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

Roza C M Opperman; Sofie Bosch; Kamran Nazmi; Floris J Bikker; Henk S Brand; Connie R Jimenez; Tim G J de Meij; Evelien Dekker; Nanne K H de Boer; Wendy E Kaman

doi:10.1021/acs.analchem.4c04586

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

Roza C M Opperman, Sofie Bosch, Kamran Nazmi, Floris J Bikker, Henk S Brand, Connie R Jimenez, Tim G J de Meij, Evelien Dekker, Nanne K H de Boer, Wendy E Kaman

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

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 ZnCl2 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 ZnCl2 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.

Original language	English
Pages (from-to)	20239-20246
Journal	Analytical chemistry
Volume	96
Issue number	51
Early online date	12 Dec 2024
DOIs	https://doi.org/10.1021/acs.analchem.4c04586
Publication status	Published - 24 Dec 2024

Funding

The authors declare the following competing financial interest(s): NdB has served as a speaker for AbbVie and MSD and has served as a consultant and/or principal investigator for TEVA Pharma BV and Takeda. He has received a research grant (unrestricted) from Dr. Falk, TEVA PharmaBV, Dutch Digestive Foundation (MLDS) and Takeda. All outside the submitted work. All other authors declare no conflicts of interest.

Funders	Funder number
TEVA PharmaBV
Maag Lever Darm Stichting
Takeda Pharmaceutical Company

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@article{1ec11cb4771343068cb7d227c56b0cdc,

title = "Detecting Colorectal Neoplasia Using Specific Fecal Fluorogenic Protease-Sensitive Substrates: A Pilot Study",

abstract = "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 ZnCl2 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 ZnCl2 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.",

author = "Opperman, {Roza C M} and Sofie Bosch and Kamran Nazmi and Bikker, {Floris J} and Brand, {Henk S} and Jimenez, {Connie R} and {de Meij}, {Tim G J} and Evelien Dekker and {de Boer}, {Nanne K H} and Kaman, {Wendy E}",

year = "2024",

month = dec,

day = "24",

doi = "10.1021/acs.analchem.4c04586",

language = "English",

volume = "96",

pages = "20239--20246",

journal = "Analytical chemistry",

issn = "0003-2700",

publisher = "American Chemical Society",

number = "51",

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T1 - Detecting Colorectal Neoplasia Using Specific Fecal Fluorogenic Protease-Sensitive Substrates

T2 - A Pilot Study

AU - Opperman, Roza C M

AU - Bosch, Sofie

AU - Nazmi, Kamran

AU - Bikker, Floris J

AU - Brand, Henk S

AU - Jimenez, Connie R

AU - de Meij, Tim G J

AU - Dekker, Evelien

AU - de Boer, Nanne K H

AU - Kaman, Wendy E

PY - 2024/12/24

Y1 - 2024/12/24

N2 - 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 ZnCl2 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 ZnCl2 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.

AB - 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 ZnCl2 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 ZnCl2 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.

U2 - 10.1021/acs.analchem.4c04586

DO - 10.1021/acs.analchem.4c04586

M3 - Article

C2 - 39665576

SN - 0003-2700

VL - 96

SP - 20239

EP - 20246

JO - Analytical chemistry

JF - Analytical chemistry

IS - 51

ER -

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

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