Noninvasive Detection of Experimental Colitis Using Folate Receptor-β-Targeted PET Imaging in Mice

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Bhowmik, Jannika Rovapalo, Erika Atencio Herre, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8410867/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Positron emission tomography (PET) is a promising noninvasive technique for detecting and monitoring inflammatory bowel disease (IBD). In IBD pathogenesis, activated macrophages – marked by overexpression of folate receptor beta (FR-β) – play a key role. Leveraging this target, folate-based PET tracers have been developed to visualize macrophage-driven inflammation. This study evaluated the feasibility of FR-β-binding aluminum-fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([ 18 F]FOL) PET/computed tomography (CT) for detecting intestinal inflammation in a murine model of acute colitis. Procedures Acute colitis was induced in female and male C57BL/6NCrl mice via 2.5% dextran sodium sulfate (DSS) in drinking water for 6 days, followed by recovery. Healthy age- and sex-matched mice served as controls. Mice underwent whole-body [ 18 F]FOL PET/CT imaging followed by ex vivo biodistribution analysis. Intestinal tissues were further analyzed by digital autoradiography and immunohistochemical staining for FR-β and Mac-3, a marker of macrophages. 2-Deoxy-2-[ 18 F]fluoro- D -glucose ([ 18 F]FDG) PET/CT was performed to evaluate metabolic activity in the gastrointestinal tract. Results DSS-treated mice developed significant clinical signs of colitis, including weight loss and increased disease activity scores. PET/CT and ex vivo analyses revealed significantly higher [ 18 F]FOL uptake in the distal colon of DSS-treated mice compared to controls. Autoradiography and immunohistochemistry confirmed the presence of inflamed regions with increased Mac-3 in the distal colon. However, only a subset of Mac-3-positive macrophages expressed FR-β. Quantitative analysis demonstrated a correlation between tracer uptake and Mac-3 staining intensity. Conclusions [ 18 F]FOL PET/CT regions enriched with activated macrophages in DSS-induced colitis, although quantitative uptake in the colon was variable. The elevated splenic uptake and strong histological correlation suggest that FR-β-targeted PET imaging can reflect systemic macrophage activation in acute IBD. These findings support the continued investigation of [ 18 F]FOL in chronic inflammation models and its translational potential for imaging macrophage-driven pathology in IBD. Inflammatory bowel disease Murine colitis Positron emission tomography Folate receptor beta [18F]FOL Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Inflammatory bowel disease (IBD), encompassing ulcerative colitis and Crohn's disease, is characterized by chronic inflammation of the gastrointestinal tract [ 1 ]. While adults with IBD typically present with symptoms such as abdominal pain, weight loss, and bloody diarrhea, pediatric patients often exhibit atypical manifestations, including growth retardation, anemia, and extraintestinal manifestation [ 2 ]. Recognizing the broad spectrum of clinical presentations is critical for timely diagnosis and effective management [ 3 , 4 ]. Although the exact etiology of IBD remains unclear, its pathogenesis involves a complex interplay between genetic susceptibility, alterations in the gut microbiota, and dysregulated mucosal immune responses [ 5 , 6 ]. Animal models have been instrumental in advancing our understanding of these mechanisms and serve as essential platforms for preclinical evaluation of novel therapeutics [ 7 ]. Currently, endoscopy is considered the gold standard for assessing disease activity in IBD. However, its invasiveness, patient discomfort, and associated cost limit its frequent use. Non-invasive biomarkers such as fecal calprotectin and C-reactive protein (CRP) offer some utility but lack sufficient specificity and sensitivity for reliable disease monitoring. This underscores the need for accurate, non-invasive imaging tools to support clinical decision-making in IBD [ 8 ]. Positron emission tomography (PET), especially when using the fluorine-18-labeled glucose analog 2-deoxy-2-[ 18 F]fluoro- D -glucose ([ 18 F]FDG), is a well-established modality for non-invasively visualizing inflammatory activity [ 9 ]. However, physiological uptake of [ 18 F]FDG by the intestinal epithelium reduces its diagnostic specificity for detecting IBD-related inflammation [ 10 ]. To address this limitation, alternative PET tracers targeting molecular markers of inflammation have been explored [ 11 ]. Promising preclinical results have been demonstrated using tracers such as [ 68 Ga]Ga-DOTA-Siglec-9, which targets vascular adhesion protein 1 (VAP-1) [ 12 ], and [ 89 Zr]Zr-DFO-infliximab, which targets tumor necrosis factor-alpha (TNF-α) [ 13 , 14 ]. Folate receptor beta (FR-β) is a cell surface protein selectively expressed on activated macrophages and has been implicated in several inflammatory disorders. Notably, FR-β expression has been found to be markedly elevated in colonic tissue from patients with Crohn’s disease and ulcerative colitis, but is absent in resting macrophages and healthy tissues, highlighting its potential as a disease-specific imaging and therapeutic target [ 15 ]. Previous studies have demonstrated high uptake of FR-β-binding aluminum-fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([ 18 F]FOL), a folate-based PET tracer, in models of atherosclerosis [ 16 ], neuroinflammation [ 17 ], and myocarditis [ 18 ]. However, its application in IBD has not yet been evaluated. In this study, we investigated the utility of [ 18 F]FOL for detecting activated macrophages in a murine model of dextran sodium sulfate (DSS)-induced colitis [ 19 ]. In addition to in vivo whole-body PET/CT imaging, we performed ex vivo biodistribution analysis, digital autoradiography, and immunohistochemical staining to assess the tracer’s specificity and spatial correlation with macrophage infiltration. Materials and Methods Mouse Model of IBD A total of 32 C57BL/6NCrl mice were obtained from Charles River Laboratories Inc. (Wilmington, MA, USA). Acute colitis (female, n = 8, aged 10-17 weeks; male, n = 8, aged 10-13 weeks) was induced by administering 2.5% DSS (40 kDa; TdB Labs AB, Uppsala, Sweden) dissolved in autoclaved drinking water for 6 days, followed by 3 days of DSS-free water to allow for recovery and monitoring of disease progression [20, 21]. PET imaging with [ 18 F]FDG was performed on day 8, followed by [ 18 F]FOL imaging on day 9. Age- and sex-matched control mice (female, n = 7; male, n = 9) were maintained under identical conditions but received only autoclaved drinking water throughout the experiment. The disease activity index (DAI) was used to quantitatively assess disease severity, incorporating body weight loss, stool consistency, and presence of blood in stool over the course of the experiment [22]. Weight loss was scored with 1 point for each 5% decrease from baseline. Stool consistency was scored from 1 (normal) to 4 (liquid), and fecal blood was assessed on a scale from 0 (none) to 4 (fresh blood on bedding or perianal area). All procedures were approved by the Finnish national Project Authorisation Board (license numbers ESAVI/4498/2023 and ESAVI/15087/2023) and complied with EU Directive 2010/EU/63 on the protection of animals used for scientific purposes. Mice were housed at the Central Animal Laboratory, University of Turku, under a 12-hour light/dark cycle with ad libitum access to chow and water. An overview of the experimental design is provided in Supplementary Table. 