Regulators of Metastasis Modulate the Migratory Response to Cell Contact under Spatial Confinement

Abstract

The breast tumor microenvironment (TMEN) is a unique niche where protein fibers help to promote invasion and metastasis. Cells migrating along these fibers are constantly interacting with each other. How cells respond to these interactions has important implications. Cancer cells that circumnavigate or slide around other cells on protein fibers take a less tortuous path out of the primary tumor; conversely, cells that turn back upon encountering other cells invade less efficiently. The contact response of migrating cancer cells in a fibrillar TMEN is poorly understood. Here, using high-aspect ratio micropatterns as a model fibrillar platform, we show that metastatic cells overcome spatial constraints to slide effectively on narrow fiber-like dimensions, whereas nontransformed MCF-10A mammary epithelial cells require much wider micropatterns to achieve moderate levels of sliding. Downregulating the cell-cell adhesion protein, E-cadherin, enables MCF-10A cells to slide on narrower micropatterns; meanwhile, introducing exogenous E-cadherin in metastatic MDA-MB-231 cells increases the micropattern dimension at which they slide. We propose the characteristic fibrillar dimension (CFD) at which effective sliding is achieved as a metric of sliding ability under spatial confinement. Using this metric, we show that metastasis-promoting genetic perturbations enhance cell sliding and reduce CFD. Activation of ErbB2 combined with downregulation of the tumor suppressor and cell polarity regulator, PARD3, reduced the CFD, in agreement with their cooperative role in inducing metastasis in vivo. The CFD was further reduced by a combination of ErbB2 activation and transforming growth factor β stimulation, which is known to enhance invasive behavior. These findings demonstrate that sliding is a quantitative property and a decrease in CFD is an effective metric to understand how multiple genetic hits interact to change cell behavior in fibrillar environments. This quantitative framework sheds insights into how genetic perturbations conspire with fibrillar maturation in the TMEN to drive the invasive behavior of cancer cells.

Positive Quantitative Relationship between EMT and Contact-Initiated Sliding on Fiber-like Tracks

“…Pairwise interactions between migrating cells with and without TGFb treatment were imaged and the extent to which interacting cells reversed direction versus sliding past each other was quantified as described in Milano et al (20).…”

“…We previously demonstrated that the CFD is an effective metric for quantifying the effects of molecular perturbations on the sliding ability of nontransformed and metastatic breast epithelial cells (20). Cells that slide poorly have a high CFD and require much wider fiberlike tracks to execute moderate levels of sliding; in contrast, cells that slide proficiently have a low CFD and achieve moderate levels of sliding at low fiberlike dimensions.…”

“…Extended treatment with a high dose of TGFb promotes EMT and enhances sliding behavior of MCF-10A cells (20). On the other hand, without inducing overt EMT, downregulation of PARD3 alongside induction of ErbB2 signaling enhanced sliding and induced metastasis in mouse models.…”

“…To begin to examine the quantitative relationship between degree of EMT and invasive cell behavior, we investigate here the effect of TGFb-induced EMT on a cell migration phenotype that is relevant to invasion in a fibrillar, crowded tumor microenvironment and that we recently demonstrated to be correlated with metastasis (20). Cancer cells invade along fibers in a highly dynamic microenvironment with a high concentration of cells (21)(22)(23).…”

“…We recently showed that metastatic MDA-MB-231 and BT-549 cells are highly effective in sliding around cellcell encounters on fiberlike adhesive tracks, even on tracks much narrower than the cell diameter (20). In contrast, nontransformed mammary epithelial MCF-10A cells largely reverse direction upon encountering another cell.…”