Single vesicle analysis of aqueous humor reveals eye specific CD63-dominant subpopulation in pediatric ocular diseases

Abstract

Purpose : Extracellular vesicles (EV) are detectable in aqueous humor (AH) from adult patients in previous studies. However, the presence of EVs in AH from pediatric diseased eyes has not been explored. Also, the expression profile of tetraspanins (CD9, CD63 and CD81) on AH EVs remains unclear. Herein, we measure the concentration of the pediatric diseased eye-derived AH EVs and analyze their size distribution and determine the phenotypic expression levels of tetraspanins of AH EV at single vesicle resolution.

Methods : Twenty-seven AH samples were obtained from 4 congenital cataract (CAT), 6 congenital glaucoma (GLC), 4 pediatric retinal disease (PRD) and 13 retinoblastoma (Rb) eyes. Rb eyes were further divided into treatment-naïve (Rb_Tn) and treatment-active (Rb_Tx) subgroups. Unprocessed AH samples were subjected to Nanoparticle Tracking Analysis (NTA) (Nanosight NS300) for size distribution and concentration, and to Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) for interferometric sizing and fluorescent-based immunophenotyping of EV marker expression (CD63, CD81, and CD9).

Results : By NTA analysis, the concentration of AH EVs is within 3.11 x 10⁹~1.38 x 10¹⁰ vesicles per mL with a major population in an average modal size of 76.8~103 nm across pediatric disease states. SP-IRIS analysis revealed that tetraspanins, CD63⁺, CD81⁺ and CD9⁺ EVs were detectable across all AH samples using the Exoview R100 platform. In addition, we identified a significant enrichment of CD63 single positive EV subpopulation in AH across 4 disease states (CAT + GLC + PRD + Rb_Tx): 54.9% of mono-CD63⁺ EV among all fluorescent positive EVs. However, a mono-CD63⁺ EV population composed only 28.3% of the AH EV population from Rb_Tn eyes (P =0.007 when compared to 4 other disease states) suggesting a higher population of tumor-derived EVs in these eyes.

Conclusions : Small EVs are readily detectable in unprocessed AH with a dominant mono-CD63⁺ EVs in AH regardless of pediatric eye disease states. Tetraspanin colocalization analysis indicates the clearance of tumor-derived EV heterogeneity by chemotherapy in Rb. This suggests an AH specific role of mono-CD63⁺ EVs. These novel findings uncover a new direction in AH EV biomarker research.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.