Extracellular Vesicles Secreted by Glioma Stem Cells Are Involved in Radiation Resistance and Glioma Progression.

Chenkai Ma,Lucy Paradiso,Zammam Areeb,Rodney B Luwor,Hong P T Nguyen,Stanley S Stylli,Jordan J Jones,Andrew H Kaye,Andrew P Morokoff,Ulrich Putz,Clarissa A Whitehead,Eric Hanssen,Ellie Cho

doi:10.3390/ijms23052770

Abstract

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.

Highlights

Glioblastoma is the most common and aggressive type of brain cancer with poor median survival of around one year
The CD9 expression level was seen to be consistent in extracellular vesicles (EVs) of all three Glioma stem cells (GSC) lines, while the expression level varied in other markers (Alix, TSG101 and CD81)
After characterisation of the GSC-derived exosomes/extracellular vesicles (GSC-EVs), we tested whether these GSC-EVs could be taken up by the recipient cells

Summary

Introduction

Glioblastoma is the most common and aggressive type of brain cancer with poor median survival of around one year. Despite maximal safe surgical resection and a combination of radiotherapy and chemotherapy [1,2], tumour recurrence is almost always seen, probably due to tumour heterogeneity both at a phenotypic and a molecular level [3]. The prognosis remains poor, despite a gross total resection at the first and at the second surgery (recurrence); overall survival is better in patients who had second surgery with local recurrence compared to non-local recurrence [4], as well as in those with a combination of bevacizumab and fractionated stereotactic radiotherapy (FSRT) treatment [5]. A small subpopulation of stem-like cancer cells (glioma stem cells, GSCs) is thought to be associated with resistance and subsequent repopulation of glioma cells post treatment [6–8]. The mechanisms of how GSCs facilitate their non-GSC glioma cells to progress are not fully elucidated [11]

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Discussion

Conclusion