A Novel Radiolabelled Polypeptide F3 Molecular Probe For SPECT/CT Imaging And Treatment For Triple Negative Breast Cancer

Part Ⅰ Novel Molecular Probe Based on Radioiodinationof Polypeptide F3Objective:To establish a methodology for the construction of novel molecular imaging probe by radioiodinating polypeptide F3 and compare it with radioiodinated cetuximab（abbreviated as Ab）to investigate the physicochemical properties of radioiodinated F3.Methods:F3 and Ab was radiolabeled with¹²⁵I using the Chloramine-T method The radiolabeling yield,radiochemical purity,and the radiolabeling stability in vitro were determined after being purified by Sephadex G-25 column.Results:Both F3 and Ab can be easily radiolabeled by¹²⁵I.The radiolabeling yield was（88.0±0.5）%and（90.8±1.9）%for F3 and Ab,respectively.After purification,the radiochemical purity can reach up to（99.1±0.2）%and（91.5±2.0）%for F3 and Ab,respectively.The radiochemical purity of the resultant¹²⁵I-F3 and¹²⁵I-Ab remained higher than 90%after being stored under 4°C for 8 h.Conclusion:Radioiodination of F3 with high radiolabeling yield,high radiochemical purity,and robust radiolabeling stability can be successfully achieved by using the Chloramine-T method,which is simple and reproducible and thus lays the foundation for the further in vivo experiments.Part Ⅱ Radioiodinated F3 as a Radiotracer for Imaging of Triple-Negative Breast Cancer by SPECT/CTObjective:To investigate the targeting ability of radioiodinated F3 to triple-negative breast cancer by the in vitro 4T1 cell binding assay and the in vivo distribution of radioiodinated F3 in nude mice bearing 4T1 tumor model.Methods:The binding ability of^125I-F3 and¹²⁵I-Ab to 4T1 cancer cells was compared by cell binding experiments.After being intravenously injected into nude mice bearing triple negative breast cancer xenografts with a dosage of 300μCi/200μL per mice,SPECT/CT images were acquired at different time points post-injection,including 0,1,2,4,6,8 and 24 h to evaluate targeting ability of¹²⁵I-F3 and¹²⁵I-Ab.In addition,the biodistribution of¹²⁵I-F3 and¹²⁵I-Ab in mice were also investigated.After being intravenously injected into female mice with a dosage of 40μCi/200μL per mice,the mice were sacrificed at 0.5,1,2,4 and 8 h post-injection and the main organs and tissues,including blood sacrificed,heart,liver,spleen,lung,kidney,stomach,intestine,bone,muscle,tumor,and skin were harvested and determined by gamma counter.Results:The cell binding ratio of¹²⁵I-F3 was（4.32±0.18）%,（7.34±0.73）%,（10.58±0.16）%,（10.76±0.35）%and（13.76±0.73）%for 1,2,4,8 and 24 h,respectively,higher than that for¹²⁵I-Ab at the same time points,which show binding ratio of（0.32±0.05）%,（0.49±0.05）%,（0.73±0.04）,（0.90±0.06）%and（1.77±0.36）%,respectively.The cell binding ratio have a significant difference for¹²⁵I-F3 after 2h compared with 1h（P<0.01）.While¹²⁵I-Ab exhibited no significant difference in the cell binding ratio between 2h and 1h（P>0.05）.SPECT/CT imaging showed that the tumor site could be displayed at the first hour after the injection of¹²⁵I-F3.With the time prolonging,the background of nude mice was reduced and the tumor site was more clearly imaged.The tumor site was most clearly visualized at the fourth hour.Then the radioactivity was gradually decreased until the 24 h when the tumor signal could hardly be detected.In remarkable difference,the¹²⁵I-Ab show highest signal at 2h post-injection.However,the maximum accumulation was much lower than that of¹²⁵I-F3.With the time went on,the radioactivity at the tumor site was rapidly decreased,and the signal at tumor site was nearly vanished at the 8h post-injection.The distribution results showed that the maximum uptake of（3.16±0.05）%for tumor site reached at 2h post-injection for¹²⁵I-F3,higher than the maximum uptake of（2.46±0.49）%for¹²⁵I-Ab that appeared at 1h post-injection.Conclusion:¹²⁵I-F3 could bind to triple-negative breast cancer cells and specifically accumulate at the site of triple-negative breast tumors,thus providing a potential targeted imaging agent for triple-negative breast cancer.Part Ⅲ Radiotherapy of Triple Negative Breast Cancer by Using Radioiodinated F3Objective:To treat the triple-negative breast cancer through radiotherapy by intratumoral administration of¹³¹I-F3,and confirm the treatment efficiency by the immunohistochemical detection of EGFR expression at the tumor site.Methods:¹³¹I labeled F3 and Ab were achieved by using the Chloramine-T method.The treatment efficiency of¹³¹I-F3 and¹³¹I-Ab to the tumor-bearing nude mice was evaluated by intratumoral injection with a dosage of 50μL（about 200μCi/body）and using saline injection as a control.The injection was repeated every other day for a total of 3 injections.HE staining and immunohistochemical staining were used to detect EGFR expression in the treated and control groups.Results:The labeling yield was（92.0±2.1）%and（89.2±0.4）%for¹³¹I-F3 and¹³¹I-Ab,respectively.The FX Pro Small Animal Imaging System showed that the two groups of tumor sites could be visualized on the fifth day of treatment,but the radioactivity was different.With the extension of treatment time,the radioactivity of the tumor sites in both groups were reduced.After 20 day treatment,the tumor inhibition rates were（11.46±7.78）%and（37.76±14.68）%for¹³¹I-F3 group and¹³¹I-Ab group,respectively.HE staining showed that the growth of tumor cells was inhibited.Immunohistochemistry showed that the expression of EGFR in tumors was reduced after treatment,and the proportion of positive cells was less than that of the control group.Conclusion:¹³¹I-F3 could inhibit the growth of triple-negative breast cancer tumor cells and may become a targeted internal irradiation therapeutic drug with clinical application value.