During morphogenesis and cancer metastasis, grouped cells migrate through tissues of dissimilar stiffness. Although the influence of matrix stiffness on cellular mechanosensitivity and motility are well-recognized, it remains unknown whether these matrix-dependent cellular features persist after cells move to a new microenvironment. Here, we interrogate whether priming of epithelial cells by a given matrix stiffness influences their future collective migration on a different matrix – a property we refer to as the ‘mechanical memory’ of migratory cells. To prime cells on a defined matrix and track their collective migration onto an adjoining secondary matrix of dissimilar stiffness, we develop a modular polyacrylamide substrate through step-by-step polymerization of different PA compositions. We report that epithelial cells primed on a stiff matrix migrate faster, display higher actomyosin expression, form larger focal adhesions, and retain nuclear YAP even after arriving onto a soft secondary matrix, as compared to their control behavior on a homogeneously soft matrix. Priming on a soft ECM causes a reverse effect. The depletion of YAP dramatically reduces this memory-dependent migration. Our results present a previously unidentified regulation of mechanosensitive collective cell migration by past matrix stiffness, in which mechanical memory depends on YAP activity.

Epithelial cells disengage from their clusters and become motile by undergoing epithelial-to-mesenchymal transition (EMT), an essential process for both embryonic development and tumor metastasis. Growing evidence suggests that high extracellular matrix (ECM) stiffness induces EMT. In reality, epithelial clusters reside in a heterogeneous microenvironment whose mechanical properties vary not only in terms of stiffness, but also topography, dimensionality, and confinement. Yet, very little is known about how various geometrical parameters of the ECM might influence EMT. Here, we adapt a hydrogel-microchannels based matrix platform to culture mammary epithelial cell clusters in ECMs of tunable stiffness and confinement. We report a previously unidentified role of ECM confinement in EMT induction. Surprisingly, confinement induces EMT even in the cell clusters surrounded by a soft matrix, which otherwise protects against EMT in unconfined environments. Further, we demonstrate that stiffness-induced and confinement-induced EMT work through cell-matrix adhesions and cytoskeletal polarization, respectively. These findings highlight that both the structure and the stiffness of the ECM can independently regulate EMT, which brings a fresh perspective to the existing paradigm of matrix stiffness-dependent dissemination and invasion of tumor cells.

Mammalian cells are exposed to complex microenvironments of varying micro-and nanoscale structural features. These multiscale extracellular cues dictate important aspects of cell behavior, including migration, proliferation and differentiation. In this study, we fabricated anodized aluminum oxide (AAO) membranes of either 80 or 40 nm pore diameters. We utilized these membranes as extracellular matrix scaffolds to culture NIH-3T3 fibroblast cells and investigated how the surface nanotopography might regulate their motility. We observed faster and more persistent fibroblast migration on AAO membranes with larger pores. Through various cell−matrix interaction markers, we found that the surfaces with higher nanoporosity enhance motility through larger focal adhesions, aligned actin fibers, and polarized cell morphology. Our findings reveal the importance of nanoscale topographical cues present in the matrix environment in regulating submicrometer-scale subcellular mechanisms of stress fiber organization and adhesion formation, micrometer-scale cell−matrix interactions, and cell motility over hundreds of micrometers.

Cell migration is a major process that drives metastatic progression of cancers, the major cause of cancer death. Existing chemotherapeutic drugs have limited efficacy to prevent and/or treat metastasis, emphasizing the need for new treatments. We focus on triple negative breast cancer (TNBC), the subtype of breast cancer with worst prognosis and no standard chemotherapy protocols. Here we demonstrate that a group of natural compounds, known as phytochemicals, effectively block migration of metastatic TNBC cells. Using a novel cell micropatterning technology, we generate consistent migration niches in standard 96-well plates where each well contains a cell-excluded gap within a uniform monolayer of cells. Over time, cells migrate into and occupy the gap. Treating TNBC cells with non-toxic concentrations of phytochemicals significantly blocks motility of cells. Using a molecular analysis approach, we show that anti-migratory property of phytochemicals is partly due to their inhibitory effects on phosphorylation of ERK1/2. This study provides a framework for future studies to understand molecular targets of phytochemicals and evaluate their effectiveness in inhibiting metastasis in animal models of cancer.