A microfluidic gradient and parallel-track system uncovers spatial control of endocytosis and adhesion formation in...

Cell migration through confined spaces is a critical step in cancer metastasis, yet the spatial regulation of endocytosis and adhesion dynamics during this process remains poorly understood. To address this, we developed a microfluidic platform that generates stable, spatially linear biochemical gradients across 5 μm-tall migration channels while limiting confounding flow-induced shear stress (<0.05 dyn/cm ² ). COMSOL simulations and optical calibration using FITC-dextran confirmed that gradients form reliably within 5 minutes. The microdevice also supports long-term live imaging and is compatible with both spinning disk confocal and total internal reflection fluorescence structured illumination microscopy modalities, enabling high-resolution visualization of adhesion and endocytic structures. Localized application of the endocytic inhibitor Dyngo-4a to the front or rear of migrating cells revealed that front-targeted inhibition significantly increased the enrichment of paxillin and the clathrin adaptor AP-2 at the leading edge, whereas rear-targeted inhibition completely abolished their front-rear asymmetry. These changes were accompanied by enhanced migration speed and persistence, particularly under front-targeted inhibition. Together, these findings highlight the critical role of spatially coordinated endocytosis in sustaining polarized adhesion and persistent cell movement. Our platform offers a powerful tool for dissecting subcellular mechanisms of migration under confinement and provides a broadly applicable framework for probing spatially localized signaling in engineered microenvironments.