The Study On The Function Of Actin Cytoskeleton To Maintain Glioma Stem Cell Phenotype

Objective: Glioblastoma(GBM)is the most common and lethal primary intracranial tumor.Actin cytoskeleton regulator Arp2/3 complex is essential for the invasion and migration of glioma cells.This study was designed to investigate whether the actin cytoskeleton is involved in the maintenance of stem cell phenotype and the detailed mechanism.Our study investigated the distribution pattern of glioma stem cells(GSCs)in primary GBM.We evaluated the expression of stem cell markers and the self-renewal ability in GSCs.This project illustrated whether the Arp2/3 complex was involved in the maintenance of glioma stem cell phenotype and the potential regulatory mechanism.Methods: We collected tissue specimens from 14 patients with glioblastoma.Immunohistochemistry staining was applied to detect the expression and distribution of stem cell markers(CD133,Nestin,Notch1 and Delta-like1)and cell differentiation markers(GFAP and Tu J1)in tumor cells.CD133 + U87-MG and U251-MG glioma stem cells were enriched by the magnetic activated cell sorting(MACS)and cultured in the stem cell medium.Western blot,immunofluorescence staining and single cell sphere formation assay were used to detect the expression of stem cell markers and the self-renewal ability of CD133 + glioma cells in vitro,respectively.Both the expression of stem cell markers and self-renewing ability were analyzed to determine the stem cell phenotype.Lentiviral sh RNAs targeting Arp2 and Delta-like1(DLL1)and CK636(a specific inhibitor of Arp2/3 complex)were involved to investigate their effect on the phenotype of glioma stem cells.The subcellular localization of DLL1 was detected by Western blot and immunofluorescence staining.The intracranial nude mouse model was established to evaluate the effect of knockdown of Arp2 on the tumorigenic ability of GSCs.Results: We showed that CD133+,Nestin+,Notch1+,and DLL1+ GSCs aggregated into the vessel-like structures,and GFAP+ and Tu J1+ tumor cells aggregated in thearea far away from the above vessel-like structures.Further immunofluorescence staining exhibited that Notch1 and DLL1 co-expressed in the above-mentioned GSCs A part of the DLL1+ GSCs also expressed endothelial markers CD31.In vitro,the percentage of CD133 + U87-MG and U251-MG cells in neurospheres after MACS was 84.70±2.70% and 74.23±2.43%,respectively.Western blot and immunofluorescence staining showed that CD133,Nestin,Notch1,NICD1,and DLL1 expression in CD133+ GSCs were significantly higher than those in the monolayer cells without MACS.And,the expression of GFAP and Tu J1 were significantly lower in GSCs than that in monolayer cells.Sh RNA targeting DLL1 significantly reduced the expression of CD133 and Nestin in U87-MG and U251-MG GSCs,decreased the activity of Notch pathway(decreased expression of NICD1 and HES1),and increased the expression of GFAP and Tu J1(p<0.05).Sh RNA targeting Arp2 and CK636 inhibited the formation of platelet pseudopodia of U87-MG and U251-MG glioma cells,and significantly decreased CD133,Nestin,NICD1,and HES1 expression levels(p<0.05).The sphere formation ability was also abolished through inhibiting Arp2/3 complex(p<0.05).However,knocking down both DLL1 and Arp2 did not further reduce CD133,Nestin,NICD1,and HES1 expression,but exogenous soluble DLL1 restored the loss of stem cell phenotype induced by the knockdown of DLL1 and Arp2.The underlying mechanism was that the Arp2/3 complex maintained the subcellular localization of DLL1.Western blot and immunofluorescence staining suggested that sh Arp2 and CK636 decreased the expression of DLL1 on the cell membrane and increased the expression of DLL1 in cytoplasm.In vivo,sh Arp2 significantly reduced the tumorigenic ability of CD133+ U87-MG GSCs in the intracranial model.Conclusion: Notch ligand DLL1 was dependent on the cytoskeleton regulator Arp2/3 complex to maintain the activated Notch pathway,thus maintaining the glioma stem cell phenotype.Cytoskeleton was involved in the regulation of phenotype of GSCs.Our study provided an innovative idea for the treatment targeting tumor invasion.