1 . Radiochemistry Following the established radiolabeling procedure [23], the radiotracer [ 18 F]FOL was synthesized with a total procedure time of 99-101 minutes starting from the end of bombardment. [ 18 F]fluoride (5-7 GBq) was trapped on a Chromafix PS-HCO 3 (45 mg) cartridge (Macherey-Nagel, Düren, Germany) and eluted with 220 μL of sterile physiological saline into a reaction vial containing NOTA-folate precursor (4 mM in 50 μL water, ABX Advanced Biochemical Compounds GmbH, Radeberg, Germany), propylene glycol (20 μL), acetonitrile (70 μL), and sodium acetate buffer containing aluminum chloride (1 M CH 3 CO 2 Na, 2 mM AlCl 3 , pH 4.0; 40 μL). The reaction mixture was heated at 100°C for 15 minutes, cooled to room temperature, and diluted with 0.1% trifluoroacetic acid (TFA) in water (800 μL) prior to purification by high-performance liquid chromatography (HPLC). Purification was performed using a semi-preparative Jupiter Proteo C18 column (4 μm, 90 Å, 250 × 10 mm, Phenomenex, Torrance, CA, USA) with UV (l 254 nm) and radioactivity detection. The product ([ 18 F]FOL) eluted at approximately 14.5 minutes into a vial containing water (20 mL), sodium bicarbonate (1 M, 150 μL), and gentisic acid (1 M, 100 μL). The collected fraction was subsequently loaded onto a tC18 cartridge (Waters Corp., Milford, MA, USA) for solid-phase extraction. The cartridge was rinsed with water (5 mL), and the product was eluted with 50% ethanol (500 μL) into a vial containing 9% propylene glycol in phosphate-buffered saline (2 mL). The final radioactivity concentration was maintained below 400 MBq/mL to prevent radiolysis. Radiochemical purity was assessed by analytical radio-HPLC to confirm the absence of radioactive impurities that could compromise imaging performance. In Vivo PET/CT Imaging In total, 32 mice underwent in vivo PET/CT imaging and were subsequently euthanized for tissue collection. Imaging was performed on two consecutive days using different tracers. Small-animal PET and CT scanners (Molecubes NV, Gent, Belgium) were used for high-resolution imaging. Anesthesia was maintained with 1-2% isoflurane. All mice received intravenous tail vein injections, and female mice were additionally catheterized to empty the bladder prior to PET/CT imaging. On day 8, mice were imaged after approximately 3 hours of fasting and received [ 18 F]FDG (3.3 ± 0.1 MBq) to evaluate metabolic activity in the gastrointestinal tract. A static 20-minute PET scan was acquired 40 minutes post-injection. On day 9, [ 18 F]FOL (9.1 ± 1.3 MBq) was administered intravenously, and dynamic PET data were acquired for 60 minutes in list-mode. Data were reconstructed using an ordered subsets expectation maximization 3D algorithm into six 10-second, four 60-second, five 300-second, and three 600-second time frames, allowing assessment of tracer kinetics. Immediately after the [ 18 F]FOL PET scan, mice received 100 µL of iodinated contrast agent (eXia 160XL; Binitio Biomedical Inc., Ottawa, ON, Canada) intravenously for 6 min CT imaging. High-resolution CT scan was acquired and reconstructed using an iterative image space algorithm to provide anatomical reference and attenuation correction. PET image analysis was performed using Carimas software (version 2.10; Turku PET Centre, Turku, Finland). Regions of interest (ROIs) were manually defined in the distal colon and other tissues on the basis of CT imaging. Tracer uptake was quantified as average standardized uptake value (SUV mean ) over 1-60 minutes post-injection and as time-activity curves (TACs). Ex Vivo Biodistribution Immediately following [ 18 F]FOL imaging, mice were euthanized, and 21 tissue samples from mice, including selected segments of the intestinal tract, were collected, weighed, and counted for radioactivity using a gamma counter (Triathler 3″; Hidex, Turku, Finland). Data were expressed as percentage of injected radioactivity dose per gram of tissue (%ID/g), corrected for decay to the time of injection and residual radioactivity in the tail and cannula. Tissue samples from the distal ileum, proximal colon, and three segments of the distal colon were embedded in TissueTek (Sakura Finetek, Alphen aan den Rijn, The Netherlands), frozen in dry ice-cooled isopentane, and stored for cryosectioning. Additional tissue samples were fixed in formalin and embedded in paraffin (FFPE). Ex V ivo Digital Auto radio graphy To determine regional tracer uptake in the intestinal tract, digital autoradiography was performed. Cryosections (5 µm and 20 µm) were mounted onto microscope slides, air-dried, and exposed to phosphor imaging plates (BAS-TR2025; Fujifilm Corp, Tokyo, Japan) for 4 hours. Plates were scanned using a Fuji Analyzer BAS-5000 (Fujifilm Corp.) at 25 µm resolution. Images were analyzed using Carimas software. The 20 µm sections were stained with hematoxylin-eosin (H&E) for histological evaluation and scanned with Pannoramic 1000 slide scanner (3DHISTECH Ltd., Budapest, Hungary). The 5 µm sections were stored at −70°C for subsequent immunohistochemistry. Autoradiographs were aligned with H&E-stained micrographs, ROIs were defined in the distal colon. Results were expressed as photostimulated luminescence per square millimeter (PSL/mm 2 ), decay-corrected, background-subtracted, and normalized to the injection dose. Histological and Immunohistochemical Staining Histological examination was performed on 20 µm cryosections of distal colon, proximal colon, and ileum stained with H&E, and scanned with a Pannoramic Midi digital slide scanner (3DHISTECH Ltd., Budapest, Hungary). Immunohistochemistry was performed on 4 µm FFPE sections using a LabVision Autostainer (Thermo Fisher Scientific, USA). Primary antibodies included monoclonal anti-mouse FR-β (working dilution 1:200; catalog number orb317614, Biorbyt Ltd, Cambridge, United Kingdom) and Mac-3 (working dilution 1:50, catalog number 550292, BD Biosciences, NJ, USA). For detection, goat anti-mouse HRP (catalog number DPVR110HRP, WellMed, China) and rat-on-mouse HRP-polymer kit (catalog number RT517, Biocare Medical, Concord, CA, USA) were used as secondary reagents. Slides were scanned using Pannoramic 1000 slide scanner (3DHISTECH Ltd.). Digital images were analyzed using CaseViewer (version 2.2; 3DHISTECH Ltd). Positive staining areas for Mac-3 and FR-β in the distal colon were quantified using Visiopharm software version 2025.02 (Visiopharm Integrator System, Hoersholm, Denmark). Statistical Analysis Data are presented as mean ± standard deviation (SD). Differences between two groups were analyzed using an independent-samples t -test. Correlation between tracer uptake and Mac-3 staining was evaluated using Pearson’s correlation coefficient ( r ). Statistical analysis was conducted in Microsoft Excel (Microsoft 365, Redmond, WA, USA), with significance set at P < 0.05. Results Radiochemistry The average purity of [ 18 F]FOL was 95.79% ± 1.87 and decay-corrected radiochemical yield was 13.50% ± 5.53 ( n = 9). Furthermore, the molar activity was 1218.85 ± 184.52 MBq/nmol ( n = 3), indicating high potency of the radiotracer for application in molecular imaging. DSS Treatment Induces Colitis and Weight Loss in Mice Mice treated with 2.5% DSS for 6 days developed signs of colitis, which were monitored daily. On day 9, at the time of PET imaging with [ 18 F]FOL tracer, the DAI was significantly elevated in DSS-treated mice compared to healthy controls (pooled results 6.62 ± 1.98 vs. 1.12 ± 0.34, P < 0.001; females: 7.80 ± 2.05 vs. 1.29 ± 0.49, P < 0.001; males 5.88 ± 1.54 vs. 1.00 ± 0.01, P < 0.001; Fig. 1a ). DSS-treated mice also exhibited significant weight loss compared to healthy controls (pooled results 14.44% ± 7.39% vs. −1.59% ± 5.29%, P < 0.001; females: 15.41% ± 8.86% vs. −0.22% ± 3.78%, P = 0.001; males: 13.50% ± 4.69% vs. −2.78% ± 6.34%, P < 0.001; Fig. 1b ) . [ 18 F]FDG Uptake in Intestinal Inflammation [ 18 F]FDG PET/CT showed increased tracer accumulation in the distal colon of the DSS-treated mice compared to controls (SUV mean , pooled results 0.85 ± 0.30 vs. 0.53 ± 0.19, P = 0.003; females: 0.94 ± 0.44 vs. 0.61 ± 0.20, P = 0.110; males 0.79 ± 0.18 vs. 0.43 ± 0.11, P = 0.001). [ 18 F]FOL Uptake in Intestinal Inflammation [ 18 F]FOL PET/CT imaging showed increased tracer accumulation in the distal colon of the DSS-treated mice compared to controls (SUV mean , pooled results 1.15 ± 0.25 vs. 0.81 ± 0.20, P = 0.002; females: 1.15 ± 0.31 vs. 0.78 ± 0.17, P = 0.015; males: 1.05 ± 0.17 vs. 0.47 ± 0.08, P = 0.080; Fig. 2a-b ) Time-activity curves show higher distal colon [ 18 F]FOL uptake