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S. Di Cesare, P. Logan, J.C. Marshall, F.J. Gregoire, D. Faingold, M.N. Burnier, Jr.; Migratory and Immunohistochemical Analysis of 5 Human Uveal Melanoma Cell Lines Towards CXCL–12, CXCL–8, CXCL–1, and HGF . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2229.
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The migratory ability of Human Uveal Melanoma cell lines towards specific chemo–attractants plays an important role in tumorigenesis in vivo. Chemokines increase the cells ability to migrate out of the primary tumor site due to an autocrine action. The aim of this project is to determine the migratory ability of 5 Human Uveal Melanoma cell lines towards the chemokines CXCL–12, CXCL–8, CXCL–1 & HGF, which have previously shown to increase migration in other cancers.
The migratory properties of 5 Human Uveal Melanoma cell lines (92.1, MKT–BR, OCM–1, SP6.5, and UW–1) were investigated using a QCMTM 24–Well Colorimetric Cell Migration Assay. This assay is based on the Boyden chamber system that contains smaller well inserts. These well inserts have a PET membrane made of a polycarbonate material perforated with 8µm pores. 1.0x106 cells were placed in the upper chamber and re–suspended in RPMI serum–free media. The lower chamber contained the chemo–attractants also re–suspended in serum free media. Appropriate positive and negative controls (10% FBS [+], Serum Free Media [–]) were also used. These plates were incubated for 24h at 37oC in a 5% CO2 supplemented atmosphere. Following incubation, cells that did not migrate were removed from the top of the insert with sterile cotton swabs. Cells that migrated were then stained, eluted from the bottom of the wells, and the optical density of the stain released from the well was read at an OD of 560nm. Immunohistochemistry was performed against CXCL–8 and CXCL–1, along with its receptor CXCR1 to confirm expression of these chemokines and their receptors in the cell lines.
The chemo–attractants induced migration for all five cell lines. The 10% FBS (positive control) gave the highest levels of migration. The serum–free medium (negative control) caused little to no migration. On average CXCL–8 resulted in the most migration for all 5 cell lines, followed by CXCL–1>CXCL–12>HGF. All cell lines stained highly positive for CXCL–1, CXCL–8 and its receptor CXCR–1.
The migratory ability of human uveal melanoma cell lines has been established in response to these four chemokines, particularly CXCL–1 and CXCL–8, which are expressed in other primary tumors during tumor extravasation in the metastatic cascade. In addition we showed the presence of an autocrine loop for CXCL–1, CXCL–8 and its receptor CXCR–1. Further studies should investigate the role of these chemokines in different steps of the metastatic cascade in uveal melanoma.
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