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 (30-150 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 × 1012 ± 0.1 and 5.8 × 1011 ± 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.