Experimental study of internal irradiation with a novel targeted FAPI molecular probe for the treatment of human pancreatic cancer xenografts

Experimental study of internal irradiation with a novel targeted FAPI molecular probe for the treatment of human pancreatic cancer xenografts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Experimental study of internal irradiation with a novel targeted FAPI molecular probe for the treatment of human pancreatic cancer xenografts Weiwei Ren, Yixuan Zhou, Yushan Li, Qingyi Lai, Kaixuan Lv, Jiangyuan Chen, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7145341/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Objective: The purpose of this study was to analyze and compare the therapeutic effects of 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 on pancreatic cancer cell xenografts. Methods: ① The in vitro antitumor effects of FAPI-Y4, FAPI-caerin 1.1 and FAPI-46 were verified by CCK8 and colony formation assays. ② FAPI-Y4, FAPI-caerin1.1 and FAPI-46 were labeled by a radioiodine demethylation reaction, and their basic properties were determined. The uptake and elution of 131 I-FAPI-Y4, 131 I-FAPI-caerin 1.1 and 131 I-FAPI-46 in human pancreatic cancer cells were studied by cell uptake and elution assays. ③ A PANC-1 nude mouse model was established to compare the efficacy of internal irradiation with that of a novel targeted molecular probe in the treatment of pancreatic cancer.H&E staining was used to detect dead cells in the tumor tissue sections, and immunohistochemical staining was used to analyze the expression of the FAP antibody in the tumor cells. Results: ① In vitro, FAPI-caerin1.1 and FAPI-46 inhibited the proliferation of human pancreatic cancer cells and normal pancreatic ductal epithelial cells in vitro in a concentration-dependent manner, and their inhibitory effects were significantly different ( P < 0.05), whereas FAPI-Y4 had no significant inhibitory effect on the proliferation of human pancreatic cancer cells or normal pancreatic ductal epithelial cells in vitro, indicating potential biological safety. ② In vivo experiments revealed that 131 I-labeled FAPI-Y4 had better antitumor activity than 131 I-labeled FAPI-caerin1.1 and FAPI-46. Conclusion: 131 I-labeled FAPI-Y4, FAPI-caerin1.1 and FAPI-46 significantly inhibited the proliferation and growth of PANC-1 cells ( P < 0.05), and 131 I-FAPI-Y4 has a more significant therapeutic effect than the other compounds and may be potential therapeutic drugs for human pancreatic cancer. pancreatic cancer PANC-1 cells 131I-labeling FAPI-Y4 FAPI-caerin 1.1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Pancreatic ductal adenocarcinoma is the world's deadliest cancer and will soon be the second leading cause of cancer death in the United States, with a 5-year overall survival (OS) rate of only 9% [ 1 , 2 ] . Diagnosis often occurs at an advanced stage, with high rates of malignancy and a poor prognosis. Over the past century, significant advances have been made in the treatment of pancreatic cancer, including improvements in surgical techniques and adjuvant and neoadjuvant therapies [ 3 ] . Despite these advances, the incidence of pancreatic cancer continues to increase. Therefore, it is important to develop new treatments that can improve survival. Fibroblast activation protein (FAP) is a type II transmembrane glycoprotein that is overexpressed in cancer-associated fibroblasts, such as those in lung, liver, stomach, breast, colorectal, pancreatic, and ovarian cancers [ 4 , 5 ] , but has low expression levels in normal fibroblasts. Therefore, FAP is considered a promising target for tumor imaging and therapy [ 6 ] . Radiolabeled fibroblast activation protein (FAP) inhibitors (FAPIs) have shown promise as cancer diagnostic drugs. However, owing to the rapid kinetics of FAPI, 89% of the injected dose is excreted through the urine 3 hours after injection, resulting in low ligand residues in the body [ 7 ] . One of the characteristics of FAPI is that it is rapidly distributed and has a lower retention rate in tumors than other compounds. Lindner et al compared a series of FAPI derivatives and reported that FAPI-46 has a relatively good retention rate [ 8 ] , but it is still very low. Therefore, we hypothesized that connecting small-molecule polypeptides through linkers would improve the retention time of FAPI-46 in tumors. Studies have shown that 177 Lu-FAPI-46 plays a role in the treatment of human pancreatic cancer xenografts [ 9 ] , but its therapeutic effect is limited. The host defense peptide Caerin 1.1 (GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH 2) is a polypeptide from the glandular secretions of Australian tree frogs [ 10 ] . Over the past few decades, several host defense peptides have been isolated and identified from the skin secretions of Australian tree frogs and toads. Many of these peptides have antibacterial or neuropeptide activities, and some peptides have high activity against cancer cells [ 11 ] . Our team's previous studies revealed that caerin1.1 has inhibitory effects on thyroid cancer cells, lung cancer cells, esophageal cancer cells and other cancer cells. 131 I-labeled caerin 1.1 ( 131 I-caerin 1.1) has a stronger antitumor effect than caerin 1.1 does.However, with the gradual increase in the drug concentration to a certain extent, caerin 1.1 began to produce inhibitory effects on normal cells. To improve safety, we modified the caerin 1.1 polypeptide and replaced part of the amino acid sequence to obtain a new small-molecule polypeptide, TFMP-Y4 (YGLFGVLGSAKHVLPHVVPVIAEHL-NH2). Our team's previous studies revealed that 131 I-TFMP-Y4 has a significant inhibitory effect on tumor cell proliferation. To improve the effectiveness of tumor therapy, we hypothesized that FAPI-46, TFMP-Y4 and caerin 1.1 were connected through linkers to construct new targeted molecular probes, namely, 131 I-FAPI-TFMP-Y4 and 131 I-FAPI-caerin 1.1, to improve the effectiveness of tumor therapy. 2. Materials and methods 2.1 Cell lines and cell cultures Human pancreatic cancer cells (PANC-1 cells) were obtained from the Chinese Academy of Sciences Stem Cell Bank. The medium used for PANC-1 cells was (by volume) 89% DMEM (GIBCO, USA), 10% heat-inactivated fetal bovine serum (FBS, Corning, USA) and 1% penicillin‒streptomycin solution (GIBCO, USA). The cells were cultured in an incubator (Thermo, USA) at 37°C with 5% CO 2 . 2.2 FAPI new probe component FAPI-TFMP-Y4, FAPI-caerin1.1, and FAPI-46 were synthesized by Jiangxi Tanzhen Biotechnology Co., Ltd., and their purities were determined to be > 98% by reversed-phase high-performance liquid chromatography. For use, FAPI-TFMP-Y4, FAPI-caerin1.1, and FAPI-46 were dissolved in a mixed solution of DMSO and phosphate-buffered saline (PBS) (DMSO:PBS = 1:1) at different concentrations (5 mg/mL, 1 mg/mL and 0.1 mg/mL). 2.3 Nude mice Four- to six-week-old SPF-grade BALB/c adult female nude mice were purchased from the Guangdong Provincial Medical Experimental Animal Center and raised under SPF-grade conditions at the Animal Resource Center of the First Affiliated Hospital of Guangdong Pharmaceutical University. No mice became ill or died before the end of the experiment. Nude mice were euthanized by cervical dislocation according to the "Animal Management Measures" issued by the Ministry of Health of the People's Republic of China (a document of the Ministry of Health of the People's Republic of China). All experiments were approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University and followed its guiding principles (ethical approval number: FAHGPU 20160316). 2.4 CCK-8 assay The cytotoxicity of different polypeptides was examined using the CCK-8 assay. For the experiments, PANC-1, PANC-1-FAP, and HPDE6-7 cells were selected as experimental cell lines. Using the P3 polypeptide as a positive control, the cytotoxicities of FAPI-46, FAPI-TFMP-Y4, and FAPI-caerin1.1 were compared. After the cells had grown to a confluence of more than 80%, a uniform cell suspension was prepared and inoculated into a 96-well plate, with 100 µL of cell suspension in each well. A total of 5×10 3 cells/well were inoculated and preincubated at 37°C and 5% CO 2 for 24 hours in an incubator. Sample solutions treated with different concentrations were subsequently added, and the medium was replaced, followed by continued culture for 24 hours. The enhanced CCK-8 solution was added by replacing 10 µL of the medium in each well, followed by incubation for 0.5 to 2 hours. The absorbance at 450 nm was measured using a microplate reader (Thermo Scientific, USA). The half-maximal inhibitory concentration (IC 50 ) values and cell viability (S%) were calculated for each drug using GraphPad Prism. 2.5 Colony formation experiments PANC-1 and PANC-1 FAP cells were seeded in four 6-well plates at a density of 800 cells/well, gently shaken uniformly, uniformly distributed in 6-well plates under a microscope and incubated at 37°C in a 5% CO 2 incubator for 24 hours. After the cells were uniformly adherent and had grown well under the microscope, different concentrations of FAPI-TFMP-Y4 and FAPI-caerin 1.1 were added (0 µg/ml, 3 µg/ml, 6 µg/ml, 9 µg/ml, 12 µg/ml and 15 µg/ml). After mixing, the samples were incubated at 37°C in a 5% CO 2 incubator for 24 hours. The medium was changed every 2 days, the cell status and colony number were observed, and the culture was terminated when most cell clusters consisted of approximately 50 cells per well with a drug concentration of 0 µg/ml. After 10 days of coculture, the cells were fixed with 4% paraformaldehyde (Sigma, USA) for 20 minutes, stained with crystal violet solution (Beyotime, China) for 15 minutes, washed with PBS for 3 minutes, and naturally dried. The cell colonies were counted using ImageJ analysis software and analyzed with GraphPad Prism. 2.6 Preparation of 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 I-FAPI-46 Indirect labeling of 131 I was achieved via a radioiododestannylation reaction with radioactive iodine [ 12 ] . First, a mixture of 5 µl acetic acid and 2.5 µl N-iodosuccinimide (NIS) was added to the radioactive iodine solution for activation for 1 minute. Under optimal radiolabeling conditions, methanol-dissolved solutions of FAPI-Y4, FAPI-caerin1.1 and FAPI-46 (30 µL, 5 mg/mL) were added to mixtures of 131I-NaI (37 MBq, 200 µL), acetic acid (5 µL) and NIS (2.5 µL, 4 mg/mL in MeOH), respectively. After reacting at room temperature for 30 min, the mixture was quenched with aqueous sodium ascorbate solution (2.5 µl, 8 mg/mL). The mixture was centrifuged at 12000 r/min for 15 min at 4°C, and the lower mixture was removed for later use. The mixture was analyzed by instant thin layer chromatography (iTLC), and the solution was developed with acetone. A gamma counter (Zhongjia Optoelectronics Co., Ltd., China) was used. GraphPad Prism was used to plot the γ counting curve, calculate the area under the curve and obtain the labeling rate. Labeling rate (%) = area under the radioactive peak of 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 I-FAPI-46/total radioactive peak area. 2.7 Stability determination of 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 I-FAPI-46 Radiochemical purity was determined by paper chromatography at different time points (0 h, 24 h) to determine the stability of the three labeled products ( 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 I-FAPI-46) at different temperatures (25°C, 37°C) in different solutions (FBS, NS). The radiochemical purity was calculated using GraphPad Prism as an index of stability. 2.8 Determination of the lipid‒water partition coefficient Five hundred microliters of n-octanol (Macklin, China), 500 µL of NS, and 50 µL of 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 were added to three 1.5 mL Eppendorf tubes, which were sealed and shaken for 2 min and then centrifuged at 4000 rpm/min for 5 min to obtain an equilibrium state between n-octanol and NS. Fifty microliter lipid and aqueous phase samples were collected, and the gamma counts of each tube were determined. This procedure was repeated 3 times. The lipid‒water partition coefficient (log P) was calculated as follows: logP = log [ (lipid phase gamma count‒background gamma count)/(aqueous phase gamma count‒background gamma count)]. 2.9 Cell-binding assays for 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 PANC-1, PANC-1-FAP, and HPDE6-C7 cells were seeded in 8 24-well cell culture plates (Corning, USA) at 5×10 4 (500 µL)/well, with 3 replicate wells and 1 positive control well for each drug. After the 24-well plates were cultured in the incubator for 24 h, the supernatant was discarded, and 0.5 mL of serum-free medium and 2 µL of Na 131 I, 131 I-FAPI-Y4, 131 I-FAPI-caerin 1.1, or 131 I-FAPI-46 solution (7 µCi/well) were added to each well. The plates were cultured in the incubator for 3, 6, 24 and 48 h. The supernatant of the experimental group was discarded, and the experimental wells were rinsed with PBS 2 times (the supernatant of each well of the positive control was collected in the corresponding test tubes). After 200 µL of trypsin (GIBCO, USA) was added to each well for digestion, the experimental wells were rinsed with PBS three times, and the samples were collected in the corresponding test tubes. The gamma count of each tube was determined. The drug binding rate was calculated using GraphPad Prism software. 