Radiosynthesis and Biological Evaluation of N-[18F]Labeled Glutamic Acid as a Tumor Metabolic Imaging Tracer

We have previously reported that N-(2-[18F]fluoropropionyl)-L-methionine ([18F]FPMET) selectively accumulates in tumors. However, due to the poor pharmacokinetics of [18F]FPMET in vivo, the potential clinical translation of this observation is hampered. In this study, we rationally designed and synthesized [18F] or [11C]labeled N-position L-glutamic acid analogues for tumor imaging. N-(2-[18F]fluoropropionyl)-L-glutamic acid ([18F]FPGLU) was synthesized with a 30±10% (n = 10, decay-corrected) overall radiochemical yield and a specific activity of 40±25 GBq/μmol (n = 10) after 130 min of radiosynthesis. In vitro cell experiments showed that [18F]FPGLU was primarily transported through the XAG – system and was not incorporated into protein. [18F]FPGLU was stable in urine, tumor tissues, and blood. We were able to use [18F]FPGLU in PET imaging and obtained high tumor to background ratios when visualizing tumors tissues in animal models.


Introduction
Positron emission tomography (PET) provides noninvasive images of physiologic function and is now regularly used in the diagnosis and staging of cancer [1]. Targeting of tumor specific metabolic pathways represents a useful strategy for early tumor detection and characterization. At present, a glucose analog, [ 18 F]fluoro-2-deoxy-D-glucose ([ 18 F]FDG) as a widely used PET tumor tracer, is based on increased glucose utilization in tumor cells [2]. However, [ 18 F]FDG has several disadvantages and limitations. [ 18 F]FDG has a high uptake into the brain and nonmalignant, inflammatory cellular elements [3]. In addition, a noticeable portion of tumors with a low metabolic rate, such as prostate [4,5], bladder, renal cancer, and other [ 18 F]FDG nonavid cancers [6], are [ 18 F]FDG PET-negative and cannot be detected by [ 18 F]FDG PET. To be able to overcome these disadvantages, work has been conducted to identify new tracers for tumor imaging that has greater sensitivity and better specificity for tumor imaging. Since tumor growth and proliferation require a high amount of energy and specific amino acids, positron-emitting radionuclide-labeled amino acids can be potential alternatives for tumor imaging. For example, S-[ 11 C]methyl-L-methionine ([ 11 C]MET) and O-2-[ 18 F]fluoroethyl-L-tyrosine ([ 18 F]FET) are commonly used amino acid tracer for the in vivo targeting of brain tumors [7]. Furthermore, [ 18 F]FET might be able to distinguish between tumor and inflammatory tissues. Unfortunately, one of the major limitations of extracranial tumor imaging with [ 11 C]MET or [ 18 F]FET has relative poor tumor uptake, producing low to moderate tumor to background ratios [8].
Tumor cells use glucose and glutamine as their main sources of energy for both growth and proliferation. It has been reported that [ 18 F]FDG-negative tumors may use a different metabolic pathway called-glutaminolysis [9][10][11][12]. Hence, in the adapted intermediary metabolism of tumors, it is possible that key roles are played by glutamine and glutamate. Recently, some [ 18 F]labeled glutamate and glutamine derivatives have been reported [2,[13][14][15][16] Figure 1) [2,14] have demonstrated high uptake in tumor cells that undergo high growth and proliferation, but both of them become defluorinated in animals models, resulting in suboptimal images. Another derivative of glutamic acid, (4S)-4-(3-[ 18 F]fluoropropyl)-L-glutamate (BAY 94-9392, 3; see Figure 1) can be useful for detecting system X C -(SLC7A11) activity in vivo, which is a potential biomarker for tumor oxidative stress. Animal models and use in humans have confirmed these observations [15,16].
Our previous studies proposed the hypothesis that radiolabeled N-position amino acid analogues could function as potential PET tracers, which was confirmed by our early studies of N-(2-[ 18 F]fluoropropionyl)-L-methionine ([ 18 F]FPMET, 4, see Figure 1) as a typical tool for tumor imaging [17]. Although intriguing uptake tumor was seen, its potential use in a clinical setting was hampered by poor stability and a high background signal in vivo.
In the present study, we design and radiosynthesize N-(2-

