Establishment Of Lymph Node Metastasis Model In Mouse Breast Cancer And Visual Evaluation Of X-ray Radiotherapy

Part I Establishment of lymph node metastasis model of mouse breast cancerObjective: Establish a method for constructing lymphatic metastasis model of mouse breast cancer.Methods: 4T1 breast cancer cells were subcutaneously injected into the foot pad of BALB/c nude mice to establish lymph metastasis model of mouse breast cancer.The tumor of pad,popliteal lymph node,liver,and other organs were collected on the thirtieth day after injection.HE staining was performed to determine the metastasis of4T1 breast cancer cells.Results: HE staining confirmed that the lymph nodes had diffuse metastasis of cancer cells.The metastasis rate was 100%.Conclusion: Subcutaneous injection of mouse breast cancer cell line 4T1 in nude mice successfully established a lymphatic metastasis model of mouse breast cancer.The method is rapid,simple,and high transfer rate,thus provides an ideal animal model for the investigation of lymphatic metastasis mechanism and cancer therapy.Part II In vivo bioluminescence imaging of lymph node metastasis modelObjective: Establish subcutaneous lymph node metastasis model by using luciferase labeled mouse breast cancer cell line(4T1-Luc).Non-invasive,real-time,and continuous monitoring the lymph node metastasis of mouse breast cancer cells by in vivo fluorescence imaging system.Methods: The 4T1-Luc cells were inoculated into the foot pad of nude mice to establish subcutaneous lymph node metastasis model.The small animal in vivo fluorescence imaging system was used to determine the metastasis of tumor cells in lymph nodes.Mice were sacrificed on the twenty-fourth day,and the pad primary lesion,popliteal lymph node metastasis,and other organs were collected for HE staining and microscopic evaluation.Results: Lymph node metastasis model was successfully established using4T1-Luc cells.The bioluminescence intensity positively correlates with the pad primary tumor volume.The lymph node metastasis was observed on the sixth day.Popliteal lymph node metastasis rate was reached 100% on the twelfth day.In addition,the bioluminescence intensity gradually increased with the prolonging feeding time.The nude mice were sacrificed on the twenty-fourth day.HE staining results showed the metastasis of cancer cells into popliteal lymph node and liver.Conclusion: The lymph node metastasis model was successfully established using luciferase-labeled mouse breast cancer cells.The lymph node metastasis can be real-time monitored in vivo by bioluminescence,thus providing an ideal experimental platform and imaging evaluation method for the investigation of tumor lymphatic metastasis mechanism and anti-tumor drug research.Keywords: Luciferase;Breast cancer;Lymph node metastasis;In vivoPart III Bioluminescence imaging evaluation for X-ray radiotherapy of cancerObjective: Aim to evaluate the effect of X-ray irradiation on the early stage of lymph node metastasis in breast cancer using bioluminescence imaging.Methods: 4T1-Luc early lymphatic metastasis models were randomly divided into treatment group and control group according to random number table method.The control group didn't do any treatment.For the treatment group,the lymph node metastasis of each popliteal fossa and footpad tumor were treated with 5Gy and 3Gy,respectively.The irradiation was performed every other day for 4 times.The growth of footpad tumor and the early metastasis tumor cells in lymph nodes were imaged before and after radiotherapy using the small animal in vivo fluorescence imaging system.The tumor bearing mice were sacrificed on the twenty-fourth day and the transplanted tumor,popliteal lymph node,and other organs were collected for evaluating the pathological changes.Results: After X-ray treatment,the bioluminescence intensity of transplanted tumor in the treatment group was significantly decreased than that of the control group.The tumor inhibition rate was 95%.The results also indicate that X-ray irradiation caneffectively inhibit the metastasis of tumor.HE staining showed that the tumor necrosis in the treatment group was significantly lower than that in the control group.Conclusion: The bioluminescence imaging characteristics of the transplanted tumor can be obtained by using intravital fluorescence imaging technique,which can be used to evaluate the effect of X-ray on the tumor suppressor effect of mouse breast cancer.