Detecting Colorectal Neoplasia Using Specific Fecal Fluorogenic Protease-Sensitive Substrates: A Pilot Study

identification and removal of advanced adenomas (AA) reduce colorectal cancer (CRC) incidence and potentially mortality. CRC screening often uses fecal immunochemical testing to select high-risk individuals for colonoscopy, despite its low sensitivity for AA and relatively high false-positivity rate. Previous studies have linked proteases to CRC development through their ability to facilitate angiogenesis and immunoregulation. This study aims to identify colorectal neoplasia-associated proteases and their substrates as a potential noninvasive screening test, introducing an innovative application of fecal protease profiling, which has previously been limited to tissue samples.

eighteen fluorogenic substrates were designed based on literature. Proteolytic degradation of these substrates was measured in fecal samples of patients with CRC (n = 12), AA (n = 9), nonadvanced adenomas (n = 10), and controls (n = 14). Substrate degradation was correlated to a matched human proteome data set, and underlying proteases were identified based on their recognition patterns. Experiments with protease inhibitors and ZnCl₂ were performed to further characterize the involved proteases.

in total, 7 of the 18 substrates tested showed a significantly decreased proteolytic degradation in feces from patients with any colorectal neoplasia compared to the control group. The l-aspartic acid-l-glutamic acid substrate (ED) showed significantly decreased degradation in AA and CRC patients. ED degradation significantly decreased with the addition of ZnCl₂ and the cysteine protease inhibitor NEM.

we successfully developed colorectal neoplasia-specific fluorogenic substrates, highlighting the ED substrate as a potential substrate for the detection of AA and CRC. Although the responsible proteases require further identification, our results suggest an association with calcium-dependent cysteine proteases.