Multivariate relationships among nucleus and Golgi properties during fibrillar migration are robust to and unchanged by epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) and maturation of a fibrillar tumor microenvironment play important roles in breast cancer progression. A better understanding of how these events promote cancer cell migration and invasion could help identify new strategies to curb metastasis. The nucleus and Golgi affect migration in a microenvironment-dependent manner. Nucleus size and mechanics influence the ability of a cell to squeeze through confined tumor microenvironments. Golgi positioning determines front-rear polarity necessary for migration. While the roles of individual attributes of nucleus and Golgi in migration are being clarified, how their manifold features are inter-related and work together remains to be understood at a systems level. Here, to elucidate relationships among nucleus and Golgi properties, we quantified twelve morphological and positional properties of these organelles during fibrillar migration of human mammary epithelial cells. Principal component analysis (PCA) reduced the twelve-dimensional space of measured properties to three principal components that capture 75% of the variations in organelle features. Unexpectedly, nucleus and Golgi properties that co-varied in a PCA model built with data from untreated cells were largely similar to co-variations identified using data from TGFß-treated cells. Thus, while TGFß-mediated EMT significantly alters gene expression and motile phenotype, it did not significantly affect the relationships among nucleus size, aspect ratio and orientation with migration direction and among Golgi size and nucleus-Golgi separation distance. Indeed, in a combined PCA model incorporating data from untreated and TGFß-treated cells, scores of individual cells occupy overlapping regions in principal component space, indicating that TGFß-mediated EMT does not promote a unique "Golgi-nucleus phenotype" during fibrillar migration. These results suggest that migration along spatially-confined fiber-like tracks employs a conserved nucleus-Golgi arrangement that is independent of EMT state.

Golgi Stabilization, Not Its Front-Rear Bias, Is Associated with EMT-Enhanced Fibrillar Migration.

Epithelial-to-mesenchymal transition (EMT) and maturation of collagen fibrils in the tumor microenvironment play a significant role in cancer cell invasion and metastasis. Confinement along fiber-like tracks enhances cell migration. To what extent and in what manner EMT further promotes migration in a microenvironment already conducive to migration is poorly understood. Here, we show that TGFß-mediated EMT significantly enhances migration on fiber-like micropatterned tracks of collagen, doubling migration speed and tripling persistence relative to untreated mammary epithelial cells. Thus, cell-intrinsic EMT and extrinsic fibrillar tracks have nonredundant effects on motility. To better understand EMT-enhanced fibrillar migration, we investigated the regulation of Golgi positioning, which is involved in front-rear polarization and persistent cell migration. Confinement along fiber-like tracks has been reported to favor posterior Golgi positioning, whereas anterior positioning is observed during 2-day wound healing. Although EMT also regulates cell polarity, little is known about its effect on Golgi positioning. Here, we show that EMT induces a 2:1 rearward bias in Golgi positioning; however, positional bias explains less than 2% of single-cell variability in migration speed and persistence. Meanwhile, EMT significantly stabilizes Golgi positioning. Cells that enhance migration in response to TGFß maintain Golgi position for 2- to 4-fold longer than nonresponsive counterparts irrespective of whether the Golgi is ahead or behind the nucleus. In fact, 28% of TGFß-responsive cells exhibit a fully committed Golgi phenotype with the organelle either in the anterior or posterior position for over 90% of the time. Furthermore, single-cell differences in Golgi stability capture up to 18% of variations in migration speed. These results suggest a hypothesis that the Golgi may be part of a core physical scaffold that affects how cell-generated forces are distributed during migration. A stable scaffold would be expected to more consistently and therefore more productively distribute forces over time, leading to efficient migration.

Label-Free Automated Cell Tracking: Analysis of the Role of E-cadherin Expression in Collective Electrotaxis.

Collective cell migration plays an important role in wound healing, organogenesis, and the progression of metastatic disease. Analysis of collective migration typically involves laborious and time-consuming manual tracking of individual cells within cell clusters over several dozen or hundreds of frames. Herein, we develop a label-free, automated algorithm to identify and track individual epithelial cells within a free-moving cluster. We use this algorithm to analyze the effects of partial E-cadherin knockdown on collective migration of MCF-10A breast epithelial cells directed by an electric field. Our data show that E-cadherin knockdown in free-moving cell clusters diminishes electrotactic potential, with empty vector MCF-10A cells showing 16% higher directedness than cells with E-cadherin knockdown. Decreased electrotaxis is also observed in isolated cells at intermediate electric fields, suggesting an adhesion-independent role of E-cadherin in regulating electrotaxis. In additional support of an adhesion-independent role of E-cadherin, isolated cells with reduced E-cadherin expression reoriented within an applied electric field 60% more quickly than control. These results have implications for the role of E-cadherin expression in electrotaxis and demonstrate proof-of-concept of an automated algorithm that is broadly applicable to the analysis of collective migration in a wide range of physiological and pathophysiological contexts.

Collective migration exhibits greater sensitivity but slower dynamics of alignment to applied electric fields.

During development and disease, cells migrate collectively in response to gradients in physical, chemical and electrical cues. Despite its physiological significance and potential therapeutic applications, electrotactic collective cell movement is relatively less well understood. Here, we analyze the combined effect of intercellular interactions and electric fields on the directional migration of non-transformed mammary epithelial cells, MCF-10A. Our data show that clustered cells exhibit greater sensitivity to applied electric fields but align more slowly than isolated cells. Clustered cells achieve half-maximal directedness with an electric field that is 50% weaker than that required by isolated cells; however, clustered cells take ∼2-4 fold longer to align. This trade-off in greater sensitivity and slower dynamics correlates with the slower speed and intrinsic directedness of collective movement even in the absence of an electric field. Whereas isolated cells exhibit a persistent random walk, the trajectories of clustered cells are more ballistic as evidenced by the superlinear dependence of their mean square displacement on time. Thus, intrinsically-directed, slower clustered cells take longer to redirect and align with an electric field. These findings help to define the operating space and the engineering trade-offs for using electric fields to affect cell movement in biomedical applications.

Cell chemotaxis on paper for diagnostics.

Microfluidic chemotaxis platforms have historically been utilized to probe phenomena such as neutrophil migration and are beginning to be developed for diagnostic applications; however, current microfluidic chemotaxis systems require specialized engineering equipment such as syringe pumps and long time frames (hours) to develop a chemokine gradient, and cell chemotaxis typically requires multiple additional hours. The paperfluidic device described in this work is a low-cost, sharp (2 mm wide), quasi-stable (at least 20 min) and rapidly generated (<1 s) chemokine gradient system capable of examining cell migration response over short time frames (20 min) that can be easily assembled. A proof-of-concept experiment on human pan-T cells showed significant (p ≪ 0.01) directed migration to the chemokine gradient over the control condition. This new technique for cell migration studies provides a foundational step in designing microfluidic chemotactic platforms for point-of-care diagnostics.