Cryogenic Electron Microscopy of Extracellular Vesicles from Temozolomide-Treated Glioblastoma Cells Reveals Great Morphological Heterogeneity

Extracellular vesicles (EVs) are attracting considerable interest due to their important role in cell signaling. However, their nanosized scale, complexity, and heterogeneity make even morphological characterization challenging. Only with the recent advances in cryogenic electron microscopy (cryo-EM), together with the increasing availability of cryo-electron microscopes, has it become possible to visualize the native structure of EVs. In this study we performed an in-depth cryo-EM analysis of EVs derived from four glioblastoma multiforme (GBM) cell lines (U87MG, U373MG, U251MG, and T98G), highlighting the morphological changes induced by temozolomide (TMZ) chemotherapeutic treatment. Size, shape, circularity, concentricity, membrane thickness, and electron density of EVs were analyzed. The key characteristic revealed by cryo-EM was that EVs can be enclosed not only by one membrane bilayer, but also by two or more bilayers (double-layered vesicles, DVs; and multilayered vesicles, MVs). Overall, TMZ treatment substantially modified both the morphology and production of EVs, decreasing the percentage of single-layered vesicles (SVs) while increasing that of DVs and MVs, as well as reducing the electron density of the EV cargo. Morphometric and morphological information can shed light on the contribution of EVs to tumor progression, metabolism, drug resistance, and immune evasion.