General
All chemicals obtained commercially were used without further purification unless otherwise indicated. [ 18 F]FDG was prepared as previously reported [18]. Sep-Pak light QMA, Plus C18, and Oasis HLB cartridges were obtained from Waters Corporation (Milford, MA, USA). Sep-Pak light QMA cartridges were preconditioned with 5 mL NaHCO 3 aqueous (8.4%) and 10 mL water before use. Plus C18 and Oasis HLB cartridges were preconditioned with 10 mL ethanol and water before use. HPLC separation was carried out at the PET-MF-2V-IT-I synthesizer module (PET Co. Ltd., Beijing, China) built-in HPLC system with a semi-preparative reverse-phase C18 column (106250 mm) equipped with a UV detector (Alltech 201, USA) and a radioactivity detector (PET Co. Ltd., China). The mobile phase was 55% solvent A (0.1% TFA in water): 45% solvent B (0.1% TFA in MeCN) with the flow rate of 4 mL/min. Analytical HPLC was performed using a Agilent 1200 Series HPLC system equipped with a ZORBAX Eclipse XDB-C18 analytic column (4.66150 mm, 5 mm) using the flow rate of 1 mL/min. The gradient program started from 98% solvent A (0.1% TFA in water): 2% solvent B (0.1% TFA in MeCN) ramped to 90% solvent A: 10% solvent B at 8 min, and ramped to 20% solvent A: 80% solvent B at 20 min. The elution profile was detected with an ultraviolet detector (Agilent interface 35900E, Agilent Technologies, USA) at 210 nm and a B-FC-3200 high energy PMT Detector (Bioscan. Inc, Washington DC, USA). Radioactivity was measured by a calibrated ion chamber (Capintec CRC-15R) or a gamma counter (c-counter) (GC-1200, USTC Chuangxin Co. Ltd. Zonkia Branch, China). No-carrier-added 18 F-fluoride was obtained through the nuclear reaction 18 O(p, n) 18 F by irradiation of more than 95% [ 18 O]enriched water target with 10-MeV proton beam on the Cyclone 10/5 cyclotron (IBA Technologies, Belgium).

Cell Culture and Animal Models
The S-180 Cell line and mice were obtained from the Laboratory Animal Center of Sun Yat-Sen University (Guangzhou, China). LTEP-a-2 and SPC-A-1 (SPCA-1) human lung adenocarcinoma cell lines were obtained from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences (Shanghai, China). SPCA-1 cell line was originally isolated from the surgical specimens of a Chinese man with advanced lung adenocarcinoma by Shanghai Chest Hospital and Cellular Institute of Chinese Academy of Science in 1980 [19,20]. The cells were cultured in RPMI 1640 medium with a physiologic glucose concentration (1.0 g/L) containing 5% fetal calf serum at 37uC in a humidified atmosphere of 5% CO 2 and 95% air. Two days before the experiments in vitro, human lung adenocarcinoma SPCA-1 cell lines were trypsinized and 2610 5 cells per well were seeded into 12-well plates. Mice were housed 5 animals per cage under standard laboratory conditions at 25uC and 50% humidity. Tumor cells (122610 7 ) were injected subcutaneously and allowed to grow for 1 to 3 weeks. At the time of the experiments, the tumor reached 6212 mm (diameter), the mice were 5-8 wk old, and weighed 18-28 g. All work studies were approved by the Institutional Animal Care and Utilization Committee (IACUU) of the First Affiliated Hospital, Sun Yat-Sen University (approval no.2012.001). All efforts were made to minimize animal suffering, to reduce the number of animals used and to utilize alternatives to in vivo techniques, if available.

Synthesis of N-(2-fluoropropionyl)-L-glutamic Acid and Nmethyl-L-glutamic Acid
The synthesis of N-(2-fluoropropionyl)-L-glutamic acid (FPGLU) was performed by a similar method as previously described [17]. Detail synthesis procedures for the FPGLU are included in File S1. The synthesis of N-methyl-L-glutamic acid (MGLU) was prepared based on previously procedures [21].

