1 could significantly reduce the activation of astrocytes in astrocytes,which was characterized by decreased expression of GFAP,VIM,CHI3L1 protein and normal cell morphology.2.Astrocyte phenotypic transformation promotes TMZ resistance:1)The phenotypic transformation of astrocytes significantly inhibited TMZ sensitivity.The higher the activation of astrocytes,the stronger the inhibition of TMZ sensitivity;2)Knocking down astrocyte NKCC1 in vitro and in vivo can significantly enhance the sensitivity of TMZ,promote GBM cell apoptosis,reduce the volume of tumor in situ and prolong the survival period of tumor-bearing mice.3.TAAs promote TMZ resistance through the release of EXOs:1)TAA-EXOs can induce TMZ resistance:exosome inhibitor GW4869 can significantly enhance the sensitivity of co-cultured GBM tumor cells to TMZ,which was reversed after adding exosomes;2)NKCC1-mediated TAA-EXOs affect TMZ drug resistance:knocking down NKCC1mediated TAA-EXOs can significantly enhance the killing effect of TMZ on GBM tumor cells,and reduce the volume of subcutaneous tumor in vivo.4.TAA-EXOs activate NKCC1 regulatory DNA damage repair gene ATRX in GBM tumor cells:1)TAA-EXOs can significantly activate NKCC1 in GBM tumor cells:exosome inhibitor GW4869 can significantly inhibit the phosphorylation level of NKCC1 in GBM tumor cells,and the phosphorylation level of NKCC1 increases significantly after adding EXOs;2)Knocking down tumor cell NKCC1 can reduce TAA-EXOs induced TMZ resistance:GBM tumor cells knocking down NKCC1 can significantly reduce TAA-EXOs mediated tumor NKCC1 phosphorylation levels,significantly increase cleaved caspase-3 and y-H2AX expression,inhibits cell proliferation and enhances its sensitivity to TMZ;3)NKCC1 is positively correlated with DNA damage repair gene ATRX:database analysis and in vitro and in vivo experiments show that inhibition of NKCC1 in GBM tumor cells can significantly reduce the expression level of ATRX;4)Inhibition of astrocyte NKCC1 can significantly reduce the phosphorylation of NKCC1 and the increase of ATRX induced by TAA-EXOs.5.TAA-EXOs deliver SPAK/OSR1 to activate NKCC1 in GBM tumor cells:1)The expression of SPAK/OSR1 was promoted by the phenotype transformation of astrocytes,and the protein content of SPAK/OSR1 increased with the activation intensity of astrocytes;2)The content of SPAK/OSR1 in EXOs secreted by TAAs was significantly higher than that in EXOs secreted by normal astrocytes;3)TAAs can transmit SPAK/OSR1 to GBM tumor cells by secreting EXOs;4)Inhibition of NKCC1 can significantly reduce the transmission of SPAK/OSR1 from TAA-EXOs to GBM tumor cells.ConclusionsThis study revealed the mechanism of TAAs in tumor microenvironment inhibiting the sensitivity of GBM to TMZ,and found that astrocyte transformed into TAAs with high expression of NKCC1 under the action of GBM tumor cells.The exosomes secreted by TAAs are transmitted to GBM tumor cells through NKCC1 carrying SPAK/OSR1 on the membrane.Promote the phosphorylation of NKCC1 in tumor cells,regulate the DNA damage repair gene ATRX to induce TMZ resistance.This is of great significance in finding new therapeutic targets and drugs,and is expected to provide new strategies for the clinical treatment of glioblastoma patients with drug resistance.