Chemistry
All reagents and solvents were purchased commercially, were of analytical grade and used without further purification. High-resolution mass spectrometry (HRMS) data were acquired using a Thermo Fisher Scientific Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The synthesis of non-radioactive compounds, which includes reference compounds 19F-P-FAPI and 19F-FAPI-42, and their precursors are described in detail in Supplementary Schemes S1 and S2.
Radiochemistry and Quality Control
No-carrier-added [18F]fluoride was synthesized using an 18 MeV proton bombardment of a high pressure [18O]H2O target utilizing a General Electric PET trace biomedical cyclotron (PET 800; GE, USA). Sep-Pak Plus QMA and Sep-Pak C18-Light cartridges were purchased from Waters Associates. Radioactivity was quantified through the use of a Capintec CAPRAC-R dose calibrator (NJ, USA).
18F-labeled FAP tracers labeling via Al18F-chelation were produced on a modified AllInOne synthesis module (Trasis, Ans, Belgium), as was previously reported [9,17]. After production in a cyclotron, [18F]F− were transfer into the module and trapped on a Sep-Pak Plus QMA, which was preconditioned with 5 mL of 0.5 M sodium acetate buffer pH 3.9 and 10 mL of water. Subsequently, [18F]F− (37~74 GBq) was eluted using 0.35 mL of 0.5 M sodium acetate buffer pH 3.9 to a mixture of AlCl3 (40.0 nmol, 20.0 μL, 2.0 mM in 0.2 M sodium acetate buffer pH 4.0) and precursor (P-FAPI or NOTA-FAPI-42, 80.0 nmol) in 300 μL DMSO. After being heated for 15 min at 105 °C, the mixture was then cooled and diluted with 5 mL of water. Next, the diluted solution was transferred over an activated C18 cartridge, which was preconditioned with 5 mL ethanol (EtOH) and 10 mL water. Afterwards, the cartridge was washed with 20 mL of water and flushed with nitrogen sequentially. The desired radiolabeled compound was eluted from the C18 cartridge with 1 mL of ethanol/water (1/1, v/v), and the C18 cartridge was flushed using 10 mL of NaCl 0.9%. The eluate was passed through a sterile Millipore filter (0.22 μm) into a sterile vial. The final drug product solution was evaluated quality control, which is described in more detail in Supplementary Method.
Surface Plasmon Resonance (SPR) Binding Assays
The experiments were performed using PlexAray HT (Plexera Bioscience, Seattle, WA, USA). The FAP ligands (10 mM in ddH2O) were prepared in 384 wells and spotted on 3D photocrosslinked (PCL) chip in a UV-free room with a humidity of 45%. The obtained PCL chips were then dried in a vacuum chamber in order to evaporate the solvent, and the chips were irradiated for 15 min using a 365-nm UV cross-linker instrument (UVJLY-1; Beijing BINTA Instrument Technology Company). The irradiated energy on the surface then amounted to 2.8 J/cm2. After undergoing UV treatment, the chips were rinsed in N,N-dimethylformamide (DMSO), EtOH, and ddH2O for 15 min each. Finally, the chips were dried under nitrogen gas for further use. In order to quantify the interaction between immobilized biomolecules and flowing proteins, an SPR imaging instrument (Kx5; Plexera) was utilized to monitor the whole procedure in real-time. In brief, a chip with well-prepared biomolecular microarrays was assembled using a plastic flow cell for sample loading. The optical architecture and operation details of the PlexArray HT were previously described elsewhere [18]. The protein sample was then prepared at the appropriate concentration in PBS running buffer while a 10 mM glycine-HCl buffer (pH 2.0) was used as a regeneration buffer. A typical binding curve was acquired using a flowing protein sample at 2 μL/s for 300-s association, and then the flowing running buffer for 300-s dissociation, followed by a 200-s regeneration buffer at 3 μL/s. In order to obtain results for binding affinity, seven gradient concentrations of the flowing phase (2000 nM, 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.3 nM) (protein sample) were prepared and flowed, respectively. All binding signals were converted to standard refractive units (RU) by calibrating every spot with 1% glycerol (wt/vol) in running buffer with a known refractive index change (1,200 RU). Binding data was collected and evaluated using a commercial SPR imaging analysis software (Plexera SPR Data Analysis Model; Plexera).
