EDB-FN-Targeted Positron Emission Tomography Imaging of Breast Cancer in Mice

EDB-FN-Targeted Positron Emission Tomography Imaging of Breast Cancer in Mice | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article EDB-FN-Targeted Positron Emission Tomography Imaging of Breast Cancer in Mice Yun Zhang, Xiao-Bin Zheng, Yan-Fang Huang, Si-Jia Li, Xiao-Chun Yang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3492654/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 The Breast Imaging Reporting and Data System (BI-RADS) and 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]F-FDG) positron emission tomography (PET) have some limitations in the diagnosis of breast cancer. The extra domain B splice variant of fibronectin (EDB-FN), which is overexpressed in several cancer types, including breast cancer, is an approved diagnostic and therapeutic target of EDB-FN-positive cancers. Herein, we provide the EDB-FN-targeted peptide EDBp as a noninvasive oncological imaging modality for the molecular imaging of breast cancer. Procedures The binding of the peptide EDBp-Cy5 to EDB-positive breast cancer cells was investigated by flow cytometry and confocal microscopy. Near-Infrared Fluorescence (NIRF) imaging was performed at 48 hours after 1 µmol EDBp-Cy5 was intravenously injected into subcutaneous MDA-MB-231 breast cancer model mice or pulmonary metastasis model mice. Model mice were intravenously injected with approximately 100 µCi of [ 18 F]-EDBp, and PET/CT scanning was performed after 2 hours of in vivo circulation. Results Our results demonstrate that EDBp-Cy5 had a strong binding ability to the MDA-MB-231 cells. EDBp-Cy5 exhibited specific tumor accumulation in MDA-MB-231 subcutaneous and pulmonary metastasis model mice. Importantly, we proposed and validated an EDBp peptide-based radiotracer [ 18 F]-EDBp for PET imaging. [ 18 F]-EDBp provided excellent diagnostic value for PET imaging of breast cancer, especially in subcutaneous tumors. The uptake of [ 18 F]-EDBp in subcutaneous tumors (6.53 ± 0.89%, ID/g) was unexpectedly higher than that in the kidney (4.96 ± 0.20, %ID/g), which is the main metabolic organ of peptides. In the pulmonary metastasis model mice, PET/CT imaging showed that [ 18 F]-EDBp moderately accumulated in the pulmonary metastases. Conclusion The high tumor uptake of this radiotracer in mice suggests its potential for application in PET imaging of EDB-FN-positive breast cancer for disease staging of regional and distant metastases and relapse monitoring. PET Breast cancer Molecular imaging EDB-FN-targeted peptide NIRF Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction According to the latest data released by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO), breast cancer has replaced lung cancer as the most common cancer in the world[ 1 ]. Factors such as population aging, delayed childbearing, and fewer births are contributing to the continuing rise in breast cancer cases[ 2 ]. Although the gold standard for the diagnosis of breast cancer is pathological assessment[ 3 ], which requires pathological tissue obtained by puncture or surgical biopsy, it is an invasive diagnostic method that can easily cause pathogenic microbial infection, damage to the surrounding tissues and bleeding[ 4 ]. Therefore, noninvasive imaging techniques are often carried out before pathological assessment. At present, ultrasound, mammography and magnetic resonance imaging (MRI) are the main clinical diagnostic techniques for breast cancer[ 5 ]. For these techniques, the American College of Radiology reached a consensus and established a Breast Imaging Reporting and Data System (BI-RADS) classification, and as a result, biopsy is considered only if the patient reaches level 4 or above[ 6 ]. Moreover, molecular imaging technology such as 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]F-FDG) positron emission tomography (PET) can provide real-time monitoring of tumor functional molecular processes[ 6 ]. Therefore, molecular imaging may be the frontier of breast cancer diagnosis. The extra domain B splice variant of fibronectin (EDB-FN) is an extracellular matrix (ECM) protein deposited by tumor-associated fibroblasts and is associated with tumor growth, angiogenesis, and invasion[ 7 , 8 ]. EDB-FN, as a tumor-associated ECM protein, is overexpressed in breast cancer[ 9 , 10 ], prostate cancer[ 11 , 12 ], small cell lung cancer[ 13 ], and colorectal cancer[ 14 ]. An EDB-FN-targeted peptide, Thr-Val-Arg-Thr-Ser-Ala-Asp (ZD2), was initially reported by Lu et al[ 12 ]. Lu et al. used phage display to screen cyclic heptapeptides that specifically bind to EDB-FN and designed fluorescent probes for near-infrared fluorescence (NIRF) imaging of subcutaneous prostate cancer model mice[ 12 ]. ZD2 was developed for imaging prostate[ 12 , 15 ], breast[ 16 , 17 ], pancreatic[ 18 ] and hepatocellular[ 19 ] cancers. Coincidentally, the amino acid sequence of ZD2 was reported by Feng et al. in an earlier study[ 20 ]. In that study, ZD2 was also identified by phage display screening of the triple-negative breast cancer cell line MDA-MB-231[ 20 ] Therefore, ZD2, as a EDB-FN-targeted peptide, can be used as an important tool for the molecular imaging of breast cancer. Recently, Feng et al. optimized ZD2 using alanine scanning technology to increase its binding affinity to EDB-FN from 4.8 µM to 14 nM. The optimized peptide was named EDBp, and its NIRF and PET molecular probes were applied to the molecular imaging of thyroid cancer[ 21 ]. In this study, we first examined the expression levels of EDB-FN in several types of breast cancer cells and their binding to EDBp. Next, we used EDBp molecular probes to perform NIRF and PET imaging of subcutaneous breast cancer model mice and pulmonary metastases model mice. Materials and Methods Cell Culture. Breast cancer cell lines, including MCF7, SKBR3, 4T1, EMT6, MDA-MB-231 and MDA-MB-453, were obtained from the cell bank of the State Key Laboratory of Oncology in South China. Cells were grown in aseptic conditions at 37°C and 5% CO 2 . Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were regularly authenticated and confirmed to be mycoplasma-negative. To facilitate tumor monitoring, MDA-MB-231 cells were stably transfected with luciferase using a lentivirus (OBiO Technology). Protein Extraction and Western Blotting. Breast cancer cells were washed with phosphate-buffered saline (PBS), and total proteins were isolated using RIPA lysis buffer (Beyotime, #P0013B). The total protein concentrations were determined by the Bradford assay (Thermo Fisher, #23225). The proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes, which were then incubated with anti-EDB-FN monoclonal antibody (1:1000, Abcam, #ab268021) overnight at 4°C. Finally, goat anti-rabbit IgG HRP antibody (1:10000, Thermo Fisher, #G-21234) was used as the secondary antibody. Synthesis of the Peptides. The red fluorescence peptide EDBp-Cy5 (chemical structure: AVRTSAD-PEG 4 -K-Cy5) and CG7C-Cy5 (chemical structure: CGGGGGGGC-Cy5) and the PET precursor peptide EDBp-NOTA (chemical structure: AVRTSAD-PEG 4 -K-NOTA) were designed by Feng et al[ 21 ]. (Fig. S1 ). They were synthesized and analyzed through high-performance liquid chromatography (HPLC) and mass spectrometry (MS) by the Chinese Peptide Company (Fig. S2 ). Flow Cytometry. To investigate the binding of peptide EDBp-Cy5 to cells, cells were seeded at a density of 1×10 6 cells/ml in a 6-well plate and then stained with 1 µM EDBp-Cy5 or CG7C-Cy5 peptides at 37°C. After incubating for 60 min, the cells were washed with PBS three times and analyzed on a CytoFLEX S cytometer. Immunofluorescence. To investigate the binding of peptide EDBp-Cy5 to cells, cells were seeded at a density of 1×10 5 cells/ml in a confocal dish and added to 1 µM EDBp-Cy5 peptide. After 2 hours incubation at 37°C, the samples were washed with PBST six times, fixed with 4% paraformaldehyde, incubated with PBS containing 0.25% Triton X-100 (Sigma-Aldrich, #V900502) and blocked with 5% BSA for 30 min. Antibody incubation was performed with anti-EDB-FN monoclonal antibody (1:1000, Abcam, #ab268021) overnight at 4°C. To detect fluorescence, the samples were incubated with goat anti-rabbit Alexa Fluor 488 secondary antibody (Thermo Fisher, #A-11008) at a concentration of 4 µg/mL in 1% BSA for 1 hour at room temperature in the dark. Cell nuclei were stained with DAPI in ProLong Gold Antifade Mountant (Thermo Fisher, #P36930). Finally, fluorescence images were visualized and captured by a confocal microscopy (Olympus FV1000, Japan). Animal Models. The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangdong Pharmaceutical University. All mice were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China). To obtain subcutaneous tumor model mice, 4-week-old