Radiomics analysis of 3D spheroids captures irradiation-induced structural and textural changes

Although radiotherapy (RT) is one of the main approaches used in cancer treatment, differences in clinical response among patients have not been eliminated, underscoring the need for improved methods to analyze radiobiological responses. Elucidation of variable responses requires a reliable preclinical model, among which three-dimensional (3D) cell aggregates have emerged as a new gold standard due to their physiological relevance and close resemblance to tumor microenvironments. Non-destructive assessment of the effect of RT or other treatments typically involves brightfield imaging to quantify spheroid volume, diameter, and circularity. However, a radiomics-based analytical framework has the potential to assess a greater range of extracted features from brightfield images beyond volume, diameter and circularity. To contribute to a better understanding of the variability observed in clinical radiation responses and to assess the potential of emerging 3D models in radiobiology, radiation-induced effects were investigated in a 3D culture model of human embryonic kidney (HEK-293) cells. As a proof of concept for the radiomics-based analysis framework, dose-related radiation-induced responses in HEK-293 spheroids were, for the first time, quantified longitudinally using a label-free, live morpho-textural radiomics approach integrating voxel-calculated volume, sphericity, entropy, energy and contrast. The findings showed that HEK-293 spheroid populations were quantitatively monitored using the label-free approach, effectively capturing both early and progressive radiation-induced changes, including subtle volume, sphericity, entropy, energy and contrast variations over time. Workflow robustness and generalizability were further assessed by analyzing brightfield images of dose-related radiation-induced responses in head and neck squamous cell carcinoma-derived FaDu tumor spheroids. Comparable trends were observed in FaDu tumor spheroids, showing the robustness and cross-model applicability of the radiomics workflow. A limitation of the present study is that the direct association between the extracted radiomic features and radiation-induced biological effects was not validated. Therefore, the clinical translation potential of the proposed approach requires further investigation by subsequent experiments integrating biological endpoints. Nevertheless, the present results demonstrated that the brightfield imaging-based radiomics approach could sensitively detect dose-dependent structural and textural changes in HEK-293 and FaDu spheroids following irradiation, offering a low-cost and non-destructive monitoring tool for subsequent radiobiology research.