Is morphological heterogeneity of extracellular vesicles from temozolomide-treated glioblastoma multiforme cells driven by lipids?

Glioblastoma multiforme (GBM) poses a major challenge in health care being the most common malignant brain tumour in adults. One of the causes of the tumour’s unresponsiveness to therapy is the establishment of the tumour microenvironment in which extracellular vesicles (EVs) play an integral role. EVs are nano- and microsized membrane-enclosed vesicles that are released by almost all types of cells. EVs carry bioactive cargo and can be internalized in recipient cells provoking a specific response. Formerly, we reported significant differences between EVs derived from four GBM cell lines (U87MG, U251MG, U373MG, T98G). We showed that treatment of GBM cells with a chemotherapeutic drug temozolomide (TMZ) altered the size, number, and protein cargo of released EVs, as well as the functional response in recipient GBM cells in terms of cell migration and cell death. Since, proteomics (LC-MS/MS) of EVs released from TMZ-treated cells showed an increase in the proteins involved in lipid biology, we performed an in-depth investigation of the EVs morphology and the role of lipids in the EVs-mediated cell-cell communication. Cryogenic electron microscopy indicated a great morphological heterogeneity (i.e. unilamellar, multilamellar vesicles; shape, membrane thickness) within EVs population and a modulation of their relative number. The dual role of lipids, as structural and functional molecules, was also evidenced by the fact that theinactivation of proteins and/or RNAs present in EVs did not alter the EVs-mediated cell death. Therefore, we characterised neutral lipids by thin-layer chromatography indicating that TMZ-treatment affects lipid metabolism and profile in GBM cells and in the conditioned media. The data acquired so far will be extended to the analysis of GBM-EVs. Since the role of lipids in EVs is one of the most overlooked aspects, our preliminary data provides an initial valuable insight into the different contribution of EVs in cell communication.