One-step Radiosynthesis and Preclinical Evaluation of Molecular Probe [18F]FEtO-CHC Targeting Monocarboxylate Transporters for PET Imaging in Tumor-bearing Mice

One-step Radiosynthesis and Preclinical Evaluation of Molecular Probe [18F]FEtO-CHC Targeting Monocarboxylate Transporters for PET Imaging in Tumor-bearing Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article One-step Radiosynthesis and Preclinical Evaluation of Molecular Probe [ 18 F]FEtO-CHC Targeting Monocarboxylate Transporters for PET Imaging in Tumor-bearing Mice Dongmei Shi, Ling Liu, Kaixin Qin, Yuzhou Zheng, Wenhao Hu, Jiarui You, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4020948/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Jun, 2025 Read the published version in Molecular Imaging and Biology → Version 1 posted 5 You are reading this latest preprint version Abstract Purpose The visualization and quantitative analysis of monocarboxylate transporters (MCTs) hold significant application value in comprehending the metabolic symbiosis, acid resistance, and invasion mechanisms of tumors. Thus, we designed and synthesized a novel MCTs-targeting radiotracer [ 18 F]FEtO-CHC and gave a comprehensive evaluation in vitro and in vivo experiments for it. Procedures The preparations for the precursor and reference of [ 18 F]FEtO-CHC were encompassed. In vitro evaluation included compound identification, purity, stability, liposolubility, and assays in BxPC3 and 4T1 tumor cell lines. Dynamic Micro-PET imaging was performed in tumor-bearing mice to determine its in vivo characteristics. Results The synthesis of [ 18 F]FEtO-CHC, a derivative of α-cyano-4-hydroxycinnamic acid (CHC), was achieved using a one-step method with the MCTs inhibitor ( E )-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate as the lead compound. The yield obtained was 52.08 ± 6.74% (n = 7, decay corrected). The cell uptake characteristics and targeting ability towards MCTs were confirmed through cell uptake and competitive inhibition experiments conducted on BxPC3 pancreatic cancer cell line and 4T1 breast cancer cell line. The biodistribution and Micro-PET/CT imaging of tumor-bearing mice revealed the hepatic and renal metabolism-mediated excretion characteristics of [ 18 F]FEtO-CHC, with radioactive uptake in tumors being consistent with MCTs expression levels. Conclusions Through the aforementioned studies, a one-step method was employed to successfully synthesize [ 18 F]FEtO-CHC, which has been validated as a small molecule PET probe specifically targeting MCTs. Monocarboxylate transporters Metabolism of symbiosis α-cyano-4-hydroxy-cinnamic acid PET imaging probe Preclinical evaluation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Cancer remains a formidable global health challenge, and targeting tumor metabolism for anti-tumor therapy represents a pivotal research strategy. Tumors exhibit the Warburg effect[ 1 ], resulting in excessive lactic acid production through glycolysis. To prevent intracellular accumulation, proton-coupled monocarboxylate transporters (MCTs) facilitate the extracellular transportation of lactic acid [ 2 – 3 ]. As integral membrane proteins [ 4 ], MCTs play crucial roles in diverse physiological processes such as lactic acid metabolism, energy supply, and pH regulation [ 5 – 7 ]. They are widely expressed in normal tissues including kidney, heart, eye, liver, and retina [ 8 – 12 ]. In addition, MCT1 and MCT4 have been identified in various human cancer cell lines and primary tumors, playing a crucial role in tumor metastasis and invasion[ 13 ], including breast cancer, head and neck cancer, lung cancer neuroblastoma, as well as brain and colon tumors[ 14 – 15 ]. Specifically, the MCT4 transporters predominantly facilitate the transportation of lactic acid generated through glycolysis in hypoxic regions within the tumor. These regions are distantly located from the tumor vascular area but serve to transport lactic acid towards extracellular spaces and oxygen-rich regions. Conversely, MCT1 primarily facilitates the uptake of produced lactic acid into cells for subsequent oxidation and energy provision in oxygen-rich areas [ 15 – 16 ]. By effectively utilizing diverse energy substrates in different oxygen-containing regions through the transport mechanisms mediated by monocarboxylic acid transporters (MCTs), tumor tissues achieve their proliferation and invasion. The metabolic characteristics of tumors present an opportunity to impede tumor progression by selectively inhibiting MCTs, thereby establishing MCTs as a promising biological target relevant to tumors [ 17 – 20 ]. Although the small molecule inhibitor AZD3965, which targets MCT1, has progressed to the clinical trial stage [ 4 , 19 ], only two MCT inhibitors have been developed as PET probes. One of them is a coumarin analogue labeled with 11 C; however, its short half-life and complex preparation process render it unsuitable for further clinical translation [ 21 – 22 ]. The other probe is an α-cyano-cinnamic acid derivative labeled with 18 F ([ 18 F]FACH), which has demonstrated radio-safety in pig studies and attempted to enhance blood-brain barrier permeability by increasing lipid solubility to improve probe uptake in intracranial tumors with high MCT expression. Nevertheless, no further research reports are available regarding this probe [ 23 – 27 ]. Therefore, our team independently designed and synthesized a 18 F-radiolabelled derivative of α-cyano-4-hydroxycinnamic acid ([ 18 F]FEtO-CHC), as a PET molecular probe, targeting MCTs. Subsequently, we evaluated its tumor imaging characteristics through in vitro and in vivo experiments as well as dynamic Micro-PET imaging. 2. Material and Methods 2.1. General Equipment and Methods The cyclotron (HM-10) was obtained from Sumitomo Corporation, Japan. The ultraflex MALDI-TOF/TOF instrument employed for mass analysis was provided by Bruker Corporation, Germany. Nuclear magnetic resonance spectroscopy was performed using the Bruker Avance III 400 MHz NMR spectrometer. High performance liquid chromatography (HPLC) was the Series III system from Lab Alliance, Tianjin Lab Alliance Company, equipped with dual detectors including the B-FC-3600 high-energy radioactivity detector from Bioscan, USA and the 201 UV detector from Tianjin Lab Alliance Company. CRC-25R radioactivity detector was purchased from Capintec, USA. A semi-prepared C18 column (5 µm, 250 mm×10mm) manufactured by YMC in Japan was employed. Sep Pak QMA and HLB columns supplied by Waters, USA. Thin layer chromatography (TLC) scanner was equipped with a B-FC-3600 high-energy radioactivity detector from Bioscan, USA. The compound 4,7,13,16,21,24-hexaoxo-diazabicyclo[8.8.8]-tridecane (Kryptofix 2.2.2, K2.2.2) was obtained from ABX advanced biochemical compounds GmbH, Germany. Anhydrous acetonitrile (CH 3 CN), and potassium carbonate (K 2 CO 3 ) were purchased from Aldrich, USA. All other reagents used were of analytical or chemical purity; experimental water was deionized purified water. BxPC3 pancreatic cancer cells and 4T1 breast cancer cells were procured from the Shanghai Cell Bank of Chinese Academy of Sciences. 2.2. Chemical Synthesis The synthetic routes to ( E )-ethyl 2-cyano-3-(4-(2-(tosyloxy)ethoxy)phenyl)acrylate (precursor compound 2 ), ( E )-2-cyano-3-(4-(2-[ 18 F]-fluoroethoxy)phenyl)acrylic acid ([ 18 F]FEtO-CHC, [ 18 F] 4 ), compound ( E )-ethyl 2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylate ([ 19 F] 3 ) and the reference compound ( E )-2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylic acid ([ 19 F]FEtO-CHC, [ 19 F] 4 ), are shown in Fig. 1 . 2.2.1. Preparation of ( E )-ethyl 2-cyano-3-(4-(2-(tosyloxy)ethoxy)phenyl)acrylate (compound 2) To a solution of ( E )-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate (compound 1 ) (200 mg, 0.92 mmol) in dry CH 3 CN (40mL) was added 18-crown-6 (48.71 mg, 0.18 mmol) and anhydrous K 2 CO 3 (254.68 mg, 1.84 mmol), ethylene glycol di-p-toluene sulfonate (511.42 mg, 1.38 mmol) was then added into the reaction mixture after being stirred for 20 min and the resulting mixture was refluxed at 90°C for about 13 hours. The reaction progress was monitored by TLC. The solvent was removed under reduced pressure and distilled water (50mL) was added. The mixture was extracted with CH 3 CN (4×50mL). The combined CH 3 CN extracts were dried with Na 2 SO 4 and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate/petroleum ether (1/4, v/v) as the eluent to afford compound 2 (98.44 mg, 49.22%). High resolution MS: [M + Na] for C 21 H 21 NO 6 SNa, calculated, 438.11, found, 438.096 (Suppl. Figure 1A). 1 H NMR(400MHz, Suppl. Figure 1D): δ 8.32 (s, 1H), 8.06 (d, J = 9.0 Hz, 2H), 7.79 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.05 (d, J = 9.0 Hz, 2H), 4.36 (d, J = 1.5 Hz, 2H), 4.35–4.27 (m, 4H), 2.41 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). 2.2.2. Preparation of ( E )-ethyl 2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylate ([ 19 F]3) To a solution of ( E )-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate (compound 1 ) (530mg, 2.44mmol) in anhydrous CH 3 CN (40 mL) was added 18-crown-6 (67.14 mg, 0.25 mmol) and anhydrous K 2 CO 3 (351 mg, 2.54mmol), fluoroethyl p-toluene sulfonate (830 mg, 3.81 mmol) was added into the reaction mixture after being stirred for 20 min and the resulting mixture was refluxed at 90°C for about 13 hours. The reaction progress was monitored by TLC. After removal of the solvent via reduced pressure, distilled water (50 mL) was added and the mixture was extracted using CH 3 CN (4×50 mL). The combined CH 3 CN extracts were dried with Na 2 SO 4 and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate/petroleum ether (1/2, v/v) as the eluent to afford [ 19 F] 3 (325.85mg, 61.48%). High resolution MS: M for C 14 H 14 FNO 3, calculated, 263.1, found, 263.086(Suppl. Figure 1B). 1 H NMR (400 MHz, Suppl. Figure 1E): δ 8.33 (s, 1H), 8.10 (d, J = 8.8 Hz, 2H), 7.20 (d, J = 8.7 Hz, 2H), 4.84 (dd, J = 4.6, 3.0 Hz, 1H), 4.72 (dd, J = 4.6, 2.9 Hz, 1H), 4.45–4.39 (m, 1H), 4.38–4.26 (m, 3H), 1.30 (td, J = 7.1, 1.1 Hz, 3H). 2.2.3. Preparation of ( E )-2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylic acid ([ 19 F]FEtO-CHC, [ 19 F]4) To a solution of [ 19 F] 3 in CH 3 CN (1 mL) was added 2N NaOH for being stirred 5 min at room temperature. The solvent was removed under reduced pressure and obtained the reference compound [ 19 F] 4 as a white solid. High resolution MS: [M + Na] for C 12 H 10 FNO 3 Na, calculated, 258.06, found, 258.045(Suppl. Figure 1C). 2.3. Radiosynthesis [ 18 F]FEtO-CHC was synthesized in one-step procedure involving nucleophilic substitution reaction and deprotection reaction. No-carrier-added [ 18 F] fluoride was trapped using 1.5 mL of H 2 O on a Sep Pak light QMA cartridge. NaHCO 3 (10 mL, 0.5 M) and H 2 O (10 mL) were used for cartridge preconditioning. The activity was eluted into the reaction flask using 1 mL of an aqueous solution of K2.2.2/K 2 CO 3 (5.91 mg of K2.2.2 and 1.36 mg of K 2 CO 3 in CH 3 CN/H 2 O 10/1, v/v). The reaction flask was evaporated at 110℃ under reduced pressure. A solution of compound 2 (4 mg) in 1 mL of CH 3 CN was added, sealed and heated at 110°C for 15 min. Semi-preparative HPLC was performed on C18-column (5 µm, 250×100 mm) using CH 3 CN/H 2 O as the eluent at a flow rate of 3 mL/min (CH 3 CN: 55%) with UV detection at 254 nm. [ 18 F]FEtO-CHC was collected at a retention time of 12-14min into a distillation flask. The collected solution was evaporated under reduced pressure and diluted with 2mL water and passed through a 0.22 µm filter to obtain a solution of [ 18 F]FEtO-CHC. TLC analysis was used for the analysis of radiochemistry of the probe using 85% CH 3 CN as eluent. The analysis of the reference compound and [ 18 F]FEtO-CHC was used HPLC (C18 chromatographic column, CH 3 CN/H 2 O = 70/30, v = 3 mL/min, UV = 254 nm). 2.4. In Vitro and Vivo Stability The in vitro stability of [ 18 F]FEtO-CHC was tested in phosphate-buffered saline (PBS) and fetal bovine serum (FBS) at 25℃ for 60 min and 120 min incubation. The in vivo stability was using blood and urine samples from a healthy male SD rat at 60 min post-injection of [ 18 F]FEtO-CHC (0.5mL, 8MBq). All the samples were analyzed by HPLC (C18 chromatographic column, CH 3 CN/H 2 O = 55/45, v = 3 mL/min, UV = 254 nm) or TLC under the same condition used in the above. 2.5. Lipophilicity The partition coefficient of [ 18 F]FEtO-CHC was detected in the solution of n-octanol/saline (logP) or n-octanol/PBS (logD7.4) at room temperature with conventional shake-flask method. Approximately 500 kBq of the probe was added to a centrifuge tube containing 3 mL of n-octanol/ saline or n -octanol/PBS (1:1, v:v) and shaken for 20 min to ensure complete mixing. The solution was then centrifuged at 5000 rpm for 5 min. Both aliquots of 1 mL of the organic phase and the inorganic phase were respectively taken to measure gamma counts. 