Ultrasensitive Detection of Uveal Melanoma Using [18F]AlF-NOTA-PRGD2 PET Imaging

Ultrasensitive Detection of Uveal Melanoma Using [18F]AlF-NOTA-PRGD2 PET Imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ultrasensitive Detection of Uveal Melanoma Using [18F]AlF-NOTA-PRGD2 PET Imaging Ling Wang, Xue Zhu, Yan Xue, Zhihong Huang, Wenjun Zou, Zhengwei Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3998432/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background: Uveal melanoma (UM) is the most common primary intraocular tumor in adults, and early detection is critical to improve the clinical outcome of this disease. In this study, the diagnostic effectiveness of [ 18 F]AlF-NOTA-PRGD2 (an investigational medicinal product) positron emission tomography (PET) imaging in UM xenografts and UM patients were evaluated. The cell uptake, cell binding ability and in vitro stability of [ 18 F]AlF-NOTA-PRGD2 were evaluated in 92-1 UM cell line. MicroPET imaging and biodistribution study of [ 18 F]AlF-NOTA-PRGD2 were conducted in 92-1 UM xenografts. Then, UM patients were further recruited for evaluating the diagnostic effectiveness of [ 18 F]AlF-NOTA-PRGD2 PET imaging (approval no.NCT02441972 in clinicaltrials.gov). In addition, comparison of [ 18 F]AlF-NOTA-PRGD2 and 18 F-labelled fluorodeoxyglucose ([ 18 F]FDG) PET imaging in UM xenografts and UM patients were conducted. Results: The in vitro data showed that [ 18 F]AlF-NOTA-PRGD2 had a high cell uptake, cell binding ability and in vitro stability in 92-1 UM cell line. The in vivo data indicated that 92-1 UM tumors were clearly visualized with the [ 18 F]AlF-NOTA-PRGD2 tracer in the subcutaneous and ocular primary UM xenografts model at 60 min post-injection. And the tumor uptake of the tracer was 2.55±0.44%ID/g and 1.73±0.15%ID/g at these two tissue locations respectively, at 7 days after animal model construction. The clinical data showed that tumors in UM patients were clearly visualized with the [ 18 F]AlF-NOTA-PRGD2 tracer at 60 min post-injection. In addition, [ 18 F]AlF-NOTA-PRGD2 tracer showed higher sensitivity and specificity for PET imaging in UM xenografts and UM patients compared to [ 18 F]FDG tracer. Conclusion: [ 18 F]AlF-NOTA-PRGD2 PET imaging may be a more preferred approach in the diagnosis of UM compared to [ 18 F]FDG PET imaging. Trial registration ClinicalTrials.gov: NCT02441972, Registered 1 January 2012, https://clinicaltrials.gov/study/NCT02441972 Uveal Melanoma [18F]AlF-NOTA-PRGD2 [18F]FDG PET imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Although rare, uveal melanoma (UM) is the primary intraocular tumor worldwide [ 1 , 2 ]. Control of local UM seems effective through non-pharmacological treatment strategies, but up to 50% of patients still develop metastasis. Overall, with a very poor prognosis [ 3 , 4 ], early detection and timely treatment are critical to improve the clinical outcome of UM [ 5 ]. Currently, UM diagnosis relies on clinical examination, the slit lamp and indirect ophthalmoscope, ocular ultrasonography, magnetic resonance imaging (MRI) and positron emission tomography/computerized tomography (PET/CT) of whole body [ 6 , 7 ]. However, the primary challenge is to distinguish small ocular tumors with a thickness of less than 3 mm, from presumed naevi as well as to detect metastasis in the whole body. PET/CT as a traditional non-invasive technique, plays a critical role in oncologic imaging and has been widely used for the diagnosis and staging of a variety of malignancies [ 8 – 10 ]. The sensitivity and specificity of PET scanning largely relies on the imaging tracer. In the past 40 years, [ 18 F]FDG is the dominant PET tracer used in the imaging of neurology, cardiology, and oncology [ 11 – 14 ]. However, the efficacy of [ 18 F]FDG PET imaging in the detection of UM is very poor with a high incidence of false-negative results. The limited sensitivity of [ 18 F]FDG PET imaging in detection of small size tumor is due to low metabolic nature of UM. In addition, it is also not sensitive enough for the detection of UM metastases into the liver and other tissues. In contrast, liver metastases from cutaneous melanoma are shown to be FDG sensitive [ 15 , 16 ]. Therefore, to develop a more sensitive PET imaging tracer is helpful for a better differential diagnosis and staging of UM, which can be used as a supplement routine examination. Integrins (consisting of two noncovalently bound transmembrane subunits ( α and β)) are large, membrane-spanning, heterodimeric proteins [ 17 , 18 ]. Integrin alphaVbeta3 (integrin αvβ3) is one member of integrins and contributes to biological processes such as cell adhesion and cell migration [ 19 ]. In the past decade, integrin αvβ3 was adopted as an important molecular target for early and differential diagnosis of rapidly growing solid tumors due to its role in tumor angiogenesis [ 20 , 21 ]. Integrin αvβ3 is a receptor for arginine-glycine-aspartic (RGD) tripeptide, which also includes linear and cyclic RGD peptide antagonists. Thus, targeting integrin αvβ3 is a viable approach to develop radiotracers for PET/CT [ 22 ]. Notni et al have conducted a preclinical evaluation of [ 68 Ga]TRAP(RGD) 3 in human melanoma xenografts and showed that there is a high uptake of such radiotracer [ 23 ]. Chen et al develop a new PET imaging probe [ 18 F]AlF-NOTA-PRGD2 (NOTA-PEG4-E[c(RGDfK)]2) and successfully applied to lung cancer patients, with longer tumor retention and simpler labeling process [ 24 ]. Up to date, [ 18 F]AlF-NOTA-PRGD2 tracer has not been applied to PET imaging of UM patients yet. The previous studies and our data have confirmed integrin αvβ3 is highly expressed in UM cells and UM tissues [ 25 ]. And the current study is the first to evaluate the efficacy of [ 18 F]AlF-NOTA-PRGD2 for PET imaging of UM, in comparison to that of [ 18 F]FDG. Materials and methods General materials All commercially obtained chemicals were analytical grade and used without further purification. FDG (fluorodeoxyglucose) was provided from the Wuxi Jiangyuan Industrial Technology And Trade Corporation and reconstituted with sterile saline. PRGD2 (PEG4-E[c(RGDyK)]2) and NOTA-PRGD2 were provided from Chinese Peptide Company (Hangzhou, China). 18 F-fluoride was obtained from an in-house PET trace cyclotron (HM-7, Sumitomo Heavy Industries Ltd, Japan) via the 18 O (p, n) 18 F nuclear reaction. Radiosyntheses of [ 18 F]FDG and [ 18 F]AlF-NOTA-PRGD2 [ 18 F]FDG and [ 18 F]AlF-NOTA-PRGD2 were synthesized as the previous report [ 26 ]. The preparation of [ 18 F]AlF-NOTA-PRGD2 was solved as follows: NOTA-PRGD2 (50 µg contains 1.6 µg AlCl 3 ) add 20 µL water to dissolved in the reaction bottle and add 5 µL of glacial acetic acid to adjust PH (PH ≈ 3). Then, 20 µL [ 18 F] fluorinated water (~ 1110 MBq) and 0.2 mL acetonitrile were added to the reaction flask. The above solution was reacted at 95°C for 10 min, the product was diluted with 9 mL water and hung on an activated Varian BOND ELUT C18 column. Wash the C18 column three times with 10ml water. Finally, 300 µL of 10 mM hydrochloric acid was used to elute the product in ethanol. The mixture was purified by the C18 cartridge using High Performance Liquid Chromatography (HPLC). The final product was formulated in saline for the subsequent studies. Cell culture and UM xenografts Human UM cell lines (92 − 1, OCM-1A and MEL270) were obtained from OcuTech Co,Ltd (Wuxi, China) and cultured in RPMI1640 medium with 10% FBS (fetal bovine serum) and 1% P/S (penicillin/streptomycin). The expression of integrin αvβ3 on 92 − 1 cells were confirmed by western blot analysis and immunofluorescence. All experiment protocols were approved by the Animal Care and Use Committee of Jiangsu Institute of Nuclear Medicine (JSINM-2022-061). The male BALB/c nude mice (4 ~ 5 weeks old, 18 ~ 22 g body weight) were purchased from Cavens Laboratory Animal Technology Co. Ltd. (Jiangsu, China). The nude mice were injected with 5 ×10 6 92-1-Luc cells in subcutaneous or 1 ×10 5 92-1-Luc cells in ocular, when the tumor size reaches about 200 mm 3 , about 3 ~ 4 weeks after the cell inoculation, the tumor model could be used for further study. Western blot analysis Cells were lysed with RIPA (Radio Immunoprecipitation Assay) buffer and protein concentration was determined with BCA assay (Beyotime, Nantong, China). Protein extract (20 µg) was separated by SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with the primary antibody of integrin alpha V (ab179475, Abcam, MA, USA) and integrin beta 3 (ab179473, Abcam, MA, USA) followed by the horseradish peroxidase-conjugated secondary antibody (ab97051, Abcam, MA, USA). The expression of target protein was verified by chemiluminescence (ECL) detection kit. Band densities was analyzed by Image J (NIH, MD, USA) with normalization to that of GAPDH. Immunofluorescence analysis Cells were fixed by 4% paraformaldehyde (15 min) and closed by QuickBlock™ Blocking Buffer for Immunol Staining (1 h) (P0260, Beyotime, Nantong, China) at room temperature. Then, cells were incubated by integrin αvβ3 antibody (abs122318, absin, China) at 4℃ for 24h, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (1 h) at room temperature. DAPI was used for nuclei staining. Fluorescence was observed by microscope (Olympus IX53, Olympus Corporation, Tokyo, Japan). Hematoxylin-eosin staining The