细胞外囊泡 (EV) 在癌症液体活检中变得越来越重要，因为它们含有多种生物标志物，包括蛋白质和 RNA，并且在全身循环。癌细胞衍生的 EV 具有高度异质性，需要多重生物标志物检测技术来提高诊断的准确性。此外，原位 EV 生物标志物检测提高了检测过程的效率，因为 EV 难以处理。在本研究中，开发了通过传统流式细胞术分析的 EV 表面蛋白、程序性细胞死亡配体 1 (PD-L1) 和内部 miRNA-21 (miR-21) 的原位同步检测，用于乳腺癌液体活检。然而，大多数 EV 无法被流式细胞仪识别用于生物标志物检测，因为 EV 的尺寸低于流式细胞仪的可检测尺寸范围。为了解决这个问题，在生物标志物检测过程中，通过1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺（DSPE）-聚乙二醇-DSPE诱导EV簇的形成。因此，来自癌细胞来源的 EV 的 PD-L1 和 miR-21 检测信号均大幅增加，使它们与正常细胞来源的 EV 区分开来。通过流式细胞术分析的 EV 簇进行原位同步癌症生物标志物检测有助于提高基于 EV 的癌症液体活检的灵敏度和准确性。

Extracellular vesicles (EVs) are becoming increasingly important in liquid biopsy for cancer because they contain multiple biomarkers, including proteins and RNAs, and circulate throughout the body. Cancer cell-derived EVs are highly heterogeneous, and multiplexed biomarker detection techniques are required to improve the accuracy of diagnosis. In addition, in situ EV biomarker detection increases the efficiency of the detection process because EVs are difficult to handle. In this study, in situ simultaneous detection of EV surface proteins, programmed cell death-ligand 1 (PD-L1), and internal miRNA-21 (miR-21) analyzed by conventional flow cytometry was developed for a breast cancer liquid biopsy. However, the majority of EVs were not recognized by flow cytometry for biomarker detection because the size of EVs was below the detectable size range of the flow cytometer. To solve this problem, the formation of EV clusters was induced by 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-polyethylene glycol-DSPE during biomarker detection. Consequently, both PD-L1 and miR-21 detection signals from cancer cell-derived EVs were drastically increased, making them distinguishable from normal cell-derived EVs. The in situ simultaneous cancer biomarker detection from EV clusters analyzed by flow cytometry contributes to an increase in the sensitivity and accuracy of the EV-based liquid biopsy for cancer.