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 clear cornea which enables use of AH as a liquid biopsy source of biomarkers for intraocular disease. Extracellular vesicles are detectable in the AH and small extracellular vesicles (sEVs) are present in the AH from adults. However, EVs in AH from pediatric eyes in vivo have never previously been explored. We know very little about the heterogeneity of AH EV populations in ocular disease. Twenty-seven processing-free AH samples from 19 patients across four different pediatric ocular diseases were subjected to Nanoparticle Tracking Analysis (NTA) and Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) analysis. NTA demonstrated the concentration of AH EV/EPs is 3.11 × 10 9 -1.38 × 10 10 particles/ml; the majority sized 76.8-103 nm. SP-IRIS revealed distinct patterns of tetraspanin expression of AH sEVs. An enriched mono-CD63+ sEV subpopulation identified in AH indicates this is a potential AH-specific biomarker. In the setting of retinoblastoma there was a more heterogeneous population of sEVs which normalized with treatment. This suggests a potential clinical application of direct measurement of sEV subpopulations in AH samples to monitor successful tumor response to therapy.

Gene expression profiling (GEP) is clinically validated to stratify the risk of metastasis by assigning uveal melanoma (UM) patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy, which is limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding, or tumor dissemination. Thus, ocular liquid biopsy has emerged as a less-invasive alternative. In this study, we seek to determine the aqueous humor (AH) proteome related to the advanced GEP class 2 using diagnostic AH liquid biopsy specimens. Twenty AH samples were collected from patients with UM, grouped by GEP classes. Protein expression levels of 1472 targets were analyzed, compared between GEP classes, and correlated with clinical features. Significant differentially expressed proteins (DEPs) were subjected to analysis for cellular pathway and upstream regulator identification. The results showed that 45 DEPs detected in the AH could differentiate GEP class 1 and 2 at diagnosis. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy.