Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal fibrostenotic strictures in patients with Crohn’s disease

Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal fibrostenotic strictures in patients with Crohn’s disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal fibrostenotic strictures in patients with Crohn’s disease Joachim Høg Mortensen, Majken Lindholm, Lasse Langholm, Paolo Giuffridda, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5882259/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Fibrostenotic stricturing disease affects 30–50% of patients with Crohn’s disease (CD) leading to intestinal resection. Currently, there exists a great medical need to identify biomarkers related to fibrostenotic strictures for optimized patient management. Thus, we investigated PRO-C16 as a biomarker for intestinal fibrosis in patients with CD. Methods Human serum from two independent cohorts of CD patients (cohort 1: n = 44, cohort 2:n = 52), healthy subjects(n = 37), and serum from a chronic rat dextran sodium sulfate(DSS) colitis model were included. The Montreal classification for CD disease behavior was applied for patient phenotyping. Results PRO-C16 was significantly elevated in patients with CD compared to healthy donors (P < 0.001), and in CD patients with fibrostenotic strictures in both cohorts. Furthermore, PRO-C16 was able to separate CD patients with strictures(B2) from CD patients without strictures (B1 and B3) (Cohort 1 [P < 0.01, AUC:0.75], and Cohort 2 [P < 0.05, AUC:0.71). In the chronic DSS rat colitis model, PRO-C16 was significantly elevated after the second and fourth cycle of DSS, reflective of collagen deposition in that model Conclusion The biomarker PRO-C16 was significantly associated with stricturing disease phenotype, indicating that PRO-C16 may be employed as a marker of intestinal fibrosis in CD, with the potential to aid in the clinical development of novel stromal-immune therapeutic agents. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 STUDY HIGHLIGHTS What is known? Intestinal fibrosis in inflammatory bowel disease is not well characterized and only a few tools are available for diagnosing and monitoring intestinal fibrosis. Intestinal fibrosis may lead to the development of intestinal fibrostenotic strictures in patients with Crohn’s disease. What is new? The PRO-C16 biomarker is elevated in serum from patients with Crohn’s disease and in the serum from rodent chronic DSS model, a murine model reflective of intestinal fibrosis. High serum levels of PRO-C16 are associated with fibrostenotic strictures and can discriminate Crohn’s disease with and without fibrostenotic strictures. Thus, the PRO-C16 serum assay may be a potential biomarker for intestinal fibrosis in inflammatory bowel disease, enabling disease monitoring and clinical development of novel anti-fibrotic agents Introduction Crohn’s Disease is a lifelong inflammatory intestinal disease, impacting various parts of the gastrointestinal tract. Crohn’s Disease is characterized by transmural inflammation, which over time can result in irreversible fibrosis. Between 30–50% of Crohn’s disease patients develop severe intestinal fibrosis, which results in fibrostenotic strictures 1–3 . Indeed, fibrostenotic strictures are the leading cause of intestinal resection in Crohn’s disease patients 4 . Currently, diagnosis of fibrostenotic strictures is achieved by endoscopy assessments and imaging techniques e.g., CT or MRE scans. However, these methods are not suitable for sequential monitoring of fibrosis including longitudinal sampling and disease progression. Since collagens are highly expressed in fibrostenotic strictures 5 , serum biomarkers reflecting the remodeling of intestinal extracellular matrix (ECM), may be instrumental for monitoring the development of fibrostenotic strictures 6 and for assisting in the development of anti-fibrotic treatments 1,7 . In this context, markers associated with both fibrogenesis and resolution of fibrosis are needed 7–9 . Intestinal fibrogenesis is strictly associated with chronic inflammation characterized by leukocyte proliferation, increased T-Cell, and leukocyte adhesion to collagens and intestinal microvasculature, in addition to increased endothelial permeability 10,11 . Activated intestinal subepithelial fibroblasts and myofibroblasts are the most prominent contributors to fibrogenesis in IBD, being the main effector cells responsible for collagen synthesis and deposition 9,12,13 . Collagen turnover reflecting degradation or formation can be quantified in serum with the Nordic-Protein FingerPrint assays (Nordic-PFP-assays), which have been previously validated in serum from IBD patients 14–20 . Nordic-PFP-assays reflect specific tissue remodeling processes including epithelium turnover by measuring specific collagen fragments of the basement membrane (type IV, VI, and XXIII collagens), but also remodeling of the lamina propria (type I, III, V collagen) 14–20 Type XVI collagen belongs to the fibril-associated collagen with interrupted helices (FACITs) family 6,21 . Type XVI collagen tissue expression is increased in intestinal tissue from CD patients compared to normal intestinal tissue 6 . FACIT collagens are crucial for the correct structure of the ECM and type XVI is co-expressed with collagen fibrils where they serve as anchors connecting the fibrils of the interstitial matrix (type I, III, V, and XI collagens) to the basement membrane (type IV and type VI collagen) 21–23 . As such FACIT collagens are often overexpressed in the fibrotic tissue along with an overexpression of fibrillar collagens 5,21 . In addition, type XVI collagen is expressed not only by subepithelial myofibroblasts but also by epithelial cells 5 . Recently, Jensen et al. demonstrated that the PRO-C16 biomarker, which measures the formation of type XVI collagen by targeting the C-terminus, was elevated in the serum of patients with IBD and colorectal cancers 24 . Based on these findings by Ratzinger et al. 5 and Jensen et al. 24 , demonstrating type XVI collagen’s relevance in patients with IBD, we wanted to focus on PRO-C16 and investigate the applicability of PRO-C16 as a marker of intestinal fibrostenotic strictures in Crohn’s disease patients. Materials and methods Clinical cohort Two cross-sectional cohorts including patients diagnosed with CD were employed for this study to investigate PRO-C16 as a marker for intestinal fibrosis. Montreal classification of disease behavior were applied to stratify the patients into luminal (B1), fibrostenotic strictures (B2), and fistulizing disease (B3). Cohort 1 included serum samples from CD patients (n = 44) either diagnosed with luminal disease (n = 20), stricturing disease (n = 11), or fistulizing disease (n = 13) collected from San Matteo Hospital Foundation in Pavia, Italy. Cohort 2 included serum from CD patients (n = 52) either diagnosed with luminal disease (n = 12), stricturing disease (n = 27), or fistulizing disease (n = 3) collected from IRCCS Policlinico San Donato in San Donato Milanese, Italy. All patients filed informed consent, and the study was approved by the local ethical committee (Ethics committee of the Fondazione IRCCS, protocol number 20100039131, approval no. E_20100039131). Cohort 3 containing serum from healthy donors (n = 37) were purchased from BioIVT, to compare PRO-C16 levels, measured in CD patients to the levels measured in the healthy subjects. Non-IBD patients, CD patients with extraintestinal manifestation, and any diagnosis of malignancies were excluded from the studies. PRO-C16 assay protocol The competitive ELISA procedure was as follows: a 96-well streptavidin-coated microtiter plate was coated with 100 µL of biotinylated peptide (Biotin- K- PMKTMKGPFG) dissolved in assay buffer (50 mmol/L phosphate-buffered saline with bovine serum albumin (1% w/v), Tween- 20 (0.1% w/v), and bronidox (0.36% v/v) (PBS- BTB), 4 g/L NaCl, pH 7.4) (final concentration of 3.1 ng/mL). The plate was incubated for 30 minutes at 20°C with shaking (300 rpm) and then washed five times in washing buffer (20 mmol/L TRIS, 50 mmol/L NaCl, pH 7.2). A volume of 20 µL of sample/control/selection peptide (PMKTMKGPFG) was added followed by im- mediately addition of 100 µL of monoclonal antibody diluted in assay buffer (final concentration of 62.5 ng/mL). The plate was incubated for 1 hour at 20°C with shaking followed by five washes in washing buffer. Then, 100 µL of goat anti-mouse horseradish peroxidase (HRP)- conjugated IgG antibody (Thermo Scientific, Waltham, MA, USA; cat. #31437) diluted in assay buffer (final concentration of 130 ng/mL) was added to each well. The plate was incubated for 1 hour at 20°C with shaking and subsequently washed five times in a washing buffer. Next, 100 µL Tetramethylbenzidine (TMB, Kem-En-Tec Diagnostics, Taastrup, Denmark) was added and incubated for 15 minutes at 20°C with shaking in the dark. To stop the reaction of TMB, 100 µL of 1% sulfuric acid (H2SO4) was added and the