Extracellular Vesicle Biomarkers in the Aqueous Humor Correlate with Retinoblastoma Seeding
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Purpose
To investigate the correlation of retinoblastoma (RB) aqueous humor (AH)–derived extracellular vesicle (EV) surface markers, particularly CD133, with clinical features of RB.
Methods
AH samples (n=22) collected at diagnosis (n=17) or primary enucleation (n=5) from RB eyes were profiled using magnetic bead-based flow cytometry. Median fluorescence intensities (MFI) of tetraspanins (CD9, CD63, CD81) and CD133 were quantified and correlated with International Intraocular RB Classification (IIRC) stage, seeding location, seeding morphology, and enucleation.
Results
CD133 + EVs were detected in 16 of 22 (73%) samples. While CD133 levels showed a non-significant increasing trend with advanced IIRC stages, CD133 expression was significantly elevated in eyes with vitreous seeding vs. subretinal only (P = 0.010). Cuzick’s trend analysis revealed a significant association between rising CD133 % MFI and complex seeding (P < 0.001). Conversely, CD63 displayed a significant inverse trend, with decreasing % MFI associated with complex seeding (P < 0.001). CD133 high eyes (MFI>10,000, n=9) exhibited increased enucleation (P = 0.007) and predominantly sphere/cloud seeding patterns (P = 0.001). CD133 high eyes demonstrated a coordinated CD63/CD81 co-expression pattern (r = 0.80), absent in the CD133 low group.
Conclusions
Elevated CD133 level and percentage were associated with vitreous involvement and complex seeding morphology, suggesting potential as a minimally invasive indicator of disease burden. The inverse relationship between CD63 and seeding severity, combined with CD63/CD81 remodeling in CD133 high eyes, suggests an EV profile indicative of aggressive disease. These findings support the association of CD133 + EVs with advanced RB seeding and warrant further prospective validation.