female BALB/c-nude mice were subcutaneously inoculated with 1×10 6 MDA-MB-231-luc cells. Similarly, for pulmonary metastasis model mice, 4-week-old female BALB/c-nude mice were intravenously inoculated with 1×10 6 MDA-MB-231-luc cells. When MDA-MB-231-luc cells could be observed in the lung by luminescence imaging approximately 3 weeks later, the pulmonary metastases mouse model was considered successfully constructed. NIRF Imaging. NIRF imaging was performed using an in vivo imaging system (IVIS) Spectrum (PerkinElmer, USA) at 48 hours after 1 µmol EDBp-Cy5 was intravenously injected into subcutaneous model mice or pulmonary metastasis model mice. NIRF images were acquired with excitation at 640 nm and emission at 680 nm, and luminescence images were obtained starting 10 min after intraperitoneal injection of 150 mg/kg luciferin (Promega, #P1041). After NIRF imaging, the mice were euthanized by cervical dislocation and immediately dissected. Luminescence and NIRF signals from the vital organs and tumors were recorded. Synthesis of [ 18 F]-EDBp. The PET precursor peptides EDBp-NOTA (50 µg) and AlCl 3 (3 µg) were previously freeze-dried in the reaction vial. Ethanol (330 µL), acetic acid (5 µL) and 2.5 mCi fluoride ion (65 µL) were added to the reaction vial. The mixture was heated at 100°C for 10 min and cooled to room temperature. Subsequently, [ 18 F]-EDBp was captured by a C18 light column (Waters Sep-Pak, #WAT023501) and eluted with ethanol (400 µL) after washing with saline. Finally, the eluted solution was evaporated with nitrogen and dissolved with saline. PET/CT Imaging. Mice were intravenously injected with approximately 100 µCi of [ 18 F]-EDBp. PET/CT scanning was performed after 1 hour or 2 hours of in vivo circulation. Medical imaging data PMOD analysis software was utilized to analyze the enrichment intensity of [ 18 F]-EDBp in the tumor. Biodistribution. To investigate the biodistribution of [ 18 F]-EDBp in vivo, the mice were euthanized and immediately dissected after PET/CT imaging. The normal organs and tumors were weighed and measured by a γ counter. Radioactivity was expressed as a percentage of the injected dose per gram (%ID/g). Hematoxylin and Eosin (HE) Staining and Immunohistochemistry (IHC). Tumors and pulmonary metastases from model mice were carefully dissected, fixed with formalin, and embedded in paraffin. Paraffin blocks were cut into 3 mm sections. One of the sections was stained with HE according to routine histological procedures. One of the sections was used for IHC with an anti-EDB-FN monoclonal antibody (1:1000, Abcam, #ab268021). IHC was performed following the conventional procedure as previously reported [ 21 ]. Finally, images were visualized and captured by a confocal microscopy confocal scanning system (Olympus FV1000, Japan). Statistical analysis. All statistical analyses were performed using GraphPad Prism 6.0 software. Statistical evaluations were performed using one-way ANOVA, and the results are shown as the mean ± standard deviation (SD). Statistical analyses of the survival experiments were performed using log-rank tests. p values are denoted as follows: ns, p > 0.05; * p < 0.05; ** p < 0.01; and *** p < 0.001. Results Binding of EDBp to Breast Cancer Cells Before investigating the binding ability, we first used western blotting to examine the expression levels of EDB-FN in several breast cancer cells, including MCF7, SK-BR-3, 4T1, EMT6, MDA-MB-231 and MDA-MB-453 cells. High expression of EDB-FN was observed in EMT6 cells, medium expression in MDA-MB-231 cells, and low expression in MCF7, SK-BR-3, and 4T1 cells (Fig. 1 a). Then, we used flow cytometry to test the binding ability of EDBp-Cy5 to these breast cancer cells. Similar to the different expression levels of EDB-FN, the cell binding ability of EDBp-Cy5 was high in MDA-MB-231 cells, moderate in MCF7 and EMT6 cells and low in SK-BR-3, 4T1 and MDA-MB-453 cells (Fig. 1 b). Furthermore, we used confocal microscopy to scan the fluorescence images, which showed that the EDBp-Cy5 peptide colocalized with EDB-FN in MDA-MB-231 cells (Fig. 1 c). NIRF Imaging of EDBp-Cy5 To verify the tumor homing of EDBp-Cy5, we injected MDA-MB-231 cells to form subcutaneous EDB-FN-positive breast cancer model mice and pulmonary metastasis model mice. 48 hours after injection of EDBp-Cy5 into mice, EDBp-Cy5 was significantly enriched in the subcutaneous tumor, while the uptake in other normal organs was low (Fig. 2 a and b). At the end of the imaging experiment, the organs of the mice were dissected, and EDBp-Cy5 was observed mainly in the kidney and subcutaneous tumor (Fig. 2 c-e). For the pulmonary metastases model mice, luminescence images showed that tumor cells had expanded in the lungs after 3 weeks of tail vein injection (Fig. 3 a). Similar to the subcutaneous tumors, the red fluorescence of EDBp-Cy5 and luminescence of MDA-MB-231-luc cells showed strong colocalization in pulmonary metastases (Fig. 3 b). When the organs of mice were collected, the red fluorescence of EDBp-Cy5 in the pulmonary metastases colocalized with the luminescence of MDA-MB-231-luc cells (Fig. 3 c-e). PET/CT Imaging in Subcutaneous Model Mice In the subcutaneous MDA-MB-231-luc breast cancer model mice, PET/CT imaging showed that[ 18 F]-EDBp was highly accumulated in the tumor, kidney and bladder after 2 hours of in vivo circulation (Fig. 4 a). In the 3D images, [ 18 F]-EDBp displayed excellent tumor enrichment, and the signal intensity of [ 18 F]-EDBp in the subcutaneous tumor was as strong as that in the kidney (Fig. 5 ). Subsequently, the mice were immediately dissected, and the percentage of injected dose per gram (%ID/g) in subcutaneous tumors and normal organs were calculated by a γ counter. Quantification indicated that the tissue uptake of [ 18 F]-EDBp was highest in the tumor (6.53 ± 0.89%, ID/g), which was higher than that in the kidney (4.96 ± 0.20, %ID/g) and liver (1.39 ± 0.12%ID/g) (Fig. 4 b). The tumor was subsequently dissected and subjected to HE staining and IHC. IHC images showed that EDB-FN was highly expressed in the subcutaneous MDA-MB-231 tumors (Fig. 4 c). PET/CT Imaging in Pulmonary Metastases Model Mice In the MDA-MB-231-luc breast cancer pulmonary metastasis model mice, PET/CT imaging showed that [ 18 F]-EDBp moderately accumulated in the pulmonary metastases 2 hours post intravenous injection (Fig. 6 a). The %ID/g in pulmonary metastases was 2.92 ± 0.10%ID/g (Fig. 6 b). HE staining confirmed the presence of pulmonary metastases in the lung. IHC images showed relatively high expression of EDB-FN in pulmonary metastases compared to adjacent lung tissues (Fig. 6 c). Discussion The use of BI-RADS has several limitations of varying clinical importance. For example, the definition of the “regional distribution” of an abnormal finding varies between mammography and MRI[ 22 ]. Therefore, it is necessary to explore cancer biomarkers that reflect tumor information at the cellular and subcellular levels long before anatomical analysis[ 23 ]. [ 18 F]F-FDG is the most widely used molecular imaging radiotracer for the diagnosis, staging and treatment monitoring of tumors[ 24 ], including Hodgkin and non-Hodgkin lymphomas[ 25 ], melanoma[ 26 ], colorectal cancer[ 27 ], breast cancer[ 28 ] and lung cancer[ 29 ]. Based on the available clinical data on breast cancer, [ 18 F]F-FDG imaging is substantially beneficial for patients with staging starting from clinical stage IIB and possibly useful value to patients with clinical stage IIA (T1N1 or T2N0) but is not helpful for patients with clinical stage I (T1N0) [ 30 ]. In addition, FDG metabolism reflects the energy metabolism of the body, so the malignant area can be easily confused with inflammation and local infection[ 31 ]. Estrogen receptor (ER) is highly expressed in 70% of breast cancers and plays a central role in prognosis and treatment selection for patients with breast cancer. The ER-targeted radiotracers 16α-[ 18 F]-fluoro-17β-fluoroestradiol ([ 18 F]-FES) were approved by the Food and Drug Administration (FDA) in 2020 as diagnostic agents for patients with recurrent or metastatic breast cancer. The limitations of [ 18 F]-FES are that ER-negative tumors are unlikely to be detected and false-positives may also occur [ 32 , 33 ]. In addition to the small molecular compounds FDG and FES, peptides are often used as molecular imaging radiotracers in breast cancer, and most of these peptides are identified by phage display[ 34 – 36 ]. Compared to common small molecule compounds, peptides have higher activity and selectivity and have fewer side effects on the human body because their metabolites are amino acids[ 37 , 38 ]. Compared with antibodies, peptides have the advantages of suitable stability, high purity, low production cost, and low immunogenicity[ 