2.1 Cells and mice
The HCC cell line used in this study is HCC-LM3 cells purchased from the American Type Culture Collection (ATCC). This cell line was cultured with DMEM containing 10% foetal bovine serum. BALB/c mice and C57BL6 mice in this study were purchased from Vital River, Charles River Laboratories China (Beijing, China). The animal experiments were accredited through the Institutional Animal Care and Use Committee (IACUC) in Sun Yat-sen University Cancer Center.
To facilitate monitoring of tumorigenesis, HCC-LM3 cells were stably transfected with luciferase to form HCC-LM3-luciferase cells. Anesthetized mice were surgically exposing their livers in a sterile environment. Then the HCC-LM3-luciferase cells were injected orthotopically to the left lobe of the liver under direct observation. One week after surgery, the tumorigenesis of mice was confirmed by bioluminescence imaging.
For the construction of the chemical induced HCC mouse model, a single dose of diethynitrosamine (DEN) (200 mg/kg) was administered into male C57BL6 mice to induce HCC. Mice were scanned via MRI to verify the presence of HCC lesions in the livers 36 weeks after injection.
H11LNL-Myc knock-in HCC mouse model was developed by Shanghai Model Organisms Center, Inc. This model was generated by CRISPR/Cas9 system in C57BL/6 J mouse background. Technical details are the same as described in previous literature (Feng et al. 2019). F0 mice were crossed with C57BL/6 J mice to gain H11LNL-Myc heterozygous mice. H11LNL-Myc; Alb-Cre double positive mice used in this study were generated by crossing H11LNL-Myc heterozygous mice with Alb-Cre transgenic mice.
2.2 Preparation of 18F-S3
NOTA-CSISSLTHC (NOTA-S3) were synthesized by the Chinese Peptide Company (Hangzhou, China) as following procedure: NOTA bis(tBu)ester and full covered peptide was once dissolved in N,N-Dimethylformamide (DMF). Then 2-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-methylmorpholine (NMM) were introduced to the reaction mixture. The response mixture was stirred at room temperature for 3 hours, then workup to supply the NOTA-modified peptide which was cleavage with the aid of tallow fatty acid to provide the crude peptide. Last, it was purified via HPLC to provide final product. The peptide is available as a freeze-dried powder freely soluble in saline solution. Then, the radiolabeling reaction procedure was as follows: the vials containing 8 nmol of NOTA-S3 and 6 nmol of AlCl3·6H2O was added with 65 μl of 18F− (about 10 mCi) in DI water, 3 μl of acetic acid and 324 μl acetonitrile, heated at 100 °C for 10 min. After the vial was cooled to room temperature, 10 ml of water was added to dilute the mixture. Then the mixture was trapped on a Varian Bond Elut C18 column (100 mg) using a 20-ml syringe. After washed with 10 ml of water, the C18 column was eluted with 0.4 ml of ethanol twice. The solution was dried by a vacuum dryer. The final product 18F-ALF-NOTA-S3 was dissolved with saline solution before injection.
2.3 PET/CT imaging
The mice were fasted from food but not water for 6 hours prior to the imaging experiments. Mice were anesthetized with 1.2% tribromoethanol solution at a dose of 250mg/kg and then intravenously injected with approximately 60~100 μCi (2.22~3.7MBq) of 18F-S3. PET/CT scans were performed after 1~1.5 hours of circulation. While waiting, the mice were kept warm by an electric blanket. After acquiring the images, regions of interest (ROIs) were drawn on the tumor and normal organs. The maximum uptake value was calculated automatically, and the tumor boundary was differentiated by a low threshold of 30% of the maximum uptake. The radiation intensity of tumor and normal organs was calculated as percentage of the injected dose per gram (%ID/g).
2.4 Biodistribution
In biodistribution analysis from static PET/CT images, ROIs were placed over the tumor and major organs (liver, heart, lung, intestine, kidneys, brain and muscle) through layered sketching with the assistance of corresponding CT images. The results were expressed in percentage of the injected dose per gram (%ID/g).
2.5 Immunohistochemistry
Samples were fixed with formalin, embedded in paraffin, sectioned, and stained with H&E in accordance to standard histopathological techniques. Paraffin sections were de-paraffinized with xylene and rehydrated. The sections were processed in citrate buffer (pH 6) and microwaved for antigen retrieval. To block nonspecific binding, the sections were incubated with 1% bovine serum albumin after a treatment with 3% hydrogen peroxide in methanol and quenching the endogenous peroxidase activity. The sections were then stained with an NKAα1 antibody (Abcam, M7-PB-E9, 1:150) for 12 h at 4 °C. After washing, the sections were incubated with an HRP-conjugated polyclonal secondary antibody (1:200). The sections were immersed in 3-amino-9-ethyl carbazole, counterstained with 10% Mayer's hematoxylin, dehydrated, and mounted in crystal mount.
2.6 Statistical analysis
Statistical analyses were performed using R 4.0.3. Data were presented as means ± SD. Means were compared using Student’s t test. The significance is given as < 0.05 (⁎ p < .05, ⁎⁎p < .01, ⁎⁎⁎p < .001).