Sandwich Culture Platforms to Investigate the Roles of Stiffness Gradients and Cell–Matrix Adhesions in Cancer Cell Migration

SIMPLE SUMMARY: Metastasis is the primary cause of death in cancer patients, yet there is a shortage of highly potent anti-metastatic drugs. Screening for anti-metastatic compounds involves studying cell migration by primarily using two-dimensional platforms, which makes migration along one plane possible. However, the spatial arrangement of biological signals in vivo is more complicated, especially when migration is guided by matrix stiffness gradients. In this work, we sought to develop a sandwich culture platform where cells are placed at the interface between a stiff tissue culture plate and soft alginate gel to study the role that transitions in stiffness have in cell migration. We specifically looked at the effects of matrix stiffness and mechanotransductive signaling on both adhesion-dependent and -independent modes of cell migration. The results reported could facilitate the development of optimal in vitro platforms to discover therapeutic strategies against tumor cell motility and invasion. ABSTRACT: Given the key role of cell migration in cancer metastasis, there is a critical need for in vitro models that better capture the complexities of in vivo cancer cell microenvironments. Using both two-dimensional (2D) and three-dimensional (3D) culture models, recent research has demonstrated the role of both matrix and ligand densities in cell migration. Here, we leveraged our previously developed 2.5D sandwich culture platform to foster a greater understanding of the adhesion-dependent migration of glioblastoma cells with a stiffness gradient. Using this model, we demonstrated the differential role of stiffness gradients in migration in the presence and absence of adhesion moieties. Furthermore, we observed a positive correlation between the density of cell adhesion moieties and migration, and a diminished role of stiffness gradients at higher densities of adhesion moieties. These results, i.e., the reduced impact of stiffness gradients on adhesion-dependent migration relative to adhesion-independent migration, were confirmed using inhibitors of both mechanotransduction and cell adhesion. Taken together, our work demonstrates the utility of sandwich culture platforms that present stiffness gradients to study both adhesion-dependent and -independent cell migration and to help expand the existing portfolio of in vitro models of cancer metastasis.