Characterization of Extracellular Vesicles purified from plasma, aqueous humor and vitreous from patients with uveal melanoma

Abstract

Purpose : : The major objective was characterize extracellular vesicles (EVs) purified from plasma, aqueous humor (AH) and vitreous humor (VH) from patients with uveal melanoma(UM) and cataract

Methods : AH, VH and plasma were collected from patients with UM (N=7) and AH and plasma from patients with cataract (control group N=7) at the Department of Ophthalmology, UNIFESP/EPM, Brazil. All patients were clinically evaluated. The EVs were isolated using the exoRNeasy Serum/Plasma Maxi Kit membrane affinity column method (Qiagen, Valencia, CA, USA) and size (nm) and concentration (particles/mL) were measured using Nanoparticles Tracking Analysis (NTA).Western blotting (WB) was performed for detection CD63 and TSG101. The Mass spectrometry was then performed to characterize the proteomic profile of EVs from samples.

Results : NTA data showed that mean size of EVs in VH, AH and plasma samples of UM patients was 232 nm, 226 nm and 220 nm respectively. No differences were observed compared to cataract patients (238 nm and 250nm in AH and plasma-derived EVs, respectively).
The mean concentration of EVs in VH from UM patients (6.6 x 10¹⁰ particles/mL) was higher compared to plasma (2.7 x10¹⁰particles/mL) and AH (9.9 x 10⁹particles/mL) (P< 0.05). Differences were noticed in the concentration of EVs from plasma and AH from UM patients compared to cataract patients (4.1 x 10⁸ and 6.7 x10⁸particles/ml in plasma and AH, respectively) (P< 0.05). WB was performed with VH, AH and plasma-derived EVs samples from patients with UM and expression of CD63 was increased. UM-derived EVs had also high expression for TSG101 in VH and plasma, but not in AH. Plasma-derived EVs from cataract patients had high expression to CD63 and TSG101, butAH-derived EVs in this group showed low expression for both markers. Proteomics analysis revealed hundreds of EV-derived proteins in each sample. Proteins counts were approximately 285-315 in plasma, 129-277 in AH and 199-367 in VH.

Conclusions : This is the first study to successfully identify and characterize EVs in aqueous humor, vitreous humor and plasma from patients with uveal melanoma. The results are promising to find a biomarker (EVs) in patients with uveal melanoma

This is a 2020 ARVO Annual Meeting abstract.