Endothelial cell-derived SDF-1α elicits stemness traits of glioblastoma <i>via</i> dual-regulation of GLI1

<b>Background:</b> Glioma stem cells (GSCs) play a critical role in the poor treatment outcomes observed in glioblastoma (GBM) patients. A primary focus of current glioma research is understanding the maintenance of stemness in GSCs and their interactions with the tumor microenvironment. In GBMs, the perivascular niche serves as a protective environment for GSCs, contributing to tumor recurrence. However, the molecular mechanisms that sustain this reservoir remain poorly understood. <b>Methods:</b> The analysis of single-cell transcriptional data in GBM was conducted to identify signaling pathways in endothelial cells (ECs) that promote stemness traits in glioma cells. Histological staining and the IvyGAP dataset were utilized to evaluate the anatomical microenvironment of glioma. The molecular mechanisms underlying the maintenance of stemness in GSCs, influenced by ECs, were assessed using ELISA, Western blotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), in vivo ubiquitination assays, and other molecular biology experiments. An orthotopic xenograft model was employed to examine the stemness phenotype of GBM cells in the presence of ECs, as well as the synergistic effects of GSK690693 and AMD3100 in inhibiting GBM cells. <b>Results:</b> We found that GSCs are located in close proximity to microvessels, and we identified the CXCL12-CXCR4 signaling pathway in ECs as a promoter of stemness traits in glioma cells. GBM cells can transition to a stem-like state in response to stromal cell-derived factor-1α (SDF-1α) secreted by ECs. This transition activates the CXCR4-mediated AKT/NF-κB signaling pathway, leading to the subsequent upregulation of glioma-associated oncogene homolog 1 (GLI1), a key transcription factor for maintaining stemness. Furthermore, we discovered that SDF-1α influences the turnover of GLI1 protein in GBM cells by modulating GLI1-associated polyubiquitin chains through the phosphorylation of the deubiquitinase USP28 at serine 67. This modification enhances the stemness-maintaining properties of GLI1 via both transcriptional regulation and protein quality control mechanisms. Preclinical studies indicated that the combination of the CXCR4 antagonist AMD3100 and the AKT inhibitor GSK690693 synergistically inhibits GBM cell progression. <b>Conclusions:</b> Our findings unveil a novel signaling axis between ECs and tumor cells that directly impacts the acquisition of stemness traits, suggesting that targeting this pathway could represent a promising therapeutic strategy against GBM.