Microinterfaces in biopolymer-based bicontinuous hydrogels guide rapid 3D cell migration.

Cell migration is critical for tissue development and regeneration but requires extracellular environments that are conducive to motion. Cells may actively generate migratory routes in vivo by degrading or remodeling their environments or instead utilize existing extracellular matrix microstructures or microtracks as innate pathways for migration. While hydrogels in general are valuable tools for probing the extracellular regulators of 3-dimensional migration, few recapitulate these natural migration paths. Here, we develop a biopolymer-based bicontinuous hydrogel system that comprises a covalent hydrogel of enzymatically crosslinked gelatin and a physical hydrogel of guest and host moieties bonded to hyaluronic acid. Bicontinuous hydrogels form through controlled solution immiscibility, and their continuous subdomains and high micro-interfacial surface area enable rapid 3D migration, particularly when compared to homogeneous hydrogels. Migratory behavior is mesenchymal in nature and regulated by biochemical and biophysical signals from the hydrogel, which is shown across various cell types and physiologically relevant contexts (e.g., cell spheroids, ex vivo tissues, in vivo tissues). Our findings introduce a design that leverages important local interfaces to guide rapid cell migration.

3D Traction Force Microscopy in Biological Gels: From Single Cells to Multicellular Spheroids.

Cell traction force plays a critical role in directing cellular functions, such as proliferation, migration, and differentiation. Current understanding of cell traction force is largely derived from 2D measurements where cells are plated on 2D substrates. However, 2D measurements do not recapitulate a vital aspect of living systems, that is, cells actively remodel their surrounding extracellular matrix (ECM), and the remodeled ECM, in return, can have a profound impact on cell phenotype and traction force generation. This reciprocal adaptivity of living systems is encoded in the material properties of biological gels. In this review, we summarize recent progress in measuring cell traction force for cells embedded within 3D biological gels, with an emphasis on cell-ECM cross talk. We also provide perspectives on tools and techniques that could be adapted to measure cell traction force in complex biochemical and biophysical environments. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 26 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

CD44 and ß1-integrin are both engaged in cell traction force generation in hyaluronic acid-rich extracellular matrices.

Mechanical properties of the extracellular matrices (ECMs) critically regulate a number of important cell function including growth, differentiation and migration. Type I collagen and glycosaminoglycans (GAGs) are two primary components of ECMs that contribute to tissue mechanics with the collagen fiber network sustaining tension and GAGs withstanding compression. Collagen stiffness as well as its architecture are known to be important role players in cell-ECM mechanical interactions, however, much less is known about how GAGs within ECMs regulate cell force generation and invasion. Inspired by a recent theoretical work from the Shenoy lab that GAGs play important roles in cell - ECM interactions, we hereby present experimental studies on the role of hyaluronic acid (HA, an unsulfated GAG) in single tumor cell traction force generation within HA collagen cogels using a recently developed 3D cell traction force microscopy. Our work revealed that CD44, a cell surface adhesion receptor to HA, was engaged in cell traction force generation in conjunction with ß1-integrin. Furthermore, we found that HA significantly modified the architecture and mechanics of the collagen fiber network, decreased tumor cells' propensity to remodel the collagen network, decreased traction force generation and transmission distance, and attenuated tumor invasion in agreement with theoretical predictions. Our findings highlighted the significance of CD44 and HA engagement in cell-ECM mechanical interactions, providing new insights on the mechanical model of cellular force transmission.

Spatial and temporal dynamics of RhoA activities of single breast tumor cells in a 3D environment revealed by a machine learning-assisted FRET technique.

One of the hallmarks of cancer cells is their exceptional ability to migrate within the extracellular matrix (ECM) for gaining access to the circulatory system, a critical step of cancer metastasis. RhoA, a small GTPase, is known to be a key molecular switch that toggles between actomyosin contractility and lamellipodial protrusion during cell migration. Current understanding of RhoA activity in cell migration has been largely derived from studies of cells plated on a two-dimensional (2D) substrate using a FRET biosensor. There has been increasing evidence that cells behave differently in a more physiologically relevant three-dimensional (3D) environment. However, studies of RhoA activities in 3D have been hindered by low signal-to-noise ratio in fluorescence imaging. In this paper, we present a a machine learning-assisted FRET technique to follow the spatiotemporal dynamics of RhoA activities of single breast tumor cells (MDA-MB-231) migrating in a 3D as well as a 2D environment. We found that RhoA activity is more polarized along the long axis of the cell for single cells migrating on 2D fibronectin-coated glass versus those embedded in 3D collagen matrices. In particular, RhoA activities of cells in 2D exhibit a distinct front-to-back and back-to-front movement during migration in contrast to those in 3D. Finally, regardless of dimensionality, RhoA polarization is found to be moderately correlated with cell shape.