Cell shape is linked to cell function. The significance of cell morphodynamics, namely the temporal fluctuation of cell shape, is much less understood. Here we study the morphodynamics of MDA-MB-231 cells in type I collagen extracellular matrix (ECM). We systematically vary ECM physical properties by tuning collagen concentrations, alignment, and gelation temperatures. We find that morphodynamics of 3D migrating cells are externally controlled by ECM mechanics and internally modulated by Rho/ROCK-signaling. We employ machine learning to classify cell shape into four different morphological phenotypes, each corresponding to a distinct migration mode. As a result, we map cell morphodynamics at mesoscale into the temporal evolution of morphological phenotypes. We characterize the mesoscale dynamics including occurrence probability, dwell time and transition matrix at varying ECM conditions, which demonstrate the complex phenotype landscape and optimal pathways for phenotype transitions. In light of the mesoscale dynamics, we show that 3D cancer cell motility is a hidden Markov process whereby the step size distributions of cell migration are coupled with simultaneous cell morphodynamics. Morphological phenotype transitions also facilitate cancer cells to navigate non-uniform ECM such as traversing the interface between matrices of two distinct microstructures. In conclusion, we demonstrate that 3D migrating cancer cells exhibit rich morphodynamics that is controlled by ECM mechanics, Rho/ROCK-signaling, and regulate cell motility. Our results pave the way to the functional understanding and mechanical programming of cell morphodynamics as a route to predict and control 3D cell motility.
Dysregulation of extracellular matrix (ECM) synthesis, organization, and mechanics are hallmark features of diseases like fibrosis and cancer. However, most in vitro models fail to recapitulate the three‐dimensional (3D) multi‐scale hierarchical architecture of collagen‐rich tissues and as a result, are unable to mirror native or disease phenotypes. Herein, using primary human fibroblasts seeded into custom fabricated 3D non‐adhesive agarose molds, a novel strategy is proposed to direct the morphogenesis of engineered 3D ring‐shaped tissue constructs with tensile and histological properties that recapitulate key features of fibrous connective tissue. To characterize the shift from monodispersed cells to a highly‐aligned, collagen‐rich matrix, a multi‐modal approach integrating histology, multiphoton second‐harmonic generation, and electron microscopy is employed. Structural changes in collagen synthesis and alignment are then mapped to functional differences in tissue mechanics and total collagen content. Due to the absence of an exogenously added scaffolding material, this model enables the direct quantification of cell‐derived changes in 3D matrix synthesis, alignment, and mechanics in response to the addition or removal of relevant biomolecular perturbations. To illustrate this, the effects of nutrient composition, fetal bovine serum, rho‐kinase inhibitor, and pro‐ and anti‐fibrotic compounds on ECM synthesis, 3D collagen architecture, and mechanophenotype are quantified.more » « less
- NSF-PAR ID:
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Science
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
Collagen fibers in the 3D tumor microenvironment (TME) exhibit complex alignment landscapes that are critical in directing cell migration through a process called contact guidance. Previous in vitro work studying this phenomenon has focused on quantifying cell responses in uniformly aligned environments. However, the TME also features short‐range gradients in fiber alignment that result from cell‐induced traction forces. Although the influence of graded biophysical taxis cues is well established, cell responses to physiological alignment gradients remain largely unexplored. In this work, fiber alignment gradients in biopsy samples are characterized and recreated using a new microfluidic biofabrication technique to achieve tunable sub‐millimeter to millimeter scale gradients. This study represents the first successful engineering of continuous alignment gradients in soft, natural biomaterials. Migration experiments on graded alignment show that human umbilical vein endothelial cells (HUVECs) exhibit increased directionality, persistence, and speed compared to uniform and unaligned fiber architectures. Similarly, patterned MDA‐MB‐231 breast cancer cell aggregates exhibit biased migration toward increasing fiber alignment, suggesting a role for alignment gradients as a taxis cue. This user‐friendly approach, requiring no specialized equipment, is anticipated to offer new insights into the biophysical cues that cells interpret as they traverse the extracellular matrix (ECM), with broad applicability in healthy and diseased tissue environments.
Abstract The collagen-rich tumor microenvironment plays a critical role in directing the migration behavior of cancer cells. 3D collagen architectures with small pores have been shown to confine cells and induce aggressive collective migration, irrespective of matrix stiffness and density. However, it remains unclear how cells sense collagen architecture and transduce this information to initiate collective migration. Here, we tune collagen architecture and analyze its effect on four core cell-ECM interactions: cytoskeletal polymerization, adhesion, contractility, and matrix degradation. From this comprehensive analysis, we deduce that matrix architecture initially modulates cancer cell adhesion strength, and that this results from architecture-induced changes to matrix degradability. That is, architectures with smaller pores are less degradable, and degradability is required for cancer cell adhesion to 3D fibrilar collagen. The biochemical consequences of this 3D low-attachment state are similar to those induced by suspension culture, including metabolic and oxidative stress. One distinction from suspension culture is the induction of collagen catabolism that occurs in 3D low-attachment conditions. Cells also upregulate Snail1 and Notch signaling in response to 3D low-attachment, which suggests a mechanism for the emergence of collective behaviors.more » « less
Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non‐mammalian and non‐avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (
), turkeys ( Lithobates catesbeianus ), alligators ( Meleagris gallopavo ), cane toads ( Alligator mississippiensis ), laboratory mice ( Rhinella marina ), and carp ( Mus musculus ). All muscles studied contained a collagen‐reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross‐species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research. Cyprinus carpio
Many cell responses that underlie the development, maturation, and function of tissues are guided by the architecture and mechanical loading of the extracellular matrix (ECM). Because mechanical stimulation must be transmitted through the ECM architecture, the synergy between these two factors is important. However, recapitulating the synergy of these physical microenvironmental cues in vitro remains challenging. To address this, a 3D magnetically actuated collagen hydrogel platform is developed that enables combined control of ECM architecture and mechanical stimulation. With this platform, it is demonstrated how these factors synergistically promote cell alignment of C2C12 myoblasts and enhance myogenesis. This promotion is driven in part by the dynamics of Yes‐associated protein and structure of cellular microtubule networks. This facile platform holds great promises for regulating cell behavior and fate, generating a broad range of engineered physiologically representative microtissues in vitro, and quantifying the mechanobiology underlying their functions.