Nanoagents for controlled manipulation of cell-ECM interaction

Invasion of epithelial cells from an aggregate into the surrounding extracellular matrix is strongly influenced by cell behavior as well as the properties of the extracellular matrix. The goal of this project is to gain a better understanding of how the local mechanical properties of the environment influence such collective invasion. In the previous funding period, we developed techniques to manipulate the mechanical properties of the cell environment and methods to control cell behavior, specifically the mechanosensing behavior of cells. We will now apply these techniques to gain deeper insight into collective invasion using cancer cell spheroids in collagen gels as model systems. In particular, we will use the photo-activatable YAP (optoYAP), which we developed in the last funding period. We showed that spatially-selective activation of optoYAP on one side of a spheroid drives invasion of the spheroid into the surrounding matrix on that side. YAP is known to activate proliferation and the mechanical force apparatus of cells. Based on our findings, we will now investigate whether proliferation or mechanical forces exerted by the cell – or a combination of both – drive the YAP-induced invasion. To this end, we will investigate the induced downstream-signaling in collaboration with B08 using the RNA-FISH probes developed by A05 in the previous funding period as well as immuno-fluorescence staining of the proteins. We will further use small molecule inhibitors of both pathways to find the prerequisites of YAP-induced invasion, and we will investigate the influence of different mechanical properties of the matrix employing the techniques that we applied to study single cells in the previous funding period. Furthermore, using nanoagents developed by B09, we will determine to what extent mechanical activation of the cell by photo-activation of actin or myosin is able to induce invasion. Since YAP can be controlled by mechanical alterations of the extracellular matrix, we will also investigate whether invasion can be induced via mechanical structures in the matrix. YAP is activated by nuclear translocation in cells, which are located on stiff substrates. We will therefore use structuring techniques – which we developed in the last funding period and will refine in collaboration with A08 – to create local, stiff collagen fiber bundles on one side of a cell spheroid. Subsequently, we will observe which kind of fiber bundles trigger translocation of YAP into the nucleus followed by invasion of the cell spheroid into the matrix. Together with the photo-activatable YAP, this will allow us to investigate the influence of mechanical signals on invasion, starting all the way upstream at the mechanical signal, via its mechanotransduction within the cell, down to the induced downstream signaling. Ultimately, the proposed experiments will show to what extent alterations in the mechanical properties of the extracellular matrix can trigger the onset of invasion. More generally, we will gain an understanding of how the mechanical properties of the extracellular matrix influence cell behavior, specifically cell shape.