The death signals mediated by extracellular vesicles between glioblastoma multiforme (GBM) cells during temozolomide treatment depend on the bioactive cargo specifically loaded by the different GBM lines

Introduction: Extracellular vesicles (EVs) of glioblastoma multiforme (GBM), a highly malignant primary brain tumour, may contribute to cancer cell proliferation and survival by shaping the tumour microenvironment. Resistance of GBM to the chemotherapeutic drug temozolomide (TMZ) leads to poor survival of patients, highlighting the importance of further research. We have previously demonstrated cell-specific properties of EVs isolated from various TMZ-treated or TMZ-untreated GBM cell lines. Here, we tested the ability of EVs to promote or inhibit the induction of cell death in recipient GBM cells and determined which class of molecules, i.e. proteins, lipids, or nucleic acids, of the EV cargo might be primarily responsible. Methods: UF/SEC isolated EVs from untreated and TMZ-treated U87MG and U251MG cells were characterized by proteomics, NTA, TEM, and cryo-EM analysis. Non-TMZ treated GBM cells were challenged with EVs in which proteins and RNAs of EVs were simultaneously or separately inactivated by heat and RNase A. Cell response was assayed by WB of cell death markers for apoptosis, ferroptosis, and autophagy. Results: EVs released from both sensitive (U87MG) and chemotherapy-resistant (U251MG) GBM cells differ in their properties, which are further influenced by TMZ treatment and when incubated with recipient GBM cells modify their physiological response. For example, EVs from TMZ-untreated U87MG cells significantly increase caspases 3/8/9 and CD71 and GPX4 apoptotic and ferroptotic markers in recipient U251MG cells. Heat and/or RNA-inactivated EVs modulate the effect described for non-inactivated EVs, and the intensity of modulation is related to the specific GBM cell. Conclusion: Our data further confirms the physiological heterogeneity between GBM cells. Indeed, the EVs produced by the two GBM cell lines not only differ in size, size distribution, shape, and cargo, but also in the spread of death signals with different contributions of proteins, lipids, and RNA depending on the specific GBM cell. Funding information: 000301_RICER CA_SAPIENZA_ 2022_Grande Progetto di Ateneo to LD