in DSS-treated mice than in controls; Fig. 2c ) Ex vivo gamma counting showed increased distal colon [ 18 F]FOL uptake in DSS-treated mice compared to controls, consistent with the in vivo PET findings (%ID/g, pooled results 2.70 ± 1.50 vs. 1.53 ± 0.76, P = 0.008; females: 3.58 ± 1.70 vs. 1.83 ± 0.44, P = 0.021; males1.83 ± 0.43 vs. 1.29 ± 0.89, P = 0.135; Fig. 3a ). Biodistribution of [ 18 F]FOL in DSS-Treated vs. Healthy Mice The ex vivo biodistribution results are summarized in Supplementary Table 2 . A notable finding was significantly elevated [ 18 F]FOL uptake in the spleens of DSS-treated mice compared to controls (%ID/g, pooled results 0.39 ± 0.13 vs. 0.21 ± 0.09, P < 0.001; females: 0.44 ± 0.14 vs. 0.25 ± 0.11, P = 0.001; males: 0.33 ± 0.09 vs. 0.21 ± 0.12, P = 0.037). In DSS-treated mice, the highest tracer uptake was observed in the liver, rectum, and content of distal colon (%ID/g, pooled results 5.82 ± 2.46, 4.39 ± 2.74, and 2.71 ± 1.37; females: 7.09 ± 2.79, 6.05 ± 2.90, and 3.06 ± 1.57; males: 4.54 ± 1.53, 2.73 ± 1.19, and 2.36 ± 1.13, respectively, Fig. 3b ). Ex Vivo Digital Autoradiography, Histology, and Immunohistochemistry Representative images of [ 18 F]FOL autoradiographs, H&E staining, and immunohistochemistry for FR-β and Mac-3 from adjacent distal colon sections are shown in Fig. 4 . Digital autoradiography demonstrated heterogeneous [ 18 F]FOL distribution in the distal colon. In DSS-treated mice, there was a marked increase in Mac-3 positive macrophages compared with controls (area-%, 1.31 ± 0.09 vs. 0.05 ± 0.04, P < 0.001). However, only a subset of Mac-3-positive macrophages expressed FR-β. Quantitative analysis in DSS-treated mice demonstrated a correlation between distal colon [ 18 F]FOL uptake and Mac-3 staining intensity ( r = 0.694, P = 0.026). Discussion Inspired by the promising results of [ 18 F]FOL in various previous preclinical studies targeting macrophage-rich inflammation—such as atherosclerosis, myocarditis, and neuroinflammation—and by reports of FR-β overexpression in the inflamed intestines of patients with IBD [ 15 ], we evaluated [ 18 F]FOL PET/CT for detecting macrophage-driven inflammation in a murine model of DSS-induced colitis [ 24 ]. Activated macrophages play a key role in IBD pathogenesis, and FR-β is selectively expressed on a subset of these cells. Our results showed increased [ 18 F]FOL uptake in the distal colon of DSS-treated mice by PET/CT and ex vivo gamma counting, although FR-β immunohistochemistry and autoradiography did not reveal statistically significant differences between DSS and control mice. This suggests that [ 18 F]FOL uptake may be heterogeneous and localized, reflecting patchy macrophage activation rather than a uniform increase across the colon. Importantly, distal colon [ 18 F]FOL autoradiography signal correlated significantly with the amount of Mac-3-positive macrophages, indicating that [ 18 F]FOL selectively localizes to regions enriched with activated macrophages, even if group-level differences were modest. This finding supports the tracer’s specificity for macrophage-rich areas and highlights the value of complementary histological validation. In our study, [ 18 F]FDG also showed elevated uptake in the distal colon of DSS-treated mice; however, its specificity is limited by physiological glucose metabolism in the intestinal epithelium, which can confound interpretation [ 10 ]. In contrast, [ 18 F]FOL targets a molecular marker specific to activated macrophages, providing a potentially more precise indicator of inflammatory activity. While [ 18 F]FOL uptake was variable, it generally correlated with Mac-3-positive macrophage density, supporting its specificity. These findings suggest that [ 18 F]FOL may complement or even improve upon [ 18 F]FDG-PET in the context of IBD imaging, particularly when assessing macrophage-driven inflammation. A particularly noteworthy finding was the significantly elevated [ 18 F]FOL uptake in the spleen of DSS-treated mice, likely reflecting systemic immune activation—a hallmark of DSS-induced inflammation [ 29 ]. The spleen may therefore serve as a surrogate indicator of generalized macrophage activity, which could have implications for monitoring systemic inflammation in IBD. This study has some limitations that may impact on the interpretation of the findings. First, the DSS-induced colitis model has inherent variability in inflammation severity between mice and among colon regions [ 25 ]. Unlike human IBD, DSS colitis is chemically induced, self-limiting, and largely independent of the gut microbiota, which complicates direct comparisons [ 26 ]. Although clinical symptoms are readily observable, they may not accurately reflect the true severity of inflammation [ 27 ]. Nevertheless, the current study confirms that DSS effectively induces colitis-like inflammation compared to controls. Second, PET image analysis presented challenges. Manual delineation of intestinal ROIs was complicated by high radioactivity in the urinary bladder, potentially causing spillover effects and affecting quantitative accuracy. In addition, the limited number of tissue sections used for autoradiography analysis may have missed small or focal inflammatory regions detectable by more comprehensive in vivo imaging or ex vivo gamma counting of larger tissue samples. Third, tissue sampling was limited to three consecutive sections from the same distal colon region per mouse. Given the heterogeneous and patchy nature of DSS-induced colitis, the focal severity can differ along the colon and between the mice [ 28 ]. Consequently, the selected sections may capture regions with relatively higher inflammation in some mice but less affected areas in others. The ability to non-invasively visualize activated macrophages has broad implications for IBD management. FR-β-targeted PET could facilitate earlier detection of inflammatory flares, guide therapeutic decisions, and serve as a biomarker for response to macrophage-directed treatments. Moreover, [ 18 F]FOL specific binding to activated macrophages may allow longitudinal monitoring with reduced background signal compared to [ 18 F]FDG. Future studies should investigate its performance in chronic and relapsing IBD models, as well as in human patients, to fully assess its translational potential. Conclusion In conclusion, [ 18 F]FOL PET/CT enables non-invasive detection of activated macrophage-rich inflammation in acute DSS-induced colitis, correlates with histological measures of macrophage infiltration, and shows promise as a more specific alternative to [ 18 F]FDG for imaging IBD. These findings support further investigation of [ 18 F]FOL in chronic colitis models and its potential clinical translation as a tool for assessing macrophage-driven pathology in patients with IBD. Declarations Supplementary Information The online version contains supplementary material available at Acknowledgments We are grateful to Aake Honkaniemi (Turku PET Centre), Marja-Riitta Kaajala, Erica Nyman (University of Turku Histocore Facility), and Markus Peurla (University of Turku) for their valuable assistance, to Xiaoqing Zhuang and Putri Andriana for their contribution to tracer radiosynthesis, and to Timo Kattelus for finalizing the figures. Author contributions Conception and design: M.K., A.A.B., M.S., P.S.L., X-G.L., J.K., D.M.T., and A.R.; acquisition, analysis and interpretation of data: M.K., A.A.B., J.R., E.A.H., J.V., H.L., J.R., X-G.L., J.K., D.M.T., and A.R. All authors were involved in drafting or critically reviewing the manuscript for important intellectual content, and all approved the final manuscript for submission. Funding The study was financially supported by grants from the Research Council of Finland (grant no. 350117). M.G. is a PhD student supported by the ImmuDocs Doctoral Pilot Program of the University of Turku. This research was also partially supported by the Research Council of Finland’s Flagship program InFLAMES (grant decision numbers 337531, 337530, 359346, and 357910). Data Availability The data supporting the conclusions of this article are included within the article and it’s the supplementary materials. The raw datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. Ethical Approval All animal experiments were approved by the National Project Authorization Board of Finland (license numbers ESAVI/4498/2023 and ESAVI/15087/2023) and were conducted in accordance with the European Union Directive 2010/EU/63 on the protection of animals used for scientific purposes. Conflicts of Interest The authors declare that they have no conflicts of interests regarding to this study. 