39 ]. Peptides can also improve the affinity, solubility, pharmacokinetic properties, stability and toxicity of drug candidates through chemical modification to support the rapid screening of drug candidates[ 40 ]. The integrin αvβ3-targeted radiotracer Arg-Gly-Asp (RGD) peptide is widely used in PET, single-photon emission computed tomography (SPECT), MRI and ultrasound imaging of breast cancer[ 31 ]. Moreover, integrin α6-targeted radiotracers, such as the CRWYDENAC peptide, have potential value in the diagnosis, staging and prognosis of human breast cancer[ 10 ]. However, in RGD or CRWYDENAC imaging of breast cancer patients, the maximum standardized uptake value (SUV max ) was not as large as that in preclinical mouse trials[ 41 ]. Moreover, the neuropilin-1-targeted peptide CLKADKAKC[ 20 ] and the sodium pump Na/K ATPase a1 subunit-targeted peptide CSISSLTHC[ 42 ] showed suitable tumor targeting in the imaging of triple-negative breast cancer-bearing mice, but their effect on patients is unpredictable. Therefore, peptides are an excellent drug form for the diagnosis of breast cancer, but it is necessary to explore more molecular probes that monitor receptors that are overexpressed on breast cancer cells. EDBp was optimized from the classical EDB-FN-targeting peptide ZD2 by alanine scanning, and its affinity with EDB-FN substantially increased from 4.8 µM to 14 nM[ 21 ]. PET/CT imaging showed that the radiotracer [ 18 F]-EDBp had high accumulation in bilateral primary thyroid lesions and bone metastases in two patients with thyroid cancer[ 21 ]. Since the preoptimized ZD2 had a suitable tumor imaging effect on breast cancer[ 16 , 17 ], we applied the optimized EDBp to the molecular imaging diagnosis of breast cancer. In this study, [ 18 F]-EDBp provided excellent diagnostic value for PET/CT imaging of breast cancer, especially in subcutaneous tumors. The uptake of [ 18 F]-EDBp in subcutaneous tumors (6.53 ± 0.89%, ID/g) was unexpectedly higher than that in the kidney (4.96 ± 0.20, %ID/g), which is the main metabolic organ of the peptide (Fig. 4 a). This phenomenon, in which tumor uptake was higher than in the kidney, was hard-won in most radiotracers, including small molecular compounds, peptides and antibodies. We also acknowledge that the uptake of [ 18 F]-EDBp in pulmonary metastases was not as high as that in subcutaneous tumors (Fig. 6 a), which may be due to the relatively weaker blood supply to the lungs. Nevertheless, this study has several limitations. First, as we can see in the results, blocking experiments of EDBp with NIRF and PET/CT imaging were not performed in mice, which hindered the validation of EDB-FN targeting. We eliminated the blocking experiments because they have already been performed in thyroid cancer studies[ 21 ]. Second, the uptake of EDBp in pulmonary metastases was not sufficient. The addition of an albumin binding domain (ABD)[ 43 ] group to alter EDBp radiotracer metabolism may prolong its half-life in vivo and increase its accumulation in pulmonary metastases. Third, the real-world effectiveness of EDBp in the diagnosis of breast cancer still needs to be verified by small-scale preclinical studies. Conclusion In this study, we provided an EDB-FN-targeted peptide for the noninvasive molecular imaging of breast cancer. The high tumor uptake of this radiotracer in mice bearing EDB-FN-positive breast cancer suggests its potential for application in PET imaging of EDB-FN-positive breast cancer, which may improve the disease staging of regional and distant metastases and relapse monitoring. Abbreviations BI-RADS Breast Imaging Reporting and Data System [ 18 F]F-FDG 2-deoxy-2-[ 18 F]fluoro-D-glucose PET positron emission tomography CT computed tomography EDB-FN extra domain B splice variant of fibronectin IARC International Agency for Research on Cancer WHO World Health Organization MRI magnetic resonance imaging ECM extracellular matrix NIRF near-infrared fluorescence PBS phosphate-buffered saline HPLC high-performance liquid chromatography MS mass spectrometry IACUC Institutional Animal Care and Use Committee Cy5 Cyanine5 NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid IVIS in vivo imaging system HE hematoxylin and eosin IHC immunohistochemistry SD standard deviation [ 18 F]-FES 16α-[ 18 F]-fluoro-17β-fluoroestradiol FDA Food and Drug Administration RGD Arg-Gly-Asp SPECT single-photon emission computed tomography SUV max maximum standardized uptake value ABD albumin binding domain. Declarations Supplementary Information The online version contains supplementary material available at https: Acknowledgements We thank G.F., X. L. and Q. W. for their excellent technical assistance. Author Contribution Y.Z., X.Z. and Y.H. are co-first authors, and they collected, analyzed and interpreted the data obtained from all experiments. Y.Z., X.Y. and Y.Y. provided resources and helps. Y.Z. wrote the manuscript. X.Y. and Y.Y. are co-corresponding authors, and they supervised the experiments and reviewed the manuscript. All authors read and approved the final manuscript. Funding This work has been supported by grants from Young Innovative Talents Project of Guangdong Provincial Education Department (2020KQNCX036) and Guangzhou Basic and Applied Basic Research Project (202201010663). Conflict of Interest The authors declare no competing interests. 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Res (Washington DC) 6:77 Supplementary Files Fig.S1.jpg Fig.S2.jpg SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted 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 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-3492654","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":243444121,"identity":"9b3b935e-d0ce-43ca-b03a-1dbb17c8c166","order_by":0,"name":"Yun Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACCSjNz8BgAKIZG4jWItlAshaDA8RqkZ/d/Ozh17bDecbnF297zMNgI7vhAPOzB/i0MM45Zm4s23a42OzGs3JjHoY04w0H2MwN8Glhlkgwk5ZsO5y47cYZM2kehsOJGw7wsEng08Imkf4NrGXzDLCW/4S18EjkmEl+BGrZwN8D0nKAsBYJiZwyaYZz6YkzbrCVG84xSDaeeZjNDK8W+Rnp2yR/lFkn9vcf3vbgTYWdbN/x5md4tYAAMy8byL4ENkgCYCakHggYf/wBkvwH2IhQOwpGwSgYBSMRAAAgd0l1YnE20gAAAABJRU5ErkJggg==","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Zhang","suffix":""},{"id":243444122,"identity":"61919966-c39b-4996-932a-0bb6e5bc2e22","order_by":1,"name":"Xiao-Bin Zheng","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center Biotherapy Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiao-Bin","middleName":"","lastName":"Zheng","suffix":""},{"id":243444123,"identity":"a5dbf002-4684-4381-ad3d-afa15282d50a","order_by":2,"name":"Yan-Fang Huang","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan-Fang","middleName":"","lastName":"Huang","suffix":""},{"id":243444124,"identity":"1d8daed9-171f-4cc0-912a-ef4e8807644b","order_by":3,"name":"Si-Jia Li","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Si-Jia","middleName":"","lastName":"Li","suffix":""},{"id":243444125,"identity":"695058c6-7bf7-4a58-b766-cce363e256e7","order_by":4,"name":"Xiao-Chun Yang","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiao-Chun","middleName":"","lastName":"Yang","suffix":""},{"id":243444126,"identity":"d35379b2-fc59-4668-adde-6dd91f8a0ae3","order_by":5,"name":"Yi-Ling Yang","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi-Ling","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2023-10-26 02:44:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3492654/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3492654/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":45486442,"identity":"7125bcfd-8042-4068-a496-b8525889059c","added_by":"auto","created_at":"2023-10-30 21:54:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":220922,"visible":true,"origin":"","legend":"\u003cp\u003eBinding of EDB-FN targeted peptide EDBp. \u003cstrong\u003ea \u003c/strong\u003eWestern blot analysis of EDB-FN in breast cancer cell lines. \u003cstrong\u003eb \u003c/strong\u003eFlow cytometry determined the binding between EDBp-Cy5 and breast cancer cell lines. \u003cstrong\u003ec\u003c/strong\u003eCellular fluorescence imaging of MDA-MB-231 cells incubated with EDBp-Cy5 at 37 °C for 2 hours. Blue, DAPI; red, EDBp-Cy5; green, EDB-FN.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/6fdd40fb8b5bd04df59c36c4.jpg"},{"id":45486445,"identity":"fbf6464a-a74e-47de-a37f-46fd4a8b0d63","added_by":"auto","created_at":"2023-10-30 21:54:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":274346,"visible":true,"origin":"","legend":"\u003cp\u003eTumor-specific homing of EDBp-Cy5in subcutaneous MDA-MB-231-luc breast cancer model mice. Luminescence imaging \u003cstrong\u003e(a)\u003c/strong\u003e and NIRF imaging \u003cstrong\u003e(b)\u003c/strong\u003eof the subcutaneous model mice after treatment for 48 hours with EDBp-Cy5 showing tumor-specific homing. Luminescence imaging \u003cstrong\u003e(c)\u003c/strong\u003e and NIRF imaging \u003cstrong\u003e(d)\u003c/strong\u003e of the subcutaneous model mice after treatment for 48 hours with EDBp-Cy5 showing the tumor and normal organs. \u003cstrong\u003ee\u003c/strong\u003e Quantitative analysis of the fluorescence intensity in the tumor and normal organs, n = 3.