plate was analyzed in a VersaMax ELISA microplate reader at 450 nm with 650 nm as reference. A standard curve was plotted using a 4- 4-parametric mathematical fit model, and data were analyzed using the Softmax Pro v. 6.3 software. Chronic DSS model Chronic DSS colitis was induced by administrating 5% DSS in the drinking water for 4 cycles for 7 days with 7 7-day recovery period with drinking water without DSS. The rats were fasted over-night before blood was drawn from the tail vein on day 0 (n = 48), 7 (n = 48), 14 (n = 42), 21 (n = 39), 28 (n = 36), 35 (n = 33), 42 (n = 30), 49 (n = 27), 56 (n = 24). Disease progression for both acute and chronic DSS colitis models was evaluated using the Disease Activity Index (DAI), which was scored each day of the study and has been described previously 25 . The DSS in vivo study's ethical guidelines were followed in accordance with the legislation and under the ethical approval of the “Dyreforsøgstilsynet” (agreement number: 2017-15-0201-01171). Statistics Statistical analysis was performed using MedCalc version 14 and GraphPad Prism version 9.2. The biomarker levels were presented as mean values and standard error of the mean (SEM). Key data was represented as Tukey plots with interquartile range (IQR). Mixed-effects analysis with Sidak’s test for multiple comparisons was applied to test the differences in changes in PRO-C16 levels between DSS rats and controls. Pearson r correlation was applied to test the association between serum PRO-C16 and DAI in DSS rats and controls. In human cohorts, age and gender were compared using a Kruskal-Wallis test. The differences in PRO-C16 between patients and healthy controls were determined by the Kruskal-Wallis one-way ANOVA test and Dunn’s multiple comparisons test. The diagnostic power of biomarkers was investigated by the area under the receiver-operating characteristics (ROC) curve (AUC) with a 95% confidence interval (CI). Sensitivity and specificity were determined for appropriate cut-off values based on the ROC curves. The significance threshold was set at p < 0.05. Results Patient demographics Two independent cohorts were included to investigate PRO-C16 as a marker of intestinal fibrosis. The cohorts demonstrated similar gender distribution, age range, and disease phenotype (Table 1 ). Differences observed between the cohorts were: disease activity, disease location, and intestinal resection. PRO-C16 did not correlate with disease activity, disease location, gender, or age but PRO-C16 was numerically elevated in CD patients with active disease compared to inactive disease (data not shown). Table 1 Patient demographics Cohort 1 Crohn’s disease Cohort 2 Crohn’s disease Healthy donors General - Total samples 44 52 37 - Gender : n (%) female 16 (37%) 12 (23%) 17 (47%) - Age (years, mean [range]) 36 [19–73] 46 [29–74] 38 [18–76] - Crohn’s Disease Activity Index (CDAI) > 150 24 (54%) 2 (4%) NA Age at diagnosis (n(%)) NA - A1 ( 40) 13 (29%) 14 (27%) Disease location (n(%)) NA - L1 (n, %) 5 (11%) 17 - L2 (n, %) 14 (32%) 0 - L3 (n, %) 25 (57%) 11 Disease behavior (n(%)) NA - B1: Luminal disease 20 (45%) 21 - B2: Stricturing 11 (25%) 27 - B3: Penetrating 13 (30%) 3 Peri-anal disease 0 0 NA Surgery (n(%)) 0 12 NA PRO-C16 is elevated in Crohn’s disease patients vs. healthy donors We initially compared the PRO-C16 levels of CD patients and healthy donors (cohorts 1 and 2) and investigated PRO-C16 serum levels in CD patients with inactive and active disease activity (cohort 1). PRO-C16 serum levels were elevated in Crohn’s disease patients compared to healthy donors (cohort 1, P < 0.0001; cohort 2, P < 0.01) (Fig. 1 ). INSERT FIGURE 1 HERE PRO-C16 is associated with fibrostenotic strictures in Crohn’s disease Patients were then stratified according to the Montreal classification of disease behavior to assess PRO-C16 as a serum marker of intestinal fibrosis. PRO-C16 was significantly associated with Crohn’s disease patients diagnosed with fibrostenosis and was significantly elevated compared to healthy donors, luminal disease, and fistula (Fig. 2 A). This was confirmed in cohort 2, where PRO-C16 was demonstrated to be elevated in fibrostenotic patients compared to healthy donors and luminal patients (Fig. 2 B). The discriminate power of PRO-C16 to identify fibrostenotic CD was evaluated in cohort 1 (B2 vs. B1/B3[P < 0.01, AUC: 0.75]) and validated in cohort 2 (B2 vs. B1/B3 [P < 0.05, AUC: 0. 71]) (Fig. 3 )]. INSERT FIGURE 2 HERE INSERT FIGURE 3 HERE PRO-C16 serum levels are elevated in the chronic DSS model Finally, the PRO-C16 findings from CD clinical cohorts were verified in the chronic DSS colitis rat model. Masson trichrome staining revealed that increased collagen deposition was present during the entire DSS study and intestinal fibrosis was accumulating after each cycle mimicking the ongoing fibrogenesis (Fig. 4 ). To verify PRO-C16 as a marker of intestinal fibrosis, PRO-C16 was also measured in serum from an in vivo chronic DSS colitis study in rats. PRO-C16 serum levels were highest from the second DSS cycle and were significantly elevated at day 21 (end of second DSS cycle) and day 49 (end of fourth DSS cycle) compared to control rats. Disease activity (DAI) peaked after the end of DSS cycle 2 (day 21) and was comparable to the end of DSS cycle 3 (day 35) and 4 (day 49) (Fig. 5 E). Body weight was lower in DSS rats (Fig. 5 A) and DSS rats also demonstrated the highest DAI after the second cycle (Fig. 5 B). Water consumption was increased in DSS rats (Fig. 5 C) and food consumption was similar between the groups (Fig. 5 D). INSERT FIGURE 4 HERE INSERT FIGURE 5 HERE Discussion Based on two independent patient cohorts, the results presented herein provide evidence that PRO-C16 could be a clinically relevant biomarker for identifying CD patients with fibrostenotic strictures and have utility for clinical development in patients treated with novel anti-fibrotic agents. This assumption was also supported by results obtained in the chronic DSS colitis model, where PRO-C16 was also demonstrated to be elevated in the DSS rats compared to healthy rats (vehicle control). Furthermore, these results are in line with a previous report by Jensen et al. demonstrating that PRO-C16 is elevated in patients with UC 24 , where, despite the rarity of fibrostenotic strictures, there is active ECM remodeling resulting in intestinal fibrosis 6,26–28 . Recently Bourgonje et al, demonstrated that CD patients with stricturing disease had significantly less degradation of type I, III, and IV collagens, which fits with the overall notion that the tissue balance in stricturing CD patients has increased accumulation of collagen and reduced collagen fibrolysis 29 . PRO-16 did not correlate with disease activity, which could be explained by the disease activity scores reflecting ongoing inflammation. Thus, it further strengthens the notion that the PRO-C16 biomarker is associated with ECM remodeling and ongoing fibrogenesis and is a potential candidate biomarker for supporting the clinical development of novel anti-fibrotic agents e.g., anti-TL1A, ALK5 inhibitors and ROCK inhibitors. The results obtained in this study are in line with the current biology and knowledge on the biology of type XVI collagen and its possible implication in tissue fibrosis 5 . Overexpression of type XVI collagen is believed to promote and support chronic inflammation thus contributing to fibrogenesis 6,21 . Type XVI collagen has been shown to increase the expression and activation of MMP-9 locally in tissue at the site of insult 30 . This could explain the elevated levels of PRO-C16 observed in Crohn’s disease patients with active disease, as MMP-9 tissue expression leads to chronic intestinal mucosal damage 14–20,31,32 . This process is thought to be facilitated by type XVI collagen’s ability to maintain adhesion of intestinal subepithelial myofibroblasts, thus increasing the pathological development of intestinal fibrostenotic strictures 5 . As type XVI collagen belongs to the FACIT collagen family, it is conceivable that other FACIT collagens, e.g. type IX, XII, XIV, XIX, XX, XI, and XXII collagens, could also be implicated and promoting intestinal fibrogenesis, due to their association with fibrillar collagens such as type I, III, and V collagens whose excessive deposition is the hallmark of tissue fibrosis, which is in line with the elevated serum levels of PRO-C16 in fibrostenotic CD patients observed in this study. As a result, luminal CD patients with elevated PRO-C16 serum levels could likely have increased ECM remodeling and intestinal fibrogenesis with premature and undiagnosed fibrostenotic stricture. We also observed differences between the two cohorts. The B3 group from Cohort 1 has a sample size of n = 13, whereas Cohort 2 B3 group has a sample size of n = 3. While CD patients with the B3 often are presented with concomitant intestinal fibrosis, we also believe that the PRO-C16 marker measures active fibrogenesis. As such, the low levels of PRO-C16 in the B3 groups could indicate non-active intestinal fibrogenesis. This is speculative, as to why future studies are warranted to further expand upon the PRO-C16 marker and its capabilities concerning intestinal fibrosis. Thus, longitudinal prospective studies are essential for further development of PRO-C16 as a marker of fibrostenotic stricture development. PRO-C16 was elevated in the DSS colitis model compared to the control rats, could indicate that the fibrogenesis was successfully induced, as increased collagen deposition and fibrogenesis were observed in the submucosa and mucosa. Furthermore, it seemed that the PRO-C16 serum level peak followed the same degree of fibrosis that could be observed from the Masson trichrome staining. Especially after the second cycle of DSS at day 21 and after the fourth cycle of DSS at day 49 severe fibrogenesis was present in the mucosa and submucosa space of the DSS rats. PRO-C16 serum levels were also lower in the DSS rats at cycle 3 at day 35, which also seemed to follow the pattern observed from the Masson trichrome staining revealing only mild to moderate fibrogenesis in the DSS rats (Fig. 4 G) compared to the control rats. However, while we don’t know the exact course of the variability of PRO-C16 measurements after the DSS cycles, then we believe it could indicate that continuous tissue destruction and remodeling will increase the fibrogenesis and intestinal fibrosis which is reflected by the elevated levels of PRO-C16 after the second cycle. The drop in PRO-C16 levels after cycle 3 could be an indication of change in the phenotype with less fibrosis but increased tissue destruction, where we see that the PRO-C16 is elevated again after cycle 4. Two independent cohorts were included where PRO-C16 serum levels were proven to be significantly elevated in Crohn’s disease patients with fibrostenotic strictures, and the elevated PRO-C16 serum levels in the chronic rat DSS model suggests that PRO-C16 could be related to intestinal fibrogenesis. While this strengthens the overall robustness of the study, there are also some limitations. Even though MRE is not preferred for consecutive evaluation of fibrostenotic stricture development, it would still be relevant to evaluate PRO-C16 serum levels and their association with the MRE findings. The two human cohorts included were cross-sectional studies and some discrepancies were observed e.g. disease activity and diagnosis of fistula. However, PRO-C16 was not demonstrated to be related to either disease activity or fistulas indicating that PRO-C16 was affected by this, but for future studies, it would be relevant to test PRO-C16 longitudinal studies for the potential to monitor intestinal fibrosis development. In addition, it would also be relevant to evaluate PRO-C16 in patients with CD who have a quiescent inflammatory disease but with a progressing stricturing phenotype in a longitudinal study. Given PRO-C16’s association to intestinal fibrosis it would also be relevant to evaluate how PRO-C16 relates to the risk of recurrence in a post-operative CD patient population and treatment response would provide additional clarity in this patient population with unmet clinical needs. Since disease location and creeping fat are relevant factors for intestinal fibrosis, future studies should also aim at a bigger sample size where meaningful stratification is based on disease location and the presence of creeping fat. Finally, the PRO-C16 marker is not specific for CD and for future studies, non-IBD controls should be included for the investigations of how PRO-C16 is regulated in other diseases. But in the context of CD and intestinal fibrosis, PRO-C16 could be a relevant marker. Conclusion There is an urgent need for biomarkers able to identify CD patients with fibrostenotic strictures. Our data demonstrates that PRO-C16 biomarker could represent a potential biomarker for existing intestinal fibrosis in CD. The data also opens the perspective for further investigations PRO-C16 and other FACIT collagens and their association to fibrostenotic CD and intestinal fibrosis development. Abbreviations AUC: area under the curve, CD: Crohn’s disease, CDAI: Crohn’s disease activity index, DAI: disease activity index, DSS: dextran sodium sulfate ECM: extracellular matrix, ELISA: enzyme-linked immunosorbent assay, FACIT: fibril associated collagen with interrupted helices, IBD: inflammatory bowel disease, IQR: interquartile range, ROC-curve: receiver operator, PFP: Protein FingerPrint Declarations Guarantor of the article The leading author, Joachim H. Mortensen, is the guarantor of the article specific author Contributions J.H. Mortensen: Concept and design of the study, acquisition of data, analysis and interpretation of data, drafting the article and revising it critically for important intellectual content., M. Lindholm, L.L. Langholm, T. Manon-Jensen, A-C. Bay-Jensen, M.A. Karsdal, G. Mazza, P. Giuffrida, L. Pastorelli, F. Caprioli, M. Pinzani, and A. Di Sabatino: Concept and design of the study, interpretation of data, revising critically for important intellectual content. G. Mazza, P. Giuffrida, F. Caprioli, L. Pastorelli, M. Pinzani, and A. Di Sabatino: Collecting IBD patient samples. Funding declaration No funding was applied for this study. Disclosures J.H. Mortensen, M. Lindholm, L.L. Langholm, T. Manon-Jensen, A-C. Bay-Jensen, and M.A. Karsdal are employed at Nordic Bioscience A/S which is a company involved in the discovery and development of biochemical biomarkers. T. Manon-Jensen, A-C. Bay-Jensen, and M.A. Karsdal own stocks in Nordic Bioscience. D. Ruane is employed at Janssen Immunology which is a company involved in drug development. G. Mazza, P. Giuffrida, F. Caprioli, L. Pastorelli, M. Pinzani, and A. Di Sabatino have no competing interests with the content of this publication. References Bos S, Laukens D. Metabolic modulation during intestinal fibrosis. 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Mol Aspects Med . 2019;65(September 2018):100–109. doi:10.1016/j.mam.2018.10.003 Latella G, Rogler G, Bamias G, et al. Results of the 4th scientific workshop of the ECCO (I): Pathophysiology of intestinal fibrosis in IBD. J Crohns Colitis . Published online April 11, 2014. doi:10.1016/j.crohns.2014.03.008 Bourgonje AR, Alexdottir MS, Otten AT, et al. Serological biomarkers of type I, III and IV collagen turnover are associated with the presence and future progression of stricturing and penetrating Crohnʼs disease. Aliment Pharmacol Ther . 2022;(May):1–19. doi:10.1111/apt.17063 Bedal KB, Gr??ssel S, Oefner PJ, Reinders J, Reichert TE, Bauer R. Collagen XVI induces expression of MMP9 via modulation of AP-1 transcription factors and facilitates invasion of oral squamous cell carcinoma. PLoS One . 2014;9(1). doi:10.1371/journal.pone.0086777 Gao Q, Meijer MJW, Kubben FJGM, et al. Expression of matrix metalloproteinases (MMP)-2 and MMP-9 in intestinal tissue of patients with inflammatory bowel diseases (IBD). Dig Liver Dis . 2005;37:584–592. Porter AC, Aubrecht J, Birch C, et al. Biomarkers of Crohn’s Disease to Support the Development of New Therapeutic Interventions. Inflamm Bowel Dis . 2020;26(10):1498–1508. doi:10.1093/ibd/izaa215 Additional Declarations Competing interest reported. J.H. Mortensen, L. Langholm, T. Manon-Jensen, A-C. Bay-Jensen, and M.A. Karsdal are employed at Nordic Bioscience A/S which is a company involved in the discovery and development of biochemical biomarkers. T. Manon-Jensen, A-C. Bay-Jensen, and M.A. Karsdal own stocks in Nordic Bioscience. D. Ruane is employed at Janssen Immunology which is a company involved in drug development Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Feb, 2025 Editor assigned by journal 23 Jan, 2025 Submission checks completed at journal 23 Jan, 2025 First submitted to journal 22 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5882259","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":406178197,"identity":"913f8ed1-1f5f-4076-bd11-cb6a0c4fd4d1","order_by":0,"name":"Joachim Høg Mortensen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYBACxhkMjA9AdBsDD8MBYrUwG5CmhUGCgU0CpKUBqIU4wDy7O626oOKebB/72YMHPjDYyek2EHLYnLPbbs84U2zcxpOXcHAGQ7KxGSHnMc7I3Xabty0hsU2Cx+Aw0DuJ24jRUky6FmaStWyW5jmTAPWLARF+MZyRu/EzT0WC7Pz2s4c/fKiwkyOspQGFa0BAOQjIE6FmFIyCUTAKRjoAAKadQfMT6zDKAAAAAElFTkSuQmCC","orcid":"","institution":"Nordic Bioscience (Denmark)","correspondingAuthor":true,"prefix":"","firstName":"Joachim","middleName":"Høg","lastName":"Mortensen","suffix":""},{"id":406178198,"identity":"f5f0a64a-1908-49f0-9ddd-77210146028e","order_by":1,"name":"Majken Lindholm","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Majken","middleName":"","lastName":"Lindholm","suffix":""},{"id":406178199,"identity":"eb4927c5-65a0-4cd9-b358-f9876a9041ae","order_by":2,"name":"Lasse Langholm","email":"","orcid":"","institution":"Nordic Bioscience (Denmark)","correspondingAuthor":false,"prefix":"","firstName":"Lasse","middleName":"","lastName":"Langholm","suffix":""},{"id":406178200,"identity":"b90e64a2-2792-47fb-b11d-d745187b6e4d","order_by":3,"name":"Paolo