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Nasrin Refaian, Konrad R Koch, Simona Luise Schlereth, Deniz Hos, Jacobus J Bosch, Claus Cursiefen, Ludwig M. Heindl; Comparing the lymphangiogenic impact of conjunctival and uveal melanoma cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5064.
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© ARVO (1962-2015); The Authors (2016-present)
A clinically relevant difference between malignant melanomas within the eye (uveal melanoma, UM) and those on the ocular surface (conjunctival melanoma, CM) consists in their distinct metastatic behaviour. CM has a propensity to first spread into regional lymph nodes. Contrarily, UM almost exclusively spreads into the liver via the hematogenic path, whereas lymphatic metastases have only been described in advanced cases with extrascleral tumor extension. The outgrowth of new lymphatics (lymphangiogenesis) is a major prerequisite for lymphatic spread (LS), which is driven by tumor-derived VEGF-C/-D secretion. The goal of this study was to evaluate whether diverging prolymphangiogenic potentials of CM and UM cells could contribute to the higher rate of LS in CM, as assessed via VEGF-C/-D expression, and the proliferation of lymphatic endothelial cells (LECs) cultivated in CM/UM cell conditioned medium.
Human primary UM cells (OCM1, Mel270) and CM cells (CM2005.1) cultured in RPMI medium (10% FCS) were harvested for mRNA isolation followed by cDNA synthesis. Quantitative real-time PCR was performed to measure VEGF-C/-D expression. For MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide) based proliferation assays, LECs were seeded on a 96-well-plate (4000 cells/well), left to attach overnight, and then cultivated for 48 hours in 25%, 50%, or 75% UM/CM cell conditioned medium (1% FCS). MTT was added resulting in intracellular formazan deposition. The reaction was stopped after 3 hours. Absorbances were measured at 570 nm.
VEGF-C and -D were expressed by all three cell lines. VEGF-C expression was significantly higher in Mel270 (p<0.01) compared with OCM1 and CM2005.1. VEGF-D expression was similar in all analyzed cell lines (p>0.05). The proliferation rate of LECs was positively correlated with increasing proportions of Mel270-, OCM1-, CM2005.1-conditioned medium (p<0.05 for each melanoma cell line). CM2005.1-conditioned medium stimulated LEC proliferation to a significantly lower extent than UM cell lines OCM1 and Mel270 (p<0.05).
CM cells did not show a higher prolymphaniogenic potential than UM cells. Accordingly, the higher rate of LS in CM is more likely attributable to the specific microenvironment at the ocular surface, which includes preexisting lymph vessels as potential access point for malignant cells, as opposed to the lymphatic free intraocular vicinity of UM.
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