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/5acbef3f9968410f5c22bad2.jpg"},{"id":45486443,"identity":"13b9377e-2c82-4d35-8bf6-a3a3c83d81fc","added_by":"auto","created_at":"2023-10-30 21:54:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":459074,"visible":true,"origin":"","legend":"\u003cp\u003eTumor-specific homing of EDBp-Cy5 in MDA-MB-231-luc breast cancer pulmonary metastasismodel mice. Luminescence imaging \u003cstrong\u003e(a)\u003c/strong\u003eand NIRF imaging \u003cstrong\u003e(b)\u003c/strong\u003e of pulmonary metastasis modelmice after treatment for 48 hours with EDBp-Cy5 showing tumor-specific homing. Luminescence imaging \u003cstrong\u003e(c) \u003c/strong\u003eand NIRF imaging \u003cstrong\u003e(d)\u003c/strong\u003e of the pulmonary metastases model mice after treatment for 48 hours with EDBp-Cy5 showing the pulmonary metastases and normal organs. \u003cstrong\u003ee\u003c/strong\u003e Quantitative analysis of the fluorescence intensity in the pulmonary metastases and normal organs, n = 3.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/0bae79e24dd66df728b65a11.jpg"},{"id":45488912,"identity":"de824961-ff4e-4f5a-9c48-f708f31b8949","added_by":"auto","created_at":"2023-10-30 22:02:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":290315,"visible":true,"origin":"","legend":"\u003cp\u003ePET/CT imaging with [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in MDA-MB-231-luc breast cancer subcutaneous model mice. \u003cstrong\u003ea\u003c/strong\u003e Representative PET/CT imaging of subcutaneous model mice after injection of 100 μCi of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp. \u003cstrong\u003eb\u003c/strong\u003e The biological distribution of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in the tumor and normal organs, n = 3. \u003cstrong\u003ec\u003c/strong\u003e HE and IHC images of tumors isolated from the subcutaneous model mice. The expression of EDN-FN was analyzed by IHC. Scale bar, 50 μm (left) and 10 μm (right).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/e82b1d1702694b91696d0db0.jpg"},{"id":45486447,"identity":"85778652-4410-4537-8c2d-74b7746e1e5d","added_by":"auto","created_at":"2023-10-30 21:54:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":218631,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional images of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in MDA-MB-231-luc breast cancer subcutaneous model mice.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/23154222cddcbdcca8ef5c42.jpg"},{"id":45486448,"identity":"c95f777b-1e64-43e8-86f5-597d92102cd0","added_by":"auto","created_at":"2023-10-30 21:54:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":277052,"visible":true,"origin":"","legend":"\u003cp\u003ePET/CT imaging with [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in MDA-MB-231-luc breast cancer pulmonary metastasis model mice. \u003cstrong\u003ea\u003c/strong\u003e Representative PET/CT imaging of pulmonary metastases model mice after injection of 100 μCi of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp. \u003cstrong\u003eb\u003c/strong\u003e The biological distribution of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in the pulmonary metastases (lung) and normal organs, n = 3. \u003cstrong\u003ec\u003c/strong\u003e HE and IHC images of pulmonary metastases isolated from pulmonary metastases model mice. The expression of EDN-FN was analyzed by IHC. Scale bar, 50 μm (left) and 10 μm (right).\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/aafc22ac45af6cf3ec66f0d0.jpg"},{"id":54296933,"identity":"47ff6f43-2f60-400e-932b-116ce3e165ef","added_by":"auto","created_at":"2024-04-08 13:34:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1091857,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/7d9f6150-560b-43c5-aafe-ba92d5cc4bf1.pdf"},{"id":45486441,"identity":"13ecd708-fd80-4a20-8bd2-392e34aa34a9","added_by":"auto","created_at":"2023-10-30 21:54:20","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":101554,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/345c7ca11fe41b41fe90a338.jpg"},{"id":45488913,"identity":"4884d143-03ee-4658-8326-b72947a54134","added_by":"auto","created_at":"2023-10-30 22:02:21","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":131520,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/95a70dbee2aa38db7854d4b4.jpg"},{"id":45486444,"identity":"6b843783-bce7-4949-a944-2f7b0f845c13","added_by":"auto","created_at":"2023-10-30 21:54:20","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":218653,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3492654/v1/aafa155faf2df488e397a94b.docx"}],"financialInterests":"","formattedTitle":"EDB-FN-Targeted Positron Emission Tomography Imaging of Breast Cancer in Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording to the latest data released by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO), breast cancer has replaced lung cancer as the most common cancer in the world[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Factors such as population aging, delayed childbearing, and fewer births are contributing to the continuing rise in breast cancer cases[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although the gold standard for the diagnosis of breast cancer is pathological assessment[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which requires pathological tissue obtained by puncture or surgical biopsy, it is an invasive diagnostic method that can easily cause pathogenic microbial infection, damage to the surrounding tissues and bleeding[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, noninvasive imaging techniques are often carried out before pathological assessment. At present, ultrasound, mammography and magnetic resonance imaging (MRI) are the main clinical diagnostic techniques for breast cancer[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For these techniques, the American College of Radiology reached a consensus and established a Breast Imaging Reporting and Data System (BI-RADS) classification, and as a result, biopsy is considered only if the patient reaches level 4 or above[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Moreover, molecular imaging technology such as 2-deoxy-2-[\u003csup\u003e18\u003c/sup\u003eF]fluoro-D-glucose ([\u003csup\u003e18\u003c/sup\u003eF]F-FDG) positron emission tomography (PET) can provide real-time monitoring of tumor functional molecular processes[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, molecular imaging may be the frontier of breast cancer diagnosis.\u003c/p\u003e \u003cp\u003eThe extra domain B splice variant of fibronectin (EDB-FN) is an extracellular matrix (ECM) protein deposited by tumor-associated fibroblasts and is associated with tumor growth, angiogenesis, and invasion[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. EDB-FN, as a tumor-associated ECM protein, is overexpressed in breast cancer[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], prostate cancer[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], small cell lung cancer[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and colorectal cancer[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. An EDB-FN-targeted peptide, Thr-Val-Arg-Thr-Ser-Ala-Asp (ZD2), was initially reported by Lu et al[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Lu et al. used phage display to screen cyclic heptapeptides that specifically bind to EDB-FN and designed fluorescent probes for near-infrared fluorescence (NIRF) imaging of subcutaneous prostate cancer model mice[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. ZD2 was developed for imaging prostate[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], breast[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], pancreatic[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and hepatocellular[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] cancers. Coincidentally, the amino acid sequence of ZD2 was reported by Feng et al. in an earlier study[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In that study, ZD2 was also identified by phage display screening of the triple-negative breast cancer cell line MDA-MB-231[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Therefore, ZD2, as a EDB-FN-targeted peptide, can be used as an important tool for the molecular imaging of breast cancer.