2.10 Cell elution assays for 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 PANC-1, PANC-1-FAP and HPDE6-C7 cells were seeded in 8 24-well cell culture plates at 5×10 4 cells (500 µL)/well and cultured for 24 h. Each drug was added to three experimental wells and one positive control well. After culture, the supernatant was discarded, and 0.5 mL of serum-free medium and 2 µL of Na 131 I, 131 I-FAPI-Y4, 131 I-FAPI-caerin 1.1 or 131 I-FAPI-46 solution (7 µCi/well) were added to each well. After 24 hours of incubation in the incubator, the supernatant from all the experimental wells was discarded, each well was rinsed twice with PBS, and 0.5 mL of serum-free medium was added to each well. The plates were incubated in the incubator for 3, 6, 24 or 48 h and then removed. The supernatant from the experimental group was discarded, and the experimental wells were rinsed twice with PBS (the supernatant from each positive control well was collected in the corresponding test tube). After 200 µL of trypsin digestion was added to each well, the experimental wells were rinsed three times with PBS, and the samples in the corresponding test tubes were collected. The gamma count of each tube was determined, and the drug retention rate was calculated with GraphPad Prism. 2.11 Analysis of tumor formation in nude mice Two hundred microliters of PANC-1 cells (5×10 6 cells) were resuspended in PBS and subcutaneously inoculated under the axillary region of nude mice to establish a subcutaneous xenograft model. When the tumors reached 10 mm in size, they were used for subsequent experiments. A digital caliper (CD-15 APX, Mitutoyo, Japan) was used to monitor tumor size every 3 days, and the tumor volume was calculated using the following formula: tumor volume = width × length × height × Π/6. To reduce iodine uptake by the thyroid gland, all nude mice were fed 0.1% potassium iodide (Macklin, China) for 3 days before the in vivo experiment to block thyroid uptake of iodine. PANC-1-bearing nude mice were randomly divided into 8 groups (n = 4 per group): PBS (control group), 131 I, FAPI-caerin1.1, 131 I-FAPI-caerin1.1, FAPI-46, FAPI-Y4, 131 I-FAPI-46, and 131 I-FAPI-Y4. The volume of each intratumoral injection in the nude mice was 50 µL, with the PBS (control group) mice receiving pure PBS. The mice in the FAPI-caerin 1.1, FAPI-46, and FAPI-Y4 groups received freshly prepared PBS mixed with methanol containing 30 µg of peptide (V PBS : V MeOH = 1:1). The 131 I group was injected with 7.4×10 6 Bq Na 131 I solution. The 131 I-FAPI-caerin1.1, 131 I-FAPI-46, and 131 I-FAPI-Y4 groups were injected with 30 µg of polypeptide and 7.4×10 6 Bq Na 131 I-labeled products, all in a volume of 50 µL. Injections were administered every 3 days for a total of 4 injections. On the 7th day after the last administration, the nude mice were euthanized, and the tumors were isolated and weighed. GraphPad Prism was used to analyze the body weight, tumor size and tumor weight of the nude mice. 2.12 Statistical analysis All the experiments were repeated at least three times. Statistical analysis was performed using GraphPad Prism. The labeling efficiency was analyzed via t tests. Other data were analyzed using the analysis of variance method. P < 0.05 was considered to indicate a statistically significant difference. 3. Results 3.1 FAPI-caerin1.1 and FAPI-46 inhibit cell proliferation in vitro, but FAPI-Y4 has no inhibitory effect on cell proliferation In this study, the inhibitory effects of FAPI-caerin 1.1 and FAPI-46 on the three cell lines were not obvious at concentrations less than 5 μg/mL. At higher concentrations, FAPI-caerin1.1 and FAPI-46 strongly inhibited the proliferation of the three cell lines, and there was a significant difference in their inhibitory effects compared with those of the control treatment ( P < 0.05). As shown in Figure 1-A, B, and C, the survival rates of PANC-1 cells were 64.37% ± 1.20%, 23.85% ± 1.03%, and 10.49% ± 0.47% at 20 μg/mL, 30 μg/mL, and 40 μg/mL FAPI-caerin 1.1, respectively; the survival rates of PANC-1-FAP cells were 33.41% ± 0.28%, 16.63% ± 0.48%, and 8.78% ± 0.22%, respectively; and the survival rates of HPDE6-7 cells were 9.35% ± 0.39%, 7.03% ± 0.17%, and 3.15% ± 0.35%, respectively. When the FAPI-46 concentration was 20 μg/mL, the survival rates of PANC-1 cells were 49.53% ± 0.49%, 35.21% ± 0.24%, and 20.56% ± 0.12% at 30 μg/mL and 40 μg/mL, respectively; the corresponding survival rates of PANC-1-FAP cells were 38.10% ± 0.16%, 23.27% ± 0.22%, and 17.34% ± 0.28, and the corresponding survival rates of HPDE6-7 cells were 62.26% ± 0.11%, 38.95% ± 0.17%, and 26.95% ± 0.16%, respectively. At high concentrations (20 µg/mL, 30 µg/mL, and 40 µg/mL) of the nonspecific control peptide P3, the survival rates were high. As shown in Figure 1-D, E, and F, the IC 50 for FAPI-caerin 1.1 was 21.15 µg/mL for PANC-1 cells, 15.08 µg/mL for PANC-1-FAP cells, and 9.648 µg/mL for HPDE6-7 cells. The IC 50 for FAPI-46 was 18.83 µg/mL for PANC-1 cells, 18.68 µg/mL for PANC-1-FAP cells, and 29.82 µg/mL for HPDE6-7 cells. 3.2 Plate colony formation experiments revealed that FAPI-caerin1.1 inhibited cell proliferation, but FAPI-Y4 had no inhibitory effect on cell proliferation Plate colony formation experiments revealed that the proliferation ability and number of PANC-1 and PANC-1 FAP cells decreased with increasing FAPI-caerin 1.1 concentrations (Figure 2-C,2-D,Figure 3). Compared with that in the control group (0 μg/mL), the difference in the number of colonies was statistically significant ( P < 0.05). However, with increasing FAPI-Y4 concentration (Figure 2-A,2-B,Figure 3), the proliferation ability of PANC-1 and PANC-1 FAP cells did not decrease, and the difference in the number of colonies was not statistically significant ( P > 0.05). 3.3 Labeling rates of 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 FAPI-Y4, FAPI-caerin1.1, and FAPI-46 were labeled by the demethylation reaction of radioactive iodine. The labeling efficiency was determined by paper chromatography. Figure 4 shows that the radiolabeling rates of 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 I-FAPI-46 were 95.17% ± 0.05%, 95.4% ± 0.16%, and 95.23% ± 1.11%, respectively. There was no significant difference among the three peptides ( P >0.05). 3.4 Stability of 131 I-FAPI-caerin1.1, 131 I-FAPI-46, and 131 I-FAPI-Y4 Figure 5 shows that the labeled product still had high stability after 24 h in fetal bovine serum (FBS) or physiological saline (NS) at room temperature (25°C or 37°C). After 24 hours at 25°C, the RCPs of 131 I-FAPI-caerin1.1+FBS and 131 I-FAPI-caerin1.1+NS were 86.23%± 1.84% and 85.83%±1.69%, respectively; at 37°C, their RCPs were 86.23%±1.01% and 84.89%±1.57%, respectively. The RCPs of 131 I-FAPI-46+FBS and 131 I-FAPI-46+NS after 24 h at 25°C were 85.96% ±1.71% and 84.17%±0.37%, and the RCPs at 37°C were 84.54%±2.36% and 83.92%±0.44%, respectively. The RCPs of 131 I-FAPI-Y4+FBS and 131 I-FAPI-Y4+NS after 24 h at 25°C were 86.08% ±0.8% and 85.85%±1.33%, and the RCPs at 37°C were 84.99%±0.84% and 84.13%±0.52%, respectively. These data indicated that 131 I-FAPI-caerin1.1, 131 I-FAPI-46, and 131 I-FAPI-Y4 and their mixtures with FBS or NS were relatively stable at 25°C and 37°C. 3.5 Lipid‒water partition coefficient LogP is the solubility index of the labeled product in the organic phase or aqueous phase. A logP>0 indicates a trend toward lipid solubility, with a higher LogP being associated with higher solubility in the organic phase (Tables 1, 2, 3). In this study, LogP = -0.538±0.045 (n=4) for 131 I-FAPI-caerin1.1, LogP = -0.601±0.025 (n=4) for 131 I-FAPI-Y4, and LogP =0.342±0.059 (n=4) for 131 I-FAPI-46, indicating that the prepared 131 I-FAPI-46 was fat soluble, whereas 131 I-FAPI-caerin1.1 and 131 I-FAPI-Y4 were water soluble. Table 1 CPM counts and lipid‒water partition coefficients of 131 I-FAPI-caerin1.1 in the lipid and aqueous phases No. Lipid phase γ count（CPM） Aqueous phase γ count （CPM） LogP（mean±SD） 1 2 635502 694103 2149272 2735442 -0.538±0.045 3 619122 1837012 （n=4) 4 667802 2401458 Mean 654132 2280796 Table 2 CPM counts and lipid‒water partition coefficients of 131 I-FAPI-Y4 in the lipid and aqueous phases No. Lipid phase γ count（CPM） Aqueous phase γ count （CPM） LogP（mean±SD） 1 544344 2040660 2 495512 2011320 3 450952 1957128 -0.601±0.025 4 528624 2030068 （n=4) Mean 504858 2009794 Table 3 CPM counts and lipid‒water partition coefficients of 131 I-FAPI-46 in the lipid and aqueous phases No. Lipid phase γ count（CPM） Aqueous phase γ count（CPM） LogP（mean±SD） 1 3039432 1464924 2 2498624 901500 3 3012616 1439370 0.342±0.059 5 2994848 1543926 （n=4) Mean 2886380 1337430 3.6 Uptake and elution of PANC-1, PANC-1-FAP and HPDE6-C7 cells Cell uptake and elution experiments revealed that all three radioisotope-labeled products could be taken up and retained by pancreatic cancer cells and normal pancreatic ductal epithelial cells (Figure 6). The uptake of 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 decreased with time, and the uptake rate peaked at 3 hours. The binding rates of PANC-1 were 10.57 ± 0.05%, 2.2 ± 0.36%, and 2.67 ± 0.12%, and the binding rates of PANC-1-FAP were 8.8 ± 0.08%, 2.76 ± 0.22%, and 2.5 ± 0.08%, respectively, while the binding rates of HPDE6-C7 cells to the three radiopharmaceuticals were significantly lower than those of the first two pancreatic cancer cell lines. The binding rates of caerin 1.1 and 131 I-FAPI-46 were 3.42 ± 0.21%, 3.33 ±0.10%, and 4.72±0.26%, respectively. However, PANC-1, PANC-1-FAP and HPDE6-C7 cells essentially did not have the ability to take up Na 131 I, and the binding rates at 3 h were only 0.57 ± 0.05%, 0.53 ± 0.05%, and 0.88 ± 0.03%, respectively. In the cell elution experiment, when 131 I-FAPI-Y4, 131 I-FAPI-caerin 1.1 and 131 I-FAPI-46 were incubated with the three cell lines for 24 h before elution, the binding rates of the three drugs to the three cell lines were greater. Pancreatic cancer cells had the highest binding rates after 6 hours of culture. The binding rates of the PANC-1 cells were (87.65±1.42) %, (89.05±5.40) %, and (17.71± 1.07) %, and the binding rates of the PANC-1-FAP cells were (87.41± 2.86) %, (90.69±5.33) %, and (16.20±1.09) %, respectively, whereas the binding rates of the HPDE6-C7 cells were (39.31 ± 1.38) %, (45.68 ± 1.77) %, (13.24 ± 0.43) %, and rapid elution occurred at 3 h of culture. 3.7 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 I-FAPI-46 inhibit the growth of xenograft PANC-1 tumors in nude mice We investigated whether 131 I-FAPI-caerin1.1, 131 I-FAPI-46, and 131 I-FAPI-Y4 could inhibit the growth of PANC-1 cells in vivo. There was no significant difference in tumor size between the groups of nude mice at the beginning of treatment (P > 0.05). The body weights of the nude mice before death were 22.69±1.55 g in the PBS group; 21.96±1.84 g in the FAPI-caerin 1.1 group; 23.74±1.44 g in the FAPI-Y4 group; 23.85±2.09 g in the FAPI-46 group; 24.60±1.68 g in the Na 131 I group; 22.46±2.11 g in the 131 I-FAPI-caerin 1.1 group; 22.55±1.24 g in the 131 I-FAPI-Y4 group; and 22.76±1.69 g in the 131 I-FAPI-46 group. In the PBS, FAPI-caerin 1.1, FAPI-Y4, and FAPI-46,Na 131 I groups, the tumor sizes were significantly smaller than those before treatment (Figure 7-A). The tumor size before treatment was 208.19±16.52 mm 3 in the PBS group, 194.28± 16.81 mm 3 in the FAPI-caerin 1.1 group, 205.01±15.29 mm 3 in the FAPI-Y4 group, 205.55±10.79 mm 3 in the FAPI-46 group, 199.10±9.42 mm 3 in the 131 I-FAPI-46 group, 204.48±8.63 mm 3 in the 131 I-caerin 1.1 group, 210.29±11.69 mm 3 in the 131 I-FAPI-Y4 group, and 210.77±22.31 mm 3 in the 131 I-FAPI-46 group. The tumor size after administration and before humane death was 455.67±157.93 mm 3 in the PBS group, 352.88±67.93 mm 3 in the FAPI-caerin 1.1 group, 349.03±53.34 mm 3 in the FAPI-Y4 group, 352.69±58.64 mm 3 in the FAPI-46 group, 360.58±55.54 mm 3 in the Na 131 I group, 104.18±76.83 mm 3 in the 131 I-FAPI-caerin 1.1 group, 39.45±10.48 mm 3 in the 131 I-FAPI-Y4 group, and 84.19±27.29 mm 3 in the 131 I-FAPI-46 group. After the nude mice were euthanized, the tumors were isolated and weighed. Figure 7-B shows that the tumor weights in the PBS group were 59.57±10.21 mg, 56.18±10.23 mg in the FAPI-caerin 1.1 group, 48.18±20.15 mg in the FAPI-Y4 group, 53.61±17.18 mg in the FAPI-46 group, 48.68±16.03 mg in the Na 131 I group, 19.91±6.98 mg in the 131 I-FAPI-caerin 1.1 group, 8.62±5.83 mg in the 131 I-FAPI-Y4 group, and 20.15±5.97 mg in the 131 I-FAPI-46 group. Therefore, 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 I-FAPI-46 can inhibit the growth of human pancreatic cancer cells in vivo, whereas 131 I-FAPI-Y4 has a more significant inhibitory effect. At the end of treatment, the tumor volume and tumor weight of the 131 I-FAPI-Y4, 131 I-FAPI-caerin 1.1 and 131 I-FAPI-46 groups were significantly lower than those of the PBS, Na 131 I, FAPI-caerin 1.1, FAPI-Y4 and FAPI-46 groups, and the difference was statistically significant ( P < 0.05).Figure 8. 