Department of Chemistry, The University of Memphis, Memphis, TN 38152.
Diagnostics Imaging Department, St Jude Children's Research Hospital, Memphis, TN 38105.
Theranostics. 2018 Apr 9;8(10):2722-2738. doi: 10.7150/thno.21358. eCollection 2018.
Exosomes are a potential source of cancer biomarkers. Probing tumor-derived exosomes can offer a potential non-invasive way to diagnose cancer, assess cancer progression, and monitor treatment responses. Novel molecular methods would facilitate exosome analysis and accelerate basic and clinical exosome research. A standard gold-coated glass microscopy slide was used to develop a miniaturized affinity-based device to capture exosomes in a target-specific manner with the assistance of low-cost 3-D printing technology. Gold nanorods coated with QSY21 Raman reporters were used as the label agent to quantitatively detect the target proteins based on surface enhanced Raman scattering spectroscopy. The expressions of several surface protein markers on exosomes from conditioned culture media of breast cancer cells and from HER2-positive breast cancer patients were quantitatively measured. The data was statistically analyzed and compared with healthy controls. A miniaturized 17 × 5 Au array device with 2-mm well size was fabricated to capture exosomes in a target-specific manner and detect the target proteins on exosomes with surface enhanced Raman scattering gold nanorods. This assay can specifically detect exosomes with a limit of detection of 2×10 exosomes/mL and analyze over 80 purified samples on a single device within 2 h. Using the assay, we have showed that exosomes derived from MDA-MB-231, MDA-MB-468, and SKBR3 breast cancer cells give distinct protein profiles compared to exosomes derived from MCF12A normal breast cells. We have also showed that exosomes in the plasma from HER2-positive breast cancer patients exhibit significantly (P ≤ 0.01) higher level of HER2 and EpCAM than those from healthy donors. We have developed a simple, inexpensive, highly efficient, and portable Raman exosome assay for detection and protein profiling of exosomes. Using the assay and model exosomes from breast cancer cells, we have showed that exosomes exhibit diagnostic surface protein markers, reflecting the protein profile of their donor cells. Through proof-of-concept studies, we have identified HER2 and EpCAM biomarkers on exosomes in plasma from HER2-positive breast cancer patients, suggesting the diagnostic potential of these markers for breast cancer diagnostics. This assay would accelerate exosome research and pave a way to the development of novel cancer liquid biopsy for cancer detection and monitoring.
外泌体是癌症生物标志物的潜在来源。探测肿瘤衍生的外泌体可以提供一种潜在的非侵入性方法来诊断癌症、评估癌症进展和监测治疗反应。新型分子方法将促进外泌体分析并加速基础和临床外泌体研究。 使用标准的镀金玻璃显微镜载玻片，结合低成本的 3D 打印技术，开发了一种小型化的基于亲和力的装置，以特异性捕获外泌体。用 QSY21 拉曼报告分子涂覆的金纳米棒被用作标记物，基于表面增强拉曼散射光谱定量检测靶蛋白。定量测量了乳腺癌细胞条件培养基和 HER2 阳性乳腺癌患者外泌体中几种表面蛋白标志物的表达。对数据进行了统计分析，并与健康对照组进行了比较。 制造了一个小型化的 17×5 Au 阵列装置，具有 2mm 井尺寸，以特异性捕获外泌体，并使用表面增强拉曼散射金纳米棒检测外泌体上的靶蛋白。该测定法可以特异性检测到 2×10 个外泌体/mL 的下限，并在单个装置上 2 小时内分析 80 多个纯化样品。使用该测定法，我们表明与源自 MCF12A 正常乳腺细胞的外泌体相比，源自 MDA-MB-231、MDA-MB-468 和 SKBR3 乳腺癌细胞的外泌体具有不同的蛋白谱。我们还表明，HER2 阳性乳腺癌患者血浆中外泌体的 HER2 和 EpCAM 水平明显高于健康供体（P≤0.01）。 我们开发了一种简单、廉价、高效且便携式的拉曼外泌体测定法，用于检测和对外泌体进行蛋白谱分析。使用该测定法和源自乳腺癌细胞的模型外泌体，我们表明外泌体表现出诊断性表面蛋白标志物，反映了其供体细胞的蛋白谱。通过概念验证研究，我们确定了 HER2 和 EpCAM 标志物在 HER2 阳性乳腺癌患者血浆中外泌体上，提示这些标志物对乳腺癌诊断具有诊断潜力。该测定法将加速外泌体研究，并为开发用于癌症检测和监测的新型癌症液体活检铺平道路。
