Cancer Protrusions on a Tightrope: Nanofiber Curvature Contrast Quantitates Single Protrusion Dynamics

Abstract

Cell migration is studied with the traditional focus on protrusion-driven cell body displacement, while less is known on morphodynamics of individual protrusions themselves, especially in fibrous environments mimicking extracellular matrix. Here, using suspended fibers, we report integrative and multiscale abilities to study protrusive behavior independent of cell body migration. By manipulating the diameter of fibers in orthogonal directions, we constrain cell migration along large diameter (2 μm) base fibers, while solely allowing cells to sense, initiate, and mature protrusions on orthogonally deposited high-curvature/low diameter (∼100, 200, and 600 nm) protrusive fibers and low-curvature (∼300 and 600 nm width) protrusive flat ribbons. In doing so, we report a set of morphodynamic metrics that precisely quantitate protrusion dynamics. Protrusion growth and maturation occur by rapid broadening at the base to achieve long lengths, a behavior dramatically influenced by curvature. While flat ribbons universally induce the formation of broad and long protrusions, we quantitatively protrutype protrusive behavior of two highly invasive cancer cell lines and find breast adenocarcinoma (MDA-MB-231) to exhibit sensitivity to fiber curvature higher than that of brain glioblastoma DBTRG-05MG. Furthermore, while actin and microtubules localize within protrusions of all sizes, we quantify protrusion size-driven localization of vimentin and, contrary to current understanding, report that vimentin is not required to form protrusions. Using multiple protrusive fibers, we quantify high coordination between hierarchical branches of individual protrusions and describe how the spatial configuration of multiple protrusions regulates cell migratory state. Finally, we describe protrusion-driven shedding and collection of cytoplasmic debris.