A Comprehensive Framework for Spatio-Temporal Analysis of DNA Damage Foci in Tumor Spheroids

SUMMARY The evaluation of DNA damage response, particularly DNA damage foci formation, is crucial for understanding tumor biology and assessing the impacts of various drugs. We have developed a sophisticated semi-automated image analysis pipeline which generates quantitative map of the spatiotemporal distribution of DNA damage foci within live tumor spheroids. Our framework seamlessly integrates live imaging of tumor spheroids via Light Sheet Fluorescence Microscopy with a DNA damage foci formation assay using a genetically encoded fluorescently labeled DNA damage sensor. By combining advanced imaging techniques with computational tools, our framework offers a powerful tool for studying DNA damage response mechanisms in complex 3D cellular environments. MOTIVATION The motivation of this work is to propose a comprehensive framework that facilitates the study of DNA repair mechanisms within 3D contexts, specifically using tumor spheroid models. By integrating advanced imaging technologies and genetically encoded fluorescent sensors, our goal is to offer researchers a robust methodology for observing and analyzing DNA damage dynamics in realistic tissue-like environments. This framework is designed to enhance accessibility and streamline data processing, thereby empowering the scientific community to investigate DNA repair processes in 3D with greater precision and efficiency. Competing Interest Statement The authors have declared no competing interest.