Department of Anesthesiology and Surgical Intensive Care Unit School of Medicine and School of Biomedical Engineering, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai, China.
State Key Laboratory of Oncogenes and Related Genes, Institute for Personalized Medicine, Shanghai Jiao Tong University, Shanghai, China.
J Extracell Vesicles. 2023 Sep;12(9):e12364. doi: 10.1002/jev2.12364.
Extracellular vesicle (EV) surface proteins, expressed by primary tumours, are important biomarkers for early cancer diagnosis. However, the detection of these EV proteins is complicated by their low abundance and interference from non-EV components in clinical samples. Herein, we present a MEmbrane-Specific Separation and two-step Cascade AmpLificatioN (MESS2CAN) strategy for direct detection of EV surface proteins within 4 h. MESS2CAN utilises novel lipid probes (long chains linked by PEG2K with biotin at one end, and DSPE at the other end) and streptavidin-coated magnetic beads, permitting a 49.6% EV recovery rate within 1 h. A dual amplification strategy with a primer exchange reaction (PER) cascaded by the Cas12a system then allows sensitive detection of the target protein at 10 EV particles per microliter. Using 4 cell lines and 90 clinical test samples, we demonstrate MESS2CAN for analysing HER2, EpCAM and EGFR expression on EVs derived from cells and patient plasma. MESS2CAN reports the desired specificity and sensitivity of EGFR (AUC = 0.98) and of HER2 (AUC = 1) for discriminating between HER2-positive breast cancer, triple-negative breast cancer and healthy donors. MESS2CAN is a pioneering method for highly sensitive in vitro EV diagnostics, applicable to clinical samples with trace amounts of EVs.
细胞外囊泡 (EV) 表面蛋白由原发性肿瘤表达，是早期癌症诊断的重要生物标志物。然而，由于其丰度低，以及临床样本中非 EV 成分的干扰，这些 EV 蛋白的检测较为复杂。在此，我们提出了一种膜特异性分离和两步级联扩增 (MESS2CAN) 策略，用于在 4 小时内直接检测 EV 表面蛋白。MESS2CAN 利用新型脂质探针（一端带有生物素、另一端带有 DSPE 的长链通过 PEG2K 连接）和链霉亲和素包被的磁性珠，在 1 小时内可实现 49.6%的 EV 回收率。然后，通过 Cas12a 系统级联的引物交换反应 (PER) 进行双重扩增策略，可实现以 10 个 EV 颗粒/微升为检测限的目标蛋白的灵敏检测。我们使用 4 种细胞系和 90 个临床测试样本，演示了 MESS2CAN 用于分析源自细胞和患者血浆的 EV 上 HER2、EpCAM 和 EGFR 的表达。MESS2CAN 报告了 EGFR（AUC=0.98）和 HER2（AUC=1）用于区分 HER2 阳性乳腺癌、三阴性乳腺癌和健康供体的理想特异性和灵敏度。MESS2CAN 是一种用于高灵敏度体外 EV 诊断的开创性方法，适用于具有痕量 EV 的临床样本。
