Minibeam radiation therapy does not induce canonical immunogenic cell death or cGAS–STING activation in glioblastoma and astrocyte monocultures

Minibeam Radiation Therapy (MBRT) uses sub-millimetric beams to generate highly heterogeneous dose distributions and has shown promising results in preclinical glioblastoma by preserving normal tissue while eliciting immune responses. However, the initiating molecular mechanisms remain unclear. We hypothesised that immunogenic cell death or cGAS-STING pathway activation could be involved. We evaluated cell death, damage-associated molecular patterns (calreticulin exposure and HMGB1 release), RT1a exposure, and cGAS-STING activation 24 h after irradiation. MBRT was compared with conventional radiation therapy (CRT; X-rays or protons) in glioblastoma cell lines (F98, RG2) and normal astrocytes using flow cytometry and qPCR. CRT induced dose-dependent tumour cell death, whereas MBRT caused lower, non-dose-dependent effects. Astrocytes showed similar responses to both modalities. Immunogenic markers were limited: CRT increased calreticulin and RT1a exposure more than MBRT, while HMGB1 was undetectable. No transcriptional activation of the cGAS-STING pathway was detected, although interferon-stimulated genes were partially upregulated in F98 cells. RG2 cells showed no immune activation. CRT selectively induced Cxcl10 expression in astrocytes. MBRT and CRT induce distinct biological and immune-related responses in tumour cells and astrocytes in vitro, suggesting different tumour-intrinsic mechanisms; whether these translate to broader immunogenic effects in vivo remains to be determined.