A Noninvasive Approach to Study Localized Forces in 3D Epithelia

Epithelial tissue integrity and remodeling rely on mechanical coupling between cytoskeletal organization and adhesion complexes, but their orchestration in three-dimensional (3D) epithelia remains unresolved. Here, we introduced a noninvasive strategy to locally probe forces within 3D epithelial tissues. We engineered elastic polyacrylamide (PAAm) microbeads with cell-like size and mechanical properties, enabling their integration into tissues. PAAm microbeads were spontaneously engulfed by the tissue, establishing an intrinsic interface. This allowed bead deformation to be directly correlated with local cytoskeletal architecture and adhesion organization, visualized through high-resolution imaging and quantitative 3D computational reconstruction. Using this approach, we demonstrated that localized mechanical perturbations trigger cytoskeletal remodeling while preserving tissue polarity. We further identified extracellular matrix composition as a key determinant of bead-tissue interactions. At the bead-cell interface, cells assembled tension-bearing focal adhesions and actin stress fibers. Quantitative analysis of bead deformation revealed sub-cellular scale patterns of cell-bead interaction forces, which correlated with the localization of cytoskeletal elements. Our findings extend previous traction force microscopy observations to 3D, revealing coordinated, spatially heterogeneous forces specifically at the cell-bead interface. Taken together, this enabled us to dissect the interplay between cellular processes and tissue mechanics in a noninvasive way in 3D model systems.