人肺癌细胞原位移植瘤小鼠模型免疫细胞的多色流式细胞术检测方法的建立

doi:10.3969/j.issn.1006-7795.2023.01.015

首都医科大学学报 ›› 2023, Vol. 44 ›› Issue (1): 99-106.doi: 10.3969/j.issn.1006-7795.2023.01.015

人肺癌细胞原位移植瘤小鼠模型免疫细胞的多色流式细胞术检测方法的建立

杨敏¹, 燕翔^2*, 刘锡娟³, 安国⁴, 丁慧荣³, 蔡英⁵, 葛会争⁵

1.首都医科大学附属北京世纪坛医院干部综合科, 北京 100038;
2.解放军总医院第五医学中心肿瘤医学部肿瘤内科,北京100039;
3.北京大学肿瘤医院暨北京市肿瘤防治研究所恶性肿瘤发病机制及转化研究教育部重点实验室中心实验室,北京 100142;
4.北京大学肿瘤医院暨北京市肿瘤防治研究所恶性肿瘤发病机制及转化研究教育部重点实验室实验动物室,北京 100142;
5.碧迪医疗北京卓越中心(COE), 北京 100176

收稿日期:2022-10-18 出版日期:2023-02-21 发布日期:2023-01-13
基金资助:
国家自然科学基金(81902910)。

Establishment and characterization of immune cells in an orthotopic cell-derived xenograft mouse model of human lung cancer using multi-color flow cytometry

Yang Min¹, Yan Xiang^2*, Liu Xijuan³, An Guo⁴, Ding Huirong³, Cai Ying⁵, Ge Huizheng⁵

1. Department of Gerontology, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China;
2. Department of Medical Oncology, The Fifth Medical Center of PLA General Hospital,Beijing 100039, China;
3. Department of Central Laboratory, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Peking University Cancer Hospital & Institute, Beijing 100142, China;
4. Department of Central Laboratory, Department of Laboratory Animal Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Peking University Cancer Hospital & Institute,Beijing 100142, China;
5. Center of Excellence(Beijing), Becton Dickinson Medical Devices (Shanghai) Co Ltd., Beijing 100176,China

Received:2022-10-18 Online:2023-02-21 Published:2023-01-13
Contact: *E-mail:yxiang301@sina.com
Supported by:
National Natural Science Foundation of China (81902910).

摘要/Abstract

摘要： 目的对肺癌原位接种的小鼠模型,设计并建立了11色的免疫细胞分类方法,对肿瘤微环境中的T细胞、巨噬细胞、自然杀伤(natural killer,NK)细胞进行更加细致的分型,从而获得有关影响肿瘤细胞进展的研究手段。方法建立11色的上皮细胞、T细胞、巨噬细胞和NK细胞的标志物荧光基团的搭配方案,对有免疫功能的小鼠血液样本进行鉴定。进一步原位接种PC-9细胞于裸鼠体内,通过该搭配方案分析鉴定M1型、M2型巨噬细胞,以及不同成熟程度的NK细胞。结果 11色免疫细胞的搭配方案能够使各种标志物发挥到最大的荧光效应,减少荧光串色。对小鼠血液标本,通过流式细胞学分析可以得到准确的淋巴细胞、巨噬细胞和NK细胞的分类和分型。对原位移植瘤样本,能够更精准的区分肿瘤细胞和各种免疫细胞及细胞亚型。采用溶瘤病毒干预措施,发现干预后的免疫细胞各个分型之间差异有统计学意义。结论在原位移植瘤的模型中,该11色免疫细胞的流式荧光抗体搭配方案可以对肿瘤微环境中的多种免疫细胞进行精确分类,为研究不同免疫细胞和肿瘤细胞之间的相互作用提供了有效的手段,并可以应用于大量的肿瘤微环境研究中。

关键词: 多色流式细胞术, 肺癌,原位移植, 免疫微环境, 免疫细胞, 溶瘤病毒

Abstract: Objective To establish a method to classify T cells, macrophages, and natural killer (NK) cells with detail in tumor microenvironment are needed to address the factors affecting tumor cell progression. Methods The combination of 11-color multiple fluorescence labeling markers for epithelial cells, T cells, macrophages, and NK cells was established to identify blood samples from immunized mice. Luciferase labeling PC-9 cells were further inoculated in the lung of nude mice. More real M1 and M2 macrophages and NK cells of different maturity levels were identified with penal analysis. Results This strategy can maximize the fluorescence effect of various markers and reduce the color cross-talking. Flow cytometry analysis of mouse blood samples allows accurate classification and typing of lymphocytes, macrophages, and NK cells. In orthotopic cell derived xenograft(CDX) samples, tumor cells, various immune cells, and cell subtypes can be distinguished. After the oncolytic virus treatment, the immune cell types were found significantly different. Conclusion In the orthotopic CDX model, the 11-color flow cytometry(FCM) scheme can accurately classify various immune cells in the tumor microenvironment, showing potential in the study of the interaction between different immune cells and tumor cells, and further apply to tumor microenvironment studies.

Key words: multi-color flow cytometry, lung cancer,orthotopic transplantation, immune microenvironment, immune cells, oncolytic virus

中图分类号:

杨敏, 燕翔, 刘锡娟, 安国, 丁慧荣, 蔡英, 葛会争. 人肺癌细胞原位移植瘤小鼠模型免疫细胞的多色流式细胞术检测方法的建立[J]. 首都医科大学学报, 2023, 44(1): 99-106.

Yang Min, Yan Xiang, Liu Xijuan, An Guo, Ding Huirong, Cai Ying, Ge Huizheng. Establishment and characterization of immune cells in an orthotopic cell-derived xenograft mouse model of human lung cancer using multi-color flow cytometry[J]. Journal of Capital Medical University, 2023, 44(1): 99-106.

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人肺癌细胞原位移植瘤小鼠模型免疫细胞的多色流式细胞术检测方法的建立

Establishment and characterization of immune cells in an orthotopic cell-derived xenograft mouse model of human lung cancer using multi-color flow cytometry

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