California NanoSystems Institute, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA, 90095, USA.
Key Laboratory for Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Suzhou, 215123, P. R. China.
Adv Sci (Weinh). 2022 May;9(14):e2105853. doi: 10.1002/advs.202105853. Epub 2022 Mar 9.
Well-preserved molecular cargo in circulating extracellular vesicles (EVs) offers an ideal material for detecting oncogenic gene alterations in cancer patients, providing a noninvasive diagnostic solution for detection of disease status and monitoring treatment response. Therefore, technologies that conveniently isolate EVs with sufficient efficiency are desperately needed. Here, a lipid labeling and click chemistry-based EV capture platform ("Click Beads"), which is ideal for EV message ribonucleic acid (mRNA) assays due to its efficient, convenient, and rapid purification of EVs, enabling downstream molecular quantification using reverse transcription digital polymerase chain reaction (RT-dPCR) is described and demonstrated. Ewing sarcoma protein (EWS) gene rearrangements and kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutation status are detected and quantified using EVs isolated by Click Beads and matched with those identified in biopsy specimens from Ewing sarcoma or pancreatic cancer patients. Moreover, the quantification of gene alterations can be used for monitoring treatment responses and disease progression.
循环细胞外囊泡 (EVs) 中保存良好的分子货物为检测癌症患者致癌基因改变提供了理想的材料，为疾病状态的检测和治疗反应的监测提供了一种非侵入性的诊断解决方案。因此，迫切需要能够方便地高效分离 EVs 的技术。本文描述并展示了一种基于脂质标记和点击化学的 EV 捕获平台（“Click Beads”），由于其高效、方便和快速的 EV 纯化，非常适合 EV 信使核糖核酸 (mRNA) 分析，可使用逆转录数字聚合酶链反应 (RT-dPCR) 进行下游分子定量。使用 Click Beads 分离的 EVs 检测和定量了尤文肉瘤蛋白 (EWS) 基因重排和克氏鼠肉瘤病毒致癌基因同源物 (KRAS) 基因突变状态，并与尤文肉瘤或胰腺癌患者活检标本中鉴定的基因改变进行匹配。此外，基因改变的定量可用于监测治疗反应和疾病进展。