\u003c/p\u003e \u003cp\u003eRecently, Feng et al. optimized ZD2 using alanine scanning technology to increase its binding affinity to EDB-FN from 4.8 \u0026micro;M to 14 nM. The optimized peptide was named EDBp, and its NIRF and PET molecular probes were applied to the molecular imaging of thyroid cancer[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this study, we first examined the expression levels of EDB-FN in several types of breast cancer cells and their binding to EDBp. Next, we used EDBp molecular probes to perform NIRF and PET imaging of subcutaneous breast cancer model mice and pulmonary metastases model mice.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eCell Culture.\u003c/b\u003e Breast cancer cell lines, including MCF7, SKBR3, 4T1, EMT6, MDA-MB-231 and MDA-MB-453, were obtained from the cell bank of the State Key Laboratory of Oncology in South China. Cells were grown in aseptic conditions at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 \u0026micro;g/mL streptomycin. Cells were regularly authenticated and confirmed to be mycoplasma-negative. To facilitate tumor monitoring, MDA-MB-231 cells were stably transfected with luciferase using a lentivirus (OBiO Technology).\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein Extraction and Western Blotting.\u003c/b\u003e Breast cancer cells were washed with phosphate-buffered saline (PBS), and total proteins were isolated using RIPA lysis buffer (Beyotime, #P0013B). The total protein concentrations were determined by the Bradford assay (Thermo Fisher, #23225). The proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes, which were then incubated with anti-EDB-FN monoclonal antibody (1:1000, Abcam, #ab268021) overnight at 4\u0026deg;C. Finally, goat anti-rabbit IgG HRP antibody (1:10000, Thermo Fisher, #G-21234) was used as the secondary antibody.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of the Peptides.\u003c/b\u003e The red fluorescence peptide EDBp-Cy5 (chemical structure: AVRTSAD-PEG\u003csub\u003e4\u003c/sub\u003e-K-Cy5) and CG7C-Cy5 (chemical structure: CGGGGGGGC-Cy5) and the PET precursor peptide EDBp-NOTA (chemical structure: AVRTSAD-PEG\u003csub\u003e4\u003c/sub\u003e-K-NOTA) were designed by Feng et al[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). They were synthesized and analyzed through high-performance liquid chromatography (HPLC) and mass spectrometry (MS) by the Chinese Peptide Company (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow Cytometry.\u003c/b\u003e To investigate the binding of peptide EDBp-Cy5 to cells, cells were seeded at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml in a 6-well plate and then stained with 1 \u0026micro;M EDBp-Cy5 or CG7C-Cy5 peptides at 37\u0026deg;C. After incubating for 60 min, the cells were washed with PBS three times and analyzed on a CytoFLEX S cytometer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence.\u003c/b\u003e To investigate the binding of peptide EDBp-Cy5 to cells, cells were seeded at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/ml in a confocal dish and added to 1 \u0026micro;M EDBp-Cy5 peptide. After 2 hours incubation at 37\u0026deg;C, the samples were washed with PBST six times, fixed with 4% paraformaldehyde, incubated with PBS containing 0.25% Triton X-100 (Sigma-Aldrich, #V900502) and blocked with 5% BSA for 30 min. Antibody incubation was performed with anti-EDB-FN monoclonal antibody (1:1000, Abcam, #ab268021) overnight at 4\u0026deg;C. To detect fluorescence, the samples were incubated with goat anti-rabbit Alexa Fluor 488 secondary antibody (Thermo Fisher, #A-11008) at a concentration of 4 \u0026micro;g/mL in 1% BSA for 1 hour at room temperature in the dark. Cell nuclei were stained with DAPI in ProLong Gold Antifade Mountant (Thermo Fisher, #P36930). Finally, fluorescence images were visualized and captured by a confocal microscopy (Olympus FV1000, Japan).\u003c/p\u003e \u003cp\u003e\u003cb\u003eAnimal Models.\u003c/b\u003e The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangdong Pharmaceutical University. All mice were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China). To obtain subcutaneous tumor model mice, 4-week-old female BALB/c-nude mice were subcutaneously inoculated with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e MDA-MB-231-luc cells. Similarly, for pulmonary metastasis model mice, 4-week-old female BALB/c-nude mice were intravenously inoculated with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e MDA-MB-231-luc cells. When MDA-MB-231-luc cells could be observed in the lung by luminescence imaging approximately 3 weeks later, the pulmonary metastases mouse model was considered successfully constructed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNIRF Imaging.\u003c/b\u003e NIRF imaging was performed using an in vivo imaging system (IVIS) Spectrum (PerkinElmer, USA) at 48 hours after 1 \u0026micro;mol EDBp-Cy5 was intravenously injected into subcutaneous model mice or pulmonary metastasis model mice. NIRF images were acquired with excitation at 640 nm and emission at 680 nm, and luminescence images were obtained starting 10 min after intraperitoneal injection of 150 mg/kg luciferin (Promega, #P1041). After NIRF imaging, the mice were euthanized by cervical dislocation and immediately dissected. Luminescence and NIRF signals from the vital organs and tumors were recorded.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of [\u003c/b\u003e \u003csup\u003e \u003cb\u003e18\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eF]-EDBp.\u003c/b\u003e The PET precursor peptides EDBp-NOTA (50 \u0026micro;g) and AlCl\u003csub\u003e3\u003c/sub\u003e (3 \u0026micro;g) were previously freeze-dried in the reaction vial. Ethanol (330 \u0026micro;L), acetic acid (5 \u0026micro;L) and 2.5 mCi fluoride ion (65 \u0026micro;L) were added to the reaction vial. The mixture was heated at 100\u0026deg;C for 10 min and cooled to room temperature. Subsequently, [\u003csup\u003e18\u003c/sup\u003eF]-EDBp was captured by a C18 light column (Waters Sep-Pak, #WAT023501) and eluted with ethanol (400 \u0026micro;L) after washing with saline. Finally, the eluted solution was evaporated with nitrogen and dissolved with saline.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePET/CT Imaging.\u003c/b\u003e Mice were intravenously injected with approximately 100 \u0026micro;Ci of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp. PET/CT scanning was performed after 1 hour or 2 hours of in vivo circulation. Medical imaging data PMOD analysis software was utilized to analyze the enrichment intensity of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in the tumor.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiodistribution.\u003c/b\u003e To investigate the biodistribution of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in vivo, the mice were euthanized and immediately dissected after PET/CT imaging. The normal organs and tumors were weighed and measured by a γ counter. Radioactivity was expressed as a percentage of the injected dose per gram (%ID/g).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHematoxylin and Eosin (HE) Staining and Immunohistochemistry (IHC).\u003c/b\u003e Tumors and pulmonary metastases from model mice were carefully dissected, fixed with formalin, and embedded in paraffin. Paraffin blocks were cut into 3 mm sections. One of the sections was stained with HE according to routine histological procedures. One of the sections was used for IHC with an anti-EDB-FN monoclonal antibody (1:1000, Abcam, #ab268021). IHC was performed following the conventional procedure as previously reported [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Finally, images were visualized and captured by a confocal microscopy confocal scanning system (Olympus FV1000, Japan).