Comprehensive optimization of urinary exfoliated tumor cells tests in bladder cancer with a promising microfluidic platform

BACKGROUND: Enrichment of urinary exfoliated tumor cells (UETCs) is a noninvasive way of bladder cancer diagnosis, but the lack of specific capture and identification of tumor cells from the urine remains a limitation that impedes the development of liquid biopsy. METHODS: The CytoBot(®) 2000, a novel circulating cell isolation and enrichment platform, was used for UETCs isolation after comprehensive optimization. The commercial cell lines of bladder cancer were used in spiking assay for cell recovery test. The flow cytometry and immunofluorescent staining assays were performed for expression validation of capture target and identification markers. The performance of optimized platform was validated by 159 clinical samples and analyzed using receiver operator characteristic curve. RESULTS: The chip that had a pore diameter of 15*20 μm could reduce the background residues while maintaining a higher cell recovery rate. We found that the cell capture ability of chip significantly improved after anti‐EpCam antibody encapsulation, but not with T4L6FM1. In identification system optimization, the spiking assay and validation of clinical sample showed that the performance of CK20 and DBC‐1 were better that pan‐CK in tumor cell identification, in addition, the staining quality is more legible with CK20. CONCLUSION: The optimized capture chip is more specific for UETCs isolation. CK20 and DBC‐1 are both sensitive biomarkers of UETCs in bladder cancer diagnosis. The performance of this optimized platform is excellent in clinical test that improves the accuracy of urine cell testing and provides a new alternative for the clinical application of BLCA liquid biopsy assessment.