Extracellular Vesicle-Based Characterization of Stem Cell Phenotype in Glioblastomas

Glioblastoma (GB) is the most common brain malignancy occurring in adult patients having an extremely low overall survival. Therefore, it is paramount to establish reliable and accurate diagnostic and prognostic markers to guide a personalized and more effective treatment. Molecular characterization of the tumor is the ultimate goal in GB management and comprises, among others, the study of the extracellular vesicles (EVs). Not only do they carry within their cargo molecules involved in shaping a favorable microenvironment for GB development, but EVs also present surface markers mirroring the phenotype of the donor cells. Our study aims to assess the dynamic evolution of EV-positive surface biomarkers and EV-derived proteins involved in maintaining and transferring a stem cell phenotype to the cells from GB surroundings. We performed a prospective observational study on GB patients operated on in the Neurosurgery Clinic of the Emergency Clinical County Hospital of Târgu Mureș, Romania. GB-derived EVs were isolated from the patients' plasma using a density gradient ultracentrifugation protocol. The expression of EVs positive to four epitopes specific to stem cells (CD44, CD133, CD326/EpCAM, and SSEA4) was followed in three moments in time, preoperatively, seven days, and three months postoperatively, respectively, and quantified by a bead-based multiple analysis using flow cytometry. Moreover, NANOGP8, a protein within GB cargo capable of promoting a stem cell phenotype, was dynamically evaluated using the Western blot technique. Our study showed a statistically significant decrease of all surface markers and NANOGP8 immediately after tumor ablation. Nonetheless, the long-term follow-up of the patients revealed an extremely variable evolutionary pattern reflecting the high heterogeneity of GB. Further studies are necessary to either confirm or infirm the accuracy of these markers in early diagnosing GB, in predicting the outcome of this disease, and in guiding an individualized therapy.