CD63/81 Small Extracellular Vesicles in the Aqueous Humor are Retinoblastoma Associated

Abstract

Purpose: Although biopsy is contraindicated in retinoblastoma (RB), the aqueous humor (AH) is a robust liquid biopsy source of molecular tumor information, facilitating biomarker discovery. Small extracellular vesicles (sEVs), promising biomarker candidates across multiple cancers, were recently identified in RB AH, but relationships between sEVs and RB clinical features are unknown.

Methods: We analyzed sEVs in 37 AH samples from 18 RB eyes of varying International Intraocular Retinoblastoma Classification (IIRC) groups and explored clinical correlations. Ten samples were collected at diagnosis (DX) and 27 during treatment (Tx). Unprocessed AH underwent Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequentially converted to percentages for analysis.

Results: Comparing DX and Tx samples, a higher percentage of CD63/81+ sEVs was found in DX AH (16.3 ± 11.6% vs. 5.49 ± 3.67% P = 0.0009), with a more homogenous mono-CD63+ sEV population seen in Tx AH (43.5 ± 14.7% vs. 28.8 ± 9.38%, P = 0.0073). Among DX samples, CD63/81+ sEVs were most abundant in group E eyes (n = 2) compared to group D (n = 6) by count (2.75 × 105 ± 3.40 × 105 vs. 5.95 × 103 ± 8.16 × 103, P = 0.0006), and to group A + B (n = 2) by count (2.75 × 105 ± 3.40 × 105 vs. 2.73 × 102 ± 2.59 × 102, P = 0.0096) and percentage (32.1 ± 7.98% vs. 7.79 ± 0.02%, P = 0.0187).

Conclusions: CD63/81+ sEVs enrich AH from RB eyes before treatment and those with more significant tumor burden, suggesting they are tumor-derived. Future research into their cargo may reveal mechanisms of cellular communication via sEVs in RB and novel biomarkers.

Figure 1.

Analytical optimization for SP-IRIS analysis of aqueous humor (AH) by the ExoviewR100 system for tetraspanin expression profiling using 2 unprocessed AH samples at varying input volumes. Representative fluorescent images detected by fluorescent-conjugated antibodies (red = CD63-AF647, green = CD81-AF555, and blue = CD9-AF488). (A) ChipView of AH from case 57 with total sample saturation. (B) ChipView of AH from case 54 with titrated sample saturation. (C) Bar plot displaying EV enumeration (Y axis) based on AH input volume (X axis) for both samples, showing that EV enumeration is linear relative to input volume when the chip is unsaturated, as in case 54 (blue line).

Figure 2.

Clinical demographic information for each retinoblastoma eye included in analysis.

Figure 3.

Quantitative comparison of small extracellular vesicle (sEV) tetraspanin subpopulation expression profiles in diagnostic (DX) and treatment active (Tx) unprocessed retinoblastoma (RB) aqueous humor (AH) samples analyzed with SP-IRIS by ExoView R100. (A) Mean total sEV counts compared between AH samples taken from DX and Tx eyes. (B) Mean sEV subpopulation counts compared between DX and Tx AH. (C) Mean sEV subpopulation percentages compared between DX and Tx AH. The yellow bar represents the CD63/81+ sEV subpopulation, which is the dominant sEV subtype in AH from RB eyes at diagnosis, and the red bar represents the mono-CD63+ sEV subpopulation, which is the dominant sEV subtype in AH from RB eyes collected during treatment. Comparisons made with nonparametric Mann–Whitney U testing. Error bars represent ± one SEM.

Figure 4.

Small extracellular vesicle (sEV) tetraspanin expression profiles in unprocessed diagnostic (DX) retinoblastoma (RB) aqueous humor (AH) samples analyzed using SP-IRIS by ExoView R100. (A) Comparison of mean sEV total counts in DX AH samples stratified by International Intraocular Retinoblastoma Classification (IIRC) group of the eye using non-parametric Mann–Whitney U analysis. (B) Comparison of mean sEV subpopulation counts in DX AH samples stratified by IIRC using ANOVA and Tukey's tests for multiple comparisons. (C) Comparison of mean sEV subpopulation percentages between DX AH samples stratified by IIRC using ANOVA and Tukey's tests for multiple comparisons. Note that the CD63/81+ sEV subpopulation percentage (yellow bar) increases in a stepwise manner from lower to higher disease burden (A + B to D to E). (D) Comparison of mean sEV subpopulation counts in DX AH samples stratified by eye-survival outcome, enucleated versus salvaged, using nonparametric Mann–Whitney U analysis. (E) Comparison of mean EV subpopulation percentages in DX AH samples stratified by eye-survival outcome, enucleated versus salvaged, using nonparametric Mann–Whitney U analysis. As denoted by the yellow bar, the CD63/81+ subpopulation is larger in AH collected from eyes which required enucleation due to tumor burden. Error bars represent ± one SEM.