tumor and ocular tissues were dehydrated by using increased ethanol concentrations. Next, the tissue paraffin blocks were embedded. For the experiments, the paraffin sections (4 µm) were stained using hematoxylin for 5 min, and eosin for another 2 min. Cell uptake and binding assays For cell uptake assay, cells (12-well plates, 1×10 5 cells/well) were cultured for 24 h and then the culture medium were replaced by [ 18 F]AlF-NOTA-PRGD2 (74 kBq/mL) containing medium (1 mL). The cells were incubated for 0.25h, 0.5h, 1h, 2 h, washed with PBS three times and lysed by 0.1 M NaOH (1 mL). The radioactivity in the cells was measured by a γ-counter (Perkin-Elmer, MA, USA) and cell uptake was calculated. For cell binding assay, cells (24-well plates, 1×10 5 cells/well) were cultured for 24 h, then the culture medium were replaced 2mL medium of [ 18 F]AlF-NOTA-PRGD2 (74 kBq/well) and unlabeled PRGD2 (the concentration ranges from 10 − 2 mM to 10 − 10 nM ). After incubation for 2 h, cells were washed with PBS three times and lysed by NaOH (0.1 M, 1 mL) solution, and a γ-counter (Perkin-Elmer, MA, USA) was used for measuring the radioactivity. The value of IC50 was calculated by nonlinear regression analysis. In vitro stability test The stability of [ 18 F]AlF-NOTA-PRGD2 in different media was tested. [ 18 F]AlF-NOTA-PRGD2 (37 MBq) was added to PBS or FBS (500 µL) solution, and incubated for 0, 1, 2 and 4 h at 37℃, respectively. At the preselected time points, PBS samples and FBS samples (10 µL of 370 KBq) were directly analyzed with HPLC to measure radioactivity. MicroPET imaging and analysis microPET scans and image analysis were performed using an Inveon microPET scanner (Siemens Medical Solutions, Germany). Mice were injected intravenously with 200 µL 3.7 MBq [ 18 F]FDG or [ 18 F]AlF-NOTA-PRGD2 (n = 4 per group). Under isoflurane anesthesia, ten-minute static PET images were acquired at 1 h post-injection. The radioactivity value within the tumors, eye, brain, lung, heart, liver, spleen and kidney were obtained and ROI (regions of interest) was analyzed (%ID/g, percentage of injected dose per gram of tissues) using vendor software (ASI Pro 5.2.4.0). The value of tumor-to-organ was also calculated. Biodistribution study and analysis The tumor-bearing mice (n = 4 per group) injected with [ 18 F]FDG or [ 18 F]AlF-NOTA-PRGD2 (3.7 MBq, 200µL) were immediately sacrificed after microPET scan, tumors and major organs of tumor-bearing mice were collected and wet-weighed. A γ-counter (PerkinElmer, MA, USA) was used for measuring the radioactivity. The data were calculated and expressed as %ID/g. Clinical patients and PET imaging This clinical study was approved by the ethics committee of The Hospital Affiliated to Jiangnan University (LS2011051). Four patients were enrolled in the clinical trial of [ 18 F]AlF-NOTA-PRGD2 for cancer diagnostics (approval no.NCT02441972 in clinicaltrials.gov). All patients signed a written informed consent form. Two of the patients were clinically diagnosed as benign tumor by MRI. The other two patients were clinically diagnosed as suspected uveal melanoma by MRI. After PET/CT scan, two UM patients underwent eye enucleation within 7 days. Immunohistochemical staining data demonstrated that S100, melanoma gp100 (HMB45), MelanA were positive. Statistical analysis Data are expressed as mean ± SD. The data among different groups were compared using Student’s t test and one-way analysis of variance (ANOVA). As p < 0.05, statistically significant was considered. Results High expression of integrin αvβ3 in UM cells and UM xenografts The expressions of integrin αvβ3 in UM cell lines (92 − 1, OCM-1A and MEL270) were assessed with western blot analysis (Fig. 1 A), and then its cellular localization in 92 − 1 cells was evaluated with immunofluorescent staining (Fig. 1 B). The results showed that integrin αvβ3 was highly expressed in UM cell lines and mainly located at cell membrane in 92 − 1 UM cells. Moreover, high expressions of integrin αvβ3 were also observed in subcutaneous and ocular tumor tissues of the UM xenograft mice (Fig. 1 C). Cell uptake, cell binding assays and in vitro stability test of [ 18 F]AlF-NOTA-PRGD2 The cell uptake of [ 18 F]AlF-NOTA-PRGD2 was examined in 92 − 1 UM cells, which was gradually increased from 0.65 ± 0.04%AD (15 min after incubation) to 1.22 ± 0.08%AD (120 min after incubation) (Fig. 2 A). Moreover, the uptake was effectively blocked in the presence of non-radiolabeled PRGD2. In cell binding assay, the unlabeled PRGD2 inhibited the binding of [ 18 F]AlF-NOTA-PRGD2 to 92 − 1 UM cells in a dose-dependent manner with an IC 50 value of 231.4 ± 1.95 nM (Fig. 2 B). The in vitro stability of [ 18 F]AlF-NOTA-PRGD2 was evaluated in PBS or FBS at 37℃. [ 18 F]AlF-NOTA-PRGD2 exhibited a good stability in both PBS or FBS for up to 4 h post-labelling, and no statistically significant change in radiochemical purity was observed throughout the duration (Fig. 2 C). All the radiochemical purity were > 99%. [ 18 F]AlF-NOTA-PRGD2 microPET imaging in UM xenografts and biodistribution study MicroPET study was performed using subcutaneous and ocular UM xenografts injected with 92-1-Luc cells, and 92-1-Luc tumor-bearing mice was used as a positive control. The subcutaneous and ocular UM xenografts were confirmed at Day 7 post injection by fluorescent imaging. MicroPET imaging was performed and UM tumors were clearly visualized with the [ 18 F]AlF-NOTA-PRGD2 tracer at 60 min post-injection in both subcutaneous and ocular UM xenografts (Fig. 3 ). The tumor uptake is 2.55 ± 0.44%ID/g and 1.73 ± 0.15%ID/g, respectively. The 92-1-Luc UM tumor was clearly visualized with a good tumor-to-background. And the ROI analysis showed the quantitative tumor uptake and the accumulation in the liver and kidney as to microPET images. To confirm the localization of [ 18 F]AlF-NOTA-PRGD2 in the 92-1-Luc UM tumor xenografts, biodistribution study was performed following microPET imaging. The tracer accumulation in tumors was 1.91 ± 0.25%ID/g and 1.44 ± 0.23%ID/g at 60 min post-injection. Its uptake in brain, lung, heart, liver, spleen and kidney in subcutaneous UM xenografts were 0.31 ± 0.17, 1.10 ± 0.20, 0.47 ± 0.26, 1.90 ± 0.32, 1.11 ± 0.24, 2.67 ± 0.37%ID/g, and which in ocular UM xenografts were 0.28 ± 0.06, 1.17 ± 0.24, 0.39 ± 0.03, 1.17 ± 0.12, 1.01 ± 0.15, 2.34 ± 0.39%ID/g. The biodistribution data were consistent with the ROI analysis of microPET imaging. Comparison of [ 18 F]AlF-NOTA-PRGD2 and [ 18 F]FDG microPET imaging in UM xenografts The comparison of [ 18 F]AlF-NOTA-PRGD2 and [ 18 F]FDG microPET imaging in subcutaneous and ocular 92 − 1 UM xenografts was further conducted. Both of the two radiotracers were clearly visualized in subcutaneous tumors (Fig. 4 ). The signals of [ 18 F]AlF-NOTA-PRGD2 tracer were distinctively observed in ocular 92 − 1 UM xenografts with very low background; while that of [ 18 F]FDG was hard to be distinguished due to a strong brain uptake. The tumor-to-brain, -lung, -heart, -liver, -spleen and -kidney uptake ratios of [ 18 F]AlF-NOTA-PRGD2 in subcutaneous UM xenografts were 6.45 ± 0.89, 1.71 ± 0.29, 4.18 ± 0.83, 1.01 ± 0.19, 1.74 ± 0.52 and 0.70 ± 0.11, respectively, while the corresponding ratios for [ 18 F]FDG were 1.02 ± 0.33, 2.93 ± 0.65, 5.73 ± 1.84, 6.58 ± 1.97, 2.52 ± 0.54 and 6.32 ± 1.64, respectively (Table. 1). The tumor-to-brain, -lung, -heart, -liver, -spleen and -kidney uptake ratios of [ 18 F]AlF-NOTA-PRGD2 in ocular UM xenografts were 5.28 ± 0.49, 1.25 ± 0.28, 3.73 ± 0.48, 1.22 ± 0.12, 1.42 ± 0.06 and 0.61 ± 0.06, respectively, while the corresponding ratios for [ 18 F]FDG were 0.89 ± 0.12, 2.09 ± 0.27, 2.71 ± 1.10, 4.06 ± 1.42, 2.18 ± 0.43 and 4.01 ± 0.68, respectively (Table. 2). Table 1 Subcutaneous Tumor to Organ Ratios of [ 18 F]AlF-NOTA-PRGD2 and [ 18 F]FDG in UM xenografts Organ [ 18 F]FDG [ 18 F]AlF-NOTA-PRGD2 Tumor to Brain 1.02 ± 0.33 6.45 ± 0.89 Tumor to Lung 2.93 ± 0.65 1.71 ± 0.29 Tumor to Heart 5.73 ± 1.84 4.18 ± 0.83 Tumor to Liver 6.58 ± 1.97 1.01 ± 0.19 Tumor to Spleen 2.52 ± 0.54 1.74 ± 0.52 Tumor to Kidney 6.32 ± 1.64 0.70 ± 0.11 Table 2 Ocular Tumor to Organ Ratios of [ 18 F]AlF-NOTA-PRGD2 and [ 18 F]FDG in UM xenografts Organ [ 18 F]FDG [ 18 F]AlF-NOTA-PRGD2 Tumor to Left eye 1.72 ± 0.22 2.71 ± 0.05 Tumor to Brain 0.89 ± 0.12 5.28 ± 0.49 Tumor to Lung 2.09 ± 0.27 1.25 ± 0.28 Tumor to Heart 2.71 ± 1.10 3.73 ± 0.48 Tumor to Liver 4.06 ± 1.42 1.22 ± 0.12 Tumor to Spleen 2.18 ± 0.43 1.42 ± 0.06 Tumor to Kidney 4.01 ± 0.68 0.61 ± 0.06 Comparison of [ 18 F]AlF-NOTA-PRGD2 and [ 18 F]FDG PET imaging in UM patient. The comparison of [ 18 F]AlF-NOTA-PRGD2 and [ 18 F]FDG PET imaging in two patients with primary UM was further determined. In the two patients with benign tumor, both two radiotracers had no detectable uptake (Fig. 5 A. C and E). In the patients with UM, [ 18 F]AlF-NOTA-PRGD2 demonstrated a lower background in the brain region than that of [ 18 F]FDG, which was preferred for the detection of primary ocular UM tumor with a favorable target-to-background ratio (Fig. 5 B .D and F). Such finding was consistent with the observation in UM xenografts. In addition, the tumor-to-brain, -lung, -heart, -liver, -spleen and -kidney uptake ratios of [ 18 F]AlF-NOTA-PRGD2 were 46.50, 18.60, 7.15, 3.10, 1.33 and 0.14 for patient 3, and 14.33, 7.17, 2.87, 1.65, 0.67 and 0.44 for patient 4, respectively, while the corresponding ratios for [ 18 F]FDG were 0.29, 6.43, 0.45, 1.41, 1.36 and 0.70 for patient 3, and 0.11, 2.86, 0.43, 0.56, 0.77 and 0.38 for patient 4, respectively (Table. 