Abstract 3163: Transendothelial migration of uveal melanoma cells
Authors: Onken Michael D. , Mooren Olivia L. , Li Jinmei , Cooper John A. 
Published: May 2015

Abstract
Uveal melanoma, cancer arising from the pigmented uveal layer of the eye, is highly metastatic despite being limited to spreading only through the bloodstream. Almost half of patients develop distant metastatic disease, most often in the liver, even after the tumor-bearing eye is completely removed. The key to understanding and targeting uveal melanoma metastasis is in understanding how circulating tumors cells exit the bloodstream at distant sites of metastasis, and then invade and colonize distant organs. To address the first process, transendothelial migration, we modeled the blood vessel wall using primary human microvascular endothelial monolayers grown on polyacrylamide substrates that mimic the physiological stiffness of normal tissue. To these monolayers, we added 92.1 and OCM-1A uveal melanoma cells, and then collected time-lapse movies using spinning-disc confocal and differential interference contrast microscopy to follow the transmigration of the uveal melanoma cells. We found that both cell lines transmigrate through endothelial monolayers by a route distinctly different from the paracellular migration we see for trafficking immune cells. During this multistep process, uveal melanoma cells first identify paracellular junctions and then intercalate between endothelial cells, taking on a flattened morphology and maintaining contacts with their adjacent endothelial cells; reminiscent of vasculogenic mimicry. Tumor cells then extend invasive projections beneath adjacent endothelial cells, before eventually migrate under the endothelium. Immunofluorescence revealed cortical actin networks with strong cortactin staining within these invasive projections. To visualize these structures better, we transfected uveal melanoma cells with F-tractin, a fluorescent fusion protein that specifically binds filamentous actin without disrupting actin function, that allows imaging of actin dynamics in live cells. Time-lapse confocal images revealed complex actin-rich projections invading and sampling the interface between the endothelial monolayer and the substrate. Dynamic actin cytoskeletal reorganization was also apparent as cells migrated under the monolayer. Previous studies have linked inactivating mutations in BAP1 to metastatic spread of uveal melanoma in patients. To test whether BAP1 loss plays a role in transendothelial migration, we knocked down BAP1 expression in our uveal melanoma cell lines. We found that BAP1-depleted cells transmigrated significantly faster through endothelial monolayers in transwell assays than control knockdown cells. Time-lapse movies showed no significant differences in the first steps of initiation or intercalation between BAP1 knockdown and control cells. These studies identify a novel mechanism of transendothelial migration in uveal melanoma cells and suggest that BAP1 depletion specifically affects the ability of cells to invade under and exit the monolayer.
Citation Format: Michael D. Onken, Olivia L. Mooren, Jinmei Li, John A. Cooper. Transendothelial migration of uveal melanoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3163. doi:10.1158/1538-7445.AM2014-3163