ConfluentFUCCI for fully-automated analysis of cell-cycle progression in a highly dense collective of migrating cells

Understanding mechanisms underlying various physiological and pathological processes requires accurate and fully automated analysis of dense cell populations that collectively migrate, and specifically, relations between biophysical features and cell cycle progression aspects. A seminal tool that led to a leap in real-time study of cell cycle is the fluorescent ubiquitination-based cell cycle indicator (FUCCI). Here, we introduce ConfluentFUCCI, an open-source graphical user interface-based framework designed for fully automated analysis of cell cycle progression, cellular dynamics, and cellular morphology, in highly dense migrating cell collectives. Leveraging state-of-the-art tools, some incorporate deep learning, ConfluentFUCCI offers accurate nuclear segmentation and tracking using FUCCI tags, enabling comprehensive investigation of cell cycle progression at both the tissue and single-cell levels. We compare ConfluentFUCCI to the most recent relevant tool, showcasing its accuracy and efficiency in handling large datasets. Furthermore, we demonstrate the ability of ConfluentFUCCI to monitor cell cycle transitions, dynamics, and morphology within densely packed epithelial cell populations, enabling insights into mechanotransductive regulation of cell cycle progression. The presented tool provides a robust approach for investigating cell cycle-related phenomena in complex biological systems, offering potential applications in cancer research and other fields. ### Competing Interest Statement The authors have declared no competing interest.