结直肠癌中自然杀伤细胞表型及功能初探

doi:10.3969/j.issn.1674-8115.2024.06.006

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (6): 713-722.doi: 10.3969/j.issn.1674-8115.2024.06.006

• 论著 · 基础研究 • 上一篇

结直肠癌中自然杀伤细胞表型及功能初探

冯昫皎¹(), 刘健悦¹, 戚炀炀¹, 孙晶², 沈蕾¹^,³()

^1.上海交通大学基础医学院免疫学与微生物学系，上海市免疫学研究所，上海 200025
^2.上海交通大学医学院附属瑞金医院普外科，上海市微创外科临床医学中心，上海 200025
^3.上海交通大学医学院附属瑞金医院免疫与疾病研究中心，上海 200025

收稿日期:2023-12-18 接受日期:2024-03-06 出版日期:2024-06-28 发布日期:2024-06-28
通讯作者: 沈蕾 E-mail:xujiao_feng@163.com;lshen@shsmu.edu.cn
作者简介:冯昫皎（1987—），女，硕士生；电子信箱：xujiao_feng@163.com。
基金资助:
国家重点研发计划(2020YFA0509203);上海市科学技术委员会“科技创新行动计划”基础研究领域项目(20JC1410100)

Phenotype and function of NK cell in colorectal cancer

FENG Xujiao¹(), LIU Jianyue¹, QI Yangyang¹, SUN Jing², SHEN Lei¹^,³()

^1.Shanghai Institute of Immunology; Department of Immunology and Microbiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^2.Department of General Surgery, Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^3.Center for Immune-Related Diseases at Shanghai Institute of Immunology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2023-12-18 Accepted:2024-03-06 Online:2024-06-28 Published:2024-06-28
Contact: SHEN Lei E-mail:xujiao_feng@163.com;lshen@shsmu.edu.cn
Supported by:
National Key Research and Development Program of China(2020YFA0509203);Shanghai Science and Technology Commission Science and Technology Innovation Program in Basic Research Area(20JC1410100)

摘要/Abstract

摘要：

目的·初步探究结直肠癌（colorectal cancer，CRC）患者肿瘤组织中免疫微环境的组成，分析自然杀伤细胞（natural killer cell，NK细胞）在CRC中的比例、表型及功能特征。方法·收集CRC患者肿瘤组织及配对的癌旁组织和匹配的CRC患者外周血样本，消化组织制备成单细胞悬液，使用流式细胞术检测各类免疫细胞比例，通过t-SNE（t-distributed stochastic neighbor embedding）降维与统计学分析，描述CRC肿瘤免疫微环境的特征。运用流式细胞术检测肿瘤组织和癌旁组织中NK细胞表面活化分子的表达情况，包括CD16、CD27、CD69、晚期活化标志人类白细胞抗原-DR（human leukocyte antigen-DR，HLA-DR）、细胞耗竭标志T细胞免疫球蛋白与黏蛋白结构域3（T cell immunoglobulin domain and mucin domain-3，TIM-3）。运用流式细胞术探究CD38分子在NK细胞上的表达水平，描述CD38^highNK细胞的表型特征。利用细胞刺激试剂盒激活NK细胞，通过流式细胞术胞内染色检测NK细胞的功能，包括细胞因子γ干扰素（interferon-γ，IFN-γ）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor，GM-CSF）的表达水平。结果·收集25例CRC患者肿瘤组织及配对的癌旁组织和15例匹配的CRC患者外周血样本。CRC肿瘤组织中包含T淋巴细胞、B淋巴细胞、NK细胞及髓系细胞等免疫细胞。与癌旁组织相比，肿瘤组织中T淋巴细胞（P=0.000）和髓系细胞（P=0.026）的比例显著升高，NK细胞（P=0.007）的比例显著降低，B淋巴细胞比例差异无统计学意义。与癌旁组织相比，肿瘤组织中NK细胞表面CD27（P=0.000）、CD69（P=0.001）表达水平显著降低，同时CD16（P=0.008）、HLA-DR（P=0.000）和TIM-3（P=0.024）的表达水平显著升高，呈现晚期活化和耗竭表型。NK细胞按CD38的表达水平可分为CD38^highNK和CD38^lowNK 2个亚群。与癌旁组织相比，肿瘤组织CD38^highNK（P=0.003）比例显著降低，CD38^lowNK比例差异无统计学意义。相比CD38^lowNK，CD38^highNK的CD27高表达，CD16、NKp46、CD57、CD94、HLA-DR和CD158a低表达（均P=0.000），处于早期分化和未活化的状态。肿瘤浸润的NK细胞与癌旁组织相比，分泌细胞因子IFN-γ（P=0.032）、TNF-α（P=0.042）和GM-CSF（P=0.019）的水平显著降低，表明NK细胞杀伤功能受损。结论·CRC患者肿瘤组织中NK细胞浸润减少，早期分化状态的CD38^highNK细胞亚群下降，分泌细胞因子的能力下降并呈现耗竭表型。这些结果提示NK细胞在CRC中功能受损，介导的抗肿瘤免疫应答下降。

