Work

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

Year: 2024

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide Background: 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. Methods: 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 ZnCl 2 were performed to further characterize the involved proteases. Results: 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 ZnCl 2 and the cysteine protease inhibitor NEM. Conclusion: 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.

Source: Analytical Chemistry

DOI: 10.1021/acs.analchem.4c04586