Cell Lines
TC cells were obtained from the cell bank of the State Key Laboratory of Oncology in South China. Specific culture methods and related experiments are provided in the Supplementary materials.
Mice
A total of 1×106 BHT-101 cells were injected into the cervical thyroid position of each BALB/c nude mouse to construct TC tumor-bearing mouse models. The mice were divided into a surgical excision group, imaging group and therapy group. When the tumors reached a diameter of 0.5-1.0 cm, in vivo imaging experiments and treatment were performed. All animal experiments were approved by The Institutional Animal Care and Use Committee at Sun Yat-sen Cancer Center (Ethical code: L102012021080H).
Immunohistochemistry(Ihc)
A total of 134 thyroid patients were recruited from September 1, 2003 to June 22, 2021 in our hospital, including 44 cases of PTC, 31 cases of FTC, 34 cases of MTC, and 25 cases of ATC (including poorly differentiated carcinoma). Paraffin sections of human thyroid carcinoma were obtained from the pathology department. The experiment was approved by the Ethics Committee of Cancer Center, Sun Yat-sen University (Ethical code: B2022-434-01). The details are shown in the Supplementary materials.
Acquired Of Edbp
All non-alanine residues in EDB-FN were mutated using the Alanine scan strategy. We obtained a new set of peptides by the alanine scan mutation technique (in which alanine is substituted by another amino acid in the peptide). Micro-Scale Thermophoresis (MST) was used to evaluate the affinity between alanine scanned peptides and EDB-FN proteins, and the peptide with strongest affinity was selected, namely EDBp. For details are showen in the Supplementary materials.
Cy5-edbp Fluorescent Imaging And Directed Resection Of Mass
Cy5-PEG4-EDBp was synthesized by the Chinese Peptide Company (Lot#: CU-09-00019, Hangzhou, China). Mice in the blocking group (2000 µM EDBp was injected via the tail vein and circle for 1 h, n = 4) and the unblocking group (n = 3) were injected with 2 µM Cy5 at the same time, and NIRF imaging was performed after a 1-h cycle. The fluorescence intensity of the orthotopic tumors was monitored by an IVIS 200 imaging system. Then, the tumors of the mice were removed as cleanly as possible and imaged to observe whether there was any tumor tissue remaining after removal. After the mice were sacrificed, the main organs, such as the kidney and heart, were collected for imaging, and the corresponding ROIs were recorded. The imaging data were analyzed using Living Image 4.7.3 (IVIS 200 Imaging Systems) software.
Preparation Of [18f]-edbp And [177lu]-edbp
EDBp-PEG4-NOTA and EDBp-PEG4-DOTA were synthesized by Chinese Peptide Company (Lot#: CU-07-01467/Lot#: CU-12-00969, Hangzhou, China). EDBp-PEG4-NOTA was mixed with AlCl3.6H2O in a sterile vacuum bottle to produce freeze-dried powder using a lyophilizer (AdVantage EL-85, SP Scientific). We mixed EDBp-PEG4-NOTA (100 µM/bottle) with anhydrous ethanol (330 µl), acetic acid (6.5 µl, pH = 4–5), and [18F]ion (65 µl, 74 MBq/2 mCi) in a 100°C metal bath for 10 min, and cooled it at room temperature for 5 min. Then, we diluted the mixture to 10 ml and passed it through a C18 column twice after washing with 10 ml sterilized water. The [18F]-EDBp was eluted with 400µl 70% ethanol twice and drained with nitrogen.
[177Lu]Cl3 (100 µl, 49.95 MBq/1.35 mCi) was added to a 1 ml solution (pH = 5) containing 100 µM precursor EDBp-PEG4-DOTA and incubated at 95°C for 10 min, followed by 5 min at room temperature. Then, the mixture was diluted to 10 ml and passed through a C18 column twice. After washing with 10 ml sterilized water, [177Lu]-EDBp was eluted with 200 µl anhydrous ethanol twice and drained with nitrogen.
Both [18F]-EDBp and [177Lu]-EDBp were directly used for high-performance liquid chromatography (HPLC, 10 µCi) and diluted with normal saline for cell and animal experiments.
