DNA repair mechanisms are essential for maintaining cell viability following radiation exposure, and impairments can trigger cell-cycle arrest, senescence, or apoptosis. Morphological changes in nuclei, mitochondria, and the actin cytoskeleton provide readouts of the pathways leading to these outcomes. To investigate how radiation dose rate influences these processes, we employed a human mammary epithelial progression model that supports growth of both normal and malignant epithelial cells in 2D and 3D cultures. Single cells were embedded in Matrigel for one week allowing formation of spheroids. Spheroids were irradiated with either conventional X-rays (XRAD 320) or ultra-high dose rate synchrotron X-rays. Following exposure, spheroids were re-embedded and maintained in culture for up to 10 days. High-content confocal imaging (CellInsight CX7) was used to assess DNA damage, nuclear and mitochondrial morphology, actin architecture, and cell-cycle arrest. Normal mammary epithelial cells exposed to conventional radiation showed higher levels of DNA damage, cell-cycle arrest, and cell death compared with those irradiated at ultra-high dose rates. In contrast, malignant spheroids did not exhibit the same degree of normal-tissue sparing. These findings are consistent with the FLASH effect, in which dose rates above ∼40 Gy/s preferentially spare normal tissue without compromising tumor control. Our results reveal distinct morphological and functional responses to conventional versus ultra-high dose rate radiation. These differences provide insight into normal tissue protection observed with FLASH radiotherapy and highlight morphological readouts as valuable biomarkers for studying radiation responses.
Subramanian et al. (Sun,) studied this question.