The Effects of Melanoma Inhibitory Activity (MIA) on the Invasive Properties of Uveal Melanoma Cell Lines

Abstract

Abstract: : Purpose: Melanoma inhibitory activity (MIA) is a protein which has been correlated with development of metastatic disease in skin melanoma patients. MIA is known to inhibit tumor cell attachment to extracellular matrix by acting as an anti-adhesive protein that alters cellular anchorage to fibronectin and laminin, the latter being a major component of Matrigel. Invasion, which leads to metastasis, is a very complex process which includes coordinated detachments and re-attachments of cell adhesions to neighboring cells and to extracellular matrix followed by invasion. The purpose of this study was to determine the effect of the addition of MIA on the detachment and invasion of uveal melanoma (UM) cells in vitro. Methods: Five UM cell lines (92.1, MKT-BR, OCM-1, SP6.5, and UW-1) were seeded in a Matrigel coated modified Boyden chamber at a concentration of 1x10⁵ cells per well. Wells were incubated for up to 72 hours. Every 24 hours, samples were taken from each well and stained with trypan blue to confirm viability of the cells. At the end of 72 hours, cells invading the Matrigel were counted. The assay was repeated adding 11.5 ng/ml of recombinant human MIA to the top layer of each well. The MIA concentration of 11.5ng/ml represents the average of a predetermined level of MIA production the UM cell lines used in this study, measured by ELISA. Results: In cell line cultures not treated with MIA, the vast majority of cells (>95%) were found to adhere to the upper surface of the Matrigel, although an average of 13 cells (0.001%) invaded the Matrigel. No cells were found suspended in the media above the Matrigel. Instead of adhering to the Matrigel, cells exposed to MIA became suspended in the media above the Matrigel and no invasion was observed. Viability between the two groups was comparable at each time point and remained above 90% throughout the experiment. Conclusions: The addition of MIA to cell cultures significantly inhibited attachment of UM cells to the Matrigel. In order to invade, uveal melanoma cells need to attach to the Matrigel. By inhibiting attachment, the first step of the invasive process, cells remained suspended in the media and were unable to invade into the Matrigel. Further assays need to be performed with varying MIA concentrations.

Keywords: cell adhesions/cell junctions • melanoma