Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during <i>in vitro</i> cultivation

Glioblastomas are characterized by extensive intra‑ and intertumoral heterogeneity, which drives their rapid invasive growth and frequent recurrence. As a result, prognosis remains poor, and developing effective treatments continues to be a major challenge. The survival and growth of glioblastomas are supported by a specific, protective tumor microenvironment (TME) that influences tumor morphology, growth rate, therapy resistance and recurrence. To better represent the complex TME with its immune cells and inflammatory mediators and the tumor heterogeneity, including glioma stem‑like cells (GSCs), realistic <i>in vitro</i> preclinical models are required. Patient‑derived glioblastoma organoids (GBOs), directly derived from tumor tissue obtained after surgical resection, were generated in the present study. Preserving the original tumor state in organoids, particularly regarding the TME and heterogeneity, is crucial. The present study evaluates the long‑term preservation of key tumor properties in cultivated GBOs, including glial structural proteins, GSCs, immune cells, endothelial cells, proliferation marker, and inflammatory mediators such as cytokines and chemokines. Expression levels of these components were measured at five defined time points in 10 independent GBO preparations, at both the mRNA and protein level, and statistical correlation analyses were performed. The findings of the present study indicate that the key components were generally maintained over a one‑month cultivation period. However, some markers showed reduced expression at later time points or recovered after an initial drop at early cultivation time points. Therefore, the optimal time window for employing patient‑derived organoids as an advanced preclinical model for glioblastoma research appears to be between 7‑14 days of cultivation.