Nanofiber geometry directs cancer cell proliferation and invasion in a minimalist dipeptide extracellular matrix model.

The complex extracellular matrix (ECM) is known to influence tumor progression; however, investigating the contribution of ECM physical properties from intertwined biochemical and mechanical cues in native ECM remains challenging. Here, we developed a minimalist dipeptide-based ECM model with reduced biochemical complexity, providing a controlled platform to systematically investigate how enzymatic matrix remodeling is associated with cancer cell behavior. Using N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) peptide hydrogels, we employed proteinase K to modulate nanofiber morphology and hydrogel mechanical properties. In two-dimensional (2D) culture, moderate enzymatic remodeling was associated with enhanced cell proliferation. In three-dimensional (3D) tumor spheroid invasion assays, cancer cell invasion progressively decreased with increasing enzymatic treatment, with longer and more continuous nanofibers associated with greater invasion under comparable bulk rheological conditions. Immunofluorescence analysis revealed greater vinculin recruitment at the invasion front in matrices containing longer fibers, whereas matrices containing shorter fibers showed reduced vinculin recruitment, suggesting altered cell-matrix mechanical coupling. Together, these results highlight associations among nanofiber morphology, matrix mechanical properties, and cancer cell behavior. This work provides a controllable reductionist biomaterial platform for investigating the interplay between nanofiber morphology, matrix mechanical properties, and cancer cell behavior.