Radiosynthesis of [ 18 F]FPGLU
[ 18 F]NFP (8) was prepared and dried based on previously procedures [22]. Anhydrous [ 18 F]NFP was added a solution of diethyl L-glutamate hydrochloride (9, 100 mg) in DMSO (200 mL) and DIPEA (20 mL). The reaction mixture was heated for 5 min at 40uC and was quenched by adding of 5% acetic acid (600 mL) and was diluted with water (10 mL). The dilution was passed through a Plus C18 cartridge, and then the cartridge was washed with water (10 mL). The ester 10 was eluted with ether (5 mL) from the Plus C18 cartridge. The ether was evaporated under nitrogen flow at room temperature. To the residue was added NaOH aqueous (1 M, 50 mL). The ester 10 was hydrolyzed at room temperature for 8 min and the solution was neutralized with HCl (1 M, 50 mL). The product 11 was formulated in 0.9% saline and passed through a 0.22 mm Millipore filter for studies.

Radiosynthesis of [ 11 C]MGLU
11 CO 2 was produced by 14 N(p, a) 11 C nuclear reactions using a Cyclone 10/5 cyclotron (IBA) and was delivered to the radiochemical laboratory. 11 CO 2 was trapped in a loop ring cooled with liquid nitrogen. 11 CH 3 I was prepared from reduction of 11 CO 2 with LiAlH 4 , hydrolysis of the intermediately formed organmetallic complex, and subsequent iodination of 11 C-methanol with hydrogen iodide. 11 CH 3 I was translated to 11 CH 3 OTf with AgOTf column [23]. 11 CH 3 OTf was passed into a solution of diethyl L-glutamate hydrochloride (9, 2 mg) in acetone (1 mL) and DIPEA (40 mL). The reaction was reacted for 5 min and diluted with water (10 mL). The compound ester 12 was purified by plus C18 and eluted with ether (5 mL) from the Plus C18 cartridge. The ether was evaporated under nitrogen flow at room temperature. To the residue was added NaOH aqueous (1 M, 100 mL) and reacted at room temperature for 10 min. The

Octanol-Water Partition Coefficient Study (logP)
Octanol-water partition coefficient for [ 18 F]FPGLU was determined by measuring the distribution of radiolabeled compound in n-octanol and phosphate-buffered saline (pH = 7.4). A 20 mL sample of [ 18 F]FPGLU (740 KBq, 20 mCi) in saline was added to a vial containing 5 mL each of n-octanol and phosphate-buffered saline. After being stirred in a vortex mixer for 1 min, the vial was centrifuged for 4 min to ensure complete separation of layers. Three hundred milliliters of each layer were measured using a ccounter. logP value was calculated using the following formula: log 10 P = log 10 (counts in 0.3 mL of octanol/counts in 0.3 mL of water) [24].

Competitive Inhibition Studies and Protein Incorporation
The methods of competitive inhibition and protein incorporation were similar as reported previously [17,25,26]. Detailed procedures for studying [ 18 F]FPGLU using human lung adenocarcinoma SPCA-1 cells are included in File S1.

In Vivo Biodistribution of [ 18 F]FPGLU
For single-isotope ( 18 F) biodistribution studies, Kunming mice (body weight range, 18-22 g) were anesthetized with 5% chloral hydrate solution (6 mL/kg) before injection of radiotracer. They were injected with 0.7421.48 MBq (20240 mCi) of [ 18 F]FPGLU in 1002200 mL of saline through the tail vein. Radioactivity in the syringe before and after administration was measured in a calibrated ion chamber. The animals were killed by cervical dislocation at various times after injection, blood was obtained through the eyeball, and the organs of interest (blood, brain, heart, lung, liver, kidneys, pancreas, stomach, and intestine) were rapidly dissected and weighed, and 18 F radioactivity was counted with a ccounter. All measurements were background-subtracted and decay-corrected to the time of injection, then averaged together.

Small-Animal PET Imaging
Small-Animal PET-CT imaging studies with tumor-bearing mice were carried out using the Inveon small-animal PET/ computed tomography (CT) scanner (Siemens). 3.727.4 MBq of [ 18 F]FPGLU were injected intravenously in conscious animals via the tail vein. Ten minutes later, the mice were anesthetized with 5% chloral hydrate solution (6 mL/kg) and were then placed on a heating pad to maintain body temperature throughout the procedure. Animals were visually monitored for breathing and any other signs of distress throughout the entire imaging period. Ten-minute static PET images were acquired at four time points (30, 60, 90 and 120 min) postinjection. For a comparative study, mice were kept fasting for 4 h and were anesthetized with 5% chloral hydrate solution (6 mL/kg) and imaged with [ 18 F]FDG (3.7 MBq) at 60 min after intravenous injection. Imaging started with a low-dose CT scan, immediately followed by a PET scan. The CT scan was used for attenuation correction and localization of the lesion site. The images were reconstructed by twodimensional ordered-subsets expectation maximum (OSEM). For each small-animal PET scan, ROIs were drawn over the tumor and major organs on decay-corrected whole-body coronal images using Inevon Research Workplace 4.1 software. The radioactivity concentration (accumulation) within a tumor or an organ was obtained from mean pixel values within the multiple ROI volume, which were converted to MBq/mL by using a conversion factor. Assuming a tissue density of 1 g/mL, the ROIs were converted to MBq/g and then divided by the administered activity to obtain an imaging ROI-derived %ID/g.

