Abstract
Purpose :
Uveal melanoma (UM) is often asymptomatic and well controlled; unfortunately 50% of patients develop metastasis plummeting survival to 15%. There is an urgency to identify mechanisms by which these melanocytic tumors are disseminating. Recent studies have focused on extracellular vesicles (EVs); biomolecule containing nanoparticles with propensity to mediate metastasis. We have previously shown UM cell line-derived EVs can induce oncogenic transformation in fibroblasts, resulting in tumor formation in vivo. Further, our group has characterized proteomic profiles of EVs from UM cell lines. The purpose of this study was to characterize EV proteomic profiles in a rabbit model of human UM. In addition, through species classification of EV protein content, to differentiate tumor originating proteins from homeostatic EV proteins.
Methods :
Peripheral blood was obtained from 7 rabbits inoculated intraocularly with human UM cells (line 92.1) in a 20-week animal model. EVs were isolated by differential ultracentrifugation from rabbit plasma at week 0 and week following tumor (weeks 10+). Confirmation of EV yield was demonstrated by nanoparticle tracking analysis (NTA). EV proteins were isolated and subjected to mass spectrometry analysis. Raw proteomics data was matched against rabbit and human databases then loaded into Q+ Scaffold_4.4.8 software for visualization, statistical comparison and quantification.
Results :
NTA data confirmed presence of EVs ranging in size of 165-210nm. EV protein concentrations showed an increase in protein content correlating with increased tumor size. Statistical analysis in scaffold highlighted significant differences in human and rabbit protein profiles between week 0 and weeks 10+ in all rabbits. Lists of differentially expressed human and rabbit proteins were compiled separately, referenced against literature and loaded into DAVID Bioinformatics database for functional gene ontology analysis. Interestingly, trends seen in human proteins were mirrored by their rabbit homologs.
Conclusions :
Here we provided a unique insight into primary UM derived EVs through a rabbit model. Furthermore, the data suggests rabbit host EV compositions may be influenced and adapting to primary UM. This experimental model proves promising for future assessments of primary UM EV contents with hopes to eventually broaden our understanding of metastasis.
This is a 2021 ARVO Annual Meeting abstract.