The rise of 3D spheroids in radiobiology for assessing tumour radioresistance

BACKGROUND AND PURPOSE: Particle therapy (PT), including proton (PRT) and carbon ion radiotherapy (CIRT), offers physical and biological advantages over photon radiotherapy (XRT), particularly for radioresistant tumours such as glioblastoma and osteosarcoma. However, systematic preclinical comparisons using physiologically relevant models remain limited. MATERIAL AND METHODS: T98G (glioblastoma), Saos-2 and U2-OS (osteosarcoma) cells were cultured as two-dimensional (2D) monolayers and three-dimensional (3D) spheroids and irradiated with XRT, PRT or CIRT at 2, 4 or 6 Gy. Clonogenic survival, metabolic activity (PrestoBlue), invasion and spheroid growth kinetics were quantified. Relative biological effectiveness (RBE) was derived from survival data, and spheroid sections were analysed histologically (H&E). RESULTS: CIRT induced the strongest cytotoxic and anti-invasive effects across all models. In 2D cultures, the surviving fraction at 2 Gy decreased from 0.62 to 0.69 after XRT to 0.16-0.28 following CIRT (RBE = 2.1-2.4 vs. 1.1 for PRT; p < 0.0001). 3D spheroids exhibited overall higher radioresistance, yet CIRT markedly reduced growth and invasion, lowering normalised indices to 0.52 ± 0.12 (T98G) and 0.58 ± 0.09 (Saos-2) at 6 Gy, while photons often promoted invasion (> 1.2; p < 0.001). RBE values in 3D reached 3.6-4.0. H&E staining confirmed dose-dependent architectural disruption, with carbon ions inducing extensive necrosis and cellular degeneration. INTERPRETATION: This study introduces a robust 3D preclinical platform for radiobiological assessment of particle therapy. CIRT consistently overcame intrinsic and microenvironment-mediated resistance, outperforming photons and protons in suppressing viability, invasion and spheroid integrity, thus reinforcing the translational relevance of 3D models and the therapeutic promise of carbon ion therapy for resistant malignancies.