Spatial Regulation of Endocytosis and Adhesion Formation Governs Breast Cancer Cell Migration Under Confinement.

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 investigate this, we adapted a microfluidic platform that generates stable, spatially linear biochemical gradients across 5 μm-tall migration channels. COMSOL simulations and optical calibration using FITC-dextran confirmed that gradients form reliably within 5 min. 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. By leveraging this platform for spatially restricted drug delivery, we locally applied the endocytic inhibitor Dyngo-4a to either the front or rear of migrating cells. This revealed that front-targeted endocytic inhibition preserved or increased leading-edge enrichment of paxillin and the clathrin adaptor AP-2, whereas rear-targeted inhibition eliminated paxillin polarity and reduced AP-2 polarity. These changes were accompanied by a significant increase in cell migration speed under front-targeted inhibition, while rear-targeted inhibition had no significant effect on speed and neither treatment altered persistence. Together, these findings suggest that endocytic polarity regulates adhesion dynamics and cell migration under confinement, offering a mechanistic insight into processes relevant to cancer cell invasion.