Cancer Protrusions on a Tightrope - Suspended Fiber Platform Reveals Protrusion Dynamics Independent of Cell Migration

Abstract

Indispensable to all modes of migration used during single cell metastasis, cytoplasmic protrusions are pivotal in surveying cells local surroundings which ultimately initiates migration of the cell body. Cancer cell migration is fairly well studied with the traditional focus on protrusion driven cell body displacement, while less is known on the role of protrusions in sensing cellular microenvironments. Here, we present a suspended and aligned fiber platform capable of high spatio-temporal imaging of protrusions capable of sensing fiber curvature contrasts independent of cell migration. By varying the diameter of suspended fibers, we are able to maintain cell migration along low curvature-large diameter (2μm) fibers, while solely allowing cells to sense, initiate, and mature protrusions on orthogonally deposited high curvature-low diameter (~100, 200 and 600 nm) fibers. Using highly aggressive breast MDA-MB-231 and brain glioblastoma DBTRG-05MG model systems, we find that MDA-MB-231 protrusion maturation dynamics are more sensitive to changes in fiber curvature and fibronectin ligand coating concentration compared to DBTRG-05MG. Furthermore, we find that vimentin intermediate filaments localize within 70% of mature protrusions, which normally form on larger diameter fibers. Additionally, protrusion lengths fluctuate continuously until the protrusion is either terminated or stabilized, and occasionally protrusions are observed to shed cytoplasmic fragments. Through manipulation of curvature contrasts, we demonstrate single protrusive hierarchical decomposition and coordination in zeroth (main), first and second order branches. The fiber curvature platform presented here uniquely allows cancer cells to sense nanofiber curvature contrasts, thus providing new mechanistic insights in protrusion initiation, maturation, and hierarchical coordination.