The NOTA was purchased from Macrocyclic, Inc; protected amino acids, other peptide synthesis reagents, and resins were obtained from Nanchang tanzhenbio Co., Ltd; anhydrous aluminum chloride (AlCl₃) and sodium acetate were purchased from Alfa Aesar (China) Chemicals Co., Ltd; and trifluoroacetic acid (TFA) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). Reagents, including phosphate-buffered saline (PBS) and cell culture medium, were obtained from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from Biological Industries (BI) company. All reagents and solvents were commercial products and used without further purification. The Siemens Cyclotron produced [¹⁸F] fluoride by bombarding a [¹⁸O] H₂O target with 12.5 MeV protons. Analytical HPLC (high-performance liquid chromatography) was performed on an Agilent 1200 system with a reversed-phase column (Agilent Zorbax ODS, 250 × 4.6 mm). Mass spectra were obtained on a Q-T of a premier UPLC system equipped with an electrospray interface (ESI) (Waters, USA). A γ-counter (CAPRA-R, Capintec, Inc., Ramsey, New Jersey, USA) was used to measure the radiation value of the biological distribution experiment. Small animal imaging was performed using a micro-PET/CT scanner (Siemens Inveon Multimodality System, Germany).

Scheme 1 describes the peptides synthesized by the solid-phase synthesis method (26). The purity of the peptides, including NOTA-ADH-1 and Cy3-ADH-1, was identified by high-performance liquid chromatography (HPLC), and the molecular weight was identified by mass spectrometry. They were stored at -20°C after freeze-drying.

Human colorectal carcinoma SW480 and pancreatic BxPC3 cell lines were purchased from the Institute of Biochemistry and Cell Biology, the Shanghai Institute for Biological Sciences, and the Chinese Academy of Sciences. Specific binding studies were evaluated on SW480 and BxPC3 tumor cells. SW480 and BxPC3 cells were cultured in a medium containing high glucose and supplemented with fetal bovine serum, glutamine, and penicillin-streptomycin, and the culture medium was changed every other day. Cells were expanded in tissue culture pans in humidified air containing 5% carbon dioxide at 37°C and detached with trypsin and phosphate-buffered saline for further cell culture when cells were confluent.

To assess the binding ability of the peptide, the Cy3-ADH-1 peptide was synthesized. An equal number (1×10⁵) of SW480 and BxPC3 cells were seeded in 6-well plates. After overnight incubation, 0.5 ml of culture medium and 0.5 ml of Cy3-ADH-1 solution with concentrations 5 μM, 20 μM, 50 μM, 100 μM, and 200 μM were added to the cell chambers in the dark, respectively, and incubated at 37°C in a 5% CO₂ incubator for half an hour. Subsequently, the solution in the 6-well plates was discarded, and the cells were fixed with paraformaldehyde for 10 min. After washing with PBS three times, the cells were mounted in a mounting medium containing DAPI for 15 minutes and then visualized by an Olympus IX71 fluorescence microscope (Olympus, Japan).

A competitive binding assay was performed on SW480 tumor cells. Therefore, 1×10⁵ SW480 cells were placed in 3-well plates, and 0.5 ml of unlabeled ADH-1 solution of 0 μM, 50 μM, and 200 μM concentration were added to each well and incubated at 37°C in binding buffer. After 30 minutes of reaction, 0.5 ml of 10 μM Cy3-ADH-1 solution was added and then incubated in binding buffer at 37°C for another 1 hour to allow fluorescently-labeled polypeptides and excess non-labeled polypeptides to combine with tumor cells. The cells were then washed 3 times with 1ml PBS solution for 5 min each time and were mounted in a mounting medium containing DAPI for 15 minutes. The uptake intensity of SW480 colon cancer cells to fluorescently-labeled polypeptides was observed and measured by a fluorescence microscope.

Scheme 2 describes the radiosynthesis routine of [¹⁸F]AlF-NOTA-ADH-1 (27–31). 100ug of NOTA-ADH-1 was dissolved in 2ml axygen in a centrifuge tube with 400ul pure water; AlCl₃ (26.0 nmol; 13.0 μL; 2.0 mM) in sodium acetate buffer solution (pH 4; 0.2 M) and 1mL acetonitrile was added to the reactor and mixed well. 500μl ¹⁸F target water (1000-1500mCi) was added to the reactor and heated at 100°C for 10min; 5uL of crude production point samples were collected for Thin-layer chromatography (TLC) detection. The remaining crude product was slightly cooled and then transferred to the pre-conditioned HLB cartridge with 15ml water and washed with 10ml PBS and 20ml water, respectively. The product was finally eluted with 2.0 mL of ethanol and water at a ratio of 1:1 (V/V), filtered using a sterile filtration membrane, and collected in the receiving bottle. The product (310-500mCi) was then diluted into a solution containing 5% ethanol using normal saline for injection, and samples were taken for quality control.