2.6. Cell Culture and Animal Models The BxPC3 and 4T1 cell lines were cultivated respectively in RPMI-1640 and DMEM supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/ streptomycin under humidified conditions (37°C, 5% CO 2 /95% air). The animal experimental procedures were conducted in accordance with the approved animal care and use guidelines by the ethics committee of Shanxi Medical University. BxPC3 human pancreatic cancer cells and 4T1 breast cancer cells in logarithmic growth phase were collected, followed by the preparation of a single cell suspension using PBS. After adjusting the cell concentration to 1×10 7 /mL, subcutaneous injection of 0.1mL was performed into the right shoulder of BALB/c nude male mice to process tumor xenografts. The mice were utilized when the tumor diameter reached 6–10 mm. 2.7. In Vitro Cell Assays 2.7.1. In vitro cell uptake BxPC3 pancreatic cancer cells and 4T1 breast cancer cells in logarithmic growth phase were incubated in 24-well plates for 24 hours (at a density of 1×10 5 cells/well). The cell samples were divided into six groups, with four replicate wells per group. Subsequently, the medium was discarded, and the cells were washed twice with warm PBS. Novel probes (0.3 MBq, 200 µL/well) were added to each well and incubated for different time intervals of 2, 5, 15, 30, 60, and 120 min respectively. After that, the probes were removed, and the cells were washed twice with ice-cold PBS followed by cleavage using a solution of 1N NaOH (0.5 mL/well) for duration of 20 min. The resulting cleavage liquid was collected, and its radioactivity was measured using a γ-counter. The obtained data underwent standardization procedures and recorded as percentages of radioactivity. 2.7.2. In vitro cell inhibition The initial treatment was conducted in the same manner as that for cell uptake. Subsequently, each well was administered 200 µL of MCT1 inhibitor CHC solution (8 mg CHC dissolved in 200 µL DMSO and diluted to 6 mL with complete medium) along with probes (0.3 MBq, 200 µL/well) and incubated for 30 minutes for four replicate wells. Following this, the inhibitors and probes were discarded, and the wells were washed twice with ice-cold PBS before cleavage using 1N NaOH (0.5 mL/well) for 20 minutes. The subsequent treatments and calculations followed the same protocol as the uptake experiment. 2.7.3. In vitro cell efflux The initial treatment was identical to that of the cell uptake. Following the addition of the radio probes (0.3MBq, 200µL/well) to each well, they were incubated at 37°C for 1 hour with four replicate wells per group. Subsequently, the radioactive liquid was discarded, and warm PBS was used for two washes. Then, 0.5 mL medium was added and co-cultured at 37°C for time intervals of 5, 15, 30, 60, 90 and 120 min. After discarding the supernatant and washing twice with PBS, cleavage was performed using 1N NaOH (0.5mL/well) for 20 minutes. The subsequent treatments and calculations followed the same protocol as the uptake experiment. 2.7.4. Western Blotting The cells were lysed with radioimmunoprecipitation assay lysis buffer (ServiceBio) containing protease inhibitor (ServiceBio), and 6 µg of proteins were separated by electrophoresis in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (ServiceBio). 2.8. Biodistribution Eighteen healthy ICR mice (25 ~ 30 g, male, clean) were provided by the Shanxi Medical University Experimental Animal Center and divided into six groups with three mice in each group. The mice were intravenously injected with [ 18 F]FEtO-CHC (200 µL, 4 MBq) solution via the tail vein. At time points of 5, 15, 30, 60, 90 and 120 min post-injection, blood samples and major organs were collected and weighed. The radioactivity was measured using a γ-counter and the percentage injection dose rate (%ID/g) after attenuation correction was calculated. 2.9. Micro-PET/CT Imaging The tumor-bearing nude male mice were placed on the micro-PET/CT scanning bed, which was maintained at a constant temperature of 37°C, following gas induction anesthesia (2.0% isoflurane air). In the experimental group, [ 18 F]FEtO-CHC (0.2 mL, 4 MBq) was injected via the tail vein and dynamic Micro-PET imaging was performed for a duration of 2 hours. In the inhibition group, MCT1 inhibitor CHC (0.2 mL, 1.6 mg) was injected via the tail vein followed by injection of [ 18 F]FEtO-CHC (0.2 mL, 4 MBq) for 2h-dynamic Micro-PET imaging. For CT image scanning, tube voltage of 80 kV and tube current of 1 mA were used with an exposure time of 80 s. A layer thickness of 160 µm was selected for pretreatment and reconstruction utilized the Feldkamp filter back projection algorithm. The PMOD software facilitated image analysis to delineate volumes-of-interest (VOI) corresponding to mouse tumors and left hind leg muscles; SUVmean values were quantitatively calculated. The ratio between tumor and muscle uptake recorded as T/M. 2.10. Immunohistochemistry The primary antibody used was a polyclonal MCT1 antibody (Proteintech, China), and secondary antibody was a horseradish peroxidase-conjugated goat anti-rabbit antibody (ServiceBio, China). Tumor and muscle tissues were fixed in 10% formalin solution, embedded, and sectioned. Subsequently, the sections were baked at 60°C followed by dewaxing and hydration steps. Antigen retrieval was performed using citrate repair solution with pH = 6.0 in an autoclave. To block endogenous peroxidase activity, 3% hydrogen peroxide was incubated at room temperature for 25 min. Non-specific background reactions were blocked by using 3% BSA (bovine serum albumin) at room temperature for 90 min. The primary antibody was then added and incubated overnight at 4°C in a humidified chamber. Afterward, the sections were incubated with the secondary antibody at room temperature for 50 min. Hematoxylin was used to stain nucleus, dehydrate and seal the cells for microscopy observation. 2.11. Statistical Analysis All statistical analysis was conducted using IBM SPSS 26.0 software. Data visualization was processed using GraphPad Prism 8. The data were presented as mean ± SEM, and using independent sample t-test to assess statistical significance. P-value < 0.05 was considered statistically significant. The cell experiments were independently repeated three times on different dates. 3. Results 3.1. Chemistry and Radiochemistry The precursor compound 2 was synthesized through the reaction between ( E )-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate (compound 1 ) and ethylene glycol di-p-toluene sulfonate, and confirmed by mass spectrometry and 1 H NMR (Suppl. Figure 1A, D). The compound ( E )-ethyl 2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylate ([ 19 F] 3 ) was synthesized by reacting ( E )-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate (compound 1 ) with fluoroethyl p-toluene sulfonate, and was confirmed with mass spectrometry and 1 H NMR (Suppl. Figure 1B, E). The reference compound [ 19 F] 4 was obtained by ( E )-ethyl 2-cyano-3-(4-(2-fluoroethoxy) phenyl) acrylate ([ 19 F] 3 ) after hydrolytic reaction and confirmed by mass spectrometry (Suppl. Figure 1C). The preparation of [ 18 F] 4 involved a nucleophilic substitution reaction with [ 18 F] − and deprotection in one-step procedure to give a retention time of 10-12min in semi-preparative HPLC (CH 3 CN /H 2 O = 55/45). The retention time (Rt) of [ 19 F] 3 in the UV spectrum of HPLC (CH 3 CN /H 2 O = 70/30) was determined to be 8.9 min, and the Rt of the reference compound [ 19 F] 4 in the UV spectrum was 5.6 min. Comparatively, [ 18 F] 4 can be verified with the same retention time (Rt) as [ 19 F] 4 under the same analytical HPLC method conditions (CH 3 CN /H 2 O = 70/30, Suppl. Figure 2A). Furthermore, after radiochemical reaction, following removal of [ 18 F] − using a C18 cartridge, two radioactive product peaks were observed at 5.7 min and 9.1 min by HPLC analysis (CH 3 CN/H 2 O = 70/30), while the proportion of the former product peak gradually increased with increasing the reaction temperature and time from 100°C for 10 min to 110°C for 15 min (Suppl. Figure 2A). After adding 50 µL of 2N NaOH into this radioactive solution for hydrolysis at room temperature for 5 min, only a single radioactive peak was observed at 5.7 min. Hence, under the reaction conditions of 110°C for 15 min and using CH 3 CN as the solvent, [ 18 F] 4 was synthesized in a one-step reaction with a total time of approximately 60 min. The radiochemical yield was determined to be 52.08 ± 6.74% (n = 7, decay corrected), while the radiochemical purity was > 97% in TLC analytical results (Suppl. Figure 2B). 3.2. In Vitro and Vivo Stability The in vitro stability of [ 18 F] 4 in PBS and FBS at room temperature within 120 min using HPLC (CH 3 CN/H 2 O = 55/45) exhibited a purity of > 95% (Suppl. Figure 3A), which indicated no degradation occurred in vitro. For the in vivo stability in a rat at 60 min postinjection, HPLC analysis revealed the decomposition product of [ 18 F] 4 was mainly retained in the urine (Suppl. Figure 3A) and TLC analysis of the blood indicated that [ 18 F] 4 stayed the predominant compound with a purity of about 91% (Suppl. Figure 3B). 3.3. Lipophilicity The lipophilicity of [ 18 F]FEtO-CHC was determined by conventional shake-flask method, the logP or logD7.4 value of [ 18 F]FEtO-CHC was calculated to be 0.19 ± 0.03 or 0.29 ± 0.03 (n = 3). 3.4. In Vitro Cell Assays 3.4.1. In vitro cell uptake To explore the uptake characteristics of [ 18 F] 4 , BxPC3 pancreatic cancer cells and 4T1 breast cancer cells were selected for the uptake experiment. The uptake of [ 18 F] 4 by BxPC3 pancreatic cancer cells exhibited a rapid initial increase within the first 5 min, reaching a peak value of 0.76 ± 0.074% at 15 min, followed by a gradual decrease. Similarly, in 4T1 breast cancer cells, the uptake reached 0.78 ± 0.04% at 15 min and peaked at 0.93 ± 0.06% after 60 min before gradually declining (Fig. 2 A). 3.4.2. In vitro cell inhibition To ascertain the target specificity of [ 18 F] 4 , the specific MCT1 inhibitor CHC was used as a competitive inhibitor. After adding CHC, the uptake of [ 18 F] 4 was decreased by 66.77 ± 1.98% and 33.88 ± 5.51% in BxPC3 and 4T1 cell lines respectively, verifying CHC significantly blocked the uptake of [ 18 F] 4 (Fig. 2 B). 3.4.3. In vitro cell efflux To assess the stability of [ 18 F] 4 binding to the target, a cell efflux experiment was conducted following incubation of BxPC3 pancreatic cancer cells and 4T1 breast cancer cells with the [ 18 F] 4 probe for 1 hour. The combined probe exhibited gradual and steady release within 2 hours (Fig. 2 C). 3.4.4. Western Blotting The image of western blot was illustrated in Fig. 2 D. The ratio of integrated density val of MCT1/β-actin calculated from AIWBwell™ was 2.93 and 2.30 in 4T1 and BxPC3 cell lines respectively, suggesting the expression of MCT1 in 4T1 cell line was higher than that in BxPC3. 