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e All statistical analyses were performed using GraphPad Prism 6.0 software. Statistical evaluations were performed using one-way ANOVA, and the results are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses of the survival experiments were performed using log-rank tests. \u003cem\u003ep\u003c/em\u003e values are denoted as follows: ns, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; and ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBinding of EDBp to Breast Cancer Cells\u003c/h2\u003e \u003cp\u003eBefore investigating the binding ability, we first used western blotting to examine the expression levels of EDB-FN in several breast cancer cells, including MCF7, SK-BR-3, 4T1, EMT6, MDA-MB-231 and MDA-MB-453 cells. High expression of EDB-FN was observed in EMT6 cells, medium expression in MDA-MB-231 cells, and low expression in MCF7, SK-BR-3, and 4T1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Then, we used flow cytometry to test the binding ability of EDBp-Cy5 to these breast cancer cells. Similar to the different expression levels of EDB-FN, the cell binding ability of EDBp-Cy5 was high in MDA-MB-231 cells, moderate in MCF7 and EMT6 cells and low in SK-BR-3, 4T1 and MDA-MB-453 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Furthermore, we used confocal microscopy to scan the fluorescence images, which showed that the EDBp-Cy5 peptide colocalized with EDB-FN in MDA-MB-231 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNIRF Imaging of EDBp-Cy5\u003c/h2\u003e \u003cp\u003eTo verify the tumor homing of EDBp-Cy5, we injected MDA-MB-231 cells to form subcutaneous EDB-FN-positive breast cancer model mice and pulmonary metastasis model mice. 48 hours after injection of EDBp-Cy5 into mice, EDBp-Cy5 was significantly enriched in the subcutaneous tumor, while the uptake in other normal organs was low (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b). At the end of the imaging experiment, the organs of the mice were dissected, and EDBp-Cy5 was observed mainly in the kidney and subcutaneous tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-e). For the pulmonary metastases model mice, luminescence images showed that tumor cells had expanded in the lungs after 3 weeks of tail vein injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Similar to the subcutaneous tumors, the red fluorescence of EDBp-Cy5 and luminescence of MDA-MB-231-luc cells showed strong colocalization in pulmonary metastases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). When the organs of mice were collected, the red fluorescence of EDBp-Cy5 in the pulmonary metastases colocalized with the luminescence of MDA-MB-231-luc cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-e).\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePET/CT Imaging in Subcutaneous Model Mice\u003c/h2\u003e \u003cp\u003eIn the subcutaneous MDA-MB-231-luc breast cancer model mice, PET/CT imaging showed that[\u003csup\u003e18\u003c/sup\u003eF]-EDBp was highly accumulated in the tumor, kidney and bladder after 2 hours of in vivo circulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In the 3D images, [\u003csup\u003e18\u003c/sup\u003eF]-EDBp displayed excellent tumor enrichment, and the signal intensity of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in the subcutaneous tumor was as strong as that in the kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Subsequently, the mice were immediately dissected, and the percentage of injected dose per gram (%ID/g) in subcutaneous tumors and normal organs were calculated by a γ counter. Quantification indicated that the tissue uptake of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp was highest in the tumor (6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89%, ID/g), which was higher than that in the kidney (4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20, %ID/g) and liver (1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12%ID/g) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The tumor was subsequently dissected and subjected to HE staining and IHC. IHC images showed that EDB-FN was highly expressed in the subcutaneous MDA-MB-231 tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePET/CT Imaging in Pulmonary Metastases Model Mice\u003c/h2\u003e \u003cp\u003eIn the MDA-MB-231-luc breast cancer pulmonary metastasis model mice, PET/CT imaging showed that [\u003csup\u003e18\u003c/sup\u003eF]-EDBp moderately accumulated in the pulmonary metastases 2 hours post intravenous injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The %ID/g in pulmonary metastases was 2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10%ID/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). HE staining confirmed the presence of pulmonary metastases in the lung. IHC images showed relatively high expression of EDB-FN in pulmonary metastases compared to adjacent lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe use of BI-RADS has several limitations of varying clinical importance. For example, the definition of the \u0026ldquo;regional distribution\u0026rdquo; of an abnormal finding varies between mammography and MRI[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, it is necessary to explore cancer biomarkers that reflect tumor information at the cellular and subcellular levels long before anatomical analysis[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. [\u003csup\u003e18\u003c/sup\u003eF]F-FDG is the most widely used molecular imaging radiotracer for the diagnosis, staging and treatment monitoring of tumors[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], including Hodgkin and non-Hodgkin lymphomas[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], melanoma[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], colorectal cancer[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], breast cancer[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and lung cancer[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Based on the available clinical data on breast cancer, [\u003csup\u003e18\u003c/sup\u003eF]F-FDG imaging is substantially beneficial for patients with staging starting from clinical stage IIB and possibly useful value to patients with clinical stage IIA (T1N1 or T2N0) but is not helpful for patients with clinical stage I (T1N0) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, FDG metabolism reflects the energy metabolism of the body, so the malignant area can be easily confused with inflammation and local infection[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Estrogen receptor (ER) is highly expressed in 70% of breast cancers and plays a central role in prognosis and treatment selection for patients with breast cancer. The ER-targeted radiotracers 16α-[\u003csup\u003e18\u003c/sup\u003eF]-fluoro-17β-fluoroestradiol ([\u003csup\u003e18\u003c/sup\u003eF]-FES) were approved by the Food and Drug Administration (FDA) in 2020 as diagnostic agents for patients with recurrent or metastatic breast cancer. The limitations of [\u003csup\u003e18\u003c/sup\u003eF]-FES are that ER-negative tumors are unlikely to be detected and false-positives may also occur [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to the small molecular compounds FDG and FES, peptides are often used as molecular imaging radiotracers in breast cancer, and most of these peptides are identified by phage display[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Compared to common small molecule compounds, peptides have higher activity and selectivity and have fewer side effects on the human body because their metabolites are amino acids[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Compared with antibodies, peptides have the advantages of suitable stability, high purity, low production cost, and low immunogenicity[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Peptides can also improve the affinity, solubility, pharmacokinetic properties, stability and toxicity of drug candidates through chemical modification to support the rapid screening of drug candidates[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The integrin αvβ3-targeted radiotracer Arg-Gly-Asp (RGD) peptide is widely used in PET, single-photon emission computed tomography (SPECT), MRI and ultrasound imaging of breast cancer[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Moreover, integrin α6-targeted radiotracers, such as the CRWYDENAC peptide, have potential value in the diagnosis, staging and prognosis of human breast cancer[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, in RGD or CRWYDENAC imaging of breast cancer patients, the maximum standardized uptake value (SUV\u003csub\u003emax\u003c/sub\u003e) was not as large as that in preclinical mouse trials[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Moreover, the neuropilin-1-targeted peptide CLKADKAKC[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and the sodium pump Na/K ATPase a1 subunit-targeted peptide CSISSLTHC[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] showed suitable tumor targeting in the imaging of triple-negative breast cancer-bearing mice, but their effect on patients is unpredictable. Therefore, peptides are an excellent drug form for the diagnosis of breast cancer, but it is necessary to explore more molecular probes that monitor receptors that are overexpressed on breast cancer cells.