Extracellular Vesicle Biomarkers in the Aqueous Humor Correlate with Retinoblastoma Seeding

PurposeTo investigate the correlation of retinoblastoma (RB) aqueous humor (AH)-derived extracellular vesicle (EV) surface markers, particularly CD133, with clinical features of RB. MethodsAH 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. ResultsCD133+ 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). Cuzicks 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). CD133high eyes (MFI>10,000, n=9) exhibited increased enucleation (P = 0.007) and predominantly sphere/cloud seeding patterns (P = 0.001). CD133high eyes demonstrated a coordinated CD63/CD81 co-expression pattern (r = 0.80), absent in the CD133low group. ConclusionsElevated 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 CD133high 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.