3.5. Biodistribution The biodistribution of [ 18 F]FEtO-CHC in healthy male mice is illustrated in Fig. 3 . As the primary metabolic organs, the liver and kidneys exhibited significant radioactivity accumulation, which gradually decreased over time. The bone consistently displayed elevated levels of radioactivity throughout each period. The heart, muscle, spleen, and lung initially demonstrated high radioactivity at 5 min but then followed a gradual decline. In contrast, the gastrointestinal tract exhibited a progressive increase in radioactivity with time, particularly in small bowel where it reached its peak 6.06%ID/g at 120 min, which may be resulted from the high expression of MCT1 in gastrointestinal tract as main absorption organs of short-chain fatty acid [ 28 ]. Brain uptake peaked 0.98%ID/g at 5 min and then rapidly declined, which may originate from the probe’s transport mechanism of blood-brain barrier (BBB) correlated with lipophilicity. Furthermore, blood radioactivity gradually decreased and reached 0.65%ID/g after 15 min, which was lower than half of its initial value of 1.91%ID/g, confirming its rapid clearance rate. 3.6. Micro-PET/CT Imaging The 2h-dynamic micro-PET imaging results of [ 18 F] 4 in BxPC3 pancreatic cancer and 4T1 breast cancer tumor-bearing mice are depicted in Fig. 4 . After intravenous administration of [ 18 F] 4 , whole-body organ perfusion imaging was performed in both BxPC3 and 4T1 mice. The uptake of liver and kidney showed a slight decrease over time, while the uptake of bladder gradually increased, which indicated that both the liver and kidney were the metabolic organs of [ 18 F] 4 . The radioactive uptake in other organs and tissues such as heart, lung, and muscle are quite low and comparable to background levels. Consistent with the forementioned results in biodistribution (Fig. 3 ), there was a gradual accumulation of radioactivity in the gastrointestinal tract and axial bone. The BxPC3 pancreatic cancer tumor-bearing mice exhibited peak tumor-to-muscle ratio approximately 40 min after [ 18 F] 4 injection, followed by a gradual decline in uptake. And the 4T1 breast cancer tumor displayed a similar tumor-to-muscle ratio as the BxPC3 pancreatic cancer tumor within the initial 40 min but demonstrated slow radioactive accumulation subsequently (Fig. 3 A, B). These uptake trends in BxPC3 and 4T1 tumor showed no difference with cell uptake assay. After the administration of MCT1 inhibitor CHC, a significant reduction in tumor uptake was observed in BxPC3 tumor-bearing mice within the first 5 minutes (Fig. 4 C), confirming that our probe's uptake can be inhibited by CHC, and the probe potentially targets MCT1 specifically in vivo. 3.7. Immunohistochemistry To investigate whether the [ 18 F]FEtO-CHC-uptake was consistent with the levels of MCT1 expression in forementioned in vitro and in vivo assays, immunohistochemistry staining and half-quantitative analysis were conducted. The representative images of MCT1 in tumor xenografts and left hind leg muscles of nude mice are depicted in Fig. 5 . Both the image (Fig. 5 A) and the positive scores (Fig. 5 B) indicated a low MCT1 expression level in muscles and a high expression level in both tumor xenografts, which was consistent with the uptake results in these two kinds of BxPC3 and 4T1 cell lines (Fig. 2 A), the micro-PET image in tumor-bearing mice and the curves of T/M in image analysis (Fig. 4 A, B). 4. Discussions MCT1 is a critical biological target for glycolysis of tumor. Several MCT1-targeting radiotracers have been developed in recent years [ 21 , 23 – 27 ]. However, one series of MCT1-targeting probe [ 18 F]FACH is still not evaluated in tumor PET imaging [ 23 – 27 ]; another 11 C-labeld coumarin analog probe was restricted for further application with its low radioactive tumor accumulation and rapid clearance [ 21 ]. Herein, we designed and synthesized a novel MCTs targeting PET probe for tumor-bearing mice imaging and evaluated it in vitro and in vivo. Based on the structure of MCTs inhibitor α-cyano-4-hydroxycinnamic acid (CHC) [ 29 ], α-cyano-4-hydroxyethyl cinnamate was directly chosen as the lead compound in this study, and the precursor compound α-cyano-4-(2-p-toluenesulfonyl ethoxy)-ethyl cinnamate (compound 2 ) was obtained through a one-step chemical reaction [ 27 ]. Intriguingly, we discovered our radiotracer could be obtained in one-step procedure using the precursor compound 2 under 110℃ for 15 min with the chemical yield of 52.08 ± 6.74% (n = 7, decay corrected), indicating the hydrolysis of ester group in the nucleophilic substitution process. Additionally, the elevation of reaction temperature and time can effectively increase the yield of the target tracer by HPLC analysis. Although the report of one-step radiosynthesis of [ 18 F]FACH using CHC analog without the protection of carboxylic acid group have been published [25] , to the aim of simplifying radiolabeling steps and increasing the yield, their precursor need to be prepared for unprotection in the chemical synthesis period. Compared with that, our synthesis of [ 18 F]FEtO-CHC demonstrates the benefits of streamlining chemical and radiolabeling processes, while maintaining a high yield in radiochemical production. However, the phenomenon of hydrolysis during the nucleophilic substitution procedure lacks relevant literature reports, necessitating further exploration of its specific mechanism. The stability studies conducted in vivo and in vitro revealed a certain level of degradation in the urine of SD rats with a 60 min post-intravenous administration of [ 18 F]FEtO-CHC, which is a common occurrence in many other fluorine-labeled organic compounds due to the inherent instability of the C-F bond [ 30 ]. Furthermore, the lipophilicity measurement for [ 18 F]FEtO-CHC obtained a logD7.4 value of 0.29 ± 0.03 (n = 3), demonstrating its restricted liposolubility. By calculating logD7.4, we can give a valuable estimate for the absorption and distribution of drugs within the body, while also identifying the capacity of drug transportation across the blood-brain barrier (BBB) [ 31 ], providing us with guidance for subsequent cell experiments and tumor imaging. Given the 4T1 breast cancer cell line has been used in the study of AZD3965 and AR-C155858 inhibitors and is potently inhibited by CHC [ 32 – 33 ] and the slight overexpression of MCT1 in BxPC3 pancreatic cancer cell line [ 34 ], we applied 4T1 and BxPC3 cell lines for our evaluation. The cellular uptake analysis revealed that both BxPC3 and 4T1 cell lines exhibited a similar peak uptake at 15 min, following which the uptake in 4T1 cells continued to increase while BxPC3 cells showed a gradual decrease, matched with the distinct expression levels of MCT1 protein in these two kinds of cell lines from western blot results. Significant uptake inhibition was observed both in vitro and in vivo when using CHC as a competitive MCT1 inhibitor, confirming the specific targeting capability of [ 18 F]FEtO-CHC towards MCT1. The cellular efflux experiment demonstrated a high and similar efflux rate in BxPC3 and 4T1 cells, suggesting its short retention time in tumors. The biodistribution results were consistent with the 2h-dynamic Micro-PET/CT imaging findings in a mouse tumor-bearing model as well. The liver and kidneys exhibited predominant metabolic activity for [ 18 F]FEtO-CHC and significant radioactive accumulation was observed in bladder, confirming the metabolic organs of [ 18 F]FEtO-CHC in both liver and kidneys. In vivo dynamic Micro-PET imaging demonstrated that the tumor-to-muscle (T/M) values in both BxPC3 and 4T1 tumor-bearing mice reached approximately 1.5 at 40 min, after which the T/M gradually decreased for BxPC3 but continued to rise slowly for 4T1, consistent with the findings from the in vitro uptake experiment. Immunohistochemical analysis further confirmed the higher expression of MCTs in both BxPC3 and 4T1 tumor tissue compared to that observed in muscle. These results collectively indicate that probe uptake, both in vitro and in vivo, is associated with MCT1 expression levels. Although the uptake of our radiotracer is relatively low in tumor due to its rapid clearance, we overcome the high plasma binding rate of 11 C-labeld coumarin analog probe and enhanced the retention capacity in tumors within 2h [ 21 ]. Furthermore, it is the first time to give a comprehensive evaluation for novel MCTs targeting probes in vitro tumor cell lines and in vivo tumor-bearing mice. To enhance the radioactive accumulation in MCT1-positive tumors, we will optimize its structural design from improving the retention in u and slowing the rate of clearance from blood. Meanwhile, we will focus on further exploration of its potential applications in diagnosing and treating specific tumors. The prospects for tumor therapy targeting MCTs have been promising in recent years. AZD3965 are in advanced clinical trial, demonstrating excellent efficacy against lymphoma [ 4 , 19 ]. The probe targeting MCT not only enables the early non-invasive diagnosis of tumors, but also holds the promise of screening MCT-positive tumors and monitoring the therapeutic efficacy of MCT- targeting therapy. In addition, owing to the study of monocarboxylate transports and glycolysis mechanism is still in progress, exploring the mechanism in diversities of diseases for MCT-dependent tumors and non-tumors such as glioblastoma, Alzheimer's disease and multiple sclerosis are promising [ 35 ], which may provide an effective technical tool to explore the transport mechanism of monocarboxylic acid. 5. Conclusion The self-designed PET molecular tracer [ 18 F]FEtO-CHC ([ 18 F] 4 ), [ 18 F]-labeled CHC derivative targeting MCTs, can be obtained through a one-step chemical reaction. Manual labeling synthesis takes approximately 60 min, yielding a radiochemical yield of 52.08 ± 6.74% (n = 7, attenuation correction), with purity exceeding 97%. The tumor imaging ability and targeting for MCT1 have been validated through in vitro and in vivo experiments, establishing a crucial molecular imaging method to assess metabolic intervention diagnosis and therapy based on lactate utilization for both tumors and non-tumors in the future. Declarations Funding This work was supported by the National Natural Science Foundation (Nos. 81471695, 81971655, 82027804, 82001873). The research was also supported by the Four “Batches” Innovation Project of invigorating Medical through Science and Technology of Shanxi Province (No. 2022XM38), Central leading local science and Technology Development Fund Project (No. YDZJSX2022A058) and supported by Fundamental Research Program of Shanxi Province (No. 202303021221226). Author contributions Corresponding authors: HW, ZW and SL. DS and LL: methodology, software, writing: original draft. KQ, YZ, WH, JY and XH: data curation, formal analysis, PW, YZ and JG: visualization. HL and ZW: validation, formal analysis. GL and SL: resources. HW: writing, review and editing. SL: writing, review, supervision. Declaration of Competing Interest Conflict of interest No conflicts of interest, financial or otherwise, are declared by the author(s). Ethical approval All animal procedures were performed in according to the protocol approved by the Animal Care and Use Committee of Shanxi Medical University. Financial support and potential conflict of interest none. References Leone RD, Powell JD (2020) Metabolism of immune cells in cancer. Nature Reviews Cancer 20:516-531. Gillies RJ, Raghunand N, Karczmar GS, et al. (2002) MRI of the tumor microenvironment. Journal of magnetic resonance imaging : JMRI 16:430-450. Cardone RA, Casavola V, Reshkin SJ (2005) The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nature reviews Cancer 5:786-795. Curtis NJ, Mooney L, Hopcroft L, et al. (2017) Pre-clinical pharmacology of AZD3965, a selective inhibitor of MCT1: DLBCL, NHL and Burkitt's lymphoma anti-tumor activity. Oncotarget 8:69219-69236. Halestrap AP (2012) The monocarboxylate transporter family-Structure and functional characterization. IUBMB Life 64:1-9. Bosshart PD, Charles RP, Garibsingh RA, et al. (2021) SLC16 Family: From Atomic Structure to Human Disease. Trends Biochem Sci 46:28-40. Aoi W, Marunaka Y (2014) Importance of pH homeostasis in metabolic health and diseases: crucial role of membrane proton transport. Biomed Res Int 2014:598986. Jackson V, Halestrap AJTJobc (1996) The kinetics, substrate, and inhibitor specificity of the monocarboxylate (lactate) transporter of rat liver cells determined using the fluorescent intracellular pH indicator, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. 271:861-868. Bosshart PD, Kalbermatter D, Bonetti S, et al. (2019) Mechanistic basis of L-lactate transport in the SLC16 solute carrier family. Nat Commun 10:2649. Bongarzone S, Barbon E, Ferocino A, et al. (2020) Imaging niacin trafficking with positron emission tomography reveals in vivo monocarboxylate transporter distribution. Nuclear Medicine and Biology 88-89:24-33. Grollman E, Philp N, McPhie P, et al. (2000) Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast. 39:9351-9357. Bonen A (2001) The expression of lactate transporters (MCT1 and MCT4) in heart and muscle. European journal of applied physiology 86:6-11. Ganapathy V, Thangaraju M, Prasad PD (2009) Nutrient transporters in cancer: relevance to Warburg hypothesis and beyond. Pharmacology & therapeutics 121:29-40. Faubert B, Li KY, Cai L, et al. (2017) Lactate Metabolism in Human Lung Tumors. Cell 171:358-371 e359. Sonveaux P, Végran F, Schroeder T, et al. (2008) Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. Journal of Clinical Investigation. Dimmer KS, Friedrich B, Lang F, et al. (2000) The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells. The Biochemical journal 350 Pt 1:219-227. Wang N, Jiang X, Zhang S, et al. (2021) Structural basis of human monocarboxylate transporter 1 inhibition by anti-cancer drug candidates. 