Glioblastoma is the most aggressive brain tumour with short survival, partly due to resistance to conventional therapy. Glioma stem cells (GSC) are likely to be involved in treatment resistance, by releasing extracellular vesicles (EVs) containing specific molecular cargoes. Here, we studied the EVs secreted by glioma stem cells (GSC-EVs) and their effects on radiation resistance and glioma progression. EVs were isolated from 3 GSCs by serial centrifugation. NanoSight measurement, cryo-electron microscopy and live imaging were used to study the EVs size, morphology and uptake, respectively. The non-GSC glioma cell lines LN229 and U118 were utilised as a recipient cell model. Wound healing assays were performed to detect cell migration. Colony formation, cell viability and invadopodium assays were conducted to detect cell survival of irradiated recipient cells and cell invasion post GSC-EV treatment. NanoString miRNA global profiling was used to select for the GSC-EVs’ specific miRNAs. All three GSC cell lines secreted different amounts of EVs, and all expressed consistent levels of CD9 but different level of Alix, TSG101 and CD81. EVs were taken up by both LN229 and U118 recipient cells. In the presence of GSC-EVs, these recipient cells survived radiation exposure and initiated colony formation. After GSC-EVs exposure, LN229 and U118 cells exhibited an invasive phenotype, as indicated by an increase in cell migration. We also identified 25 highly expressed miRNAs in the GSC-EVs examined, and 8 of these miRNAs can target PTEN. It is likely that GSC-EVs and their specific miRNAs induced the phenotypic changes in the recipient cells due to the activation of the PTEN/Akt pathway. This study demonstrated that GSC-EVs have the potential to induce radiation resistance and modulate the tumour microenvironment to promote glioma progression. Future therapeutic studies should be designed to interfere with these GSC-EVs and their specific miRNAs.
OBJECTIVE Glioblastoma is the most common primary central nervous system tumor in adults. These tumors are highly invasive and infiltrative and result in tumor recurrence as well as an extremely poor patient prognosis. The current standard of care involves surgery, radiotherapy, and chemotherapy. However, previous studies have suggested that glioblastoma cells that survive treatment are potentially more invasive. The goal of this study was to investigate whether this increased phenotype in surviving cells is facilitated by actin-rich, membrane-based structures known as invadopodia. METHODS A number of commercially available cell lines and glioblastoma cell lines obtained from patients were initially screened for the protein expression levels of invadopodia regulators. Gelatin-based zymography was also used to establish their secretory protease profile. The effects of radiation and temozolomide treatment on the glioblastoma cells were then investigated with cell viability, Western blotting, gelatin-based zymography, and invadopodia matrix degradation assays. RESULTS The authors' results show that the glioma cells used in this study express a number of invadopodia regulators, secrete MMP-2, and form functional matrix-degrading invadopodia. Cells that were treated with radiotherapy and temozolomide were observed to show an increase primarily in the activation of MMP-2. Importantly, this also resulted in a significant enhancement in the invadopodia-facilitated matrix-degrading ability of the cells, along with an increase in the percentage of cells with invadopodia after radiation and temozolomide treatment. CONCLUSIONS The data from this study suggest that the increased invasive phenotype that has been previously observed in glioma cells posttreatment is mediated by invadopodia. The authors propose that if the formation or activity of these structures can be disrupted, they could potentially serve as a viable target for developing novel adjuvant therapeutic strategies that can be used in conjunction with the current treatment protocols in combatting the invasive phenotype of this deadly disease.
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