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.

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Classified as a grade IV tumor of the central nervous system, Glioblastoma multiforme (GBM) arises from the glia. A poor understanding of tumor metastasis and limited treatment options have led to increase in deaths of patients suffering from GBM. Studying glioma behavior using aligned structures that mimic native glioblastoma metastatic path is challenging. In this study, we utilize a previously described non-electrospinning platform to manufacture aligned 3D structures called STEP nanonets that not only allows the study of individual cell-nanofiber interaction, but also allows the calculation of migratory forces using beam mechanics. In particular, the blebbing dynamics, force generation, and the effect of an actin disruptor, Cytochalasin D have been investigated on a glioma cell line (DBTRG, Denver Based Tumor Research Group). It was observed that cell pulled onto the nanofibers causing measurable deflections when they were in spread and non-blebbing conditions. In non-spread configurations while attached to fibers, the cells acquired spherical configurations and resumed blebbing. The average migratory force generated by cells exposed to DMSO (control, 1:1000 dilution) using nanonets of 2μm by 400nm fibers was 0.58±0.06nN. Actin disruptor, Cytochalasin D severely compromised the ability of the glioma cells to migrate causing no deflection of the fibers. Forces exerted by tumor cells on their native microenvironment affects their ability to metastasize, invade and proliferate. While the result presents actin disruptor as a potential target to minimize metastasis, the influence of other cytoskeleton disruptors can also be studied using the platform. Moreover, the results obtained from the study can be utilized to better understand the individual cell – nanofibers interaction which can shed light on how cells interact with their native environment during metastasis.