The radiochemical yield was monitored by TLC (50% acetonitrile), and the radiochemical purity was confirmed by HPLC using a Zorbax ODS(C18) 4.6 * 250 mm analysis column. The HPLC included the mobile phase A, a 0.1% acetonitrile solution of trifluoroacetic acid (TFA), and phase B, an aqueous solution of 0.1% TFA. The mobile phases were performed to carry out a gradient elution. The ratio of phase A to phase B at 0 min was 10%:90% and gradually rose to 70%:30% at 20 min, which was maintained at a flow rate of 1mL/min.

The stability of [¹⁸F]AlF-NOTA-ADH-1 was tested in PBS and bovine serum. In brief, 3.7 MBq of[¹⁸F]AlF-NOTA-ADH-1 was pipetted into 0.5 mL of PBS and incubated in PBS at room temperature or bovine serum at 37°C. For the study, an aliquot of the solution was directly taken at 1, 6, and 8 h after incubation, and the radiochemical purity was determined by radio-TLC or radio-HPLC under identical conditions.

The partition coefficient of [¹⁸F]AlF-NOTA-ADH-1 was measured by assessing the distribution of radioactivity in 1-octanol and phosphate buffer in a 2 mL centrifuge tube. 20µL of [¹⁸F]AlF-NOTA-ADH-1 solution was added to a tube containing 0.5 mL each of 1-octanol and PBS. The mixture was vortexed and centrifuged (5,000 r/min) for 5 min. A total of 2 samples (50 μL) were collected from each layer and were assayed in a γ counter. Partition coefficients (log Po/w) are shown as the log counts in 1-octanol vs. PBS layers (n=3).

All animal experiments were performed in accordance with and approved by the Institutional Animal Care and Use Committee guidelines of the First Affiliated Hospital of Zhejiang University School of Medicine. BALB/c nu/nu mice (female, 20 ± 3 g, 4- to 6-week-old; Department of Laboratory Animal Science, Zhejiang University) were used in this study. The SW480 and BxPC3 xenografts were generated, and 5×10⁶ tumor cells were subcutaneously injected into the flanks of female athymic nude mice. The PDX-bearing mouse models were established as follows. Pancreatic cancer (diagnosed as pancreatic ductal adenocarcinoma) tissues were obtained from the surgery of a 67-year-old female patient. Written informed consent was obtained from the patient, and the research protocol was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine. The sample was sectioned into about 1mm³ pieces, and the tumor tissues were subcutaneously implanted into BALB/c nu/nu mice flanks. Mice were allowed free access to sterile water and food and were kept under strict disease-free conditions with controlled temperature (~25°C), humidity (50-70%), and circadian rhythms (12-hour light/dark cycles). All necessary procedures were carried out to minimize discomfort and avoid the waste of animals. The length and width of the tumor were measured every other day. Biodistribution studies and positron emission tomography in mice were performed after the tumor volume reached 150 to 200 mm³.

Thirty SPF Kunming mice weighing 18-20g were randomly divided into 6 groups (n=5). As described above, [¹⁸F]AlF-NOTA-ADH-1 was purified and separated by HPLC analysis. Each mouse was injected with a dose of 0.74MBq (20μCi) through a caudal vein. Blood was collected from the orbit, the mice were sacrificed, and the brain, heart, lung, liver, spleen, kidney, stomach, intestine, muscle, bone and other organs were taken at 2 min, 5 min, 15 min, 30 min, 60min and 120min after injection. The radioactivity results were recorded as injected radioactivity per gram of tissue (% ID/g) corrected for background and decay.

The BALB/c nude mice were randomized into different tumor models (SW480, BxPC3, and pancreatic PDX), including 5 mice per group. Each nude mouse was injected with 0.74MBq (20μCi) [¹⁸F]AlF-NOTA-ADH-1 through the caudal vein. Blood was collected from the orbit, and the mice were dissected to collect the brain, heart, lung, liver, liver, spleen, stomach, intestine, muscle, bone and others at 30 min, 30min, 60min, 90min and 120min after injection. In the blocked group, the study was performed with a saturating dose of ADH-1 (20 mg/kg of mouse body weight) intravenously administered 60min before the intravenous injection of [¹⁸F]AlF-NOTA-ADH-1 in the pancreatic PDX model and SW480 xenograft model. The mice in the tumor xenograft model were sacrificed 1h after [¹⁸F]AlF-NOTA-ADH-1 injection. The nude mice were dissected, and tumor tissues and related tissues (blood, brain, heart, lung, liver, spleen, kidney, stomach, intestine, muscle and bone) were weighed, and their radioactivity was measured by γ-counter. The radioactivity results were recorded as % ID/g.