In Vitro and in Vivo Stability
In vitro experiment, a sample of [ 18 F]FPGLU (0.74 MBq, 20 mL) dissolved in normal saline was added to 200 mL of mouse serum and incubated at 37uC [27]. An aliquot of the serum sample was filtered through a 0.22 mm Millipore filter and was injected into a radio-HPLC column to analyze the stability of [ 18 F]FPGLU in mouse serum within 2 h. In vivo stability experiment, Kunming mice bearing S180 fibrosarcoma were anesthetized with 5% chloral hydrate solution (6 mL/kg) and were injected with a dose of approximately 30 MBq (810 mCi) of [ 18 F]FPGLU in 200 mL of normal saline via the tail vein [8]. The mice were sacrificed at 0.5 h and 1 h after injection. The amount of unchanged tracer in tissue samples was determined by radio-HPLC. Tumors were separately homogenized in phosphate-buffered saline (PBS) and centrifugation (18000 g, 6 min). Supernatant was filtered through a 0.22 mm Millipore filter and analyzed by radio-HPLC. Blood was obtained by extirpating eyeballs, centrifuging (18000 g, 6 min). The plasma was filtered through a 0.22 mm Millipore filter and analyzed by radio-HPLC. The urine was collected from the bladder and was injected into an HPLC column to analyze the metabolic fate of [ 18 F]FPGLU in vivo.

Statistical Analysis
Data were expressed as mean 6 SD. Statistical analysis was performed with SPSS software, version 13.0 (SPSS Inc.), for Windows (Microsoft). Continuous variables were analyzed using the Student's t-test. A P value of less than 0.05 was considered to indicate statistical significance.

Radiochemistry
Nonradioactive FPGLU and MGLU were synthesized for use as a reference standard for characterizing radioactive [ 18 F]FPGLU and [ 11 C]MGLU by HPLC. [ 18 F]FPGLU (11) was synthesized as shown in Figure 2. The prosthetic group [ 18 F]NFP (8) was synthesized in an automated process from ethyl 2-bromopropionate (5) as the precursor via a three-step, one-pot procedure on the modified PET-MF-2V-IT-I synthesizer [22]. Compound 9 was coupled with dried [ 18 F]NFP and purified with a C18 cartridge. The compound 10 was further hydrolyzed with sodium hydroxide and neutralized with hydrochloric acid to afford 11 [ 18 F]FPGLU. The decay-corrected radiochemistry yield of [ 18 F]FPGLU was 9065% (n = 10) from [ 18 F]NFP for 30 min. The total decaycorrected radiochemical yield of [ 18 F]FPGLU was 30610% (n = 10) from 18 F 2 for 130 min, with a specific activity of 40625 GBq/mmol (n = 10). As can be seen from Figure 3, [ 18 F]FPGLU had a radiochemical purity of more than 95% and the tracer was initially evaluated as an epimeric mixture.

Octanol-Water Partition Coefficient Study (logP)
The lipophilicity logP value of [ 18 F]FPGLU at pH 7.4 is 2 1.5460.06. As expected, the tracer was hydrophilic, as indicated by the logP of this derivative of an amino acid.

