Image quantification technique reveals novel lung cancer cytoskeletal phenotype with partial EMT signature

Abstract

Epithelial-mesenchymal Transition (EMT) is a multi-step process that involves cytoskeletal rearrangement. Here, using novel image quantification tools, we have identified an intermediate EMT state with a specific cytoskeletal signature. We have been able to partition EMT into two steps: (1) initial formation of transverse arcs and dorsal stress fibers and (2) their subsequent conversion to ventral stress fibers with a concurrent alignment of fibers. Using the Orientational Order Parameter (OOP) as a figure of merit, we have been able to track EMT progression in live cells as well as characterize and quantify drug responses. Our technique has improved throughput and is non-destructive, making it a viable candidate for studying a broad range of biological processes. Further, owing to the increased stiffness (and hence invasiveness) of the intermediate phenotype compared to mesenchymal cells, our work can be instrumental in aiding the search for new treatment strategies that combat metastasis by specifically targeting the fiber alignment process.

Biophysical and biochemical attributes of hybrid epithelial/mesenchymal phenotypes

“…Image quantification can also reveal specific cytoskeletal attributes of hybrid E/M phenotypes. On treatment of NSCLC cells A549, H460 and H1299 with TGFβ, stress fibers increasingly aligned well among themselves, as reflected by a gradual change in the orientational order parameter [48]. Cells in partial or early EMT (14 h posttreatment) stage had fewer spots for focal adhesion kinases (FAKs) but stress fibers in late EMT (48 h post-treatment, i.e.…”

“…Atomic force microscopy measurements revealed that the Young's modulus of the 'nest-like' partial EMT cells was intermediate to that of untreated and late stage EMT. Although the time-period for which cells were treated with TGFβ may seem insufficient to induce a complete EMT, but E-cadherin was drastically reduced both at mRNA and protein levels [48]. However, how loss of E-cadherin and the cytoskeletal changes can impact each other, needs further analysis.…”