Extracellular vesicles from temozolomide treated glioblastoma multiforme cells: insight into the morphological features and the role of bioactive cargo in the spread of cell death signals

Introduction: Glioblastoma multiforme (GBM) is a highly malignant and heterogeneous type of brain tumour. A significant role of extracellular vesicles (EVs) in shaping the tumour microenvironment, promoting cell survival and resistance to chemotherapy in GBMs is supported by emerging data. Formerly we have reported significant changes in the composition of cargo and morphology of EVs isolated from GBM cell lines treated with the commonly used chemotherapeutic drug temozolomide (TMZ). In this study, we extended our research to identify the effect of isolated EVs on recipient cells and to determine to which extent this influence is related to different classes of molecules in the cargo of EVs. Methods: EVs from untreated and TMZ-treated GBM cell lines (U87MG and U251MG) were isolated by UF/SEC and characterized by MS, TEM, cryo-EM, and NTA. PKH26 stained EVs were used to investigate the process of internalization. Contribution of different classes of molecules was analysed by separate or simultaneous elimination of proteins and nucleic acids by heat and RNase A. Cell response to native or modified EVs was assessed by western blot. Results: TMZ-treatment promotes the release of multilamellar and multicompartmental EVs, affects the number and size distribution of the vesicles. The response of the two analysed GBM cell lines to EVs differs significantly. For example, EVs from TMZ-untreated U87MG cells decrease the expression of cell death markers in recipient U87MG cells, while U251MG cells treated with the same EVs display significant increase in apoptotic (caspases 3/8/9) and ferroptotic (CD71 and GPX4) markers. Treatment of cells with protein- and/or RNA-inactivated EVs modulates the effect of native EVs, but its intensity is strongly dependent on the specific GBM cell line. Conclusions: The current research gives an insight into the heterogeneity of GBM-derived EVs which is further promoted by TMZ treatment. Contribution of bioactive molecules in EVs to the spread of cell death signals greatly depends on the specific GBM cell line. Funding information: 000301_RICER CA_SAPIENZA_ 2022_Grande Progetto di Ateneo to LD