Biological evaluation of [18F]AlF-NOTA-NSC-GLU as a PET tracer for hepatocellular carcinoma

Purpose 18 F-labeled amino acids (AAs) as tumor-specic imaging agents play a critical role in hepatocellular carcinoma (HCC) imaging. In this work, we evaluated the synthesis and biological properties of a simple 18 F-labeled glutamate analogue, [ 18 F]AlF-1,4,7-triazacyclononane-1,4,7-triacetic-acid-2-S-(4-isothiocyanatobenzyl))-l-glutamate ([ 18 F]AlF-NOTA-NSC-GLU) for HCC imaging via one-step reaction sequence. Methods [ 18 F]AlF-NOTA-NSC-GLU was synthesized via the one-step reaction sequence from NOTA-NSC-GLU. In order to investigate the imaging value of [ 18 F]AlF-NOTA-NSC-GLU in HCC, we conducted PET/CT imaging and competitive binding of [ 18 F]AlF-NOTA-NSC-GLU in human Hep3B tumor-bearing mice. The transport mechanism of [ 18 F]AlF-NOTA-NSC-GLU was determined by competitive inhibition and protein incorporation experiments in vitro. Results ± 0.17 at 30 min post-injection. In vivo competitive binding experiments exhibited that the tumor-to-liver uptake ratio decreased by the addition of the inhibitors to block the system X AG- . Conclusion We have successfully synthesized [ 18 F]AlF-NOTA-NSC-GLU as a novel PET tracer with good radiochemical yield and high radiochemical purity. Our ndings indicate that [ 18 F]AlF-NOTA-NSC-GLU might have good clinical potential as a PET tumor-detecting agent for HCC imaging.


Introduction
Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related death worldwide and is the fth most frequently diagnosed malignancy on a global scale [1,2]. Also known as "the silent killer", early-stage HCC is often missed, and the 5-year survival rate of an advanced disease is less than 5%, compared to 40%-70% if diagnosed early [3]. Thus, timely diagnosis and precise staging is essential for selecting proper treatment and improving prognosis. Increasingly, medical imaging has become the primary method for noninvasive diagnosis of HCC, supported by guidelines of the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver [4,5]. Notably, the most commonly conducted examinations for diagnosing HCC consist of computed tomography (CT) and magnetic resonance imaging (MRI) [6]. However, limitations of CT or MRI, including radiation risk, cost and the high false positive signals, prompted the development of positron emission tomography/computed tomography (PET/CT), which is also a noninvasive imaging technique. It can detect and characterize tumors based on their molecular and biochemical properties [7], and plays a vital role in the evaluation of HCC, especially with the rapid development of hepatocyte-speci c PET tracers.
Tumor cells can be identi ed by abnormal proliferation and metabolic activities of nutrients, i.e., glucose, amino acids (AAs ), fatty acids, vitamins and so on [13]. To increase more speci c tumor uptake, PET tracers for the metabolism of glucose, lipids, AAs and nucleic acids in tumor have been developed. AA metabolism PET supplements the method of glucose metabolism PET and plays a crucial role in oncologic imaging. Studies have supported the use of 18 F-labeled AAs in the imaging of various tumors (e.g., gliomas, neuroendocrine tumors, prostate cancer and breast cancer) [14,15]. L-methy-[ 11 C]methionine ([ 11 C]Met) once was the most commonly used AA tracer in HCC imaging. However, the sensitivity and speci city of [ 11 C]Met for HCC imaging have been shown inadequate [16,17]. Moreover, the short half-life of carbon-11 also limits the development of [ 11 C]Met PET [18][19][20]. In recent years, dualtracer ([ 18 F]FDG and [ 11 C]Acetate) has been introduced for HCC imaging with improved sensitivity and speci city [19,21]. Unfortunately, dual-tracer PET/CT incurs more radiation burden than single-tracer PET/CT, which limits its clinical application [22,23].
Currently, we are working on a series of radiolabeled N-substituted AA analogues, which target the increased levels of AA transport by various types of malignant cells (e.g., systems L, X AG -, X C -, ASC and A), as potential PET tracers to image HCC [24].  [24,25]. Reportedly, a simple one-step procedure, which prepares 18 F-labeled peptides via chelating an aluminum-uoride (AlF) with 1,4,7-triazacyclononane-1,4,7-triaceticacid (NOTA), offers an original strategy to simplify the labeling procedure [26,27]. This has prompted the design of a simple 18 F-labeled AA tracer.
In this study, we produced a small-molecule 18

General information
All chemicals applied in the synthesis were commercially sourced and used without further puri cation unless otherwise indicated. [ 18 F]FDG was radiolabeled as previously described [28].

