Extracellular vesicles (EVs) are gaining considerable traction within the liquid biopsy arena, as carriers of information from cells in distant sites that may not be accessible for biopsy. Therefore, there is a need to develop methods to enrich for specific EV subtypes, based on their cells of origin. Here we describe the development of an automated method to enrich tumor-derived EVs from plasma using the CellSearch technology compared to Total EVs isolated using differential ultracentrifugation (DUC). We use a modified CellSearch protocol to enrich EpCAM+ EVs from the plasma of patients with non-small cell lung carcinoma (NSCLC) and triple negative breast cancer (TNBC). As a test case, we examined PD-L1, an immune checkpoint ligand known to be expressed in some tumor tissues, to demonstrate enrichment for EpCAM+ EVs. For this purpose, we developed two custom immunoassays utilizing the Simoa HD-1 analyzer (Quanterix) to detect PD-L1 in EVs and interrogate specific EV populations from human plasma. PD-L1 was present in Total EVs from the plasma of healthy individuals and cancer patients, since it is also expressed on several immune cells. However, EpCAM+ EVs were only detectable from the plasma of cancer patients, suggesting these are tumor-derived EVs. As low as 250 μL of plasma could be used to reliably detect PD-L1 from patient-derived EpCAM+ EVs. In summary, this report demonstrates the development of a robust tumor-derived EV enrichment method from human blood. Furthermore, this proof-of-concept study is extendable to other known cancer-specific proteins expressed on EVs exuded from tumors.

细胞外囊泡 (EV) 在液体活检领域获得了相当大的吸引力，因为它作为来自远距离细胞的信息载体，这些细胞可能无法进行活检。因此，需要开发方法来丰富特定 EV 亚型，基于它们的起源细胞。在这里，我们描述了一种自动化方法的开发，该方法使用 CellSearch 技术从血浆中富集肿瘤来源的 EV，与使用差速超速离心 (DUC) 分离的总 EV 相比。我们使用改进的 CellSearch 协议从非小细胞肺癌 (NSCLC) 和三阴性乳腺癌 (TNBC) 患者的血浆中富集 EpCAM+ EV。作为一个测试案例，我们检查了 PD-L1，一种已知在某些肿瘤组织中表达的免疫检查点配体，以证明 EpCAM+ EVs 的富集。以此目的，我们利用 Simoa HD-1 分析仪 (Quanterix) 开发了两种定制免疫测定法来检测 EV 中的 PD-L1 并询问人血浆中的特定 EV 群体。PD-L1 存在于来自健康个体和癌症患者血浆的总 EV 中，因为它也在几种免疫细胞上表达。然而，EpCAM+ EVs 只能从癌症患者的血浆中检测到，这表明这些是肿瘤衍生的 EVs。低至 250 μL 的血浆可用于从患者来源的 EpCAM+ EV 中可靠地检测 PD-L1。总之，本报告展示了从人类血液中开发出一种强大的肿瘤衍生 EV 富集方法。此外，这项概念验证研究可扩展到在肿瘤渗出的 EV 上表达的其他已知癌症特异性蛋白质。