Spatiotemporal Organization of PTK7 Diffusion on Cell Surface Facilitates Tumor Invasion and Migration

ABSTRACT The spatiotemporal dynamics of protein kinase diffusion govern signal activation cascades, thereby modulating fundamental cellular functions. Pseudokinases, catalytically inactive members of the protein kinase superfamily, utilize noncatalytic signaling mechanisms to exert pivotal cellular functions and are frequently dysregulated in human diseases. While nanoscale dynamics of catalytically active receptors regulate signaling integrity, the functional significance of pseudokinase spatial organization remains unknown. Here, using aptamer‐based single‐molecule tracking in living cells, we observed heterogeneous diffusion modes of pseudokinase PTK7 (confined, Brownian, and directed motion). Specifically, spatially PTK7 diffusion coefficients (D) quantitatively correlate with metastatic potential across pancreatic, colorectal and breast cancer cell lines. Functional validation demonstrates that antibody‐mediated PTK7 immobilization suppresses invasion, while Epithelial‐Mesenchymal Transition (EMT) induction accelerates diffusion kinetics to promote metastasis. Crucially, faster PTK7 mobility increases stochastic collision frequency with tyrosine kinase‐like orphan receptor 2 (ROR2), enhancing complex formation to robustly activate the WNT/PCP pathway. Moreover, in patient‐derived primary cells, accelerated PTK7 kinetics positively correlate with invasive and metastatic phenotypes, confirming the clinical relevance of this biophysical regulatory mechanism. This work establishes pseudokinase spatial dynamics as a biophysical regulator of tumor progression, revealing a non‐catalytic paradigm where receptor diffusion kinetics encode cellular behavior through stochastic signaling potentiation.