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

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.