Extracellular vesicle (EV) secretion is a dynamic process crucial to cellular communication. Temporally sorting EVs, i.e., separating the newly-produced ones from the pre-existing, can allow not only deep understanding of EV dynamics, but also the discovery of potential EV biomarkers that are related to disease progression or responsible to drug intervention. However, the high similarity between the nascent and pre-existing EVs makes temporal separation extremely challenging. Here, by co-translational introduction of azido groups to act as a timestamp for click chemistry labelling, we develop a microfluidic-based strategy to enable selective isolation of nascent EVs stimulated by an external cue. In two mouse models of anti-PD-L1 immunotherapy, we demonstrate the strategy’s feasibility and reveal the high positive correlation of nascent PD-L1⁺ EV level to tumor volume, suggesting an important role of nascent EVs in response to immunotherapy in cancer treatment.

细胞外囊泡（EV）分泌是一个对细胞通讯至关重要的动态过程。对 EV 进行临时分类，即将新产生的 EV 与已有的 EV 分开，不仅可以深入了解 EV 动态，还可以发现与疾病进展相关或负责药物干预的潜在 EV 生物标志物。然而，新生电动汽车和现有电动汽车之间的高度相似性使得时间分离极具挑战性。在这里，通过共翻译引入叠氮基团作为点击化学标记的时间戳，我们开发了一种基于微流体的策略，以实现外部线索刺激的新生 EV 的选择性分离。在两个抗 PD-L1 免疫治疗的小鼠模型中，我们证明了该策略的可行性，并揭示了新生 PD-L1 ⁺ EV 水平与肿瘤体积的高度正相关性，表明新生 EV 在癌症治疗中对免疫治疗的反应中发挥着重要作用。