3). The selectivity and sensitivity of [ 18 F]AlF-NOTA-PRGD2 tracer for PET imaging of UM patients are even better than that in UM xenografts. Table 3 Ocular Tumor to Organ Ratios of [ 18 F]AlF-NOTA-PRGD2 and [ 18 F]FDG in Patient 3 and Patient 4 Organ Patient 3 Patient 4 [ 18 F]FDG [ 18 F]AlF-NOTA-PRGD2 [ 18 F]FDG [ 18 F]AlF-NOTA-PRGD2 Tumor to Eye 2.65 46.50 1.33 10.75 Tumor to Brain 0.29 46.50 0.11 14.33 Tumor to Lung 6.43 18.60 2.86 7.17 Tumor to Heart 0.45 7.15 0.43 2.87 Tumor to Liver 1.41 3.10 0.56 1.65 Tumor to Spleen 1.36 1.33 0.77 0.67 Tumor to Kidney 0.70 0.14 0.38 0.44 Discussion In recent years, dimeric PRGD2 has been adopted as a new PET tracer in the early detection and staging of various tumors, due to its higher affinity for integrin αvβ3 receptor compared to monomeric counterpart [ 24 , 27 , 28 ]. In addition, compared with [ 18 F]FDG, the radiosynthesis of this imaging tracer is convenient and less time consuming (< 30 min). The addition of a NOTA functional group to PRGD2 enabled a chelate compatible for both 18 F-fluoride-aluminum and 68 Ga. Li et al report the effectiveness of [ 68 Ga]PRGD2 PET/CT in glioma grading and demarcation [ 29 ]. Zheng et al investigate the application of [ 68 Ga]NOTA-PRGD2 PET/CT in lung cancer diagnosis, which showed a superior performance than that of [ 18 F]FDG PET/CT due to specifically determining metastatic lymph nodes [ 30 ]. Zhang et al show that [ 18 F]AlF-NOTA-PRGD2 PET/CT noninvasively visualized GBM lesions and predicted the outcome of concurrent chemoradiotherapy as early as 3 weeks after the initiation of treatment [ 24 ]. However, there has been no report about the application of [ 18 F]AlF-NOTA-PRGD2 in UM. This study first evaluated [ 18 F]AlF-NOTA-PRGD2 PET imaging in the UM xenografts as well as a UM patients. Our data revealed that tumors in subcutaneous and ocular UM xenografts could be clearly visualized with this tracer at 60 min post-injection. Moreover, the PET imaging with this tracer in the UM patient may be more specific and sensitive than that in UM xenografts. The characteristics of [ 18 F]AlF-NOTA-PRGD2 were evaluated in in vitro and in vivo models of UM. Our in vitro studies revealed that [ 18 F]AlF-NOTA-PRGD2 showed a high specificity to integrin αvβ3 in 92 − 1 UM cells in the aspects of cell uptake, high affinity in cell binding and high stability in vitro stability. The in vivo microPET imaging and biodistribution analysis demonstrated that [ 18 F]AlF-NOTA-PRGD2 had a high tumor uptake and a very low background. In addition, integrin αvβ3 expression is upregulated during tumor angiogenesis, particularly in vascular endothelial cells. The high tumor uptake of [ 18 F]AlF-NOTA-PRGD2 in vivo might also indicate the occurrence of tumor angiogenesis in UM. And the significantly increased liver and kidney uptake of [ 18 F]AlF-NOTA-PRGD2 may suggest that this tracer is mainly metabolized in these two organs. As to PET imaging, [ 18 F]AlF-NOTA-PRGD2 showed a longer retention in tumors and a faster clearance from normal tissues [ 31 ]. This study focuses on evaluating the application of [ 18 F]AlF-NOTA-PRGD2 in the diagnosis of primary UM tumors; its application in metastatic UM xenografts needs to be assessed in the future studies. [ 18 F]FDG is the common PET tracer currently adopted in the early detection and staging of UM patients in clinical settings. However, a large variation in its sensitivity, specificity, and accuracy in UM diagnosis has been reported [ 16 , 32 ]. Comparison of [ 18 F]AlF-NOTA-PRGD2 and [ 18 F]FDG in PET imaging, the uptake of [ 18 F]AlF-NOTA-PRGD2 in UM xenografts was lower than that of [ 18 F]FDG; but this was opposite in the UM patients. Moreover, the low background of [ 18 F]AlF-NOTA-PRGD2 in the brain region promoted its application in the detection of primary UM tumor with a high target-to-background ratio. In addition, the tumor to organ ratios of [ 18 F]AlF-NOTA-PRGD2 in the UM patients were superior than that in UM xenografts, indicating [ 18 F]AlF-NOTA-PRGD2 PET imaging was more preferred for the diagnosis of clinical UM patients compared to [ 18 F]FDG PET imaging. In conclusion, our study suggests that [ 18 F]AlF-NOTA-PRGD2 tracer shows a great advantage in the diagnosis and staging of UM due to its higher sensitivity and specificity compared to [ 18 F]FDG tracer; however, this promising pilot data needs more extensive clinical evaluation. Abbreviations UM Uveal melanoma PET Positron emission tomography [ 18 F]FDG 18 F-labelled fluorodeoxyglucose %ID/g Percentage of injected dose per gram of tissues MRIMagnetic resonance imaging PET/CT Positron emission tomography/computerized tomography HPLC High Performance Liquid Chromatography FBS Fetal bovine serum P/S Penicillin/Streptomycin RIPA Radio Immunoprecipitation Assay ROI Regions of interes Declarations Acknowledgements Not applicable. Authors’ contributions LW and XZ conducted the experiment and wrote the article; YX, ZH and WZ analyzed data and provided materials; ZZ and MY supervised the project; KW and DP designed the experiment and proofed article. All authors read and approved the final manuscript. Funding This work was supported by Major Project of Wuxi Commission of Health (Grant number [Z202110]), Project of Jiangsu Administration of Traditional Chinese Medicine (Grant number [MS2022145]), The Elderly Health Research Project of Jiangsu Province (Grant number [2022043]), The Social Development Project of Jiangsu Provincial Department of Science and Technology (Grant number [BE2022699]), Chinese Medicine Science and Technology Development Project of Jiangsu Province (Grant number [MS2023166]), Scientific Research Project of Jiangsu Provincial Health Commission (H2023150) and Scientific Research Project of Wuxi Health Commission (Grant number [Z202303]). Availability of data and materials The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This clinical study was approved by the ethics committee of The Hospital Affiliated to Jiangnan University (LS2011051). the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendment. Written informed consents were obtained from all participants. All experiment protocols were approved by the Animal Care and Use Committee of Jiangsu Institute of Nuclear Medicine (JSINM-2022-061) and the study was carried out in compliance with the ARRIVE guidelines. Consent for publication All authors are consent for publication. Competing Interests The authors declare that they have no financial interests. References Kaliki S, Shields CL. Uveal melanoma: relatively rare but deadly cancer. Eye. 2017;31:241–57. 10.1038/eye.2016.275 . Jager MJ, Shields CL, Cebulla CM, Abdel-Rahman MH, Grossniklaus HE, Stern MH, et al. Uveal melanoma. 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Leroy-Freschini B, Treglia G, Argemi X, Bund C, Kessler R, Herbrecht R, et al. 18F-FDG PET/CT for invasive fungal infection in immunocompromised patients. QJM. 2018;111:613–22. 10.1093/qjmed/hcy128 . Xiang X, Wind K, Wiedemann T, Blume T, Shi Y, Briel N, et al. Microglial activation states drive glucose uptake and FDG-PET alterations in neurodegenerative diseases. Sci Transl Med. 2021;13:eabe5640. 10.1126/scitranslmed.abe5640 . Strobel K, Bode B, Dummer R, Veit-Haibach P, Fischer DR, Imhof L, et al. Limited value of 18F-FDG PET/CT and S-100B tumour marker in the detection of liver metastases from uveal melanoma compared to liver metastases from cutaneous melanoma. Eur J Nucl Med Mol Imaging. 2009;36:1774–82. 10.1007/s00259-009-1175-0 . Calcagni ML, Mattoli MV, Blasi MA, Petrone G, Sammarco MG, Indovina L, et al. A prospective analysis of ¹⁸F-FDG PET/CT in patients with uveal melanoma: comparison between metabolic rate of glucose (MRglu) and standardized uptake value (SUV) and correlations with histopathological features. Eur J Nucl Med Mol Imaging. 2013;40:1682–91. 10.1007/s00259-013-2488-6 . Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer. 2010;10:9–22. 10.1038/nrc2748 . Arnaout MA. Integrin structure: new twists and turns in dynamic cell adhesion. Immunol Rev. 2002;186:125–40. 10.1034/j.1600-065x.2002.18612.x . Rocha LA, Learmonth DA, Sousa RA, Salgado AJ. αvβ3 and α5β1 integrin-specific ligands: From tumor angiogenesis inhibitors to vascularization promoters in regenerative medicine? Biotechnol Adv. 2018;36:208–27. 10.1016/j.biotechadv.2017.11.004 . Kumar CC. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets. 2003;4:123–31. 10.2174/1389450033346830 . Cheng TM, Chang WJ, Chu HY, De Luca R, Pedersen JZ, Incerpi S, et al. Nano-Strategies Targeting the Integrin αvβ3 Network for Cancer Therapy. Cells. 2021;10. 10.3390/cells10071684 . Liu Z, Liu S, Wang F, Liu S, Chen X. Noninvasive imaging of tumor integrin expression using (18)F-labeled RGD dimer peptide with PEG (4) linkers. Eur J Nucl Med Mol Imaging. 2009;36:1296–307. 10.1007/s00259-009-1112-2 . Notni J, Pohle K, Wester HJ. Be spoilt for choice with radiolabelled RGD peptides: preclinical evaluation of ⁶⁸Ga-TRAP(RGD)₃. Nucl Med Biol. 2013;40:33–41. 