21色流式检测人非小细胞肺癌组织中免疫细胞亚群方案的建立

Establishment of a 21-color Panel for the Detection of Immune Cell Subsets in Human Non-small Cell Lung Cancer Tumor Tissues with Flow Cytometry

背景与目的肺癌组织的免疫微环境已成为关注的重点,随着多色流式的兴起,流式检测肺癌免疫微环境成为重要的手段,但多为检测细胞亚群占比或主要细胞亚群功能,无法同时对两者进行检测。因此本研究建立了一种可靠的21色流式方案,以检测人非小细胞肺癌(non-small cell lung cancer,NSCLC)肿瘤组织中免疫细胞各亚群。方法选用细胞膜表面抗体细胞分化簇(cluster of differentiation,CD)45、CD3、CD19、CD4、CD8、程序性死亡受体1(programmed cell death 1,PD-1)、CD39、CD103、CD25、CD127、趋化因子受体8(chemokine receptor8,CCR8)、CD56、CD11c、人类白细胞抗原(human leukocyte antigen,HLA)-DR、CD38、CD27、CD69、CD62L、CD45RA、CCR7和核酸染料L/D制定方案。首先对各抗体进行抗体滴定实验、电压优化、减一色染色和单色染色实验,确定各实验条件及检测方案后,采用6例健康成年志愿者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)标本验证方案的可行性;检测分析6例NSCLC患者的肿瘤组织样本。结果采用建立的21色流式方案检测了6例NSCLC患者的肿瘤组织样本,可分析出肺癌组织中各细胞亚群的占比及主要细胞群的免疫表型和分化情况。结论成功建立的21色流式方案适用于PBMCs和NSCLC组织样本检测,为监测肺癌中免疫微环境状态提供了一种有效的新思路。

Background and objective With the rise of multicolor flow cytometry,flow cytometry has become an important means to detect the immune microenvironment of lung cancer,but most of them are used to detect the proportion of cell subsets or the function of major cell subsets,and they cannot be detected at the same time.Therefore,a reliable 21-color flow cytometry protocol was established to detect the immune cell subsets in human non-small cell lung cancer(NSCLC)tumor tissues.Methods Cell membrane surface antibodies cluster of differentiation(CD)45,CD3,CD19,CD4,CD8,programmed cell death 1(PD-1),CD39,CD103,CD25,CD127,chemokine receptor 8(CCR8),CD56,CD11c,human leukocyte antigen(HLA)-DR,CD38,CD27,CD69,CD62L,CD45RA,CCR7 and nucleic acid dye L/D were used to develop the protocol.Firstly,antibody titration experiments,voltage optimization,subtraction of one color staining and single color staining experiments were carried out for each antibody,and after the experimental conditions and detection schemes were determined,the feasibility of the scheme was verified by using peripheral blood mononuclear cells(PBMCs)specimens of six healthy adult volunteers.Tumor tissue samples from 6 NSCLC patients were tested and analyzed.Results The established 21-color flow cytometry protocol was used to detect the tumor tissue samples of 6 NSCLC patients,and the proportion of each cell subset in lung cancer tissue,as well as the immunophenotype and differentiation of the main cell population,were analyzed.Conclusion The successfully established 21-color flow cytometry protocol is suitable for the detection of PBMCs and NSCLC tissue samples,which provides an effective new idea for monitoring the immune microenvironment status in lung cancer.