Deciphering Cell Polarity Through Clathrin-Mediated Endocytosis and its Implications for Breast Cancer Cell Migration
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Date
2025-05
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The Ohio State University
Abstract
Metastasis is the process by which cancer cells spread to distant organs. Cellular events that underpin metastasis include endocytosis and polarity establishment 4. These events are highly dynamic and thereby require new tools and techniques to understand these processes in live cells. In this study, we investigated the spatial regulation of clathrin-mediated endocytosis (CME), a process by which cells internalize membrane proteins and extracellular molecules through clathrin-coated vesicles, and its role in guiding polarized cell migration 4, 5. Using a custom-designed microfluidic device, we established a stable epidermal growth factor (EGF) gradient to induce directed migration of SUM159 triple-negative breast cancer cells. Through fluorescence microscopy and quantitative image analysis, we observed enhanced CME at the rear of migrating cells, correlating with increased migration speed and directional persistence. Targeted inhibition using Dyngo-4a and Pitstop 2 revealed that rearward endocytosis promotes migration.
To support high-throughput and accurate quantification, we employed a Cellpose-based machine learning model to segment and analyze migrating cells from microscopy images. This automated segmentation pipeline enabled extraction of cell trajectories and endocytic features, streamlining the analysis of large image datasets. Our findings highlight the importance of spatially controlled endocytosis in cancer cell motility and lay the foundation for future investigations into how membrane trafficking and adhesion dynamics differ between cancerous and healthy cells.