Spatially Modulated Irradiation Alters Extracellular Vesicle MicroRNA Cargo in Rat Glioma and Astrocyte Models

Spatially fractionated radiation therapy, particularly minibeam radiation therapy (MBRT), improves normal tissue sparing while maintaining tumor control compared with conventional radiotherapy (CRT). However, the mechanisms underlying these advantages remain unclear. EVs carrying miRNAs are increasingly recognized as mediators of radiation-induced intercellular communication and may contribute to non-targeted and cohort effects. We investigated the impact of MBRT versus CRT on EV-miRNA profiles in glioma and normal astrocyte cell lines. Rat glioma (RG2, F98) and astrocyte (CTX-TNA2) cell lines were irradiated with X-ray CRT or MBRT. Small EVs were isolated 24 h post-irradiation, characterized by transmission electron microscopy and nanoflow cytometry, and analyzed by small RNA sequencing. EV morphology, size, and expression of canonical markers (CD9, CD63, CD81) were preserved across conditions, indicating that irradiation primarily altered EV cargo. Glioma cells showed limited and cell-dependent changes. In RG2 cells, CRT increased miR-6319 and miR-374-5p, whereas MBRT modulated miR-151-5p in a dose-dependent manner and reduced miR-466c-5p at 5 Gy. No significant changes were observed in F98 cells. In contrast, astrocytes exhibited marked remodeling of EV-miRNA cargo after irradiation, including enrichment of miRNAs associated with stress adaptation, hypoxia-related signalling, and immune regulation, together with consistent depletion of miR-122b and miR-486 across irradiation conditions. MBRT and CRT were associated with differential remodeling of EV miRNA cargo, with a markedly stronger response in astrocytes than in glioma cells. These findings suggest altered EV signaling potential following irradiation and provide an initial molecular framework for investigating how spatial dose modulation may influence extracellular communication. Further functional validation in human-relevant systems is required to determine mechanistic and translational significance.