Competitive Inhibition Studies
To investigate the transport mechanisms involved in the uptake of [ 18 F]FPGLU, we conducted a series of competitive inhibition studies in SPCA-1 cells using specific inhibitors for system A, ASC, L, X C -, and X AG -, which are the major amino acid transport systems in mammalian cells and are potentially responsible for the uptake of glutamate and its analogs [14,28,29]. Figure 6 showed the results from these inhibition experiments when competing amounts of inhibitors were included and when Na + was either present or absent. The data showed that the system A inhibitor Nmethyl-2-amino-isobutyric acid (MeAIB) had no inhibitory effect on the uptake of [ 18 F]FPGLU. In presence of Na + , the uptake of [ 18 F]FPGLU was inhibited by 23% by the system L inhibitor 2amino-2-norbornane-carboxylic acid (BCH). The system ASC inhibitors, L-serine (Ser) and L-glutamine (Gln), inhibited uptake

Protein Incorporation
SPCA-1 cells were incubated with [ 18 F]FPGLU for 30 min, then trichloroacetic acid was used to precipitate them, which showed there was less than 1% of the radioactivity in the acid precipitable fraction. Therefore, there was no incorporation of [ 18 F]FPGLU into the protein, which was similar to many labeled non-protein-composition amino acids tracers (e.g., BAY 85-8050, [15,17,30,31].

In Vivo Biodistribution of [ 18 F]FPGLU
The in vivo biodistribution data of [ 18 F]FPGLU were summarized in Table 1. The rapid uptake of [ 18 F]FPGLU was observed within the kidneys but it was quickly excreted through the urinary bladder, with 30.0 and 1.15%ID/g in the kidneys at 5 and 120 min, respectively. Blood clearance dropped fairly quickly with time, and showed low blood activity (1.04%ID/g) at 120 min after injection. [ 18 F]FPGLU showed a moderate liver uptake with a relatively slow washout rate. Lung, spleen, intestine, heart, pancreas and stomach, showed relatively moderate uptake of radioactivity at 5 min, which decreased over the course of the 2 h study. There were negligible or very low levels (,1%ID/g) of uptake in other organs of interest, for example, the brain, bone, and muscle.

Small-Animal PET Imaging
Small-animal PET-CT imaging using [ 18 F]FPGLU was performed on S180 fibrosarcoma, SPCA-1, and LTEP-a-2 human lung adenocarcinoma mouse models (n = 3 per group) ( Figures 7A  and 7B). The tumors were clearly visible with high contrast to the contralateral background (muscle, bone, brain, neck, and other tissues) within each animal model. PET imaging provided the consistent distribution data obtained from the ex vivo biodistribution result of [ 18 F]FPGLU. During the early part of the experiment, prominent renal uptake was measured and accumulation in the bladder was observed, which suggests that the renalbladder route was the main excretory system. As shown in Figures 7A and 7B, the tumor could be readily visualized with [ 18 F]FPGLU. Regions of interest (ROIs) from the whole organ on the coronal images were measured so that the accumulation of the radioactivity in the small-animal PET scans could be quantified. The uptake between [ 18 F]FPGLU and [ 18 F]FDG in the S180 fibrosarcoma and SPCA-1 human lung adenocarcinoma has no significant difference at 1 h (5.361.0%ID/g vs. 4.860.8%ID/g for S180 fibrosarcoma, n = 3, P.0.05; 3.160.4%ID/g vs. 2.960.5%ID/g for SPCA-1 human lung adenocarcinoma, n = 3, P.0.05). In the LTEP-a-2 human lung adenocarcinoma, the uptake of [ 18 F]FPGLU was significantly higher than that of [ 18 F]FDG at 1 h (2.660.3%ID/g vs. 1.860.2%ID/g, n = 3, P, 0.05) ( Figure 7C).

In Vitro and in Vivo Stability
Radio-HPLC analysis showed that [ 18 F]FPGLU in serum was stable. [ 18 F]FPGLU in mouse serum at 37uC was more than 98% intact after 2 h. The stability of [ 18 F]FPGLU in vivo and its metabolic fate was analyzed in urine, plasma, and tumor tissue (Figure 8). For the plasma and tumor tissue collected after 1 h, it was not possible to use HPLC to detect the radioactivity, as its level was too low. Therefore, data points for up to 0.5 h were the only ones that could be analyzed for the metabolites.
Radio-HPLC analysis of the extracted activity indicated that more than 40% of the [ 18 F]FPGLU remained intact in the plasma 0.5 h post-injection. In tumor tissue, more than 65% of the tracer remained intact 0.5 h post-injection. The percentage of unchanged [ 18 F]FPGLU in urine was 90% 0.5 h post-injection, which was reduced to 78% 1 h post-injection. HPLC analysis also showed that the main radioactive metabolites in tissues were highly polar products as they were eluted very fast.