3.8 Tumor H&E staining results The tumor blocks of all the mice were stained with H&E (Figure 9). Small amounts of cell degeneration and necrosis were observed in the FAPI-Y4, FAPI-caerin 1.1, and FAPI-46 groups. Compared with that in the PBS control group, the difference was not statistically significant ( P > 0.05). The tumor cells in the 131 I-FAPI-caerin 1.1 and 131 I-FAPI-Y4 groups all exhibited extensive degeneration and necrosis, as indicated by homogeneous red staining, and the cell structure was also destroyed. There was a significant difference in the tumor necrosis area between the 131 I-FAPI-caerin 1.1 and 131 I-FAPI-Y4 groups and the PBS group and the Na 131 I group ( P < 0.05)(Figure 10). Compared with the simple peptide groups, the 131 I-FAPI-caerin 1.1 and 131 I-FAPI-Y4 groups presented significantly different areas of tumor necrosis ( P 0.05). These results showed that 131 I-FAPI-Y4 and 131 I-FAPI-caerin1.1 can inhibit tumor cells and tumor proliferation in vivo. 3.9 Immunohistochemistry results To further determine the expression of FAP in the tumor model used in the present study, immunohistochemical staining was conducted. As shown in Figure 11, FAP expression was detected in the stroma of PANC-1 tumor xenografts in the different drug treatment groups. 4. Discussion Pancreatic cancer (PC) is one of the most lethal malignancies, with a 5-year survival rate of only 9%. It is usually advanced at the time of diagnosis, and treatment options are still limited [ 13 ] . Therefore, a new treatment method is urgently needed to improve patient prognosis and survival. Radionuclide therapy was first proposed in the mid-1940s for the treatment of hyperthyroidism or differentiated thyroid cancer. Treatment of thyroid disease with 131 I is the most common radionuclide therapy in nuclear medicine. With the development of radionuclide therapy, the introduction of 131 I-labeled small-molecule products into the body to destroy cancer cells has been widely used in nuclear medicine and has become a very important imaging and treatment method [ 14 ] . In our previous study, we reported that caerin 1.1 isolated from the skin secretions of Australian tree frogs inhibits the proliferation of thyroid cancer cells, lung cancer cells, esophageal cancer cells, and other cancer cells [ 10 , 15 , 16 ] . However, when it reaches a certain concentration, it also has a significant inhibitory effect on normal cells. To minimize the adverse effects of the caerin 1.1 polypeptide on normal cells, we modified the caerin 1.1 polypeptide to obtain the small-molecule polypeptide TFMP-Y4, which has no effect on normal cell proliferation. Previous experiments have shown that even at very high drug concentrations, TFMP-Y4 has no inhibitory effect on the growth of normal cells or tumor cells, whereas 131 I-TFMP-Y4 has a significant inhibitory effect on tumor proliferation. The main purpose of this study was to construct a novel targeted molecular probe to prolong the retention time of FAPI-46 in tumors and explore its therapeutic effect on pancreatic cancer. In this study, a CCK8 assay revealed that FAPI-caerin1.1 and FAPI-46 inhibited the proliferation of pancreatic cancer cells and normal pancreatic ductal epithelial cells, but the IC 50 of FAPI-caerin1.1 in normal pancreatic ductal epithelial cells was lower than that in tumor cells. Moreover, FAPI-Y4 did not significantly inhibit the proliferation of pancreatic cancer cells or normal pancreatic ductal epithelial cells in vitro. In the in vitro colony formation test, higher concentrations of FAPI-caerin 1.1 strongly inhibited the proliferation of pancreatic cancer cells, whereas FAPI-Y4 did not significantly inhibit the proliferation of pancreatic cancer cells. This finding is consistent with the experimental results of the CCK8 assay. Three 131 I markers, 131 I-FAPI-caerin1.1, 131 I-FAPI-Y4 and 131 I-FAPI-46, were prepared by a radioactive demethylation reaction. The labeling rates of the three methods exceeded 95%. In the stability experiments, the three samples still had high stability at different temperatures (25°C and 37°C) and in different solutes (NS and FBS). On the basis of the results of the lipid‒water partition experiments, we preliminarily speculated that 131 I-FAPI-caerin 1.1 and 131 I-FAPI-Y4 were lipid soluble, whereas 131 I-FAPI-46 was water soluble. These findings suggest that 131 I-FAPI-caerin1.1 and 131 I-FAPI-Y4 may be excreted predominantly by the liver and that 131 I-FAPI-46 may be excreted predominantly by the kidney. In the cell uptake and elution assay, 131 I-FAPI-caerin1.1, 131 I-FAPI-Y4, and 131 I-FAPI-46 were rapidly distributed and then rapidly eluted, and the cell uptake rate peaked at 3 hours, which may be related to the pharmacokinetics of FAPI-46 [ 17 ] . The uptake rate of 131 I-FAPI-Y4 by PANC-1 and PANC-1-FAP cells was significantly greater than that of 131 I-FAPI-caerin1.1 and 131 I-FAPI-46, and the uptake rate of 131 I-FAPI-Y4 reached a plateau from 24 h to 48 h, which tended to be stable, but the uptake rate of 131 I-FAPI-caerin1.1 continued to decrease. This may be related to the structure of TFMP-Y4. 131 I-FAPI-Y4 can be taken up and stably retained by pancreatic cancer cells, showing obvious advantages. In the cell elution test, the binding rate of PANC-1 and PANC-1-FAP cells was the highest after 6 hours of culture. With increasing incubation time, the binding rates of PANC-1 and PANC-1-FAP cells gradually decreased, but they still had high binding rates after 24 h, and the binding rates of 131 I-FAPI-Y4 and 131 I-FAPI-caerin 1.1 to cells were significantly greater than those of 131 I-FAPI-46 and Na 131 I to cells. Previous experiments by our research group revealed that caerin 1.1 can be taken up by thyroid cancer CAL-62 cells, B-CPAP cells and lung cancer A549 cells and is enriched mainly in the cytoplasm [ 15 , 18 ] . Cell elution experiments revealed that FAPI-46 was connected with FAPI-Y4 and FAPI-caerin 1.1 through connectors. Compared with those of FAPI-46 alone, the tumor retention rate and retention time were improved, which further verified the feasibility of this study and provided a basis for subsequent in vivo treatment experiments. Moreover, we explored the binding rates of normal pancreatic ductal epithelial cells to 131 I-FAPI-caerin1.1, 131 I-FAPI-Y4 and 131 I-FAPI-46. The results revealed that the uptake rate of the three drugs was significantly lower than that of the other drugs in pancreatic cancer cells, and the binding rate after 24 hours of incubation was also significantly lower than that in pancreatic cancer cells, indicating that the three drugs could not be stably taken up and retained by normal pancreatic ductal epithelial cells, reducing the killing effect on normal pancreatic ductal epithelial cells.In the cell uptake and elution experiments, because the binding rates of 131 I-FAPI-caerin1.1, 131 I-FAPI-Y4, and 131 I-FAPI-46 in PANC-1 and PANC-1-FAPI-46-FAP cells were not significantly different, we only established a PANC-1 xenograft tumor model via in vivo experiments. The reason may be that PANC-1 cells themselves have strong FAP expression [ 19 ] , which is not significantly different from the degree of FAP expression in transfected PANC-1-FAP cells. In vivo, we established a nude mouse xenograft tumor model with PANC-1 cells and used Vernier calipers to measure the size of the tumors over time. In addition, considering the relatively low expression of FAP in the smaller xenograft tumor model, we used a relatively large tumor body model. The results showed that by the end of treatment, the tumor sizes of the 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 I-FAPI-46 groups were significantly smaller than those of the PBS group or the Na 131 I group, and the difference was statistically significant ( P < 0.05). Compared with those in the 131 I-FAPI-caerin 1.1 and 131 I-FAPI-46 groups, the tumor volume in the 131 I-FAPI-Y4 group decreased more significantly, which was consistent with the results of the cell uptake and elution experiments. Moreover, we performed H&E staining on the tumor tissue sections, observed them under a microscope, and counted the areas of cell degeneration and necrosis in each group for comparison. Owing to the high necrosis area of tumor cells in the PBS group, there was no significant difference in tumor cell degeneration or necrosis area between the FAPI-Y4, FAPI-caerin 1.1, and FAPI-46 groups and the PBS group ( P > 0.05). This may be due to the rapid growth of tumor cells in the PBS group, resulting in insufficient blood supply and cell necrosis due to nutritional deficiency. Extensive cell degeneration and necrosis were observed in the tumor sections from the 131 I-FAPI-caerin 1.1 and 131 I-FAPI-Y4 groups. Compared with those in the PBS group and the Na 131 I group, the tumor necrosis areas were significantly different ( P < 0.05). In vivo treatment experiments and H&E staining experiments revealed that 131 I-FAPI-Y4 and 131 I-FAPI-caerin1.1 significantly inhibited the proliferation of tumor cells, but the therapeutic effect of 131 I-FAPI-Y4 was more obvious, and FAPI-Y4 alone had no killing effect on normal pancreatic epithelial cells or pancreatic cancer cells, indicating its potential safety. 5.Conclusion 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 all have tumor-targeting effects, can bind to pancreatic cancer cells, and have obvious killing effects; however, FAPI-caerin1.1 also has a killing effect on normal pancreatic epithelial cells, and the IC 50 is lower than that of tumor cells, while FAPI-Y4 does not have a killing effect on normal cells, and 131 I-FAPI-Y4 has a more significant toxic effect on pancreatic cancer. Therefore, 131 I-FAPI-Y4 is a potential drug for the internal irradiation treatment of pancreatic cancer. The findings of this study provide new ideas for the treatment of pancreatic cancer. Declarations Ethics approval and consent to participate The animal research report was approved by The Animal Experiment Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University. (Ethics approval number: FAHGPU20160316). Consent for publication （ Not applicable） Availability of data and materials Data will be made available on reasonable request to corresponding author. Competing Interest The authors claim that the study was conducted without any commercial or financial relationship that could be considered a potential conflict of interest. Funding The research was supported by grants from the Foshan Science and Technology Innovation Project (No. 2320001006278). Authors' contributions Weiwei Ren, Jianwei Yuan, Peipei Zhang and Jun Jiang contributed to the study’s conception and design. Material preparation and experimental procedures were performed by Weiwei Ren, Yixuan Zhou,Yushan Li,Qingyi Lai,Kaixuan Lv,Jiangyuan Chen,Ruoqi Huang and Zewei Xiao. Data processing and analysis were carried out by Wenjuan Liu , Zhuanming Chen and Tongsheng Chen. The statistical methods used were reviewed by Peipei Zhang, and Jun Jiang. The first draft of the manuscript was written by Weiwei Ren and was revised by Jianwei Yuan. All the authors contributed to the article and approved the submitted version. Acknowledgements （ Not applicable） References MICHL P, LOHR M, NEOPTOLEMOS J P, et al. UEG position paper on pancreatic cancer. Bringing pancreatic cancer to the 21st century: Prevent, detect, and treat the disease earlier and better [J]. United European Gastroenterol J, 2021, 9(7): 860-71. KELLY K N, MACEDO F I, MERCHANT N B. Neoadjuvant Therapy: When Should It Be Used for Pancreatic Cancer? [J]. Adv Surg, 2020, 54: 49-68. TORPHY R J, FUJIWARA Y, SCHULICK R D. Pancreatic cancer treatment: better, but a long way to go [J]. Surg Today, 2020, 50(10): 1117-25. BUSEK P, MATEU R, ZUBAL M, et al. Targeting fibroblast activation protein in cancer – Prospects and caveats [J]. 2018, 23(10): 1933-68. FITZGERALD A A, WEINER L M. The role of fibroblast activation protein in health and malignancy [J]. Cancer Metastasis Rev, 2020, 39(3): 783-803. PANG Y, ZHAO L, FANG J, et al. Development of FAPI Tetramers to Improve Tumor Uptake and Efficacy of FAPI Radioligand Therapy [J]. J Nucl Med, 2023, 64(9): 1449-55. WATABE T, LIU Y, KANEDA-NAKASHIMA K, et al. Theranostics Targeting Fibroblast Activation Protein in the Tumor Stroma: (64)Cu- and (225)Ac-Labeled FAPI-04 in Pancreatic Cancer Xenograft Mouse Models [J]. J Nucl Med, 2020, 61(4): 563-9. LOKTEV A, LINDNER T, BURGER E M, et al. Development of Fibroblast Activation Protein-Targeted Radiotracers with Improved Tumor Retention [J]. J Nucl Med, 2019, 60(10): 1421-9. LIU Y, WATABE T, KANEDA-NAKASHIMA K, et al. Fibroblast activation protein targeted therapy using [(177)Lu]FAPI-46 compared with [(225)Ac]FAPI-46 in a pancreatic cancer model [J]. Eur J Nucl Med Mol Imaging, 2022, 49(3): 871-80. NI G, CHEN S, CHEN M, et al. Host-Defense Peptides Caerin 1.1 and 1.9 Stimulate TNF-Alpha-Dependent Apoptotic Signals in Human Cervical Cancer HeLa Cells [J]. Front Cell Dev Biol, 2020, 8: 676. MA B Y J, CHEN S, HUANG K, WANG Q, MA J, LIN R, ZHANG L, ZHOU Y, WANG T, WALTON SF, PAN X, CHEN G, WANG Y, NI G, LIU X. Topical application of temperature-sensitive caerin 1.1 and 1.9 gel inhibits TC-1 tumor growth in mice. [J]. Am J Transl Res, 2020 Jan 15: 12(1):191-202. MA H, LI F, SHEN G, et al. Synthesis and Preliminary Evaluation of (131)I-Labeled FAPI Tracers for Cancer Theranostics [J]. Mol Pharm, 2021, 18(11): 4179-87. MADADJIM R, AN T, CUI J. MicroRNAs in Pancreatic Cancer: Advances in Biomarker Discovery and Therapeutic Implications [J]. International Journal of Molecular Sciences, 2024, 25(7). SALIH S, ALKATHEERI A, ALOMAIM W, et al. Radiopharmaceutical Treatments for Cancer Therapy, Radionuclides Characteristics, Applications, and Challenges [J]. Molecules, 2022, 27(16). LIU N, HE T, XIAO Z, et al. (131)I-Caerin 1.1 and (131)I-Caerin 1.9 for the treatment of non-small-cell lung cancer [J]. Front Oncol, 2022, 12: 861206. HE T, DU J, ZHU K, et al. Experimental study of (131)I-caerin 1.1 and (131)I-c(RGD)(2) for internal radiation therapy of esophageal cancer xenografts [J]. Biomed Pharmacother, 2023, 164: 114891. MEYER C, DAHLBOM M, LINDNER T, et al. Radiation Dosimetry and Biodistribution of (68)Ga-FAPI-46 PET Imaging in Cancer Patients [J]. J Nucl Med, 2020, 61(8): 1171-7. LIN R, MA B, LIU N, et al. Targeted radioimmunotherapy with the iodine-131-labeled caerin 1.1 peptide for human anaplastic thyroid cancer in nude mice [J]. Ann Nucl Med, 2021, 35(7): 811-22. ROHRICH M, NAUMANN P, GIESEL F L, et al. Impact of (68)Ga-FAPI PET/CT Imaging on the Therapeutic Management of Primary and Recurrent Pancreatic Ductal Adenocarcinomas [J]. J Nucl Med, 2021, 62(6): 779-86. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 Oct, 2025 Reviews received at journal 30 Sep, 2025 Reviewers agreed at journal 20 Sep, 2025 Reviews received at journal 03 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers invited by journal 29 Aug, 2025 Editor invited by journal 20 Aug, 2025 Editor assigned by journal 25 Jul, 2025 Submission checks completed at journal 25 Jul, 2025 First submitted to journal 17 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7145341","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":510255195,"identity":"351f5deb-1e86-4285-94d5-851dca2beee3","order_by":0,"name":"Weiwei Ren","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Ren","suffix":""},{"id":510255196,"identity":"25bc1b59-c851-4861-83c4-f35ded605953","order_by":1,"name":"Yixuan Zhou","email":"","orcid":"","institution":"The First Affiliated Hospital 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Yuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIie3QsQrCMBCA4ZRAXWq7XnGwj3BSKA59E5cToZt7h6IFQcc+jFDXSKBTpWvHPILioouom1viJphvC9yfkGPMsn6SYIxy4AHnUn2RtNNBuHMzNH/I2eYBdl4ERtN4bCdKuTCKpceQFelMn4g2RvIgTuRQKNZky1KfnGoggEUifUKnlEbJ4UYI6/3GQzBNakYEHLlpEoruCiSAg3wtmUz+4vfN/HJ/rHhQSanORapPol58nEg3/jautLdalmX9vScNzEKy8pLxSAAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Guangdong Pharmaceutical University","correspondingAuthor":true,"prefix":"","firstName":"Jianwei","middleName":"","lastName":"Yuan","suffix":""}],"badges":[],"createdAt":"2025-07-17 06:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7145341/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7145341/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90668886,"identity":"42e740e5-8942-48d8-9393-41fff21a39e2","added_by":"auto","created_at":"2025-09-05 13:12:15","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":129142,"visible":true,"origin":"","legend":"\u003cp\u003eCCK-8 experiment results\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"32\" height=\"48\" src=\"file:///C:/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_bc6/AC/Temp/msohtmlclip1/01/clip_image001.gif\"/\u003eFigure 1. Figure 1-A, 1-B, 1-C: Survival rates of PANC-1, PANC-1-FAP, and HPDE6-7 cells exposed to different concentrations of P3, FAPI-46, FAPI-Y4, and FAPI-caerin1.1; Figure 1-D, 1-E, 1-F: IC\u003csub\u003e50\u003c/sub\u003e values of PANC-1, PANC-1-FAP, and HPDE6-7 cells exposed to P3, FAPI-46, FAPI-Y4, and FAPI-caerin1.1 (Ns \u003cem\u003eP\u003c/em\u003e\u0026gt; 0.05; *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05; **\u003cem\u003eP \u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001; ****\u003cem\u003eP \u003c/em\u003e\u0026lt;0.0001)\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/b34b38dc49405bf10ada1308.jpeg"},{"id":90668884,"identity":"aed92314-69b9-483d-a7df-a823e2ae35c0","added_by":"auto","created_at":"2025-09-05 13:12:15","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":124314,"visible":true,"origin":"","legend":"\u003cp\u003eCloning experiment results\u003c/p\u003e\n\u003cp\u003eFigure 2. Figure 2-A and Figure 2-B show the number of PANC-1 and PANC-1-FAP cell clones exposed to different concentrations of FAPI-Y4. Figure 2-C and Figure 2-D show the numbers of PANC-1 and PANC-1-FAP cell clones exposed to different concentrations of FAPI-caerin 1.1, respectively, compared with those in the control group (drug concentration: 0 µg/mL) (ns: \u003cem\u003eP\u0026gt;\u003c/em\u003e0.05; ***; \u003cem\u003eP\u0026lt;\u003c/em\u003e0.001; ****; \u003cem\u003eP\u0026lt;\u003c/em\u003e0.0001).\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/d95e36b2869989a6379bd7ed.jpeg"},{"id":90669173,"identity":"dfa90362-3884-4a81-b37f-7bd6fcc0eb44","added_by":"auto","created_at":"2025-09-05 13:20:15","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107793,"visible":true,"origin":"","legend":"\u003cp\u003ePhotos of Cloning experiment\u003c/p\u003e\n\u003cp\u003eFigure 3. Plate cloning experiment detecting different concentrations (0 µg/mL, 3 µg/mL, 6 µg/mL, 9 μg/mL, 12 μg/mL, and 15 μg/mL) of FAPI-Y4 and FAPI-caerin1.1 in PANC-1 and PANC-1-FAP cells, which were then stained and fixed.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/b2b3d3bc245b4ecbbe4c216c.jpeg"},{"id":90668887,"identity":"17d2ecd9-5c6b-4e98-a3cb-83e29aae0537","added_by":"auto","created_at":"2025-09-05 13:12:15","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98796,"visible":true,"origin":"","legend":"\u003cp\u003eLabeling efficiency results\u003c/p\u003e\n\u003cp\u003eFigure 4. Figures 4-A, 4-B, and 4-C show the γ count value curves of\u0026nbsp;\u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4,\u0026nbsp;\u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and\u0026nbsp;\u003csup\u003e131\u003c/sup\u003eI-FAPI-46, respectively; the labeling efficiency values of the three peptides were all greater than 95%, and the results were not significantly different (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05) (Figure 4-D).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/9307257c72aea5f20ab4c137.jpeg"},{"id":90670738,"identity":"4a65195a-954f-4e7c-b4be-768adea50419","added_by":"auto","created_at":"2025-09-05 13:28:15","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":132096,"visible":true,"origin":"","legend":"\u003cp\u003eStability of the three labeled products in different environments\u003c/p\u003e\n\u003cp\u003eFigure 5. Radioactive purity (RCP) of \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and \u003csup\u003e131\u003c/sup\u003e I-FAPI-Y4 mixed with fetal bovine serum (FBS) or physiological saline (NS) at room temperature (25°C) and 37°C for different durations (0 h, 24 h).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/60c727d837053e0b7170d6ce.jpeg"},{"id":90670740,"identity":"c96f8908-3cb8-44ae-bad9-b83a090725fa","added_by":"auto","created_at":"2025-09-05 13:28:15","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":129233,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the cell uptake and washout experiments\u003c/p\u003e\n\u003cp\u003eFigure 6-A, Figure 6-B, and Figure 6-E present the uptake of \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and Na\u003csup\u003e131\u003c/sup\u003eI by PANC-1, PANC-1-FAP, and HPDE6-C7 cells at different time points (3 h, 6 h, 24 h, and 48 h);\u003c/p\u003e\n\u003cp\u003eFig. 6-C, Fig. 6-D, and Fig. 6-F represent the uptake of \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and Na\u003csup\u003e131\u003c/sup\u003eI, respectively, at different time points (3 h, 6 h, 24 h, 48 h) after the drug was incubated with the cells for 24 h and then eluted.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/d42136139d70cf0045d3d9ee.jpeg"},{"id":90670739,"identity":"618d214a-82ab-4136-8317-926ddef4ea45","added_by":"auto","created_at":"2025-09-05 13:28:15","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":113409,"visible":true,"origin":"","legend":"\u003cp\u003eTumor volume, body weight changes and isolated tumor weights during the treatment of PANC-1 tumor-bearing nude mice\u003c/p\u003e\n\u003cp\u003eFigure 7-A shows the changes in tumor volume and size in each treatment group at different time points after tumor development.Figure 7-B shows the weights of the tumors isolated from each treatment group. Figure 7-C shows the weight changes in the nude mice in each treatment group at different time points after tumor bearing. (ns \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05; * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/ed28c5e9e0cc38d593f2804f.jpeg"},{"id":90669177,"identity":"10092d43-e553-4895-a938-e266a18a6ba2","added_by":"auto","created_at":"2025-09-05 13:20:15","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":77120,"visible":true,"origin":"","legend":"\u003cp\u003ePhotos of tumors from different treatment groups\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/b4ca579a4dd5d0cc95425303.jpeg"},{"id":90668898,"identity":"80ddf7cc-76c8-4c3d-a42a-85510b1991f7","added_by":"auto","created_at":"2025-09-05 13:12:15","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":347154,"visible":true,"origin":"","legend":"\u003cp\u003eH\u0026amp;E staining of tumor sections from different treatment groups.\u003c/p\u003e","description":"","filename":"image9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/ad75dd26a1a51ebed403858f.jpeg"},{"id":90668909,"identity":"4f01c953-6c2d-4672-bb44-32854027db78","added_by":"auto","created_at":"2025-09-05 13:12:16","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":13122,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical analysis of differences in the degree of cell degeneration and necrosis area among the different treatment groups.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/8e8f1b2af2aab55f68de589a.png"},{"id":90668910,"identity":"ff7c44cc-d8b2-48c4-b26a-6a3641c5f2e1","added_by":"auto","created_at":"2025-09-05 13:12:16","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":271468,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemica results\u003c/p\u003e\n\u003cp\u003eFigure 11. Immunohistochemical staining of fbroblast activation protein (FAP) in PANC-1 xenograft.FAP expression was observed in the tumour stroma.\u003c/p\u003e","description":"","filename":"image11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/10de936541030bd967e5364e.jpeg"},{"id":90671039,"identity":"32bd0e58-2b13-42cb-add3-150134694f4a","added_by":"auto","created_at":"2025-09-05 13:36:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2482448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7145341/v1/55765c59-19ac-43a7-9054-e76add3fa92d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Experimental study of internal irradiation with a novel targeted FAPI molecular probe for the treatment of human pancreatic cancer xenografts","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePancreatic ductal adenocarcinoma is the world's deadliest cancer and will soon be the second leading cause of cancer death in the United States, with a 5-year overall survival (OS) rate of only 9%\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Diagnosis often occurs at an advanced stage, with high rates of malignancy and a poor prognosis. Over the past century, significant advances have been made in the treatment of pancreatic cancer, including improvements in surgical techniques and adjuvant and neoadjuvant therapies \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Despite these advances, the incidence of pancreatic cancer continues to increase. Therefore, it is important to develop new treatments that can improve survival.\u003c/p\u003e\u003cp\u003eFibroblast activation protein (FAP) is a type II transmembrane glycoprotein that is overexpressed in cancer-associated fibroblasts, such as those in lung, liver, stomach, breast, colorectal, pancreatic, and ovarian cancers\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, but has low expression levels in normal fibroblasts. Therefore, FAP is considered a promising target for tumor imaging and therapy \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Radiolabeled fibroblast activation protein (FAP) inhibitors (FAPIs) have shown promise as cancer diagnostic drugs. However, owing to the rapid kinetics of FAPI, 89% of the injected dose is excreted through the urine 3 hours after injection, resulting in low ligand residues in the body\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. One of the characteristics of FAPI is that it is rapidly distributed and has a lower retention rate in tumors than other compounds. Lindner et al compared a series of FAPI derivatives and reported that FAPI-46 has a relatively good retention rate\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, but it is still very low. Therefore, we hypothesized that connecting small-molecule polypeptides through linkers would improve the retention time of FAPI-46 in tumors. Studies have shown that \u003csup\u003e177\u003c/sup\u003eLu-FAPI-46 plays a role in the treatment of human pancreatic cancer xenografts\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, but its therapeutic effect is limited.\u003c/p\u003e\u003cp\u003eThe host defense peptide Caerin 1.1 (GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH 2) is a polypeptide from the glandular secretions of Australian tree frogs\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Over the past few decades, several host defense peptides have been isolated and identified from the skin secretions of Australian tree frogs and toads. Many of these peptides have antibacterial or neuropeptide activities, and some peptides have high activity against cancer cells\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Our team's previous studies revealed that caerin1.1 has inhibitory effects on thyroid cancer cells, lung cancer cells, esophageal cancer cells and other cancer cells. \u003csup\u003e131\u003c/sup\u003eI-labeled caerin 1.1 (\u003csup\u003e131\u003c/sup\u003eI-caerin 1.1) has a stronger antitumor effect than caerin 1.1 does.However, with the gradual increase in the drug concentration to a certain extent, caerin 1.1 began to produce inhibitory effects on normal cells. To improve safety, we modified the caerin 1.1 polypeptide and replaced part of the amino acid sequence to obtain a new small-molecule polypeptide, TFMP-Y4 (YGLFGVLGSAKHVLPHVVPVIAEHL-NH2). Our team's previous studies revealed that \u003csup\u003e131\u003c/sup\u003eI-TFMP-Y4 has a significant inhibitory effect on tumor cell proliferation. To improve the effectiveness of tumor therapy, we hypothesized that FAPI-46, TFMP-Y4 and caerin 1.1 were connected through linkers to construct new targeted molecular probes, namely, \u003csup\u003e131\u003c/sup\u003eI-FAPI-TFMP-Y4 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1, to improve the effectiveness of tumor therapy.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Cell lines and cell cultures\u003c/h2\u003e\u003cp\u003eHuman pancreatic cancer cells (PANC-1 cells) were obtained from the Chinese Academy of Sciences Stem Cell Bank. The medium used for PANC-1 cells was (by volume) 89% DMEM (GIBCO, USA), 10% heat-inactivated fetal bovine serum (FBS, Corning, USA) and 1% penicillin‒streptomycin solution (GIBCO, USA). The cells were cultured in an incubator (Thermo, USA) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 FAPI new probe component\u003c/h2\u003e\u003cp\u003eFAPI-TFMP-Y4, FAPI-caerin1.1, and FAPI-46 were synthesized by Jiangxi Tanzhen Biotechnology Co., Ltd., and their purities were determined to be \u0026gt;\u0026thinsp;98% by reversed-phase high-performance liquid chromatography. For use, FAPI-TFMP-Y4, FAPI-caerin1.1, and FAPI-46 were dissolved in a mixed solution of DMSO and phosphate-buffered saline (PBS) (DMSO:PBS\u0026thinsp;=\u0026thinsp;1:1) at different concentrations (5 mg/mL, 1 mg/mL and 0.1 mg/mL).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Nude mice\u003c/h2\u003e\u003cp\u003eFour- to six-week-old SPF-grade BALB/c adult female nude mice were purchased from the Guangdong Provincial Medical Experimental Animal Center and raised under SPF-grade conditions at the Animal Resource Center of the First Affiliated Hospital of Guangdong Pharmaceutical University. No mice became ill or died before the end of the experiment. Nude mice were euthanized by cervical dislocation according to the \"Animal Management Measures\" issued by the Ministry of Health of the People's Republic of China (a document of the Ministry of Health of the People's Republic of China). All experiments were approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University and followed its guiding principles (ethical approval number: FAHGPU 20160316).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 CCK-8 assay\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of different polypeptides was examined using the CCK-8 assay. For the experiments, PANC-1, PANC-1-FAP, and HPDE6-7 cells were selected as experimental cell lines. Using the P3 polypeptide as a positive control, the cytotoxicities of FAPI-46, FAPI-TFMP-Y4, and FAPI-caerin1.1 were compared. After the cells had grown to a confluence of more than 80%, a uniform cell suspension was prepared and inoculated into a 96-well plate, with 100 \u0026micro;L of cell suspension in each well. A total of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well were inoculated and preincubated at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours in an incubator. Sample solutions treated with different concentrations were subsequently added, and the medium was replaced, followed by continued culture for 24 hours. The enhanced CCK-8 solution was added by replacing 10 \u0026micro;L of the medium in each well, followed by incubation for 0.5 to 2 hours. The absorbance at 450 nm was measured using a microplate reader (Thermo Scientific, USA). The half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values and cell viability (S%) were calculated for each drug using GraphPad Prism.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Colony formation experiments\u003c/h2\u003e\u003cp\u003ePANC-1 and PANC-1 FAP cells were seeded in four 6-well plates at a density of 800 cells/well, gently shaken uniformly, uniformly distributed in 6-well plates under a microscope and incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 hours. After the cells were uniformly adherent and had grown well under the microscope, different concentrations of FAPI-TFMP-Y4 and FAPI-caerin 1.1 were added (0 \u0026micro;g/ml, 3 \u0026micro;g/ml, 6 \u0026micro;g/ml, 9 \u0026micro;g/ml, 12 \u0026micro;g/ml and 15 \u0026micro;g/ml). After mixing, the samples were incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 hours. The medium was changed every 2 days, the cell status and colony number were observed, and the culture was terminated when most cell clusters consisted of approximately 50 cells per well with a drug concentration of 0 \u0026micro;g/ml. After 10 days of coculture, the cells were fixed with 4% paraformaldehyde (Sigma, USA) for 20 minutes, stained with crystal violet solution (Beyotime, China) for 15 minutes, washed with PBS for 3 minutes, and naturally dried. The cell colonies were counted using ImageJ analysis software and analyzed with GraphPad Prism.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Preparation of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46\u003c/h2\u003e\u003cp\u003eIndirect labeling of \u003csup\u003e131\u003c/sup\u003eI was achieved via a radioiododestannylation reaction with radioactive iodine\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. First, a mixture of 5 \u0026micro;l acetic acid and 2.5 \u0026micro;l N-iodosuccinimide (NIS) was added to the radioactive iodine solution for activation for 1 minute. Under optimal radiolabeling conditions, methanol-dissolved solutions of FAPI-Y4, FAPI-caerin1.1 and FAPI-46 (30 \u0026micro;L, 5 mg/mL) were added to mixtures of 131I-NaI (37 MBq, 200 \u0026micro;L), acetic acid (5 \u0026micro;L) and NIS (2.5 \u0026micro;L, 4 mg/mL in MeOH), respectively. After reacting at room temperature for 30 min, the mixture was quenched with aqueous sodium ascorbate solution (2.5 \u0026micro;l, 8 mg/mL). The mixture was centrifuged at 12000 r/min for 15 min at 4\u0026deg;C, and the lower mixture was removed for later use. The mixture was analyzed by instant thin layer chromatography (iTLC), and the solution was developed with acetone. A gamma counter (Zhongjia Optoelectronics Co., Ltd., China) was used. GraphPad Prism was used to plot the γ counting curve, calculate the area under the curve and obtain the labeling rate.\u003c/p\u003e\u003cp\u003eLabeling rate (%)\u0026thinsp;=\u0026thinsp;area under the radioactive peak of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46/total radioactive peak area.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Stability determination of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46\u003c/h2\u003e\u003cp\u003eRadiochemical purity was determined by paper chromatography at different time points (0 h, 24 h) to determine the stability of the three labeled products (\u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46) at different temperatures (25\u0026deg;C, 37\u0026deg;C) in different solutions (FBS, NS). The radiochemical purity was calculated using GraphPad Prism as an index of stability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Determination of the lipid‒water partition coefficient\u003c/h2\u003e\u003cp\u003eFive hundred microliters of n-octanol (Macklin, China), 500 \u0026micro;L of NS, and 50 \u0026micro;L of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 were added to three 1.5 mL Eppendorf tubes, which were sealed and shaken for 2 min and then centrifuged at 4000 rpm/min for 5 min to obtain an equilibrium state between n-octanol and NS. Fifty microliter lipid and aqueous phase samples were collected, and the gamma counts of each tube were determined. This procedure was repeated 3 times. The lipid‒water partition coefficient (log P) was calculated as follows: logP\u0026thinsp;=\u0026thinsp;log [ (lipid phase gamma count‒background gamma count)/(aqueous phase gamma count‒background gamma count)].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Cell-binding assays for \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46\u003c/h2\u003e\u003cp\u003ePANC-1, PANC-1-FAP, and HPDE6-C7 cells were seeded in 8 24-well cell culture plates (Corning, USA) at 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e (500 \u0026micro;L)/well, with 3 replicate wells and 1 positive control well for each drug. After the 24-well plates were cultured in the incubator for 24 h, the supernatant was discarded, and 0.5 mL of serum-free medium and 2 \u0026micro;L of Na\u003csup\u003e131\u003c/sup\u003eI, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1, or \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 solution (7 \u0026micro;Ci/well) were added to each well. The plates were cultured in the incubator for 3, 6, 24 and 48 h. The supernatant of the experimental group was discarded, and the experimental wells were rinsed with PBS 2 times (the supernatant of each well of the positive control was collected in the corresponding test tubes). After 200 \u0026micro;L of trypsin (GIBCO, USA) was added to each well for digestion, the experimental wells were rinsed with PBS three times, and the samples were collected in the corresponding test tubes. The gamma count of each tube was determined. The drug binding rate was calculated using GraphPad Prism software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Cell elution assays for \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46\u003c/h2\u003e\u003cp\u003ePANC-1, PANC-1-FAP and HPDE6-C7 cells were seeded in 8 24-well cell culture plates at 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells (500 \u0026micro;L)/well and cultured for 24 h. Each drug was added to three experimental wells and one positive control well. After culture, the supernatant was discarded, and 0.5 mL of serum-free medium and 2 \u0026micro;L of Na\u003csup\u003e131\u003c/sup\u003eI, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 or \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 solution (7 \u0026micro;Ci/well) were added to each well. After 24 hours of incubation in the incubator, the supernatant from all the experimental wells was discarded, each well was rinsed twice with PBS, and 0.5 mL of serum-free medium was added to each well. The plates were incubated in the incubator for 3, 6, 24 or 48 h and then removed. The supernatant from the experimental group was discarded, and the experimental wells were rinsed twice with PBS (the supernatant from each positive control well was collected in the corresponding test tube). After 200 \u0026micro;L of trypsin digestion was added to each well, the experimental wells were rinsed three times with PBS, and the samples in the corresponding test tubes were collected.