1. Synthesis and Radiolabeling
The HYNIC-FAPI probe were chemically modified and synthesized from Nanchang Tanzhen Biotechnologies Co., Ltd. (Jiangxi, China) (Fig. 1). 99mTc is obtained from the 99mTc generator obtained by China Atomic Energy Research Institute (Beijing, China). 10mg Tricine (Tricine buffer, free acid) and 20mg TPPTS (Tris(3-sulfonatophenyl) phosphine, sodium salt) dissolved in 5ml saline (95%), then the 200μl mixture above was added 50μl HYNIC‑FAPI (1μg/μl) and 50μl 99mTc (185 MBq). The mixture was heated at 100 °C for 30 min to synthesize 99mTc-HYNIC-FAPI. The radiolabeling yield of 99mTc-HYNIC-FAPI was checked by Instant thin-layer chromatography (ITLC).
2. Quality Control and Stability Study
The radiochemical purity was determined by High Performance Liquid Chromatography (HPLC) under working conditions maintained with a flow rate of 0.1% TFA/water (A) and 0.1% TFA/acetonitrile (B) flow rate of 1 mL/min. The injection volume was 20 μL and detection time determined as 20 min (12%-32% B). ITLC was also carried out for the assessment of 99mTc-HYNIC-FAPI stability in vitro at 0,2,4,6h. 99mTc-HYNIC-FAPI was incubated in saline and fetal bovine serum at room temperature and 37 °C respectively (99mTc complexes: saline/serum=1:1).
3. Cell culture and transfection
MCF-7 human breast cells were purchased from Procell Life Science&Technology CO.,Ltd. (Wuhan, China), The cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum and 1% penicillin−streptomycin solution at 37°C/5% carbon dioxide. Lentiviruses encoding the human FAP-gene were obtained from Genechem Technology Co., Ltd. (Shanghai, China). All transfections were implemented using according to manufacturer’s instructions. The lentiviral vector was transiently transfected into MCF-7 cells. Stable cell lines were screened with puromycin.
4. Western blot
Total protein was extracted using ice-cold whole cell lysis assay (KeyGEN BioTECH). After centrifugation at 14,000×g for 4°C,15 min and protein quantification by using BCA protein assay kit (KeyGEN BioTECH). Then, the samples were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore) for 1.5h. The membranes were blocked with 5% bovine serum albumin (BSA) for 1.5h at room temperature. Next, the target proteins were incubated with the anti-Fibroblast activation protein (1:1000, Abcam) and the anti-GAPDH (1:5000, Bioss) overnight at 4 °C and subsequently washed three times by Tris Buffered Saline with Tween 20(TBST-1 ×). After incubation with the secondary antibody (1: 5000, Bioss) at room temperature for 1h, and washed thrice. The protein was visualized in the Bio-Rad Imager.
5. Cellular uptake
MCF-7-FAP and MCF-7 cells were incubated 5 105 cells/1mL in 12-well plate overnight. The cells were divided into 99mTc-HYNIC-FAPI, block and control groups. The control group was considered that MCF-7 cells were added to 99mTc-HYNIC-FAPI. Then, 1μL HYNIC-FAPI (1 μg/μL) was added to block group. After 30 min,2μL 99mTc-HYNIC-FAPI (37kBq) was added directly into 99mTc-HYNIC-FAPI and control groups. The cells were incubated with the radiolabeled compound for 3h at 37 °C. All wells were pashed three times by 500μL PBS. Next, trypsin containing EDTA was used to lyse the cells, and the cells were pashed thrice. Radioactivity was evaluated by Automatic Gamma Counter (Perkinmer). The radioactivity counts of cells and medium were measured respectively.
6. Cell wound scratch assay
PANC-1 cells were seeded in six-well plates. When the cell density reached 90%, the monolayer was scratched using a 200 μL plastic pipette tip, and the cells were washed with PBS to remove the floating cells. Then, the experiment was divided into 99mTc-HYNIC-FAPI,block and 99mTc. Images were acquired by inverted microscopy at 0h and 24h later. Calculation formula: Migration area (%) = (0h scratch area-24h scratch area)/ 0h scratch area100%.
7. Statistical analysis
All results are presented as the mean ± standard deviation (SD). Compared with the means of different groups was performed using Independent-sample t test by GraphPad Prism 7. Statistical significance was defined when p < 0.05.