Discussion
Glutamate and its derivative play key roles in the adapted intermediary metabolism of tumors. Glutamate can be converted into and functions as a substitute for glutamine. Glutamate's transamination product, a-ketoglutarate is directly channeled into the truncated tricarboxylic acid (TCA) cycle [13]. Its derivative Nacetyl-L-glutamate is the essential allosteric activator of the first urea cycle enzyme (carbamoyl phosphate synthetase I), which is a  Malignant tumor cells accumulate amino acids and their close analogs because increased levels of their transporters are produced by the tumor, which also has a greater rate of protein synthesis. Adaptations of amino acid transport systems in cancer cells can offer opportunities to provide novel diagnostic and therapeutic targets. The BAY 94-9392 targeting system X C is considered to be a potential tracer for tumor imaging [15,16]. In our experiments, [ 18 F]FPGLU transport in SPCA-1 cells consisted of three processes, two saturable Na + -dependent activities and a nonsaturable Na + -independent route. In the presence of Na + , [ 18 F]FPGLU uptake was moderately inhibited by serine since the Na + -dependent system ASC contributed to [ 18 F]FPGLU transport. In the presence and absence of Na + , [ 18 F]FPGLU uptake was moderately inhibited by BCH since the Na +independent system L also contributed to [ 18 F]FPGLU transport. Glu and Asp peotently inhibited the uptake of [ 18 F]FPGLU in the presence of Na + . However, when Na + was absent, the uptake of [ 18 F]FPGLU was not further reduced by Glu. The X AG system is a Na + -dependent transport system for anionic amino acids [33]. Therefore, [ 18 F]FPGLU uptake was primarily transported through Na + -dependent X AG -. Since [ 18 F]FPGLU was initially evaluated as an epimeric mixture, it was possible that the amino acid transport mechanism of this epimeric mixture was different from that of its single epimer, primarily due to its specificity for the various amino acid transport systems. A more detailed study on the biological transport properties of its single epimer in vivo and in vitro would require further study.
The biodistribution of [ 18 F]FPGLU in mice showed that its radioactivity rapidly accumulated in kidneys and was quickly excreted through the urinary-bladder route. The radioactivity in other tissues was relatively low during the entire observation time.
In comparison with [ 18 F]FPMET [17], [ 18 F]FPGLU had a lower uptake in the organs except in the kidneys at 30 and 60 min. It was suggested that [ 18 F]FPGLU had a lower background to signal ratio than that of [ 18 F]FPMET in vivo. The in vivo biodistribution results were confirmed by small-animal PET imaging. Small-animal PET studies using [ 18 F]FPGLU exhibited the expected high uptake and retention in S180 fibrosarcoma, SPCA-1, and LTEP-a-2 human lung adenocarcinoma mice models. Future studies would be further observed the tumor uptake of [ 18 F]FPGLU in other subcutaneous human tumor mouse models.
[ 18 F]FPGLU was stable in mouse serum at 37uC for 2 h. However, the tracer was metabolized in vivo, which could be due to the lack of peptidases for peptide bond cleavage in the serum [34]. The level in the tumor tissue of the intact [ 18 F]FPGLU was greater than that in the plasma 0.5 h post-injection. Perhaps [ 18 F]FPGLU was rapidly accumulated in specific tumors through amino acid transporters and therefore maintained long-time stability in the tumor. It has been reported that the stability of [ 18 F]FPMET was 25% of the unchanged [ 18 F]FPMET in plasma and 8% of the unchanged [ 18 F]FPMET in urine 0.25 h postinjection [17]. In these tissues studied, [ 18 F]FPGLU was more stable than [ 18 F]FPMET.

Conclusion
In this study, we successfully designed and prepared the new tracer [ 18 F] and [ 11 C]labeled N-position L-glutamic acid analogues, and examined its potential application in imaging analysis of solid tumors in vivo. [ 18 F]FPGLU showed good uptake and good tumor-to-background contrast in S180 fibrosarcoma, SPCA-1, and LTEP-a-2 human lung adenocarcinoma mouse models. [ 18 F]FPGLU was primarily transported through the Na + -dependent X AG system. In addition, [ 18 F]FPGLU showed relatively favorable pharmacokinetics in vivo. This work has shown that it is possible to use [ 18 F]FPGLU as a tumor metabolic imaging tracer with PET.

Supporting Information
File S1 The synthesis of nonradioactive N-(2-fluoropropionyl)-Lglutamate (FPGLU); the detail procedures of PET imaging of