\u003c/p\u003e\u003cp\u003eThe gamma count of each tube was determined, and the drug retention rate was calculated with GraphPad Prism.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Analysis of tumor formation in nude mice\u003c/h2\u003e\u003cp\u003eTwo hundred microliters of PANC-1 cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells) were resuspended in PBS and subcutaneously inoculated under the axillary region of nude mice to establish a subcutaneous xenograft model. When the tumors reached 10 mm in size, they were used for subsequent experiments. A digital caliper (CD-15 APX, Mitutoyo, Japan) was used to monitor tumor size every 3 days, and the tumor volume was calculated using the following formula: tumor volume\u0026thinsp;=\u0026thinsp;width \u0026times; length \u0026times; height\u0026thinsp;\u0026times;\u0026thinsp;Π/6. To reduce iodine uptake by the thyroid gland, all nude mice were fed 0.1% potassium iodide (Macklin, China) for 3 days before the in vivo experiment to block thyroid uptake of iodine. PANC-1-bearing nude mice were randomly divided into 8 groups (n\u0026thinsp;=\u0026thinsp;4 per group): PBS (control group), \u003csup\u003e131\u003c/sup\u003eI, FAPI-caerin1.1, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, FAPI-46, FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4. The volume of each intratumoral injection in the nude mice was 50 \u0026micro;L, with the PBS (control group) mice receiving pure PBS. The mice in the FAPI-caerin 1.1, FAPI-46, and FAPI-Y4 groups received freshly prepared PBS mixed with methanol containing 30 \u0026micro;g of peptide (V\u003csub\u003ePBS\u003c/sub\u003e: V\u003csub\u003eMeOH\u003c/sub\u003e = 1:1). The \u003csup\u003e131\u003c/sup\u003eI group was injected with 7.4\u0026times;10\u003csup\u003e6\u003c/sup\u003e Bq Na\u003csup\u003e131\u003c/sup\u003eI solution. The \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 groups were injected with 30 \u0026micro;g of polypeptide and 7.4\u0026times;10\u003csup\u003e6\u003c/sup\u003e Bq Na\u003csup\u003e131\u003c/sup\u003eI-labeled products, all in a volume of 50 \u0026micro;L. Injections were administered every 3 days for a total of 4 injections. On the 7th day after the last administration, the nude mice were euthanized, and the tumors were isolated and weighed. GraphPad Prism was used to analyze the body weight, tumor size and tumor weight of the nude mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll the experiments were repeated at least three times. Statistical analysis was performed using GraphPad Prism. The labeling efficiency was analyzed via t tests. Other data were analyzed using the analysis of variance method. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003e3.1 \u0026nbsp;FAPI-caerin1.1 and FAPI-46 inhibit cell proliferation in vitro, but FAPI-Y4 has no inhibitory effect on cell proliferation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the inhibitory effects of FAPI-caerin 1.1 and FAPI-46 on the three cell lines were not obvious at concentrations less than 5 \u0026mu;g/mL. At higher concentrations, FAPI-caerin1.1 and FAPI-46 strongly inhibited the proliferation of the three cell lines, and there was a significant difference in their inhibitory effects compared with those of the control treatment (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05). As shown in\u0026nbsp;Figure 1-A, B, and C, the survival rates of PANC-1 cells were 64.37% \u0026plusmn; 1.20%, 23.85% \u0026plusmn; 1.03%, and 10.49% \u0026plusmn; 0.47% at 20 \u0026mu;g/mL, 30 \u0026mu;g/mL, and 40 \u0026mu;g/mL FAPI-caerin 1.1, respectively; the survival rates of PANC-1-FAP cells were 33.41% \u0026plusmn; 0.28%, 16.63% \u0026plusmn; 0.48%, and 8.78% \u0026plusmn; 0.22%, respectively; and the survival rates of HPDE6-7 cells were 9.35% \u0026plusmn; 0.39%, 7.03% \u0026plusmn; 0.17%, and 3.15% \u0026plusmn; 0.35%, respectively. When the FAPI-46 concentration was 20 \u0026mu;g/mL, the survival rates of PANC-1 cells were 49.53% \u0026plusmn; 0.49%, 35.21% \u0026plusmn; 0.24%, and 20.56% \u0026plusmn; 0.12% at 30 \u0026mu;g/mL and 40 \u0026mu;g/mL, respectively; the corresponding survival rates of PANC-1-FAP cells were 38.10% \u0026plusmn; 0.16%, 23.27% \u0026plusmn; 0.22%, and 17.34% \u0026plusmn; 0.28, and the corresponding survival rates of HPDE6-7 cells were 62.26% \u0026plusmn; 0.11%, 38.95% \u0026plusmn; 0.17%, and 26.95% \u0026plusmn; 0.16%, respectively. At high concentrations (20 \u0026micro;g/mL, 30 \u0026micro;g/mL, and 40 \u0026micro;g/mL) of the nonspecific control peptide P3, the survival rates were high. As shown in Figure 1-D, E, and F, the IC\u003csub\u003e50\u003c/sub\u003e for FAPI-caerin 1.1 was 21.15 \u0026micro;g/mL for PANC-1 cells, 15.08 \u0026micro;g/mL for PANC-1-FAP cells, and 9.648 \u0026micro;g/mL for HPDE6-7 cells. The IC\u003csub\u003e50\u003c/sub\u003e for FAPI-46 was 18.83 \u0026micro;g/mL for PANC-1 cells, 18.68 \u0026micro;g/mL for PANC-1-FAP cells, and 29.82 \u0026micro;g/mL for HPDE6-7 cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2 \u0026nbsp;Plate colony formation experiments revealed that FAPI-caerin1.1 inhibited cell proliferation, but FAPI-Y4 had no inhibitory effect on cell proliferation\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePlate colony formation experiments revealed that the proliferation ability and number of PANC-1 and PANC-1 FAP cells decreased with increasing FAPI-caerin 1.1 concentrations (Figure 2-C,2-D,Figure 3). Compared with that in the control group (0 \u0026mu;g/mL), the difference in the number of colonies was statistically significant (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). However, with increasing FAPI-Y4 concentration (Figure 2-A,2-B,Figure 3), the proliferation ability of PANC-1 and PANC-1 FAP cells did not decrease, and the difference in the number of colonies was not statistically significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3 Labeling\u0026nbsp;\u003c/em\u003e\u003cem\u003erates of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFAPI-Y4, FAPI-caerin1.1, and FAPI-46 were labeled by the demethylation reaction of radioactive iodine. The labeling efficiency was determined by paper chromatography. Figure 4 shows that the radiolabeling rates of \u0026nbsp;\u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46\u0026nbsp;were 95.17% \u0026plusmn; 0.05%, 95.4% \u0026plusmn; 0.16%, and 95.23% \u0026plusmn; 1.11%, respectively. There was no significant difference among the three peptides (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4 \u0026nbsp;Stability of \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 5 shows that the labeled product still had high stability after 24 h in fetal bovine serum (FBS) or physiological saline (NS) at room temperature (25\u0026deg;C or 37\u0026deg;C). After 24 hours at 25\u0026deg;C, the RCPs of \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1+FBS and \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1+NS were 86.23%\u0026plusmn; 1.84% and 85.83%\u0026plusmn;1.69%, respectively; at 37\u0026deg;C, their RCPs were 86.23%\u0026plusmn;1.01% and 84.89%\u0026plusmn;1.57%, respectively. The RCPs of \u003csup\u003e131\u003c/sup\u003eI-FAPI-46+FBS and\u003csup\u003e\u0026nbsp;131\u003c/sup\u003eI-FAPI-46+NS after 24 h at 25\u0026deg;C were 85.96% \u0026plusmn;1.71% and 84.17%\u0026plusmn;0.37%, and the RCPs at 37\u0026deg;C were 84.54%\u0026plusmn;2.36% and 83.92%\u0026plusmn;0.44%, respectively. The RCPs of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4+FBS and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4+NS after 24 h at 25\u0026deg;C were 86.08% \u0026plusmn;0.8% and 85.85%\u0026plusmn;1.33%, and the RCPs at 37\u0026deg;C were 84.99%\u0026plusmn;0.84% and 84.13%\u0026plusmn;0.52%, respectively. These data indicated that \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 and their mixtures with FBS or NS were relatively stable at 25\u0026deg;C and 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5 \u0026nbsp;Lipid‒water partition coefficient\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLogP is the solubility index of the labeled product in the organic phase or aqueous phase. A logP\u0026gt;0 indicates a trend toward lipid solubility, with a higher LogP being associated with higher solubility in the organic phase (Tables 1, 2, 3). In this study, LogP = -0.538\u0026plusmn;0.045 (n=4) for \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, LogP = -0.601\u0026plusmn;0.025 (n=4) for \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, and LogP =0.342\u0026plusmn;0.059 (n=4) for \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, indicating that the prepared\u003csup\u003e131\u003c/sup\u003e I-FAPI-46 was fat soluble, whereas \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 were water soluble.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTable 1 CPM counts and lipid‒water partition coefficients of \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 in the lipid and aqueous phases\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"589\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eLipid phase \u0026gamma; count（CPM）\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003eAqueous phase \u0026gamma; \u0026nbsp;count （CPM）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 195px;\"\u003e\n \u003cp\u003eLogP（mean\u0026plusmn;SD）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e635502\u003c/p\u003e\n \u003cp\u003e694103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e2149272\u003c/p\u003e\n \u003cp\u003e2735442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e-0.538\u0026plusmn;0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e619122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e1837012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e（n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e667802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e2401458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e654132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e2280796\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTable 2 CPM counts and lipid‒water partition coefficients of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 in the lipid and aqueous phases\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"597\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003eLipid phase \u0026gamma; count（CPM）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 164px;\"\u003e\n \u003cp\u003eAqueous phase \u0026gamma; \u0026nbsp;count （CPM）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eLogP（mean\u0026plusmn;SD）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e544344\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 164px;\"\u003e\n \u003cp\u003e2040660\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e495512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 164px;\"\u003e\n \u003cp\u003e2011320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e450952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 164px;\"\u003e\n \u003cp\u003e1957128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e-0.601\u0026plusmn;0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e528624\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 164px;\"\u003e\n \u003cp\u003e2030068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e（n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e504858\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 164px;\"\u003e\n \u003cp\u003e2009794\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTable 3 CPM counts and lipid‒water partition coefficients of \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 in the lipid and aqueous phases\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"583\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003eLipid phase \u0026gamma; count（CPM）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003eAqueous phase \u0026gamma; \u0026nbsp;count（CPM）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eLogP（mean\u0026plusmn;SD）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e3039432\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e1464924\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e2498624\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e901500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e3012616\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e1439370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e0.342\u0026plusmn;0.059\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e2994848\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e1543926\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e（n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e2886380\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e1337430\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.6 \u0026nbsp;Uptake and elution of PANC-1, PANC-1-FAP and HPDE6-C7 cells\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCell uptake and elution experiments revealed that all three radioisotope-labeled products could be taken up and retained by pancreatic cancer cells and normal pancreatic ductal epithelial cells (Figure 6). The uptake of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 decreased with time, and the uptake rate peaked at 3 hours. The binding rates of PANC-1 were 10.57 \u0026plusmn; 0.05%, 2.2 \u0026plusmn; 0.36%, and 2.67 \u0026plusmn; 0.12%, and the binding rates of PANC-1-FAP were 8.8 \u0026plusmn; 0.08%, 2.76 \u0026plusmn; 0.22%, and 2.5 \u0026plusmn; 0.08%, respectively, while the binding rates of HPDE6-C7 cells to the three radiopharmaceuticals were significantly lower than those of the first two pancreatic cancer cell lines.