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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

29卷

期数:

2023年01期

页码:

9-14

栏目:

肿瘤基础研究专题

出版日期:

2023-01-20

文章信息/Info

Title:

Isolation and identification of spontaneously tumorigenic primary breast cancer cells from MMTV-PyMT transgenic mice

文章编号:

1006-8147(2023)01-0009-06

作者:

颜文欣; 冯玉梅

（天津医科大学肿瘤医院肿瘤研究所生物化学与分子生物学研究室；国家肿瘤临床医学研究中心；天津市“肿瘤防治”重点实验室；天津市恶性肿瘤临床医学研究中心；乳腺癌防治教育部重点实验室，天津300060）

Author(s):

YAN Wen-xin;  FENG Yu-mei

（Department of Biochemistry and Molecular Biology，Cancer Institute and Hospital，Tianjin Medical University，National Clinical Research Center for Cancer，Key Laboratory of Cancer Prevention and Therapy，Tianjin′s Clinical Research Center for Cancer，Key Laboratory of Breast Cancer Prevention and Therapy，Tianjin Medical University，Ministry of Education，Tianjin 300060，China）

关键词:

小鼠乳腺癌细胞系; 原代培养; HER2过表达分子亚型

Keywords:

mouse-derived breast cancer cell line; cell primary culture; HER2-over expression molecular subtype

分类号:

R737.9

DOI:

-

文献标志码:

A

摘要:

目的：分离培养小鼠乳腺肿瘤病毒介导多瘤病毒中间T抗原（MMTV-PyMT）转基因小鼠自发成瘤的乳腺癌细胞，初步研究其生长特性、分子表型、体内成瘤和转移能力。方法：取MMTV-PyMT小鼠自发成瘤的乳腺癌组织（n=3），采用酶解贴壁培养法进行原代细胞分离并传代培养，倒置显微镜下观察细胞形态特征；采用CCK-8检测细胞的增殖能力；通过Western印迹检测细胞的雌激素受体（ER）、孕激素受体（PR）和人表皮生长因子受体2（HER2）表达以鉴定细胞分子亚型。将分离培养的细胞接种于C57BL/6雌性小鼠（n=4）乳腺脂肪垫，观察细胞成瘤能力和生长特性；取肿瘤组织和内脏器官，制备组织切片后采用苏木精-伊红（HE）染色，观察组织形态学特点；采用免疫组织化学（IHC）染色检测ER、PR、HER2及Ki-67蛋白表达，分析分子分型。结果：成功分离得到MMTV-PyMT转基因小鼠自发成瘤的乳腺癌细胞（MMTV-PyMT细胞）并可稳定生长和传代，细胞倍增时间为56.0 h；MMTV-PyMT细胞可在C57BL/6雌性小鼠乳腺脂肪垫成瘤并生长，但未见远处内脏转移。该细胞在细胞学和组织学中的分子分型皆为HER2过表达亚型，肿瘤组织中Ki-67阳性表达率为34%。结论：成功构建得到可体外稳定传代并具有体内成瘤能力的HER2过表达分子亚型小鼠乳腺癌细胞系，该细胞可作为HER2过表达亚型乳腺癌体内、外实验研究的细胞模型。

Abstract:

Objective： To isolate and identify primary breast cancer cells from MMTV-PyMT（mouse mammary tumor virus-polyoma middle T antigen） transgenic mice，explore its growth characteristics，molecular phenotype，tumorigenic and metastatic abilities in vivo preliminary. Methods： Breast cancer tissues from MMTV-PyMT transgenic mice（n=3） were collected，and primary breast cancer cells were isolated and passaged by enzymatic-adherence-culture method. An inverted microscope was used to observe the morphological characteristics of cells；Cell-Counting-Kit 8（CCK-8） was used to detect cell proliferation characteristics；Western blotting was used to detect estrogen receptor（ER），progesterone receptor（PR） and human epidermal growth factor receptor 2（HER2） protein expression to identify its subtypes. The cells were orthotopic injected into the mammary fat pads of C57BL/6 female mice（n=4），and the tumorigenic ability and growth characteristics of the cells were observed；tumor tissues and visceral organ tissues were taken and stained with hematoxylin-eosin staining（HE） to observe the morphological characteristics；Immunohistochemistry staining（IHC） was used to detect ER，PR，HER2 and Ki-67 protein expression，and the tumor molecular type was analyzed. Results： Tumor cells derived from MMTV-PyMT mice primary breast tumors （MMTV-PyMT cells） were successfully isolated，it could expand and be passaged stably in vitro，and the cell doubling time was 56.04 hours. It could grow in the mammary fat pad of C57BL/6 mice，but no distant visceral metastasis. The molecular subtype of MMTV-PyMT cells in both cytology and histology was HER2-overexpression subtype（ER-negative/PR-negative/HER2-positive），and the positive expression rate of Ki-67 in tumor tissue was 34%. Conclusion：A HER2-overexpressing molecular subtype mouse-derived breast cancer cell line that can be stably passaged in vitro and has tumorigenic ability in vivo is successfully constructed. It can be used as a HER2-overexpression cell model for breast cancer in vitro and in vivo experiments.

