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 antimigration 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 by using drugs, biasing toward 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.

The epithelial-mesenchymal transition and the cytoskeleton in bioengineered systems

“…In order to resolve these cell state transitions, there is an increased need to map molecular and subcellular changes within a heterogeneous population over space and time [ 148 ]. Bioengineering approaches allow direct visualization of how cells apply forces to a compliant biomaterial, both on planar 2D substrates [ 86 ] as well as tri-dimensional 3D matrix [ 143 ], which we summarize in Table 1 . In 3D, epithelial cells are initially compact and round with uniformly distributed tractions around the periphery.…”

“…However, cell migration occurs randomly and will vary with the specific matrix conditions selected. Moreover, preparing 3D matrix requires specialized training and experimental optimization [ 12 , 14 , 24 , 28 , 47 , 50 , 98 , 105 , 115 – 117 , 125 – 131 , 133 – 135 , 138 – 140 , 142 , 143 , 145 – 147 ] Spheroid/organoid invasion in 3D matrix Generate multicellular spheroids or isolate tumor fragments and embed into a crosslinked 3D matrix; observe spheroid/tumor size and outward invasion Diverse approaches to prepare larger tissues with multiple cell types and investigate collective invasion; however, transferring spheroids and tissue into 3D matrix must be done carefully and can be tedious. 3D matrix properties are tunable, requiring additional optimization for larger spheroids that can settle to the well bottom [ 4 , 11 , 132 , 136 , 141 , 144 ] Animal models of cancer (mouse, zebrafish, chick CAM assay) Generate fluorescently labeled or luciferese-labeled tumors in an animal model; implant cancer cell lines into immunodeficient organisms, or use a genetically engineered cancer model, anesthetize and image over time using intravital microscopy, or bioluminescence imaging Can be used to monitor tumor growth, invasion, intravasation/extravasation, transmigration, and EMT in an in vivo tumor microenvironment.…”

“…b Multicellular clusters also exert spatially non-uniform patterns of protrusive and contractile tractions for epithelial, transitory (EMT), and mesenchymal states induced via Snail. Reproduced from [ 143 ]. c Multicellular clusters respond to dynamically stiffened substrates via dissemination and EMT activation via Twist, TGF- β , and YAP signaling.…”

“…Leggett et al [ 143 , 145 ] investigated the spatial patterns of 3D matrix deformations of multicellular clusters (MCF-10A) at varying stages of inducible Snail expression within a composite silk fibroin-collagen I hydrogel. Epithelial clusters (with no Snail induction) exhibited a spherical morphology and applied both protrusive and contractile tractions to the surrounding matrix (Fig.…”

Single-Cell Mechanophenotyping in Microfluidics to Evaluate Behavior of U87 Glioma Cells

“…Several different technologies have been developed to measure mechanical properties of cells including flow cytometry [ 15 , 16 , 17 ], atomic force microscopy (AFM) [ 18 , 19 ], magnetic twisting cytometry (MTC) [ 20 ], parallel-plate rheometry [ 11 , 21 ], optical stretching (OS) [ 22 ], optical tweezer [ 23 , 24 ], microfluidic ektacytometry [ 25 , 26 ], and micropipette aspiration [ 27 , 28 ]. Utilizing these modern tools for delineating mechanophenotypic properties of cells including stiffness, adhesiveness, viscosity, deformation (ratio of the area to volume), morphology, and migration trajectories of cells have been extensively investigated in cancer biology [ 29 , 30 , 31 ].…”