Preparation And Evaluation Of ⁹⁹Tc^m-Z_HER2:V2 And Imaging In Breast Cancer Xenografts Using Athymic Mice

Objective: To prepare the molecular probe ⁹⁹Tc^m-Z_HER2:V2 using the bi-functional chelator-G（Gly）GGC（Cys）,forming a N3 S structure,and to evaluate its binding specificity to human epidermal growth factor receptor type 2（HER2）and its correlation between ⁹⁹Tc^m-Z_HER2:V2 and HER2 expression levels in vivo and vitro.Build two kinds of athymic mice xenografts models,they are HER2 high expression BT474 xenografts and HER2 low expression MDA-MB-231 xenografts,whose HER2 expression levels are different,visualization were performed in those two models and compared to determine the feasibility in diagnosing primary and specific HER2-expressing positive breast carcinoma.Finally biodistribution was only performed in MDA-MB-231 xenografts and Immunohistochemistry technique was applied to testify the results of imaging and biodistribution.Methods:1 Molecular probe synthesis:The HER2-binding ZHER2:V2 Affibody molecule with a C-terminal chelating sequence-G（Gly）GGC（Cys）,forming a similar N3 S structure,was assembled using Fmoc/tBu solid phase peptide synthesis.Affibody molecule ZHER2:V2 was labeled with 99 Tcm using the ligand exchange method.2 Labeling efficiency was analyzed by reverse-phase high performance liquid chromatography（RP-HPLC）.3 To evaluate the labeling stability in vitro,⁹⁹Tc^m-Z_HER2:V2 was incubated with normal saline and fresh human serum at 37℃ respectively,and the radiochemistry purity was detected by RP-HPLC at 1,2,4,6,12 and 24 h.4 Cell culture: In logarithmic growth phase,the BT474 cells and MDA-MB-231 cells were cultivated in Roswell Park Memorial Institute 1640（RPMI 1640）medium and harvested using trypsin EDTA was included withthe concentration of 0.02%.5 Cellular uptake,retention kinetics,internalization,and blocking studies of the probe were investigated in the BT474 and MDA-MB-231 cell lines.6 Build tumor model:Cell suspension were injected subcutaneously into the right anterior superior limbs of the athymic mice,with nearly 1x107 cells in 0.2ml every mouse including the two models,and all of the mice were fed in specific pathogen free（SPF）environment.The tumors were allowed to be used in experiment when grew to reach above 1cm diameter for about 4-6weeks.7 To evaluate the stability in vivo of ⁹⁹Tc^m-Z_HER2:V2,the urine of the BALB/c athymic mice bearing BT474 tumor xenografts was collected to evaluate the metabolic stability in vivo of ⁹⁹Tc^m-Z_HER2:V2 within 1.5-2h after injecting the molecular probe through tail vein.8 Tumor imaging: In molecular imaging studies,five BALB/c athymic mice with BT474 or MDA-MB-231 xenografts were randomly chosen and injected with ⁹⁹Tc^m-Z_HER2:V2 via the tail vein.In addition,1 athymic mice bearing the BT474 tumor xenografts were pre-injected with excess of non-labelled ZHER2:V2,to saturate the HER2 receptors of tumors.At 1,2,4 and6 h after injection,the mice were anesthetized and imaged.The ratios of radioactive counts in the tumor to the place in the contralateral equivalent region（target to nontarget ratio [T/NT]）were calculated by drawing regions of interest（ROI）at each time point.9 In biodistribution studies,16 BALB/c nude mice bearing MDAMB-231 xenografts were randomly divided into 4 groups with 4 mice in each group,and each one was injected with labeled conjugates.At 1,2,4 and 6h after injection,one group of the mice were euthanized.Blood,heart,liver,lung,spleen,kidney,stomach,intestinum tenue,brain,bone,muscle and tumor were collected,weighed and measured for radioactivty.The tissue or organs uptake values