Mechanophenotyping of 3D Multicellular Clusters using Displacement Arrays of Rendered Tractions

Abstract

Epithelial tissues mechanically deform the surrounding extracellular matrix during embryonic development, wound repair, and tumor invasion. Ex vivo measurements of such multicellular tractions within three-dimensional (3D) biomaterials could elucidate collective dissemination during disease progression, and enable preclinical testing of targeted anti-migration therapies. However, past 3D traction measurements have been low throughput due to the challenges of imaging and analyzing information-rich 3D material deformations. Here, we demonstrate a method to profile multicellular clusters in a 96-well plate format based on spatially heterogeneous contractile, protrusive, and circumferential tractions. As a case study, we profile multicellular clusters across varying states of the epithelial-mesenchymal transition, revealing a successive loss of protrusive and circumferential tractions, as well as the formation of localized contractile tractions with elongated cluster morphologies. These cluster phenotypes were biochemically perturbed using drugs, biasing towards traction signatures of different epithelial or mesenchymal states. This higher-throughput analysis is promising to systematically interrogate and perturb aberrant mechanobiology, which could be utilized with human patient samples to guide personalized therapies.

Epifluorescence-based three-dimensional traction force microscopy

“…To investigate cellular and inter-cellular force generation, traction force microscopy (TFM) has become a well-established technique in mechanobiology that quantifies tractions produced during cell–cell or cell–matrix interactions, i.e., cell-imposed forces acting on interfaces between a cell or cells and the microenvironment 5 – 7 . TFM has been cast in both two-dimensional (2D) and three-dimensional (3D) variants 8 – 11 , from single cells 8 – 10 , 12 to multicellular clusters and sheets 13 – 16 . While the original development of TFM started with 2D images acquired from phase contrast and epifluorescence microscopy and accounted for only small material deformations, most recent TFM approaches have the capability to fully reconstruct 3D motion and traction fields in a variety of linear, non-linear, and viscoelastic material systems 6 , 11 , 17 – 19 using confocal, multiphoton, or superresolution techniques 17 , 20 – 22 .…”

Neural cell injury pathology due to high-rate mechanical loading

“…The advantage here is that both strain rate and strain are commonly-employed quantification metrics of mechanical deformations used in cell mechanics, biophysics [6, 30–33] and computational modeling of brain injury [22, 34–36]. Conveniently, the logarithmic, or Hencky, strain is simply defined as the natural log of the material stretch, i.e., E θθ = E ϕϕ = ln( λ θ ), and E rr = −2 ln( λ θ ), where both strain and stretch are functions of position r 0 and time t .…”

“…The advantage here is that both strain rate and strain are commonly-employed quantification metrics of mechanical deformations used in cell mechanics, biophysics [6,[29][30][31][32] and computational modeling of brain injury [21,[33][34][35]. Conveniently, the logarithmic, or Hencky, strain is simply defined as the natural log of the material stretch, i.e., E θθ = E φφ = ln(λ θ ), and E rr = −2 ln(λ θ ), where both strain and stretch are functions of position r 0 and time t. As neural cells are commonly considered to be susceptible to tensile deformation [33,[36][37][38][39], we concern ourselves primarily with the tensile hoop strain components E θθ = E φφ , just as we did in the case for the hoop stretch, λ θ .…”

Light-sheet photonic force optical coherence elastography for high-throughput quantitative 3D micromechanical imaging

“…LS-pfOCE has the potential to enable numerous novel research studies in the field of mechanobiology. Recent developments in traction force microscopy (TFM) have enabled the study of time-varying cell forces and cell-mediated ECM deformations in 3D, including both isolated 8,44,51,52 and collective cellular behaviours (such as stromal-cell-mediated dissemination of cancer cells from co-cultured tumour spheroids 6,53,54 and epithelial-mesenchymal transitions in multicellular epithelial clusters 55 ). LS-pfOCE has the potential to significantly elevate such studies by providing crucial (albeit currently missing) information on the 4D spatiotemporal dynamics of the ECM micromechanical properties (our correlative analysis of the changes in ECM viscoelasticity and matrix deformation in Fig.…”