Brachytherapy enriches tumor-derived extracellular vesicles in aqueous humor of uveal melanoma eyes

Abstract

Purpose : Small extracellular vesicles (sEVs) have been shown to promote tumorigenesis, treatment resistance, and metastasis, leading to the discovery of clinically-relevant biomarkers for multiple cancer types; however sEVs in the aqueous humor (AH) of uveal melanoma (UM) patients have never previously been profiled. In this study, we used single particle analysis to characterize sEV subpopulations in the AH of UM patients by quantifying their size, concentration, and phenotypes based on cell surface markers, specifically tetraspanin co-expression patterns.

Methods : sEVs were analyzed from paired pre- and post-brachytherapy AH samples collected from 19 UM patients. AH samples from 5 glaucoma patients served as the control cohort. Unprocessed AH underwent Nanoparticle Tracking Analysis for size quantification and Single Particle-Interferometric Reflectance Imaging Sensor analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequently converted to percentages for analysis. Statistics were performed using Mann-Whitney U, ANOVA, and Tukey’s tests.

Results : AH derived from UM eyes (pre-brachytherapy) displayed a broader sEV size distribution than glaucoma eyes, with more diverse sEV subpopulations. Both tumor and nontumor AH samples demonstrated a dominant CD63+ sEV subpopulation. A significantly higher mean percentage of CD63/81+ sEVs (pre AH: 5.719% vs. post AH: 9.061%, P< 0.001) and CD9/63/81+ sEVs (pre AH: 8.846% vs. post AH: 13.32%, P= 0.001) were found in post-brachytherapy samples compared to pre-brachytherapy.

Conclusions : sEV subpopulations in UM eyes demonstrate increased heterogeneity in comparison to nontumor eyes. Our results are consistent with prior studies reporting that the CD63+ sEV subtype is an AH-specific biomarker enriched across multiple ocular diseases. The increase in CD63/81+ sEVs and CD9/63/81+ sEVs after brachytherapy is presumably a result of radiation-induced release of these vesicles, suggesting a tumor-derived origin of these subpopulations. CD63/81+ sEV subpopulation was previously reported to also be a tumor-derived subpopulation in AH of retinoblastoma eyes. Further study of the cargo carried by these sEV subpopulations may uncover biomarkers of clinical utility in UM and other ocular cancers.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.