10.1016/j.nucmedbio.2012.08.006 . Zhang H, Liu N, Gao S, Hu X, Zhao W, Tao R, et al. Can an ¹⁸F-ALF-NOTA-PRGD2 PET/CT Scan Predict Treatment Sensitivity to Concurrent Chemoradiotherapy in Patients with Newly Diagnosed Glioblastoma? Journal of nuclear medicine: official publication. Soc Nuclear Med. 2016;57:524–9. 10.2967/jnumed.115.165514 . Marshall JF, Rutherford DC, Happerfield L, Hanby A, McCartney AC, Newton-Bishop J, et al. Comparative analysis of integrins in vitro and in vivo in uveal and cutaneous melanomas. Br J Cancer. 1998;77:522–9. 10.1038/bjc.1998.85 . Lang L, Li W, Fau - Guo N, Guo N, Fau - Ma Y, Ma Y, Fau - Zhu L, Zhu L, Fau - Kiesewetter DO. Kiesewetter Do Fau - Shen B, Comparison study of [18F]FAl-NOTA-PRGD2, [18F]FPPRGD2, and [68Ga]Ga-NOTA-PRGD2 for PET imaging of U87MG tumors in mice. Xie F, Zheng K, Liu L, Jin X, Fu L, Zhu Z. A Pilot Study of Radiomics Models Combining Multi-Probe and Multi-Modality Images of (68)Ga-NOTA-PRGD2 and (18)F-FDG PET/CT for Differentiating Benign and Malignant Pulmonary Space-Occupying Lesions. Front Oncol. 2022;12:877501. 10.3389/fonc.2022.877501 . Guo N, Lang L, Li W, Kiesewetter DO, Gao H, Niu G, et al. Quantitative analysis and comparison study of [18F]AlF-NOTA-PRGD2, [18F]FPPRGD2 and [68Ga]Ga-NOTA-PRGD2 using a reference tissue model. PLoS ONE. 2012;7:e37506. 10.1371/journal.pone.0037506 . Cancer risks in BRCA2 mutation carriers. J Natl Cancer Inst. 1999;91:1310–6. 10.1093/jnci/91.15.1310 . Zheng K, Liang N, Zhang J, Lang L, Zhang W, Li S et al. 68Ga-NOTA-PRGD2 PET/CT for Integrin Imaging in Patients with Lung Cancer. Journal of nuclear medicine: official publication, Society of Nuclear Medicine. 2015;56:1823–7. 10.2967/jnumed.115.160648 . Liu S, Liu Z, Fau - Chen K, Chen K, Fau - Yan Y, Yan Y, Fau - Watzlowik P, Watzlowik P. Fau - Wester H-J, Wester Hj Fau - Chin FT, 18F-labeled galacto and PEGylated RGD dimers for PET imaging of αvβ3 integrin expression. Kato K, Kubota T, Ikeda M, Tadokoro M, Abe S, Nakano S, et al. Low efficacy of 18F-FDG PET for detection of uveal malignant melanoma compared with 123I-IMP SPECT. J Nucl Med. 2006;47:404–9. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision 03 May, 2024 Reviewers agreed at journal 30 Mar, 2024 Reviewers invited by journal 27 Mar, 2024 Editor assigned by journal 27 Mar, 2024 First submitted to journal 25 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3998432","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284474090,"identity":"04cd42c5-b8f9-479d-8bd4-ba9c82aea571","order_by":0,"name":"Ling Wang","email":"","orcid":"","institution":"Jiangsu Institute of Nuclear Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Wang","suffix":""},{"id":284474091,"identity":"a5b769c8-6064-40f9-a317-e220fac5621c","order_by":1,"name":"Xue Zhu","email":"","orcid":"","institution":"Jiangsu Institute of Nuclear Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Zhu","suffix":""},{"id":284474092,"identity":"5888e7ba-3aa1-470b-8d1d-e6ed4703eb29","order_by":2,"name":"Yan Xue","email":"","orcid":"","institution":"Jiangsu Institute of Nuclear Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Xue","suffix":""},{"id":284474093,"identity":"a53faf6f-d737-4314-8ee0-25d9ff766431","order_by":3,"name":"Zhihong Huang","email":"","orcid":"","institution":"Jiangsu Institute of Nuclear Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhihong","middleName":"","lastName":"Huang","suffix":""},{"id":284474094,"identity":"643e6d2c-b272-479d-b16d-442a57ab9c7a","order_by":4,"name":"Wenjun Zou","email":"","orcid":"","institution":"Wuxi No 2 People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenjun","middleName":"","lastName":"Zou","suffix":""},{"id":284474095,"identity":"b43da39f-60c9-434b-8e6e-a9e8fe2bfa57","order_by":5,"name":"Zhengwei Zhang","email":"","orcid":"","institution":"Wuxi No 2 People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhengwei","middleName":"","lastName":"Zhang","suffix":""},{"id":284474096,"identity":"6aa9c48c-f06e-48a8-8331-8b6772afe949","order_by":6,"name":"Mengxi Yu","email":"","orcid":"","institution":"Wuxi No 2 People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mengxi","middleName":"","lastName":"Yu","suffix":""},{"id":284474097,"identity":"079b6b4b-f214-44e3-9b13-74fc90dd0993","order_by":7,"name":"Donghui Pan","email":"","orcid":"","institution":"Jiangsu Institute of Nuclear Medicine","correspondingAuthor":false,"prefix":"","firstName":"Donghui","middleName":"","lastName":"Pan","suffix":""},{"id":284474098,"identity":"e29c584f-170e-46a3-9ca8-276f060c080f","order_by":8,"name":"Ke Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYJACxgYGG2YGZhK1pJGu5TAJyg2Onz38ckbFeXb5dt6jGxgq7tk1ENRyJi/NcsOZ28wGh/nSbjCcKU4mqMXsQI6Z4cM2oBZmHrMbjG0JyQQdZnb+DVDLv3PM8s1Ea7mRY/xwY8MBZobDEC12BLXY33hjxjjjWDLQL0AtCWcSEghqkezPMf7YU2OXLN9/xuzGh4oEe4JagIBNAkhAvAC0IrGBCC3MH4AE3AtE2TIKRsEoGAUjCwAAIFk/G9hw5koAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3812-7018","institution":"Jiangsu Institute of Nuclear Medicine","correspondingAuthor":true,"prefix":"","firstName":"Ke","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-02-29 03:58:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3998432/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3998432/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53881599,"identity":"502e75c4-655a-47cf-8de7-01f4f6ff6386","added_by":"auto","created_at":"2024-04-01 17:53:48","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":417390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expressions of integrin αvβ3 in UM cells and UM xenografts. A: \u003c/strong\u003eWestern blot analysis of integrin αvβ3 in UM cell lines (92-1, OCM-1A and MEL270). \u003cstrong\u003eB:\u003c/strong\u003e Immunofluorescent staining of integrin αvβ3 in 92-1 UM cells (integrin αvβ3: red fluorescence). \u003cstrong\u003eC:\u003c/strong\u003e HE, Ki67 and IHC analysis of integrin αvβ3 in subcutaneous and ocular tumor tissues of 92-1 UM xenograft mice.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3998432/v1/b0789cf139e539d6476448d9.jpeg"},{"id":53881598,"identity":"a002d945-2a84-443f-8f44-4f1ed4cf5908","added_by":"auto","created_at":"2024-04-01 17:53:48","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":138415,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell uptake, cell binding assays and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e stability test of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]AlF-NOTA-PRGD2. A: \u003c/strong\u003eCell uptake and block assays of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in 92-1 UM cells.\u003cstrong\u003e B: \u003c/strong\u003eCompetitive binding of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 with unlabeled PRGD2.\u003cstrong\u003e C:\u003c/strong\u003e \u003cem\u003eIn vitro\u003c/em\u003e stability assessment of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in PBS or FBS at different time points\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3998432/v1/226e183d72650635fa1e879d.jpeg"},{"id":53881597,"identity":"4a1e4ec4-2c56-4b01-ba8f-a9645e36d091","added_by":"auto","created_at":"2024-04-01 17:53:48","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":299863,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]AlF-NOTA-PRGD2 microPET imaging in UM xenografts and biodistribution study. A: \u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e microPET imaging of subcutaneous tumor in 92-1 UM xenografts with [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2, and biodistribution analysis in whole body.\u003cstrong\u003e B: \u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e microPET imaging of ocular tumor in 92-1 UM xenografts with [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and biodistribution analysis in eye. **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 \u003cem\u003evs.\u003c/em\u003eRight eye/Tumor.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3998432/v1/72af3112e65ef9ffdfa1a994.jpeg"},{"id":53881600,"identity":"b12f40ef-f655-4fee-99c5-e9c58883f6eb","added_by":"auto","created_at":"2024-04-01 17:53:48","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":278987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]AlF-NOTA-PRGD2 and [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]FDG microPET imaging in UM xenografts. A: \u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e microPET imaging of subcutaneous tumor in 92-1 UM xenografts using [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG in whole body.\u003cstrong\u003e B: \u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e microPET imaging of ocular tumor in 92-1 xenografts using [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG. **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 \u003cem\u003evs.\u003c/em\u003eRight eye/Tumor.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3998432/v1/a47e5ed58caded734c7a0c2f.jpeg"},{"id":53881601,"identity":"18952a18-3236-4045-a010-89c946f3e79b","added_by":"auto","created_at":"2024-04-01 17:53:48","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":268387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe PET imaging of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]AlF-NOTA-PRGD2 and [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]FDG in patients. A: \u003c/strong\u003eOcular PET imaging in Patient 1 and Patient 2 with benign tumor using [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG PET tracers. \u003cstrong\u003eB: \u003c/strong\u003e\u0026nbsp;Ocular PET imaging and tumor uptake in the Patient 3 and Patient 4 with UM using [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG PET tracers.