were calculated as the percent injected dose per gram tissue（%ID/g）.10 All experimental variables were measurement data,expressed as mean± SD and were performed with Two-sample t（or t′）test,Wilcoxon rank sum test,S-N-K and Krustal-Wallis H test,using SPSS（version 21.0）and WPS sheet（version 2016）.P values of less than 0.05 were considered significant.Results:1 The molecule probe ⁹⁹Tc^m-Z_HER2:V2 was identified by RP-HPLC,the results showed a single radiation peak with retention time at 13.68 min and a high labeling rate（99.49±0.01）%（n=6）.The radiochemical purity of 99TcmZHER2:V2 displayed a stabled labeling rate（>91%）within 24 h and need-ed no purity,indicating that no significant degradation and off-labeling occur-red in fresh human serum or normal saline at 37 ℃.RP-HPLC analysis of the urine samples from athymic mice administered ⁹⁹Tc^m-Z_HER2:V2 at 1.5-2h showed 2peaks and without even a peak corresponding to free 99 Tcm.The peak detected before the retention time may be the degradation fragments of the peptide.2 The cellular uptake of the molecular probe ⁹⁹Tc^m-Z_HER2:V2 in BT474 cells and in MDA-MB-231 cells at 1,2,4,6,12 and 24 h were（4.86±0.60）%、（5.62±0.37）%、（44.18±6.40）%、（60.85±1.51）%、（58.76±6.32）%、（58.35±2.08）%;（2.46±2.60）%、（3.75±0.71）%、（3.80±1.60）%、（5.38±1.21）%、（4.29±1.60）%、（3.56±0.60）%respectively,which demonstrated that the cellular uptake of the molecular probe in BT474 were higher than in MDA-MB-231 cells,indicating that the uptake ratios were related to HER2 expression level.The cellular retention kinetics demonstrated that ⁹⁹Tc^m-Z_HER2:V2 residual in the BT474 cells was as high as 89.74% at 1h.The internalization studies showed that ⁹⁹Tc^m-Z_HER2:V2combined with the HER2 receptors were specified.In the cellular blocked studies an excess amount（500 times and 1000 times）of unlabeled ZHER2:V2was medicated at the same time with the ⁹⁹Tc^m-Z_HER2:V2.The blocked studies showed that the uptake rates of BT474 cells were decreased from（44.18±0.06）% to（5.28±0.02）%、（2.29±0.01）%,at 500 times and 1000 times,respectively.3 The results of biodistribution showed that radioactive accumulations in MDA-MB-231 xenografts were more obvious than in the other parts relatively,and the tumor uptake was（1.73±0.17）,（1.94±0.13）,（3.11±0.11）,（2.56±0.12）% ID/g at 1,2,4 and 6h,respectively.The ratio of T/M increased from 1.73±0.17 at 1h to 3.11±0.11 at 4h.In addition,a rapid clearance of⁹⁹Tc^m-Z_HER2:V2 from most of the normal organs besides the kidneys.4 In vivo SPECT imaging,BT474 xenografts were visualized as early as1 h and were shown most clearly at 4h after the administration of⁹⁹Tc^m-Z_HER2:V2.There was no obvious tumor uptake in low HER2-expressing MDA-MB-231 xenografts at 1h and afterwards dim radioactivity uptake were visualized.The tumors in BT474 tumor-bearing athymic mice accumulated radioactivity more obvious than in the MDA-MB-231 xenografts or the blocked BT474 xenografts,with significantly difference T/NT ratios between the two types of xenografts at all time points（P<0.05,n=5）.Conclusions: This study showed that ⁹⁹Tc^m-Z_HER2:V2is a promising radiopharmaceutical candidate for HER2 expression of molecular imaging in tumors.The label method was simple,and the labeled efficiency was high.Besides,it had a good stability in vivo and in vitro,so this molecular probe could be used in animal imaging directly and showed high specificity for HER2 high expressing tumor cells,and could be used in tumor-burdened athymic mice imaging.⁹⁹Tc^m-Z_HER2:V2 molecular probe may be a promising candidate radiopharmaceutical for diagnosis of high HER2-expressing human breast carcinoma tumors.