184:370-383.e313. Becker HM, Deitmer JW (2020) Transport Metabolons and Acid/Base Balance in Tumor Cells. Cancers 12. Silva A, Antunes B, Batista A, et al. (2022) In Vivo Anticancer Activity of AZD3965: A Systematic Review. Molecules 27. Takenaga K, Koshikawa N, Akimoto M, et al. (2021) MCT4 is induced by metastasis-enhancing pathogenic mitochondrial NADH dehydrogenase gene mutations and can be a therapeutic target. Sci Rep 11:13302. Tateishi H, Tsuji AB, Kato K, et al. (2017) Synthesis and evaluation of 11C-labeled coumarin analog as an imaging probe for detecting monocarboxylate transporters expression. Bioorganic & Medicinal Chemistry Letters 27:4893-4897. Draoui N, Schicke O, Fernandes A, et al. (2013) Synthesis and pharmacological evaluation of carboxycoumarins as a new antitumor treatment targeting lactate transport in cancer cells. Bioorganic & Medicinal Chemistry 21:7107-7117. Sadeghzadeh M, Wenzel B, Gündel D, et al. (2020) Development of Novel Analogs of the Monocarboxylate Transporter Ligand FACH and Biological Validation of One Potential Radiotracer for Positron Emission Tomography (PET) Imaging. Molecules 25. Sattler B, Kranz M, Wenzel B, et al. (2020) Preclinical Incorporation Dosimetry of [18F]FACH—A Novel 18F-Labeled MCT1/MCT4 Lactate Transporter Inhibitor for Imaging Cancer Metabolism with PET. Molecules 25. Sadeghzadeh M, Moldovan R-P, Teodoro R, et al. (2019) One-step radiosynthesis of the MCTs imaging agent [18F]FACH by aliphatic 18F-labelling of a methylsulfonate precursor containing an unprotected carboxylic acid group. Scientific Reports 9. Gündel D, Sadeghzadeh M, Deuther-Conrad W, et al. (2021) Preclinical Evaluation of [18F]FACH in Healthy Mice and Piglets: An 18F-Labeled Ligand for Imaging of Monocarboxylate Transporters with PET. International Journal of Molecular Sciences 22. Sadeghzadeh M, Moldovan RP, Fischer S, et al. (2019) Development and radiosynthesis of the first 18F‐labeled inhibitor of monocarboxylate transporters (MCTs). Journal of Labelled Compounds and Radiopharmaceuticals 62:411-424. Kirat D, Inoue H, Iwano H, et al. (2005) Expression and distribution of monocarboxylate transporter 1 (MCT1) in the gastrointestinal tract of calves. Research in Veterinary Science 79:45-50. Gurrapu S, Jonnalagadda SK, Alam MA, et al. (2015) Monocarboxylate transporter 1 inhibitors as potential anticancer agents. ACS Med Chem Lett 6:558-561. Yu YJ, Zhang FL, Peng TY, et al. (2021) Sequential C-F bond functionalizations of trifluoroacetamides and acetates via spin-center shifts. Science 371:1232-1240. Odi R, Bibi D, Wager T, et al. (2020) A perspective on the physicochemical and biopharmaceutic properties of marketed antiseizure drugs—From phenobarbital to cenobamate and beyond. Epilepsia 61:1543-1552. Guan X, Rodriguez-Cruz V, Morris MJTAj (2019) Cellular Uptake of MCT1 Inhibitors AR-C155858 and AZD3965 and Their Effects on MCT-Mediated Transport of L-Lactate in Murine 4T1 Breast Tumor Cancer Cells. 21:13. Guan X, Bryniarski MA, Morris ME (2018) In Vitro and In Vivo Efficacy of the Monocarboxylate Transporter 1 Inhibitor AR-C155858 in the Murine 4T1 Breast Cancer Tumor Model. AAPS J 21:3. Sandforth L, Ammar N, Dinges LA, et al. (2020) Impact of the Monocarboxylate Transporter-1 (MCT1)-Mediated Cellular Import of Lactate on Stemness Properties of Human Pancreatic Adenocarcinoma Cells dagger. Cancers 12. Kim D, Ko HY, Chung JI, et al. (2023) Visualizing Cancer-Originating Acetate Uptake Through MCT1 in Reactive Astrocytes in the Glioblastoma Tumor Microenvironment. Neuro Oncol. Supplementary Files ElectronicSupplementaryMaterialESM.docx Cite Share Download PDF Status: Published Journal Publication published 11 Jun, 2025 Read the published version in Molecular Imaging and Biology → Version 1 posted Editorial decision: Major revisions 15 Jul, 2024 Reviewers agreed at journal 20 Jun, 2024 Reviewers invited by journal 10 Apr, 2024 Editor assigned by journal 07 Mar, 2024 First submitted to journal 06 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4020948","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289571121,"identity":"ecd2ad61-29e6-46cd-99e1-2935bb10de67","order_by":0,"name":"Dongmei Shi","email":"","orcid":"","institution":"First Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dongmei","middleName":"","lastName":"Shi","suffix":""},{"id":289571122,"identity":"74618fb9-7d26-4aa2-8a35-48aa54fb2346","order_by":1,"name":"Ling Liu","email":"","orcid":"","institution":"First Hospital of Shanxi Medical 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12:46:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4020948/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4020948/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11307-025-02024-1","type":"published","date":"2025-06-11T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54929272,"identity":"e7ff0d76-02d2-4175-8236-d3b0eda69dbe","added_by":"auto","created_at":"2024-04-18 17:47:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56933,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis routes of precursor compound \u003cstrong\u003e2\u003c/strong\u003e, \u003csup\u003e18\u003c/sup\u003eF-radiolabeled CHC probe [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC, [\u003csup\u003e19\u003c/sup\u003eF] \u003cstrong\u003e3\u003c/strong\u003e and the reference compound [\u003csup\u003e19\u003c/sup\u003eF]FEtO-CHC.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4020948/v1/ba700ea4d95fb3ba671f0a5f.png"},{"id":54928620,"identity":"56779307-de28-4c74-af3a-76f7a53cc8e7","added_by":"auto","created_at":"2024-04-18 17:39:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95347,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Plots of cell uptake results for BxPC3 and 4T1 cell lines; (\u003cstrong\u003eB\u003c/strong\u003e) bar graphs of cell inhibition results in BxPC3 and 4T1 cell lines after co-incubation with CHC for 30 min; (\u003cstrong\u003eC\u003c/strong\u003e) plots of cell efflux results for BxPC3 and 4T1 cell lines; (\u003cstrong\u003eD\u003c/strong\u003e) western blot for BxPC3 and 4T1 cell lines. p\u0026lt;0.001(***)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4020948/v1/8c1f5b4c88a1cecc023ce45d.png"},{"id":54928621,"identity":"fe59ff1e-7325-422a-8c18-6efb6df04a75","added_by":"auto","created_at":"2024-04-18 17:39:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58385,"visible":true,"origin":"","legend":"\u003cp\u003eThe biodistribution of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e in ICR mice\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4020948/v1/0a40d348d8215d3cadb6c916.png"},{"id":54928624,"identity":"8771f351-1b4d-4230-9c7a-0bb7a697b6de","added_by":"auto","created_at":"2024-04-18 17:39:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":480776,"visible":true,"origin":"","legend":"\u003cp\u003ePET/CT images (\u003cstrong\u003eA\u003c/strong\u003e) and time-activity curves of tumor-to-muscle (T/M) (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ein BxPC3 and 4T1 tumor-bearing mice after injection of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e; (\u003cstrong\u003eC\u003c/strong\u003e) PET/CT images and SUVmax values in BxPC3 tumor-bearing mice after co-injection of MCT1 inhibitor CHC and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e. Arrows represent the locations of the BxPC3 or 4T1 tumor.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4020948/v1/5d479b2be2ac44eb2f91fb18.png"},{"id":54928622,"identity":"9a3386c3-f1ef-4d08-a540-d5b2f4dd2f43","added_by":"auto","created_at":"2024-04-18 17:39:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":556782,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative images of MCT1 immunochemical staining of xenograft tumors and the left hind leg muscles in nude mice; (\u003cstrong\u003eB\u003c/strong\u003e) the positive scores of MCT1 immunochemical staining (n=3).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4020948/v1/eb64f9c207798c8a88fcd9a4.png"},{"id":84726606,"identity":"7436b07e-c6e3-4109-af48-3a964093d0d5","added_by":"auto","created_at":"2025-06-16 16:07:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2169605,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4020948/v1/39713b0b-4c06-4eb2-ac05-d3f8fe34557b.pdf"},{"id":54928625,"identity":"ea6a642e-f09c-49b4-b189-06112bd81af0","added_by":"auto","created_at":"2024-04-18 17:39:33","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":595629,"visible":true,"origin":"","legend":"","description":"","filename":"ElectronicSupplementaryMaterialESM.docx","url":"https://assets-eu.researchsquare.com/files/rs-4020948/v1/5479e350dcc57ec40d887e18.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eOne-step Radiosynthesis and Preclinical Evaluation of Molecular Probe [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC Targeting Monocarboxylate Transporters for PET Imaging in Tumor-bearing Mice\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer remains a formidable global health challenge, and targeting tumor metabolism for anti-tumor therapy represents a pivotal research strategy. Tumors exhibit the Warburg effect[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], resulting in excessive lactic acid production through glycolysis. To prevent intracellular accumulation, proton-coupled monocarboxylate transporters (MCTs) facilitate the extracellular transportation of lactic acid [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As integral membrane proteins [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], MCTs play crucial roles in diverse physiological processes such as lactic acid metabolism, energy supply, and pH regulation [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. They are widely expressed in normal tissues including kidney, heart, eye, liver, and retina [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, MCT1 and MCT4 have been identified in various human cancer cell lines and primary tumors, playing a crucial role in tumor metastasis and invasion[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], including breast cancer, head and neck cancer, lung cancer neuroblastoma, as well as brain and colon tumors[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Specifically, the MCT4 transporters predominantly facilitate the transportation of lactic acid generated through glycolysis in hypoxic regions within the tumor. These regions are distantly located from the tumor vascular area but serve to transport lactic acid towards extracellular spaces and oxygen-rich regions. Conversely, MCT1 primarily facilitates the uptake of produced lactic acid into cells for subsequent oxidation and energy provision in oxygen-rich areas [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. By effectively utilizing diverse energy substrates in different oxygen-containing regions through the transport mechanisms mediated by monocarboxylic acid transporters (MCTs), tumor tissues achieve their proliferation and invasion. The metabolic characteristics of tumors present an opportunity to impede tumor progression by selectively inhibiting MCTs, thereby establishing MCTs as a promising biological target relevant to tumors [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough the small molecule inhibitor AZD3965, which targets MCT1, has progressed to the clinical trial stage [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], only two MCT inhibitors have been developed as PET probes. One of them is a coumarin analogue labeled with \u003csup\u003e11\u003c/sup\u003eC; however, its short half-life and complex preparation process render it unsuitable for further clinical translation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The other probe is an α-cyano-cinnamic acid derivative labeled with \u003csup\u003e18\u003c/sup\u003eF ([\u003csup\u003e18\u003c/sup\u003eF]FACH), which has demonstrated radio-safety in pig studies and attempted to enhance blood-brain barrier permeability by increasing lipid solubility to improve probe uptake in intracranial tumors with high MCT expression. Nevertheless, no further research reports are available regarding this probe [\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, our team independently designed and synthesized a \u003csup\u003e18\u003c/sup\u003eF-radiolabelled derivative of α-cyano-4-hydroxycinnamic acid ([\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC), as a PET molecular probe, targeting MCTs. Subsequently, we evaluated its tumor imaging characteristics through in vitro and in vivo experiments as well as dynamic Micro-PET imaging.