单细胞转录组数据揭示PHLDA1是卵巢癌T细胞耗竭的关键分子

doi:10.11904/j.issn.1002-3070.2024.02.002

实用肿瘤学杂志 ›› 2024, Vol. 38 ›› Issue (2): 79-87.doi: 10.11904/j.issn.1002-3070.2024.02.002

单细胞转录组数据揭示PHLDA1是卵巢癌T细胞耗竭的关键分子

高燕, 韩晓阳, 程瑾, 侯丽莎, 岳文涛

首都医科大学附属北京妇产医院中心实验室(北京 100026)

收稿日期:2023-12-26 修回日期:2024-04-08 出版日期:2024-04-28 发布日期:2024-07-12
通讯作者: 岳文涛,E-mail:yuewt@ccmu.edu.cn
作者简介:高燕,女,(1986-),博士,助理研究员,从事妇科肿瘤的相关研究。
基金资助:
首都医科大学附属北京妇产医院中青年学科骨干培养专项(编号:FCYY201915);北京市医院管理中心创新梦工场经费资助(编号:202131)

Single cell sequencing data reveal PHLDA1 as a critical molecule responsible for T cell exhaustion in ovarian cancer

GAO Yan, HAN Xiaoyang, CHENG Jin, HOU Lisha, YUE Wentao

Central Laboratory,Beijing Obstetrics and Gynecology Hospital,Capital Medical University,Beijing Maternal and Child Health Care Hospital,Beijing 100026,China

Received:2023-12-26 Revised:2024-04-08 Online:2024-04-28 Published:2024-07-12

摘要/Abstract

摘要： 目的通过绘制高级别浆液性卵巢癌(High-grade serous ovarian cancer,HGSOC)单细胞转录组图谱,筛选并验证与CD8⁺T细胞耗竭相关的关键基因。方法利用实验室前期的单细胞测序数据(SRA数据库:PRJNA756768),并整合数据库中5例HGSOC测序数据,分析肿瘤微环境中T细胞的具体亚型,拟时序分析探究T细胞亚群分化轨迹,差异基因富集确定免疫抑制的CD8⁺IL-2^Low和CD8⁺IFN-γ^Low细胞亚群及差异基因,再结合患者预后筛选出与CD8⁺T细胞耗竭密切相关的候选分子。流式分析人PBMC中的T细胞激活到耗竭过程中Pleckstrin同源样结构域家族A成员1(Pleckstrin homology-like domain,family A,member 1,PHLDA1)在CD8⁺T、CD4⁺T及Treg细胞上的表达,ELISA检测PHLDA1^High和PHLDA1^Low的CD8⁺T细胞分泌IFN-γ和IL-2的水平,最后流式数据分析PHLDA1与耗竭标志物PD-1、TIM-3的关联。结果将T细胞按三种方式分群:(1)IL-2^High和IL-2^Low;(2)IFN-γ^High和IFN-γ^Low;(3)耗竭和杀伤T细胞。随后取其差异基因的交集,最终筛选到关键基因PHLDA1。流式分析提示T细胞激活到耗竭的过程中,PHLDA1在CD8⁺T、CD4⁺T及Treg细胞上的表达持续升高;ELISA结果显示CD8⁺PHLDA1^High的T细胞分泌IFN-γ和IL-2的水平显著低于CD8⁺PHLDA1^LowT细胞。同时,CD8⁺PHLDA1^HighT细胞亚群可同时覆盖CD8⁺TIM-3⁺和CD8⁺PD-1⁺的耗竭T细胞类型。结论本研究基于单细胞测序数据筛选到PHLDA1是卵巢癌CD8⁺T细胞耗竭的关键分子,该研究为卵巢癌的免疫治疗提供了新的思路。

