遗传 ›› 2019, Vol. 41 ›› Issue (2): 175-184.doi: 10.16288/j.yczz.18-230

• 技术与方法 • 上一篇    

肺癌恶性胸腔积液中稀有肿瘤细胞的鉴定与单细胞测序分析

吴保军,王卓,董宇,邓宇亮,施奇惠()   

  1. 上海交通大学系统生物医学研究院,系统生物医学教育部重点实验室,上海 200240
  • 收稿日期:2018-10-31 修回日期:2018-12-27 出版日期:2019-02-25 发布日期:2019-01-14
  • 作者简介:吴保军,硕士研究生,专业方向：循环肿瘤细胞单细胞测序。E-mail: wbaojun@sjtu.edu.cn
  • 基金资助:
    国家自然科学基金项目(21775103);国家自然科学基金项目(81501613);上海交通大学“医工交叉基金”项目(YG2017QN54)

Identification and single-cell sequencing analysis of rare tumor cells in malignant pleural effusion of lung cancer patients

Baojun Wu,Zhuo Wang,Yu Dong,Yuliang Deng,Qihui Shi()   

  1. Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, China
  • Received:2018-10-31 Revised:2018-12-27 Online:2019-02-25 Published:2019-01-14
  • Supported by:
    [Supported by the National Natural Science Foundation of China(21775103);[Supported by the National Natural Science Foundation of China(81501613);Medical Engineering Cross Research Fund of Shanghai Jiao Tong University](YG2017QN54)

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摘要：

肿瘤细胞的异质性是指肿瘤细胞在基因组或表型水平上具有不同特征。在外界环境压力或人为杀伤因素刺激下,肿瘤细胞具有不同的响应方式,从而导致它们在细胞增殖、侵袭转移及耐药能力等方面产生差别,尤其是一部分具有转移能力的肿瘤细胞能脱离原位组织并在远处器官形成转移灶。因此,肿瘤细胞的异质性为其发生转移和耐药提供了可能。传统的肿瘤异质性研究主要基于不同位置原位组织样本中的群体细胞,缺乏单细胞层面的解析,尤其是对具有转移能力的肿瘤细胞的异质性研究。本研究建立了基于肺癌恶性胸腔积液中转移性肿瘤细胞的单细胞研究路线,首先利用代谢标志物鉴定恶性胸腔积液中高代谢活性的肿瘤细胞,其次通过单细胞Sanger测序揭示这些肿瘤细胞具有一致的驱动基因突变特征,然后利用高通量测序技术对肿瘤细胞染色体拷贝数变异进行分析,从而揭示同一患者具有转移能力的肿瘤细胞即使具有相同的驱动基因突变特征,但在基因组层面上仍具有异质性,可进一步细分为若干子群。本研究结果对于更深入地理解肿瘤转移机制具有重要意义。

关键词: 肺癌, 胸腔积液, 肿瘤细胞, 单细胞测序

Abstract:

Tumor heterogeneity refers to distinct genomic or phenotypic characteristics of tumor cells. Under the environmental or drug stress, tumor cells exhibit different responses, corresponding to different properties of cell proliferation, invasion, metastasis and drug resistance. In particular, a small fraction of tumor cells are capable of detaching from primary tumor sites and initiating distant metastases. Thus, tumor heterogeneity sets the basis for tumor resistance and metastasis. Traditional methods in studying tumor heterogeneity are mainly based on bulk cells from different locations in primary tumors, lacking analysis at the single-cell level and of metastatic tumor cells. This study establishes a single-cell method to study metastatic tumor cells in malignant pleural effusions of lung cancer patients. Metabolically active tumor cells in malignant pleural effusions are firstly identified with a metabolic marker 2-NBDG, a fluorescent glucose analog. These metabolically active tumor cells are confirmed to harbor the same driver oncogenic mutations by Sanger sequencing, followed by high-throughput sequencing to analyze copy number variation profiles. Our results show metastatic tumor cells in pleural effusion have the same driver mutations but different features in copy number variation patterns. The study provides new insights to understand the mechanism of tumor metastasis.

Key words: lung cancer, pleural effusion, tumor cells, single-cell sequencing