Giuffridda","email":"","orcid":"","institution":"University of Pavia","correspondingAuthor":false,"prefix":"","firstName":"Paolo","middleName":"","lastName":"Giuffridda","suffix":""},{"id":406178202,"identity":"2634a6a0-7e6b-4db3-b6ad-a9e7b8b659b8","order_by":4,"name":"Darren Ruane","email":"","orcid":"","institution":"Janssen Immunology","correspondingAuthor":false,"prefix":"","firstName":"Darren","middleName":"","lastName":"Ruane","suffix":""},{"id":406178203,"identity":"e26779fd-1514-497f-b4f7-d946cd1ab288","order_by":5,"name":"Tina Manon-Jensen","email":"","orcid":"","institution":"Nordic Bioscience (Denmark)","correspondingAuthor":false,"prefix":"","firstName":"Tina","middleName":"","lastName":"Manon-Jensen","suffix":""},{"id":406178204,"identity":"aa86667f-9d4d-4749-a95b-2e4e5049235b","order_by":6,"name":"Giuseppe Mazza","email":"","orcid":"","institution":"The Royal Free Hospital","correspondingAuthor":false,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Mazza","suffix":""},{"id":406178205,"identity":"72858c03-dd95-4a92-b0a7-5d6151ac1446","order_by":7,"name":"Flavio Caprioli","email":"","orcid":"","institution":"University of Milan","correspondingAuthor":false,"prefix":"","firstName":"Flavio","middleName":"","lastName":"Caprioli","suffix":""},{"id":406178207,"identity":"3d67584c-507f-4d31-973f-82d704ac035e","order_by":8,"name":"Luca Pastorelli","email":"","orcid":"","institution":"University of Milan","correspondingAuthor":false,"prefix":"","firstName":"Luca","middleName":"","lastName":"Pastorelli","suffix":""},{"id":406178213,"identity":"0a7bcd26-4871-4a1b-8b0e-c91818490e7b","order_by":9,"name":"Anne-Christine Bay-Jensen","email":"","orcid":"","institution":"Nordic Bioscience (Denmark)","correspondingAuthor":false,"prefix":"","firstName":"Anne-Christine","middleName":"","lastName":"Bay-Jensen","suffix":""},{"id":406178215,"identity":"dc1dcb61-d629-41f6-b184-cfa3c642aaea","order_by":10,"name":"Massimo Pinzani","email":"","orcid":"","institution":"The Royal Free Hospital","correspondingAuthor":false,"prefix":"","firstName":"Massimo","middleName":"","lastName":"Pinzani","suffix":""},{"id":406178217,"identity":"1da30f91-7424-4bb2-a3da-098a232b5ced","order_by":11,"name":"Morten Asser Karsdal","email":"","orcid":"","institution":"Nordic Bioscience (Denmark)","correspondingAuthor":false,"prefix":"","firstName":"Morten","middleName":"Asser","lastName":"Karsdal","suffix":""},{"id":406178219,"identity":"7d6d27b5-8403-4a3f-b7e0-628785733a4a","order_by":12,"name":"Antonio Di Sabatino","email":"","orcid":"","institution":"University of Pavia","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"Di","lastName":"Sabatino","suffix":""}],"badges":[],"createdAt":"2025-01-22 15:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5882259/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5882259/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75310846,"identity":"8c54d8c5-3608-452b-969f-e5ab911310f9","added_by":"auto","created_at":"2025-02-03 09:07:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":769654,"visible":true,"origin":"","legend":"\u003cp\u003eSerum levels of PRO-C16 (type XVI collagen), in healthy donors and Crohn’s disease (CD) patients (A: cohort 1, B: cohort 2). Asterisk (*) indicates significant differences: *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001. Data are depicted as interquartile range [IQR] with 10–90 percentile. PRO-C16 levels are associated with disease activity in Crohn’s disease.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5882259/v1/fc212ce13d09c7c124f864e1.png"},{"id":75310847,"identity":"0f470544-5951-4aff-970e-442537a6e730","added_by":"auto","created_at":"2025-02-03 09:07:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":990962,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in PRO-C16 serum levels in Crohn’s disease (CD) phenotypes and healthy donors (HD). Montreal classification of disease behavior were applied to stratify the patients in to luminal (B1), fibrostenotic strictures (B2), and fistulizing disease (B3). A) cohort 1 and B) cohort 2, Asterisk (*) indicates significant differences: *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001. Data are depicted as interquartile range [IQR] with 10–90 percentile.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5882259/v1/f5ef27de17c93cbd3b6730ac.png"},{"id":75312588,"identity":"7e77694d-ce79-4667-8843-65d8c96cbeda","added_by":"auto","created_at":"2025-02-03 09:15:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":892389,"visible":true,"origin":"","legend":"\u003cp\u003eSerum levels of PRO-C16 (type XVI collagen), in healthy donors (HD) and Crohn’s disease (CD) stratified according to the Montreal classification of disease behavior was applied: luminal (B1), fibrostenotic strictures (B2), and fistulizing disease (B3) for cohort 1 and cohort 2. Receiver operator characteristics-curve (ROC-curve) analysis demonstrates that the biomarker PRO-C16 significantly separates stricturing CD (B2) vs. luminal/penetrating CD (B1/B3). Asterisk (*) indicates significant differences: *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001. Error bars depict the standard error of the mean (SEM).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5882259/v1/9aff7232e3ebfb803eb3c68b.png"},{"id":75310853,"identity":"3e4e975a-2a73-4a9d-8630-7f7c792d179f","added_by":"auto","created_at":"2025-02-03 09:07:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1656755,"visible":true,"origin":"","legend":"\u003cp\u003eResults from Chronic DSS colitis study with four cycles of DSS. A) Differences in body weight between DSS rats and control rats, B) Disease activity index (DAI) between DSS rats and control rats, C) Water consumption, D) food consumption, E) PRO-16 levels depicted as the change from baseline between DSS rats and control rats. Asterisk (*) indicates significant difference: *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001. Error bars depict the standard error of the mean (SEM).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5882259/v1/c49a31484f6c18e3a8be8416.png"},{"id":75310857,"identity":"65805ec8-112c-4409-badb-9be479a8884b","added_by":"auto","created_at":"2025-02-03 09:07:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4796727,"visible":true,"origin":"","legend":"\u003cp\u003eCollagen deposition in healthy control rats (A-B) and DSS rats (C-J). The tissue was stained with Masson Trichrome, where blue stains collagens, red/pink stains muscle fibers and cytoplasm of e.g., the epithelium. A) control rat at day 7, B) control rat at day 56, C) Cycle 1 DSS rat at day 7, D) Cycle 1 DSS rat at day 14, E) Cycle 2 DSS rat at day 21, F) Cycle 2 DSS rat at day 28, G) Cycle 3 DSS rat at day 35, H) Cycle 3 DSS rat at day 42, I) Cycle 4 DSS rat at day 49, J) Cycle 4 DSS rat at day 56. Asterisks depict the intestinal lumen.\u003c/p\u003e","description":"","filename":"Figure4revised.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5882259/v1/9c991c33b1587cf417265ade.jpg"},{"id":75313415,"identity":"2cae8b96-b9ef-40da-b0ee-db54ba1277d7","added_by":"auto","created_at":"2025-02-03 09:23:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10388159,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5882259/v1/bfb7a562-0566-468a-b47f-d9c97c77b189.pdf"}],"financialInterests":"Competing interest reported. J.H. Mortensen, L. Langholm, T. Manon-Jensen, A-C. Bay-Jensen, and M.A. Karsdal are employed at Nordic Bioscience A/S which is a company involved in the discovery and development of biochemical biomarkers. T. Manon-Jensen, A-C. Bay-Jensen, and M.A. Karsdal own stocks in Nordic Bioscience. D. Ruane is employed at Janssen Immunology which is a company involved in drug development","formattedTitle":"Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal fibrostenotic strictures in patients with Crohn’s disease","fulltext":[{"header":"STUDY HIGHLIGHTS","content":"\u003ch4\u003eWhat is known?\u003c/h4\u003e\n\u003cul\u003e\n \u003cli\u003eIntestinal fibrosis in inflammatory bowel disease is not well characterized and only a few tools are available for diagnosing and monitoring intestinal fibrosis.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eIntestinal fibrosis may lead to the development of intestinal fibrostenotic strictures in patients with Crohn\u0026rsquo;s disease.\u003c/li\u003e\n\u003c/ul\u003e\n\u003ch4\u003eWhat is new?\u003c/h4\u003e\n\u003cul\u003e\n \u003cli\u003eThe PRO-C16 biomarker is elevated in serum from patients with Crohn\u0026rsquo;s disease and in the serum from rodent chronic DSS model, a murine model reflective of intestinal fibrosis.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHigh serum levels of PRO-C16 are associated with fibrostenotic strictures and can discriminate Crohn\u0026rsquo;s disease with and without fibrostenotic strictures.