Synthesis of [ 18 F]AlF-NOTA-NSC-GLU
The NOTA-NSC-GLU was synthesized by Shanghai Apeptide Co. Ltd. (Shanghai, China), with > 95% purity. 18 F dissolved in water was passed through a preconditioned Sep-pak QMA cartridge. Then, 18 F was eluted from the QMA cartridge with 0.9% NaCl. Next, 90 μL of eluate was added to a vial containing 6 μL of 2 mM aluminum chloride, 5 mL of glacial acetic acid, 325 μL of acetonitrile and 50 μL of 50 μg NOTA-NSC-GLU in 50 μL of deionized water. The resulting solution was performed at 100℃for 10 min. The cooled crude reaction mixture was diluted with 10 mL of water and passed through a preconditioned C-18 Sep-Pak cartridge. The radioactivity trapped in the C-18 cartridge was eluted with 1.5 mL of ethanol. The ethanol solution was evaporated with argon ow, and the nal product was reconstituted in normal saline for further studies (Fig. 1).

In vivo biodistribution studies
The biodistribution experiment in vivo was performed on twenty healthy male Kunming mice and four animals were used at each time point. The mice were injected intravenously (IV) with 20-40 μCi of [ 18 F]AlF-NOTA-NSC-GLU in 0.2 mL of saline. At 15,30,45,60, and 90 min after injection, the distribution of the tracer in selected organs were evaluated. Organs of interest (blood, brain, heart, lung, liver, spleen, kidneys, pancreas, stomach, intestine, muscle, and bone) were weighed and 18 F radioactivity was counted with a γ-counter. All measurements were background-subtracted and decay-corrected to the time of injection, then averaged. The results were expressed as percentage injected dose per gram of tissue (%ID/g).

Transport assays
When HCC Hep3B cells were seeded into 24-well plates and reached the logarithmic proliferation phase, we started the transport assays. The methods and transport mechanism of [ 18 F]AlF-NOTA-NSC-GLU were previously reported [24,29]. In addition, each experiment was carried out in triplicate and averaged, and repeatedly conducted on three different days. The transport experiments were implemented in the presence and absence of Na + (NaCl medium and Choline Chloride medium). For the competitive inhibition studies, α-(methylamino)isobutyric acid (MeAIB) for system A, serine (Ser) and L-glutamine (L-Gln) for system ASC, 2-amino-2-norbornane-carboxylic acid (BCH) for system L, L-glutamate (L-Glu) for system X C − and X AG − , cystine (Cyss) for system X C − , L-aspartic (L-Asp) and D-aspartic (D-Asp) for system X AG For the octanol-water partition coe cient study, 20 μL of [ 18 F]AlF-NOTA-NSC-GLU (740 KBq, 20 μCi) in saline was added to an equal volume (octanol/PBS: 5mL/5mL) mixture. The mixture could stand for complete phase separation prior to use through stirring in the vortex mixer for 2 min and centrifuging at 3,000 rpm for 5 min. Samples of 300 μL were taken from each layer and radioactivity was measured with a γ-counter. The logP value (logP = log10(counts of octanol/counts of PBS) as calculated.

Small-animal PET-CT imaging and competitive binding in vivo
Small-animal PET/CT imaging using the Inveon PET scanner was performed following tail-vein injection Imaging acquisition started with a low-dose CT scan (30 mAs), immediately followed by PET scan. The CT scan was used for attenuation correction and organ localization. Image reconstruction was performed with the two-dimensional ordered-subsets expectation maximin (2D-OSEM). Inveon Research Workplace 4.1 software was used to draw regions of interest (ROIs) of 2 mm in diameter at the same section level of each PET/CT image. The radioactivity in each volume of interest was obtained from mean pixel values and converted into MBq/mL using a conversion factor. Supposing the density of tissue was 1 g/mL, the ROIs were converted to MBq/g and then divided by the administered activity to obtain an imaging ROIderived %ID/g. Finally, an imaging ROI-derived %ID/g as well as tumor-to-background relative uptake ratio was obtained.

