E-cadherin-mediated Mechanosensing Can Induce Changes in Cell Shape and EMT Markers

Abstract

Introduction E-cadherin is a mechanosensitive adherens junction protein, and is vital in healthy tissue morphogenesis and homeostasis. Conversely, in cancer, downregulation of E-cadherin indicates poor prognosis and is a hallmark of the epithelial-to-mesenchymal transition (EMT), which is correlated with increased metastasis. Cancer cells have also been found to be generally softer than their healthy counterparts [1]. However, the effects of E-cadherin-mediated mechanosensing in EMT and cancer progression is not yet elucidated. Methods We used multiphoton-based microfabrication [2,3] to create flat protein microstructures with elastic moduli between 26.5 kPa and 60 kPa and micropillars with stiffnesses between 0.00067 pN/μm and 2.26 pN/μm. We then functionalized these structures with E-cadherin via indirect multiphoton crosslinking of Protein A/G and immobilized E-cadherin-Fc. MDCK cells were seeded on the structures and evaluated after 3 days for their EMT marker expression and cell shape characteristics. Results On E-cadherin-coated flat matrices with low moduli, cells showed increased EMT marker expression such as N-cadherin and Vimentin, as compared to E-cadherin-coated flat matrices with high moduli. In addition, cells did not form good monolayers and preferred to form multilayer clusters. However, not much difference was seen in cell shape, such as in aspect ratio and area. On micropillar surfaces with lower stiffnesses, cells showed increased EMT marker expression, decreased area, and increased aspect ratio. Although they formed a monolayer-like surface, cell-cell junctions were poor and disconnected. Discussion These results show that softer substrates lead to more EMT-like phenotypes, and are consistent with previous literature where cancer cells are generally softer than their normal counterparts [1]. Moreover, the influence of the mechanical properties on E-cadherin-coated surfaces raises an interesting possibility that low cell stiffness can lead to cancer and EMT progression, rather than simply being an effect of tumor progression. As the effects of modulus and stiffness in the cell niche are typically evaluated using matrix mechanical properties, this study reflects the importance of the mechanosensing at cell-cell junctions, which may be investigated using micropatterning of cell-cell junction proteins.