Micropillar-Arrayed Surfaces Promote Transforming Growth Factor Beta 1 Induced Epithelial to Mesenchymal Transition by Focal Adhesion Kinase-Related Signaling in A549 Cells
Substrate rigidity and topography are two critical biomechanical factors that influence the biological behavior of cancer cells, including the epithelial to mesenchymal transition (EMT). EMT is a process where epithelial cells lose their cell polarity and cell-cell adhesion, gaining migratory and invasive properties to become mesenchymal stem cells. This transition is pivotal in cancer progression, metastasis, and fibrosis. Although recent studies have started to explore how the mechanical microenvironment dictates cell fate, the exact mechanisms by which mechanical signals influence EMT remain unclear.
Matrix mechanical signals have been shown to play a significant role in regulating EMT processes associated with disease development. For instance, experiments using polyacrylamide hydrogels to alter substrate rigidity have demonstrated that substrate stiffness affects EMT. The key question is how cells convert mechanical signals into biological signals. This conversion is primarily mediated by the cytoskeleton, with the focal adhesion complex acting as the bridge between the cytoskeleton and the substrate. Focal adhesion kinase (FAK) is a crucial player at the intersection of multiple mechanical signal transduction pathways, activating various downstream signaling cascades. FAK-related pathways are also implicated in the EMT process.
Previous research has confirmed that changes in cell morphology can be induced by substrate topography. However, the specific signals driving these changes remain unknown. While it is understood that matrix topography and rigidity synergistically induce EMT, and that the phosphatidylinositol 3-kinase/protein kinase B signaling pathway may be involved, it is unclear whether individual changes in topology alone can affect EMT.
To investigate the impact of topography on the biological behavior of human adenocarcinoma A549 cells, researchers used micropillar-arrayed substrates made from polydimethylsiloxane (PDMS). These substrates were fabricated with varying micropillar diameters and heights, creating different topographical environments. A549 cells cultured on these substrates exhibited distinct morphological changes depending on the micropillar dimensions. On planar substrates and substrates with micropillars of 10 mm diameter and 2 mm height, cells were round and oriented in multiple directions. In contrast, on substrates with micropillars of 10 mm diameter and 4 mm or 7 mm height, cells became fusiform and spread laterally.
The study also examined the effect of micropillar spacing on cytoskeletal organization and focal adhesions. Immunofluorescence staining revealed that vinculin expression increased with larger micropillar spacing, indicating enhanced focal adhesion formation. Additionally, cytoskeletal depolymerization became more pronounced as micropillar spacing increased, suggesting that cells are more prone to migration on substrates with larger micropillar spacing.
The impact of micropillar spacing on EMT markers was also evaluated. Without the addition of transforming growth factor beta 1 (TGF-β1), the expression of E-cadherin (E-CAD), an epithelial marker, decreased with increasing micropillar spacing, while the expression of vimentin (VIM), a mesenchymal marker, significantly increased. This process was further enhanced by the phosphorylation of SMAD3 and FAK, indicating that both TGF-β1 signaling and FAK activation are involved in the EMT process induced by micropillar topography.
To further elucidate the role of FAK in this process, A549 cells were infected with a lentivirus expressing FAK small hairpin RNA (shRNA) to stably silence the FAK gene. In FAK-silenced cells, the expression of E-CAD was relatively increased, while VIM expression decreased, suggesting that FAK is a critical mediator of the EMT process induced by micropillar topography.
In summary, the study demonstrated that micropillar-arrayed substrates significantly influence the biological behavior of A549 cells, inducing EMT-like behaviors as micropillar spacing changes. The expression of cell adhesion-associated proteins and cytoskeletal proteins also varied with micropillar spacing. Moreover, micropillar spacing affected the TGF-β1-induced EMT process, which is associated with the phosphorylation levels of SMAD3 and FAK. These findings highlight the role of substrate topography in regulating EMT and provide new insights into potential therapeutic targets for pulmonary fibrosis.
The study was conducted in the Key Laboratory of Biorheological Science and Technology, College of Bioengineering, Chongqing University, from 2016 to 2019. The research was supported by grants from the Natural Science Foundation of China, the Chongqing Graduate Scientific Research and Innovation Foundation of China, and the Fundamental Research Funds for the Central Universities.
The findings of this study contribute to the growing body of evidence that mechanical microenvironments play a crucial role in cellular behavior and disease progression. By understanding how substrate topography influences EMT, researchers can develop novel strategies for targeting cancer metastasis and fibrosis. The use of micropillar-arrayed substrates provides a valuable tool for studying the mechanical regulation of cellular processes and identifying new therapeutic targets.
doi.org/10.1097/CM9.0000000000001139
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