Abstract 3416: Clearance of tumor-derived extracellular vesicle heterogeneity in aqueous humor after chemotherapy in retinoblastoma eyes
Authors: Peng Chen-Ching , Im Deborah , Sirivolu Shreya , Reiser Bibiana , Nagiel Aaron , Neviani Paolo , Xu Liya , Berry Jesse L. 
Published: June 2022

Abstract
Introduction: Aqueous humor (AH), the clear fluid in front of the eye, maintains the pressure and vitality of ocular tissues. This fluid is accessible via the cornea which enables use of AH as a liquid biopsy for intraocular disease. Retinoblastoma (Rb), an intraocular cancer in children, is unique in that direct tumor biopsy is prohibited, thus liquid biopsy has great clinical potential. cfDNA in the AH as a biomarker for Rb patients has been investigated and extracellular vesicles (EVs) are detectable in the AH from adults. However, AH EVs in Rb have previously never been explored. We know very little about the heterogeneity of AH EV populations in ocular cancers.
Materials and Methods: 27 AH samples from 19 patients from 13 tumor-free eyes and 11 Rb eyes were collected. Rb eyes were further divided into treatment-naïve (Rb_Tn) and treatment-active (Rb_Tx) subgroups. Unprocessed AH samples were subjected to Nanoparticle Tracking Analysis (NTA) (Nanosight NS300) for size distribution and concentration, and to Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) for fluorescent-based immunophenotyping of EV marker expression (CD63, CD81, and CD9).
Results: NTA demonstrated the concentration of AH EVs is 3.11 x 109-1.38 x 1010 vesicles/mL; majority sized 76.8-103 nm. Study group comparisons showed that non-tumor containing eyes had a smaller nanoparticle mean size compared to Rb containing eyes (P = 0.002). More EVs were significantly detected in Rb_Tn containing eyes compared to Rb_Tx containing eyes (P = 0.022), suggesting the possible presence of tumor-derived EVs in Rb_Tn which were subsequently eliminated by treatment. SP-IRIS revealed distinct patterns of tetraspanin expression of AH small EVs (sEVs). Mono-CD63+ EVs were identified to be the most dominant sEV subpopulation from AH across non-tumor and Rb_Tx eyes. However, more diverse sEV subpopulation profile was detected in Rb_Tn AH samples. Significantly lower percentage of mono-CD63+ EVs could be determined in Rb_Tn eyes compared to Rb_Tx eyes (70.3% vs. 96.1%, P = 0.001). A significant accumulation of CD9/CD63 (4.3% vs. 1.1%, P = 0.035), CD63/CD81 (20.7% vs. 2.0%, P = 0.012) and CD9/CD63/CD81 (4.8% vs. 0.8%, P = 0.022) subpopulations in Rb_Tn was also observed. An enriched mono-CD63+ sEV subpopulation identified in AH indicates this is a potential AH-specific biomarker. In the setting of Rb there was a more heterogeneous population of sEVs which normalized with treatment.
Conclusions: Small EVs are readily detectable in unprocessed AH with a dominant mono-CD63+ subpopulation in AH regardless of pediatric eye disease states. Tetraspanin colocalization analysis indicates the clearance of retinoblastoma-derived EV heterogeneity by chemotherapy. These novel finding suggests a potential clinical application of measurement of sEV subpopulations in AH samples to monitor response to therapy.
Citation Format: Chen-Ching Peng, Deborah Im, Shreya Sirivolu, Bibiana Reiser, Aaron Nagiel, Paolo Neviani, Liya Xu, Jesse L. Berry. Clearance of tumor-derived extracellular vesicle heterogeneity in aqueous humor after chemotherapy in retinoblastoma eyes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3416.