Multivalent DNA Origami Enables Single-Molecule Dissection of Integrin αvβ6–Receptor Tyrosine Kinase Crosstalk in Cancer Biology

Nanoscale organization of integrin-mediated receptor crosstalk is crucial for controlling cellular signaling in cancer biology. Previously, interactions between integrin αvβ6 and receptor tyrosine kinases (RTKs) have been implicated in cancer progression, but the spatial regulatory mechanisms remain undefined. Here, we developed a programmable DNA origami-based platform for nanoscale control of heteroligand multivalency and spacing, enabling systematic investigation of αvβ6-RTK interactions in cancer biology. We identified a spatial activation threshold for the αvβ6-specific peptide A20FMDV2 that promotes A375P β6 cell adhesion and FAK phosphorylation along with spacing- and density-dependent EGFR phosphorylation triggered by EGFR aptamers. Importantly, at an optimized peptide-to-RTK (EGFR, HER2, and Met) aptamer ratio and ligand density, αvβ6-RTK coactivation synergistically enhanced cell spreading and amplified phosphorylation of AKT and ERK, part of the PI3K-AKT and Ras-MAPK pathways. Validation in breast cancer models (MDA-MB-468 and BT-474) highlighted cell-type-specific signaling dependencies. This platform offers a framework for tumor microenvironment mimics and integrin-RTK-targeted therapies, emphasizing the critical role of nanoscale ligand patterning and multivalency in cancer progression.