Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA.
Department of Radiology, School of Medicine, Stanford University, Stanford, California, USA.
Lab Chip. 2024 Jul 10;24(14):3403-3411. doi: 10.1039/d4lc00331d.
The expression of programmed death-ligand 1 (PD-L1) on extracellular vesicles (EVs) is an emerging biomarker for cancer, and has gained particular interest for its role mediating immunotherapy. However, precise quantification of PD-L1+ EVs in clinical samples remains challenging due to their sparse concentration and the enormity of the number of background EVs in human plasma, limiting applicability of conventional approaches. In this study, we develop a high-throughput droplet-based extracellular vesicle analysis (DEVA) assay for ultrasensitive quantification of EVs in plasma that are dual positive for both PD-L1 and tetraspanin (CD81) known to be expressed on EVs. We achieve a performance that significantly surpasses conventional approaches, demonstrating 360× enhancement in the limit of detection (LOD) and a 750× improvement in the limit of quantitation (LOQ) compared to conventional plate enzyme-linked immunoassay (ELISA). Underlying this performance is DEVA's high throughput analysis of individual EVs one at a time and the high specificity to targeted EVs background. We achieve a 0.006% false positive rate per droplet by leveraging avidity effects that arise from EVs having multiple copies of their target ligands on their surface. We use parallelized optofluidics to rapidly process 10 million droplets per minute, ∼100× greater than conventional approaches. A validation study on a cohort of 14 patients with melanoma confirms DEVA's ability to match conventional ELISA measurements with reduced plasma sample volume and without the need for prior EV purification. This proof-of-concept study demonstrates DEVA's potential for clinical utility to enhance prognosis as well as guide treatment for cancer.
程序性死亡配体 1（PD-L1）在细胞外囊泡（EVs）上的表达是癌症的一种新兴生物标志物，其在介导免疫疗法方面的作用引起了特别关注。然而，由于其浓度稀疏，以及人血浆中背景 EV 的数量巨大，临床样本中 PD-L1+EV 的精确定量仍然具有挑战性，这限制了常规方法的适用性。在这项研究中，我们开发了一种基于液滴的高通量细胞外囊泡分析（DEVA）测定法，用于超灵敏定量双阳性的 EVs，这些 EVs 同时表达已知在 EV 上表达的 PD-L1 和四跨膜蛋白（CD81）。我们实现了显著优于常规方法的性能，与传统的平板酶联免疫吸附测定（ELISA）相比，检测限（LOD）提高了 360 倍，定量限（LOQ）提高了 750 倍。这种性能的基础是 DEVA 对单个 EV 的高通量分析，以及对靶向 EV 的高特异性和背景。我们通过利用 EV 表面上存在多个靶配体的亲合力效应，实现了每个液滴 0.006%的假阳性率。我们使用并行的光流控技术每分钟快速处理 1000 万个液滴，比传统方法快 100 倍。对 14 名黑色素瘤患者的队列进行的验证研究证实了 DEVA 的能力，它可以与传统的 ELISA 测量相匹配，同时减少血浆样本量，且无需事先进行 EV 纯化。这项概念验证研究表明，DEVA 具有临床应用的潜力，可以增强预后，并指导癌症的治疗。
