| In recent years,laboratory animals are widely used as models for the study of tumor diseases in clinical practice.This model has certain limitations,including high cost,animal ethics,constraints of breeding site and environment,etc.,so a new experimental model is urgently needed as a substitute for the study of tumor diseases.The Chick Embryo Chorioallantoic Membrane（CAM）is a new tumor model rising in recent years,because it has the advantages of convenient source,cheap price,short preparation cycle,no ethics,etc.,the research on blood vessel,tumor,drug screening and development is increasing day by day.At the same time,many researchers at home and abroad have successfully inoculated tumor cells on CAM to establish a transplanted tumor model.Canine Mammary Tumor（CMT）is the most common tumor in adult female dogs and one of the main causes of death in elderly dogs.About 30%of canine breast tumors express Human Epidermal Growth Factor Receptor 2（HER2）.Accurate detection of HER2 expression level in tumors is conducive to early diagnosis of HER2-positive CMT.However,it has not been reported whether the application of chicken chorioallantoic membrane can establish HER2 positive breast tumor model in canines.In this study,human breast cancer cells of HER2-positive Bcap37 and HER2-negative MDA-MB-231 were inoculated with chicken chorioallantoic membrane to establish a breast tumor model based on chicken chorioallantoic membrane.Specific cell uptake,immunofluorescence of tumor cells,western blot,Micro PET imaging and other experiments comprehensively verified the role of 68Ga-MZHER targeted HER2 probe in the imaging of chicken embryo chorioallantoic graft tumor.The conventional mouse tumor model was established and compared with the chick chorioallantoic tumor model,to verify the stability and reliability of the new chick chorioallantoic tumor model.Finally,HER2-positive breast tumor specimens were inoculated with chicken chorioallantoic membrane to establish a HER2-positive breast cancer transplant tumor model based on chicken chorioallantoic membrane.The value of targeted HER2-probe PET imaging in early diagnosis of HER2-positive breast tumors in dogs was determined through Micro PET imaging,pathology,biological distribution and other experiments.The main research contents and results are as follows:1.Construction and evaluation of a novel chick chorioallantoic breast tumor modelIn this study,HER2-positive Bcap37 and HER2-negative MDA-MB-231 breast tumor models based on chicken chorioallantoic membrane were successfully established.The tumorigenesis time was about 7 days,and the tumorigenesis rate was 80-90%.Micro PET imaging showed the rapid distribution of 68Ga-MZHER targeting HER2 probe in the body.30 min after injection of the probe,the uptake of HER2-positive Bcap37 tumor was5.36±0.26%ID/g,and the uptake of MDA-MB-231 was 1.57±0.15%ID/g,indicating a high target-to-substance ratio.It can effectively identify HER2 status in transplanted tumors.The results of cell uptake showed that the uptake rate of Bcap37 cells was2.85±0.12%AD/105 cells at 30 min and increased to 3.77±0.19%AD/105 cells at 90 min.The uptake rate of MDA-MB-231 cells was 1.15±0.12%AD/105 cells at 30 min and1.15±0.07%AD/105 cells at 90 min,which was consistent with that of Micro PET.In summary,this study successfully established HER2-negative and HER2-positive breast tumor models based on chicken chorioallantoic membrane for the first time.68Ga-MZHER Micro PET imaging of HER2-targeting drug showed significant uptake of HER2-positive tumor Bcap37,while MDA-MB-231 was negative.2.Comparison of novel chick chorioallantoic breast tumor model with conventional mouse tumor modelConventional mouse breast tumor model was established,and the tumor formation rate was 68-86%.30 min after probe injection,the tumor uptake rate of Bcap37 transplanted mice was 5.26±0.43%ID/g,and that of MDA-MB-231 transplanted mice was1.67±0.25%ID/g.The tumor uptake of Bcap37 grafted mice at 90 min was6.12±0.39%ID/g,slightly higher than that at 30 min,and the tumor uptake of MDA-MB-231 grafted mice was 1.22±0.21%ID/g,which was statistically significant compared with HER2 positive group at the same time point.Pathological experiments showed that Ki67 and HER2 expression were positive in Bcap37 tumor tissue of mice,Ki67 expression was positive in MDA-MB-231 graft tumor of mice,and HER2 expression was negative,which was consistent with the Micro PET imaging results.The reliability of the novel chicken embryo chorioallallic breast tumor model was verified by mouse tumor experiment.These results indicate that the chick chorioallantoic tumor model can be used to supplement or replace the mouse tumor model to a certain extent.3.Establishment and verification of HER2 positive canine breast tumor model based on chick chorioallantoic membraneHer2-positive breast tumor tissue was inoculated on chicken chorioallantoic membrane to establish a canine HER2-positive breast tumor model based on chicken chorioallantoic membrane.The tumor formation rate was about 86.7%.Micro PET imaging showed that the HER2-targeting probe was obviously taken up in the transplanted tumor,which could effectively monitor the HER2 status in the transplanted tumor.By delineating the area of interest,it was calculated that the tumor uptake was 4.51±0.26%ID/g 30 min after injection of the probe and 4.89±0.25%ID/g 90 min after injection.The tracer was mainly metabolized by liver and excreted by kidney,and its properties in vivo were similar to those of mice.In conclusion,this study successfully established a novel chicken chorioallantoic breast tumor model,and verified that the chicken chorioallantoic breast tumor model can be used to replace or supplement conventional mouse tumor models to a certain extent through experimental comparison.Finally,we established a HER2-positive canine breast tumor model based on chicken embryonic chorioallantoic membrane for the first time,and visualized the HER2 state in the tumor by targeting HER2 probe(68Ga-MZHER)Micro PET imaging,which is of great significance in the early accurate diagnosis,dynamic efficacy monitoring,and determination of treatment plan of canine tumors. |