\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThus, the PRO-C16 serum assay may be a potential biomarker for intestinal fibrosis in inflammatory bowel disease, enabling disease monitoring and clinical development of novel anti-fibrotic agents\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCrohn\u0026rsquo;s Disease is a lifelong inflammatory intestinal disease, impacting various parts of the gastrointestinal tract. Crohn\u0026rsquo;s Disease is characterized by transmural inflammation, which over time can result in irreversible fibrosis. Between 30\u0026ndash;50% of Crohn\u0026rsquo;s disease patients develop severe intestinal fibrosis, which results in fibrostenotic strictures\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Indeed, fibrostenotic strictures are the leading cause of intestinal resection in Crohn\u0026rsquo;s disease patients\u003csup\u003e4\u003c/sup\u003e. Currently, diagnosis of fibrostenotic strictures is achieved by endoscopy assessments and imaging techniques e.g., CT or MRE scans. However, these methods are not suitable for sequential monitoring of fibrosis including longitudinal sampling and disease progression. Since collagens are highly expressed in fibrostenotic strictures\u003csup\u003e5\u003c/sup\u003e, serum biomarkers reflecting the remodeling of intestinal extracellular matrix (ECM), may be instrumental for monitoring the development of fibrostenotic strictures\u003csup\u003e6\u003c/sup\u003e and for assisting in the development of anti-fibrotic treatments\u003csup\u003e1,7\u003c/sup\u003e. In this context, markers associated with both fibrogenesis and resolution of fibrosis are needed\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIntestinal fibrogenesis is strictly associated with chronic inflammation characterized by leukocyte proliferation, increased T-Cell, and leukocyte adhesion to collagens and intestinal microvasculature, in addition to increased endothelial permeability\u003csup\u003e10,11\u003c/sup\u003e. Activated intestinal subepithelial fibroblasts and myofibroblasts are the most prominent contributors to fibrogenesis in IBD, being the main effector cells responsible for collagen synthesis and deposition\u003csup\u003e9,12,13\u003c/sup\u003e. Collagen turnover reflecting degradation or formation can be quantified in serum with the Nordic-Protein FingerPrint assays (Nordic-PFP-assays), which have been previously validated in serum from IBD patients\u003csup\u003e14\u0026ndash;20\u003c/sup\u003e. Nordic-PFP-assays reflect specific tissue remodeling processes including epithelium turnover by measuring specific collagen fragments of the basement membrane (type IV, VI, and XXIII collagens), but also remodeling of the lamina propria (type I, III, V collagen)\u003csup\u003e14\u0026ndash;20\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eType XVI collagen belongs to the fibril-associated collagen with interrupted helices (FACITs) family\u003csup\u003e6,21\u003c/sup\u003e. Type XVI collagen tissue expression is increased in intestinal tissue from CD patients compared to normal intestinal tissue\u003csup\u003e6\u003c/sup\u003e. FACIT collagens are crucial for the correct structure of the ECM and type XVI is co-expressed with collagen fibrils where they serve as anchors connecting the fibrils of the interstitial matrix (type I, III, V, and XI collagens) to the basement membrane (type IV and type VI collagen)\u003csup\u003e21\u0026ndash;23\u003c/sup\u003e. As such FACIT collagens are often overexpressed in the fibrotic tissue along with an overexpression of fibrillar collagens\u003csup\u003e5,21\u003c/sup\u003e. In addition, type XVI collagen is expressed not only by subepithelial myofibroblasts but also by epithelial cells\u003csup\u003e5\u003c/sup\u003e. Recently, Jensen et al. demonstrated that the PRO-C16 biomarker, which measures the formation of type XVI collagen by targeting the C-terminus, was elevated in the serum of patients with IBD and colorectal cancers\u003csup\u003e24\u003c/sup\u003e. Based on these findings by Ratzinger et al.\u003csup\u003e5\u003c/sup\u003e and Jensen et al.\u003csup\u003e24\u003c/sup\u003e, demonstrating type XVI collagen\u0026rsquo;s relevance in patients with IBD, we wanted to focus on PRO-C16 and investigate the applicability of PRO-C16 as a marker of intestinal fibrostenotic strictures in Crohn\u0026rsquo;s disease patients.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical cohort\u003c/h2\u003e \u003cp\u003eTwo cross-sectional cohorts including patients diagnosed with CD were employed for this study to investigate PRO-C16 as a marker for intestinal fibrosis. Montreal classification of disease behavior were applied to stratify the patients into luminal (B1), fibrostenotic strictures (B2), and fistulizing disease (B3). Cohort 1 included serum samples from CD patients (n\u0026thinsp;=\u0026thinsp;44) either diagnosed with luminal disease (n\u0026thinsp;=\u0026thinsp;20), stricturing disease (n\u0026thinsp;=\u0026thinsp;11), or fistulizing disease (n\u0026thinsp;=\u0026thinsp;13) collected from San Matteo Hospital Foundation in Pavia, Italy. Cohort 2 included serum from CD patients (n\u0026thinsp;=\u0026thinsp;52) either diagnosed with luminal disease (n\u0026thinsp;=\u0026thinsp;12), stricturing disease (n\u0026thinsp;=\u0026thinsp;27), or fistulizing disease (n\u0026thinsp;=\u0026thinsp;3) collected from IRCCS Policlinico San Donato in San Donato Milanese, Italy. All patients filed informed consent, and the study was approved by the local ethical committee (Ethics committee of the Fondazione IRCCS, protocol number 20100039131, approval no. E_20100039131). Cohort 3 containing serum from healthy donors (n\u0026thinsp;=\u0026thinsp;37) were purchased from BioIVT, to compare PRO-C16 levels, measured in CD patients to the levels measured in the healthy subjects. Non-IBD patients, CD patients with extraintestinal manifestation, and any diagnosis of malignancies were excluded from the studies.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePRO-C16 assay protocol\u003c/h3\u003e\n\u003cp\u003eThe competitive ELISA procedure was as follows: a 96-well streptavidin-coated microtiter plate was coated with 100 \u0026micro;L of biotinylated peptide (Biotin- K- PMKTMKGPFG) dissolved in assay buffer (50 mmol/L phosphate-buffered saline with bovine serum albumin (1% w/v), Tween- 20 (0.1% w/v), and bronidox (0.36% v/v) (PBS- BTB), 4 g/L NaCl, pH 7.4) (final concentration of 3.1 ng/mL). The plate was incubated for 30 minutes at 20\u0026deg;C with shaking (300 rpm) and then washed five times in washing buffer (20 mmol/L TRIS, 50 mmol/L NaCl, pH 7.2). A volume of 20 \u0026micro;L of sample/control/selection peptide (PMKTMKGPFG) was added followed by im- mediately addition of 100 \u0026micro;L of monoclonal antibody diluted in assay buffer (final concentration of 62.5 ng/mL). The plate was incubated for 1 hour at 20\u0026deg;C with shaking followed by five washes in washing buffer. Then, 100 \u0026micro;L of goat anti-mouse horseradish peroxidase (HRP)- conjugated IgG antibody (Thermo Scientific, Waltham, MA, USA; cat. #31437) diluted in assay buffer (final concentration of 130 ng/mL) was added to each well. The plate was incubated for 1 hour at 20\u0026deg;C with shaking and subsequently washed five times in a washing buffer. Next, 100 \u0026micro;L Tetramethylbenzidine (TMB, Kem-En-Tec Diagnostics, Taastrup, Denmark) was added and incubated for 15 minutes at 20\u0026deg;C with shaking in the dark. To stop the reaction of TMB, 100 \u0026micro;L of 1% sulfuric acid (H2SO4) was added and the plate was analyzed in a VersaMax ELISA microplate reader at 450 nm with 650 nm as reference. A standard curve was plotted using a 4- 4-parametric mathematical fit model, and data were analyzed using the Softmax Pro v. 6.3 software.\u003c/p\u003e\n\u003ch3\u003eChronic DSS model\u003c/h3\u003e\n\u003cp\u003eChronic DSS colitis was induced by administrating 5% DSS in the drinking water for 4 cycles for 7 days with 7 7-day recovery period with drinking water without DSS. The rats were fasted over-night before blood was drawn from the tail vein on day 0 (n\u0026thinsp;=\u0026thinsp;48), 7 (n\u0026thinsp;=\u0026thinsp;48), 14 (n\u0026thinsp;=\u0026thinsp;42), 21 (n\u0026thinsp;=\u0026thinsp;39), 28 (n\u0026thinsp;=\u0026thinsp;36), 35 (n\u0026thinsp;=\u0026thinsp;33), 42 (n\u0026thinsp;=\u0026thinsp;30), 49 (n\u0026thinsp;=\u0026thinsp;27), 56 (n\u0026thinsp;=\u0026thinsp;24).\u003c/p\u003e \u003cp\u003eDisease progression for both acute and chronic DSS colitis models was evaluated using the Disease Activity Index (DAI), which was scored each day of the study and has been described previously \u003csup\u003e25\u003c/sup\u003e. The DSS in vivo study's ethical guidelines were followed in accordance with the legislation and under the ethical approval of the \u003cem\u003e\u0026ldquo;Dyrefors\u0026oslash;gstilsynet\u0026rdquo;\u003c/em\u003e (agreement number: 2017-15-0201-01171).