参考文献/References:

[1] PASCHALL A V, LIU K. An orthotopic mouse model of spontaneous breast cancer metastasis[J]. J Vis Exp，2016，（114）：54040.
[2] GAO J J，SWAIN S M. Luminal A breast cancer and molecular assays： a review [J]. Oncologist，2018，23（5）： 556-565.
[3] GOLDHIRSCH A，WOOD W C，COATES A S，et al. Strategies for subtypes-dealing with the diversity of breast cancer： highlights of the St gallen international expert consensus on the primary therapy of early breast cancer 2011[J]. Ann Oncol，2011，22（8）：1736-1747.
[4] SZYMICZEK A，LONE A，AKBARI M R. Molecular intrinsic versus clinical subtyping in breast cancer：a comprehensive review[J]. Clin Genet，2021，99（5）：613-637.
[5] HOLLIDAY D L，SPEIRS V. Choosing the right cell line for breast cancer research[J]. Breast Cancer Res，2011，13（4）：215.
[6] TAKADA M，TOI M. Neoadjuvant treatment for HER2-positive breast cancer[J]. Chin Clin Oncol，2020，9（3）：32.
[7] HARBECK N，GNANT M. Breast cancer[J]. Lancet，2017，389（10074）：1134-1150.
[8] SUGIE T. Immunotherapy for metastatic breast cancer[J]. Chin Clin Oncol，2018，7（3）：28.
[9] NAGINI S. Breast cancer：current molecular therapeutic targets and new players[J]. Anticancer Agents Med Chem，2017，17（2）：152-163.
[10] PULASKI B A，OSTRAND-ROSENBERG S. Mouse 4T1 breast tumor model[J]. Curr Protoc Immunol，2001，Chapter 20：Unit 20.2.
[11] LE NAOUR A，KOFFI Y，DIAB M，et al. EO771，the first luminal B mammary cancer cell line from C57BL/6 mice[J]. Cancer Cell Int，2020，20：328.
[12] SEUNG E，XING Z，WU L，et al. A trispecific antibody targeting HER2 and T cells inhibits breast cancer growth via CD4 cells[J]. Nature，2022，603（7900）：328-334.
[13] SINGH S，LEE N，PEDROZA D A，et al. Chemotherapy coupled to macrophage inhibition induces T-cell and B-cell infiltration and durable regression in triple-negative breast cancer[J]. Cancer Res，2022，82（12）：2281-2297.
[14] CHELI Y，TULIC M K，EL HACHEM N，et al. ITGBL1 is a new immunomodulator that favors development of melanoma tumors by inhibiting natural killer cells cytotoxicity[J].Mol Cancer，2021， 20（1）：12.
[15] THOMSEN M，YACOUB-YOUSSEF H，MARCHEIX B. Reconstitution of a human immune system in immunodeficient mice：models of human alloreaction in vivo[J]. Tissue Antigens，2005，66（2）：73-82.
[16] PARTRIDGE A H，HUGHES M E，WARNER E T，et al. Subtype-dependent relationship between young age at diagnosis and breast cancer survival[J]. J Clin Oncol，2016，34（27）：3308-3314.
[17] CRONIN K A，HARLAN L C，DODD K W，et al. Population-based estimate of the prevalence of HER-2 positive breast cancer tumors for early stage patients in the US[J]. Cancer Invest，2010，28（9）：963-968.
[18] LING Y，LIANG G，LIN Q，et al. circCDYL2 promotes trastuzumab resistance via sustaining HER2 downstream signaling in breast cancer [J]. Mol Cancer，2022，21（1）：8.
[19] MAO S P H，PARK M，CABRERA R M，et al. Loss of amphiregulin reduces myoepithelial cell coverage of mammary ducts and alters breast tumor growth[J]. Breast Cancer Res，2018，20（1）：131.
[20] SUNDARAM S，YAN L. High-fat diet enhances mammary tumorigenesis and pulmonary metastasis and alters inflammatory and angiogenic profiles in MMTV-PyMT Mice[J]. Anticancer Res，2016， 36（12）：6279-6287.
[21] OWYONG M，CHOU J，VAN DEN BIJGAART R J，et al. MMP9 modulates the metastatic cascade and immune landscape for breast cancer anti-metastatic therapy[J]. Life Sci Alliance，2019，2（6）：e201800226.
[22] FU A，YAO B，DONG T，et al. Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer[J]. Cell，2022， 185（8）：1356-1372.e26.
[23] LIU Y，QI C，ZHENG L，et al. （1）H-NMR based metabolic study of MMTV-PyMT mice along with pathological progress to screen biomarkers for the early diagnosis of breast cancer[J]. Mol Omics，2022，18（2）：167-177.
[24] LIU T L，UPADHYAYULA S，MILKIE D E，et al. Observing the cell in its native state：imaging subcellular dynamics in multicellular organisms[J]. Science，2018，360（6386）：eaaq1392.
[25] ZWICKER J I，PROFFITT R T，REYNOLDS C P. A microcomputer program for calculating cell population doubling time in vitro and in vivo[J]. Cancer Chemother Pharmacol，1996，37（3）：203-210.
[26] ONG S E，BLAGOEV B，KRATCHMAROVA I，et al. Stable isotope labeling by amino acids in cell culture，SILAC，as a simple and accurate approach to expression proteomics[J]. Mol Cell Proteomics，2002，1（5）：376-386.
[27] SHISHIDO S，DELAHAYE A，BECK A，et al. The MMTV-PyVT transgenic mouse as a multistage model for mammary carcinoma and the efficacy of antineoplastic treatment[J]. J Cancer Ther，2013，4（7）： 1187-1197.
[28] LIN E Y，JONES J G，LI P，et al. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases[J]. Am J Pathol，2003，163（5）：2113-2126.

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备注/Memo

备注/Memo:

作者简介:颜文欣(1997-)，女，硕士在读，研究方向：肿瘤与分子生物学；通信作者：冯玉梅，E-mail：ymfeng@tmu.edu.cn。

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更新日期/Last Update: 2023-02-01