Glioblastoma organoids derived from patient tumor tissueas a model for studying extracellular communication and microRNAs in the tumor microenvironment.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adult patients and is characterized by significant cellular heterogeneity and complex interactions between tumor cells and the tumor microenvironment. Extracellular vesicles (EVs) and the microRNAs (miRNAs) they carry are important mediators of intercellular signaling and, at the same time, a potential source of biomarkers useful in the diagnosis of gliomas. Experimental models capable of preserving the biological properties of the original tumor are essential for studying these processes. In this study, we prepared glioblastoma organoids (GBOs) derived from patient tumor tissue and analyzed extracellular miRNAs present in EVs isolated from conditioned media. Concurrently, we analyzed miRNAs contained in EVs isolated from cerebrospinal fluid (CSF) of patients with gliomas of varying degrees of malignancy. A total of 12 GBOs cultures were successfully established, long-term cultured, and cryopreserved. Immunohistochemical analysis confirmed the presence of markers typical of glioblastoma cells (Nestin, GFAP, Sox2). In early-stage organoids, cell populations representing the tumor microenvironment were also detected, including endothelial cells (CD31) and microglia (CD163). In some cases, reactive astrocytes and neurons were also present. Highly abundant miRNAs were identified in EVs isolated from conditioned media of GBO and primary GBM cultures, including hsa-miR-21-5p, members of the let-7 family, hsa-miR-16-5p, hsa-miR-125b-5p, and others. These miRNAs were simultaneously detected in EVs isolated from the CSF of patients with GBM and high-grade gliomas. In contrast, miR-204-5p, which was predominantly present in CSF samples, was minimally represented in EVs from tumor cultures, suggesting a possible origin of this miRNA in non-tumor brain tissue. The presented data support the use of GBOs derived from patient tumor tissue as a suitable model for studying intercellular communication and extracellular signaling in GBM and simultaneously demonstrate the potential of EV-associated miRNAs as biomarkers of the tumor microenvironment.