\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eStatistical analysis was performed using MedCalc version 14 and GraphPad Prism version 9.2. The biomarker levels were presented as mean values and standard error of the mean (SEM). Key data was represented as Tukey plots with interquartile range (IQR). Mixed-effects analysis with Sidak\u0026rsquo;s test for multiple comparisons was applied to test the differences in changes in PRO-C16 levels between DSS rats and controls. Pearson r correlation was applied to test the association between serum PRO-C16 and DAI in DSS rats and controls. In human cohorts, age and gender were compared using a Kruskal-Wallis test. The differences in PRO-C16 between patients and healthy controls were determined by the Kruskal-Wallis one-way ANOVA test and Dunn\u0026rsquo;s multiple comparisons test. The diagnostic power of biomarkers was investigated by the area under the receiver-operating characteristics (ROC) curve (AUC) with a 95% confidence interval (CI). Sensitivity and specificity were determined for appropriate cut-off values based on the ROC curves. The significance threshold was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePatient demographics\u003c/h2\u003e \u003cp\u003eTwo independent cohorts were included to investigate PRO-C16 as a marker of intestinal fibrosis. The cohorts demonstrated similar gender distribution, age range, and disease phenotype (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Differences observed between the cohorts were: disease activity, disease location, and intestinal resection. PRO-C16 did not correlate with disease activity, disease location, gender, or age but PRO-C16 was numerically elevated in CD patients with active disease compared to inactive disease (data not shown).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient demographics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCohort 1\u003c/p\u003e \u003cp\u003eCrohn\u0026rsquo;s disease\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCohort 2\u003c/p\u003e \u003cp\u003eCrohn\u0026rsquo;s disease\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHealthy donors\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGeneral\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- Total samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eGender : n (%) female\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e16 (37%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (23%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e17 (47%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eAge (years, mean [range])\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e36 [19\u0026ndash;73]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e46 [29\u0026ndash;74]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e38 [18\u0026ndash;76]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eCrohn\u0026rsquo;s Disease Activity Index (CDAI)\u0026thinsp;\u0026gt;\u0026thinsp;150\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e24 (54%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e2 (4%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge at diagnosis (n(%))\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eA1 (\u0026lt;\u0026thinsp;16)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0 (0%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e3 (6%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eA2 (16\u0026ndash;40)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e31 (71%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e35(67%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eA3 (\u0026gt;\u0026thinsp;40)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e13 (29%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14 (27%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDisease location (n(%))\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eL1 (n, %)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e5 (11%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eL2 (n, %)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e14 (32%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eL3 (n, %)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e25 (57%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDisease behavior (n(%))\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eB1: Luminal disease\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e20 (45%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eB2: Stricturing\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e11 (25%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- \u003cb\u003eB3: Penetrating\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e13 (30%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePeri-anal disease\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSurgery (n(%))\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePRO-C16 is elevated in Crohn’s disease patients vs. healthy donors\u003c/h3\u003e\n\u003cp\u003eWe initially compared the PRO-C16 levels of CD patients and healthy donors (cohorts 1 and 2) and investigated PRO-C16 serum levels in CD patients with inactive and active disease activity (cohort 1). PRO-C16 serum levels were elevated in Crohn\u0026rsquo;s disease patients compared to healthy donors (cohort 1, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; cohort 2, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eINSERT FIGURE \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e HERE\u003c/p\u003e\n\u003ch3\u003ePRO-C16 is associated with fibrostenotic strictures in Crohn’s disease\u003c/h3\u003e\n\u003cp\u003ePatients were then stratified according to the Montreal classification of disease behavior to assess PRO-C16 as a serum marker of intestinal fibrosis. PRO-C16 was significantly associated with Crohn\u0026rsquo;s disease patients diagnosed with fibrostenosis and was significantly elevated compared to healthy donors, luminal disease, and fistula (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This was confirmed in cohort 2, where PRO-C16 was demonstrated to be elevated in fibrostenotic patients compared to healthy donors and luminal patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The discriminate power of PRO-C16 to identify fibrostenotic CD was evaluated in cohort 1 (B2 vs. B1/B3[P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, AUC: 0.75]) and validated in cohort 2 (B2 vs. B1/B3 [P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, AUC: 0. 71]) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eINSERT FIGURE \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e HERE\u003c/p\u003e \u003cp\u003eINSERT FIGURE \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e HERE\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePRO-C16 serum levels are elevated in the chronic DSS model\u003c/h2\u003e \u003cp\u003eFinally, the PRO-C16 findings from CD clinical cohorts were verified in the chronic DSS colitis rat model. Masson trichrome staining revealed that increased collagen deposition was present during the entire DSS study and intestinal fibrosis was accumulating after each cycle mimicking the ongoing fibrogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To verify PRO-C16 as a marker of intestinal fibrosis, PRO-C16 was also measured in serum from an in vivo chronic DSS colitis study in rats. PRO-C16 serum levels were highest from the second DSS cycle and were significantly elevated at day 21 (end of second DSS cycle) and day 49 (end of fourth DSS cycle) compared to control rats. Disease activity (DAI) peaked after the end of DSS cycle 2 (day 21) and was comparable to the end of DSS cycle 3 (day 35) and 4 (day 49) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Body weight was lower in DSS rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and DSS rats also demonstrated the highest DAI after the second cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Water consumption was increased in DSS rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and food consumption was similar between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eINSERT FIGURE \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e HERE\u003c/p\u003e \u003cp\u003eINSERT FIGURE \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e HERE\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBased on two independent patient cohorts, the results presented herein provide evidence that PRO-C16 could be a clinically relevant biomarker for identifying CD patients with fibrostenotic strictures and have utility for clinical development in patients treated with novel anti-fibrotic agents. This assumption was also supported by results obtained in the chronic DSS colitis model, where PRO-C16 was also demonstrated to be elevated in the DSS rats compared to healthy rats (vehicle control). Furthermore, these results are in line with a previous report by Jensen et al. demonstrating that PRO-C16 is elevated in patients with UC\u003csup\u003e24\u003c/sup\u003e, where, despite the rarity of fibrostenotic strictures, there is active ECM remodeling resulting in intestinal fibrosis\u003csup\u003e6,26\u0026ndash;28\u003c/sup\u003e. Recently Bourgonje et al, demonstrated that CD patients with stricturing disease had significantly less degradation of type I, III, and IV collagens, which fits with the overall notion that the tissue balance in stricturing CD patients has increased accumulation of collagen and reduced collagen fibrolysis\u003csup\u003e29\u003c/sup\u003e. PRO-16 did not correlate with disease activity, which could be explained by the disease activity scores reflecting ongoing inflammation. Thus, it further strengthens the notion that the PRO-C16 biomarker is associated with ECM remodeling and ongoing fibrogenesis and is a potential candidate biomarker for supporting the clinical development of novel anti-fibrotic agents e.g., anti-TL1A, ALK5 inhibitors and ROCK inhibitors.