\u003c/p\u003e\n\u003cp\u003eThe binding rates of caerin 1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 were 3.42 \u0026plusmn; 0.21%, 3.33 \u0026plusmn;0.10%, and 4.72\u0026plusmn;0.26%, respectively. However, PANC-1, PANC-1-FAP and HPDE6-C7 cells essentially did not have the ability to take up Na\u003csup\u003e131\u003c/sup\u003eI, and the binding rates at 3 h were only 0.57 \u0026plusmn; 0.05%, 0.53 \u0026plusmn; 0.05%, and 0.88 \u0026plusmn; 0.03%, respectively. In the cell elution experiment, when\u003csup\u003e\u0026nbsp;131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 were incubated with the three cell lines for 24 h before elution, the binding rates of the three drugs to the three cell lines were greater. Pancreatic cancer cells had the highest binding rates after 6 hours of culture. The binding rates of the PANC-1 cells were (87.65\u0026plusmn;1.42) %, (89.05\u0026plusmn;5.40) %, and (17.71\u0026plusmn; 1.07) %, and the binding rates of the PANC-1-FAP cells were (87.41\u0026plusmn; 2.86) %, (90.69\u0026plusmn;5.33) %, and (16.20\u0026plusmn;1.09) %, respectively, whereas the binding rates of the HPDE6-C7 cells were (39.31 \u0026plusmn; 1.38) %, (45.68 \u0026plusmn; 1.77) %, (13.24 \u0026plusmn; 0.43) %, and rapid elution occurred at 3 h of culture.\u003c/p\u003e\n\u003cp\u003e3.7 \u0026nbsp;\u003cem\u003e\u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4,\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u003csup\u003e131\u003c/sup\u003e\u003c/em\u003e\u003cem\u003eI-FAPI-caerin1.1\u0026nbsp;\u003c/em\u003e\u003cem\u003eand\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u003csup\u003e131\u003c/sup\u003e\u003c/em\u003e\u003cem\u003eI-FAPI-46\u003c/em\u003e\u003cem\u003e\u0026nbsp;inhibit the growth of xenograft PANC-1 tumors in nude mice\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated whether \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 could inhibit the growth of PANC-1 cells in vivo. There was no significant difference in tumor size between the groups of nude mice at the beginning of treatment \u003cem\u003e(P\u003c/em\u003e \u0026gt; 0.05). The body weights of the nude mice before death were 22.69\u0026plusmn;1.55 g in the PBS group; 21.96\u0026plusmn;1.84 g in the FAPI-caerin 1.1 group; 23.74\u0026plusmn;1.44 g in the FAPI-Y4 group; 23.85\u0026plusmn;2.09 g in the FAPI-46 group; 24.60\u0026plusmn;1.68 g in the Na\u003csup\u003e131\u003c/sup\u003eI group; 22.46\u0026plusmn;2.11 g in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 group; 22.55\u0026plusmn;1.24 g in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 group; and 22.76\u0026plusmn;1.69 g in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 group. In the PBS, FAPI-caerin 1.1, FAPI-Y4, and FAPI-46,Na\u003csup\u003e131\u003c/sup\u003eI groups, the tumor sizes were significantly smaller than those before treatment (Figure 7-A). The tumor size before treatment was 208.19\u0026plusmn;16.52 mm\u003csup\u003e3\u003c/sup\u003e in the PBS group, 194.28\u0026plusmn; 16.81 mm\u003csup\u003e3\u003c/sup\u003e in the FAPI-caerin 1.1 group, 205.01\u0026plusmn;15.29 mm\u003csup\u003e3\u003c/sup\u003e in the FAPI-Y4 group, 205.55\u0026plusmn;10.79 mm\u003csup\u003e3\u003c/sup\u003e in the FAPI-46 group, 199.10\u0026plusmn;9.42 mm\u003csup\u003e3\u003c/sup\u003e in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 group, 204.48\u0026plusmn;8.63 mm\u003csup\u003e3\u003c/sup\u003e in the \u003csup\u003e131\u003c/sup\u003eI-caerin 1.1 group, 210.29\u0026plusmn;11.69 mm\u003csup\u003e3\u003c/sup\u003e in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 group, and 210.77\u0026plusmn;22.31 mm\u003csup\u003e3\u003c/sup\u003e in the\u003csup\u003e\u0026nbsp;131\u003c/sup\u003eI-FAPI-46 group. The tumor size after administration and before humane death was 455.67\u0026plusmn;157.93 mm\u003csup\u003e3\u003c/sup\u003e in the PBS group, 352.88\u0026plusmn;67.93 mm\u003csup\u003e3\u003c/sup\u003e in the FAPI-caerin 1.1 group, 349.03\u0026plusmn;53.34 mm\u003csup\u003e3\u003c/sup\u003e in the FAPI-Y4 group, 352.69\u0026plusmn;58.64 mm\u003csup\u003e3\u003c/sup\u003e in the FAPI-46 group, 360.58\u0026plusmn;55.54 mm\u003csup\u003e3\u003c/sup\u003e in the Na\u003csup\u003e131\u003c/sup\u003eI group, 104.18\u0026plusmn;76.83 mm\u003csup\u003e3\u003c/sup\u003e in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 group, 39.45\u0026plusmn;10.48 mm\u003csup\u003e3\u003c/sup\u003e in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 group, and 84.19\u0026plusmn;27.29 mm\u003csup\u003e3\u003c/sup\u003e in the\u003csup\u003e\u0026nbsp;131\u003c/sup\u003eI-FAPI-46 group. After the nude mice were euthanized, the tumors were isolated and weighed. Figure 7-B shows that the tumor weights in the PBS group were 59.57\u0026plusmn;10.21 mg, 56.18\u0026plusmn;10.23 mg in the FAPI-caerin 1.1 group, 48.18\u0026plusmn;20.15 mg in the FAPI-Y4 group, 53.61\u0026plusmn;17.18 mg in the FAPI-46 group, 48.68\u0026plusmn;16.03 mg in the Na\u003csup\u003e131\u003c/sup\u003eI group, 19.91\u0026plusmn;6.98 mg in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 group, 8.62\u0026plusmn;5.83 mg in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 group, and 20.15\u0026plusmn;5.97 mg in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 group. Therefore, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 can inhibit the growth of human pancreatic cancer cells in vivo, whereas \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 has a more significant inhibitory effect. At the end of treatment, the tumor volume and tumor weight of the \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 and\u003csup\u003e\u0026nbsp;131\u003c/sup\u003eI-FAPI-46 groups were significantly lower than those of the PBS, Na\u003csup\u003e131\u003c/sup\u003eI, FAPI-caerin 1.1, FAPI-Y4 and FAPI-46 groups, and the difference was statistically significant (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).Figure 8.\u003c/p\u003e\n\u003cp\u003e3.8 \u0026nbsp;\u003cem\u003eTumor\u0026nbsp;\u003c/em\u003e\u003cem\u003eH\u0026amp;E\u0026nbsp;\u003c/em\u003e\u003cem\u003estaining results\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe tumor blocks of all the mice were stained with H\u0026amp;E (Figure 9). Small amounts of cell degeneration and necrosis were observed in the FAPI-Y4, FAPI-caerin 1.1, and FAPI-46 groups. Compared with that in the PBS control group, the difference was not statistically significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). The tumor cells in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 groups all exhibited extensive degeneration and necrosis, as indicated by homogeneous red staining, and the cell structure was also destroyed. There was a significant difference in the tumor necrosis area between the \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 groups and the PBS group and the Na\u003csup\u003e131\u003c/sup\u003eI group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)(Figure 10). Compared with the simple peptide groups, the \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 groups presented significantly different areas of tumor necrosis (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). There was no significant difference in the tumor necrosis area between the \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 group and the \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 group (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). These results showed that \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 can inhibit tumor cells and tumor proliferation in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.9\u003c/em\u003e \u003cem\u003eImmunohistochemistry results\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo further determine the expression of FAP in the tumor model used in the present study, immunohistochemical staining was conducted. As shown in Figure 11, FAP expression was detected in the stroma of PANC-1 tumor xenografts in the different drug treatment groups.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePancreatic cancer (PC) is one of the most lethal malignancies, with a 5-year survival rate of only 9%. It is usually advanced at the time of diagnosis, and treatment options are still limited\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Therefore, a new treatment method is urgently needed to improve patient prognosis and survival. Radionuclide therapy was first proposed in the mid-1940s for the treatment of hyperthyroidism or differentiated thyroid cancer. Treatment of thyroid disease with \u003csup\u003e131\u003c/sup\u003eI is the most common radionuclide therapy in nuclear medicine. With the development of radionuclide therapy, the introduction of \u003csup\u003e131\u003c/sup\u003eI-labeled small-molecule products into the body to destroy cancer cells has been widely used in nuclear medicine and has become a very important imaging and treatment method\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In our previous study, we reported that caerin 1.1 isolated from the skin secretions of Australian tree frogs inhibits the proliferation of thyroid cancer cells, lung cancer cells, esophageal cancer cells, and other cancer cells\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. However, when it reaches a certain concentration, it also has a significant inhibitory effect on normal cells. To minimize the adverse effects of the caerin 1.1 polypeptide on normal cells, we modified the caerin 1.1 polypeptide to obtain the small-molecule polypeptide TFMP-Y4, which has no effect on normal cell proliferation. Previous experiments have shown that even at very high drug concentrations, TFMP-Y4 has no inhibitory effect on the growth of normal cells or tumor cells, whereas \u003csup\u003e131\u003c/sup\u003eI-TFMP-Y4 has a significant inhibitory effect on tumor proliferation. The main purpose of this study was to construct a novel targeted molecular probe to prolong the retention time of FAPI-46 in tumors and explore its therapeutic effect on pancreatic cancer.\u003c/p\u003e\u003cp\u003eIn this study, a CCK8 assay revealed that FAPI-caerin1.1 and FAPI-46 inhibited the proliferation of pancreatic cancer cells and normal pancreatic ductal epithelial cells, but the IC\u003csub\u003e50\u003c/sub\u003e of FAPI-caerin1.1 in normal pancreatic ductal epithelial cells was lower than that in tumor cells. Moreover, FAPI-Y4 did not significantly inhibit the proliferation of pancreatic cancer cells or normal pancreatic ductal epithelial cells in vitro. In the in vitro colony formation test, higher concentrations of FAPI-caerin 1.1 strongly inhibited the proliferation of pancreatic cancer cells, whereas FAPI-Y4 did not significantly inhibit the proliferation of pancreatic cancer cells. This finding is consistent with the experimental results of the CCK8 assay.\u003c/p\u003e\u003cp\u003eThree \u003csup\u003e131\u003c/sup\u003eI markers, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, were prepared by a radioactive demethylation reaction. The labeling rates of the three methods exceeded 95%. In the stability experiments, the three samples still had high stability at different temperatures (25\u0026deg;C and 37\u0026deg;C) and in different solutes (NS and FBS). On the basis of the results of the lipid‒water partition experiments, we preliminarily speculated that \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 were lipid soluble, whereas \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 was water soluble. These findings suggest that \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 may be excreted predominantly by the liver and that \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 may be excreted predominantly by the kidney.