Delineating the Effects of Reactive Oxygen Species on Confined Cancer Cell Migration

In the normal progression of cancer within a human, the establishment of secondary tumor sites in the patient’s body, or metastasis, is associated with much lower survival rates and treatment efficacy. This transition, incited by the detachment of cancer cells from the primary tumor site, sees cells move through confined spaces; channel-like tracks that vary from 3-30 micrometers in width. Understanding the mechanisms of a cancer cell’s movement from a primary tumor through these small passages in our tissues and blood vessels is critical in any clinical attempts to improve the efficacy of patient treatment.

How cells sense and respond to confinement is not well understood. Reactive Oxygen Species (ROS), a class of molecules associated with cellular respiration, can cause DNA damage in excess with profound consequences on genomic integrity. Although ROS levels increase in confinement, the impact of this increase on the modes and mechanisms of confined cell migration remains unclear.

To address this gap in knowledge, human fibrosarcoma cells and human dermal fibroblasts were seeded into polydimethylsiloxane-based microfluidic devices, consisting of arrayed, parallel channels with a fixed length and width (200 µm and 10 µm) and a variable height of 3 or 10 µm (confined/unconfined). N-acetylcysteine (NAC) in 10 µM was used to inhibit ROS. Time-lapse microscopy was used to image cells on an inverted Nikon Ti-2 microscope. Images were taken of cell migration, after fixing and staining of cells to visualize actin polarization and nuclear integrity, and of ROS localization. Results were analyzed using ImageJ and MATLAB.

Our results revealed that inhibition of ROS increased cellular migration speeds and markedly suppressed membrane bleb formation, causing cells to preferentially exhibit protrusive-based migration phenotypes. This was observed in confined but not unconfined channels. Furthermore, treatment with NAC reduced the extent of nuclear blebbing, suggesting that ROS promotes nuclear deformation and presumably nuclear envelope rupture which may have detrimental effects on cell survival. Our future studies will focus on delineating how ROS regulates the efficiency and mechanisms of confined cell locomotion.

Collectively, these findings reveal the key role of ROS in confined cell migration and suggest that overactivation of ROS may represent a novel approach to suppress cancer cell migration in vivo.