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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-8410867","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":575124373,"identity":"bdeb7414-ce00-4b26-9a2f-0fbc30fcda45","order_by":0,"name":"Mitra Gerami","email":"","orcid":"","institution":"Turku PET Centre: Turun PET keskus","correspondingAuthor":false,"prefix":"","firstName":"Mitra","middleName":"","lastName":"Gerami","suffix":""},{"id":575124374,"identity":"d81789b4-ccd2-49b7-8e1d-b52c9d6f1b36","order_by":1,"name":"Achol A. 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04:00:27","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96849,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8410867/v1/80432855fc9efa11be928a3a.html"},{"id":100748210,"identity":"09f8cd27-1cb7-4b36-8a3e-915e85ad5dee","added_by":"auto","created_at":"2026-01-21 04:01:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":678303,"visible":true,"origin":"","legend":"\u003cp\u003eProgression of symptoms in DSS-induced acute colitis. (\u003cstrong\u003ea\u003c/strong\u003e) The disease activity index (DAI) shows a significant difference between DSS-treated mice and controls at day 9.\u0026nbsp; (\u003cstrong\u003eb\u003c/strong\u003e) Weight loss in female and male mice compared to their respective controls. DSS-treated mice showed significantly greater weight loss compared with healthy control mice in both sexes. # [\u003csup\u003e18\u003c/sup\u003eF]FDG-PET/CT was performed on day 8; ## [\u003csup\u003e18\u003c/sup\u003eF]FOL-PET/CT was performed on day 9. *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Values are presented as mean ± SD.\u003cem\u003e P\u003c/em\u003e values were determined using independent-samples \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8410867/v1/6bc8eba176f954049f7460f0.png"},{"id":100748126,"identity":"7989cc62-d648-415a-8fa9-4ddc5b64715a","added_by":"auto","created_at":"2026-01-21 04:00:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17143453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e [\u003csup\u003e18\u003c/sup\u003eF]FOL PET/CT imaging of DSS-treated and control mice.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Representative PET/CT images of a DSS-treated mouse and a control.\u003cem\u003e In vivo\u003c/em\u003e\u0026nbsp;[\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in the distal colon is indicated by white arrows. Red dashed lines = distal colon; C = cecum; K = kidney; B = urinary bladder; SUV = standardized uptake value. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of \u003cem\u003ein vivo\u003c/em\u003e [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in the distal colon of DSS-treated and control mice. Data are presented as mean ± SD. \u003cem\u003eP\u003c/em\u003e value was determined using independent-samples \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003ec\u003c/strong\u003e) Decay-corrected time–activity curves of [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in the distal colon across different groups.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8410867/v1/c41260149b46b821327b2583.png"},{"id":100748088,"identity":"c4e6b7ba-20b7-4d44-b2f6-03f480968472","added_by":"auto","created_at":"2026-01-21 04:00:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1040729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEx vivo\u003c/em\u003e uptake of [\u003csup\u003e18\u003c/sup\u003eF]FOL in distal colon and different tissues of DSS and control. (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eEx vivo \u003c/em\u003e[\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in the distal colon shows significantly higher values in DSS-treated mice compared to controls. (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eEx vivo \u003c/em\u003e[\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in different tissues of female and male DSS-treated and control mice. Results are presented as percentage injected radioactivity dose per gram of tissue (%ID/g) mean ± SD. \u003cem\u003eP\u003c/em\u003e values were determined using independent-samples \u003cem\u003et\u003c/em\u003e-test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. \u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8410867/v1/dafaea3e53eb708b6363e6d0.png"},{"id":100748089,"identity":"b9fcb754-8022-47ad-9e39-72b4a38f0ed3","added_by":"auto","created_at":"2026-01-21 04:00:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":19199910,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative\u003cem\u003e ex vivo \u003c/em\u003eautoradiographs, hematoxylin-eosin (H\u0026amp;E) staining, and FR-β and Mac-3 immunohistochemical staining of distal colon cryosections from DSS-treated and control mice. The scale bars are 500 µm and 25 µm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8410867/v1/4c90873c1c083c97569da820.png"},{"id":104400444,"identity":"fb9ce725-75a1-45c4-9990-0d207f3b0739","added_by":"auto","created_at":"2026-03-11 12:09:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36129621,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8410867/v1/af04ff69-4899-4e39-b9a9-0db9360bd4a6.pdf"},{"id":100748081,"identity":"03a80ba8-3a8a-4133-9c4f-a8c28ededc20","added_by":"auto","created_at":"2026-01-21 04:00:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":55654,"visible":true,"origin":"","legend":"","description":"","filename":"Geramisupplementarymaterial201225.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8410867/v1/d56a0a48465b96cbba8cdd88.pdf"}],"financialInterests":"","formattedTitle":"Noninvasive Detection of Experimental Colitis Using Folate Receptor-β-Targeted PET Imaging in Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInflammatory bowel disease (IBD), encompassing ulcerative colitis and Crohn's disease, is characterized by chronic inflammation of the gastrointestinal tract [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While adults with IBD typically present with symptoms such as abdominal pain, weight loss, and bloody diarrhea, pediatric patients often exhibit atypical manifestations, including growth retardation, anemia, and extraintestinal manifestation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Recognizing the broad spectrum of clinical presentations is critical for timely diagnosis and effective management [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough the exact etiology of IBD remains unclear, its pathogenesis involves a complex interplay between genetic susceptibility, alterations in the gut microbiota, and dysregulated mucosal immune responses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Animal models have been instrumental in advancing our understanding of these mechanisms and serve as essential platforms for preclinical evaluation of novel therapeutics [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, endoscopy is considered the gold standard for assessing disease activity in IBD. However, its invasiveness, patient discomfort, and associated cost limit its frequent use. Non-invasive biomarkers such as fecal calprotectin and C-reactive protein (CRP) offer some utility but lack sufficient specificity and sensitivity for reliable disease monitoring. This underscores the need for accurate, non-invasive imaging tools to support clinical decision-making in IBD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePositron emission tomography (PET), especially when using the fluorine-18-labeled glucose analog 2-deoxy-2-[\u003csup\u003e18\u003c/sup\u003eF]fluoro-\u003cem\u003eD\u003c/em\u003e-glucose ([\u003csup\u003e18\u003c/sup\u003eF]FDG), is