THE ROLE OF THE CYTOSKELETON IN 3D CANCER CELL MIGRATION

Arp2/3 is a protein complex that nucleates actin filament assembly in the lamellipodium in adherent cells crawling on planar two-dimensional (2D) substrates. However, in patho-physiological situations, cell migration typically occurs within a three-dimensional (3D) environment and little is known about the role of Arp2/3 and associated proteins in 3D cell migration. Using time resolved live-cell imaging and a fibrosarcoma cell line, HT1080, commonly used to study cell migration, we find that the Arp2/3 complex and associated proteins N-WASP, WAVE1, Cortactin, and Cdc42 regulate 3D cell migration. This regulation is caused by formation of multi-generation dendritic protrusions, which mediate traction forces on the surrounding matrix and effective cell migration. The primary protrusions emanating directly from the cell body and prolonging the nucleus form independent of Arp2/3 and dependent on focal adhesion proteins FAK, talin, and p130Cas. The Arp2/3 complex, N-WASP, WAVE1, Cortactin, and Cdc42 regulate the secondary protrusions branching off from the primary protrusions. In 3D matrices, fibrosarcoma cells as well as migrating breast, pancreatic, and prostate cancer cells do not display lamellipodial structures. This study characterizes the unique topology of protrusions made by cells in a 3D matrix and show that these dendritic protrusions play a critical role in 3D cell motility and matrix deformation. The relative contribution of these proteins to 3D migration is significantly different from their role in 2D migration. Microtubules have long been targeted to control tumor growth and, more recently, metastatic disease, for which a critical step is the local invasion of tumor cells into the 3D collagen-rich stromal matrix. To migrate in collagen matrices human fibrosarcoma and breast cancer cells exploit the dynamic formation of highly branched protrusions, which are composed of a microtubule-filled core surrounded by actin filaments that is largely absent in the same cells flattened on 2D substrates. Microtubule plus-end tracking protein EB1 and microtubule-associated motor protein dynein critically modulate 3D cell migration, not by regulating vesicular trafficking, but by regulating both speed and persistence through regulation of protrusion branching itself regulated by differential assembly dynamics of microtubules in the protrusions. These proteins do not regulate conventional 2D migration. An important consequence of the prominent role of microtubules in 3D migration is that the treatment of fibrosarcomas by commonly used cancer drug paclitaxel, which stabilizes microtubules, is dramatically more effective in 3D than in 2D, uniformly and completely blocking 3D cell migration. This work reveals the central role that microtubule dynamics plays in cell migration in more pathologically relevant 3D collagen matrices and suggests that cancer drugs targeting microtubule dynamics to mitigate migration should be further tested in 3D microenvironments. Cell migration through three-dimensional (3D) extra-cellular matrices is critical to the normal development of tissues and organs and in disease processes, yet adequate analytical tools to characterize 3D migration are lacking. We quantified the migration patterns of individual fibrosarcoma cells on 2D substrates and in 3D collagen matrices and found that 3D migration does not follow a random walk. Both 2D and 3D migration feature a non-Gaussian, exponential mean cell velocity distribution, which we show is primarily a result of cell-to-cell variations. Unlike in the 2D case, 3D cell migration is anisotropic: velocity profiles display different speed and self-correlation processes in different directions, rendering the classical persistent random walk (PRW) model of cell migration inadequate. By incorporating cell heterogeneity and local anisotropy to the PRW model, 3D cell motility is predicted over a wide range of matrix densities, which identifies density-independent emerging migratory properties. This analysis also reveals the unexpected robust relation between cell speed and persistence of migration over a wide range of matrix densities