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.

Biomechanical changes in the tumor microenvironment influence tumor progression and metastases. Collagen content and fiber organization within the tumor stroma are major contributors to biomechanical changes (e., tumor stiffness) and correlated with tumor aggressiveness and outcome. What signals and in what cells control collagen organization within the tumors, and how, is not fully understood. We show in mouse breast tumors that the action of the collagen receptor DDR2 in CAFs controls tumor stiffness by reorganizing collagen fibers specifically at the tumor-stromal boundary. These changes were associated with lung metastases. The action of DDR2 in mouse and human CAFs, and tumors in vivo, was found to influence mechanotransduction by controlling full collagen-binding integrin activation via Rap1-mediated Talin1 and Kindlin2 recruitment. The action of DDR2 in tumor CAFs is thus critical for remodeling collagen fibers at the tumor-stromal boundary to generate a physically permissive tumor microenvironment for tumor cell invasion and metastases.

Grouped cells often leave large cell colonies in the form of narrow multicellular streams. However, it remains unknown how collective cell streaming exploits specific matrix properties, like stiffness and fiber length. It is also unclear how cellular forces, cell-cell adhesion and velocities are coordinated within streams. To independently tune stiffness and collagen fiber length, we developed new hydrogels and discovered invasion-like streaming of normal epithelial cells on soft substrates coated with long collagen fibers. Here, streams arise owing to a surge in cell velocities, forces, YAP activity and expression of mesenchymal marker proteins in regions of high-stress anisotropy. Coordinated velocities and symmetric distribution of tensile and compressive stresses support persistent stream growth. Stiff matrices diminish cell-cell adhesions, disrupt front-rear velocity coordination and do not promote sustained fiber-dependent streaming. Rac inhibition reduces cell elongation and cell-cell cooperation, resulting in a complete loss of streaming in all matrix conditions. Our results reveal a stiffness-modulated effect of collagen fiber length on collective cell streaming and unveil a biophysical mechanism of streaming governed by a delicate balance of enhanced forces, monolayer cohesion and cell-cell cooperation.

In fibrosis and cancer, degradation of basement membrane (BM) and cell invasion are considered as key outcomes of a cellular transformation called epithelial-mesenchymal transition (EMT). Here, we pose a converse question - can preexisting physical defects in the BM matrix cause EMT in normal epithelial cells? On a BM-mimicking matrix of collagen-IV-coated polyacrylamide (PA) gel, we have discovered a reverse phenomenon in which preexisting defects trigger EMT in normal epithelial cells. Through spatiotemporal measurements and simulations in silico, we demonstrate that the EMT precedes cellular mechanoactivation on defective matrices, but they occur concurrently on stiff matrices. The defect-dependent EMT caused cell invasion though a stroma-mimicking collagen-I layer, which could be disabled through MMP9 inhibition. Our findings reveal that the known BM degradation caused by cellular EMT and invasion is not a one-way process. Instead, normal epithelial cells can exploit physical defects in the BM matrix to undergo disease-like cellular transformations.

Microcontact printing of extracellular matrix (ECM) proteins in defined regions of a substrate allows spatial control over cell attachment and enables the study of cellular response to irregular ECM geometries. Over the past decade, numerous micropatterning techniques have emerged that conjugate ECM proteins on hydrogel substrates of tunable stiffness, which have revealed a range of cellular responses to varying matrix stiffness and geometry. However, micropatterning of ECM proteins on polyacrylamide (PA) hydrogel remains inconsistent due to its unreliable conjugation with the commonly used protein cross-linkers, particularly at low stiffness. To address these problems, we developed a micropatterning technique in which the PA gel is functionalized by incorporating oxidized N-hydroxyethylacrylamide, which allows direct protein binding through reactive aldehyde groups without any exogenous cross-linkers. As a result, a uniform and consistent protein transfer onto the hydrogel substrates of defined geometries is achieved, even for soft PA gels. We formed square, rectangular, and triangular patterns of two constant areas on soft and stiff PA gels that provide large and small adhesive areas for the MCF10A human mammary epithelial cell pairs. We measured intercellular E-cadherin (E-cad) expression and found that cell–cell junctions could be deteriorated independently by either the stiff ECM of any shape or the elongated cell morphology, accompanied by increased cell-generated tractions, on rectangular soft ECM patterns. Inhibition of nonmuscle myosin II reduced the E-cad junctional localization in cell pairs. When the cell spreading was restricted by reducing the adhesive area of the patterns, we observed an overall rise in E-cad expression at cell–cell junctions. Our findings present an improved micropatterning technique which reveals a geometric regulation of cell–cell junctions in epithelial cell pairs.