(339d) The Mechanobiology of Cancer Cell Motility Under Vertical and Lateral Confinement

Cell migration is a fundamental cellular phenomenon that plays a pivotal role during cancer metastasis. Our current understanding of cell migration relies mainly on studies using two-dimensional (2D) substrates or 3D gels. However, in vivo, cancer cells migrate not only within 3D extracellular matrices but also through longitudinal tissue microtracks, which impose varying degrees of confinement on cells. Evidence suggests that physical confinement alters intracellular signaling pathways, thereby affecting the mechanisms and phenotypes of cell migration. Recent work reveals that compression on the top and bottom of cells (vertical confinement), as opposed to compression on the sides (lateral confinement), induces significantly higher levels of nuclear rupture. This suggests a fundamentally different mechano-sensing mechanism which depends on the direction of confinement. Thus, we hypothesized that cells employ distinct migration mechanisms under vertical vs lateral confinement. To address this hypothesis, we developed PDMS- (stiffness of ~1MPa) and hydrogel- (10-50 kPa) based microchannel devices of prescribed cross-sectional areas: width(W)=3μm and height(H)=10μm for lateral confinement and W=10μm and H=3μm for vertical confinement. Using HT-1080 fibrosarcoma cells as a model, we demonstrate that vertically confined cells migrate more slowly than laterally confined cells. This observation holds true for a number of different normal-like (fibroblasts and smooth muscle cells) and cancer cells (HOS osteosarcoma cells). We also report that the vertically confined cancer cells employ a slower bleb-based mode of migration, whereas they switch to the faster protrusion-based mode of migration in lateral confinement. In addition, our work explores the molecular mechanisms responsible for the differential cell migration speed in vertical vs lateral confinement. We employ a multi-disciplinary approach involving state-of-the-art bioengineering (Micropipette Aspiration, Atomic Force Microscopy), imaging (confocal, FRET) and molecular biology techniques, to study the role of perinuclear actin (ARP2/3, FMN2), nuclear stiffness (LMNA, EMD), the nucleo-cytoskeleton linker (SUN1, SUN2) and small GTPases during vertically and laterally confined migration. Collectively, our work enhances our understanding of the complex process of confined cell migration and provides a novel perspective on how cells sense and respond to different types of confinement.