The Vision Center, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.
USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Int J Mol Sci. 2024 Oct 30;25(21):11660. doi: 10.3390/ijms252111660.
Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics.
最近在房水（AH）无细胞 DNA（cfDNA）基因组学方面的进展为视网膜母细胞瘤（RB）的体外分子分析开辟了新途径，RB 是最常见的小儿眼内恶性肿瘤，通常禁止进行活检。虽然这些研究为肿瘤提供了遗传蓝图，但它们缺乏多组学分子表型分析，这对于了解肿瘤的功能状态至关重要。细胞外囊泡（EVs）在 AH 中自然存在，为提供时间分辨的表型信息提供了希望。我们使用多重珠基流式细胞术和单外泌体纳米镜（SEVEN）分析了来自五个 RB 患者、三个脉络膜黑色素瘤（UM）和两个年龄匹配的青光眼（GLC）患者的 AH 中的 EV 表型。研究发现，RB AH 中存在独特的 CD133 富集的 EV，而在 GLC 和 UM AH 中则不存在。这通过对五个 RB 细胞系的进一步分析得到了证实，包括两个商业（Y79、Weri）和三个内部开发的细胞系，证实了 CD133 的富集，并支持其作为 RB 特异性 EV 标志物的作用。单囊泡分析表明 CD133 与 CD81 和 CD63 强烈相关，而 CD9 存在较少。这些通过互补技术验证的结果，使 CD133 成为 RB 衍生 EV 中的关键标志物，为增强 RB 的多组学特征分析和潜在的临床诊断进展铺平了道路。