关键词: 自然杀伤细胞, 结直肠癌, CD38

Abstract:

Objective ·To investigate the composition of immune cells in tumor microenvironment of colorectal cancer (CRC), and examine the proportion, phenotype and effector function of natural killer (NK) cells in CRC. Methods ·Fresh tumor tissues, paired normal tissues adjacent to tumor, and peripheral blood samples in the same cohort were collected from CRC patients. Tissues were digested and prepared into single cell suspension. The major immune cell lineages were detected by flow cytometry. t-Distributed stochastic neighbor embedding (t-SNE) and statistical analysis were used to analyze the composition of immune cells in tumor microenvironment of CRC. To analyze the phenotype of NK cells, the expression levels of activation markers, including CD16, CD27, CD69, human leukocyte antigen-DR (HLA-DR), and T cell immunoglobulin domain and mucin domain-3 (TIM-3), were detected by flow cytometry. NK cell subsets: CD38^lowNK cells and CD38^highNK cells were also examined by flow cytometry. To assess the effector function of NK cells, they were stimulated with cell stimulation cocktail and the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured by flow cytometry. Results ·Twenty-five pairs of fresh tumor tissues and normal tissues adjacent to tumor, and 15 peripheral blood samples in the same cohort were collected from CRC patients. The tumor microenvironment of CRC included diverse immune cell types, including T cells, B cells, NK cells and myeloid cells. The proportions of T cells (P=0.000) and myeloid cells (P=0.026) in tumor tissues were significantly higher than those in normal tissues. By contrast, the proportion of NK cells (P=0.007) in tumor was significantly reduced. The proportion of B cells was comparable between tumor and normal tissues. Compared to normal tissues, NK cells in tumor tissues expressed significantly lower CD27 (P=0.000) and CD69 (P=0.001), while the expression levels of CD16 (P=0.008), HLA-DR (P=0.000) and TIM-3 (P=0.024) were significantly elevated. The results indicated that NK cells in CRC tumor exhibited a phenotype of late activation and exhaustion. According to the expression level of CD38, NK cells could be divided into two subsets, CD38^highNK cells and CD38^lowNK cells. The proportion of CD38^highNK cells (P=0.003) in tumor tissues was significantly lower than that in normal tissues, while the proportion of CD38^lowNK cells was unaffected. Compared to CD38^lowNK cells, CD38^highNK cells expressed higher CD27, meanwhile significantly less CD16, NKp46, CD57, CD94, HLA-DR and CD158a (P=0.000). These results suggested that CD38^highNK cells were at early differentiation state. The secretion of cytokines IFN-γ (P=0.032), TNF-α (P=0.042), and GM-CSF (P=0.019) by tumor-infiltrated NK cells was significantly decreased compared to that in normal tissues. The results showed that the function of tumor-infiltrated NK cells was impaired. Conclusion ·Together, these data suggest that NK cell compartment is disrupted in tumor tissues of CRC, leading to the impaired anti-tumor immunity mediated by NK cells.

Key words: natural killer cell (NK cell), colorectal cancer (CRC), CD38

中图分类号:

R392.12

冯昫皎, 刘健悦, 戚炀炀, 孙晶, 沈蕾. 结直肠癌中自然杀伤细胞表型及功能初探[J]. 上海交通大学学报（医学版）, 2024, 44(6): 713-722.

FENG Xujiao, LIU Jianyue, QI Yangyang, SUN Jing, SHEN Lei. Phenotype and function of NK cell in colorectal cancer[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(6): 713-722.

