胶质母细胞瘤（GBM）是最常见的恶性脑肿瘤，预后不佳。诊断和治疗反应的影像学结果是非特异性的。开发非侵入性检测方法以增强影像学会有所帮助。血浆细胞外囊泡（EVs）是一个有前途的生物标志物来源。我们在这里开发了光谱流式细胞术技术，证明了GBM患者与正常供体之间血浆EV总体表型的差异，这可作为液体活检的基础。血浆EV被染色与EV相关的四跨膜蛋白（CD9/CD63/CD81），细胞起源标记物（CD11b/CD31/CD41a/CD45），以及肌动蛋白/吡劳定（以排除细胞碎片）。使用光谱流式细胞术对EV进行分析。使用t-分布随机邻域嵌入（t-SNE）和自组织映射（FlowSOM）的多参数分析，对比GBM和正常供体（ND）的血浆EV。尺寸排除色谱加上基于光谱的流式细胞仪门限值设定，可以富集血浆EV，同时减少背景噪音。GBM患者具有增加的CD9+，CD63+，CD81+和髓系来源的（CD11b+）EV。多参数分析显示，在GBM血浆EV中，表面标记物的表达配置有所不同。鉴别了15个血浆EV亚种群，它们在大小和表面标记物表达上有所不同，其中6个富集在GBM患者中，2个富集在正常供体中。多参数分析表明，GBM患者的非肿瘤血浆EV表型与正常供体有所不同。这种简便快速的分析可以在不纯化肿瘤EV的情况下进行，可能构成液体活检的基础。© 作者 2023年。由牛津大学出版社、神经肿瘤学会和欧洲神经肿瘤学协会发表。

Glioblastoma (GBM) is the most common malignant brain tumor and has a poor prognosis. Imaging findings at diagnosis and in response to treatment are nonspecific. Developing noninvasive assays to augment imaging would be helpful. Plasma extracellular vesicles (EVs) are a promising biomarker source for this. Here, we develop spectral flow cytometry techniques that demonstrate differences in bulk plasma EV phenotype between GBM patients and normal donors that could serve as the basis of a liquid biopsy.Plasma EVs were stained for EV-associated tetraspanins (CD9/CD63/CD81), markers indicating cell of origin (CD11b/CD31/CD41a/CD45), and actin/phalloidin (to exclude cell debris). EVs were analyzed using spectral flow cytometry. Multiparametric analysis using t-distributed stochastic neighbor embedding (t-SNE) and self-organizing maps on flow cytometry data (FlowSOM) was performed comparing GBM and normal donor (ND) plasma EVs.Size exclusion chromatography plus spectral-based flow cytometer threshold settings enriched plasma EVs while minimizing background noise. GBM patients had increased CD9+, CD63+, CD81+, and myeloid-derived (CD11b+) EVs. Multiparametric analysis demonstrated distinct surface marker expression profiles in GBM plasma EVs compared to ND EVs. Fifteen plasma EV sub-populations differing in size and surface marker expression were identified, six enriched in GBM patients and two in normal donors.Multiparametric analysis demonstrates that GBM patients have a distinct nonneoplastic plasma EV phenotype compared to ND. This simple rapid analysis can be performed without purifying tumor EVs and may serve as the basis of a liquid biopsy.© The Author(s) 2023. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.