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1. General Equipment and Methods\u003c/h2\u003e\n\u003cp\u003eThe cyclotron (HM-10) was obtained from Sumitomo Corporation, Japan. The ultraflex MALDI-TOF/TOF instrument employed for mass analysis was provided by Bruker Corporation, Germany. Nuclear magnetic resonance spectroscopy was performed using the Bruker Avance III 400 MHz NMR spectrometer. High performance liquid chromatography (HPLC) was the Series III system from Lab Alliance, Tianjin Lab Alliance Company, equipped with dual detectors including the B-FC-3600 high-energy radioactivity detector from Bioscan, USA and the 201 UV detector from Tianjin Lab Alliance Company. CRC-25R radioactivity detector was purchased from Capintec, USA. A semi-prepared C18 column (5 \u0026micro;m, 250 mm\u0026times;10mm) manufactured by YMC in Japan was employed. Sep Pak QMA and HLB columns supplied by Waters, USA. Thin layer chromatography (TLC) scanner was equipped with a B-FC-3600 high-energy radioactivity detector from Bioscan, USA. The compound 4,7,13,16,21,24-hexaoxo-diazabicyclo[8.8.8]-tridecane (Kryptofix 2.2.2, K2.2.2) was obtained from ABX advanced biochemical compounds GmbH, Germany. Anhydrous acetonitrile (CH\u003csub\u003e3\u003c/sub\u003eCN), and potassium carbonate (K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e) were purchased from Aldrich, USA. All other reagents used were of analytical or chemical purity; experimental water was deionized purified water. BxPC3 pancreatic cancer cells and 4T1 breast cancer cells were procured from the Shanghai Cell Bank of Chinese Academy of Sciences.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2. Chemical Synthesis\u003c/h2\u003e\n\u003cp\u003eThe synthetic routes to (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-(2-(tosyloxy)ethoxy)phenyl)acrylate (precursor compound \u003cstrong\u003e2\u003c/strong\u003e), (\u003cem\u003eE\u003c/em\u003e)-2-cyano-3-(4-(2-[\u003csup\u003e18\u003c/sup\u003eF]-fluoroethoxy)phenyl)acrylic acid ([\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e), compound (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylate ([\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e3\u003c/strong\u003e) and the reference compound (\u003cem\u003eE\u003c/em\u003e)-2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylic acid ([\u003csup\u003e19\u003c/sup\u003eF]FEtO-CHC, [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e), are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n\u003ch2\u003e2.2.1. Preparation of (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-(2-(tosyloxy)ethoxy)phenyl)acrylate (compound 2)\u003c/h2\u003e\n\u003cp\u003eTo a solution of (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate (compound \u003cstrong\u003e1\u003c/strong\u003e) (200 mg, 0.92 mmol) in dry CH\u003csub\u003e3\u003c/sub\u003eCN (40mL) was added 18-crown-6 (48.71 mg, 0.18 mmol) and anhydrous K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (254.68 mg, 1.84 mmol), ethylene glycol di-p-toluene sulfonate (511.42 mg, 1.38 mmol) was then added into the reaction mixture after being stirred for 20 min and the resulting mixture was refluxed at 90\u0026deg;C for about 13 hours. The reaction progress was monitored by TLC. The solvent was removed under reduced pressure and distilled water (50mL) was added. The mixture was extracted with CH\u003csub\u003e3\u003c/sub\u003eCN (4\u0026times;50mL). The combined CH\u003csub\u003e3\u003c/sub\u003eCN extracts were dried with Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate/petroleum ether (1/4, v/v) as the eluent to afford compound \u003cstrong\u003e2\u003c/strong\u003e (98.44 mg, 49.22%). High resolution MS: [M\u0026thinsp;+\u0026thinsp;Na] for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eNO\u003csub\u003e6\u003c/sub\u003eSNa, calculated, 438.11, found, 438.096 (Suppl. Figure\u0026nbsp;1A). \u003csup\u003e1\u003c/sup\u003eH NMR(400MHz, Suppl. Figure\u0026nbsp;1D): \u0026delta; 8.32 (s, 1H), 8.06 (d, J\u0026thinsp;=\u0026thinsp;9.0 Hz, 2H), 7.79 (d, J\u0026thinsp;=\u0026thinsp;8.3 Hz, 2H), 7.47 (d, J\u0026thinsp;=\u0026thinsp;8.1 Hz, 2H), 7.05 (d, J\u0026thinsp;=\u0026thinsp;9.0 Hz, 2H), 4.36 (d, J\u0026thinsp;=\u0026thinsp;1.5 Hz, 2H), 4.35\u0026ndash;4.27 (m, 4H), 2.41 (s, 3H), 1.30 (t, J\u0026thinsp;=\u0026thinsp;7.1 Hz, 3H).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n\u003ch2\u003e2.2.2. Preparation of (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylate ([\u003csup\u003e19\u003c/sup\u003eF]3)\u003c/h2\u003e\n\u003cp\u003eTo a solution of (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate (compound \u003cstrong\u003e1\u003c/strong\u003e) (530mg, 2.44mmol) in anhydrous CH\u003csub\u003e3\u003c/sub\u003eCN (40 mL) was added 18-crown-6 (67.14 mg, 0.25 mmol) and anhydrous K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (351 mg, 2.54mmol), fluoroethyl p-toluene sulfonate (830 mg, 3.81 mmol) was added into the reaction mixture after being stirred for 20 min and the resulting mixture was refluxed at 90\u0026deg;C for about 13 hours. The reaction progress was monitored by TLC. After removal of the solvent via reduced pressure, distilled water (50 mL) was added and the mixture was extracted using CH\u003csub\u003e3\u003c/sub\u003eCN (4\u0026times;50 mL). The combined CH\u003csub\u003e3\u003c/sub\u003eCN extracts were dried with Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate/petroleum ether (1/2, v/v) as the eluent to afford [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e3\u003c/strong\u003e (325.85mg, 61.48%). High resolution MS: M for C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eFNO\u003csub\u003e3,\u003c/sub\u003e calculated, 263.1, found, 263.086(Suppl. Figure\u0026nbsp;1B). \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, Suppl. Figure\u0026nbsp;1E): \u0026delta; 8.33 (s, 1H), 8.10 (d, J\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 7.20 (d, J\u0026thinsp;=\u0026thinsp;8.7 Hz, 2H), 4.84 (dd, J\u0026thinsp;=\u0026thinsp;4.6, 3.0 Hz, 1H), 4.72 (dd, J\u0026thinsp;=\u0026thinsp;4.6, 2.9 Hz, 1H), 4.45\u0026ndash;4.39 (m, 1H), 4.38\u0026ndash;4.26 (m, 3H), 1.30 (td, J\u0026thinsp;=\u0026thinsp;7.1, 1.1 Hz, 3H).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n\u003ch2\u003e2.2.3. Preparation of (\u003cem\u003eE\u003c/em\u003e)-2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylic acid ([\u003csup\u003e19\u003c/sup\u003eF]FEtO-CHC, [\u003csup\u003e19\u003c/sup\u003eF]4)\u003c/h2\u003e\n\u003cp\u003eTo a solution of [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e3\u003c/strong\u003e in CH\u003csub\u003e3\u003c/sub\u003eCN (1 mL) was added 2N NaOH for being stirred 5 min at room temperature. The solvent was removed under reduced pressure and obtained the reference compound [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e as a white solid. High resolution MS: [M\u0026thinsp;+\u0026thinsp;Na] for C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eFNO\u003csub\u003e3\u003c/sub\u003eNa, calculated, 258.06, found, 258.045(Suppl. Figure\u0026nbsp;1C).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3. Radiosynthesis\u003c/h2\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC was synthesized in one-step procedure involving nucleophilic substitution reaction and deprotection reaction. No-carrier-added [\u003csup\u003e18\u003c/sup\u003eF] fluoride was trapped using 1.5 mL of H\u003csub\u003e2\u003c/sub\u003eO on a Sep Pak light QMA cartridge. NaHCO\u003csub\u003e3\u003c/sub\u003e (10 mL, 0.5 M) and H\u003csub\u003e2\u003c/sub\u003eO (10 mL) were used for cartridge preconditioning. The activity was eluted into the reaction flask using 1 mL of an aqueous solution of K2.2.2/K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (5.91 mg of K2.2.2 and 1.36 mg of K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e in CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO 10/1, v/v). The reaction flask was evaporated at 110℃ under reduced pressure. A solution of compound \u003cstrong\u003e2\u003c/strong\u003e (4 mg) in 1 mL of CH\u003csub\u003e3\u003c/sub\u003eCN was added, sealed and heated at 110\u0026deg;C for 15 min. Semi-preparative HPLC was performed on C18-column (5 \u0026micro;m, 250\u0026times;100 mm) using CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO as the eluent at a flow rate of 3 mL/min (CH\u003csub\u003e3\u003c/sub\u003eCN: 55%) with UV detection at 254 nm. [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC was collected at a retention time of 12-14min into a distillation flask. The collected solution was evaporated under reduced pressure and diluted with 2mL water and passed through a 0.22 \u0026micro;m filter to obtain a solution of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC. TLC analysis was used for the analysis of radiochemistry of the probe using 85% CH\u003csub\u003e3\u003c/sub\u003eCN as eluent. The analysis of the reference compound and [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC was used HPLC (C18 chromatographic column, CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;=\u0026thinsp;70/30, v\u0026thinsp;=\u0026thinsp;3 mL/min, UV\u0026thinsp;=\u0026thinsp;254 nm).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4. In Vitro and Vivo Stability\u003c/h2\u003e\n\u003cp\u003eThe in vitro stability of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC was tested in phosphate-buffered saline (PBS) and fetal bovine serum (FBS) at 25℃ for 60 min and 120 min incubation. The in vivo stability was using blood and urine samples from a healthy male SD rat at 60 min post-injection of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC (0.5mL, 8MBq). All the samples were analyzed by HPLC (C18 chromatographic column, CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;=\u0026thinsp;55/45, v\u0026thinsp;=\u0026thinsp;3 mL/min, UV\u0026thinsp;=\u0026thinsp;254 nm) or TLC under the same condition used in the above.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003e2.5. Lipophilicity\u003c/h2\u003e\n\u003cp\u003eThe partition coefficient of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC was detected in the solution of n-octanol/saline (logP) or n-octanol/PBS (logD7.4) at room temperature with conventional shake-flask method. Approximately 500 kBq of the probe was added to a centrifuge tube containing 3 mL of n-octanol/ saline or \u003cem\u003en\u003c/em\u003e-octanol/PBS (1:1, v:v) and shaken for 20 min to ensure complete mixing. The solution was then centrifuged at 5000 rpm for 5 min. Both aliquots of 1 mL of the organic phase and the inorganic phase were respectively taken to measure gamma counts.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e2.6. Cell Culture and Animal Models\u003c/h2\u003e\n\u003cp\u003eThe BxPC3 and 4T1 cell lines were cultivated respectively in RPMI-1640 and DMEM supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/ streptomycin under humidified conditions (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e/95% air).\u003c/p\u003e\n\u003cp\u003eThe animal experimental procedures were conducted in accordance with the approved animal care and use guidelines by the ethics committee of Shanxi Medical University. BxPC3 human pancreatic cancer cells and 4T1 breast cancer cells in logarithmic growth phase were collected, followed by the preparation of a single cell suspension using PBS. After adjusting the cell concentration to 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e/mL, subcutaneous injection of 0.1mL was performed into the right shoulder of BALB/c nude male mice to process tumor xenografts. The mice were utilized when the tumor diameter reached 6\u0026ndash;10 mm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e2.7. In Vitro Cell Assays\u003c/h2\u003e\n\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n\u003ch2\u003e2.7.1. In vitro cell uptake\u003c/h2\u003e\n\u003cp\u003eBxPC3 pancreatic cancer cells and 4T1 breast cancer cells in logarithmic growth phase were incubated in 24-well plates for 24 hours (at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well). The cell samples were divided into six groups, with four replicate wells per group. Subsequently, the medium was discarded, and the cells were washed twice with warm PBS. Novel probes (0.3 MBq, 200 \u0026micro;L/well) were added to each well and incubated for different time intervals of 2, 5, 15, 30, 60, and 120 min respectively. After that, the probes were removed, and the cells were washed twice with ice-cold PBS followed by cleavage using a solution of 1N NaOH (0.5 mL/well) for duration of 20 min. The resulting cleavage liquid was collected, and its radioactivity was measured using a \u0026gamma;-counter. The obtained data underwent standardization procedures and recorded as percentages of radioactivity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n\u003ch2\u003e2.7.2. In vitro cell inhibition\u003c/h2\u003e\n\u003cp\u003eThe initial treatment was conducted in the same manner as that for cell uptake. Subsequently, each well was administered 200 \u0026micro;L of MCT1 inhibitor CHC solution (8 mg CHC dissolved in 200 \u0026micro;L DMSO and diluted to 6 mL with complete medium) along with probes (0.3 MBq, 200 \u0026micro;L/well) and incubated for 30 minutes for four replicate wells. Following this, the inhibitors and probes were discarded, and the wells were washed twice with ice-cold PBS before cleavage using 1N NaOH (0.5 mL/well) for 20 minutes. The subsequent treatments and calculations followed the same protocol as the uptake experiment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n\u003ch2\u003e2.7.3. In vitro cell efflux\u003c/h2\u003e\n\u003cp\u003eThe initial treatment was identical to that of the cell uptake. Following the addition of the radio probes (0.3MBq, 200\u0026micro;L/well) to each well, they were incubated at 37\u0026deg;C for 1 hour with four replicate wells per group. Subsequently, the radioactive liquid was discarded, and warm PBS was used for two washes. Then, 0.5 mL medium was added and co-cultured at 37\u0026deg;C for time intervals of 5, 15, 30, 60, 90 and 120 min. After discarding the supernatant and washing twice with PBS, cleavage was performed using 1N NaOH (0.5mL/well) for 20 minutes. The subsequent treatments and calculations followed the same protocol as the uptake experiment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n\u003ch2\u003e2.7.4. Western Blotting\u003c/h2\u003e\n\u003cp\u003eThe cells were lysed with radioimmunoprecipitation assay lysis buffer (ServiceBio) containing protease inhibitor (ServiceBio), and 6 \u0026micro;g of proteins were separated by electrophoresis in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (ServiceBio).