\u003c/p\u003e \u003cp\u003eEDBp was optimized from the classical EDB-FN-targeting peptide ZD2 by alanine scanning, and its affinity with EDB-FN substantially increased from 4.8 \u0026micro;M to 14 nM[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. PET/CT imaging showed that the radiotracer [\u003csup\u003e18\u003c/sup\u003eF]-EDBp had high accumulation in bilateral primary thyroid lesions and bone metastases in two patients with thyroid cancer[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Since the preoptimized ZD2 had a suitable tumor imaging effect on breast cancer[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we applied the optimized EDBp to the molecular imaging diagnosis of breast cancer. In this study, [\u003csup\u003e18\u003c/sup\u003eF]-EDBp provided excellent diagnostic value for PET/CT imaging of breast cancer, especially in subcutaneous tumors. The uptake of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in subcutaneous tumors (6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89%, ID/g) was unexpectedly higher than that in the kidney (4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20, %ID/g), which is the main metabolic organ of the peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This phenomenon, in which tumor uptake was higher than in the kidney, was hard-won in most radiotracers, including small molecular compounds, peptides and antibodies. We also acknowledge that the uptake of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in pulmonary metastases was not as high as that in subcutaneous tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), which may be due to the relatively weaker blood supply to the lungs.\u003c/p\u003e \u003cp\u003eNevertheless, this study has several limitations. First, as we can see in the results, blocking experiments of EDBp with NIRF and PET/CT imaging were not performed in mice, which hindered the validation of EDB-FN targeting. We eliminated the blocking experiments because they have already been performed in thyroid cancer studies[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Second, the uptake of EDBp in pulmonary metastases was not sufficient. The addition of an albumin binding domain (ABD)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] group to alter EDBp radiotracer metabolism may prolong its half-life in vivo and increase its accumulation in pulmonary metastases. Third, the real-world effectiveness of EDBp in the diagnosis of breast cancer still needs to be verified by small-scale preclinical studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we provided an EDB-FN-targeted peptide for the noninvasive molecular imaging of breast cancer. The high tumor uptake of this radiotracer in mice bearing EDB-FN-positive breast cancer suggests its potential for application in PET imaging of EDB-FN-positive breast cancer, which may improve the disease staging of regional and distant metastases and relapse monitoring.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBI-RADS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBreast Imaging Reporting and Data System\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e[\u003csup\u003e18\u003c/sup\u003eF]F-FDG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-deoxy-2-[\u003csup\u003e18\u003c/sup\u003eF]fluoro-D-glucose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epositron emission tomography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecomputed tomography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDB-FN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextra domain B splice variant of fibronectin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIARC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInternational Agency for Research on Cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWorld Health Organization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emagnetic resonance imaging\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNIRF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enear-infrared fluorescence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIACUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInstitutional Animal Care and Use Committee\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCy5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCyanine5\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNOTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1,4,7-triazacyclononane-1,4,7-triacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIVIS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ein vivo imaging system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e[\u003csup\u003e18\u003c/sup\u003eF]-FES\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e16α-[\u003csup\u003e18\u003c/sup\u003eF]-fluoro-17β-fluoroestradiol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFood and Drug Administration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRGD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArg-Gly-Asp\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPECT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esingle-photon emission computed tomography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSUV\u003csub\u003emax\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emaximum standardized uptake value\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eABD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealbumin binding domain.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003eThe online version contains supplementary material available at https:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe thank G.F., X. L. and Q. W. for their excellent technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e Y.Z., X.Z. and Y.H. are co-first authors, and they collected, analyzed and interpreted the data obtained from all experiments. Y.Z., X.Y. and Y.Y. provided resources and helps. Y.Z. wrote the manuscript. X.Y. and Y.Y. are co-corresponding authors, and they supervised the experiments and reviewed the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work has been supported by grants from Young Innovative Talents Project of Guangdong Provincial Education Department (2020KQNCX036) and Guangzhou Basic and Applied Basic Research Project (202201010663).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u0026nbsp;\u003c/strong\u003eThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article\u0026apos;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026apos;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL et al (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71:209\u0026ndash;249\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoerjomataram I, Bray F (2021) Planning for tomorrow: global cancer incidence and the role of prevention 2020\u0026ndash;2070. 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Eur J Cancer 34:1081\u0026ndash;1085\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto E, Yoshida T, Kawarada Y, Sakakura T (1999) Expression of fibronectin isoforms in human breast tissue: production of extra domain A+/extra domain B + by cancer cells and extra domain A + by stromal cells. Jpn J Cancer Res 90:320\u0026ndash;325\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrini I, Barachini S, Carnicelli V et al (eds) (2017) ED-B fibronectin expression is a marker of epithelial-mesenchymal transition in translational oncology. Oncotarget 8:4914\u0026ndash;4921\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan Z, Zhou Z, Shi X et al (2015) EDB Fibronectin Specific Peptide for Prostate Cancer Targeting. 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ACS Med Chem Lett 9:730\u0026ndash;735\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe X-X, Zhao Y-Y, Wang Q et al (2017) EDB Fibronectin-Specific SPECT Probe (99m)Tc-HYNIC-ZD2 for Breast Cancer Detection. ACS omega 2:2459\u0026ndash;2468\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Liu C, Gao Y et al (2018) ZD2-Engineered Gold Nanostar@Metal-Organic Framework Nanoprobes for T(1)-Weighted Magnetic Resonance Imaging and Photothermal Therapy Specifically Toward Triple-Negative Breast Cancer. Adv Healthc Mater 7:e1801144\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao S, Qin J, Sergeeva O et al (2019) Synthesis and assessment of ZD2-((68)Ga-NOTA) specific to extradomain B fibronectin in tumor microenvironment for PET imaging of pancreatic cancer. Am J Nucl Med Mol Imaging 9:216\u0026ndash;229\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSergeeva O, Zhang Y, Gao S et al (2023) PET Imaging of Hepatocellular Carcinoma Using ZD2-((68)Ga-NOTA). J Hepatocell carcinoma 10:291\u0026ndash;301\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng GK, Liu R, Bin, Zhang MQ et al (2014) SPECT and near-infrared fluorescence imaging of breast cancer with a neuropilin-1-targeting peptide. J Control Release 192:236\u0026ndash;242\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi R, He H, Li X et al (2023) EDB-FN targeted probes for the surgical navigation, radionuclide imaging, and therapy of thyroid cancer. Eur J Nucl Med Mol Imaging.