ARC Excellence Centre for Nanoscale BioPhotonics (CNBP), Department of Molecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.
Department of Biomedical Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.
ACS Sens. 2020 Mar 27;5(3):764-771. doi: 10.1021/acssensors.9b02377. Epub 2020 Mar 17.
Circulating cancer-derived small extracellular vesicles (EVs) are nanoscale membranous vesicles shed from cancer cells that are released into surrounding body fluids. Small EVs contain biomolecules associated with cancer such as DNA and proteins for cell-to-cell communication. Therefore, small EVs have been regarded as important cancer biomarkers for liquid biopsy-based cancer diagnosis and drug treatment monitoring. However, because of the high heterogeneity and low level of small EVs in body fluids, there is a high demand for sensitive detection and characterization of such vesicles at a molecular level. In this study, we have developed a sensitive and effective approach to simultaneously profile multiple protein biomarkers expressed on cancer-derived small EVs using surface-enhanced Raman spectroscopy (SERS) nanotags in a single test, without complex isolation steps. Rapid and multiplexed phenotypic profiling of small EVs is achieved by mixing specific detection antibody-coated SERS nanotags, filtered conditioned EV-suspended medium (conditioned EVs), and capture antibody (CD63)-conjugated magnetic beads to form a sandwich immunoassay. As a proof-of-concept demonstration, we applied this approach to characterize pancreatic cancer-derived EVs by simultaneously detecting three specific EV surface receptors including Glypican-1, epithelial cell adhesion molecules (EpCAMs), and CD44 variant isoform 6 (CD44V6). The sensitivity of this method was measured down to 2.3 × 10 particles/mL, which is more sensitive and shows higher multiplexing capability than most other reported EV profiling techniques, such as western blot, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, phenotypic profiling of small EVs from colorectal cancer and bladder cancer cell lines (SW480 and C3) was conducted and compared to those derived from pancreatic cancer (Panc-1), highlighting the significant difference in EV phenotypes for various cancer cell types suspended in both phosphate-buffered saline and plasma. Thus, we believe that this technology enables a comprehensive evaluation of small secreted EV heterogeneity with high sensitivity, offering strong potential for accurate noninvasive cancer diagnosis and monitoring of drug treatment. In addition, this assay provides point-of-care use because of the easy sample preparation and portable nature of the Raman spectrometer.
循环肿瘤来源的小细胞外囊泡 (EVs) 是从癌细胞脱落并释放到周围体液中的纳米级膜囊泡。小 EVs 包含与癌症相关的生物分子，如 DNA 和蛋白质，用于细胞间通讯。因此，小 EVs 已被视为液体活检为基础的癌症诊断和药物治疗监测的重要癌症生物标志物。然而，由于体液中小 EVs 的高度异质性和低水平，因此需要在分子水平上对这些囊泡进行灵敏的检测和特征分析。在本研究中，我们开发了一种灵敏有效的方法，通过表面增强拉曼光谱 (SERS) 纳米标签在单次测试中同时对癌症来源的小 EV 上表达的多种蛋白质生物标志物进行分析，而无需复杂的分离步骤。通过混合特定检测抗体包被的 SERS 纳米标签、过滤条件化 EV 悬浮介质（条件化 EVs）和捕获抗体 (CD63) 偶联磁珠，形成三明治免疫测定，快速且多重表型分析小 EVs。作为概念验证演示，我们应用该方法通过同时检测三种特定的 EV 表面受体，包括 Glypican-1、上皮细胞黏附分子 (EpCAMs) 和 CD44 变体 6 (CD44V6)，来表征胰腺癌细胞来源的 EVs。该方法的灵敏度可低至 2.3×10 个颗粒/ml，比大多数其他报道的 EV 分析技术（如 Western blot、酶联免疫吸附测定和流式细胞术）更灵敏且具有更高的多重分析能力。此外，对来自结直肠癌和膀胱癌细胞系（SW480 和 C3）的小 EVs 的表型进行了分析，并与来自胰腺癌细胞系 (Panc-1) 的 EVs 进行了比较，突出了不同癌症细胞类型在磷酸盐缓冲液和血浆中悬浮时 EV 表型的显著差异。因此，我们相信该技术能够以高灵敏度全面评估小分泌 EV 的异质性，为准确的非侵入性癌症诊断和药物治疗监测提供强大的潜力。此外，由于拉曼光谱仪易于进行样本制备且具有便携性，该检测方法可用于即时护理。
