School of Pharmacy, Fudan University, Shanghai, 200438, China.
Sinopec Shanghai Research Institute of Petrochemical Technology, Shanghai, 201208, China.
Analyst. 2022 Sep 26;147(19):4237-4248. doi: 10.1039/d2an01268e.
Exosomes have been extensively studied as liquid biopsy biomarkers in the past decade. However, the origin and molecular heterogeneity of exosomes hinder the research development moving from proof-of-concept to clinical applications. Herein, we report an integrated microfluidic platform termed Sub-ExoProfile chip, to achieve the selective isolation and subsequent proteomic profiling of multiplex exosome subpopulations simultaneously. The Sub-ExoProfile chip comprises three cylindrical self-assembled nanopillars, on which specific exosome capture antibodies (CD81, EpCAM, HER2) were immobilized to capture and sort different exosome subpopulations. It is worth noting that the 3D porous nanopillars afford enhanced interface binding efficiency; thus, a tumor-specific exosome subpopulation with lowly-expressed surface marker was still isolated with satisfactory capture efficiency. Moreover, the amphiphilic mesoporous silica nanoparticle self-assembled nanopillars also served as a nanoreactor for the enrichment and digestion of exosomal proteins, providing improved performance for the mass-spectrometry based molecular characterization of exosome subpopulations. The Sub-ExoProfile chip was investigated on standard exosome samples from different breast cancer cell lines. The isolation and quantitative detection of exosome subpopulations were in line with the molecular subtype of breast cancer cell lines, and the molecular makeup was confirmed using classic microplate ELISA. Clinical samples from HER2-positive and triple-negative breast cancer patients were also examined using the Sub-ExoProfile chip. The quantitative results of three exosome subpopulations distinguished the three subtypes of breast cancer significantly. Most importantly, the molecular characterization of three exosome subpopulations revealed that the distinct exosome subpopulation participated in a different signaling pathway and performed distinct biological functions. It is envisioned that the analysis of the exosome subpopulation on the Sub-ExoProfile chip will facilitate the screening of tumor-specific exosomal biomarkers and open a new avenue for exosome-based liquid biopsy.
过去十年，外泌体作为液体活检生物标志物得到了广泛研究。然而，外泌体的起源和分子异质性阻碍了从概念验证到临床应用的研究进展。在此，我们报告了一种集成的微流控平台，称为 Sub-ExoProfile 芯片，可同时实现多种外泌体亚群的选择性分离和随后的蛋白质组学分析。Sub-ExoProfile 芯片由三个自组装的纳米柱组成，其中固定了特定的外泌体捕获抗体（CD81、EpCAM、HER2），以捕获和分选不同的外泌体亚群。值得注意的是，3D 多孔纳米柱提供了增强的界面结合效率；因此，即使是表面标志物低表达的肿瘤特异性外泌体亚群，仍能以令人满意的捕获效率分离出来。此外，两亲性介孔硅纳米颗粒自组装纳米柱还可用作外泌体蛋白的富集和消化的纳米反应器，为外泌体亚群的基于质谱的分子特征提供了改进的性能。Sub-ExoProfile 芯片用于来自不同乳腺癌细胞系的标准外泌体样本的研究。外泌体亚群的分离和定量检测与乳腺癌细胞系的分子亚型一致，并用经典的微量板 ELISA 进行了分子组成确认。还使用 Sub-ExoProfile 芯片检查了 HER2 阳性和三阴性乳腺癌患者的临床样本。三种外泌体亚群的定量结果显著区分了三种乳腺癌亚型。最重要的是，三种外泌体亚群的分子特征表明，不同的外泌体亚群参与了不同的信号通路，并发挥了不同的生物学功能。可以预见，Sub-ExoProfile 芯片上对外泌体亚群的分析将有助于筛选肿瘤特异性外泌体生物标志物，并为基于外泌体的液体活检开辟新途径。