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3998432/v1/f0d52ad4d45346bb6b327612.jpeg"},{"id":53882260,"identity":"cc905a10-8c2f-4f28-89ae-a377c4df57ec","added_by":"auto","created_at":"2024-04-01 18:01:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":989381,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3998432/v1/67d0c87a-d615-4c2c-927d-cf113fbee4d4.pdf"}],"financialInterests":"","formattedTitle":"Ultrasensitive Detection of Uveal Melanoma Using [18F]AlF-NOTA-PRGD2 PET Imaging","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlthough rare, uveal melanoma (UM) is the primary intraocular tumor worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Control of local UM seems effective through non-pharmacological treatment strategies, but up to 50% of patients still develop metastasis. Overall, with a very poor prognosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], early detection and timely treatment are critical to improve the clinical outcome of UM [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Currently, UM diagnosis relies on clinical examination, the slit lamp and indirect ophthalmoscope, ocular ultrasonography, magnetic resonance imaging (MRI) and positron emission tomography/computerized tomography (PET/CT) of whole body [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the primary challenge is to distinguish small ocular tumors with a thickness of less than 3 mm, from presumed naevi as well as to detect metastasis in the whole body. PET/CT as a traditional non-invasive technique, plays a critical role in oncologic imaging and has been widely used for the diagnosis and staging of a variety of malignancies [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The sensitivity and specificity of PET scanning largely relies on the imaging tracer. In the past 40 years, [\u003csup\u003e18\u003c/sup\u003eF]FDG is the dominant PET tracer used in the imaging of neurology, cardiology, and oncology [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the efficacy of [\u003csup\u003e18\u003c/sup\u003eF]FDG PET imaging in the detection of UM is very poor with a high incidence of false-negative results. The limited sensitivity of [\u003csup\u003e18\u003c/sup\u003eF]FDG PET imaging in detection of small size tumor is due to low metabolic nature of UM. In addition, it is also not sensitive enough for the detection of UM metastases into the liver and other tissues. In contrast, liver metastases from cutaneous melanoma are shown to be FDG sensitive [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, to develop a more sensitive PET imaging tracer is helpful for a better differential diagnosis and staging of UM, which can be used as a supplement routine examination.\u003c/p\u003e \u003cp\u003eIntegrins (consisting of two noncovalently bound transmembrane subunits (\u003cem\u003eα\u003c/em\u003e and β)) are large, membrane-spanning, heterodimeric proteins [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Integrin alphaVbeta3 (integrin αvβ3) is one member of integrins and contributes to biological processes such as cell adhesion and cell migration [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the past decade, integrin αvβ3 was adopted as an important molecular target for early and differential diagnosis of rapidly growing solid tumors due to its role in tumor angiogenesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Integrin αvβ3 is a receptor for arginine-glycine-aspartic (RGD) tripeptide, which also includes linear and cyclic RGD peptide antagonists. Thus, targeting integrin αvβ3 is a viable approach to develop radiotracers for PET/CT [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Notni \u003cem\u003eet al\u003c/em\u003e have conducted a preclinical evaluation of [\u003csup\u003e68\u003c/sup\u003eGa]TRAP(RGD)\u003csub\u003e3\u003c/sub\u003e in human melanoma xenografts and showed that there is a high uptake of such radiotracer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Chen \u003cem\u003eet al\u003c/em\u003e develop a new PET imaging probe [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 (NOTA-PEG4-E[c(RGDfK)]2) and successfully applied to lung cancer patients, with longer tumor retention and simpler labeling process [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Up to date, [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 tracer has not been applied to PET imaging of UM patients yet.\u003c/p\u003e \u003cp\u003eThe previous studies and our data have confirmed integrin αvβ3 is highly expressed in UM cells and UM tissues [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. And the current study is the first to evaluate the efficacy of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 for PET imaging of UM, in comparison to that of [\u003csup\u003e18\u003c/sup\u003eF]FDG.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneral materials\u003c/h2\u003e \u003cp\u003eAll commercially obtained chemicals were analytical grade and used without further purification. FDG (fluorodeoxyglucose) was provided from the Wuxi Jiangyuan Industrial Technology And Trade Corporation and reconstituted with sterile saline. PRGD2 (PEG4-E[c(RGDyK)]2) and NOTA-PRGD2 were provided from Chinese Peptide Company (Hangzhou, China). \u003csup\u003e18\u003c/sup\u003eF-fluoride was obtained from an in-house PET trace cyclotron (HM-7, Sumitomo Heavy Industries Ltd, Japan) via the \u003csup\u003e18\u003c/sup\u003eO (p, n)\u003csup\u003e18\u003c/sup\u003eF nuclear reaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRadiosyntheses of [\u003csup\u003e18\u003c/sup\u003eF]FDG and [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2\u003c/h2\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FDG and [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 were synthesized as the previous report [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The preparation of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 was solved as follows: NOTA-PRGD2 (50 \u0026micro;g contains 1.6 \u0026micro;g AlCl\u003csub\u003e3\u003c/sub\u003e) add 20 \u0026micro;L water to dissolved in the reaction bottle and add 5 \u0026micro;L of glacial acetic acid to adjust PH (PH\u0026thinsp;\u0026asymp;\u0026thinsp;3). Then, 20 \u0026micro;L [\u003csup\u003e18\u003c/sup\u003eF] fluorinated water (~\u0026thinsp;1110 MBq) and 0.2 mL acetonitrile were added to the reaction flask. The above solution was reacted at 95\u0026deg;C for 10 min, the product was diluted with 9 mL water and hung on an activated Varian BOND ELUT C18 column. Wash the C18 column three times with 10ml water. Finally, 300 \u0026micro;L of 10 mM hydrochloric acid was used to elute the product in ethanol. The mixture was purified by the C18 cartridge using High Performance Liquid Chromatography (HPLC). The final product was formulated in saline for the subsequent studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and UM xenografts\u003c/h2\u003e \u003cp\u003eHuman UM cell lines (92\u0026thinsp;\u0026minus;\u0026thinsp;1, OCM-1A and MEL270) were obtained from OcuTech Co,Ltd (Wuxi, China) and cultured in RPMI1640 medium with 10% FBS (fetal bovine serum) and 1% P/S (penicillin/streptomycin). The expression of integrin αvβ3 on 92\u0026thinsp;\u0026minus;\u0026thinsp;1 cells were confirmed by western blot analysis and immunofluorescence. All experiment protocols were approved by the Animal Care and Use Committee of Jiangsu Institute of Nuclear Medicine (JSINM-2022-061). The male BALB/c nude mice (4\u0026thinsp;~\u0026thinsp;5 weeks old, 18\u0026thinsp;~\u0026thinsp;22 g body weight) were purchased from Cavens Laboratory Animal Technology Co. Ltd. (Jiangsu, China). The nude mice were injected with 5 \u0026times;10\u003csup\u003e6\u003c/sup\u003e 92-1-Luc cells in subcutaneous or 1 \u0026times;10\u003csup\u003e5\u003c/sup\u003e 92-1-Luc cells in ocular, when the tumor size reaches about 200 mm\u003csup\u003e3\u003c/sup\u003e, about 3\u0026thinsp;~\u0026thinsp;4 weeks after the cell inoculation, the tumor model could be used for further study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCells were lysed with RIPA (Radio Immunoprecipitation Assay) buffer and protein concentration was determined with BCA assay (Beyotime, Nantong, China). Protein extract (20 \u0026micro;g) was separated by SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with the primary antibody of integrin alpha V (ab179475, Abcam, MA, USA) and integrin beta 3 (ab179473, Abcam, MA, USA) followed by the horseradish peroxidase-conjugated secondary antibody (ab97051, Abcam, MA, USA). The expression of target protein was verified by chemiluminescence (ECL) detection kit. Band densities was analyzed by Image J (NIH, MD, USA) with normalization to that of GAPDH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence analysis\u003c/h2\u003e \u003cp\u003eCells were fixed by 4% paraformaldehyde (15 min) and closed by QuickBlock\u0026trade; Blocking Buffer for Immunol Staining (1 h) (P0260, Beyotime, Nantong, China) at room temperature. Then, cells were incubated by integrin αvβ3 antibody (abs122318, absin, China) at 4℃ for 24h, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (1 h) at room temperature. DAPI