Spect/ct Imaging Of ¹⁷⁷Lu-labeled Peptide Nucleic Acid?PNA?Targeting Cited1 And Its Therapeutic Evaluation In Tumor-bearing Nude Mice And In Vitro

Background:Papillary thyroid carcinoma?PTC?prognosis is good,but there are still 25%of the patients with local lymph node metastasis and relapse,while the fine needle aspiration biopsy?FNAB?often presents negative diagnosis results and is invasive.Therefore,it is a difficult problem that we are trying to solve now to find a specific early diagnosis method to make the patients get timely treatment.Some studies have confirmed that the expression of CITED1 in PTC is significantly up-regulated and can promote the proliferation of thyroid papillary cancer cells by regulating relevant pathways,which is a potential target for the treatment of PTC.Objectives:This study intends to use both radiological imaging and treatment characteristics of radionuclide ¹⁷⁷Lu of antisense peptide nucleic acid?asPNA?,appraisal the physical and chemical properties of¹⁷⁷Lu-DOTA-anti-CITED1-PNA and evaluate its for PTC in nude mice model of SPECT/CT imaging and the feasibility of the treatment,especially for the loss of iodine taken ability cannot benefit from ¹3131 I treatment of patients,to provide early diagnosis and treatment of recurrence or distant metastasis.Methods:1.Preparation,characterization of ¹⁷⁷Lu-DOTA-anti-CITED1-PNA:Real-time fluorescence quantitative PCR?RT-PCR?was used to identify the targeting specificity of PNA.The ¹⁷⁷Lu antisense probe was obtained by indirect labeling method,and the labeling rate,radiochemical purity and in vivo and in vitro physiological stability were determined by TLC and HPLC.2.In vitro efficacy evaluation of ¹⁷⁷Lu-DOTA-anti-CITED1-PNA:The uptake and retention of antisense probes were determined by using the Gamma counter,and the proliferation inhibition and pro-apoptosis effect of antisense probes on K1 cells cultured in vitro were detected by CCK-8 method and flow cytometry.3.Biodistribution,SPECT imaging and efficacy evaluation of¹⁷⁷Lu-DOTA-anti-CITED1-PNA in tumor-bearing nude mice:A nude mouse model of K1 cells in papillary carcinoma of the thyroid gland was established.Twenty nude mice were randomly divided into four groups according to the time of sacrifice,and¹⁷⁷Lu-DOTA-anti-CITED1-PNA was injected into the tail vein,tumor tissues and vital tissues and organs were collected at 4,12,24,and 48 hours respectively for biological distribution study.At the same time,three tumor-bearing nude mice were injected with ¹⁷⁷Lu-DOTA-anti-CITED1-PNA via caudal vein,and SPECT imaging was performed at 4,12,24,and48h after injection.Another 30 tumor-bearing nude mice were divided into3 groups,namely saline group,¹⁷⁷Lu-DOTA-anti-CITED1-PNA group and anti-CITED1-PNA group.Then tumor volume was measured,tumor growth curve was plotted and survival analysis was performed in nude mice to evaluate the therapeutic effect of ¹⁷⁷Lu-DOTA-anti-CITED1-PNA on nude mice with tumor.Results:1.RT-PCR showed that the expression of CITED1 was significantly decreased after the action of antisense peptide nucleic acid on K1 cells.The radiochemical purity of the antisense probe was?96.5±0.15?%,and the specific activity was?8.7±0.53?MBq/?g.The radiochemical purity of the marker incubated in PBS and human serum for 48h was more than 91%.2.In vitro uptake and retention experiments showed that the antisense probe could be specifically ingested by human K1 cells and stay in cells for a longer time than the nonsense probe.CCK-8 test showed that¹⁷⁷Lu-DOTA-anti-CITED1-PNA significantly inhibited the proliferation of K1 cells compared with uncoupled ¹⁷⁷Lu and anti-CITED1-PNA?P<0.01?.Flow cytometry showed that ¹⁷⁷Lu-DOTA-anti-CITED1-PNA significantly induced apoptosis compared with anti-CITED1-PNA?P<0.001?and control group?P<0.0001?.3.Biological distribution in tumor-bearing nude mice showed that 4h after injection of ¹⁷⁷Lu-DOTA-anti-CITED1-PNA,the concentration of radioactivity in tumor tissue,liver,spleen,kidney and blood was higher,but no obvious absorption was observed in brain tissue,and the tumor/muscle ratio was the highest?4.98±0.34?at 24h.SPECT plane and fusion images showed that at 24h after injection,¹⁷⁷Lu-DOTA-anti-CITED1-PNA was clearly developed at the tumor site,and the T/NT value reached a peak of 5.11,which was significantly compared with normal tissue.By measuring the tumor volume of tumor-bearing nude mice,the tumor size of the ¹⁷⁷Lu-DOTA-anti-CITED1-PNA group was significantly different from 9 days after injection?P<0.05?.The median survival of ¹⁷⁷Lu-DOTA-anti-CITED1-PNA group,anti-CITED1-PNA group and normal saline group was 43 days,30 days and 27days,respectively.Moreover,the overall survival rate of the¹⁷⁷Lu-DOTA-anti-CITED1-PNA group was significantly higher than that of the DOTA-anti-CITED1-PNA groups?P=0.002,log-rank test?and saline groups?P=0.002,log-rank test?.Conclusions:1.The ¹⁷⁷Lu-labeled CITED1mRNA antisense PNA probe was successfully prepared,with a simple labeling process and high purity.Internal and external stability,meet the requirements of radioactive drugs.2.¹⁷⁷Lu-DOTA-anti-CITED1-PNA can be specifically absorbed by K1cells,the proliferation ability of K1 cells is significantly decreased,and the apoptosis rate is significantly increased.3.¹⁷⁷Lu-DOTA-anti-CITED1-PNA has good imaging effect and tumor inhibition effect in nude mice model,and it is expected to provide a new way for the early diagnosis and treatment of recurrence or distant metastasis of PTC,especially for patients with iodine-resistant thyroid carcinoma.