图/表 6

表1 试剂配置

Tab 1 Reagent formulation

Reagent name	System
Tumor wash buffer
PBS (1×)	Up to 20 mL (for 1 g biopsies)
DTT (2 mol·L^-1)	65 μL (final 6.5 mmol·mL^-1)
FBS	2 mL (10%)
Normal wash buffer
PBS (1×)	Up to 20 mL (for 1 g biopsies)
HEPES (1 mol·L^-1)	300 μL (final 15 mmol·mL^-1)
EDTA (0.5 mol·L^-1)	200 μL (final 5 mmol·mL^-1)
DTT (2 mol·L^-1)	10 μL (final 1 mmol·mL^-1)
FBS	2 mL (10%)
Tissue digestion buffer
RPMI1640 (+10%FBS, +1%P/S)	Up to 40 mL (for 2 g biopsies)
Col Ⅷ	45.7 μL (final 0.38 mg·mL^-1)
DNase Ⅰ	26.8 μL (final 0.1 mg·mL^-1)

表2 25例结直肠癌患者的临床和病理特征

Tab 2 Clinical and pathological characteristics of 25 colorectal cancer patients

Characteristic	Colorectal cancer patient (n=25)
Age/year	62.4±11.8
Gender/n(%)
Male	9 (36.0)
Female	16 (64.0)
Pathological T stage/n(%)
T2	3 (12.0)
T3	18 (72.0)
T4	4 (16.0)
Pathological N stage/n(%)
N0	14 (56.0)
N1	6 (24.0)
N2	5 (20.0)
Pathological M stage/n(%)
M0	24 (96.0)
M1	1 (4.0)
TNM stage/n(%)
Ⅰ	2 (8.0)
Ⅱ	12 (48.0)
Ⅲ	10 (40.0)
Ⅳ	1 (4.0)

图1 结直肠癌免疫微环境的组成分析Note： A. Gating strategy of T cells, B cells, NK cells, macrophages, dendritic cells and monocytes in colorectal cancer sample. B. t-SNE dimensionality reduction analysis of immune cell components in colorectal cancer samples. C. Percentage of T cells, B cells, NK cells, myeloid cells and other cells in CD45+ immune cells in tumor and normal tissues (n=25). ①P=0.000, ②P=0.007, ③P=0.026.

Fig 1 Analysis of the immune microenvironment composition in colorectal cancer

图2 肿瘤组织中NK细胞比例减少且呈现耗竭表型Note： A. Representative flow cytometry analysis and percentage of NK cells in normal and tumor tissues (n=25). B. Representative flow cytometry analysis and percentage of CD16+NK cells in PBMC, normal and tumor tissues (nPBMC=15, nnormal=25, and nTumor=25). C. Representative flow cytometry analysis of the expression of CD27, CD69, HLA-DR and TIM-3 on NK cells in normal and tumor tissues. D. The expression levels of CD27, CD69, HLA-DR and TIM-3 on NK cells in normal and tumor tissues (n=25). ①P=0.007, ②P=0.000, ③P=0.008, ④P=0.001, ⑤P=0.024. MFI—mean fluorenscence intensity.

Fig 2 NK cells in CRC tumor tissues were reduced and showed an exhaustion phenotype

图3 CD38highNK细胞在肿瘤组织中显著减少Note： A. Representative flow cytometry analysis and percentage of CD38highNK cells in normal and tumor tissues (n=25). B. Representative flow cytometry analysis of the expression of CD16, NKp46, CD57, CD94, HLA-DR and CD158a in CD38highNK cells and CD38lowNK cells in tumor tissues. C. The expression levels of CD16, NKp46, CD57, CD94, HLA-DR and CD158a in CD38highNK cells and CD38lowNK cells in tumor tissues (n=25). ①P=0.003, ②P=0.000.

Fig 3 CD38highNK cells were dramatically reduced in tumor tissues

图4 肿瘤组织中NK细胞分泌细胞因子的能力减弱Note：A. Representative flow cytometry analysis and percentage of IFN-γ+NK cells in normal and tumor tissues (n=25). B. Representative flow cytometry analysis and percentage of TNF-α+NK cells in normal and tumor tissues (n=25). C. Representative flow cytometry analysis and percentage of GM-CSF+NK cells in normal and tumor tissues (n=25). ①P=0.032, ②P=0.042, ③P=0.019.

Fig 4 Impaired cytokine production of NK cells in tumors

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结直肠癌中自然杀伤细胞表型及功能初探

Phenotype and function of NK cell in colorectal cancer

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