\u003c/p\u003e \u003cp\u003eThe results obtained in this study are in line with the current biology and knowledge on the biology of type XVI collagen and its possible implication in tissue fibrosis\u003csup\u003e5\u003c/sup\u003e. Overexpression of type XVI collagen is believed to promote and support chronic inflammation thus contributing to fibrogenesis\u003csup\u003e6,21\u003c/sup\u003e. Type XVI collagen has been shown to increase the expression and activation of MMP-9 locally in tissue at the site of insult\u003csup\u003e30\u003c/sup\u003e. This could explain the elevated levels of PRO-C16 observed in Crohn\u0026rsquo;s disease patients with active disease, as MMP-9 tissue expression leads to chronic intestinal mucosal damage\u003csup\u003e14\u0026ndash;20,31,32\u003c/sup\u003e. This process is thought to be facilitated by type XVI collagen\u0026rsquo;s ability to maintain adhesion of intestinal subepithelial myofibroblasts, thus increasing the pathological development of intestinal fibrostenotic strictures\u003csup\u003e5\u003c/sup\u003e. As type XVI collagen belongs to the FACIT collagen family, it is conceivable that other FACIT collagens, e.g. type IX, XII, XIV, XIX, XX, XI, and XXII collagens, could also be implicated and promoting intestinal fibrogenesis, due to their association with fibrillar collagens such as type I, III, and V collagens whose excessive deposition is the hallmark of tissue fibrosis, which is in line with the elevated serum levels of PRO-C16 in fibrostenotic CD patients observed in this study. As a result, luminal CD patients with elevated PRO-C16 serum levels could likely have increased ECM remodeling and intestinal fibrogenesis with premature and undiagnosed fibrostenotic stricture.\u003c/p\u003e \u003cp\u003eWe also observed differences between the two cohorts. The B3 group from Cohort 1 has a sample size of n\u0026thinsp;=\u0026thinsp;13, whereas Cohort 2 B3 group has a sample size of n\u0026thinsp;=\u0026thinsp;3. While CD patients with the B3 often are presented with concomitant intestinal fibrosis, we also believe that the PRO-C16 marker measures active fibrogenesis. As such, the low levels of PRO-C16 in the B3 groups could indicate non-active intestinal fibrogenesis. This is speculative, as to why future studies are warranted to further expand upon the PRO-C16 marker and its capabilities concerning intestinal fibrosis. Thus, longitudinal prospective studies are essential for further development of PRO-C16 as a marker of fibrostenotic stricture development.\u003c/p\u003e \u003cp\u003ePRO-C16 was elevated in the DSS colitis model compared to the control rats, could indicate that the fibrogenesis was successfully induced, as increased collagen deposition and fibrogenesis were observed in the submucosa and mucosa. Furthermore, it seemed that the PRO-C16 serum level peak followed the same degree of fibrosis that could be observed from the Masson trichrome staining. Especially after the second cycle of DSS at day 21 and after the fourth cycle of DSS at day 49 severe fibrogenesis was present in the mucosa and submucosa space of the DSS rats. PRO-C16 serum levels were also lower in the DSS rats at cycle 3 at day 35, which also seemed to follow the pattern observed from the Masson trichrome staining revealing only mild to moderate fibrogenesis in the DSS rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) compared to the control rats. However, while we don\u0026rsquo;t know the exact course of the variability of PRO-C16 measurements after the DSS cycles, then we believe it could indicate that continuous tissue destruction and remodeling will increase the fibrogenesis and intestinal fibrosis which is reflected by the elevated levels of PRO-C16 after the second cycle. The drop in PRO-C16 levels after cycle 3 could be an indication of change in the phenotype with less fibrosis but increased tissue destruction, where we see that the PRO-C16 is elevated again after cycle 4.\u003c/p\u003e \u003cp\u003eTwo independent cohorts were included where PRO-C16 serum levels were proven to be significantly elevated in Crohn\u0026rsquo;s disease patients with fibrostenotic strictures, and the elevated PRO-C16 serum levels in the chronic rat DSS model suggests that PRO-C16 could be related to intestinal fibrogenesis. While this strengthens the overall robustness of the study, there are also some limitations. Even though MRE is not preferred for consecutive evaluation of fibrostenotic stricture development, it would still be relevant to evaluate PRO-C16 serum levels and their association with the MRE findings. The two human cohorts included were cross-sectional studies and some discrepancies were observed e.g. disease activity and diagnosis of fistula. However, PRO-C16 was not demonstrated to be related to either disease activity or fistulas indicating that PRO-C16 was affected by this, but for future studies, it would be relevant to test PRO-C16 longitudinal studies for the potential to monitor intestinal fibrosis development. In addition, it would also be relevant to evaluate PRO-C16 in patients with CD who have a quiescent inflammatory disease but with a progressing stricturing phenotype in a longitudinal study. Given PRO-C16\u0026rsquo;s association to intestinal fibrosis it would also be relevant to evaluate how PRO-C16 relates to the risk of recurrence in a post-operative CD patient population and treatment response would provide additional clarity in this patient population with unmet clinical needs.\u003c/p\u003e \u003cp\u003eSince disease location and creeping fat are relevant factors for intestinal fibrosis, future studies should also aim at a bigger sample size where meaningful stratification is based on disease location and the presence of creeping fat. Finally, the PRO-C16 marker is not specific for CD and for future studies, non-IBD controls should be included for the investigations of how PRO-C16 is regulated in other diseases. But in the context of CD and intestinal fibrosis, PRO-C16 could be a relevant marker.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThere is an urgent need for biomarkers able to identify CD patients with fibrostenotic strictures. Our data demonstrates that PRO-C16 biomarker could represent a potential biomarker for existing intestinal fibrosis in CD. The data also opens the perspective for further investigations PRO-C16 and other FACIT collagens and their association to fibrostenotic CD and intestinal fibrosis development.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eAUC: area under the curve, CD: Crohn\u0026rsquo;s disease, CDAI: Crohn\u0026rsquo;s disease activity index, DAI: disease activity index, DSS: dextran sodium sulfate ECM: extracellular matrix, ELISA: enzyme-linked immunosorbent assay, FACIT: fibril associated collagen with interrupted helices, IBD: inflammatory bowel disease, IQR: interquartile range, ROC-curve: receiver operator, PFP: Protein FingerPrint\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eGuarantor of the article\u003c/h3\u003e\n\u003cp\u003eThe leading author, Joachim H. Mortensen, is the guarantor of the article\u003c/p\u003e\n\u003ch3\u003especific author Contributions\u003c/h3\u003e\n\u003cp\u003eJ.H. Mortensen: Concept and design of the study, acquisition of data, analysis and interpretation of data, drafting the article and revising it critically for important intellectual content., M. Lindholm, L.L. Langholm, T. Manon-Jensen, A-C. Bay-Jensen, M.A. Karsdal,\u0026nbsp;G. Mazza, P. Giuffrida, L. Pastorelli, F. Caprioli, M. Pinzani, and A. Di Sabatino: Concept and design of the study, interpretation of data, revising critically for important intellectual content.\u0026nbsp;G. Mazza, P. Giuffrida, F. Caprioli, L. Pastorelli, M. Pinzani, and A. Di Sabatino: Collecting IBD patient samples.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eFunding declaration\u003c/h3\u003e\n\u003cp\u003eNo funding was applied for this study.