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Juxtacrine signaling interactions between the EphA2 receptor tyrosine kinase and its ephrin-A1 ligand contribute to healthy tissue maintenance and misregulation of this system is observed in at least 40% of human breast cancer. Hybrid live cell-supported membrane experiments in which membrane-linked ephrin-A1 displayed in supported membranes interacts with EphA2 in living cells have revealed large scale clustering of EphA2/ephrin-A1 complexes as well as their lateral transport across the cell surface during triggering. Here, we utilize 100 nm spaced hexagonally ordered arrays of gold nanodots embedded within supported membranes to present defined obstacles to the movement and assembly of EphA2 clusters. By functionalizing both the supported membrane and the nanodots with ephrin-A1, we perform a type of affinity chromatography on EphA2 signaling clusters in live cell membranes. Analysis of 10 different breast cancer cell lines reveals that EphA2 transport is most frustrated by nanodot arrays in the most diseased cell lines. These observations suggest that strong physical association among EphA2 receptors, as well as their assembly into larger clusters, correlates with and may contribute to the pathological misregulation of the EphA2/ephrin-A1 pathway in breast cancer.

Nanoscale Obstacle Arrays Frustrate Transport of EphA2–Ephrin-A1 Clusters in Cancer Cell Lines