Poster V14 - Controlling Directional Cell Migration Decisions in Multi-cue Environments

Introduction: Cell migration is important for many biological processes, including development, immune response, tissue repair and regeneration and cancer metastasis. When an injury occurs, gradients in stiffness and collagen alignment direct cell migration toward the wound site in order to facilitate the wound healing process. Multiple directional cues are present in nearly all examples of cell migrating toward targets. Numerous engineered environments have been designed to control the presentation of directional cues to cells and cellular responses as well as intracellular mechanisms associated with directional cues such as contact guidance and durotaxis are known. However, there is very little understanding of how multiple directional cues compete or cooperate to direct cell migration. How the cell integrates directional signals from cues that may be directing cell migration towards different targets is completely unknown. Are the directional signals additive or synergistic? Are distinct signaling or cytoskeletal regulatory networks used or is there a competition for the activation of similar networks? In this study, we use a unique approach to present both aligned collagen fibers and gradients in stiffness in different orientations to understand which cues dominate in directed cells to migrate.

Materials and

Methods: Collagen fibrils were assembled on the surface of the mica (10 g/ml collagen in 50 mM Tris-HCl, 200 mM KCl at pH 9.2 for 12 h). These fibrils can be transferred to various substrates including sulfo-SANPAH-functionalized polyacrylamide gels by pouring 30% warm gelatin onto the fibers, allowing it to solidify, stamping it to the new substrate and melting and washing the gelatin away. Gradients of stiffness can be generated in the polyacrylamide using photopolymerization techniques. Cells that use different motility modes were placed on these substrates and morphology, cell migration and traction forces were assessed in areas where the collagen alignment and stiffness gradients were oriented to different extents.

Results, Conclusions, and Discussions: Radial stiffness gradients ranging from 2 kPa at the edge and 20 kPa at the center were generated in polyacrylamide and aligned collagen fibers were attached to the flexible substrate. When collagen fiber alignment and stiffness gradients were oriented in the same direction, cell alignment was robust (figure 1A), but when they were oriented in different directions, cells were directionally confused (figure 1B). Alignment was assessed circumferentially around the stiffness gradient (figure 1C). Unlike conditions containing only contact guidance or durotaxis, cell alignment quantified through a directionality index showed a sinusoidal behavior (figure 1D). When contact guidance and durotactic cues are aligned, cell alignment was greater than either of the single cues. Durotactic gradients perpendicularly oriented with respect to aligned fibers diminished the alignment usually seen on collagen fibers. Furthermore, when directionality was examined radially (figure 1E), higher stiffnesses appeared to diminish contact guidance as well, but the effect was only detected when directional cues were aligned. This indicates that while contact guidance cues dominate directional responses, durotactic gradients can attenuate the response if oriented orthogonally. We are beginning to examine how traction forces are oriented differently in response to aligned or unaligned directional cues and what cytoskeletal pathways lead to cue dominance. Cell type differences in multi-cue sensing will open avenues to designing extracellular environments to direct migration of multiple cell types to distinct targets, particularly in the context of traumatic or chronic wounds.

Acknowledgements (Optional): This work was supported by R01GM143302.