a well-established modality for non-invasively visualizing inflammatory activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, physiological uptake of [\u003csup\u003e18\u003c/sup\u003eF]FDG by the intestinal epithelium reduces its diagnostic specificity for detecting IBD-related inflammation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To address this limitation, alternative PET tracers targeting molecular markers of inflammation have been explored [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Promising preclinical results have been demonstrated using tracers such as [\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-Siglec-9, which targets vascular adhesion protein 1 (VAP-1) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-infliximab, which targets tumor necrosis factor-alpha (TNF-α) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFolate receptor beta (FR-β) is a cell surface protein selectively expressed on activated macrophages and has been implicated in several inflammatory disorders. Notably, FR-β expression has been found to be markedly elevated in colonic tissue from patients with Crohn\u0026rsquo;s disease and ulcerative colitis, but is absent in resting macrophages and healthy tissues, highlighting its potential as a disease-specific imaging and therapeutic target [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Previous studies have demonstrated high uptake of FR-β-binding aluminum-fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([\u003csup\u003e18\u003c/sup\u003eF]FOL), a folate-based PET tracer, in models of atherosclerosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], neuroinflammation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and myocarditis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, its application in IBD has not yet been evaluated.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the utility of [\u003csup\u003e18\u003c/sup\u003eF]FOL for detecting activated macrophages in a murine model of dextran sodium sulfate (DSS)-induced colitis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition to \u003cem\u003ein vivo\u003c/em\u003e whole-body PET/CT imaging, we performed \u003cem\u003eex vivo\u003c/em\u003e biodistribution analysis, digital autoradiography, and immunohistochemical staining to assess the tracer\u0026rsquo;s specificity and spatial correlation with macrophage infiltration.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eMouse Model of IBD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 32 C57BL/6NCrl mice were obtained from Charles River Laboratories Inc. (Wilmington, MA, USA). Acute colitis (female, \u003cem\u003en\u0026nbsp;\u003c/em\u003e= 8, aged 10-17 weeks; male, \u003cem\u003en\u003c/em\u003e = 8, aged 10-13 weeks) was induced by administering 2.5% DSS (40 kDa; TdB Labs AB, Uppsala, Sweden) dissolved in autoclaved drinking water for 6 days, followed by 3 days of DSS-free water to allow for recovery and monitoring of disease progression\u0026nbsp;[20, 21]. PET imaging with [\u003csup\u003e18\u003c/sup\u003eF]FDG was performed on day 8, followed by [\u003csup\u003e18\u003c/sup\u003eF]FOL imaging on day 9. Age- and sex-matched control mice (female, \u003cem\u003en\u003c/em\u003e = 7; male, \u003cem\u003en\u003c/em\u003e = 9) were maintained under identical conditions but received only autoclaved drinking water throughout the experiment.\u003c/p\u003e\n\u003cp\u003eThe disease activity index (DAI) was used to quantitatively assess disease severity, incorporating body weight loss, stool consistency, and presence of blood in stool over the course of the experiment [22]. Weight loss was scored with 1 point for each 5% decrease from baseline. Stool consistency was scored from 1 (normal) to 4 (liquid), and fecal blood was assessed on a scale from 0 (none) to 4 (fresh blood on bedding or perianal area).\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by the Finnish national Project Authorisation Board (license numbers ESAVI/4498/2023 and ESAVI/15087/2023) and complied with EU Directive 2010/EU/63 on the protection of animals used for scientific purposes. Mice were housed at the Central Animal Laboratory, University of Turku, under a 12-hour light/dark cycle with \u003cem\u003ead libitum\u003c/em\u003e access to chow and water. An overview of the experimental design is provided in \u003cstrong\u003eSupplementary Table. 1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRadiochemistry\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the established radiolabeling procedure [23], the radiotracer [\u003csup\u003e18\u003c/sup\u003eF]FOL was synthesized with a total procedure time of 99-101 minutes starting from the end of bombardment. [\u003csup\u003e18\u003c/sup\u003eF]fluoride (5-7 GBq) was trapped on a Chromafix PS-HCO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(45 mg) cartridge (Macherey-Nagel, D\u0026uuml;ren, Germany) and eluted with 220 \u0026mu;L of sterile physiological saline into a reaction vial containing NOTA-folate precursor (4 mM in 50 \u0026mu;L water, ABX Advanced Biochemical Compounds GmbH, Radeberg, Germany), propylene glycol (20 \u0026mu;L), acetonitrile (70 \u0026mu;L), and sodium acetate buffer containing aluminum chloride (1 M CH\u003csub\u003e3\u003c/sub\u003eCO\u003csub\u003e2\u003c/sub\u003eNa, 2 mM AlCl\u003csub\u003e3\u003c/sub\u003e, pH 4.0; 40 \u0026mu;L). The reaction mixture was heated at 100\u0026deg;C for 15 minutes, cooled to room temperature, and diluted with 0.1% trifluoroacetic acid (TFA) in water (800 \u0026mu;L) prior to purification by high-performance liquid chromatography (HPLC). Purification was performed using a semi-preparative Jupiter Proteo C18 column (4 \u0026mu;m, 90 \u0026Aring;, 250 \u0026times; 10 mm, Phenomenex, Torrance, CA, USA) with UV (l\u0026nbsp;254 nm) and radioactivity detection. The product ([\u003csup\u003e18\u003c/sup\u003eF]FOL) eluted at approximately 14.5 minutes into a vial containing water (20 mL), sodium bicarbonate (1 M, 150 \u0026mu;L), and gentisic acid (1 M, 100 \u0026mu;L). The collected fraction was subsequently loaded onto a tC18 cartridge (Waters Corp., Milford, MA, USA) for solid-phase extraction. The cartridge was rinsed with water (5 mL), and the product was eluted with 50% ethanol (500 \u0026mu;L) into a vial containing 9% propylene glycol in phosphate-buffered saline (2 mL). The final radioactivity concentration was maintained below 400 MBq/mL to prevent radiolysis. Radiochemical purity was assessed by analytical radio-HPLC to confirm the absence of radioactive impurities that could compromise imaging performance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn Vivo\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003ePET/CT Imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn total, 32 mice underwent \u003cem\u003ein vivo\u003c/em\u003e PET/CT imaging and were subsequently euthanized for tissue collection. Imaging was performed on two consecutive days using different tracers. Small-animal PET and CT scanners (Molecubes NV, Gent, Belgium) were used for high-resolution imaging. Anesthesia was maintained with 1-2% isoflurane. All mice received intravenous tail vein injections, and female mice were additionally catheterized to empty the bladder prior to PET/CT imaging.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn day 8, mice were imaged after approximately 3 hours of fasting and received [\u003csup\u003e18\u003c/sup\u003eF]FDG (3.3 \u0026plusmn; 0.1 MBq) to evaluate metabolic activity in the gastrointestinal tract. A static 20-minute PET scan was acquired 40 minutes post-injection.\u003c/p\u003e\n\u003cp\u003eOn day 9, [\u003csup\u003e18\u003c/sup\u003eF]FOL (9.1 \u0026plusmn; 1.3 MBq) was administered intravenously, and dynamic PET data were acquired for 60 minutes in list-mode. Data were reconstructed using an ordered subsets expectation maximization 3D algorithm into six 10-second, four 60-second, five 300-second, and three 600-second time frames, allowing assessment of tracer kinetics.