[¹⁸F]AlF-NOTA-ADH-1 was evaluated by micro-PET imaging on nude mice bearing a PDX tumor, SW-480 and BxPC3 tumor xenografts. Each tumor-bearing nude mouse was injected with 3.7MBq [¹⁸F]AlF-NOTA-ADH-1 via the tail vein. After being anesthetized with isoflurane, tumor-bearing nude mice were imaged using a micro-PET/CT scanner at 60 minutes post-injection under continuous isoflurane inhalation using a nasal mask with a connecting tube. Micro-PET/CT scanning was performed after low-dose 3D acquisition CT scanning at 10 minutes per bed. A blocking study was conducted, in which a saturating dose of unlabeled ADH-1 (20 mg/kg of mouse body weight) was intravenously administered 60 min before the intravenous injection of [¹⁸F]AlF-NOTA-ADH-1.

The three-dimensional volume of interest (VOI) was used to assess the standard uptake value (SUV) of selected organs. The Seimens Inveon analysis software configured with micro-PET/CT was used to delineate the region of interest along the edge of the tumor and each normal tissue on the PET image. Subsequently, the SUV of each region of interest was measured, and the tumor/non-tumor ratio (T/NT) between the tumor and normal tissue was measured.

Tumor tissues were fixed in 10% methanol for 4 hours. The samples were then dehydrated in graded ethanol, embedded in paraffin, and sliced into 5 μm sections. Sections were incubated with N-cadherin (mouse monoclonal antibody, 1:200, Abcam) and then goat anti-mouse secondary antibodies (Abcam diluted 1:200). The secondary antibodies were formed by binding horseradish peroxidase (HRP) and Goat anti-mouse IgG. Then, the sections were stained with diaminobenzidine (DAB )staining solution. DAB generates brown precipitates under the catalysis of HRP, thereby amplifying signals and developing colors. After a series of routine processing, immunohistochemical images were finally obtained.

SPSS 26.0 software was used for analysis, and all quantitative data in the experiment were expressed as mean ± SD. t-test analysis was used for the comparison of differences between two groups; analysis of variance was used for comparison between multiple organizations. A p-value less than 0.05 was considered statistically significant.

A standard FMOC solid-phase synthesis was used to synthesize NOTA-ADH-1 and Cy3-ADH-1 ( Scheme 1 ). The synthesized peptide was characterized by High Resolution Mass Spectrometry (HRMS) ( Supplementary Figures S1 , S2 ), and its purity was determined by HPLC, revealing a chemical purity of greater than 95%.

The in vitro results showed that the degree of SW480 cell uptake of Cy3-ADH-1 was concentration-dependent, exhibiting an increasing fluorescence intensity with increasing Cy3-ADH-1 concentration. In contrast, no obvious fluorescence signal was found in the blank control tube ( Figure 1 ). Competitive inhibition assays demonstrated that unlabeled ADH-1 significantly affected the uptake of Cy3-ADH-1 in SW480 cells, with the 200nM inhibition group showing a substantially higher uptake than the 50nM inhibition group ( Figure 2 ). No significant uptake (or fluorescence signal) of Cy3-ADH-1 by BXPC3 cells was observed in the concentration range of 5 μM to 200 μM ( Figure 3 ).

[¹⁸F]AlF-NOTA-ADH-1 was successfully prepared using the [¹⁸F]AlF method ( Scheme 2 ). Thin-layer chromatography was performed on the reaction solution. As shown in Figure 4 , the fluoride ion peak is at the origin, and the Rf of product [¹⁸F]AlF-NOTA-ADH-1 is 0.5. The non-decay corrected radiochemical yield of [¹⁸F]AlF-NOTA-ADH-1 was 28.07 ± 2.42% (n=20), the radioactivity of the radiotracer was 310-500mCi, and HPLC analysis showed that the retention time of the drug was 9.4 min. The radio molar activity of the drug was 153.55 ± 28.25 GBq/mmol, and the radiochemical purity was 98.1 ± 0.6% ( Figure 5 ). The total time of the radiosynthesis reaction was about 30 minutes. The [¹⁸F]AlF-NOTA-ADH-1 product was colorless and transparent, with a pH meter of 6.5. The drug is sterile, with bacterial endotoxin levels per 1mL less than 10 EU. The measured partition coefficient of the product was -2.45 ± 0.09, indicating that the probe was hydrophilic. The drug was stable at 37 °C in PBS and calf serum, and the radiochemical purity was about 95.5 and 95.0%, respectively, after 8 h. The results indicated that [¹⁸F]AlF-NOTA-ADH-1 was stable in vitro.

