The epithelial-mesenchymal transition (EMT) is intrinsically linked to alterations of the intracellular cytoskeleton and the extracellular matrix. After EMT, cells acquire an elongated morphology with front/back polarity, which can be attributed to actin-driven protrusion formation as well as the gain of vimentin expression. Consequently, cells can deform and remodel the surrounding matrix in order to facilitate local invasion. In this review, we highlight recent bioengineering approaches to elucidate EMT and functional changes in the cytoskeleton. First, we review transitions between multicellular clusters and dispersed individuals on planar surfaces, which often exhibit coordinated behaviors driven by leader cells and EMT. Second, we consider the functional role of vimentin, which can be probed at subcellular length scales and within confined spaces. Third, we discuss the role of topographical patterning and EMT via a contact guidance like mechanism. Finally, we address how multicellular clusters disorganize and disseminate in 3D matrix. These new technologies enable controlled physical microenvironments and higher-resolution spatiotemporal measurements of EMT at the single cell level. In closing, we consider future directions for the field and outstanding questions regarding EMT and the cytoskeleton for human cancer progression.

Biophysical aspects of in vivo tissue microenvironments include microscale mechanical properties, fibrillar alignment, and architecture or topography of the extracellular matrix (ECM). These aspects act in concert with chemical signals from a myriad of diverse ECM proteins to provide cues that drive cellular responses. Here, we used a bottom-up approach to build fibrillar architecture into 3D amorphous hydrogels using magnetic-field driven assembly of paramagnetic colloidal particles functionalized with three types of human ECM proteins found in vivo. We investigated if cells cultured in matrices comprised of fibrils of the same size and arranged in similar geometries will show similar behavior for each of the ECM proteins tested. We were able to resolve spatial heterogeneities in microscale mechanical properties near aligned fibers that were not observed in bulk tissue mechanics. We then used this platform to examine factors contributing to cell alignment in response to topographical cues in 3D laminin-rich matrices. Multiple human cell lines extended protrusions preferentially in directions parallel or perpendicular to aligned fibers independently of the ECM coating. Focal adhesion proteins, as measured by paxillin localization, were mainly diffuse in the cytoplasm, with few puncta localized at the protrusions. Integrin β1 and fascin regulated protrusion extension but not protrusion alignment. Myosin II inhibition did not reduce observed protrusion length. Instead, cells with reduced myosin II activity generated protrusions in random orientations when cultured in hydrogels with aligned fibers. Similarly, myosin II dependence was observed in vivo, where cells no longer aligned along the abluminal surfaces of blood vessels upon treatment with blebbistatin. These data suggest that myosin II can regulate sensing of topography in 3D engineered matrices for both normal and transformed cells.

Biophysical aspects of in vivo tissue microenvironments include microscale mechanical properties, fibrillar alignment, architecture or topography of the extracellular matrix (ECM), and the repertoire of ECM ligands present, all of which provide cues to drive cellular response. Cell-ECM interactions are important regulators of both normal tissue homeostasis and malignancy.Thus, understanding both extracellular cues and the cellular responses they elicit is fundamental to developing therapeutic strategies. Various in vitro platforms for 3D cell culture and tissue engineering have been used to study cellular response to the microenvironment. However, recapitulating the diversity of tissue architectures present in vivo in a controlled manner in threedimensional tissue mimetics is challenging using naturally derived ECM hydrogels. Here, we use a bottom-up approach to build fibrillar architecture into 3D amorphous hydrogels using selfassembly of magnetic colloidal particles functionalized with human ECM proteins. Human ECM proteins associated with organ-specific pathological states were used. We determined that, while the bulk tissue mechanics of hydrogels containing either aligned fibers or randomly distributed colloidal particles were similar, aligned hydrogels exhibited spatial heterogeneities in microscale mechanical properties near aligned fibers. We then used this platform in combination with 2D substrates of defined elastic modulus to decouple the role of topography from microscale tissue mechanics for normal and tumor cells. We determined that topographical cues dominate cellular response for human and normal cells, which responded independently of microscale mechanics and ECM composition in 3D hydrogels. These data suggest that topography alone can drive fundamental cellular responses such as polarization and migration for both normal and transformed cells.

Tumor cells invade individually or in groups, mediated by mechanical interactions between cells and their surrounding matrix. These multicellular dynamics are reminiscent of leader-follower coordination and epithelial-mesenchymal transitions (EMT) in tissue development, which may occur via dysregulation of associated molecular or physical mechanisms. However, it remains challenging to elucidate such phenotypic heterogeneity and plasticity without precision measurements of single cell behavior. The convergence of technological developments in live cell imaging, biophysical measurements, and 3D biomaterials are highly promising to reveal how tumor cells cooperate in aberrant microenvironments. Here, we highlight new results in collective migration from the perspective of cancer biology and bioengineering. First, we review the biology of collective cell migration. Next, we consider physics-inspired analyses based on order parameters and phase transitions. Further, we examine the interplay of metabolism and heterogeneity in collective migration. We then review the extracellular matrix, and new modalities for mechanical characterization of 3D biomaterials. We also explore epithelial-mesenchymal plasticity and implications for tumor progression. Finally, we speculate on future directions for integrating mechanobiology and cancer cell biology to elucidate collective migration.

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