Detecting Colorectal\nNeoplasia Using Specific Fecal\nFluorogenic Protease-Sensitive Substrates: A Pilot Study

Background: identification and removal of advanced adenomas\n(AA)\nreduce colorectal cancer (CRC) incidence and potentially mortality.\nCRC screening often uses fecal immunochemical testing to select high-risk\nindividuals for colonoscopy, despite its low sensitivity for AA and\nrelatively high false-positivity rate. Previous studies have linked\nproteases to CRC development through their ability to facilitate angiogenesis\nand immunoregulation. This study aims to identify colorectal neoplasia-associated\nproteases and their substrates as a potential noninvasive screening\ntest, introducing an innovative application of fecal protease profiling,\nwhich has previously been limited to tissue samples. Methods: eighteen\nfluorogenic substrates were designed based on literature. Proteolytic\ndegradation of these substrates was measured in fecal samples of patients\nwith CRC (<i>n</i> = 12), AA (<i>n</i> = 9), nonadvanced\nadenomas (<i>n</i> = 10), and controls (<i>n</i> = 14). Substrate degradation was correlated to a matched human proteome\ndata set, and underlying proteases were identified based on their\nrecognition patterns. Experiments with protease inhibitors and ZnCl<sub>2</sub> were performed to further characterize the involved proteases.\nResults: in total, 7 of the 18 substrates tested showed a significantly\ndecreased proteolytic degradation in feces from patients with any\ncolorectal neoplasia compared to the control group. The l-aspartic acid–l-glutamic acid substrate (ED) showed\nsignificantly decreased degradation in AA and CRC patients. ED degradation\nsignificantly decreased with the addition of ZnCl<sub>2</sub> and\nthe cysteine protease inhibitor NEM. Conclusion: we successfully developed\ncolorectal neoplasia-specific fluorogenic substrates, highlighting\nthe ED substrate as a potential substrate for the detection of AA\nand CRC. Although the responsible proteases require further identification,\nour results suggest an association with calcium-dependent cysteine\nproteases.