\u003c/p\u003e\n\u003cp\u003eImmediately after the [\u003csup\u003e18\u003c/sup\u003eF]FOL PET scan, mice received 100 \u0026micro;L of iodinated contrast agent (eXia 160XL; Binitio Biomedical Inc., Ottawa, ON, Canada) intravenously for 6 min CT imaging. High-resolution CT scan was acquired and reconstructed using an iterative image space algorithm to provide anatomical reference and attenuation correction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePET image analysis was performed using Carimas software (version 2.10; Turku PET Centre, Turku, Finland). Regions of interest (ROIs) were manually defined in the distal colon and other tissues on the basis of CT imaging. Tracer uptake was quantified as average standardized uptake value (SUV\u003csub\u003emean\u003c/sub\u003e) over 1-60 minutes post-injection and as time-activity curves (TACs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEx Vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Biodistribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmediately following [\u003csup\u003e18\u003c/sup\u003eF]FOL imaging, mice were euthanized, and 21 tissue samples from mice, including selected segments of the intestinal tract, were collected, weighed, and counted for radioactivity using a gamma counter (Triathler 3\u0026Prime;; Hidex, Turku, Finland). Data were expressed as percentage of injected radioactivity dose per gram of tissue (%ID/g), corrected for decay to the time of injection and residual radioactivity in the tail and cannula.\u003c/p\u003e\n\u003cp\u003eTissue samples from the distal ileum, proximal colon, and three segments of the distal colon were embedded in TissueTek (Sakura Finetek, Alphen aan den Rijn, The Netherlands), frozen in dry ice-cooled isopentane, and stored for cryosectioning. Additional tissue samples were fixed in formalin and embedded in paraffin (FFPE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEx\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eV\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Digital Auto\u003c/strong\u003e\u003cstrong\u003eradio\u003c/strong\u003e\u003cstrong\u003egraphy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine regional tracer uptake in the intestinal tract, digital autoradiography was performed. Cryosections (5 \u0026micro;m and 20 \u0026micro;m) were mounted onto microscope slides, air-dried, and exposed to phosphor imaging plates (BAS-TR2025; Fujifilm Corp, Tokyo, Japan) for 4 hours. Plates were scanned using a Fuji Analyzer BAS-5000 (Fujifilm Corp.) at 25 \u0026micro;m resolution. Images were analyzed using Carimas software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 20 \u0026micro;m sections were stained with hematoxylin-eosin (H\u0026amp;E) for histological evaluation and scanned with Pannoramic 1000 slide scanner (3DHISTECH Ltd., Budapest, Hungary). The 5 \u0026micro;m sections were stored at \u0026minus;70\u0026deg;C for subsequent immunohistochemistry.\u003c/p\u003e\n\u003cp\u003eAutoradiographs were aligned with H\u0026amp;E-stained micrographs, ROIs were defined in the distal colon. Results were expressed as photostimulated luminescence per square millimeter (PSL/mm\u003csup\u003e2\u003c/sup\u003e), decay-corrected, background-subtracted, and normalized to the injection dose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological and Immunohistochemical Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistological examination was performed on 20 \u0026micro;m cryosections of distal colon, proximal colon, and ileum stained with H\u0026amp;E, and scanned with a Pannoramic Midi digital slide scanner (3DHISTECH Ltd., Budapest, Hungary).\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry was performed on 4 \u0026micro;m FFPE sections using a LabVision Autostainer (Thermo Fisher Scientific, USA). Primary antibodies included monoclonal anti-mouse FR-\u0026beta; (working dilution 1:200; catalog number orb317614, Biorbyt Ltd, Cambridge, United Kingdom) and Mac-3 (working dilution 1:50, catalog number 550292, BD Biosciences, NJ, USA). For detection, goat anti-mouse HRP (catalog number DPVR110HRP, WellMed, China) and rat-on-mouse HRP-polymer kit (catalog number RT517, Biocare Medical, Concord, CA, USA) were used as secondary reagents. Slides were scanned using Pannoramic 1000 slide scanner (3DHISTECH Ltd.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDigital images were analyzed using CaseViewer (version 2.2; 3DHISTECH Ltd). Positive staining areas for Mac-3 and FR-\u0026beta; in the distal colon were quantified using Visiopharm software version 2025.02 (Visiopharm Integrator System, Hoersholm, Denmark).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; standard deviation (SD). Differences between two groups were analyzed using an independent-samples \u003cem\u003et\u003c/em\u003e-test. Correlation between tracer uptake and Mac-3 staining was evaluated using Pearson\u0026rsquo;s correlation coefficient (\u003cem\u003er\u003c/em\u003e). Statistical analysis was conducted in Microsoft Excel (Microsoft 365, Redmond, WA, USA), with significance set at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eRadiochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe average purity of [\u003csup\u003e18\u003c/sup\u003eF]FOL was 95.79% \u0026plusmn; 1.87 and decay-corrected radiochemical yield was 13.50% \u0026plusmn; 5.53 (\u003cem\u003en\u003c/em\u003e = 9). Furthermore, the molar activity was 1218.85 \u0026plusmn; 184.52 MBq/nmol (\u003cem\u003en\u003c/em\u003e = 3), indicating high potency of the radiotracer for application in molecular imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDSS Treatment Induces Colitis and Weight Loss in Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice treated with 2.5% DSS for 6 days developed signs of colitis, which were monitored daily. On day 9, at the time of PET imaging with [\u003csup\u003e18\u003c/sup\u003eF]FOL tracer, the DAI was significantly elevated in DSS-treated mice compared to healthy controls (pooled results 6.62 \u0026plusmn; 1.98 vs. 1.12 \u0026plusmn; 0.34, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; females: 7.80 \u0026plusmn; 2.05 vs. 1.29 \u0026plusmn; 0.49, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; males 5.88 \u0026plusmn; 1.54 vs. 1.00 \u0026plusmn; 0.01, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; \u003cstrong\u003eFig. 1a\u003c/strong\u003e). DSS-treated mice also exhibited significant weight loss compared to healthy controls (pooled results 14.44% \u0026plusmn; 7.39% vs. \u0026minus;1.59% \u0026plusmn; 5.29%, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; females: 15.41% \u0026plusmn; 8.86% vs. \u0026minus;0.22% \u0026plusmn; 3.78%, \u003cem\u003eP\u003c/em\u003e = 0.001; males: 13.50% \u0026plusmn; 4.69% vs. \u0026minus;2.78% \u0026plusmn; 6.34%, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; \u003cstrong\u003eFig. 1b\u003c/strong\u003e)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]FDG Uptake in Intestinal Inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FDG PET/CT showed increased tracer accumulation in the distal colon of the DSS-treated mice compared to controls (SUV\u003csub\u003emean\u003c/sub\u003e, pooled results 0.85 \u0026plusmn; 0.30 vs. 0.53 \u0026plusmn; 0.19, \u003cem\u003eP\u003c/em\u003e = 0.003; females: 0.94 \u0026plusmn; 0.44 vs. 0.61 \u0026plusmn; 0.20, \u003cem\u003eP\u003c/em\u003e = 0.110; males 0.79 \u0026plusmn; 0.18 vs. 0.43 \u0026plusmn; 0.11, \u003cem\u003eP\u003c/em\u003e = 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]FOL Uptake in Intestinal Inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FOL PET/CT imaging showed increased tracer accumulation in the distal colon of the DSS-treated mice compared to controls (SUV\u003csub\u003emean\u003c/sub\u003e, pooled results 1.15 \u0026plusmn; 0.25 vs. 0.81 \u0026plusmn; 0.20, \u003cem\u003eP\u003c/em\u003e = 0.002; females: 1.15 \u0026plusmn; 0.31 vs. 0.78 \u0026plusmn; 0.17, \u003cem\u003eP\u003c/em\u003e = 0.015; males: 1.05 \u0026plusmn; 0.17 vs. 0.47 \u0026plusmn; 0.08, \u003cem\u003eP\u003c/em\u003e = 0.080; \u003cstrong\u003eFig. 2a-b\u003c/strong\u003e)\u0026nbsp;Time-activity curves show higher distal colon [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in DSS-treated mice than in controls; \u003cstrong\u003eFig. 2c\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEx vivo\u003c/em\u003e gamma counting showed increased distal colon [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in DSS-treated mice compared to controls, consistent with the \u003cem\u003ein vivo\u003c/em\u003e PET findings (%ID/g, pooled results 2.70 \u0026plusmn; 1.50 vs. 1.53 \u0026plusmn; 0.76, \u003cem\u003eP\u003c/em\u003e = 0.008; females: 3.58 \u0026plusmn; 1.70 vs. 1.83 \u0026plusmn; 0.44, \u003cem\u003eP\u003c/em\u003e = 0.021; males1.83 \u0026plusmn; 0.43 vs. 1.29 \u0026plusmn; 0.89, \u003cem\u003eP\u003c/em\u003e = 0.135; \u003cstrong\u003eFig. 3a\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiodistribution of [\u003csup\u003e18\u003c/sup\u003eF]FOL in DSS-Treated vs. Healthy Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eex vivo\u003c/em\u003e biodistribution results are summarized in \u003cstrong\u003eSupplementary Table\u003c/strong\u003e \u003cstrong\u003e2\u003c/strong\u003e.