Incorporation of [ 18 F]AlF-NOTA-NSC-GLU into Protein
The method of determining the extent of protein incorporation of [ 18 F]AlF-NOTA-NSC-GLU was previously reported [31]. Brie y, 400 μL(185-296 KBq) [ 18 F]AlF-NOTA-NSC-GLU was added to the Hep3B cells and incubated at 37℃ for 30 min. Upon removal of the radioactive medium, the cells were washed three times with ice cold PBS (1.0 mL, pH = 7.4), separated by 0.5 mL of 0.25% trypsin and resuspended in PBS. After centrifuging (13,000 rpm, 5 min), the supernatant removed and the cells suspended in 0.2 mL of Triton-X 100 (1%) prior to transferring into new vessels and adding 0.5 mL of 20% trichloroacetic acid (TCA). Kept in ice-cold water for 30 min, the mixture was then centrifuged (13,000 rpm) for 5 min. The supernatant was removed and the pellet was washed thrice with ice-cold PBS. Radioactivity in both the supernatant and the pellet was counted with a γ-counter. Protein incorporation was calculated as the percentage of acid precipitable radioactivity. The experiment was repeated on three different days.

Histochemical studies
After the PET/CT scans, liver tissue and tumor samples were collected and performed with histochemical studies. Formalin-xed, para n-embedded 3-μm-thick sections of tumor and liver were stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed with the method previously reported [32,33]. The immunohistochemical staining of excitatory amino acid carrier 1 (EAAC1) was performed with a rabbit anti EAACI monoclonal antibody (Abcam, 1:1000).

Statistical analysis
Statistical analysis was performed with the Prism 6 Software (GraphPad Software, La Jolla, CA). Data were presented as mean ± standard deviations (SDs). Comparisons between conditions were made using unpaired, 2-tailed Student t-test. P 0.05 was considered statistically signi cant, and P 0.0001 was considered to indicate meaningful differences.

Radiosynthesis of [ 18 F]AlF-NOTA-NSC-GLU
The overall radiochemical yield of [ 18 F]AlF-NOTA-NSC-GLU from 18  interest (e.g. the liver, spleen, muscle and brain); and the brain was the organ with the lowest uptake level (< 1%ID/g).

Competitive inhibition studies
Results of the competitive inhibition experiments are shown in Fig 3. In the presence of Na + , the uptake of [ 18 F]AlF-NOTA-NSC-GLU was inhibited by 20.51 ± 4.77% and 20.07 ± 2.07% (P < 0.05) by Ser and Gln, respectively, substrate of system ASC. The uptake of tracer was suppressed by BCH, inhibitor for system B0 + , by 23.2 ± 13.5% (P < 0.05). MeAIB, a speci c inhibitor for system A, did not markedly suppress the uptake of [ 18 F]AlF-NOTA-NSC-GLU. The addition of system X AG inhibitor L-Asp and L-Glu (an inhibitor for system X C or X AG -), inhibited the uptake of [ 18 F]AlF-NOTA-NSC-GLU by 41.7 ± 0.76% and 44.14 ± 5.2%, respectively (P < 0.05). The speci c inhibitor for system X AG -, D-Asp, suppressed the uptake of the tracer by 50.83 ± 5% (P < 0.05) (Fig 3b) In addition, Cyss suppressed the uptake by 12.53 ± 8.23% (P < 0.05) (Fig  3b).
In the absence of Na + , the addition of L-Glu and L-Asp, decreased the uptake of [ 18 (Fig 5a). The data of uptake in organs of interest in small animal PET imaging were shown in Table 1. The highly uptake of [ 18 F]AlF-NOTA-NSC-GLU in the tumor was observed at 30 min post-injection. During the experiment, most radioactivity accumulations were found in the kidney and bladder, suggesting that the tracer is mainly cleared through the urinary system (Fig 5a). The uptake of [ 18 F]AlF-NOTA-NSC-GLU in the tumor was 1.9 ± 0.057 %ID/g, 1.33 ± 0.15 %ID/g and 0.99 ± 0.096 %ID/g, respectively, at 30 min, 60 min and 90 min post-injection; but in the liver was 0.92 ± 0.025% ID/g, 0.75 ± 0.028 %ID/g and 0.62 ± 0.035% ID/g, respectively, at 30 min, 60 min and 90 min post-injection (Fig 5b). The tumor-to -liver uptake ratio for [ 18  imaging. In addition, the tumor-to -liver and tumor-to -muscle uptake ratio for [ 18 F]AlF-NOTA-NSC-GLU decreased after the injection of L-Glu, L-Asp and D-Asp, respectively (Fig 5d). The results of the competitive binding showed that the uptake of [ 18 F]AlF-NOTA-NSC-GLU in vivo was also involved in transport of the X AG − system.

