Applying CRISPR/Cas13 to Construct Exosomal PD-L1 Ultrasensitive Biosensors for Dynamic Monitoring of Tumor Progression in Immunotherapy

Abstract

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.