was used for nuclei staining. Fluorescence was observed by microscope (Olympus IX53, Olympus Corporation, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-eosin staining\u003c/h2\u003e \u003cp\u003eThe tumor and ocular tissues were dehydrated by using increased ethanol concentrations. Next, the tissue paraffin blocks were embedded. For the experiments, the paraffin sections (4 \u0026micro;m) were stained using hematoxylin for 5 min, and eosin for another 2 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell uptake and binding assays\u003c/h2\u003e \u003cp\u003eFor cell uptake assay, cells (12-well plates, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured for 24 h and then the culture medium were replaced by [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 (74 kBq/mL) containing medium (1 mL). The cells were incubated for 0.25h, 0.5h, 1h, 2 h, washed with PBS three times and lysed by 0.1 M NaOH (1 mL). The radioactivity in the cells was measured by a γ-counter (Perkin-Elmer, MA, USA) and cell uptake was calculated. For cell binding assay, cells (24-well plates, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured for 24 h, then the culture medium were replaced 2mL medium of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 (74 kBq/well) and unlabeled PRGD2 (the concentration ranges from 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e mM to 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e nM ). After incubation for 2 h, cells were washed with PBS three times and lysed by NaOH (0.1 M, 1 mL) solution, and a γ-counter (Perkin-Elmer, MA, USA) was used for measuring the radioactivity. The value of IC50 was calculated by nonlinear regression analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003estability test\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe stability of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in different media was tested. [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 (37 MBq) was added to PBS or FBS (500 \u0026micro;L) solution, and incubated for 0, 1, 2 and 4 h at 37℃, respectively. At the preselected time points, PBS samples and FBS samples (10 \u0026micro;L of 370 KBq) were directly analyzed with HPLC to measure radioactivity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMicroPET imaging and analysis\u003c/h2\u003e \u003cp\u003emicroPET scans and image analysis were performed using an Inveon microPET scanner (Siemens Medical Solutions, Germany). Mice were injected intravenously with 200 \u0026micro;L 3.7 MBq [\u003csup\u003e18\u003c/sup\u003eF]FDG or [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 (n\u0026thinsp;=\u0026thinsp;4 per group). Under isoflurane anesthesia, ten-minute static PET images were acquired at 1 h post-injection. The radioactivity value within the tumors, eye, brain, lung, heart, liver, spleen and kidney were obtained and ROI (regions of interest) was analyzed (%ID/g, percentage of injected dose per gram of tissues) using vendor software (ASI Pro 5.2.4.0). The value of tumor-to-organ was also calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBiodistribution study and analysis\u003c/h2\u003e \u003cp\u003eThe tumor-bearing mice (n\u0026thinsp;=\u0026thinsp;4 per group) injected with [\u003csup\u003e18\u003c/sup\u003eF]FDG or [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 (3.7 MBq, 200\u0026micro;L) were immediately sacrificed after microPET scan, tumors and major organs of tumor-bearing mice were collected and wet-weighed. A γ-counter (PerkinElmer, MA, USA) was used for measuring the radioactivity. The data were calculated and expressed as %ID/g.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClinical patients and PET imaging\u003c/h2\u003e \u003cp\u003eThis clinical study was approved by the ethics committee of The Hospital Affiliated to Jiangnan University (LS2011051). Four patients were enrolled in the clinical trial of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 for cancer diagnostics (approval no.NCT02441972 in clinicaltrials.gov). All patients signed a written informed consent form. Two of the patients were clinically diagnosed as benign tumor by MRI. The other two patients were clinically diagnosed as suspected uveal melanoma by MRI. After PET/CT scan, two UM patients underwent eye enucleation within 7 days. Immunohistochemical staining data demonstrated that S100, melanoma gp100 (HMB45), MelanA were positive.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The data among different groups were compared using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test and one-way analysis of variance (ANOVA). As \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, statistically significant was considered.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHigh expression of integrin αvβ3 in UM cells and UM xenografts\u003c/h2\u003e \u003cp\u003eThe expressions of integrin αvβ3 in UM cell lines (92\u0026thinsp;\u0026minus;\u0026thinsp;1, OCM-1A and MEL270) were assessed with western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and then its cellular localization in 92\u0026thinsp;\u0026minus;\u0026thinsp;1 cells was evaluated with immunofluorescent staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The results showed that integrin αvβ3 was highly expressed in UM cell lines and mainly located at cell membrane in 92\u0026thinsp;\u0026minus;\u0026thinsp;1 UM cells. Moreover, high expressions of integrin αvβ3 were also observed in subcutaneous and ocular tumor tissues of the UM xenograft mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCell uptake, cell binding assays and\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003estability test of [\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]AlF-NOTA-PRGD2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe cell uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 was examined in 92\u0026thinsp;\u0026minus;\u0026thinsp;1 UM cells, which was gradually increased from 0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04%AD (15 min after incubation) to 1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08%AD (120 min after incubation) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, the uptake was effectively blocked in the presence of non-radiolabeled PRGD2. In cell binding assay, the unlabeled PRGD2 inhibited the binding of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 to 92\u0026thinsp;\u0026minus;\u0026thinsp;1 UM cells in a dose-dependent manner with an IC\u003csub\u003e50\u003c/sub\u003e value of 231.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The \u003cem\u003ein vitro\u003c/em\u003e stability of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 was evaluated in PBS or FBS at 37℃. [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 exhibited a good stability in both PBS or FBS for up to 4 h post-labelling, and no statistically significant change in radiochemical purity was observed throughout the duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). All the radiochemical purity were \u0026gt;\u0026thinsp;99%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 microPET imaging in UM xenografts and biodistribution study\u003c/h2\u003e \u003cp\u003eMicroPET study was performed using subcutaneous and ocular UM xenografts injected with 92-1-Luc cells, and 92-1-Luc tumor-bearing mice was used as a positive control. The subcutaneous and ocular UM xenografts were confirmed at Day 7 post injection by fluorescent imaging. MicroPET imaging was performed and UM tumors were clearly visualized with the [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 tracer at 60 min post-injection in both subcutaneous and ocular UM xenografts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The tumor uptake is 2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44%ID/g and 1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15%ID/g, respectively. The 92-1-Luc UM tumor was clearly visualized with a good tumor-to-background. And the ROI analysis showed the quantitative tumor uptake and the accumulation in the liver and kidney as to microPET images. To confirm the localization of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in the 92-1-Luc UM tumor xenografts, biodistribution study was performed following microPET imaging. The tracer accumulation in tumors was 1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25%ID/g and 1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23%ID/g at 60 min post-injection. Its uptake in brain, lung, heart, liver, spleen and kidney in subcutaneous UM xenografts were 0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, 1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20, 0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, 1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32, 1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24, 2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37%ID/g, and which in ocular UM xenografts were 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24, 0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15, 2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39%ID/g. The biodistribution data were consistent with the ROI analysis of microPET imaging.