Cancer cells release extracellular vesicles known as extracellular vesicles (EVs), containing tumor-derived DNA, RNA and proteins within their cargo, into the circulation. Circulating tumor-derived extracellular vesicles (TEV) can be used in the context of serial “liquid biopsies” for early detection of cancer, for monitoring disease burden in patients, and for assessing recurrence in the post-resection setting. Nonetheless, isolating sufficient TEV by ultracentrifugation-based approaches, in order to enable molecular assessment of EVs cargo, can be an arduous, time-consuming process and is inconsistent in the context of yield and purity among institutions. Herein, we describe a microfluidic platform, which we have named MITEV (Microfluidic Isolation of Tumor-derived Extracellular Vesicles) for the rapid isolation of TEV from the plasma of pancreatic cancer patients. The device, which has ~100,000 pillars placed in a zigzag pattern and is coated with antibodies against generic EV surface proteins (anti-CD63, -CD9, and -CD81 antibodies) or the TEV specific anti-Epithelial Cell Adhesion Molecule (EpCAM) antibody, is capable of high-throughput EVs isolation and yields sufficient DNA (total of ~2–14 ng from 2-ml plasma) for downstream genomic analysis. Using two independent quantitative platforms, droplet digital polymerase chain reaction (ddPCR) and molecular barcoding using nanoString nCounter® technology, we can reliably identify KRAS mutations within isolated TEV of treatment-naïve metastatic pancreatic cancer patients. Our study suggests that the MITEV device can be used for point-of-care applications, such as in the context of monitoring residual or recurrent tumor presence in pancreatic cancer patients undergoing therapy.

中文翻译：

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使用微流体平台分离和突变评估胰腺癌细胞外囊泡


癌细胞将称为细胞外囊泡 (EV) 的细胞外囊泡释放到循环中，其中含有肿瘤来源的 DNA、RNA 和蛋白质。循环肿瘤来源的细胞外囊泡 (TEV) 可用于连续“液体活检”，以早期检测癌症、监测患者的疾病负担以及评估切除后的复发情况。尽管如此，通过基于超速离心的方法分离足够的 TEV，以便对 EV 货物进行分子评估，可能是一个艰巨、耗时的过程，并且在各机构的产量和纯度方面不一致。在此，我们描述了一个微流体平台，我们将其命名为 MITEV（肿瘤源性细胞外囊泡的微流体分离），用于从胰腺癌患者的血浆中快速分离 TEV。该装置具有大约 100,000 个呈锯齿形排列的柱子，并涂有针对通用 EV 表面蛋白的抗体（抗 CD63、-CD9 和 -CD81 抗体）或 TEV 特异性抗上皮细胞粘附分子 (EpCAM) 抗体，能够高通量分离 EV，并产生足够的 DNA（从 2 ml 血浆中总共约 2-14 ng）用于下游基因组分析。通过使用两个独立的定量平台、液滴数字聚合酶链式反应 (ddPCR) 和使用 nanoString nCounter® 技术的分子条形码，我们可以可靠地识别未经治疗的转移性胰腺癌患者分离的 TEV 中的KRAS突变。我们的研究表明，MITEV 设备可用于即时护理应用，例如监测接受治疗的胰腺癌患者的残留或复发肿瘤。