Comprehensive Detection of PD-L1 Protein and mRNA in Tumor Cells and Extracellular Vesicles through a Real-Time qPCR Assay

A growingnumber of studies have shown that tumor cells secreteextracellular vesicles (EVs) containing programmed death-ligand 1(PD-L1) protein. These vesicles can travel to lymph nodes and remotelyinactivate T cells, thereby evading immune system attack. Therefore,the simultaneous detection of PD-L1 protein expression in cells andEVs is of great significance in guiding immunotherapy. Herein, wedeveloped a method based on qPCR for the simultaneous detection ofPD-L1 protein and mRNA in EVs and their parental cells (PREC-qPCRassay). Lipid probes immobilized on magnetic beads were used to captureEVs directly from samples. For RNA assay, EVs were directly brokenby heating and quantified with qPCR. As to protein assay, EVs wererecognized and bound with specific probes (such as aptamers), whichwere used as templates in subsequent qPCR analysis. This method wasused to analyze EVs of patient-derived tumor clusters (PTCs) and plasmasamples from patients and healthy volunteers. The results revealedthat the expression of exosomal PD-L1 in PTCs was correlated withtumor types and significantly higher in plasma-derived EVs from tumorpatients than that of healthy individuals. When extended to cellsand PD-L1 mRNAs, the results showed that the expression of PD-L1 proteinwas consistent with mRNA in cancer cell lines, while PTCs demonstratedsignificant heterogeneity. This comprehensive detection of PD-L1 atfour levels (cell, EVs, protein, and mRNA) is believed to enhanceour understanding of the relationship among PD-L1, tumors, and theimmune system and to provide a promising tool for predicting the benefitsof immunotherapy.

Automated summary

A method for simultaneous detection of PD-L1 protein and mRNA in extracellular vesicles (EVs) and their parental cells was developed using qPCR. The expression of exosomal PD-L1 in patient-derived tumor clusters (PTCs) was found to be correlated with tumor types and significantly higher in plasma-derived EVs from tumor patients compared to healthy individuals. This comprehensive detection of PD-L1 at four levels provides a promising tool for predicting the benefits of immunotherapy.