Suppression of cofilin-1 promotes invasion in 3D hyaluronic acid matrices by promoting actin-based protrusions.

Contributions of the actin turnover machinery to cell motility have been extensively studied in traditional two-dimensional (2D) culture paradigms. However, much remains unknown about how these proteins contribute to three-dimensional (3D) motility, particularly in matrices lacking strong contact guidance cues. Here, we explore this question in the context of glioblastoma (GBM) cell invasion through 3D hyaluronic acid (HA) hydrogels. We begin with a CRISPR screen to identify contributions of core actin turnover proteins to migration speed in 2D and 3D. Although suppression of most proteins reduced motility in both 2D and 3D, suppression of cofilin-1 (CFL) increased migration speed in 3D. CFL knockout cells uniquely formed longer and more protrusions in 3D compared with non-targeting control cells. Consistent with the screen, targeted CFL short hairpin RNA-mediated knockdown (KD) decreased motility on 2D HA but increased motility in 3D HA. This effect appears HA-specific, as CFL KD did not increase motility in 3D collagen or in a transwell assay. Myosin X, CD44, and hyaluronidase-2 all localized to a subset of protrusions irrespective of cellular CFL status, implying that CFL suppression promotes filopodia and microtentacle extension. We propose that loss of CFL promotes the actin filaments in these protrusions, enabling GBM cells to penetrate 3D HA matrices.