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e2.8. Biodistribution\u003c/h2\u003e\n\u003cp\u003eEighteen healthy ICR mice (25\u0026thinsp;~\u0026thinsp;30 g, male, clean) were provided by the Shanxi Medical University Experimental Animal Center and divided into six groups with three mice in each group. The mice were intravenously injected with [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC (200 \u0026micro;L, 4 MBq) solution via the tail vein. At time points of 5, 15, 30, 60, 90 and 120 min post-injection, blood samples and major organs were collected and weighed. The radioactivity was measured using a \u0026gamma;-counter and the percentage injection dose rate (%ID/g) after attenuation correction was calculated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e2.9. Micro-PET/CT Imaging\u003c/h2\u003e\n\u003cp\u003eThe tumor-bearing nude male mice were placed on the micro-PET/CT scanning bed, which was maintained at a constant temperature of 37\u0026deg;C, following gas induction anesthesia (2.0% isoflurane air). In the experimental group, [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC (0.2 mL, 4 MBq) was injected via the tail vein and dynamic Micro-PET imaging was performed for a duration of 2 hours. In the inhibition group, MCT1 inhibitor CHC (0.2 mL, 1.6 mg) was injected via the tail vein followed by injection of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC (0.2 mL, 4 MBq) for 2h-dynamic Micro-PET imaging. For CT image scanning, tube voltage of 80 kV and tube current of 1 mA were used with an exposure time of 80 s. A layer thickness of 160 \u0026micro;m was selected for pretreatment and reconstruction utilized the Feldkamp filter back projection algorithm. The PMOD software facilitated image analysis to delineate volumes-of-interest (VOI) corresponding to mouse tumors and left hind leg muscles; SUVmean values were quantitatively calculated. The ratio between tumor and muscle uptake recorded as T/M.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e2.10. Immunohistochemistry\u003c/h2\u003e\n\u003cp\u003eThe primary antibody used was a polyclonal MCT1 antibody (Proteintech, China), and secondary antibody was a horseradish peroxidase-conjugated goat anti-rabbit antibody (ServiceBio, China). Tumor and muscle tissues were fixed in 10% formalin solution, embedded, and sectioned. Subsequently, the sections were baked at 60\u0026deg;C followed by dewaxing and hydration steps. Antigen retrieval was performed using citrate repair solution with pH\u0026thinsp;=\u0026thinsp;6.0 in an autoclave. To block endogenous peroxidase activity, 3% hydrogen peroxide was incubated at room temperature for 25 min. Non-specific background reactions were blocked by using 3% BSA (bovine serum albumin) at room temperature for 90 min. The primary antibody was then added and incubated overnight at 4\u0026deg;C in a humidified chamber. Afterward, the sections were incubated with the secondary antibody at room temperature for 50 min. Hematoxylin was used to stain nucleus, dehydrate and seal the cells for microscopy observation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003e2.11. Statistical Analysis\u003c/h2\u003e\n\u003cp\u003eAll statistical analysis was conducted using IBM SPSS 26.0 software. Data visualization was processed using GraphPad Prism 8. The data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and using independent sample t-test to assess statistical significance. P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. The cell experiments were independently repeated three times on different dates.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1. Chemistry and Radiochemistry\u003c/h2\u003e\n\u003cp\u003eThe precursor compound \u003cstrong\u003e2\u003c/strong\u003e was synthesized through the reaction between (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate (compound \u003cstrong\u003e1\u003c/strong\u003e) and ethylene glycol di-p-toluene sulfonate, and confirmed by mass spectrometry and \u003csup\u003e1\u003c/sup\u003eH NMR (Suppl. Figure\u0026nbsp;1A, D). The compound (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-(2-fluoroethoxy)phenyl)acrylate ([\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e3\u003c/strong\u003e) was synthesized by reacting (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate (compound \u003cstrong\u003e1\u003c/strong\u003e) with fluoroethyl p-toluene sulfonate, and was confirmed with mass spectrometry and \u003csup\u003e1\u003c/sup\u003eH NMR (Suppl. Figure\u0026nbsp;1B, E). The reference compound [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e was obtained by (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-(2-fluoroethoxy) phenyl) acrylate ([\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e3\u003c/strong\u003e) after hydrolytic reaction and confirmed by mass spectrometry (Suppl. Figure\u0026nbsp;1C).\u003c/p\u003e\n\u003cp\u003eThe preparation of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e involved a nucleophilic substitution reaction with [\u003csup\u003e18\u003c/sup\u003eF]\u003csup\u003e\u0026minus;\u003c/sup\u003e and deprotection in one-step procedure to give a retention time of 10-12min in semi-preparative HPLC (CH\u003csub\u003e3\u003c/sub\u003eCN /H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;=\u0026thinsp;55/45). The retention time (Rt) of [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e3\u003c/strong\u003e in the UV spectrum of HPLC (CH\u003csub\u003e3\u003c/sub\u003eCN /H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;=\u0026thinsp;70/30) was determined to be 8.9 min, and the Rt of the reference compound [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e in the UV spectrum was 5.6 min. Comparatively, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e can be verified with the same retention time (Rt) as [\u003csup\u003e19\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e under the same analytical HPLC method conditions (CH\u003csub\u003e3\u003c/sub\u003eCN /H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;=\u0026thinsp;70/30, Suppl. Figure\u0026nbsp;2A). Furthermore, after radiochemical reaction, following removal of [\u003csup\u003e18\u003c/sup\u003eF]\u003csup\u003e\u0026minus;\u003c/sup\u003e using a C18 cartridge, two radioactive product peaks were observed at 5.7 min and 9.1 min by HPLC analysis (CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;=\u0026thinsp;70/30), while the proportion of the former product peak gradually increased with increasing the reaction temperature and time from 100\u0026deg;C for 10 min to 110\u0026deg;C for 15 min (Suppl. Figure\u0026nbsp;2A). After adding 50 \u0026micro;L of 2N NaOH into this radioactive solution for hydrolysis at room temperature for 5 min, only a single radioactive peak was observed at 5.7 min. Hence, under the reaction conditions of 110\u0026deg;C for 15 min and using CH\u003csub\u003e3\u003c/sub\u003eCN as the solvent, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e was synthesized in a one-step reaction with a total time of approximately 60 min. The radiochemical yield was determined to be 52.08\u0026thinsp;\u0026plusmn;\u0026thinsp;6.74% (n\u0026thinsp;=\u0026thinsp;7, decay corrected), while the radiochemical purity was \u0026gt;\u0026thinsp;97% in TLC analytical results (Suppl. Figure\u0026nbsp;2B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2. In Vitro and Vivo Stability\u003c/h2\u003e\n\u003cp\u003eThe in vitro stability of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e in PBS and FBS at room temperature within 120 min using HPLC (CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;=\u0026thinsp;55/45) exhibited a purity of \u0026gt;\u0026thinsp;95% (Suppl. Figure\u0026nbsp;3A), which indicated no degradation occurred in vitro. For the in vivo stability in a rat at 60 min postinjection, HPLC analysis revealed the decomposition product of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e was mainly retained in the urine (Suppl. Figure\u0026nbsp;3A) and TLC analysis of the blood indicated that [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e stayed the predominant compound with a purity of about 91% (Suppl. Figure\u0026nbsp;3B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3. Lipophilicity\u003c/h2\u003e\n\u003cp\u003eThe lipophilicity of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC was determined by conventional shake-flask method, the logP or logD7.4 value of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC was calculated to be 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 or 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4. In Vitro Cell Assays\u003c/h2\u003e\n\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n\u003ch2\u003e3.4.1. In vitro cell uptake\u003c/h2\u003e\n\u003cp\u003eTo explore the uptake characteristics of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e, BxPC3 pancreatic cancer cells and 4T1 breast cancer cells were selected for the uptake experiment. The uptake of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e by BxPC3 pancreatic cancer cells exhibited a rapid initial increase within the first 5 min, reaching a peak value of 0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.074% at 15 min, followed by a gradual decrease. Similarly, in 4T1 breast cancer cells, the uptake reached 0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04% at 15 min and peaked at 0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06% after 60 min before gradually declining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n\u003ch2\u003e3.4.2. In vitro cell inhibition\u003c/h2\u003e\n\u003cp\u003eTo ascertain the target specificity of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e, the specific MCT1 inhibitor CHC was used as a competitive inhibitor. After adding CHC, the uptake of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e was decreased by 66.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98% and 33.88\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51% in BxPC3 and 4T1 cell lines respectively, verifying CHC significantly blocked the uptake of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\n\u003ch2\u003e3.4.3. In vitro cell efflux\u003c/h2\u003e\n\u003cp\u003eTo assess the stability of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e binding to the target, a cell efflux experiment was conducted following incubation of BxPC3 pancreatic cancer cells and 4T1 breast cancer cells with the [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e probe for 1 hour. The combined probe exhibited gradual and steady release within 2 hours (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\n\u003ch2\u003e3.4.4. Western Blotting\u003c/h2\u003e\n\u003cp\u003eThe image of western blot was illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD. The ratio of integrated density val of MCT1/\u0026beta;-actin calculated from AIWBwell\u0026trade; was 2.93 and 2.30 in 4T1 and BxPC3 cell lines respectively, suggesting the expression of MCT1 in 4T1 cell line was higher than that in BxPC3.