The research results showed that [¹⁸F]AlF-NOTA-ADH-1 had rapid blood clearance. After injection of [¹⁸F]AlF-NOTA-ADH-1, the radioactive concentration of [¹⁸F]AlF-NOTA-ADH-1 in blood was 18.84 ± 1.95%ID/g at 2 min, 8.77 ± 1.37%ID/g at 5 min, 3.79 ± 1.19%ID/g at 15 min, and 2.28 ± 0.81%ID/g at 30 min, which decreased significantly to 1.04 ± 0.36%ID/g at 120min after injection. [¹⁸F]AlF-NOTA-ADH-1 is widely distributed in the kidney and the intestines, indicating that [¹⁸F]AlF-NOTA-ADH-1 is mainly excreted from the urinary system and hepatobiliary systems. In addition, lower uptake was observed in the lungs, and muscle tissues and almost no uptake was seen in the brain tissue, suggesting that the probe does not efficiently cross the blood-brain barrier ( Table 1 ).

The biological distribution of normal tissues in tumor-bearing nude mice was similar to that of normal mice, with high radioactive uptake in the liver and kidneys ( Tables 2 – 4 ). To investigate the tumor uptake of [¹⁸F]AlF-NOTA-ADH-1 in the pancreatic PDX xenograft model, tumors, blood, and tissue/organs were excised to measure the radioactivity. The highest tumor uptake was observed at 60 min after injection, suggesting that this may be an appropriate time for tumor imaging. In the blocking group, the radioactive uptake of the tumor was 3.75 ± 0.07%ID/g at 60 min p.i, significantly lower than that in the non-blocking inhibition group (t=5.304, p=0.006). In SW480 tumor-bearing nude mice, the tumor/muscle (T/M) ratio was the highest at 60min (1.91 ± 0.69). In the blocking inhibition experiment, the radioactive uptake of the tumor was 1.07 ± 0.57%ID/g at 60min p.i, lower than that in the non-blocking group (p=0.004). In the BxPC3 tumor model, no significant uptake of [¹⁸F]AlF-NOTA-ADH-1 was observed in the tumor tissue.

Micro PET studies of [¹⁸F]AlF-NOTA-ADH-1 were performed on nude mice bearing pancreatic PDX, Human Colorectal Carcinoma SW480, and pancreatic BxPC3 tumor xenografts at 60 min p.i. A significantly increased radioactive uptake of tumor tissue was shown in the PDX tumor model, with a tumor/muscle ratio of 8.069±2.832. Unlabeled ADH-1 inhibitors can significantly reduce the T/NT ratios, with a T/Bone of 2.263 ± 0.465, and a T/Lung of 5.062 ± 0.805 ( Figure 6 ). The T/NT ratios showed significant differences between the non-blocked group and the blocked group, including T/Muscle (p=0.003), T/Bone (p=0.023), T/Kidney (p=0.0001), T/Liver (p=0.002), and T/Lung (p=0.001). Immunohistochemical staining showed strong positive N-calcium adhesion expression in tumor tissue, supporting the high specificity of [18F]AlF-NOTA-ADH-1 binding to tumors expressing N-calcium adhesion in vivo.

As shown in Figure 7 , micro-PET imaging of the SW480 tumor-bearing nude mouse model 60 min after injecting [¹⁸F]AlF-NOTA-ADH-1 revealed high radioactive uptake in the tumor with a tumor/muscle ratio of 1.443±0.121, T/Bone of 1.903±0.273, and T/Lung of 3.359±2.998, unlabeled ADH-1 inhibitors can significantly reduce the T/NT ratios, too. A significant difference in T/NT ratios was observed between the non-blocked group and the blocked group, including T/Muscle (p=0.019), T/Bone (p=0.002), T/Liver (p=0.001), and T/Lung (p=0.008). Immunohistochemical staining showed positive N-calcium adhesion expression in the tumor tissue. However, no significant increase in radioactive uptake was observed in the tumor tissues of BxPC3-bearing tumor model mice, with a tumor/muscle ratio of 1.018 ± 0.498, T/Bone 0.055 ± 0.022, and T/lung 0.291 ± 0.195. Immunohistochemical staining of N-cadherin showed low expression in tumor tissues ( Figure 8 ).