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eDisclosures\u003c/h3\u003e\n\u003cp\u003eJ.H. Mortensen, M. Lindholm, L.L. Langholm, T. Manon-Jensen, A-C. Bay-Jensen, and M.A. Karsdal are employed at Nordic Bioscience A/S which is a company involved in the discovery and development of biochemical biomarkers. T. Manon-Jensen, A-C. Bay-Jensen, and M.A. Karsdal own stocks in Nordic Bioscience. D. Ruane is employed at Janssen Immunology which is a company involved in drug development. G. Mazza, P. Giuffrida, F. Caprioli, L. Pastorelli, M. Pinzani, and A. Di Sabatino have no competing interests with the content of this publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBos S, Laukens D. Metabolic modulation during intestinal fibrosis. \u003cem\u003eJ Dig Dis\u003c/em\u003e. 2020;21(6):319\u0026ndash;325. doi:10.1111/1751-2980.12882\u003c/li\u003e\n\u003cli\u003eGiuffrida P, Pinzani M, Corazza GR, Sabatino A Di. 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Misbalance in type III collagen formation/degradation as a novel serological biomarker for penetrating (Montreal B3) Crohn\u0026rsquo;s disease. \u003cem\u003eAliment Pharmacol Ther\u003c/em\u003e. 2017;46:26\u0026ndash;39. doi:10.1111/apt.14092\u003c/li\u003e\n\u003cli\u003eMortensen JH, Manon-Jensen T, Jensen MD, et al. Ulcerative colitis, Crohn\u0026rsquo;s disease, and irritable bowel syndrome have different profiles of extracellular matrix turnover, which also reflects disease activity in Crohn\u0026rsquo;s disease. \u003cem\u003ePLoS One\u003c/em\u003e. 2017;12(10):1\u0026ndash;16. doi:10.1371/journal.pone.0185855\u003c/li\u003e\n\u003cli\u003eManon-Jensen T, Sun S, Lindholm M, et al. Elevated ectodomain of type 23 collagen is a novel biomarker of the intestinal epithelium to monitor disease activity in ulcerative colitis and Crohn\u0026rsquo;s disease. \u003cem\u003eUnited Eur Gastroenterol J\u003c/em\u003e. Published online 2020. doi:10.1177/2050640620977371\u003c/li\u003e\n\u003cli\u003eHolm Nielsen S, Mortensen JH, Willumsen N, et al. A Fragment of Collagen Type VI alpha-3 chain is Elevated in Serum from Patients with Gastrointestinal Disorders. \u003cem\u003eSci Rep\u003c/em\u003e. 2020;10(1):1\u0026ndash;9. doi:10.1038/s41598-020-62474-1\u003c/li\u003e\n\u003cli\u003eHaaften WT Van, Mortensen JH, Dige AK, et al. Serological Biomarkers of Tissue Turnover Identify Responders to Anti-TNF Therapy in Crohn \u0026rsquo; s Disease : A Pilot Study. \u003cem\u003eClin Transl Gastroenterol\u003c/em\u003e. 2020;11(9):1\u0026ndash;10.\u003c/li\u003e\n\u003cli\u003eMortensen JH, van Haaften WT, Karsdal MA, et al. The Citrullinated and MMP-degraded Vimentin Biomarker (VICM) Predicts Early Response to Anti-TNF\u0026alpha; Treatment in Crohn\u0026rsquo;s Disease. \u003cem\u003eJ Clin Gastroenterol\u003c/em\u003e. 2021;55(1):59\u0026ndash;66. doi:10.1097/MCG.0000000000001341\u003c/li\u003e\n\u003cli\u003eGr\u0026auml;ssel S, Ratzinger S. COL16A1 (collagen, type XVI, alpha 1). \u003cem\u003eAtlas Genet Cytogenet Oncol Haematol\u003c/em\u003e. 2011;34(7):679\u0026ndash;687. doi:10.4267/2042/44804\u003c/li\u003e\n\u003cli\u003eJensen C, Nielsen SH, Mortensen JH, et al. Serum type XVI collagen is associated with colorectal cancer and ulcerative colitis indicating a pathological role in gastrointestinal disorders. \u003cem\u003eCancer Med\u003c/em\u003e. 2018;(June):1\u0026ndash;8. doi:10.1002/cam4.1692\u003c/li\u003e\n\u003cli\u003eShaw LM, Olsen BR. FACIT collagens: diverse molecular bridges in extracellular matrices. \u003cem\u003eTrends Biochem Sci\u003c/em\u003e. 1991;16(C):191\u0026ndash;194. doi:10.1016/0968-0004(91)90074-6\u003c/li\u003e\n\u003cli\u003eJensen C, Nielsen SH, Mortensen JH, et al. Serum type XVI collagen is associated with colorectal cancer and ulcerative colitis indicating a pathological role in gastrointestinal disorders. \u003cem\u003eCancer Med\u003c/em\u003e. 2018;7(9):4619\u0026ndash;4626. doi:10.1002/cam4.1692\u003c/li\u003e\n\u003cli\u003eLindholm M, Manon-Jensen T, Madsen GI, et al. Extracellular Matrix Fragments of the Basement Membrane and the Interstitial Matrix Are Serological Markers of Intestinal Tissue Remodeling and Disease Activity in Dextran Sulfate Sodium Colitis. \u003cem\u003eDig Dis Sci\u003c/em\u003e. 2019;64:3134\u0026ndash;3142. doi:10.1007/s10620-019-05676-6\u003c/li\u003e\n\u003cli\u003eDe Bruyn JR, Meijer SL, Wildenberg ME, Bemelman WA, Van Den Brink GR, D\u0026rsquo;Haens GR. Development of fibrosis in acute and longstanding ulcerative colitis. \u003cem\u003eJ Crohn\u0026rsquo;s Colitis\u003c/em\u003e. 2015;9(11):966\u0026ndash;972. doi:10.1093/ecco-jcc/jjv133\u003c/li\u003e\n\u003cli\u003eLenti MV, Di Sabatino A. Intestinal fibrosis. \u003cem\u003eMol Aspects Med\u003c/em\u003e. 2019;65(September 2018):100\u0026ndash;109. doi:10.1016/j.mam.2018.10.003\u003c/li\u003e\n\u003cli\u003eLatella G, Rogler G, Bamias G, et al. Results of the 4th scientific workshop of the ECCO (I): Pathophysiology of intestinal fibrosis in IBD. \u003cem\u003eJ Crohns Colitis\u003c/em\u003e. Published online April 11, 2014. doi:10.1016/j.crohns.2014.03.008\u003c/li\u003e\n\u003cli\u003eBourgonje AR, Alexdottir MS, Otten AT, et al. Serological biomarkers of type I, III and IV collagen turnover are associated with the presence and future progression of stricturing and penetrating Crohnʼs disease. \u003cem\u003eAliment Pharmacol Ther\u003c/em\u003e. 2022;(May):1\u0026ndash;19. doi:10.1111/apt.17063\u003c/li\u003e\n\u003cli\u003eBedal KB, Gr??ssel S, Oefner PJ, Reinders J, Reichert TE, Bauer R. Collagen XVI induces expression of MMP9 via modulation of AP-1 transcription factors and facilitates invasion of oral squamous cell carcinoma. \u003cem\u003ePLoS One\u003c/em\u003e. 2014;9(1). doi:10.1371/journal.pone.0086777\u003c/li\u003e\n\u003cli\u003eGao Q, Meijer MJW, Kubben FJGM, et al. Expression of matrix metalloproteinases (MMP)-2 and MMP-9 in intestinal tissue of patients with inflammatory bowel diseases (IBD). \u003cem\u003eDig Liver Dis\u003c/em\u003e. 2005;37:584\u0026ndash;592.\u003c/li\u003e\n\u003cli\u003ePorter AC, Aubrecht J, Birch C, et al. Biomarkers of Crohn\u0026rsquo;s Disease to Support the Development of New Therapeutic Interventions. \u003cem\u003eInflamm Bowel Dis\u003c/em\u003e. 2020;26(10):1498\u0026ndash;1508. doi:10.1093/ibd/izaa215\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5882259/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5882259/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eFibrostenotic stricturing disease affects 30\u0026ndash;50% of patients with Crohn\u0026rsquo;s disease (CD) leading to intestinal resection. Currently, there exists a great medical need to identify biomarkers related to fibrostenotic strictures for optimized patient management. Thus, we investigated PRO-C16 as a biomarker for intestinal fibrosis in patients with CD.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman serum from two independent cohorts of CD patients (cohort 1: n\u0026thinsp;=\u0026thinsp;44, cohort 2:n\u0026thinsp;=\u0026thinsp;52), healthy subjects(n\u0026thinsp;=\u0026thinsp;37), and serum from a chronic rat dextran sodium sulfate(DSS) colitis model were included. The Montreal classification for CD disease behavior was applied for patient phenotyping.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePRO-C16 was significantly elevated in patients with CD compared to healthy donors (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and in CD patients with fibrostenotic strictures in both cohorts. Furthermore, PRO-C16 was able to separate CD patients with strictures(B2) from CD patients without strictures (B1 and B3) (Cohort 1 [P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, AUC:0.75], and Cohort 2 [P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, AUC:0.71). In the chronic DSS rat colitis model, PRO-C16 was significantly elevated after the second and fourth cycle of DSS, reflective of collagen deposition in that model\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe biomarker PRO-C16 was significantly associated with stricturing disease phenotype, indicating that PRO-C16 may be employed as a marker of intestinal fibrosis in CD, with the potential to aid in the clinical development of novel stromal-immune therapeutic agents.\u003c/p\u003e","manuscriptTitle":"Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal fibrostenotic strictures in patients with Crohn’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-03 09:07:40","doi":"10.21203/rs.3.rs-5882259/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-05T14:09:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-23T13:17:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-23T13:14:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Gastroenterology","date":"2025-01-22T15:38:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7143b85a-35e0-44c5-be93-ef35833f2b3e","owner":[],"postedDate":"February 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-08T08:23:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-03 09:07:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5882259","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5882259","identity":"rs-5882259","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}