(275f) Nuclear Mechanics and Morphology of Epithelial Cells Determine Cellular Composition in Intestinal Tissues

The crypt-villus structure of the mammalian gut epithelium is crucial for maintaining tissue function and can be replicated in vitro using intestinal stem cell (ISC)-derived organoids. Under homeostatic conditions, the ISC niche coexists with differentiated Paneth cells at the base of the crypt. Disruptions in this balance can contribute to diseases such as colorectal cancer. While biochemical signals from surrounding mesenchymal cells are known to regulate ISC differentiation and organization, the role of mechanical cues has only recently been recognized. However, the spatial and temporal role of cell nuclei during crypt formation and cellular fate determination remains largely unexplored.

To address this, we utilized an interdisciplinary approach combining murine ISC-derived organoids, in vivo tissue sections, innovative hydrogel systems, two-photon laser ablation, and advanced imaging techniques, including light-sheet and expansion microscopy. The hydrogel system was composed of poly(ethylene glycol) (PEG) chains functionalized with nitrobenzyl-azide and dibenzylcyclooctyne (DBCO). These PEG macromers underwent strain-promoted alkyne-azide cycloaddition (SPAAC) bio-click polymerization, creating a synthetic platform for organoid encapsulation and subsequent study. Notably, ortho-nitrobenzyl crosslinks within the hydrogel could be selectively cleaved using a 405 nm confocal laser, allowing precise spatial and temporal control over crypt formation. To investigate nuclear mechanics, we employed immunostaining of nuclear envelope markers alongside 3D shape analysis. Additionally, fluorescent reporter-bearing organoids were used to monitor cell population dynamics in crypts generated in vitro.

Our findings revealed a 25% decrease in nuclear aspect ratio in Paneth cells compared to adjacent non-Paneth cells, both in vivo and in organoid models. These differences were accompanied by alterations in nuclear lamina composition and morphology, suggesting distinct mechanical forces acting on the nuclei of the two cell types. To further evaluate these forces, we employed laser ablation to release cytoplasmic pressure at single-cell resolution and subsequently measured changes in nuclear morphology. To elucidate the temporal progression of these events, we used organoid lines expressing fluorescent reporters for Paneth cell precursors, along with synthetic hydrogel-based in vitro models that enabled synchronized crypt growth. Our results suggest that nuclear mechanics play an active role in crypt formation and cellular differentiation.

In summary, we present a novel biomaterial-based platform integrated with 3D organoid models to investigate previously uncharacterized aspects of nuclear mechanotransduction in intestinal crypt homeostasis. These findings provide a foundation for future studies on cancer stem cell regulation and their contribution to colorectal cancer development and progression.

Acknowledgments: This research was supported by the National Science Foundation grant RECODE-2033723, the National Institutes of Health grants R01-DK120921, 1S10OD034320 and the Helen Hay Whitney Foundation award F-1339.