Synthetic Fibrous Hydrogels as Minimal Systems to Modulate Cell Migration Modes in 3D

Cell migration in three-dimensional (3D) environments is highly plastic and regulated by extracellular matrix (ECM) cues. Engineered biomaterials provide controllable platforms to investigate how specific matrix signals regulate cell behavior in 3D, yet how defined biochemical signals control migration modes remains unclear. Here, we present tunable fibrous polyisocyanide (PIC) hydrogels functionalized with integrin-binding RGD peptides, cadherin-mimetic HAVDI peptides, or no ligands to direct mesenchymal, hybrid, or amoeboid-like migration of human adipose-derived stem cells without altering matrix mechanics. Using live-cell tracking, 3D displacement microscopy, matrix remodeling analysis, and YAP nuclear localization, we show that ligand identity governs adhesion organization, force transmission, and mechanotransduction. RGD-functionalized matrices are associated with punctate β1-integrin staining at the cell membrane, extensive matrix remodeling, strong YAP activation and upregulation of migration-related genes. In contrast, non-adhesive matrices limit adhesion formation, resulting in weak force transmission and amoeboid-like state. HAVDI-functionalized matrices are associated with punctate membrane-associated N-cadherin staining and heterogeneous cellular responses, supporting a hybrid migration mode associated with adhesion organization and force transmission. Together, these findings demonstrate that ligand identity alone is able to program migration mode in a force-responsive 3D matrix and provide a versatile platform to dissect cell-matrix interactions in complex environments. STATEMENT OF SIGNIFICANCE: Cell migration in tissues is highly adaptable, yet precise control of migration modes in defined 3D biomaterials remains challenging. We introduce fibrous PIC hydrogels presenting RGD, HAVDI, or no adhesive ligands to bias human stem cells toward mesenchymal-like, hybrid, or amoeboid-like migration states. By linking ligand identity to adhesion organization, matrix remodeling, YAP mechanotransduction, and gene expression, this work provides a minimal platform to dissect and engineer 3D cell-matrix interactions.