\u0026nbsp;A notable finding was significantly elevated [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in the spleens of DSS-treated mice compared to controls (%ID/g, pooled results 0.39 \u0026plusmn; 0.13 vs. 0.21 \u0026plusmn; 0.09, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; females: 0.44 \u0026plusmn; 0.14 vs. 0.25 \u0026plusmn; 0.11, \u003cem\u003eP\u003c/em\u003e = 0.001; males: 0.33 \u0026plusmn; 0.09 vs. 0.21 \u0026plusmn; 0.12, \u003cem\u003eP\u003c/em\u003e = 0.037).\u003c/p\u003e\n\u003cp\u003eIn DSS-treated mice, the highest tracer uptake was observed in the liver, rectum, and content of distal colon (%ID/g, pooled results 5.82 \u0026plusmn; 2.46, 4.39 \u0026plusmn; 2.74, and 2.71 \u0026plusmn; 1.37; females: 7.09 \u0026plusmn; 2.79, 6.05 \u0026plusmn; 2.90, and 3.06 \u0026plusmn; 1.57; males: 4.54 \u0026plusmn; 1.53, 2.73 \u0026plusmn; 1.19, and 2.36 \u0026plusmn; 1.13, respectively, \u003cstrong\u003eFig. 3b\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEx Vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Digital Autoradiography, Histology, and Immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative images of [\u003csup\u003e18\u003c/sup\u003eF]FOL autoradiographs, H\u0026amp;E staining, and immunohistochemistry for FR-\u0026beta; and Mac-3 from adjacent distal colon sections are shown in \u003cstrong\u003eFig. 4\u003c/strong\u003e. Digital autoradiography demonstrated heterogeneous [\u003csup\u003e18\u003c/sup\u003eF]FOL distribution in the distal colon. In DSS-treated mice, there was a marked increase in Mac-3 positive macrophages compared with controls (area-%, 1.31 \u0026plusmn; 0.09 vs. 0.05 \u0026plusmn; 0.04, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). However, only a subset of Mac-3-positive macrophages expressed FR-\u0026beta;. Quantitative analysis in DSS-treated mice demonstrated a correlation between distal colon [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake and Mac-3 staining intensity (\u003cem\u003er\u003c/em\u003e = 0.694, \u003cem\u003eP\u003c/em\u003e = 0.026).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInspired by the promising results of [\u003csup\u003e18\u003c/sup\u003eF]FOL in various previous preclinical studies targeting macrophage-rich inflammation\u0026mdash;such as atherosclerosis, myocarditis, and neuroinflammation\u0026mdash;and by reports of FR-β overexpression in the inflamed intestines of patients with IBD [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], we evaluated [\u003csup\u003e18\u003c/sup\u003eF]FOL PET/CT for detecting macrophage-driven inflammation in a murine model of DSS-induced colitis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Activated macrophages play a key role in IBD pathogenesis, and FR-β is selectively expressed on a subset of these cells. Our results showed increased [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in the distal colon of DSS-treated mice by PET/CT and \u003cem\u003eex vivo\u003c/em\u003e gamma counting, although FR-β immunohistochemistry and autoradiography did not reveal statistically significant differences between DSS and control mice. This suggests that [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake may be heterogeneous and localized, reflecting patchy macrophage activation rather than a uniform increase across the colon.\u003c/p\u003e \u003cp\u003eImportantly, distal colon [\u003csup\u003e18\u003c/sup\u003eF]FOL autoradiography signal correlated significantly with the amount of Mac-3-positive macrophages, indicating that [\u003csup\u003e18\u003c/sup\u003eF]FOL selectively localizes to regions enriched with activated macrophages, even if group-level differences were modest. This finding supports the tracer\u0026rsquo;s specificity for macrophage-rich areas and highlights the value of complementary histological validation.\u003c/p\u003e \u003cp\u003eIn our study, [\u003csup\u003e18\u003c/sup\u003eF]FDG also showed elevated uptake in the distal colon of DSS-treated mice; however, its specificity is limited by physiological glucose metabolism in the intestinal epithelium, which can confound interpretation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In contrast, [\u003csup\u003e18\u003c/sup\u003eF]FOL targets a molecular marker specific to activated macrophages, providing a potentially more precise indicator of inflammatory activity. While [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake was variable, it generally correlated with Mac-3-positive macrophage density, supporting its specificity. These findings suggest that [\u003csup\u003e18\u003c/sup\u003eF]FOL may complement or even improve upon [\u003csup\u003e18\u003c/sup\u003eF]FDG-PET in the context of IBD imaging, particularly when assessing macrophage-driven inflammation.\u003c/p\u003e \u003cp\u003eA particularly noteworthy finding was the significantly elevated [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in the spleen of DSS-treated mice, likely reflecting systemic immune activation\u0026mdash;a hallmark of DSS-induced inflammation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The spleen may therefore serve as a surrogate indicator of generalized macrophage activity, which could have implications for monitoring systemic inflammation in IBD.\u003c/p\u003e \u003cp\u003eThis study has some limitations that may impact on the interpretation of the findings. First, the DSS-induced colitis model has inherent variability in inflammation severity between mice and among colon regions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Unlike human IBD, DSS colitis is chemically induced, self-limiting, and largely independent of the gut microbiota, which complicates direct comparisons [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although clinical symptoms are readily observable, they may not accurately reflect the true severity of inflammation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Nevertheless, the current study confirms that DSS effectively induces colitis-like inflammation compared to controls. Second, PET image analysis presented challenges. Manual delineation of intestinal ROIs was complicated by high radioactivity in the urinary bladder, potentially causing spillover effects and affecting quantitative accuracy. In addition, the limited number of tissue sections used for autoradiography analysis may have missed small or focal inflammatory regions detectable by more comprehensive \u003cem\u003ein vivo\u003c/em\u003e imaging or \u003cem\u003eex vivo\u003c/em\u003e gamma counting of larger tissue samples. Third, tissue sampling was limited to three consecutive sections from the same distal colon region per mouse. Given the heterogeneous and patchy nature of DSS-induced colitis, the focal severity can differ along the colon and between the mice [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Consequently, the selected sections may capture regions with relatively higher inflammation in some mice but less affected areas in others.