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEghtedari M, Chong A, Rakow-Penner R, Ojeda-Fournier H (2021) Current Status and Future of BI-RADS in Multimodality Imaging, From the AJR Special Series on Radiology Reporting and Data Systems. AJR Am J Roentgenol 216:860\u0026ndash;873\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SY, Jeon SI, Jung S et al (2014) Targeted multimodal imaging modalities. Adv Drug Deliv Rev 76:60\u0026ndash;78\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKahle XU, Jesus FMM, De, Glaudemans AWJM et al (2020) Review Molecular imaging in lymphoma beyond \u003csup\u003e18\u003c/sup\u003eF-FDG-PET: understanding the biology and its implications for diagnostics and therapy. Lancet Haematol 7:e479\u0026ndash;e489\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarrington SF, Kluge R (2017) FDG PET for therapy monitoring in Hodgkin and non-Hodgkin lymphomas. Eur J Nucl Med Mol Imaging 44:97\u0026ndash;110\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAyati N, Sadeghi R, Kiamanesh Z et al (2021) The value of (18)F-FDG PET/CT for predicting or monitoring immunotherapy response in patients with metastatic melanoma: a systematic review and meta-analysis. Eur J Nucl Med Mol Imaging 48:428\u0026ndash;448\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMirshahvalad SA, Hinzpeter R, Kohan A et al (2022) Diagnostic performance of [(18)F]-FDG PET/MR in evaluating colorectal cancer: a systematic review and meta-analysis. Eur J Nucl Med Mol Imaging 49:4205\u0026ndash;4217\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvril S, Muzic RFJ, Plecha D et al (2016) \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT for Monitoring of Treatment Response in Breast Cancer. J Nucl Med 57(Suppl 1):34S\u0026ndash;9S\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaz SC, Adam JA, Delgado Bolton RC et al (2022) Joint EANM/SNMMI/ESTRO practice recommendations for the use of 2-[(18)F]FDG PET/CT external beam radiation treatment planning in lung cancer V1.0. Eur J Nucl Med Mol Imaging 49:1386\u0026ndash;1406\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroheux D, Hindie E (2021) Breast cancer: initial workup and staging with FDG PET / CT. Clin Transl Imaging 9:221\u0026ndash;231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChakravarty R, Chakraborty S, Dash A (2015) Molecular Imaging of Breast Cancer: Role of RGD Peptides. Mini Rev Med Chem 15:1073\u0026ndash;1094\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatzenellenbogen JA (2021) The quest for improving the management of breast cancer by functional imaging: The discovery and development of 16α-[(18)F]fluoroestradiol (FES), a PET radiotracer for the estrogen receptor, a historical review. Nucl Med Biol 92:24\u0026ndash;37\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUlaner GA (2022) 16α-18F-fluoro-17β-Fluoroestradiol (FES): Clinical Applications for Patients With Breast Cancer. Semin Nucl Med 52:574\u0026ndash;583\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelo CM, Wang H, Fujimura K et al (2021) The Heparan Sulfate Binding Peptide in Tumor Progression of Triple-Negative Breast Cancer. Front Oncol 11:697626\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeung K (2004) (99m)Tc-Diamine dioxime-Lys-Cys-Arg-Gly-Asp-Cyc-Phe-Cys-polyethylene glycol. Bethesda (MD)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarimer BM, Deutscher SL (2014) Development of a peptide by phage display for SPECT imaging of resistance-susceptible breast cancer. Am J Nucl Med Mol Imaging 4:435\u0026ndash;447\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi M, McHugh KJ (2023) Strategies for overcoming protein and peptide instability in biodegradable drug delivery systems. Adv Drug Deliv Rev 199:114904\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaranko A, Saha S, Chilkoti A (2020) Recent trends in protein and peptide-based biomaterials for advanced drug delivery. Adv Drug Deliv Rev 156:133\u0026ndash;187\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun X, Li Y, Liu T et al (2017) Peptide-based imaging agents for cancer detection. Adv Drug Deliv Rev 110\u0026ndash;111:38\u0026ndash;51\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang W, Hu Z (2019) Targeting Peptide-Based Probes for Molecular Imaging and Diagnosis. Adv Mater 31:e1804827\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoon H-J, Kang KW, Chun IK et al (2014) Correlation of breast cancer subtypes, based on estrogen receptor, progesterone receptor, and HER2, with functional imaging parameters from \u003csup\u003e68\u003c/sup\u003eGa-RGD PET/CT and \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT. Eur J Nucl Med Mol Imaging 41:1534\u0026ndash;1543\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Li S-B, Zhao Y-Y et al (2018) Identification of a sodium pump Na(+)/K(+) ATPase α1-targeted peptide for PET imaging of breast cancer. J Control release Off J Control Release Soc 281:178\u0026ndash;188\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Zhang D, An S et al (2023) Development and Characterization of Nanobody-Derived CD47 Theranostic Pairs in Solid Tumors. Res (Washington DC) 6:77\u003c/span\u003e\u003c/li\u003e\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":true,"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":"PET, Breast cancer, Molecular imaging, EDB-FN-targeted peptide, NIRF","lastPublishedDoi":"10.21203/rs.3.rs-3492654/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3492654/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003ePurpose\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe Breast Imaging Reporting and Data System (BI-RADS) and 2-deoxy-2-[\u003csup\u003e18\u003c/sup\u003eF]fluoro-D-glucose ([\u003csup\u003e18\u003c/sup\u003eF]F-FDG) positron emission tomography (PET) have some limitations in the diagnosis of breast cancer. The extra domain B splice variant of fibronectin (EDB-FN), which is overexpressed in several cancer types, including breast cancer, is an approved diagnostic and therapeutic target of EDB-FN-positive cancers. Herein, we provide the EDB-FN-targeted peptide EDBp as a noninvasive oncological imaging modality for the molecular imaging of breast cancer.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProcedures\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe binding of the peptide EDBp-Cy5 to EDB-positive breast cancer cells was investigated by flow cytometry and confocal microscopy. Near-Infrared Fluorescence (NIRF) imaging was performed at 48 hours after 1 \u0026micro;mol EDBp-Cy5 was intravenously injected into subcutaneous MDA-MB-231 breast cancer model mice or pulmonary metastasis model mice. Model mice were intravenously injected with approximately 100 \u0026micro;Ci of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp, and PET/CT scanning was performed after 2 hours of in vivo circulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results demonstrate that EDBp-Cy5 had a strong binding ability to the MDA-MB-231 cells. EDBp-Cy5 exhibited specific tumor accumulation in MDA-MB-231 subcutaneous and pulmonary metastasis model mice. Importantly, we proposed and validated an EDBp peptide-based radiotracer [\u003csup\u003e18\u003c/sup\u003eF]-EDBp for PET imaging. [\u003csup\u003e18\u003c/sup\u003eF]-EDBp provided excellent diagnostic value for PET imaging of breast cancer, especially in subcutaneous tumors. The uptake of [\u003csup\u003e18\u003c/sup\u003eF]-EDBp in subcutaneous tumors (6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89%, ID/g) was unexpectedly higher than that in the kidney (4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20, %ID/g), which is the main metabolic organ of peptides. In the pulmonary metastasis model mice, PET/CT imaging showed that [\u003csup\u003e18\u003c/sup\u003eF]-EDBp moderately accumulated in the pulmonary metastases.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe high tumor uptake of this radiotracer in mice suggests its potential for application in PET imaging of EDB-FN-positive breast cancer for disease staging of regional and distant metastases and relapse monitoring.\u003c/p\u003e","manuscriptTitle":"EDB-FN-Targeted Positron Emission Tomography Imaging of Breast Cancer in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-30 21:54:15","doi":"10.21203/rs.3.rs-3492654/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":"c327c249-87be-40c3-a29b-26f842e9edb6","owner":[],"postedDate":"October 30th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-08T13:26:32+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-30 21:54:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3492654","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3492654","identity":"rs-3492654","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}