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eComparison of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG microPET imaging in UM xenografts\u003c/h2\u003e \u003cp\u003eThe comparison of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG microPET imaging in subcutaneous and ocular 92\u0026thinsp;\u0026minus;\u0026thinsp;1 UM xenografts was further conducted. Both of the two radiotracers were clearly visualized in subcutaneous tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The signals of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 tracer were distinctively observed in ocular 92\u0026thinsp;\u0026minus;\u0026thinsp;1 UM xenografts with very low background; while that of [\u003csup\u003e18\u003c/sup\u003eF]FDG was hard to be distinguished due to a strong brain uptake. The tumor-to-brain, -lung, -heart, -liver, -spleen and -kidney uptake ratios of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in subcutaneous UM xenografts were 6.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89, 1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29, 4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83, 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19, 1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 and 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11, respectively, while the corresponding ratios for [\u003csup\u003e18\u003c/sup\u003eF]FDG were 1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33, 2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65, 5.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84, 6.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97, 2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 and 6.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64, respectively (Table. 1). The tumor-to-brain, -lung, -heart, -liver, -spleen and -kidney uptake ratios of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in ocular UM xenografts were 5.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49, 1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28, 3.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48, 1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, 1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 and 0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, respectively, while the corresponding ratios for [\u003csup\u003e18\u003c/sup\u003eF]FDG were 0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, 2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27, 2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10, 4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42, 2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 and 4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68, respectively (Table. 2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSubcutaneous Tumor to Organ Ratios of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG in UM xenografts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrgan\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FDG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Brain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Lung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Heart\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Liver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Spleen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Kidney\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOcular Tumor to Organ Ratios of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG in UM xenografts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrgan\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FDG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Left eye\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Brain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Lung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Heart\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Liver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Spleen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Kidney\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eComparison of [\u003c/b\u003e \u003csup\u003e \u003cb\u003e18\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eF]AlF-NOTA-PRGD2 and [\u003c/b\u003e \u003csup\u003e \u003cb\u003e18\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eF]FDG PET imaging in UM patient.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe comparison of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG PET imaging in two patients with primary UM was further determined. In the two patients with benign tumor, both two radiotracers had no detectable uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. C and E). In the patients with UM, [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 demonstrated a lower background in the brain region than that of [\u003csup\u003e18\u003c/sup\u003eF]FDG, which was preferred for the detection of primary ocular UM tumor with a favorable target-to-background ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB .D and F). Such finding was consistent with the observation in UM xenografts. In addition, the tumor-to-brain, -lung, -heart, -liver, -spleen and -kidney uptake ratios of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 were 46.50, 18.60, 7.15, 3.10, 1.33 and 0.14 for patient 3, and 14.33, 7.17, 2.87, 1.65, 0.67 and 0.44 for patient 4, respectively, while the corresponding ratios for [\u003csup\u003e18\u003c/sup\u003eF]FDG were 0.29, 6.43, 0.45, 1.41, 1.36 and 0.70 for patient 3, and 0.11, 2.86, 0.43, 0.56, 0.77 and 0.38 for patient 4, respectively (Table. 3). The selectivity and sensitivity of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 tracer for PET imaging of UM patients are even better than that in UM xenografts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOcular Tumor to Organ Ratios of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG in Patient 3 and Patient 4\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOrgan\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePatient 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003ePatient 4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FDG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FDG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Eye\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e46.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Brain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e46.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Lung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Heart\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Liver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Spleen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor to Kidney\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, dimeric PRGD2 has been adopted as a new PET tracer in the early detection and staging of various tumors, due to its higher affinity for integrin αvβ3 receptor compared to monomeric counterpart [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, compared with [\u003csup\u003e18\u003c/sup\u003eF]FDG, the radiosynthesis of this imaging tracer is convenient and less time consuming (\u0026lt;\u0026thinsp;30 min). The addition of a NOTA functional group to PRGD2 enabled a chelate compatible for both \u003csup\u003e18\u003c/sup\u003eF-fluoride-aluminum and \u003csup\u003e68\u003c/sup\u003eGa. Li \u003cem\u003eet al\u003c/em\u003e report the effectiveness of [\u003csup\u003e68\u003c/sup\u003eGa]PRGD2 PET/CT in glioma grading and demarcation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Zheng \u003cem\u003eet al\u003c/em\u003e investigate the application of [\u003csup\u003e68\u003c/sup\u003eGa]NOTA-PRGD2 PET/CT in lung cancer diagnosis, which showed a superior performance than that of [\u003csup\u003e18\u003c/sup\u003eF]FDG PET/CT due to specifically determining metastatic lymph nodes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Zhang \u003cem\u003eet al\u003c/em\u003e show that [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 PET/CT noninvasively visualized GBM lesions and predicted the outcome of concurrent chemoradiotherapy as early as 3 weeks after the initiation of treatment [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, there has been no report about the application of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in UM. This study first evaluated [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 PET imaging in the UM xenografts as well as a UM patients. Our data revealed that tumors in subcutaneous and ocular UM xenografts could be clearly visualized with this tracer at 60 min post-injection. Moreover, the PET imaging with this tracer in the UM patient may be more specific and sensitive than that in UM xenografts.