Tunable mechanical properties of stent-like microscaffolds for studying cancer cell recognition of stiffness gradients

Physical and mechanical properties of extracellularmatrix (ECM) have been proved to be crucial in the metastatic process. However, currently available studies on the interplay between ECM stiffness and cancer cell invasive behaviour are performed on planar assays, while the in vivo interaction takes place in three-dimensions. To take into consideration the ECM structural and mechanical complexity in the cell/structure interactions, we fabricated 3D microscaffolds through two-photon lithography (2PL) and tested how they are invaded by human colorectal adenocarcinoma (LS-174T) tumor cells, showing that it is possible to detect significant differences in cells/structure interactionwhen structural parameters are modified. In particular, both scaffold geometry and 2PL fabrication parameters were optimized to obtain 3D polymeric cylindrical structures with controlled Young's modulus and with linear stiffness gradients. The ability of LS-174T to migrate in the scaffolds was tested in different experimental conditions, including scaffolds functionalization and under β-catenin downregulation. It was observed that high Young's modulus scaffolds are always less invaded than softer ones, confirming the role of the 3D micro-environmental stiffness in mediating cells migration, including when specific functionalization or pharmacological treatments are performed.