(323f) Dimensionality and Matrix Stiffness Jointly Regulate Cell Migration from Multicellular Aggregates

While single cell studies using synthetic hydrogels have revealed key biochemical regulators that control cell migration patterns, understanding hoe cell cell and cell matrix interactions regulate multicellular migration within spatially arranged micro environment remains elusive. Here, we develop a polysaccharide based, photo crossed linked hydrogel system enabling independent modulating of adhesive cues and mechanical properties for cell culture. We also employed fabrication tools to generate multicellular aggregates based on Human Dermal Fibroblast which were cultured both on 2D surface AND 3D. This facilitated the investigation of cell migration patterns, multicellularity during invasion.

While single-cell studies using synthetic hydrogels have revealed key biochemical and biophysical regulators that control cell migration pattens, understanding how cell-cell and cell-matrix interactions regulate multicellular migration within spatially arranged microenvironment remains elusive. Here, we develop a polysaccharide-based, photo-crosslinked hydrogel system enabling independent modulation of adhesive cues and mechanical properties for cell culture. We also employed fabrication tools to generate multicellular aggregates based on human dermal fibroblasts (HDFs), which were cultured both on 2D surface and encapsulated within 3D dextran-based hydrogel. This facilitated the investigation of cell migration patterns, multicellularity during invasion, integrin activation and matrix protein deposition. Using these systems, our observation reveal that cells migrating from aggregates on 2D hydrogel substrates exhibited rapid single-cell migration characterized by large cell area and well-developed lamellipodia and filopodial cell morphology. The extent and speed of cell migration were controlled by the concentration of adhesive motif and substrate stiffness. Conversely, HDFs migrating in 3D hydrogels adopted a slower invasion speed with mesenchymal migration dependent on matrix stiffness and pore size. Collective cell migration was observed in soft matrix whereas limited cell invasion occurred in stiffer hydrogels. We also investigated the impact of aggregate size on cell migration and found that smaller size of aggregates promoted increased cell migration. Further examination revealed how changes in dimensionality influenced integrin activation profiles, mediated jointly by N-cadherin signaling and SMA activation. Depending on the material stiffness, migrating cells exhibited distinct migration modes expressing alpha-SMA, and N-cadherin, indicative of an epithelial-to-mesenchymal transition. These findings will expand the scope of fundamental adhesion biology and mechanobiology, and using tunable biomaterials with tailored mechanical properties allows to explore how multicellular interaction with the surrounding environment during wound healing, and differential migration patterns within tumor-like microenvironments paving the way for studying potential therapeutic for cancer metastasis.