| dc.description.abstract | Mechanosensors play a vital role in how cells respond to external forces, impacting cell-substrate adhesions through integrin clustering and focal adhesion formation associated with actomyosin contractility. Adhesions between cells and the substrate occur through integrin clustering and focal adhesion formation that orchestrate essential cellular processes such as cell spreading, contractility generation, migration, and cell cycle progression. Understanding how cells respond to temporal and directional cues from mechanical stimuli, such as fluid shear stress, is crucial in unraveling the diverse mechanisms of cell-substrate interactions in physiological and pathological conditions. Methods to characterize strength and changes in adhesions due to fluid shear stress are potentially useful to delineate the intricate links between mechanosensing and vital cellular functions and develop mechanodiagnostic methods to study cell adaption under mechanical stimuli. Quantifying cell-substrate adhesion strengths is important in conditions like cancer and fibrosis, where adhesion dysregulation is frequently observed and contributes to disease progression.
In this study, we used a custom fluid shear device to quantify differences in cell adhesion strengths of various cells, including breast epithelial cells (MCF-7 and MDA-MB-231), lung epithelial cells (A549 and HPL1D), HeLa, and NIH3T3 fibroblast cells. We show that invasive cells, such as A549 and MDA-MB-231, exhibit significantly lower critical adhesion strengths and higher traction stresses compared to their non-invasive counterparts (HPL1D, MCF7). These data show that mechanical properties could be potentially used as an indicator of cell invasiveness. We also show that differences in vinculin distributions and actin cytoskeleton organization between invasive and non-invasive cells further underscore the relationship between cellular mechanics and invasiveness.
We next used inhibitors to selectively disrupt focal adhesion dynamics, actin dynamics, microtubules, contractility, and TGFβ activation in highly invasive (MDA-MB-231) and less invasive (MCF7) breast cancer cells. These studies revealed differences in cell areas, de-adhesion strengths (τ50), and cell tractions as compared to untreated controls. Highly metastatic MDA-MB-231 cells showed drastic differences in cells treated with inhibitors as compared to less invasive MCF7 cells. Treatment with FAK caused a significantly higher increase in the cell area, tractions, and critical adhesion strength as compared to all other groups. Latrunculin-A treatment, which prevents actin polymerization, resulted in the lowest decrease in the cell area, tractions, and critical adhesion strength. How do cancer cells, with varying invasive potentials, respond to different inhibitors in terms of mechanodiagnostic parameters? The importance of this question can hardly be overstated because of its relevance and potential to yield critical insights into using combinational therapy for cancer management and the use of biophysical markers as compared to biochemical markers for diagnostics. Such investigations emphasize the importance of mechanobiology in cancer therapy and the potential of mechanodiagnostic parameters in personalizing treatment strategies.
We next used NIH3T3 cells to understand cell remodeling responses under shear. Our results show that fibroblasts actively remodel under constant shear stress by activating signaling molecules. Cells have increased cell area, aspect ratio, align in the flow direction, and have higher traction force when subjected to constant shear stress. α-SMA expression and genes involved in focal adhesion, ECM, and actin remodeling increased until 6 hours of shear exposure and saturated by 12 hours of shear stress exposure. Interestingly, these cells also undergo myofibroblast activation, which leads to alignment along the flow direction. Cells also responded differentially to ligand type and density, specifically in terms of adhesion strengths, area, and aspect ratio to fibronectin and collagen-1. Our results show that fibroblasts exhibit stronger adhesion to collagen-1 compared to fibronectin; interestingly, the adhesion strength, cell area, and aspect ratio increased with ligand density and plateaued at ligand density beyond 40μg/ml. The ability of the cells to dynamically remodel in response to shear and undergo myofibroblast activation just through biophysical activation, without any biochemical activation, is important in fibrosis and its management. These findings provide crucial insights into cellular remodeling under shear stress, highlighting potential therapeutic avenues for conditions like fibrosis.
Lastly, we fabricated an endothelium-on-chip device to replicate controlled disturbed shear flow patterns, which are generally reported on curved arterial regions and at bifurcations and characterized using a shear rosette that represents the spatial-temporal map of shear stress variation at each point in the geometry. We replicated disturbed shear flows using microfluidics in an endothelium-on-chip device. We cultured human aortic endothelial cells in straight channels or the custom device and subjected them to either no flow, unidirectional, oscillatory, or bidirectional oscillatory flows. Under laminar flows, cells displayed well-defined stress fibers and VE-cadherin expression. Unique cellular characteristics, including nuclear size and changes in lamin A/C and heterochromatin organization, were observed in response to different flow patterns. Elevated expressions of actin, VE-cadherin, inflammatory NF-κB, and lamin were noted in the device compared to other groups. This device has the potential to generate controlled disturbed flows, making it valuable in personalized medicine and reducing the reliance on animal trials for studying mechanobiology. The endothelium-on-chip device opens new avenues for studying cellular responses under controlled conditions, paving the way for more accurate and personalized medical approaches. Collectively, these results emphasize the importance of mechanobiology in fundamental research and clinical applications. | en_US |
