Identifying Treatment Resistance Related Pathways by Analyzing Serum Extracellular Vesicles of Patients With Resistant Versus Regressed Retinoblastoma

PURPOSE. To identify the genes and pathways responsible for treatment resistance (TR) in retinoblastoma (RB) by analyzing serum small extracellular vesicles (sEVs) of patients with TR active RB (TR-RB) and completely regressed RB (CR-RB). METHODS. Serum-derived sEVs were characterized by transmission electron microscopy and nanoparticle tracking analysis. sEV transcriptome profiles of two TR-RB and one CR-RB with good response (>20 years tumor free) were compared to their age-matched controls (n = 3). Gene expression data were analyzed by the R Bioconductor package. The CD9 protein and mRNA expression of CD9, CD63, and CD81 were studied in five RB tumors and two control retinae by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. RESULTS. The isolated serum sEVs were round shaped and within the expected size (30150 nm), and they had zeta potentials ranging from -10.8 to 15.9 mV. The mean +/- SD concentrations of sEVs for two adults and four children were 1.1 x 10(12) +/- 0.1 and 5.8 x 10(11) +/- 1.7 particles/mL. Based on log2 fold change of +/- 2 and P < 0.05 criteria, there were 492 dysregulated genes in TR-RB and 184 in CR-RB. KAT2B, VWA1, CX3CL1, MLYCD, NR2F2, USP46-AS1, miR6724-4, and LINC01257 genes were specifically dysregulated in TR-RB. Negative regulation of apoptotic signaling, cell growth, and proton transport genes were greater than fivefold expressed only in TR-RB. CD9, CD63, and CD81 mRNA levels were high in RB tumors versus control retina, with increased and variable CD9 immunoreactivity in the invasive areas of the tumor. CONCLUSIONS. Serum sEVs could serve as a potential liquid biopsy source for understanding TR mechanisms in RB.