\u003c/p\u003e \u003cp\u003eThe ability to non-invasively visualize activated macrophages has broad implications for IBD management. FR-β-targeted PET could facilitate earlier detection of inflammatory flares, guide therapeutic decisions, and serve as a biomarker for response to macrophage-directed treatments. Moreover, [\u003csup\u003e18\u003c/sup\u003eF]FOL specific binding to activated macrophages may allow longitudinal monitoring with reduced background signal compared to [\u003csup\u003e18\u003c/sup\u003eF]FDG. Future studies should investigate its performance in chronic and relapsing IBD models, as well as in human patients, to fully assess its translational potential.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, [\u003csup\u003e18\u003c/sup\u003eF]FOL PET/CT enables non-invasive detection of activated macrophage-rich inflammation in acute DSS-induced colitis, correlates with histological measures of macrophage infiltration, and shows promise as a more specific alternative to [\u003csup\u003e18\u003c/sup\u003eF]FDG for imaging IBD. These findings support further investigation of [\u003csup\u003e18\u003c/sup\u003eF]FOL in chronic colitis models and its potential clinical translation as a tool for assessing macrophage-driven pathology in patients with IBD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003eThe online version contains supplementary material available at\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eWe are grateful to Aake Honkaniemi (Turku PET Centre), Marja-Riitta Kaajala, Erica Nyman (University of Turku Histocore Facility), and Markus Peurla (University of Turku) for their valuable assistance, to Xiaoqing Zhuang and Putri Andriana for their contribution to tracer radiosynthesis, and to Timo Kattelus for finalizing the figures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eConception and design: M.K., A.A.B., M.S., P.S.L., X-G.L., J.K., D.M.T., and A.R.; acquisition, analysis and interpretation of data: M.K., A.A.B., J.R., E.A.H., J.V., H.L., J.R., X-G.L., J.K., D.M.T., and A.R. All authors were involved in drafting or critically reviewing the manuscript for important intellectual content, and all approved the final manuscript for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e The study was financially supported by grants from the Research Council of Finland (grant no. 350117). M.G. is a PhD student supported by the ImmuDocs Doctoral Pilot Program of the University of Turku. This research was also partially supported by the Research Council of Finland\u0026rsquo;s Flagship program InFLAMES (grant decision numbers 337531, 337530, 359346, and 357910).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eThe data supporting the conclusions of this article are included within the article and it\u0026rsquo;s the supplementary materials. The raw datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003eAll animal experiments were approved by the National Project Authorization Board of Finland (license numbers ESAVI/4498/2023 and ESAVI/15087/2023) and were conducted in accordance with the European Union Directive 2010/EU/63 on the protection of animals used for scientific purposes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e The authors declare that they have no conflicts of interests regarding to this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBaumgart DC, Carding SR (2007) Inflammatory bowel disease: cause and immunobiology. 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Front Immunol 14:1145473\u003c/li\u003e\n \u003cli\u003ePer\u0026scaron;e M, Cerar A (2012) Dextran sodium sulphate colitis mouse model: traps and tricks. J Biomed Biotechnol 2012:718617\u003c/li\u003e\n \u003cli\u003eDe Salvo C, Ray S, Pizarro TT (2014) Mechanisms and models for intestinal fibrosis in IBD. Dig Dis 32 Suppl 1:26-34\u003c/li\u003e\n \u003cli\u003eValatas V, Bamias G, Kolios G (2015) Experimental colitis models: Insights into the pathogenesis of inflammatory bowel disease and translational issues. Eur J Pharmacol 759:253-264\u003c/li\u003e\n \u003cli\u003eWirtz S, Neurath MF (2007) Mouse models of inflammatory bowel disease. Adv Drug Deliv Rev 59:1073-1083\u003c/li\u003e\n \u003cli\u003eTindemans I, Joosse ME, Samsom JN (2020) Dissecting the heterogeneity in T-cell mediated inflammation in IBD. Cells 9:110.\u003c/li\u003e\n \u003cli\u003eWang X, Du C, Subramanian S et al (2024) Severe gut mucosal injury induces profound systemic inflammation and spleen‑associated lymphoid organ response. Front Immunol 14:1340442.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Inflammatory bowel disease, Murine colitis, Positron emission tomography, Folate receptor beta, [18F]FOL","lastPublishedDoi":"10.21203/rs.3.rs-8410867/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8410867/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003ePositron emission tomography (PET) is a promising noninvasive technique for detecting and monitoring inflammatory bowel disease (IBD). In IBD pathogenesis, activated macrophages \u0026ndash; marked by overexpression of folate receptor beta (FR-β) \u0026ndash; play a key role. Leveraging this target, folate-based PET tracers have been developed to visualize macrophage-driven inflammation. This study evaluated the feasibility of FR-β-binding aluminum-fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([\u003csup\u003e18\u003c/sup\u003eF]FOL) PET/computed tomography (CT) for detecting intestinal inflammation in a murine model of acute colitis.\u003c/p\u003e\u003ch2\u003eProcedures\u003c/h2\u003e \u003cp\u003eAcute colitis was induced in female and male C57BL/6NCrl mice via 2.5% dextran sodium sulfate (DSS) in drinking water for 6 days, followed by recovery. Healthy age- and sex-matched mice served as controls. Mice underwent whole-body [\u003csup\u003e18\u003c/sup\u003eF]FOL PET/CT imaging followed by \u003cem\u003eex vivo\u003c/em\u003e biodistribution analysis. Intestinal tissues were further analyzed by digital autoradiography and immunohistochemical staining for FR-β and Mac-3, a marker of macrophages. 2-Deoxy-2-[\u003csup\u003e18\u003c/sup\u003eF]fluoro-\u003cem\u003eD\u003c/em\u003e-glucose ([\u003csup\u003e18\u003c/sup\u003eF]FDG) PET/CT was performed to evaluate metabolic activity in the gastrointestinal tract.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDSS-treated mice developed significant clinical signs of colitis, including weight loss and increased disease activity scores. PET/CT and \u003cem\u003eex vivo\u003c/em\u003e analyses revealed significantly higher [\u003csup\u003e18\u003c/sup\u003eF]FOL uptake in the distal colon of DSS-treated mice compared to controls. Autoradiography and immunohistochemistry confirmed the presence of inflamed regions with increased Mac-3 in the distal colon. However, only a subset of Mac-3-positive macrophages expressed FR-β. Quantitative analysis demonstrated a correlation between tracer uptake and Mac-3 staining intensity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FOL PET/CT regions enriched with activated macrophages in DSS-induced colitis, although quantitative uptake in the colon was variable. The elevated splenic uptake and strong histological correlation suggest that FR-β-targeted PET imaging can reflect systemic macrophage activation in acute IBD. These findings support the continued investigation of [\u003csup\u003e18\u003c/sup\u003eF]FOL in chronic inflammation models and its translational potential for imaging macrophage-driven pathology in IBD.\u003c/p\u003e","manuscriptTitle":"Noninvasive Detection of Experimental Colitis Using Folate Receptor-β-Targeted PET Imaging in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 03:59:38","doi":"10.21203/rs.3.rs-8410867/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c86a3daf-7b7f-4160-acb4-bb37826d0152","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T18:04:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 03:59:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8410867","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8410867","identity":"rs-8410867","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}