Invasion Of White Matter Tracts By Glioma Stem Cells Is Regulated By A NOTCH1-SOX2 Positive-feedback Loop

Glioma is the most common primary tumor of the central nervous system.Despite advances in surgery,radiation and chemotherapy treatments,the overall5-year survival rate of glioma remains<10%.Early invasive growth along white matter tracts,perivascular space and meninges is the most prominent clinicopathological characteristic of malignant glioma and is regarded as one of the main causes of a poor therapeutic outcome.It is important to understand whether glioma cells randomly reach these existing structures or whether these anatomical components attract glioma cells and then provide advantages for the survival of these cells.Unfortunately,knowledge regarding this is limited.Recently,researchers have found that Nestin⁺CD133⁺GSCs?Glioma stem cells?are located next to capillaries in malignant glioma.Abundant nutrition in the blood vessels and certain soluble factors secreted by vascular endothelial cells are released to the vicinity of GSCs that migrate to the perivascular regions,maintaining the stemness of GSCs and promoting their invasion.In turn,GSCs can also secrete vascular endothelial growth factors to induce endothelial cell migration and angiogenesis.The spatial relationship between GSCs and blood vessels might be the main reason for the early invasion of glioma along the perivascular space.However,the spatial relationship between GSCs and white matter tracts still needs further exploration.In this study,we found that CD133⁺GSCs were preferentially located along the matter tracts,and the demyelinated white matter tracts activated the NOTCH1-SOX2positive-feedback loop in GSCs through exposed Jagged1,providing a survival advantage for GSCs.This positive-feedback loop can be used as a therapeutic target for gliomas.The main findings of our study can be described as follows:1.Distribution of GSCs along white matter tracts in peritumoral tissue specimens of glioma.?1?CD133 and Tau1 immunofluorescence staining were performed on the peritumoral tissue specimens of glioma,and the staining results were quantitatively analyzed.The results showed that49.5%of CD133⁺cells were within 5?m of the nerve fibers.?2?Transmission electron microscopy observation of the peritumoral tissue of glioma found that the white matter tracts around the tumor were obviously damaged?swelling,rupture?.More importantly,some white matter tracts were demyelinated.?3?Tau1/NF200 and MBP double-immunofluorescence staining were performed on the peritumoral tissue specimens of glioma,and the staining results were co-localized quantitatively.The results showed that the signals for Tau1/NF200 and MBP did not overlap completely,and some of the Tau1/NF200 positive signals were not surrounded by MBP positive signals;the Rr value between Tau1/NF200 and MBP was 0.761,the R value was0.811,the k1 value was 0.895,the k2 value was 0.757,and the m1 value was 0.884,the m2value was 0.723.It is suggested that some of the white matter tracts have undergone demyelination.2.Activated Notch signaling pathway promotes the distribution of GSCs along white matter tracts.?1?Tau1 and Jagged1 immunofluorescence staining were performed on neurons cultured in vitro,and the results showed that neurons had co-expression of Jagged1 and Tau1.?2?Immunofluorescence staining of Tau1/Jagged1,CD133/Notch1 and NF200/Notch1was performed on the peritumoral tissue specimens of glioma and quantitative analysis.The results showed that Jagged1 signal was co-expressed on the white matter nerve fibers of tau1⁺,and Notch1 signal was co-expressed on the glioma cells of CD133⁺;the glioma cells of Notch1⁺were scattered around the white matter nerve fibers of NF200^+.Among them,54.9%of Jagged1⁺signal were within 5?m of the nerve fibers;the Rr value between CD133 and Notch1 was 0.638,the R value was 0.692?,the k1 value was 0.593,the k2 value was 0.853,and the m1 value was 0.608,the m2 value was 1.067?;24.4%of Notch1⁺cells were within 5?m of the nerve fibers.The results suggested that the white matter nerve fibers of glioma tumor tissue express Jagged1,and the GSC of CD133⁺Notch1⁺is scattered along the white matter nerve fibers ofNF200⁺.?3?GL261-NOTCH1^KD and GL261-NOTCH1^MOCK GSCs cultured in spheres were implanted into the right striatum adjacent to the corpus callosum of Thy1-EGFP mice.At 2and 4 weeks after implantation,laser confocal microscopy was performed.The results showed that after 2 weeks implantation,a small amount of CD133⁺glioma cells invaded along the white matter tracts in the control group(GL261-NOTCH1^MOCK group),while rare glioma cells were detected in the same region of the knockdown group?GL261-NOTCH1^KDD group?.Quantitative analysis showed that the number of glioma cells invading white matter tracts and CD133⁺glioma cells in the control group were 16 and 9,respectively,which were significantly higher than those in the knockdown group?4 and 4,respectively?.4 weeks after implantation,multiple tumor lesions on the opposite side of the brain midline,ipsilateral hippocampus and contralateral hippocampus were observed in the control group;the tumors grew only near the site of implantation in the knockdown group.The results suggested that down-regulation of Notch1 expression in GSCs cells can significantly inhibit GSC invasion along the