基础医学与临床 ›› 2022, Vol. 42 ›› Issue (7): 1083-1091.doi: 10.16352/j.issn.1001-6325.2022.07.1083

• 研究论文 • 上一篇    下一篇

小鼠乳腺癌肺特异性转移细胞亚系的建立和验证

王子辕, 段昭君*, 罗云萍*   

  1. 中国医学科学院基础医学研究所 北京协和医学院基础学院 免疫学系, 北京 100005
  • 收稿日期:2022-03-03 修回日期:2022-04-27 出版日期:2022-07-05 发布日期:2022-06-29
  • 通讯作者: * duanzhaojun@ibms.pumc.edu.cn;ypluo@ibms.pumc.edu.cn
  • 基金资助:
    中央高校基本科研业务费专项资金(3332020033)

Establishment and validation of lung-specific metastatic cell subline of mouse breast cancer

WANG Zi-yuan, DUAN Zhao-jun*, LUO Yun-ping*   

  1. Department of Immunology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China
  • Received:2022-03-03 Revised:2022-04-27 Online:2022-07-05 Published:2022-06-29
  • Contact: * duanzhaojun@ibms.pumc.edu.cn;ypluo@ibms.pumc.edu.cn

摘要: 目的 筛选和建立乳腺癌的自发性高器官特异性转移亚系,验证所建立的细胞亚系的细胞特征和肺转移器官特异性。方法 利用实验性肺转移模型,尾静脉注射小鼠乳腺癌细胞系4T07,回收肺转移灶细胞,体外扩增培养后,再重复2次,获得高肺转移乳腺癌细胞;再利用小鼠乳腺脂肪垫原位接种构建自发肺转移模型;在此之间,结合肺转移灶体外培养扩增,重复3次,获得肺高转移细胞亚系及自发性肺转移模型;转染筛选稳定表达荧光素酶的高转移细胞亚系,显微镜下观察细胞形态学变化;小动物活体成像和HE染色来检测肺特异性转移和体外迁移能力的变化;流式细胞测量术分析原发灶、转移灶及脾脏中免疫细胞的变化。细胞划痕实验和Transwell小室法检测细胞亚系的体外迁移能力的变化。结果 成功建立了小鼠乳腺癌4T07肺特异性转移细胞亚系4T07LuM。将稳定表达荧光素酶的4T07LuM细胞接种于小鼠乳腺脂肪垫后,小动物活体成像结果发现荧光信号主要集中于肺器官,HE切片可见肺脏中的肿瘤转移灶。4T07LuM相较于亲本细胞4T07体外迁移能力有明显提升,48 h的划痕闭合程度是亲本细胞的1.64倍(P<0.001),12 h时迁移过小室的细胞数是亲本细胞的1.91倍(P<0.001)。4T07LuM细胞荷瘤鼠的脾脏、原位灶和转移器官的免疫细胞比例发生显著变化,体内免疫微环境均更倾向于免疫抑制状态。结论 实验性转移和自发性转移的连续筛选联合体外培养扩增可以建立高器官特异性转移的细胞亚系。

关键词: 乳腺癌, 肺转移, 器官特异性, 自发转移模型

Abstract: Objective To screen and establish a spontaneously high organotrophic cell subline of metastatic breast cancer and to validate characteristic and the organotropism of the lung metastasis cell subline obtained from the screening. Methods By using the experimental lung metastasis, mouse breast cancer cells line 4T07 were injected into tail vein of mouse. Retrieving metastatic tumor cells in the lung and enriching them in vitro; By using the spontaneous metastasis model, the mammary fat pad was inoculated with tumor cells. Combined with the in vitro expansion culture of lung metastasis and repeated three times, a lung organotrophic cell sub-line and model of spontaneous lung metastasis were obtained; A highly metastatic cell subline stably expressing luciferase was obtained by transfection and screening. Cell morphology was microscopied with HE staining and an in vivo imaging system was performed to detect the migration and lung-specific metastasis. Flow cytometry was used to analyze the changes of immune cells in primary site, metastasis and spleen. Wound healing and Transwell assays were performed to detect migration of cell lines in vitro. Results A mouse breast cancer lung-specific metastatic cell subline (4T07LuM) was successfully established. in vivo imaging results validated that the fluorescent signal was mainly concentrated in the lung after injection of 4T07LuM cells labeled with luciferase in situ. HE staining microscopy results verified tumor metastasis in the lung. In addition, according to the results of Transwell and wound healing assays, 4T07LuM, compared to the parental cells 4T07, had shown a significantly more invasive migration(P<0.001). Finally, according to the results of flow cytometry detection, 4T07LuM cell had showed significantly altered the immune cell ratios as compared to that of 4T07 cell. The immune microenvironment in vivo was more inclined to an immuno-suppressed state. Conclusions Cell sublines with high organ-specific metastasis ability are obtained through combination of serial screening of experimental and spontaneous metastasis and in vitro culture expansion.

Key words: breast cancer, lung metastasis, organ-specificity, spontaneous metastasis model

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