Biodistribution
ATC tumor-bearing mice (n = 6) were blocked with 3000 µM EDBp for 1 hour. Mice in the unblocking group (n = 10) and blocking group (n = 6) were injected with 3.7 MBq (100 µCi)/150 µl of [18F]-EDBp through the tail vein. One hour later, both the unblocking group (n = 6) and blocking group (n = 6) were immediately sacrificed and dissected for the biological distribution study. Two hours after the treatment, the remaining mice from the unblocking group (n = 4) were sacrificed and examined for biological distribution. Organs and tissues, such as liver, heart, lung, and bone, were measured by a γ counter (Automatic Gamma Counter, 2480 WIZARD2), and the distribution of [18F]-EDBp in different organs was analyzed.
Pet/ct Imaging
Mice were injected with [18F]-EDBp (3.7 MBq/150 µl) through the tail vein. PET/CT scans were performed after 1 h of drug circulation. The mice were divided into an unblocking group and a blocking group. In the blocking group, each mouse was injected with 4 mg/1 EDBp in the tail vein before [18F]-EDBp was injected. PET scanning parameter setting: 600-s one-bed acquisition, MLEM algorithm, iteration number 12, scattering, decay, random correction, no attenuation correction. CT parameter settings: 0.4 mA, 45 kV. Reconstruction algorithm FBP (Small animal PET/CT, Albira II, Bruker).
[177lu]-edbp Treatment Of Tc Tumor-bearing Mice
When the diameter of the ATC tumor reached 0.5-1.0 cm, the mice were randomly divided into four groups: saline treatment group (Day 1 (D1): 150 µl, tail vein injection), DOTA-EDBp treatment group (D1: 10 µM/150 µl, tail vein injection), albumin paclitaxel (ABRAXANE) treatment group (15 mg/kg, three times a week for two weeks, intraperitoneal injection), [177Lu]-EDBp treatment group (D1: 7.4 MBq/150 µl, tail vein injection). The physiological status of the mice was observed and recorded daily. The body weight of the mice and the volumes of tumors were measured every two days. The experiment was terminated when the mice died or the tumor diameter reached 1.5 cm. The survival status of mice from treatment to death or termination of the experiment was recorded, and tumor tissues were collected for IHC analysis using Ki-67 antibodies.
Ex Vivo Autoradiography
Paraffin sections of TC tissues of different pathological types were obtained from the pathology department of SYSUCC. The dewaxed and hydrated sections were incubated in 20 µCi/mL [18F]-EDBp Tris-HCl buffer (pH = 8.2) at room temperature for 30 min. Then, the sections were washed with ultra-pure water 6 times and Tris-HCl buffer 2 times. The slices were dried in an oven at 60°C for 5 min until they became white and posted on an imaging film for 2 h. The imaging film was scanned with a phosphor screen imaging system (Typhoon FLA7000IP, GE) at a pixel size of 100 µm, and the PMT was 1000. The data were analyzed using ImageQuant TL8.1 software.
Pet/ct Imaging Of Patients With [f]-edbp
The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Jiangnan University and conducted in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards (). Each patient gave written and informed consent before the PET/CT study. No patient preparation was required for the [18F]-EDBp PET scan. At 5 min and 45 min after intravenous injection (dose of 4.07 ± 0.1 MBq/kg, a total of 296 MBq) of patient #1 and at 1 h after intravenous injection (dose of 3.7 ± 0.1 MBq/kg, a total of 281.2 MBq) of patient #2, the two patients were scanned on a Biograph 64 PET/CT scanner (Siemens). Whole-body (vertex to thigh) PET/CT images were obtained in 3D mode (2 min per bed position). A continuous low-dose CT scan for attenuation correction was acquired in spiral mode with the settings 120 kV, 170 mAs, slice thickness 2 mm, and pitch 0.8. For image analysis, ROIs were measured with a multimodal work station (Syngo.via; Siemens Medical Solutions).
Statistical analysis
Statistical analysis and mapping were performed using SPSS 22.0 and GraphPad Prism 8. Data are presented as mean ± SD. Data were compared using two-way analysis of variance (ANOVA) with Bonferroni correction and Student’s t test. Overall survival was determined by the Kaplan‒Meier method, and the P value was calculated with the Wilcoxon test. The significance is given as *, p < 0.05; **, p < 0.01; ***, p < 0.001.