white matter tracts.3.Notch1 upregulates the transcription of SOX2 via Sox9.?1?The TCGA?The Cancer Genome Atlas?and GEO?Gene expression omnibus?databases were selected to evaluate the correlation between the expression level of SOX2 and the dry fraction and NOTCH1 expression by Pearson correlation coefficient.The results showed that the expression level of SOX2 was significantly positively correlated with the dry fraction and NOTCH1 expression.?2?Immunofluorescence staining of Sox2/NF200 and Sox9/NF200 was performed on the peritumoral tissue specimens of glioma and quantitative analysis.The results showed that the glioma cells of Sox2⁺and Sox9⁺were scattered around the NF200⁺white matter tracts;among them 93.1%of the Sox2⁺cells were located within 5?m of nerve fibers,and 72%of the Sox9⁺cells were located within 5?m of nerve fibers.It is suggested that Sox2 and Sox9 is expressed in glioma cells near the white matter tracts.?3?Notch1?Sox9 and Sox2 immunofluorescence staining of GSCs cultured in spheres showed that Notch1?Sox9 and Sox2 proteins were co-expressed in GSCs.?4?The expression of NOTCH1 in primary glioblastoma cells?GBM1 and GBM2?and glioma cell line U87 was intervened,its effects on SOX2 expression were detected by RT-PCR and Western-blot.The results showed that NOTCH1 could positively regulate the expression of Sox2.The expression of SOX9 in GBM1 was intervened,and its effects on Sox2 expression was detected by Western-blot.The results showed that intervention of SOX9reversed the change of Sox2 expression induced by overexpression of NOTCH1.?5?Chromatin immunoprecipitation and gel migration assays showed that Sox9 can bind to the SOX2 promoter region?Site 1-3 site?.4.Sox2 promotes the distribution of GSCs along white matter tracts by regulating Notch1.?1?The expression of SOX2 in GBM1/2 and U87 was intervened,and the effect of NOX1expression was detected by RT-PCR and Western-blot.The results show that SOX2 can positively regulate the expression of NOTCH1.?2?GBM3-NOTCH1^KD and GBM3-NOTCH1^MOCK cells were implanted into the right striatum adjacent to the corpus callosum of NOD/SCID mice,and examined by laser confocal microscopy at 4 weeks after implantation.The results showed that after 4 weeks of implantation,multiple Sox2⁺glioma cells on the opposite side of the brain midline,ipsilateral hippocampus and contralateral hippocampus were observed in the control group(GBM3-NOTCH1^MOCK group),while the tumors grew only near the site of implantation in the knockdown group(GBM3-NOTCH1^KD group).Quantitative analysis showed that the number of glioma cells invading white matter tracts and Sox2⁺glioma cells in the control group were158 and 156,respectively,which were significantly higher than those in the knockdown group?13 and 12,respectively?.The DTI?Diffusion tensor imaging?test results were imported into TrackVis software to reconstruct the fiber bundles in ROI?Region of interest?.The FA?Fractional anisotropy?map and nerve fiber bundle reconstruction map of the mouse brain tissue were obtained and fitted with the immunofluorescence full slide scan imaging results.The results showed that Sox2⁺glioma cells were distributed along the reconstructed white matter tracts.The above results indicate that knockdown of Notch1 can inhibit the invasion of Sox2⁺glioma cells along the white matter nerve tracts.5.SOX2 up-regulates transcription of NOTCH1 via TET3.?1?After methylation inhibitor 5-Aza treatment of GBM2,MeDIP-PCR was used to detect the degree of methylation of NOTCH1 promoter region,and RT-PCR and Western-Blot were used to detect the expression level of NOTCH1.The results showed that the down-regulation of NOTCH1 promoter methylation level by SOX2 and the up-regulation of NOTCH1 transcription level were all restored by 5-Aza.?2?After knocking down the expression of TET3 in SOX2 over-expressed GBM2(GBM2-SOX2^OE)cells,RT-PCR was used to detect the expression level of NOTCH1,MeDIP-qPCR was used to detect the methylation level of NOTCH1 promoter.The results showed that knockdown of TET3 restored the up-regulation of NOTCH1 transcription and the downregulation of NOTCH1 promoter methylation induced by SOX2 overexpression.The main conclusions of this study include:1.CD133⁺GSCs has the characteristic of preferential positioning along white matter tracts.Among them,the interaction of Notch1 on the surface of Jagged1 and GSCs exposed to demyelinated white matter tracts may be a determinant of this spatial distribution.2.Notch1 upregulates the transcription of SOX2 by Sox9 acting on site 1-3 of the SOX2gene transcription promoter region.3.Sox2 upregulates the transcription of NOTCH1 by regulating the methylation level of the CpG island of the NOTCH1 promoter by TET3.In summary,our study found that in the glioma peritumoral tissue,the interaction between Jagged1 exposed to demyelinated white matter tracts and Notch1 in the adjacent GSCs activates the NOTCH1-SOX2 positive feedback loop in the GSCs,thereby promoting the invasion of GSCs along white matter tracts;interfering with NOTCH1 in the positive feedback loop could inhibit GSCs invasion along white matter tracts.Our study reveals that the positive feedback loop of NOTCH1-SOX2 plays an important role in regulating the invasion of GSCs along white matter tracts,providing a new therapeutic target for the invasion of glioma.