Glioma initiating cells (GICs), also known as glioma stem cells (GSCs), play an important role in the progression and recurrence of glioblastoma multiforme (GBM) due to their potential for self-renewal, multiple differentiation and tumor initiation. In the recent years, Notch1 has been found to be overexpressed in GICs. However, the regulatory mechanism of Notch1 in the self-renewal and invasion ability of GICs remains unclear. This study aims to explore the effect of Notch pathway on self-renewal and invasion of GICs and the underlying mechanisms. Bioinformatic analysis and immunohistochemistry (IHC) were performed to evaluate the expression of Notch1 and Hes1 in GBM samples. Immunofluorescent (IF) staining was performed to observe the distribution of Notch1 and CXCR4 in GBM and GICs. Both pharmacological intervention and RNA interference were employed to investigate the role of Notch1 in GICs self-renewal, invasion and tumor growth in vitro or in vivo. The crosstalk effect of Notch1 and CXCL12/CXCR4 system on GIC self-renewal and invasion was explored by sphere formation assay, limiting dilution assay and Transwell assay. Western blots were used to verify the activation of Notch1/CXCR4/AKT pathway in self-renewal, invasion and tumor growth of GICs. Luciferase reporter assay was used to testify the potential binding site of Notch1 signaling and CXCR4. The orthotopic GICs implantations were established to analyze the role and the mechanism of Notch1 in glioma progression in vivo. Notch1 signaling activity was elevated in GBM tissues. Notch1 and CXCR4 were both upregulated in GICs, compared to Notch1 positive glioma cells comprised a large proportion in the CD133+ glioma cell spheres, CXCR4 positive glioma cells which usually expressed Notch1 both and dispersed in the periphery of the sphere, only represent a small subset of CD133+ glioma cell spheres. Furthermore, downregulation of the Notch1 pathway by shRNA and MK0752 significantly inhibited the PI3K/AKT/mTOR signaling pathway via the decreased expression of CXCR4 in GICs, and weakened the self-renewal, invasion and tumor growth ability of GICs. These findings suggest that the cross-talk between Notch1 signaling and CXCL12/CXCR4 system could contribute to the self-renewal and invasion of GICs, and this discovery could help drive the design of more effective therapies in Notch1-targeted treatment of GBMs.

Background: Glioblastoma (GBM) is characterized by high invasiveness and metabolic heterogeneity. Protein Tyrosine Phosphatase Receptor Type Z1 (PTPRZ1) has been implicated in glioma stemness and tumor grade progression, while the molecular mechanism remains unknown. Methods: Integrated bioinformatics analysis of TCGA dataset and TMA IHC profiling were used to explore clinical relevance of PTPRZ1, and potential molecular mechanisms of PTPRZ1 involved glioma progression. Ivy Glioblastoma datasets analysis and multiregional GBM IHC were performed to spatially characterize the expression patterns of PTPRZ1 in GBM tissues. Zebrafish xenograft model was established to elucidate the role of PTPRZ1 in driving glioblastoma cell migration. Western blotting, mitochondrial functional assays, Transwell migration assays, and human GBM tissue multiplexing were employed to investigate the mechanism of PTPRZ1 mediated glioma cell mobility via maintaining mitochondrial functions. Results: Spatial profiling revealed that PTPRZ1 is significantly enriched in the infiltrating areas and peritumoral margins of GBM, and acts as a poor prognosis factor in lower grade astrocytoma, proneural and mesenchymal GBM. Mechanistically, PTPRZ1 expression was found to be strongly correlated with cell adhesion molecules (CAMs) and mitochondrial metabolism across multiple glioma subtypes. Knockdown of PTPRZ1 triggered a shift from elongated to fragmented mitochondrial morphology by specifically downregulating the mitochondrial fusion protein OPA1. This mitochondrial dysfunction led to a metabolic shift toward glycolysis and a marked accumulation of ROS, which suppressed N-cadherin expression, thereby impairing glioma cell migration. These findings were corroborated in vivo, where PTPRZ1 depletion significantly reduced disseminated tumor foci in a zebrafish model. Conclusion: Our study demonstrates that PTPRZ1 drives glioblastoma migration by sustaining an OPA1-dependent mitochondrial fusion program, which suppresses mitochondrial ROS levels and subsequently stabilizes a pro-migratory phenotype via N-cadherin expression. Targeting the PTPRZ1/OPA1/ROS axis represents a promising therapeutic strategy to inhibit the invasive expansion of GBM cells.