Cumulative microscopy reveals cellular states in fibroblasts from patients with genetic disorders

Analysis of cellular states and signaling trajectories can provide insights into causes of disease. We developed cumulative microscopy, a method to perform cyclical imaging without elution or quenching steps. Cumulative microscopy computationally extracts individual signals from accumulating fluorescence during sequential imaging. We used cumulative microscopy to quantitatively assess cell cycle and stress markers in individual primary fibroblasts from patients with rare genetic proliferative disorders with increased cancer risk. Neural network-based analysis of cumulative microscopy data suggested that cells from patients with Cartilage-hair hypoplasia (CHH), but not Mulibrey Nanism (MUL), showed replication stress. We analyzed cell states and cell trajectories and found that a subset of cells from patients with CHH showed spontaneous replication stress, followed by cell cycle exit in both G1 and G2 phase. We note that replication stress potentially could underlie both proliferative defects and increased cancer risk in CHH patients and conclude that cumulative microscopy is an efficient, quantitative, and generalizable approach to multiplex microscopy.