Quantitative Analysis of Colonic Epithelial Cell Aging in a Cell-cycle like Model: Changes in Nucleus and Cytoplasm Along the Crypt Axis.

Understanding epithelial cell aging in bowel tissue can provide critical insights into gastrointestinal (GI) health, potentially leading to improved diagnosis, treatment, and prevention strategies for various GI conditions. Despite the significance of cellular aging in the GI tract, previous studies have not quantified epithelial cell maturity development along the base-to-surface axis of colon crypts due to the labor-intensive nature of manual quantification and the lack of a clear definition of cellular age. Moreover, the process faces tremendous challenges when tissue is cut in 2D and examines results solely from one or a few biopsy cuts without reconstructing the full 3D space, as the original cell structure is 3D, leading to variability based on the cut angle. This research addresses these challenges by systematically analyzing the relationship between epithelial cell morphology and their position from the base to the surface in colon crypt tissue. Our approach involves using multiplex immunofluorescence (MxIF) imaging for cell nuclei segmentation, identifying the potential full longitudinal cut, and manually annotating the surface and base of the colon crypts. By analyzing the epithelial cells, we create density maps to investigate the relationship between nucleus size and the surface-to-base distance ratio, defining this ratio as cell age. We also examine the nucleus-to-cytoplasm (N/C) ratio in relation to cell age. For the statistical analysis, we use a robust linear regression model to remove outliers and a random effects model for the final regression. Our results show a decreasing trend in both the N/C ratio and nucleus size as epithelial cells move from the base to the surface. These findings provide a quantitative pipeline for single-cell profiling in a spatial manner, facilitating broader analysis of epithelial cell aging and its implications for GI health.