Partition Coefficient
The n-octanol/PBS partition coefficients of 18F-P-FAPI, 18F-FAPI-42, and 68Ga-FAPI-04 were determined, as previously described [19].
Cell lines
Human cell lines A549 (ATCC) was maintained in DMEM (Gibco, USA) supplemented with 10% FBS (Fetal bovine serum, Gibco, USA) and 1% streptomycin and penicillin (Gibco, USA) at 37 ℃ in 5% CO2. The A549-FAP cell line, stably expressed human FAP, was obtained by lentiviral infection, following 2 weeks screening with 2 μg puro (puromycin, Thermo Fisher, USA).
Cell Studies and Animal Models
The detailed protocols for in vitro cell studies and animal experiments, including cell uptake, efflux, internalization, and tumor transplantation, are provided in the Supplemental Information File.
In Vitro Stability and In Vivo Stability
Detailed procedures for in vitro and in vivo stability of 18F-P-FAPI and 18F-FAPI-42 are included in the Supplemental Information.
MicroPET Imaging
For dynamic micro-PET imaging studies, either 18F or 68Ga labeled tracer (5.55–11.1 MBq, n = 3) were injected through the lateral tail vein into mice bearing A549-FAP tumors xenograft utilizing an Inveon Micro-PET/CT scanner (Siemens; Erlangen, Germany). Image studies were conducted through the use of a three-dimensional ordered-subset expectation maximum (OSEM) algorithm (Siemens, Erlangen, Germany). For the blocking study, A549-FAP tumor-bearing mice were co-injected using DOTA-FAPI-04 (100 nmol/mouse, n = 3) as competitor. The images and regions of interest (ROIs) were produced through the use of a software, Inevon Research Workplace 4.1 (Siemens, Erlangen, Germany).
Biodistribution Studies
Organ distribution studies were carried out using mice with xenografted A549-FAP tumors with and without DOTA-FAPI-04 (100 nmol/mouse, n = 3). The mice were then injected through the tail vein using 18F-P-FAPI (1.11–1.85 MBq), and were euthanized at 1 h post-injection (p.i.). Organs of interest and tumor were quickly dissected, weighed and radioactivity was quantified through the use of a γ-counter and calculated as percentage of injected dose per gram of tissue (%ID/g).
PET/CT Imaging and Analysis
The PET/CT imaging study was performed on a total-body PET/CT scanner (uEXPLORER, United Imaging Healthcare; Shanghai, China) and granted approval by the Ethics Committee of Nangfang Hospital (No. NFEC-2020-205). These were conducted in accordance with Declaration of Helsinki. The patient signed an informed consent form prior to study participation. A-41-year-old patient with nasopharyngeal cancer was examined using 18F-FDG and 18F-P-FAPI on two consecutive days. The patient received an intravenous injection of 18F-FDG (3.7 MBq/Kg) while fasting for at least 6 h on the first day and 18F-P-FAPI (3.7 MBq/Kg) on the second day without fasting. After 1 h post-injection of 18F-FDG or 18F-P-FAPI, the patient was imaged and PET images were reconstructed through the use of ordered subsets expectation maximization algorithm (2 iterations, 10 subsets, 192 × 192 matrix) and corrected for CT-based attenuation, dead time, random events, and scatter [20].
Statistical Analysis
Data was found to be indicated as mean ± standard error of the mean (SEM) and significance of comparison between the two data sets was determined through the use of SPSS 22.0 software (IBM Corp., Armonk, NY). Significance was defined as P < 0.05.