\u003c/p\u003e \u003cp\u003eThe characteristics of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 were evaluated in \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models of UM. Our \u003cem\u003ein vitro\u003c/em\u003e studies revealed that [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 showed a high specificity to integrin αvβ3 in 92\u0026thinsp;\u0026minus;\u0026thinsp;1 UM cells in the aspects of cell uptake, high affinity in cell binding and high stability \u003cem\u003ein vitro\u003c/em\u003e stability. The \u003cem\u003ein vivo\u003c/em\u003e microPET imaging and biodistribution analysis demonstrated that [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 had a high tumor uptake and a very low background. In addition, integrin αvβ3 expression is upregulated during tumor angiogenesis, particularly in vascular endothelial cells. The high tumor uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 \u003cem\u003ein vivo\u003c/em\u003e might also indicate the occurrence of tumor angiogenesis in UM. And the significantly increased liver and kidney uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 may suggest that this tracer is mainly metabolized in these two organs. As to PET imaging, [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 showed a longer retention in tumors and a faster clearance from normal tissues [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This study focuses on evaluating the application of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in the diagnosis of primary UM tumors; its application in metastatic UM xenografts needs to be assessed in the future studies.\u003c/p\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FDG is the common PET tracer currently adopted in the early detection and staging of UM patients in clinical settings. However, a large variation in its sensitivity, specificity, and accuracy in UM diagnosis has been reported [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Comparison of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and [\u003csup\u003e18\u003c/sup\u003eF]FDG in PET imaging, the uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in UM xenografts was lower than that of [\u003csup\u003e18\u003c/sup\u003eF]FDG; but this was opposite in the UM patients. Moreover, the low background of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in the brain region promoted its application in the detection of primary UM tumor with a high target-to-background ratio. In addition, the tumor to organ ratios of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 in the UM patients were superior than that in UM xenografts, indicating [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 PET imaging was more preferred for the diagnosis of clinical UM patients compared to [\u003csup\u003e18\u003c/sup\u003eF]FDG PET imaging.\u003c/p\u003e \u003cp\u003eIn conclusion, our study suggests that [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 tracer shows a great advantage in the diagnosis and staging of UM due to its higher sensitivity and specificity compared to [\u003csup\u003e18\u003c/sup\u003eF]FDG tracer; however, this promising pilot data needs more extensive clinical evaluation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eUM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Uveal melanoma\u003c/p\u003e\n\u003cp\u003ePET \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Positron emission tomography\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FDG \u0026nbsp; \u0026nbsp;\u003csup\u003e18\u003c/sup\u003eF-labelled fluorodeoxyglucose\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e%ID/g \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Percentage of injected dose per gram of tissues\u003c/p\u003e\n\u003cp\u003eMRIMagnetic resonance imaging\u003c/p\u003e\n\u003cp\u003ePET/CT \u0026nbsp; \u0026nbsp; \u0026nbsp;Positron emission tomography/computerized tomography\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHPLC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; High Performance Liquid Chromatography\u003c/p\u003e\n\u003cp\u003eFBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eP/S \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Penicillin/Streptomycin\u003c/p\u003e\n\u003cp\u003eRIPA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Radio Immunoprecipitation Assay\u003c/p\u003e\n\u003cp\u003eROI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Regions of interes\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLW and XZ conducted\u0026nbsp;the experiment and wrote the article; YX, ZH and WZ analyzed data and provided materials; ZZ and MY supervised the project; KW and DP designed the experiment and\u0026nbsp;proofed article.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Major Project of Wuxi Commission of Health (Grant number [Z202110]), Project of Jiangsu Administration of Traditional Chinese Medicine (Grant number [MS2022145]), The Elderly Health Research Project of Jiangsu Province (Grant number [2022043]), The Social Development Project of Jiangsu Provincial Department of Science and Technology (Grant number [BE2022699]), Chinese Medicine Science and Technology Development Project of Jiangsu Province (Grant number [MS2023166]), Scientific Research Project of Jiangsu Provincial Health Commission (H2023150) and Scientific Research Project of Wuxi Health Commission (Grant number [Z202303]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis clinical study was approved by the ethics committee of The Hospital Affiliated to Jiangnan University (LS2011051). the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendment. Written informed consents were obtained from all participants.\u0026nbsp;All experiment protocols were approved by the Animal Care and Use Committee of Jiangsu Institute of Nuclear Medicine (JSINM-2022-061) and the study was carried out in compliance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors are consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKaliki S, Shields CL. Uveal melanoma: relatively rare but deadly cancer. 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J Nucl Med. 2006;47:404\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Uveal Melanoma, [18F]AlF-NOTA-PRGD2, [18F]FDG, PET imaging","lastPublishedDoi":"10.21203/rs.3.rs-3998432/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3998432/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eUveal melanoma (UM) is the most common primary intraocular tumor in adults, and early detection is critical to improve the clinical outcome of this disease. In this study, the diagnostic effectiveness of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 (an investigational medicinal product) positron emission tomography (PET) imaging in UM xenografts and UM patients were evaluated.\u003cstrong\u003e \u003c/strong\u003eThe cell uptake, cell binding ability and \u003cem\u003ein vitro\u003c/em\u003e stability of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 were evaluated in 92-1 UM cell line. MicroPET imaging and biodistribution study of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 were conducted in 92-1 UM xenografts. Then, UM patients were further recruited for evaluating the diagnostic effectiveness of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 PET imaging (approval no.NCT02441972 in clinicaltrials.gov). In addition, comparison of [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 and \u003csup\u003e18\u003c/sup\u003eF-labelled fluorodeoxyglucose ([\u003csup\u003e18\u003c/sup\u003eF]FDG) PET imaging in UM xenografts and UM patients were\u0026nbsp; conducted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe \u003cem\u003ein vitro\u003c/em\u003e data showed that [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 had a high cell uptake, cell binding ability and \u003cem\u003ein vitro\u003c/em\u003e stability in 92-1 UM cell line. The \u003cem\u003ein vivo\u003c/em\u003e data indicated that 92-1 UM tumors were clearly visualized with the [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 tracer in the subcutaneous and ocular primary UM xenografts model at 60 min post-injection. And the tumor uptake of the tracer was 2.55±0.44%ID/g and 1.73±0.15%ID/g at these two tissue locations respectively, at 7 days after animal model construction. The clinical data showed that tumors in UM patients were clearly visualized with the [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 tracer at 60 min post-injection. In addition, [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 tracer showed higher sensitivity and specificity for PET imaging in UM xenografts and UM patients compared to [\u003csup\u003e18\u003c/sup\u003eF]FDG tracer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-PRGD2 PET imaging may be a more preferred approach in the diagnosis of UM compared to [\u003csup\u003e18\u003c/sup\u003eF]FDG PET imaging.\u003c/p\u003e\n\u003cp\u003eTrial registration ClinicalTrials.gov: NCT02441972, Registered 1 January 2012, https://clinicaltrials.gov/study/NCT02441972\u003c/p\u003e","manuscriptTitle":"Ultrasensitive Detection of Uveal Melanoma Using [18F]AlF-NOTA-PRGD2 PET Imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 17:53:43","doi":"10.21203/rs.3.rs-3998432/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-05-03T10:40:14+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-30T13:03:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-27T10:27:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-27T07:25:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Research","date":"2024-03-25T06:21:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4bd98b24-c744-43c9-8a88-733df7bf7f0b","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-26T06:46:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-01 17:53:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3998432","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3998432","identity":"rs-3998432","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}