Applying CRISPR/Cas13 to Construct Exosomal PD-L1 Ultrasensitive Biosensors for Dynamic Monitoring of Tumor Progression in Immunotherapy
Applying CRISPR/Cas13 to Construct Exosomal PD-L1 Ultrasensitive Biosensors for Dynamic Monitoring of Tumor Progression in Immunotherapy
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DOI：
10.1002/adtp.202000093
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发表时间：
2020-07-20
影响因子：
4.6
通讯作者：
Liu, Wanli
中科院分区：
文献类型：
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作者：
He, Yi;Wu, Yetao;Liu, Wanli
外泌体上的程序性细胞死亡受体1（PD-L1）蛋白（exosomal PD-L1）是用于癌症免疫治疗监测的最有前途的生物标志物之一。然而，目前用于外泌体PD-L1检测的方法灵敏度差、费力且耗时。在此，建立了一种新的方法，称为Aptamer-RPA-TMA-Cas 13 a Assay（ARTCA），该方法能够直接检测血清中的外泌体PD-L1，下限为10个颗粒mL（-1）。在机制上，使用特异性结合外泌体PD-L1的DNA适体，通过重组酶聚合酶扩增（RPA）与转录介导的扩增（TMA）偶联来扩增适体两次，同时用CRISPR/Cas 13 a系统实时检测TMA产物。利用ARTCA，循环外泌体中的PD-L1水平似乎是肿瘤组织中PD-L1表达的可靠标志物。循环外泌体PD-L1水平在肿瘤进展患者中显著增加。ARTCA对血清外泌体PD-L1的超痕量检测为动态监测接受免疫治疗的患者的肿瘤进展提供了一种潜在的便捷方法。这些结果表明，CRISPR-Cas 13 a用于蛋白质检测，循环外泌体PD-L1水平似乎是肿瘤组织中PD-L1表达的可靠标志物，为监测肿瘤进展开辟了新途径。
Programmed cell death receptor 1 (PD-L1) protein on exosomes (exosomal PD-L1) is one of the most promising biomarkers for cancer immunotherapy monitoring. However, current approaches for exosomal PD-L1 detection are poorly sensitive, laborious, and time-consuming. Here, a new method, named Aptamer-RPA-TMA-Cas13a Assay (ARTCA) is established, which enables exosomal PD-L1 to be detected directly in serum with a lower limit of 10 particles mL(-1). Mechanistically, using DNA aptamer specifically binding to exosomal PD-L1, the aptamer is amplified twice by recombinase polymerase amplification (RPA) coupled with transcription-mediated amplification (TMA) and simultaneously the TMA products are detected in real-time with CRISPR/Cas13a system. Utilizing ARTCA, PD-L1 levels in circulating exosomes seem to be a reliable marker of PD-L1 expression in tumor tissue. The level of circulating exosomal PD-L1 increases significantly in patients with tumor progression. Ultra-trace detection of serum exosomal PD-L1 by ARTCA provides a potentially convenient way for dynamic monitoring of tumor progression for patients undergoing immunotherapy. These results demonstrate the use of CRISPR-Cas13a for protein detection, and circulating exosomal PD-L1 levels seem to be a reliable marker as well as PD-L1 expression in tumor tissue, opening up new avenues for monitoring tumor progression.