In vitro Characterization of Vascular Loops in Four Uveal Melanoma Cell Lines

Abstract

Purpose: : The formation of new capillaries from pre–existing host vessels, known as tumor–derived angiogenesis can be divided into four basic steps: basement membrane degradation, migration, proliferation, and morphogenesis of endothelial cells into tube–like structures called vascular loops. In uveal melanoma (UM), the presence of vascular loops correlates with poor prognosis. An in vitro model was established to investigate vascular loop formation by exposing endothelial cells to uveal melanoma–conditioned medium, in order to gain a better understanding of angiogenesis.

Methods: : The ability of conditioned medium from four human uveal melanoma cell lines (92.1, MKT–BR, SP6.5, OCM–1) to induce vascular loop formation was determined using an in vitro angiogenesis assay kit (Chemicon International, Temecula, CA). The bottom of a 24–well plate was coated with fibrinogen and thrombin to create a fibrin gel surface. Human umbilical vein endothelial cells (HUVEC) were suspended in uveal melanoma–conditioned medium (MCM) at a concentration of 5x10⁴ cells/ml and pipetted onto the gel surface. The MCM was prepared 24 hours beforehand by seeding a 6–well plate with 1x10⁶cells/ml per cell line. An appropriate positive control, as provided by the manufacturer, was utilized. The 24–well plate was incubated for 48 hours at 37ºC in a 5% CO₂ supplemented atmosphere. The formation of vascular loops was observed under phase contrast microscopy and the number of full and half loops were counted at 24 and 48 hours.

Results: : At 24 and 48 hours, melanoma–conditioned medium from all four–cell lines induced vascular loop formation. 92.1–conditioned medium induced the greatest number of loops followed by MKT–BR>OCM–1>SP6.5. Only 92.1 conditioned–medium produced more vascular loops than the positive control. All cell lines showed an increase in vascular loops at 48 hours compared to 24 hours of incubation whereas control levels remained unchanged.

Conclusions: : Our study indicates that UM conditioned–medium is capable of inducing vascular loop formation in vitro. It is possible that each cell line secretes different factors since vascular loop formation was different for each cell line. Moreover the finding that the most metastatic cell line, 92.1, gave the highest formation of vascular loops further supports the correlation of vascular loops and worse prognosis in uveal melanoma patients.

Keywords: melanoma • neovascularization • vascular cells