Nanoparticle Metal Mass Uptake Governs Radiosensitizing Efficacy Across 2D, 3D, and In Vivo Models

Abstract Despite extensive efforts to develop nanoparticle-based radioenhancers, clinical translation remains limited, partly due to the lack of physiologically relevant in vitro models. To address this gap, we developed a 3D spheroid model of head and neck cancer using FaDu cells and compared it directly to a corresponding in vivo model in a radiotherapy setting. The spheroids exhibited key tumor-like features, including the formation of a hypoxic core and growth kinetics comparable to in vivo tumors. Importantly, the model allowed for long-term monitoring of tumor growth and radiation response. Upon X-ray irradiation, dose–response behavior in spheroids mirrored that observed in vivo. Furthermore, TiO₂, HfO₂, and Au nanoparticles demonstrated consistent radiosensitization effects in both systems when matched for uptake mass. In contrast, conventional 2D clonogenic assays failed to predict in vivo performance, likely due to their lower radioresistance and unrealistic nanoparticle exposure conditions. This study introduces a robust, scalable, and clinically compatible 3D in vitro platform for preclinical screening of nanoparticle radioenhancers. The system may offer streamlining of development pipelines and support the 3R principles of reduction, replacement, and refinement in radiation oncology research. Competing Interest Statement The authors have declared no competing interest.