自发Basal-like乳腺癌小鼠模型研究

Mouse Model for Spontaneous Basal-Like Breast Cancer

目的:探讨TA2小鼠自发乳腺癌生物学行为、形态及免疫表型特征,比较其与人Basal-like乳腺癌的相似程度,研究TA2小鼠乳腺癌发生与妊娠及小鼠乳腺癌病毒(mouse mammary tumor virus,MMTV)的关系。方法:收集TA2自发乳腺癌小鼠84只,采用H&E染色和免疫组织化学染色检测TA2自发乳腺癌组织形态、免疫表型与转移情况,比较与人Basal-like乳腺癌的相似程度。电化学法检测TA2自发乳腺癌小鼠(n=10)、妊娠15天小鼠(n=6)与正常小鼠(n=6)血清雌二醇和孕酮水平。电镜观察肿瘤细胞内是否存在MMTV病毒颗粒,使用PCR和Real-time PCR分别检测MMTV LTR在TA2正常小鼠乳腺、妊娠小鼠乳腺和乳腺癌中基因组与mRNA表达变化。结果:TA2自发乳腺癌小鼠平均见瘤年龄(329.81±9532)天,平均分娩次数(2.70±1.82)次,且肿瘤在妊娠期内迅速增长。肿瘤多发生内脏转移,淋巴结转移未见,80.0%(64/80)发生肺转移。TA2自发乳腺癌形态一致,癌巢由胞浆稀少的小圆细胞构成,周围被发育良好的基质包绕。实性癌巢中央为未分化或分化差的癌细胞,且核分裂像多见,中央可见坏死;癌巢边缘的癌细胞多发生分化,呈腺泡状,腺管状或乳头状;腺泡状癌巢周围有单层或多层肌上皮和致密基质包绕;腺管状分化癌巢中央可见粉色分泌物积聚;实性癌巢中央可见坏死。免疫组化染色显示TA2自发乳腺癌不表达ER、PR和HER2,高表达Cyclin D1、PCNA、p53和CK5/6。电镜观察显示肿瘤细胞内存在MMTV病毒颗粒。TA2自发乳腺癌小鼠血清雌二醇和孕酮浓度及瘤组织内MMTV LTR mRNA表达均升高,其改变与妊娠小鼠相似。结论:TA2小鼠自发乳腺癌形态、免疫表型和生物学特征与人Basal-like乳腺癌极为相似,能够反映Basal-like乳腺癌发生发展的全过程,是研究Basal-like乳腺癌的良好模型。妊娠激素水平升高导致的MMTV激活在Basal-like乳腺癌发生过程中起重要作用。

Objective： Basal-like breast cancer is very invasive and highly metastatic with poor prognosis. The molecular mechanisms underlying its tumorigenesis and tumor progression remain unclear, and ideal animal models are lacking. The present study aims to identify the features of spontaneous breast cancer in TA2 mice, to determine whether the mouse model is useful and relevant for human basal-like breast cancer, and to investigate the relationship among pregnancy, mouse mammary tumor virus （ MMTV ）, and TA2breast cancer. Methods： Spontaneous breast cancers were collected from 84 female TA2 mice. The metastasis, morphology, and immunophenotype of the TA2 mice bearing breast cancer tumors were determined using hematoxylin, eosin staining, and immunohistochemistry, respectively. The similarities between the basal-like breast cancers of the animals and that of the human patients were compared. Electrochemiluminescence immunoassay was used to detect the serum levels of estradiol （ E2 ） and progesterone in the TA2 mice （ n=10 ）, the mice at d15 during the gestation period （ n = 6 ）, and the normal mice （ n = 6 ）. The MMTV in the TA2 breast cancer cells were checked under electron microscopy （ EM ）. The MMTV LTR expression in the mammary glands of the normal and the pregnant miceand the TA2 mice were determined using reverse transcriptase polymerase chain reaction. Results： The spontaneous breast cancer tissues in the TA2 mice were similar to human basal-like breast cancer in terms of biology, morphology, and phenotype. The mean age ofthe TA2 mice at tumorigenesis was （ 329.81 ± 95.32 ） d, and the mean frequency of delivery was （ 2.70± 1.8 ）, with rapid tumor growth within the gestation period. Metastasis in the visceral organs frequently occurred in TA2 tumors. However, nodal metastasis was notfound in the current research. Pulmonary metastasis was seen in 80% （ 64/80 ） of the tumors. The TA2 cancers were homologous in hisomorphology. The cancer nest consisted of roundlet cells with rare cytoplasm, surrounded by well-developed cell matrices. There were undifferentiated or poorly differentiated cancer cells at the center of the solid cancer nest. Caryomitotic figures were seen, with the ne-crosis at their centers. In the cancer cells at the edge of the cancer nest, aciniform, chrysanthemum-like, or papillar cell differentiation occurred occasionally. Around the aciniform nest, a monostratified or multilamellar myoepithelium and a pyknotic matrix were ob-served. Accumulated pink excretions were seen in the uniformly differentiated cancer nest. Immunohistochemistry revealed the absence of ER, PR, and HER2 expression, but the overexpression of cyclin D1, PCNA, p53, and CK5/6 were observed in the TA2 cancer. TheEM observation showed MMTV viral particles in the tumor cells. The serum E2 and progesterone concentrations in the TA2 mice and the intratumoral expression of MMTV LTR mRNA all increased. The change was similar to that in the pregnant mice. Conclusion： Thehistomorphology, immunophenotype, and biological features of breast cancer in TA2 mice are similar to those in human basal-like breast cancer, which reflects the full process of tumorigenesis and progression of basal-like cancer. Therefore, the mouse model is feasi-ble for use in the research of basal-like breast cancer. MMTV activation induced by the increases in E2 and progesterone levels plays a key role in the carcinogenesis of basal-like breast cancer.