Lentivirus-Mediated Gene Transfer of PEDF Results in Decreased Uveal Melanoma Transendothelial Migration

Abstract

Purpose:: The purpose of this study is to understand whether PEDF affects transendothelial migration in human uveal melanoma cells, to identify different effects of melanoma transendothial migration between exogenous PEDF and endogenous PEDF, and to elucidate the interactions between melanoma cells and vascular endothelium.

Methods:: Transendothelial migration assays were performed with the CytoSelect^TM Tumor Transendothelial Migration Assay Kit (Cell Biolabs, Inc. San Diego, CA). For human uveal melanoma cell transendothelial migration, human umbilical vascular endothelial cells (HUVECs) were plated onto 8 µm pore size inserts in 24-well-plates and allowed to form a confluent monolayer on the membrane for 72 hours. Human Mel290 and 02-1486 uveal melanoma cells were pretreated with human recombinant PEDF with concentrations of 0.1µg/ml, 1µg/ml, 10µg/ml, or RPMI1640 w/o PEDF as a control. These cells were marked with CytoTracker^TM before they were plated onto the endothelial cell monolayer in the upper wells. Media was added with or w/o TNFα in the bottom wells. For lentivirial PEDF transduced melanoma (B16LS9-PEDF), lentiviral transduced melanoma (B16LS9-LacZ) and untransduced B16LS9 transendothelial migration, murine vascular endothelial cells were plated onto the upper inserts. After B16LS9-PEDF, B16LS9-LacZ and B16LS9 cells were tracked, these cells were seeded onto the upper inserts via bottom inserts containing RPMI with or without 10%FBS. The tumor cells were allowed to transmigrate through the endothelium and membrane overnight. The non-migratory cells were removed and migratory cells were lysed. The fluorescence was determined with a fluorescence plate reader at 480 nm/520 nm.

Results:: In comparison with the control group, the percentages of human uveal melanoma cell transendothelial migration of Mel290 and 02-1486 pretreated with 10 µg/ml PEDF were decreased by 41.61% and 39.13% respectively. The transendothelial migration percentage of B16LS9 transduced by the lentivirus-mediated PEDF gene was 55.50% when compared with B16LS9. There was a significant difference in the absolute value of RFU from migratory cells between Mel290 pretreated with 10 µg/ml PEDF, 02-1486 pretreated with 10 µg/ml PEDF, B16LS9-mPEDF compared to controls(p<0.001).

Conclusions:: PEDF decreased uveal melanoma cell transendothelial migration in vitro. It appears that transferred endogenous PEDF is more effective than recombinant exogenous PEDF in inhibition of transendothelial migration. This indicates that PEDF may be used to an anti-metastasic agent.

Keywords: melanoma • uvea • gene transfer/gene therapy