关键词: 单细胞测序, 卵巢癌, Pleckstrin同源样结构域家族A成员1, T细胞耗竭

Abstract: Objective The critical genes associated with exhausted CD8⁺T cells were screened and validated by mapping the single-cell transcriptome profile of high-grade serous ovarian cancer(HGSOC). Methods The specific subtypes of T cells in the tumor microenvironment were analyzed using the single-cell sequencing data from the early stage of laboratory(SRA database:PRJNA756768)and integrating 5 HGSOC sequencing from the database,and the differentiation trajectory of T cell subsets was explored through pseudotime analysis.Differential gene enrichment was used to determine immunosuppressed CD8⁺IL-2^Low and CD8⁺IFN-γ^LowT cell subsets and differential genes,and candidate molecules closely related to exhausted CD8⁺T cells were screened based on patient prognosis.Flow cytometry was used to analyze the expression of PHLDA1 on CD8⁺T cells,CD4⁺T cells and Treg cells during the activation to exhaustion process of T cells in human PBMCs.ELISA was used to detect the levels of IFN-γ and IL-2 secreted by CD8⁺T cells in PHLDA1^High and PHLDA1^Low.Finally,flow cytometry was used to analyze the association between PHLDA1 and exhausted markers PD-1 and TIM-3. Results The results showed that T cells were grouped in three ways:(1)IL-2^High and IL-2^Low;(2)IFN-γ^High and IFN-γ^Low;and(3)exhausted and cytotoxic CD8⁺T cells.Subsequently,the intersection of its differentially expressed genes was taken,and the key gene PHLDA1 was ultimately screened.Flow cytometry analysis suggested that during the process of T cell activation to exhaustion,the expression of PHLDA1 continued to increase on CD8⁺T cells,CD4⁺T cells and Treg cells;The ELISA results showed that the levels of IFN-γ and IL-2 secreted by CD8⁺PHLDA1^HighT cells were significantly lower than those of CD8⁺ PHLDA1^LowT cells.Meanwhile,the CD8⁺PHLDA1^HighT cell subset could simultaneously cover the exhausted T cell types of CD8⁺TIM-3⁺ and CD8⁺PD-1⁺. Conclusion Based on single-cell sequencing data,this study identified PHLDA1 as a key molecule responsible for CD8⁺T cell exhaustion in OC,providing new insights for immunotherapy of OC.

Key words: Single cell sequencing, Ovarian cancer, Pleckstrin homology-like domain,family A,member 1, T cell exhaustion

中图分类号:

R737.31

高燕, 韩晓阳, 程瑾, 侯丽莎, 岳文涛. 单细胞转录组数据揭示PHLDA1是卵巢癌T细胞耗竭的关键分子[J]. 实用肿瘤学杂志, 2024, 38(2): 79-87.

GAO Yan, HAN Xiaoyang, CHENG Jin, HOU Lisha, YUE Wentao. Single cell sequencing data reveal PHLDA1 as a critical molecule responsible for T cell exhaustion in ovarian cancer[J]. Journal of Practical Oncology, 2024, 38(2): 79-87.