\u003c/p\u003e\u003cp\u003eIn the cell uptake and elution assay, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 were rapidly distributed and then rapidly eluted, and the cell uptake rate peaked at 3 hours, which may be related to the pharmacokinetics of FAPI-46\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The uptake rate of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 by PANC-1 and PANC-1-FAP cells was significantly greater than that of \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46, and the uptake rate of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 reached a plateau from 24 h to 48 h, which tended to be stable, but the uptake rate of \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 continued to decrease. This may be related to the structure of TFMP-Y4. \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 can be taken up and stably retained by pancreatic cancer cells, showing obvious advantages. In the cell elution test, the binding rate of PANC-1 and PANC-1-FAP cells was the highest after 6 hours of culture. With increasing incubation time, the binding rates of PANC-1 and PANC-1-FAP cells gradually decreased, but they still had high binding rates after 24 h, and the binding rates of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 to cells were significantly greater than those of \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 and Na\u003csup\u003e131\u003c/sup\u003eI to cells. Previous experiments by our research group revealed that caerin 1.1 can be taken up by thyroid cancer CAL-62 cells, B-CPAP cells and lung cancer A549 cells and is enriched mainly in the cytoplasm\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Cell elution experiments revealed that FAPI-46 was connected with FAPI-Y4 and FAPI-caerin 1.1 through connectors. Compared with those of FAPI-46 alone, the tumor retention rate and retention time were improved, which further verified the feasibility of this study and provided a basis for subsequent in vivo treatment experiments.\u003c/p\u003e\u003cp\u003eMoreover, we explored the binding rates of normal pancreatic ductal epithelial cells to \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46. The results revealed that the uptake rate of the three drugs was significantly lower than that of the other drugs in pancreatic cancer cells, and the binding rate after 24 hours of incubation was also significantly lower than that in pancreatic cancer cells, indicating that the three drugs could not be stably taken up and retained by normal pancreatic ductal epithelial cells, reducing the killing effect on normal pancreatic ductal epithelial cells.In the cell uptake and elution experiments, because the binding rates of \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 in PANC-1 and PANC-1-FAPI-46-FAP cells were not significantly different, we only established a PANC-1 xenograft tumor model via in vivo experiments. The reason may be that PANC-1 cells themselves have strong FAP expression\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, which is not significantly different from the degree of FAP expression in transfected PANC-1-FAP cells.\u003c/p\u003e\u003cp\u003eIn vivo, we established a nude mouse xenograft tumor model with PANC-1 cells and used Vernier calipers to measure the size of the tumors over time. In addition, considering the relatively low expression of FAP in the smaller xenograft tumor model, we used a relatively large tumor body model. The results showed that by the end of treatment, the tumor sizes of the \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 groups were significantly smaller than those of the PBS group or the Na\u003csup\u003e131\u003c/sup\u003eI group, and the difference was statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with those in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 groups, the tumor volume in the \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 group decreased more significantly, which was consistent with the results of the cell uptake and elution experiments. Moreover, we performed H\u0026amp;E staining on the tumor tissue sections, observed them under a microscope, and counted the areas of cell degeneration and necrosis in each group for comparison. Owing to the high necrosis area of tumor cells in the PBS group, there was no significant difference in tumor cell degeneration or necrosis area between the FAPI-Y4, FAPI-caerin 1.1, and FAPI-46 groups and the PBS group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This may be due to the rapid growth of tumor cells in the PBS group, resulting in insufficient blood supply and cell necrosis due to nutritional deficiency. Extensive cell degeneration and necrosis were observed in the tumor sections from the \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 groups. Compared with those in the PBS group and the Na\u003csup\u003e131\u003c/sup\u003eI group, the tumor necrosis areas were significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In vivo treatment experiments and H\u0026amp;E staining experiments revealed that \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1 significantly inhibited the proliferation of tumor cells, but the therapeutic effect of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 was more obvious, and FAPI-Y4 alone had no killing effect on normal pancreatic epithelial cells or pancreatic cancer cells, indicating its potential safety.\u003c/p\u003e"},{"header":"5.Conclusion","content":"\u003cp\u003e\u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 all have tumor-targeting effects, can bind to pancreatic cancer cells, and have obvious killing effects; however, FAPI-caerin1.1 also has a killing effect on normal pancreatic epithelial cells, and the IC\u003csub\u003e50\u003c/sub\u003e is lower than that of tumor cells, while FAPI-Y4 does not have a killing effect on normal cells, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 has a more significant toxic effect on pancreatic cancer. Therefore, \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 is a potential drug for the internal irradiation treatment of pancreatic cancer. The findings of this study provide new ideas for the treatment of pancreatic cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal research report was approved by The Animal Experiment Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University. (Ethics approval number:\u003c/p\u003e\n\u003cp\u003eFAHGPU20160316). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003eNot applicable）\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on reasonable request to corresponding author.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors claim that the study was conducted without any commercial or financial relationship that could be considered a potential conflict of interest. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by grants from the Foshan Science and Technology Innovation Project (No. 2320001006278).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeiwei Ren, Jianwei Yuan, Peipei Zhang and Jun Jiang contributed to the study\u0026rsquo;s conception and design. Material preparation and experimental procedures were performed by Weiwei Ren, Yixuan Zhou,Yushan Li,Qingyi Lai,Kaixuan Lv,Jiangyuan Chen,Ruoqi Huang and Zewei Xiao. Data processing and analysis were carried out by Wenjuan Liu , Zhuanming Chen and Tongsheng Chen. The statistical methods used were reviewed by Peipei Zhang, and Jun Jiang. The first draft of the manuscript was written by Weiwei Ren and was revised by Jianwei Yuan. All the authors contributed to the article and approved the submitted version.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003eNot applicable）\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMICHL P, LOHR M, NEOPTOLEMOS J P, et al. UEG position paper on pancreatic cancer. Bringing pancreatic cancer to the 21st century: Prevent, detect, and treat the disease earlier and better [J]. 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Molecules, 2022, 27(16).\u003c/li\u003e\n\u003cli\u003eLIU N, HE T, XIAO Z, et al. (131)I-Caerin 1.1 and (131)I-Caerin 1.9 for the treatment of non-small-cell lung cancer [J]. Front Oncol, 2022, 12: 861206.\u003c/li\u003e\n\u003cli\u003eHE T, DU J, ZHU K, et al. Experimental study of (131)I-caerin 1.1 and (131)I-c(RGD)(2) for internal radiation therapy of esophageal cancer xenografts [J]. Biomed Pharmacother, 2023, 164: 114891.\u003c/li\u003e\n\u003cli\u003eMEYER C, DAHLBOM M, LINDNER T, et al. Radiation Dosimetry and Biodistribution of (68)Ga-FAPI-46 PET Imaging in Cancer Patients [J]. J Nucl Med, 2020, 61(8): 1171-7.\u003c/li\u003e\n\u003cli\u003eLIN R, MA B, LIU N, et al. Targeted radioimmunotherapy with the iodine-131-labeled caerin 1.1 peptide for human anaplastic thyroid cancer in nude mice [J]. Ann Nucl Med, 2021, 35(7): 811-22.\u003c/li\u003e\n\u003cli\u003eROHRICH M, NAUMANN P, GIESEL F L, et al. Impact of (68)Ga-FAPI PET/CT Imaging on the Therapeutic Management of Primary and Recurrent Pancreatic Ductal Adenocarcinomas [J]. J Nucl Med, 2021, 62(6): 779-86. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pancreatic cancer, PANC-1 cells, 131I-labeling, FAPI-Y4, FAPI-caerin 1.1 ","lastPublishedDoi":"10.21203/rs.3.rs-7145341/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7145341/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e The purpose of this study was to analyze and compare the therapeutic effects of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin1.1, and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 on pancreatic cancer cell xenografts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e ① The in vitro antitumor effects of FAPI-Y4, FAPI-caerin 1.1 and FAPI-46 were verified by CCK8 and colony formation assays. ② FAPI-Y4, FAPI-caerin1.1 and FAPI-46 were labeled by a radioiodine demethylation reaction, and their basic properties were determined. The uptake and elution of \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4, \u003csup\u003e131\u003c/sup\u003eI-FAPI-caerin 1.1 and \u003csup\u003e131\u003c/sup\u003eI-FAPI-46 in human pancreatic cancer cells were studied by cell uptake and elution assays. ③ A PANC-1 nude mouse model was established to compare the efficacy of internal irradiation with that of a novel targeted molecular probe in the treatment of pancreatic cancer.H\u0026amp;E staining was used to detect dead cells in the tumor tissue sections, and immunohistochemical staining was used to analyze the expression of the FAP antibody in the tumor cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e① In vitro, FAPI-caerin1.1 and FAPI-46 inhibited the proliferation of human pancreatic cancer cells and normal pancreatic ductal epithelial cells in vitro in a concentration-dependent manner, and their inhibitory effects were significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), whereas FAPI-Y4 had no significant inhibitory effect on the proliferation of human pancreatic cancer cells or normal pancreatic ductal epithelial cells in vitro, indicating potential biological safety. ② In vivo experiments revealed that \u003csup\u003e131\u003c/sup\u003eI-labeled FAPI-Y4 had better antitumor activity than \u003csup\u003e131\u003c/sup\u003eI-labeled FAPI-caerin1.1 and FAPI-46.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e \u003csup\u003e131\u003c/sup\u003eI-labeled FAPI-Y4, FAPI-caerin1.1 and FAPI-46 significantly inhibited the proliferation and growth of PANC-1 cells (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and \u003csup\u003e131\u003c/sup\u003eI-FAPI-Y4 has a more significant therapeutic effect than the other compounds and may be potential therapeutic drugs for human pancreatic cancer.\u003c/p\u003e","manuscriptTitle":"Experimental study of internal irradiation with a novel targeted FAPI molecular probe for the treatment of human pancreatic cancer xenografts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-05 13:12:10","doi":"10.21203/rs.3.rs-7145341/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-08T12:12:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T03:25:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267982585621098544368683803161574419475","date":"2025-09-20T18:15:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-04T01:07:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292790666909403205723876700149857242605","date":"2025-09-01T07:34:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-29T08:32:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-20T13:46:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T11:47:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-25T11:45:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pharmacology and Toxicology","date":"2025-07-17T06:05:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d893297d-e9ec-4f0a-b1b2-1bd6a91f9001","owner":[],"postedDate":"September 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-12T09:08:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-05 13:12:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7145341","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7145341","identity":"rs-7145341","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}