Simple fabrication method for cancer cell migration studies on biomimetic substrates with tunable stiffness

: Glioblastoma is one of the most invasive and aggressive brain cancers, yet current therapies fail to effectively target its pronounced migratory behavior. The development of anti-migratory strategies is therefore critical to improve patient outcomes, highlighting the urgent need for reliable in vitro platforms for drug testing. Here, a simple, low-cost, and reproducible hydrogel-based system that recapitulates key mechanical features of glioblastoma microenvironment is presented. By tuning hydrogel concentration, highly flat and transparent substrates with either uniform stiffness or controlled stiffness gradients are fabricated, enabling live-cell imaging and quantitative analysis of cell migration. Substrate stiffness modulates cell morphology, spreading, and motility in U87-MG glioblastoma cells and SVG-A astrocytes. To validate the platform for the screening of anti-migratory compounds, the cyclopeptide Ala-2/5, a recently developed anti-migratory molecule, is tested, revealing a significant reduction in cell migration in both cell types, with a stronger effect observed in tumor cells. Overall, these substrates overcome the limitations of traditional 2D assays while providing an easy-to-fabricate, versatile, and scalable tool for mechanobiology studies and for the preclinical evaluation of anti-migratory therapies targeting invasive cancers.