(502d) Confinement Regulates the Migration Direction Following Cell Exposure to a Pressure Differential

Cell motility is an essential and complex phenomenon that regulates several physiological and pathological events, including embryonic development and tissue regeneration and disease progression. It is generally believed that migrating cells can adapt to a wide variety of physical stimuli such as stiffness, viscoelasticity, pressure, which in turn control the mechanisms and efficiency of cell locomotion. However, little is known about how asymmetric physical signals influence cell motility. Among these cues, pressure differentials (ΔP) are of particular interest because they are commonly encountered by cells as they migrate toward target sites.

Typically, cells experience pressure gradients during extravasation or interstitial migration because of hydrostatic and osmotic pressure differences between microvessels (e.g., capillaries or post-capillary venules) and the surrounding tissue, resulting in a net outward filtration pressure of ~0.1 to 0.3 kPa. Pressure gradients are known to generate flow fields that exert drag forces on cells in the direction of flow. While some studies show that cell exposure to ΔP results in migration towards low-pressure regions, others report the exact opposite results. These conflicting data have been generated using discrete 3D gels that may evoke divergent migratory responses to pressure gradients due to differences in pore sizes/the degree of confinement. To evaluate the effects of ΔP on confined vs. unconfined migration, we generated hydrostatic pressure gradients across microchannels of prescribed dimensions. Our results demonstrate that in confining microchannels ((W)idth x (H)eight = 10 x 3 μm²) where cells occupy the entire channel cross-sectional area, application of ΔP=-0.16 kPa (i.e., high-pressure at the cell rear and low-pressure at the cell front) causes a rapid reversal of migration direction; ~50% of cells move away from low-pressure regions and migrate in the upstream direction. In contrast, unconfined (W x H = 20 x 10 μm²) or partially confined microchannels (W x H = 10 x 10 μm²) trigger predominantly downstream migration. These observations hold true for HT-1080 fibrosarcoma cells, MDA-MB-231 breast cancer cells and bone-marrow-derived mesenchymal stem cells and are independent of seeding density.

Actomyosin contractility controls migration direction following cell exposure to ΔP. Pharmacological inhibition of actomyosin contractility (Y-27632 and blebbistatin) or Myosin IIA knockdown abolish pressure-induced migration direction reversal and promote downstream migration in confinement. Similar results are observed using a calcium inhibitor that markedly suppresses Myosin II activity. Moreover, live-cell imaging of Myosin IIA-GFP-labelled cells reveals that before exposure to ΔP, Myosin IIA primarily localizes at the rear of confined cells. Interestingly, the application of ΔP promotes the reduction of Myosin IIA levels at the trailing edge and simultaneously induces the enrichment of Myosin IIA at the opposite pole (new trailing edge). We employed optogenetic tools to evaluate whether pressure-mediated cell reversal requires Myosin IIA repolarization. While light-induced activation of actomyosin contractility at the cell rear inhibits cell reversal and induces downstream motility, upregulation of contractility at the opposite pole promotes almost exclusively migration towards high pressure regions.

Collectively, our data reveal the key role of confinement in regulating cell responses to asymmetric pressure forces and demonstrate how Myosin II triggers upstream cell migration. This work enhances our understanding of how migrating cells sense, interpret, and respond to diverse microenvironmental signals. Our future studies will focus on identifying cell surface molecules that are responsible for sensing ΔP and controlling Myosin IIA-induced upstream motility.