Measuring changes in epithelial tissue mechanics following the Epithelial-to-Mesenchymal Transition (EMT) with Cell Monolayer Deformation Microscopy (CMDM)
Amy Sutton
The mechanics of multicellular clusters are essential for processes such as collective motion, supracellular mechano-sensing, and diverse changes in physiology and pathology [1-3]. One example of multicellular pathology is the detachment of metastatic cells from their original network, whereupon they become migratory. The epithelial-mesenchymal transition (EMT) is one well known process whereby malignant cells can fragment from each other and become invasive [3-5]. However, the changes in the collective intercellular mechanics of cells as they progress through EMT are unknown.
Figure 1: Schematic of CMDM cell tissue mechanics measurement procedure.
We have developed a new methodology, Cell Monolayer Deformation Microscopy (CMDM), to locally strain cell monolayers and measure the monolayer’s planar rheology [6, 7]. In brief, we use image cross-correlation algorithms to determine the differences in poly-N-isopropylacrylamide (PNIPAm) hydrogel substrate deformation with and without an attached cell monolayer. We then quantify the resistance of the cell monolayers to lateral stretching, and calculate deformability, yielding, and time-dependent viscoelastic creep-/relaxation-associated deformation [7]. We have applied this technique to examine changes in the mechanical behavior of NMuMG cell monolayers in their epithelial phenotype, and in their Transforming Growth Factor-ß (TGFß) induced mesenchymal phenotype, to capture the differences in collective cell mechanics following EMT in this model breast epithelial cell line.
Figure 2: CMDM data output. (A) Fluorescent-labeling of the cell membranes confirms that substrate strain is transmitted to attached NMuMG cell monolayers (epithelial), which may be repeatedly subjected to strain without releasing the substrate or fragmenting. Two strain pulses with a 5 min interval are shown, playing at 3x the real speed. (B) The raw data used to extract information on the mechanical properties of the cell monolayers are image sequences of the micron-sized marker beads entrapped at the upper surface of the PNIPAm hydrogel substrate. (Top) Transmitted light image of the micron-sized, optically-absorbant beads in the relaxed PNIPAm hydrogel. (Bottom) The high bead density and image contrast ensures that micron-scale bead displacements are detectable (red arrow). (C) Laser-induced displacement field of the marker beads. Scale bars are 25 μm.
In currently ongoing work, we are characterizing the monolayer mechanics of prostate cancer cell lines of varying EMT phenotypes and metastatic potentials, and exploring the dependence on androgen receptor signaling. This CMDM methodology is further applicable to other 2D cell monolayer systems, and will help to resolve the complex mechanical functioning of multicellular structures. We expect the results of our work will provide key insight for healthcare by quantitatively characterizing epithelial tissue cohesion, providing a mechanical fingerprint of collective changes during EMT, and offering a new metric for identifying specific stages of EMT in cancer.
Figure 3: Quantitative monolayer stiffness measurements with CMDM. Comparisons of the ensemble of displacement values at all x,y coordinates between the cell monolayer and reference substrate-only strain pulses measure how much a monolayer sample resists deformation, and how stiff it is compared to other monolayer samples.
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Reveals Dose-Dependent Effects of Transforming Growth Factor- on the Epithelial-to-Mesenchymal
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in the epithelial to mesenchymal transition” PNAS in revision, 2021.
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