Self-assembly circular multi-DNA mediated rolling circle amplification for sensitive detection of PD-L1+ circulating tumor cells in the peripheral blood of cancer patients

It has been found that evaluating the status of programmed cell death ligand (PD-L1) in the circulating tumor cells (CTCs) was associated with improved efficacy to anti-PD-L1 inhibitors. However, high-quality detection of PD-L1 positive (PD-L1+) CTCs remains a huge challenge. In this study, we propose a short time, high efficiency and applicability method that fabricated by self-assembly circular multi-DNA (CMD) mediating rolling circle amplification (RCA) for highly sensitive and rapid analysis of PD-L1+ cells. Firstly, a short single-stranded circular DNA which could bind specifically with PD-L1 protein was designed. In addition, CMD were designed as self-assembly which could serve as a template in RCA process. Next, the exponential amplification of DNA was achieved by the rolling circle amplification and the fluorescence signal was detected by using the molecular beacons. The unique CMD mediated rolling circle amplification facilitated quantitative detection of PD-L1+ CTCs in the blood of different cancer patients. To visualize the results of CMD on the target cells, we used confocal microscope to assess PD-L1 expression in MCF-7 (breast cancer), OE33 (esophagus cancer) and HCC827 (lung cancer) cells. The results indicated that the CMD mediating RCA can perfectly combine with different kinds of cells, amplify the signal and the recovery rate was over 99 %. What's more, we performed clinical tests in the blood of the corresponding patient (14 cases of breast cancer, 15 esophageal cancer and 11 lung cancer). We further quantified the number of CTCs in patient blood through establishing the standard curve and the limit of detection (LOD) was calculated to be 1 cell/mL. Moreover, the ring structure was designed to have replaceable sequence and to realize combining different CTCs surface markers (such as Her-2). This method has the advantages of high sensitivity, good expansibility, low cost and can be completed in a short time as well. Therefore, it is expected to develop a new CTCs typing detection kit and might thus provide reliable diagnostic results to provide individualized treatment strategies.