General information
All chemicals applied in the synthesis were commercially sourced and used without further purification unless otherwise indicated. [18F]FDG was radiolabeled as previously described[28]. Sep-Pak light QMA and Plus C-18 cartridges were purchased from Waters Corporation (Milford, MA, USA). Sep-Pak light QMA cartridges were preconditioned with 10 mL of NaHCO3 aqueous (8.4%) and water in advance. Preconditioning of the Plus C-18 cartridge was performed with 10 mL of ethanol followed by 10 mL of water. Micro PET/CT scanner by Siemens (Germany) was used.
Animals models and cell culture
The human HCC Hep3B 2.1-7 cell line was provided by Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). The cells were cultivated in DMEM medium containing 10% fetal bovine serum and 1% penicillin streptomycin at 37℃ in a humidified atmosphere of 5% CO2 and 95% air.
Male BALB/c nude mice (4-6 weeks old and weighing 18-22 g) for tumor-bearing models were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Normal Kunming mice (male, 6-8 weeks old, 20-25 g) for biodistribution studies of [18F]AIF-NOTA-NSC-GLU were supplied by the Laboratory Animal Center of Sun Yat-Sen University (Guangzhou, China). HCC Hep3B 2.1-7 cells (1-2 × 10^7) were subcutaneously implanted in the left or right axillary and allowed to grow for 2-3 weeks. Mice were used for imaging when tumors grew to 10-15 mm in diameter.
The experiments were conducted in accordance with the recommendations and guidelines of the Institutional Animal Care and Utilization Committee (IACUU) of the First Affiliated Hospital, Sun Yat-Sen University (approval number 2018033). All animals were housed 5 animals per cage under standard laboratory condition.
Synthesis of [18F]AIF-NOTA-NSC-GLU
The NOTA-NSC-GLU was synthesized by Shanghai Apeptide Co. Ltd. (Shanghai, China), with > 95% purity. 18F dissolved in water was passed through a preconditioned Sep-pak QMA cartridge. Then, 18F 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 flow, and the final product was reconstituted in normal saline for further studies (Fig. 1).
In vivo biodistribution studies
For the biodistribution experiment in vivo, twenty Kunming mice were divided into 4 groups randomly and injected intravenously (IV) with 20-40 μci of [18F]AIF-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 18F 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 2.1-7 cells were seeded into 24-well plates and reached the logarithmic proliferation phase, we started the transport assays. The methods and transport mechanism of [18F]AIF-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 (Gln) for system ASC, 2-amino-2-norbornane-carboxylic acid (BCH) for system L, L-glutamate (L-Glu) for system XC− and XAG−, cystine (Cyss) for system XC−, L-aspartic (L-Asp) and D-aspartic (D-Asp) for system XAG− were applied. Concentration of the inhibitors was 15 mmol/L. Cells with [18F]AIF-NOTA-NSC-GLU (an average of 8 KBq per well) and the inhibitors were incubated at 37℃ for 10 min. After washing 3 times with ice-cold NaCl or Choline Chloride medium, the activity of cells was measured by γ counter (GC-1200, USTC Chuangxin Co. Ltd. Zonkia Branch, China). To further evaluate the role of system XC− in the uptake of this agent, sulfasalazine, as an inhibitor of XC− system, was used in competitive inhibition experiments in vitro. The experiment was conducted with as previously described[30] and divided into three level of concentration: 100 μM, 200 μM, 300 μM.
In vitro and in vivo stability and octanol–water partition coefficient study (logP)
For the stability tests in vivo, mice were injected with 11.1 MBq (300 μci) of the [18F]AIF-NOTA-NSC-GLU (0.2 mL) via the tail vein. The mice were sacrificed at 1 h post-injection. Blood samples were collected from the eyeballs, and then centrifuged (6,000 rpm, 4 min) to separate plasma and used for the HPLC analysis.
Additionally, a sample of [18F]AIF-NOTA-NSC-GLU (1.48 MBq, 20 μL) dissolved in normal saline was incubated with 200 μL of fetal bovine serum at 37℃ for 120 min. An aliquot of the serum sample was filtered through a 0.22 μm Millipore filter and used for the HPLC analysis.
For the octanol–water partition coefficient study, 20 μL of [18F]AIF-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 of 3.70-7.40 MBq (100-200 μci) of [18F]AIF-NOTA-NSC-GLU in 100-200 μL of saline under 5% chloral hydrate solution (6 mL/kg) anesthesia in tumor-bearing mice (HCC Hep3B 2.1-7 cells). The animals were kept fasting for at least 4 h before injection of tracer and visually monitored for breathing throughout the entire imaging period. Then, ten-minute static PET images acquisition was performed at three time points (30, 60, 90 min) post-injection. For a comparative study, experimental animals were anesthetized with 5% chloral hydrate solution prior to scanning with [18F]FDG (4–6 MBq) at 60 min post IV injection. In vivo competitive binding experiments used respective models of L-Glu, L-Asp and D-Asp (30% of Lethal Dose 50 (LD50), intraperitoneal injection, n=3). To perform the inhibition experiments for XAG− system, each inhibitor was injected 15 min prior to the injection of [18F]AIF-NOTA-NSC-GLU and small animal PET/CT imaging was conducted at 30 min after administration of the tracer.
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 man 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 ROI-derived %ID/g. Finally, an imaging ROI-derived %ID/g as well as tumor-to-background relative uptake ratio was obtained.
Incorporation of [18F]AIF-NOTA-NSC-GLU into Protein
The method of determining the extent of protein incorporation of [18F]AIF-NOTA-NSC-GLU was previously reported [31]. Briefly, 400 μL(185-296 KBq) [18F]AIF-NOTA-NSC-GLU was added to the Hep3B 2.1-7 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-fixed, paraffin-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 significant, and P<0.0001 was considered to indicate meaningful differences.