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5. Biodistribution\u003c/h2\u003e\n\u003cp\u003eThe biodistribution of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC in healthy male mice is illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. As the primary metabolic organs, the liver and kidneys exhibited significant radioactivity accumulation, which gradually decreased over time. The bone consistently displayed elevated levels of radioactivity throughout each period. The heart, muscle, spleen, and lung initially demonstrated high radioactivity at 5 min but then followed a gradual decline. In contrast, the gastrointestinal tract exhibited a progressive increase in radioactivity with time, particularly in small bowel where it reached its peak 6.06%ID/g at 120 min, which may be resulted from the high expression of MCT1 in gastrointestinal tract as main absorption organs of short-chain fatty acid [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Brain uptake peaked 0.98%ID/g at 5 min and then rapidly declined, which may originate from the probe\u0026rsquo;s transport mechanism of blood-brain barrier (BBB) correlated with lipophilicity. Furthermore, blood radioactivity gradually decreased and reached 0.65%ID/g after 15 min, which was lower than half of its initial value of 1.91%ID/g, confirming its rapid clearance rate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6. Micro-PET/CT Imaging\u003c/h2\u003e\n\u003cp\u003eThe 2h-dynamic micro-PET imaging results of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e in BxPC3 pancreatic cancer and 4T1 breast cancer tumor-bearing mice are depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eAfter intravenous administration of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e, whole-body organ perfusion imaging was performed in both BxPC3 and 4T1 mice. The uptake of liver and kidney showed a slight decrease over time, while the uptake of bladder gradually increased, which indicated that both the liver and kidney were the metabolic organs of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e. The radioactive uptake in other organs and tissues such as heart, lung, and muscle are quite low and comparable to background levels. Consistent with the forementioned results in biodistribution (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), there was a gradual accumulation of radioactivity in the gastrointestinal tract and axial bone. The BxPC3 pancreatic cancer tumor-bearing mice exhibited peak tumor-to-muscle ratio approximately 40 min after [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003e4\u003c/strong\u003e injection, followed by a gradual decline in uptake. And the 4T1 breast cancer tumor displayed a similar tumor-to-muscle ratio as the BxPC3 pancreatic cancer tumor within the initial 40 min but demonstrated slow radioactive accumulation subsequently (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). These uptake trends in BxPC3 and 4T1 tumor showed no difference with cell uptake assay.\u003c/p\u003e\n\u003cp\u003eAfter the administration of MCT1 inhibitor CHC, a significant reduction in tumor uptake was observed in BxPC3 tumor-bearing mice within the first 5 minutes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC), confirming that our probe's uptake can be inhibited by CHC, and the probe potentially targets MCT1 specifically in vivo.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7. Immunohistochemistry\u003c/h2\u003e\n\u003cp\u003eTo investigate whether the [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC-uptake was consistent with the levels of MCT1 expression in forementioned in vitro and in vivo assays, immunohistochemistry staining and half-quantitative analysis were conducted. The representative images of MCT1 in tumor xenografts and left hind leg muscles of nude mice are depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. Both the image (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA) and the positive scores (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB) indicated a low MCT1 expression level in muscles and a high expression level in both tumor xenografts, which was consistent with the uptake results in these two kinds of BxPC3 and 4T1 cell lines (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), the micro-PET image in tumor-bearing mice and the curves of T/M in image analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussions","content":"\u003cp\u003eMCT1 is a critical biological target for glycolysis of tumor. Several MCT1-targeting radiotracers have been developed in recent years [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, one series of MCT1-targeting probe [\u003csup\u003e18\u003c/sup\u003eF]FACH is still not evaluated in tumor PET imaging [\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]; another \u003csup\u003e11\u003c/sup\u003eC-labeld coumarin analog probe was restricted for further application with its low radioactive tumor accumulation and rapid clearance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Herein, we designed and synthesized a novel MCTs targeting PET probe for tumor-bearing mice imaging and evaluated it in vitro and in vivo.\u003c/p\u003e \u003cp\u003eBased on the structure of MCTs inhibitor α-cyano-4-hydroxycinnamic acid (CHC) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], α-cyano-4-hydroxyethyl cinnamate was directly chosen as the lead compound in this study, and the precursor compound α-cyano-4-(2-p-toluenesulfonyl ethoxy)-ethyl cinnamate (compound \u003cb\u003e2\u003c/b\u003e) was obtained through a one-step chemical reaction [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Intriguingly, we discovered our radiotracer could be obtained in one-step procedure using the precursor compound \u003cb\u003e2\u003c/b\u003e under 110℃ for 15 min with the chemical yield of 52.08\u0026thinsp;\u0026plusmn;\u0026thinsp;6.74% (n\u0026thinsp;=\u0026thinsp;7, decay corrected), indicating the hydrolysis of ester group in the nucleophilic substitution process. Additionally, the elevation of reaction temperature and time can effectively increase the yield of the target tracer by HPLC analysis. Although the report of one-step radiosynthesis of [\u003csup\u003e18\u003c/sup\u003eF]FACH using CHC analog without the protection of carboxylic acid group have been published \u003csup\u003e[25]\u003c/sup\u003e, to the aim of simplifying radiolabeling steps and increasing the yield, their precursor need to be prepared for unprotection in the chemical synthesis period. Compared with that, our synthesis of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC demonstrates the benefits of streamlining chemical and radiolabeling processes, while maintaining a high yield in radiochemical production. However, the phenomenon of hydrolysis during the nucleophilic substitution procedure lacks relevant literature reports, necessitating further exploration of its specific mechanism.\u003c/p\u003e \u003cp\u003eThe stability studies conducted in vivo and in vitro revealed a certain level of degradation in the urine of SD rats with a 60 min post-intravenous administration of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC, which is a common occurrence in many other fluorine-labeled organic compounds due to the inherent instability of the C-F bond [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, the lipophilicity measurement for [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC obtained a logD7.4 value of 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (n\u0026thinsp;=\u0026thinsp;3), demonstrating its restricted liposolubility. By calculating logD7.4, we can give a valuable estimate for the absorption and distribution of drugs within the body, while also identifying the capacity of drug transportation across the blood-brain barrier (BBB) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], providing us with guidance for subsequent cell experiments and tumor imaging.\u003c/p\u003e \u003cp\u003eGiven the 4T1 breast cancer cell line has been used in the study of AZD3965 and AR-C155858 inhibitors and is potently inhibited by CHC [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and the slight overexpression of MCT1 in BxPC3 pancreatic cancer cell line [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we applied 4T1 and BxPC3 cell lines for our evaluation. The cellular uptake analysis revealed that both BxPC3 and 4T1 cell lines exhibited a similar peak uptake at 15 min, following which the uptake in 4T1 cells continued to increase while BxPC3 cells showed a gradual decrease, matched with the distinct expression levels of MCT1 protein in these two kinds of cell lines from western blot results. Significant uptake inhibition was observed both in vitro and in vivo when using CHC as a competitive MCT1 inhibitor, confirming the specific targeting capability of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC towards MCT1. The cellular efflux experiment demonstrated a high and similar efflux rate in BxPC3 and 4T1 cells, suggesting its short retention time in tumors. The biodistribution results were consistent with the 2h-dynamic Micro-PET/CT imaging findings in a mouse tumor-bearing model as well. The liver and kidneys exhibited predominant metabolic activity for [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC and significant radioactive accumulation was observed in bladder, confirming the metabolic organs of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC in both liver and kidneys. In vivo dynamic Micro-PET imaging demonstrated that the tumor-to-muscle (T/M) values in both BxPC3 and 4T1 tumor-bearing mice reached approximately 1.5 at 40 min, after which the T/M gradually decreased for BxPC3 but continued to rise slowly for 4T1, consistent with the findings from the in vitro uptake experiment. Immunohistochemical analysis further confirmed the higher expression of MCTs in both BxPC3 and 4T1 tumor tissue compared to that observed in muscle. These results collectively indicate that probe uptake, both in vitro and in vivo, is associated with MCT1 expression levels. Although the uptake of our radiotracer is relatively low in tumor due to its rapid clearance, we overcome the high plasma binding rate of \u003csup\u003e11\u003c/sup\u003eC-labeld coumarin analog probe and enhanced the retention capacity in tumors within 2h [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, it is the first time to give a comprehensive evaluation for novel MCTs targeting probes in vitro tumor cell lines and in vivo tumor-bearing mice. To enhance the radioactive accumulation in MCT1-positive tumors, we will optimize its structural design from improving the retention in u and slowing the rate of clearance from blood. Meanwhile, we will focus on further exploration of its potential applications in diagnosing and treating specific tumors.\u003c/p\u003e \u003cp\u003eThe prospects for tumor therapy targeting MCTs have been promising in recent years. AZD3965 are in advanced clinical trial, demonstrating excellent efficacy against lymphoma [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The probe targeting MCT not only enables the early non-invasive diagnosis of tumors, but also holds the promise of screening MCT-positive tumors and monitoring the therapeutic efficacy of MCT- targeting therapy. In addition, owing to the study of monocarboxylate transports and glycolysis mechanism is still in progress, exploring the mechanism in diversities of diseases for MCT-dependent tumors and non-tumors such as glioblastoma, Alzheimer's disease and multiple sclerosis are promising [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which may provide an effective technical tool to explore the transport mechanism of monocarboxylic acid.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe self-designed PET molecular tracer [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC ([\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003e4\u003c/b\u003e), [\u003csup\u003e18\u003c/sup\u003eF]-labeled CHC derivative targeting MCTs, can be obtained through a one-step chemical reaction. Manual labeling synthesis takes approximately 60 min, yielding a radiochemical yield of 52.08\u0026thinsp;\u0026plusmn;\u0026thinsp;6.74% (n\u0026thinsp;=\u0026thinsp;7, attenuation correction), with purity exceeding 97%. The tumor imaging ability and targeting for MCT1 have been validated through in vitro and in vivo experiments, establishing a crucial molecular imaging method to assess metabolic intervention diagnosis and therapy based on lactate utilization for both tumors and non-tumors in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation (Nos. 81471695, 81971655, 82027804, 82001873). The research was also supported by the Four \u0026ldquo;Batches\u0026rdquo; Innovation Project of invigorating Medical through Science and Technology of Shanxi Province (No. 2022XM38), Central leading local science and Technology Development Fund Project (No. YDZJSX2022A058) and supported by Fundamental Research Program of Shanxi Province (No. 202303021221226).