CD63/81 Small Extracellular Vesicles in the Aqueous Humor are Retinoblastoma Associated

Abstract

Purpose Although biopsy is contraindicated in retinoblastoma (RB), the aqueous humor (AH) is a robust liquid biopsy source of molecular tumor information, facilitating biomarker discovery. Small extracellular vesicles (sEVs), promising biomarker candidates across multiple cancers, were recently identified in RB AH, but relationships between sEVs and RB clinical features are unknown. Methods We analyzed sEVs in 37 AH samples from 18 RB eyes of varying International Intraocular Retinoblastoma Classification (IIRC) groups and explored clinical correlations. Ten samples were collected at diagnosis (DX) and 27 during treatment (Tx). Unprocessed AH underwent Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequentially converted to percentages for analysis. Results Comparing DX and Tx samples, a higher percentage of CD63/81+ sEVs was found in DX AH (16.3 ± 11.6% vs. 5.49 ± 3.67% P = 0.0009), with a more homogenous mono-CD63+ sEV population seen in Tx AH (43.5 ± 14.7% vs. 28.8 ± 9.38%, P = 0.0073). Among DX samples, CD63/81+ sEVs were most abundant in group E eyes ( n = 2) compared to group D ( n = 6) by count (2.75 × 10 5 ± 3.40 × 10 5 vs. 5.95 × 10 3 ± 8.16 × 10 3 , P = 0.0006), and to group A + B ( n = 2) by count (2.75 × 10 5 ± 3.40 × 10 5 vs. 2.73 × 10 2 ± 2.59 × 10 2 , P = 0.0096) and percentage (32.1 ± 7.98% vs. 7.79 ± 0.02%, P = 0.0187). Conclusions CD63/81+ sEVs enrich AH from RB eyes before treatment and those with more significant tumor burden, suggesting they are tumor-derived. Future research into their cargo may reveal mechanisms of cellular communication via sEVs in RB and novel biomarkers.

Comparative Single Vesicle Analysis of Aqueous Humor Extracellular Vesicles before and after Radiation in Uveal Melanoma Eyes

“…In retinoblastoma eyes, the CD63/81+ sEV subpopulation was also hypothesized to be tumor-derived [ 18 ]. Given its association with both UM and retinoblastoma, further investigation into the cargo of these sEVs is warranted.…”

“…Single sEV analysis was performed by using SP-IRIS-based ExoView R100 platform and ExoView Human Tetraspanin Kit (Unchained Labs, Pleasanton, CA, USA) as previously published [ 15 , 18 ]. Between 0.25 and 10 µL of unprocessed AH was diluted by using buffer A to a final volume of 40 µL.…”

“…Previous reports suggest that CD63 is closely associated with lysosomal trafficking and is a key player in exosome formation and release by participating in the endosomal sorting complex required for transport (ESCART)-independent pathway [ 17 ]. In studying patients with retinoblastoma, a primary pediatric ocular malignancy, we observed a significantly dominant subpopulation of CD63/81+ sEVs [ 18 ]. This subpopulation was more enriched before treatment and in patients with more significant tumor burden, suggesting they are a tumor-derived subpopulation.…”