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.

Force-exerting perpendicular lateral protrusions in fibroblastic cell contraction

“…To partially remedy this, we recently used orthogonal arrangement of fibers of mismatched fiber diameters to constrain cell migration along large diameter fibers while studying lateral protrusions of various shapes and sizes on smaller diameter fibers 30 , 31 . We observed the protrusions formed in an integrin-dependent manner on small diameter fibers, and notably suspended flat ribbons of equivalent widths did not capture protrusive sensitivity to curvature 30 . Here, using aligned and suspended fiber nanonets that also act as force sensors 32 – 34 , we report the identification of force exerting side protrusions termed ‘3D perpendicular lateral protrusions (3D-PLPs)’ that do not require pre-existing fibers to extend upon.…”

“…Overall, the utility of lateral protrusions remains poorly understood, as classic 2D culturing methods limit the formation of lateral protrusions, while 3D gels lack the homogeneity and topographic patterning needed to study the role of fiber geometry on protrusion frequency, morphology, and dynamics. To partially remedy this, we recently used orthogonal arrangement of fibers of mismatched fiber diameters to constrain cell migration along large diameter fibers while studying lateral protrusions of various shapes and sizes on smaller diameter fibers 30,31 . We observed the protrusions formed in an integrin-dependent manner on small diameter fibers, and notably suspended flat ribbons of equivalent widths did not capture protrusive sensitivity to curvature 30 .…”

Crosshatch nanofiber networks of tunable interfiber spacing induce plasticity in cell migration and cytoskeletal response

“…Suspended fibers not only provide curvature as a cue to cells but also ensure that cells interact only with fibers. Supporting this, we have previously shown that suspended flat 2D ribbons do not capture the protrusive sensitivity to suspended round fibers (49).…”

“…Although many in vitro strategies (including by us) have demonstrated anisotropic migration using aligned geometries, the contributions of other configurations, including cross-linked networks of varied interfiber spacing, remain unclear. Here, using our previously reported nonelectrospinning spinneret-based tunable engineered parameters (STEP) technique (47)(48)(49)(50), we use suspended nanofiber crosshatch networks of tunable interfiber spacing to interrogate the plasticity of single-cell migratory behavior and cytoskeleton arrangement in the Hras1 murine cell line. We chose Hras1 because it is derived from aggressive follicular thyroid cancer, a tumor with highly invasive capacity and propensity to metastasize to distant sites, primarily the lungs (51).…”

Force-exerting lateral protrusions in fibroblastic cell contraction

“…[27][28][29] Overall, the utility of lateral protrusions remains partially understood as current cell culturing methods of classic 2D culturing limit the formation of lateral protrusions, while 3D gels lack the homogeneity and topographic patterning that are needed to study them in a reproducible manner. To this end, using fiber diameters of curvature contrast, we have recently shown the ability to study lateral protrusions of various shapes and sizes, formed in an integrin-dependent manner, on fibers of different diameters 30 . Here, using aligned and suspended fiber nanonets that also act as force sensors, we report that elongated cells (of different lineages) can form lateral protrusions that swing freely in 3D and that these attach to neighboring ECM fibers in a matter of seconds.…”