Purchase this article with an account.
Ryan P Lee, Michelle Sims, Bradley T Gao, Hans E Grossniklaus, Lawrence M Pfeffer, Matthew W Wilson, Vanessa Marie Morales; Generation of Luc2-eGFP-UM Cell Lines for Pre-Clinical Studies on Metastatic Uveal Melanoma . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5309.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Metastatic Uveal Melanoma (UM) has no available treatment. UM is the most common primary intraocular malignancy in adults. Almost half of cases metastasize to the liver, resulting in a very poor 2-year survival, but little is known about how UM invades new tissue. Of particular interest is the ability of UM to remain dormant as sub-clinical micro-metastasis. The goal of our study was to generate UM cell lines transduced with Luciferase bioluminescent protein (Luc2) and a fluorescent reporter gene (eGFP) to investigate in vivo (1) how UM migrates to, seeds, and invades the liver and (2) the kinetics of the transition from micro-metastasis to detectable metastasis.
The UM cell lines, Mel 270, 92.1, and OMM1, were transduced with a lentiviral construct (pLenti-UBC-Luc2-EGFP). Transduced cell lines were screened using IVIS® Xenogen (Caliper Life Sciences, MA) for bioluminescence and confocal microscopy for fluorescence. Validation of the transduced cell lines was performed using flow cytometry to analyze (a) cell surface expression of ICAM-1 (CD54), ICAM-2 (CD102), VCAM-1 (CD106), ALCAM (CD166), EpCAM (CD326), and VLA-4 (CD49d), (b) cell death by Annexin V and Propidium Iodide (PI), and (c) proliferative capacity by Ki-67. Cell line authentication was performed by Genetica DNA Laboratories, Inc. (Cincinnati, OH) by comparison to published material. <br />
All three UM cell lines exhibited significant bioluminescence and green fluorescence in vitro 48 hours after transduction, which was sustained through time, evidencing stable transduction. Comparing transduced to non-transduced control cells, we found no difference in (a) the cohort of surface molecules, (b) the live/dead cell ratio by Annexin V and PI, and (c) proliferative capacity by Ki-67. Authentication analysis showed cell line properties were consistent with published characterization data.
We have generated a valuable tool for the in vivo study of metastatic UM, allowing for extremely sensitive tracking of cancer cells in animal models. Using this non-invasive tool, we are currently performing in vivo analyses of the kinetics of UM metastasis.
This PDF is available to Subscribers Only