Analysis of melanoma cell material and their effect on the brain endothelial barrier

Abstract

Introduction and Aims: Brain metastasis is the most devastating complication of metastatic disease, of which melanoma has the highest propensity to invade the brain of any other cancer. Treatments for brain metastasis are limited due to the tight structure of the blood-brain barrier, however melanoma cells harbour mechanisms to disrupt the integrity of the cerebral microvasculature and invade through the endothelial cells. The literature surrounding melanoma metastasis suggest the cells disrupt the cerebral endothelial cells and secrete prometastatic factors to aid this process, although the extent of which are not fully understood. This study examines the effects of a non-adherent sub-population released by melanoma cells in vitro on the integrity of the blood-brain barrier, to further investigate the mechanisms employed by melanoma cells to extravasate into the brain.

Methods: Electrical Cell-Substrate Impedance Sensing (ECIS) technology was used to assess the effect of the melanoma-derived subpopulation on the barrier function of human brain endothelial cells. Imaging techniques such as immunocytochemistry and fluorescent confocal microscopy were used to visualise the effects of the melanoma material on the integrity of the endothelium, and detect the mechanism of interaction in 3D. Flow cytometry and fluorescence activated cell sorting (FACS) were used to analyse the size and density of the material and distinguish the bioactive material within the sample.

Results: The melanoma material induced disruption of the endothelial barrier at a considerably higher rate than their parent melanoma line. This disruption was shared between both the paracellular and basolateral components, suggesting a transcellular route of entry. The melanoma material rapidly associated with the endothelial cell body, inducing global changes in cell structure. 3-Dimensional visualisation confirmed endothelial cell uptake of the melanoma material through a transcellular mechanism.

Discussion: The current literature surrounding melanoma extravasation at the cerebral endothelium suggests the cells use a paracellular mechanism, and release factors to enhance or prime metastatic ability. The preliminary results of this study suggest the material produced by the melanoma cells could be vesicle-like in nature, likely a result of apoptosis or membrane blebbing, that have independent effects on the integrity of the blood brain barrier, with a potential role in enhancing melanoma brain metastasis.