Protein incorporation
Protein-bound activity of [ 18 F]AlF-NOTA-NSC-GLU in Hep3B cells indicated that about 1.25 ± 0.11% of the radioactivity was in the acid precipitable fraction after co-incubating for 30 min (Fig 6). Hence, the uptake of [ 18 F]AlF-NOTA-NSC-GLU in Hep3B cells is through AA transport rather than protein incorporation.

HE staining and IHC
The results of immunohistochemical staining indicated that diffuse EAACI transporter staining was shown in Hep3B hepatoma (Fig 7c), while minimal EAAC1 staining was shown in normal hepatic tissue (Fig 7d), which suggested that the transport of [ 18 F]AlF-NOTA-NSC-GLU in the Hep3B cell line was likely to involve glutamate transporter EAAC1. In Hep3B tumor, massive cancer cells were observed by hematoxylin-and-eosin staining (Fig 7a).

Discussion
PET imaging has been used in clinical applications (e.g., detection, diagnosis, distant metastasis, and effect monitoring) for decades. Although glucose metabolism plays an important role in tumor cell growth, glutamine metabolism is considered second only to glucose in tumor [34] 18 F-labeled glutamate imaging agents. Alternatively, the method of using the 18 F-uoride-aluminum-NOTA complex for labeling peptides, which could simplify the labeling procedure and reduce the time for radiosynthesis, was reported by McBride et al [40]. Here, we presented the successful synthesis of a new 18 F-labeled glutamate imaging agent via the 18 F-uoride-aluminum-NOTA complex for labeling peptides. Process One-step Two-step Two-step Muilt-step Transport mechanism Na + -dependent system X AG -Na + -dependent system X AG and X C -System X C -Na + -dependent system X AG and X C -Bone uptake (ID/g) < 1% < 1% < 1% > 1% Biodistribution study of this tracer demonstrated that the kidney, among all organs, had the highest accumulation at 15 min after injection, suggesting that the renal-bladder route was the main excretory system. Although the uptake in the stomach and intestine was slightly high at 15 min post-injection, other tissues showed relatively low uptake during the entire observation period, suggesting that the tracer had low background signal in vivo. There were relatively low uptake levels of this agent in bone (< 1%ID/g) during the entire observation time, suggesting no de uorination of [ 18 F]AlF-NOTA-NSC-GLU in vivo. The results of biodistribution were also con rmed by small-animal PET imaging. Surprisingly, the lowest levels of activity were observed in the brain like other 18 F-labeled glutamate imaging agents. On the one hand, this suggests that[ 18 F]AlF-NOTA-NSC-GLU could be a potential agent in brain tumor imaging. Further studies are needed to determine its suitability for brain tumor imaging. On the other hand, it also indicates that [ 18 F]AlF-NOTA-NSC-GLU will not have access to brain via the blood-brain barrier.
Results of in vitro experiments also demonstrated satisfactory stability and hydrophilicity of [ 18 F]AlF-NOTA-NSC-GLU. Moreover, 95% of [ 18 F]AlF-NOTA-NSC-GLU was preserved intact 1 h post-injection in vivo, which demonstrated that the product was also relatively stable in vivo.
As many 18 F-labeled glutamate imaging agents, [ 18 F]AlF-NOTA-NSC-GLU is almost not incorporated into protein indicating that it also can re ect AA transport rate in tumor. Furthermore, AAs generally enter cells via membrane associated carrier proteins, malignant tumor cells accumulate AAs owing to increasing expression of AA transporters [42]. To investigate the transport mechanism involved in the uptake of the system X C - [29]. In addition, Na + -dependent system X AG and Na + -independent system X C were involved in the transport of [ 18  can use extracellular cystine to exchange for intracellular glutamate [44]. Also, SLC1A5 (ASCT2), SLC7A5 (LAT1), SLC7A11 (xCT) and SLC6A14 (ATB 0+ ) are positively expressed in cancer. It has been found that xCT, the member of system X C transporter, is actively expressed in HCC patients [45]. Cyss, as a substrate of X C system, did not markedly inhibit the uptake of this agent. Hence, sulfasalazine, a speci c inhibitor of X CT -mediated cystine transport, were applied in this experiment. [46]. tumor and low accumulation in healthy liver tissues. The system X AG inhibitor L-Glu, L-Asp and D-Asp was used in vivo competitive binding. Surprisingly, the tumor-to-liver and tumor-to-muscle uptake ratio for

Declarations
Ethical Approval and Consent to participate All applicable international, national, and institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted The Institutional Animal Care and Utilization Committee (IACUU) of the First A liated Hospital, Sun Yat-Sen University (approval number 2018033)

Consent for publication
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Availability of data and materials
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Competing interests
The authors declare that they have no competing interests.