Generation And Characterization Of A Breast Carcinoma Model By PyMT Overexpression Lentivirus In C57BL/6 Mouse

ObjectiveTo establish breast carcinoma model by injection of PyMT overexpression lentivirus in mammary duct of C57BL/6 mouse,and identify the pathological features and molecular classification of the tumors.MethodsFirstly,the PyMT overexpression plasmid and lentiviral packaging plasmids(MDL,REV and VSVG)were co-transfected into HEK293T cells for lentiviral package,and concentrated the lentivirus to higher concentration.Then,the lentivirus was injected into the mammary ducts of female C57BL/6 mice by microinjector.Tumorigenesis and the volume of the tumor were observed regularly.Finally,after the tumor reached 1.5cm in length,tumor and lung tissue sections were taken for HE staining to detect the tumor characteristicsand lung metastasis.The co-localization of GFP and CK14was investigated by immunofluorescence of tissue frozen sections.Immunohistochemistry was used to assess the expression of ER?,PR,HER2,Ki67,CK5 and?-SMA in tumor tissues to confirm the molecular classification of tumors.When the volume of the tumors reached 150mm~3,the mice were intraperitoneal injected with normal saline or doxorubicin to assess the reactivity to chemotherapeutic drugs.Results1.At 4th week after lentivirus injection,tiny bumps were detected among the mouse mammary gland area.As time went on,the volume of the tumor gradually increased,and the number of generated tumors mice gradually increased.During the 12-week observation period,the tumor incidence of transgenic mice was 100%,and that of wild-type mice was75%.2.Immunofluorescence results of tissue frozen sections showed that PyMT expressing GFP fluorescence co-localized with mammary epithelial cell marker CK14.3.The HE results showed that the pathological type of breast cancer induced by PyMT was papillary carcinoma without lung metastasis within 12 weeks.The results of Immunohistochemistry indicated that the expression of CK5 was missing,and?-SMA was positive of the tumors,indicating that the tumor located inside the duct.The expression of ER?,PR,HER2 and Ki67 was heterogeneous among different mice.The results of western blot revealed that tumor tissues were associated with different levels of PI3K/AKT,MAPK/ERK,JAK/STAT3 signaling pathway activation and elevated expression of c-MYC,CyclinD1,Bcl-2,and Bcl-xl.4.After 3 weeks treatment of doxorubicin,the growth of the tumors was significantly suppressed.ConclusionsAfter 4 weeks latent,a breast cancer model was successfully constructed in C57BL/6 background mice by injecting PyMT overexpressing lentivirus into the mammary duct.The tumor model presented with papillary carcinoma characteristic and elevated expression of oncogenes.