Abstract
Epithelial cells disengage from their clusters and become motile by undergoing epithelial-to-mesenchymal transition (EMT), an essential process for both embryonic development and tumor metastasis. Growing evidence suggests that high extracellular matrix (ECM) stiffness induces EMT. In reality, epithelial clusters reside in a heterogeneous microenvironment whose mechanical properties vary not only in terms of stiffness, but also topography, dimensionality, and confinement. Yet, very little is known about how various geometrical parameters of the ECM might influence EMT. Here, we adapt a hydrogel-microchannels based matrix platform to culture mammary epithelial cell clusters in ECMs of tunable stiffness and confinement. We report a previously unidentified role of ECM confinement in EMT induction. Surprisingly, confinement induces EMT even in the cell clusters surrounded by a soft matrix, which otherwise protects against EMT in unconfined environments. Further, we demonstrate that stiffness-induced and confinement-induced EMT work through cell-matrix adhesions and cytoskeletal polarization, respectively. These findings highlight that both the structure and the stiffness of the ECM can independently regulate EMT, which brings a fresh perspective to the existing paradigm of matrix stiffness-dependent dissemination and invasion of tumor cells.
Highlights
Epithelial clusters in polyacrylamide (PA) channels of tunable stiffness and confinement
We found that inhibition of focal adhesion kinase (FAK) disabled the effect of ECM stiffness on EMT
We used photolithography techniques to fabricate silicone masters of defined topography, as shown in Fig. 1, where channel width is prescribed in the mask design and the depth is dictated by the thickness of the photoresist layer
Summary
Epithelial clusters in polyacrylamide (PA) channels of tunable stiffness and confinement. After the disruption of the cytoskeletal structure by pharmacological inhibition of microtubules and nonmuscle myosin II, cells lost their ability to undergo EMT in a confinement-dependent manner. The ability of cells to generate active actomyosin forces, maintain cytoskeletal structure through microtubules, and attain elongated shapes might play a central role in enabling confinement-sensitive EMT. Cell-ECM adhesions might be key mediators for triggering a mechano-transductive signaling cascade that weakens cell-cell adhesions and induces EMT. Taken together, these results bring a fresh perspective to the existing paradigm of matrix stiffness-dependent EMT and highlight that ECM confinement alone can disrupt the integrity of epithelial entities
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