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorresponding authors: HW, ZW and SL. DS and LL: methodology, software, writing: original draft. KQ, YZ, WH, JY and XH: data curation, formal analysis, PW, YZ and JG: visualization. HL and ZW: validation, formal analysis. GL and SL: resources. HW: writing, review and editing. SL: writing, review, supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflicts of interest, financial or otherwise, are declared by the author(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were performed in according to the protocol approved by the Animal Care and Use Committee of Shanxi Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial support and potential conflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enone.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeone RD, Powell JD (2020) Metabolism of immune cells in cancer. Nature Reviews Cancer 20:516-531.\u003c/li\u003e\n\u003cli\u003eGillies RJ, Raghunand N, Karczmar GS, et al. (2002) MRI of the tumor microenvironment. Journal of magnetic resonance imaging : JMRI 16:430-450.\u003c/li\u003e\n\u003cli\u003eCardone RA, Casavola V, Reshkin SJ (2005) The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nature reviews Cancer 5:786-795.\u003c/li\u003e\n\u003cli\u003eCurtis NJ, Mooney L, Hopcroft L, et al. (2017) Pre-clinical pharmacology of AZD3965, a selective inhibitor of MCT1: DLBCL, NHL and Burkitt\u0026apos;s lymphoma anti-tumor activity. Oncotarget 8:69219-69236.\u003c/li\u003e\n\u003cli\u003eHalestrap AP (2012) The monocarboxylate transporter family-Structure and functional characterization. IUBMB Life 64:1-9.\u003c/li\u003e\n\u003cli\u003eBosshart PD, Charles RP, Garibsingh RA, et al. (2021) SLC16 Family: From Atomic Structure to Human Disease. Trends Biochem Sci 46:28-40.\u003c/li\u003e\n\u003cli\u003eAoi W, Marunaka Y (2014) Importance of pH homeostasis in metabolic health and diseases: crucial role of membrane proton transport. Biomed Res Int 2014:598986.\u003c/li\u003e\n\u003cli\u003eJackson V, Halestrap AJTJobc (1996) The kinetics, substrate, and inhibitor specificity of the monocarboxylate (lactate) transporter of rat liver cells determined using the fluorescent intracellular pH indicator, 2\u0026apos;,7\u0026apos;-bis(carboxyethyl)-5(6)-carboxyfluorescein. 271:861-868.\u003c/li\u003e\n\u003cli\u003eBosshart PD, Kalbermatter D, Bonetti S, et al. (2019) Mechanistic basis of L-lactate transport in the SLC16 solute carrier family. Nat Commun 10:2649.\u003c/li\u003e\n\u003cli\u003eBongarzone S, Barbon E, Ferocino A, et al. (2020) Imaging niacin trafficking with positron emission tomography reveals in vivo monocarboxylate transporter distribution. Nuclear Medicine and Biology 88-89:24-33.\u003c/li\u003e\n\u003cli\u003eGrollman E, Philp N, McPhie P, et al. (2000) Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast. 39:9351-9357.\u003c/li\u003e\n\u003cli\u003eBonen A (2001) The expression of lactate transporters (MCT1 and MCT4) in heart and muscle. European journal of applied physiology 86:6-11.\u003c/li\u003e\n\u003cli\u003eGanapathy V, Thangaraju M, Prasad PD (2009) Nutrient transporters in cancer: relevance to Warburg hypothesis and beyond. Pharmacology \u0026amp; therapeutics 121:29-40.\u003c/li\u003e\n\u003cli\u003eFaubert B, Li KY, Cai L, et al. (2017) Lactate Metabolism in Human Lung Tumors. Cell 171:358-371 e359.\u003c/li\u003e\n\u003cli\u003eSonveaux P, V\u0026eacute;gran F, Schroeder T, et al. (2008) Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. Journal of Clinical Investigation.\u003c/li\u003e\n\u003cli\u003eDimmer KS, Friedrich B, Lang F, et al. (2000) The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells. The Biochemical journal 350 Pt 1:219-227.\u003c/li\u003e\n\u003cli\u003eWang N, Jiang X, Zhang S, et al. (2021) Structural basis of human monocarboxylate transporter 1 inhibition by anti-cancer drug candidates. 184:370-383.e313.\u003c/li\u003e\n\u003cli\u003eBecker HM, Deitmer JW (2020) Transport Metabolons and Acid/Base Balance in Tumor Cells. Cancers 12.\u003c/li\u003e\n\u003cli\u003eSilva A, Antunes B, Batista A, et al. (2022) In Vivo Anticancer Activity of AZD3965: A Systematic Review. Molecules 27.\u003c/li\u003e\n\u003cli\u003eTakenaga K, Koshikawa N, Akimoto M, et al. (2021) MCT4 is induced by metastasis-enhancing pathogenic mitochondrial NADH dehydrogenase gene mutations and can be a therapeutic target. Sci Rep 11:13302.\u003c/li\u003e\n\u003cli\u003eTateishi H, Tsuji AB, Kato K, et al. (2017) Synthesis and evaluation of 11C-labeled coumarin analog as an imaging probe for detecting monocarboxylate transporters expression. Bioorganic \u0026amp; Medicinal Chemistry Letters 27:4893-4897.\u003c/li\u003e\n\u003cli\u003eDraoui N, Schicke O, Fernandes A, et al. (2013) Synthesis and pharmacological evaluation of carboxycoumarins as a new antitumor treatment targeting lactate transport in cancer cells. Bioorganic \u0026amp; Medicinal Chemistry 21:7107-7117.\u003c/li\u003e\n\u003cli\u003eSadeghzadeh M, Wenzel B, G\u0026uuml;ndel D, et al. (2020) Development of Novel Analogs of the Monocarboxylate Transporter Ligand FACH and Biological Validation of One Potential Radiotracer for Positron Emission Tomography (PET) Imaging. Molecules 25.\u003c/li\u003e\n\u003cli\u003eSattler B, Kranz M, Wenzel B, et al. (2020) Preclinical Incorporation Dosimetry of [18F]FACH\u0026mdash;A Novel 18F-Labeled MCT1/MCT4 Lactate Transporter Inhibitor for Imaging Cancer Metabolism with PET. Molecules 25.\u003c/li\u003e\n\u003cli\u003eSadeghzadeh M, Moldovan R-P, Teodoro R, et al. (2019) One-step radiosynthesis of the MCTs imaging agent [18F]FACH by aliphatic 18F-labelling of a methylsulfonate precursor containing an unprotected carboxylic acid group. Scientific Reports 9.\u003c/li\u003e\n\u003cli\u003eG\u0026uuml;ndel D, Sadeghzadeh M, Deuther-Conrad W, et al. (2021) Preclinical Evaluation of [18F]FACH in Healthy Mice and Piglets: An 18F-Labeled Ligand for Imaging of Monocarboxylate Transporters with PET. International Journal of Molecular Sciences 22.\u003c/li\u003e\n\u003cli\u003eSadeghzadeh M, Moldovan RP, Fischer S, et al. (2019) Development and radiosynthesis of the first 18F‐labeled inhibitor of monocarboxylate transporters (MCTs). Journal of Labelled Compounds and Radiopharmaceuticals 62:411-424.\u003c/li\u003e\n\u003cli\u003eKirat D, Inoue H, Iwano H, et al. (2005) Expression and distribution of monocarboxylate transporter 1 (MCT1) in the gastrointestinal tract of calves. Research in Veterinary Science 79:45-50.\u003c/li\u003e\n\u003cli\u003eGurrapu S, Jonnalagadda SK, Alam MA, et al. (2015) Monocarboxylate transporter 1 inhibitors as potential anticancer agents. ACS Med Chem Lett 6:558-561.\u003c/li\u003e\n\u003cli\u003eYu YJ, Zhang FL, Peng TY, et al. (2021) Sequential C-F bond functionalizations of trifluoroacetamides and acetates via spin-center shifts. Science 371:1232-1240.\u003c/li\u003e\n\u003cli\u003eOdi R, Bibi D, Wager T, et al. (2020) A perspective on the physicochemical and biopharmaceutic properties of marketed antiseizure drugs\u0026mdash;From phenobarbital to cenobamate and beyond. Epilepsia 61:1543-1552.\u003c/li\u003e\n\u003cli\u003eGuan X, Rodriguez-Cruz V, Morris MJTAj (2019) Cellular Uptake of MCT1 Inhibitors AR-C155858 and AZD3965 and Their Effects on MCT-Mediated Transport of L-Lactate in Murine 4T1 Breast Tumor Cancer Cells. 21:13.\u003c/li\u003e\n\u003cli\u003eGuan X, Bryniarski MA, Morris ME (2018) In Vitro and In Vivo Efficacy of the Monocarboxylate Transporter 1 Inhibitor AR-C155858 in the Murine 4T1 Breast Cancer Tumor Model. AAPS J 21:3.\u003c/li\u003e\n\u003cli\u003eSandforth L, Ammar N, Dinges LA, et al. (2020) Impact of the Monocarboxylate Transporter-1 (MCT1)-Mediated Cellular Import of Lactate on Stemness Properties of Human Pancreatic Adenocarcinoma Cells dagger. Cancers 12.\u003c/li\u003e\n\u003cli\u003eKim D, Ko HY, Chung JI, et al. (2023) Visualizing Cancer-Originating Acetate Uptake Through MCT1 in Reactive Astrocytes in the Glioblastoma Tumor Microenvironment. Neuro Oncol.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-imaging-and-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mibi","sideBox":"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)","snPcode":"11307","submissionUrl":"https://www.editorialmanager.com/mibi/default2.aspx","title":"Molecular Imaging and Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Monocarboxylate transporters, Metabolism of symbiosis, α-cyano-4-hydroxy-cinnamic acid, PET imaging probe, Preclinical evaluation","lastPublishedDoi":"10.21203/rs.3.rs-4020948/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4020948/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThe visualization and quantitative analysis of monocarboxylate transporters (MCTs) hold significant application value in comprehending the metabolic symbiosis, acid resistance, and invasion mechanisms of tumors. Thus, we designed and synthesized a novel MCTs-targeting radiotracer [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC and gave a comprehensive evaluation in vitro and in vivo experiments for it.\u003c/p\u003e\u003ch2\u003eProcedures\u003c/h2\u003e \u003cp\u003eThe preparations for the precursor and reference of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC were encompassed. In vitro evaluation included compound identification, purity, stability, liposolubility, and assays in BxPC3 and 4T1 tumor cell lines. Dynamic Micro-PET imaging was performed in tumor-bearing mice to determine its in vivo characteristics.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe synthesis of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC, a derivative of α-cyano-4-hydroxycinnamic acid (CHC), was achieved using a one-step method with the MCTs inhibitor (\u003cem\u003eE\u003c/em\u003e)-ethyl 2-cyano-3-(4-hydroxyphenyl)acrylate as the lead compound. The yield obtained was 52.08\u0026thinsp;\u0026plusmn;\u0026thinsp;6.74% (n\u0026thinsp;=\u0026thinsp;7, decay corrected). The cell uptake characteristics and targeting ability towards MCTs were confirmed through cell uptake and competitive inhibition experiments conducted on BxPC3 pancreatic cancer cell line and 4T1 breast cancer cell line. The biodistribution and Micro-PET/CT imaging of tumor-bearing mice revealed the hepatic and renal metabolism-mediated excretion characteristics of [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC, with radioactive uptake in tumors being consistent with MCTs expression levels.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThrough the aforementioned studies, a one-step method was employed to successfully synthesize [\u003csup\u003e18\u003c/sup\u003eF]FEtO-CHC, which has been validated as a small molecule PET probe specifically targeting MCTs.\u003c/p\u003e","manuscriptTitle":"One-step Radiosynthesis and Preclinical Evaluation of Molecular Probe [18F]FEtO-CHC Targeting Monocarboxylate Transporters for PET Imaging in Tumor-bearing Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-18 17:39:28","doi":"10.21203/rs.3.rs-4020948/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-07-15T12:46:03+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-20T19:50:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-10T07:25:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-07T23:14:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Imaging and Biology","date":"2024-03-06T07:46:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-imaging-and-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mibi","sideBox":"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)","snPcode":"11307","submissionUrl":"https://www.editorialmanager.com/mibi/default2.aspx","title":"Molecular Imaging and Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"00bfdd91-5fdc-4d7c-86da-c2678b05b9b6","owner":[],"postedDate":"April 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T16:03:53+00:00","versionOfRecord":{"articleIdentity":"rs-4020948","link":"https://doi.org/10.1007/s11307-025-02024-1","journal":{"identity":"molecular-imaging-and-biology","isVorOnly":false,"title":"Molecular Imaging and Biology"},"publishedOn":"2025-06-11 15:57:36","publishedOnDateReadable":"June 11th, 2025"},"versionCreatedAt":"2024-04-18 17:39:28","video":"","vorDoi":"10.1007/s11307-025-02024-1","vorDoiUrl":"https://doi.org/10.1007/s11307-025-02024-1","workflowStages":[]},"version":"v1","identity":"rs-4020948","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4020948","identity":"rs-4020948","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}