Tuning cell-surface affinity to direct cell specific responses to patterned proteins

Interactions with local extracellular cues direct cell migration. A versatile method to study cell response to a protein consists of patterning the protein cue on a substrate and quantifying the distribution of cells between patterned and non-patterned areas. Here, we define the concepts of (i) cell-surface affinity to describe cell choices, and of (ii) reference surface (RS) to clarify that the choice is made relative to a reference. Furthermore, we report a method to systematically tune the RS and show that it can dominate the experimental cell response to a protein cue. The cell response to a cue can be switched from strong preference to strong aversion by only changing the RS. Using microcontact printing, we patterned the extracellular matrix proteins fibronectin or netrin-1 adjacent to a series of RSs with different ratios of poly-d-lysine (PDL) and polyethylene glycol (PEG), which are of high affinity and of low-affinity for cells, respectively. C2C12 myoblasts or primary neurons seeded on substrates with a high affinity RS (high % PDL) did not respond to a printed protein of interest, and conversely on RSs of low affinity (high % PEG) the cells preferred the printed protein even in the absence of a specific interaction. However, when testing cell response to a standardized series of RSs varying from high to low affinity, a specific response curve was obtained that was unique to each cell type-protein pair. Importantly, for intermediate RSs with moderate affinity, the cell response to the cue was dependent on the activation of biologically relevant protein-specific biochemical signal transduction pathways. Our results establish that choices made by cells in response to a surface-bound cue must take into account, and be interpreted in the context of, the RS. The use of a series of RSs with varying cell-surface affinity reveals specific response curves of cells to a cue that can be compared quantitatively and that may help gain new insights into cellular responses to extracellular proteins.