Physical confinement alters tumor cell adhesion and migration phenotypes

Abstract

Cell migration on planar surfaces is driven by cycles of actin protrusion, integrin-mediated adhesion, and myosin-mediated contraction; however, this mechanism may not accurately describe movement in 3-dimensional (3D) space. By subjecting cells to restrictive 3D environments, we demonstrate that physical confinement constitutes a biophysical stimulus that alters cell morphology and suppresses mesenchymal motility in human breast carcinoma (MDA-MB-231). Dorsoventral polarity, stress fibers, and focal adhesions are markedly attenuated by confinement. Inhibitors of myosin, Rho/ROCK, or β1-integrins do not impair migration through 3-μm-wide channels (confinement), even though these treatments repress motility in 50-μm-wide channels (unconfined migration) by ≥50%. Strikingly, confined migration persists even when F-actin is disrupted, but depends largely on microtubule (MT) dynamics. Interfering with MT polymerization/depolymerization causes confined cells to undergo frequent directional changes, thereby reducing the average net displacement by ≥80% relative to vehicle controls. Live-cell EB1-GFP imaging reveals that confinement redirects MT polymerization toward the leading edge, where MTs continuously impact during advancement of the cell front. These results demonstrate that physical confinement can induce cytoskeletal alterations that reduce the dependence of migrating cells on adhesion-contraction force coupling. This mechanism may explain why integrins can exhibit reduced or altered function during migration in 3D environments.

Microtubules in 3D cell motility

“…in 3-µm-wide channels) is much more profoundly affected by the inhibition of microtubule polymerization than by that of actin polymerization, actomyosin contractility or integrin adhesion (Balzer et al, 2012). Here, confinement redirected microtubule polymerization towards the leading edge, suggesting that growing microtubules provide force for advancement of the cell edge (Balzer et al, 2012). These data support the idea that, in certain 3D situations, microtubulecortex interactions are regulated in a way that promotes their mechanical role during cell protrusion.…”

“…It is noteworthy that the movement of cancer cells cultured in strong confinement (i.e. in 3-µm-wide channels) is much more profoundly affected by the inhibition of microtubule polymerization than by that of actin polymerization, actomyosin contractility or integrin adhesion (Balzer et al, 2012). Here, confinement redirected microtubule polymerization towards the leading edge, suggesting that growing microtubules provide force for advancement of the cell edge (Balzer et al, 2012).…”

Scattering of Cell Clusters in Confinement

“…Over the years, several studies have established that stiffer ECMs cause greater subcellular forces and protrusions, stronger cell-ECM adhesions, and more polarized morphology of individual cells, which in turn lead to the rupture of cell-cell junctions followed by EMT (1)(2)(3)(4). Since cell polarization is a key precursor to the stiffness-induced cell scattering in these studies, we asked whether the cells trapped inside confined environments could undergo a similar shape polarization, as we and others have shown earlier (5)(6)(7)(8), and commence dissociation from their native cluster. This is an important open question given that tissue environments often vary in their topography and dimensionality without significant stiffness variations.…”

HER2 missense mutations have distinct effects on oncogenic signaling and migration

“…Standard photolithography and replica molding were used to create the polydimethylsiloxane (PDMS) microfluidic device as previously described (23)(24)(25)(26). See SI Materials and Methods for details.…”

“…S6). We next examined the migratory capacity of cells with and without HER2 V777L mutation in collagen-I-coated microchannels using a microfluidic device constructed of polydimethylsiloxane (PDMS) (23)(24)(25)(26). This device allows for the characterization of single-cell migration, rather than the collective migration observed during wound closure.…”