参考文献

1 Bray F,Laversanne M,Sung H,et al.Global cancer statistics 2022:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin,2024,74(3):229-263.
2 Chan JK,Brady MF,Penson RT,et al.Weekly vs.every-3-week paclitaxel and carboplatin for ovarian cancer[J].N Engl J Med,2016,374(8):738-748.
3 Drakes ML,Mehrotra S,Aldulescu M,et al.Stratification of ovarian tumor pathology by expression of programmed cell death-1(PD-1)and PD-ligand-1(PD-L1)in ovarian cancer[J].J Ovarian Res,2018,11(1):43.
4 Blank CU,Haining WN,Held W,et al.Defining 'T cell exhaustion'[J].Nat Rev Immunol,2019,19(11):665-674.
5 Normann MC,Türzer M,Diep LM,et al.Early experiences with PD-1 inhibitor treatment of platinum resistant epithelial ovarian cancer[J].J Gynecol Oncol,2019,30(4):e56.
6 Zhao HY,Gao Y,Miao JW,et al.Single-cell RNA-seq highlights a specific carcinoembryonic cluster in ovarian cancer[J].Cell Death Dis,2021,13,12(11):1082.
7 Guo JH,Han XY,Li J,et al.Single-cell transcriptomics in ovarian cancer identify a metastasis-associated cell cluster overexpressed RAB13[J].J Transl Med,2023,12,21(1):254.
8 Geistlinger L,Oh S,Ramo M,et al.Multi-omic analysis of subtype evolution and heterogeneity in high-grade serous ovarian carcinoma[J].Cancer Res,2020,80(20):4335-4345.
9 Beltra JC,Manne S,Abdel-Hakeem MS,et al.Developmental relationships of four Exhausted CD8+ T cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms[J].Immunity,2020,52:825-841.
10 Hellmann MD,Rizvi NA,Goldman JW,et al.Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer(CheckMate 012):results of an open-label,phase 1,multicohort study[J].Lancet Oncol,2017,18:31-41.
11 Park CG,Lee SY,Kandala G,et al.A novel gene product that couples tcr signaling to Fas(Cd95)expression in activation-induced cell death[J].Immunity,1996,4:583-591.
12 Fearon AE,Carter EP,Clayton NS,et al.PHLDA1 mediates drug resistance in receptor tyrosine kinase-driven Cancer[J].Cell Rep,2018,22(9):2469-2481.
13 Wang J,Yao N,Hu Y,et al.PHLDA1 promotes glioblastoma cell growth via sustaining the activation state of Ras[J].Cell Mol Life Sci,2022,79(10):520.
14 Joo JH,Liao G,Collins JB,et al.Farnesol-induced apoptosis in human lung carcinoma cells is coupled to the endoplasmic reticulum stress response[J].Cancer Res,2007,67:7929-7936.
15 Peng H,Wang JP,Song XH,et al.PHLDA1 suppresses TLR4-triggered proinflammatory cytokine production by interaction with tollip[J].Front Immunol,2022,13:731500.
16 Chen Y,Takikawa M,Tsutsumi S,et al.PHLDA1,another PHLDA family protein that inhibits Akt[J].Cancer Sci,2018,109(11):3532-3542.
17 Zhou X,Fang D,Liu H,et al.PMN-MDSCs accumulation induced by CXCL1 promotes CD8+ T cells exhaustion in gastric cancer[J].Cancer Lett,2022,532:215598.
18 Oberg H,Sipos B,Kalthoff H,et al.Regulation of T-cell death-associated gene 51(TDAG51)expression in human T-cells[J].Cell Death Differ,2004,11(6):674-684.
19 Nagai MA.Pleckstrin homology‐like domain,family A,member 1(PHLDA1)and cancer[J].Biomed Rep,2016,4(3):275-281.

[1]	高燕, 姚盟成, 李哲丰, 韩晓阳, 岳文涛. 单细胞测序数据揭示DMKN在卵巢癌中的诊断及预测价值[J]. 实用肿瘤学杂志, 2023, 37(6): 478-484.
[2]	樊婷婷, 丁丹妮, 赵子雪, 于洋, 韩凤娟. 脂肪酸代谢与卵巢癌[J]. 实用肿瘤学杂志, 2023, 37(5): 429-433.
[3]	杨帅, 王新恒, 吴佳乐, 付子彤, 魏博, 杨灵冰, 许守平. 单细胞测序技术在乳腺癌中的研究进展[J]. 实用肿瘤学杂志, 2023, 37(3): 287-292.
[4]	孙阜圣, 徐冶, 张永健, 娄阁. 卵巢癌类器官模型的研究进展和治疗新策略[J]. 实用肿瘤学杂志, 2022, 36(4): 346-350.
[5]	尹翼, 欧朝阳, 何爱琴. GAB2在卵巢癌组织中的表达及其与预后的相关性[J]. 实用肿瘤学杂志, 2022, 36(2): 150-155.
[6]	董丽娜, 梁田, 张献, 张广美. 卵巢癌对富脂大网膜的器官趋向性转移的研究进展[J]. 实用肿瘤学杂志, 2022, 36(1): 54-58.
[7]	范琳, 田霖丽, 刘鸣. 单细胞测序技术在头颈部恶性肿瘤中的研究与发展[J]. 实用肿瘤学杂志, 2021, 35(5): 472-475.
[8]	张文文,姜亮亮(综述),王晶(审校). CAR-T疗法在卵巢癌中的应用前景[J]. 实用肿瘤学杂志, 2021, 35(1): 59-63.
[9]	侯建英, 朱巍. 免疫抑制剂PD-1/PD-L1治疗卵巢癌的研究进展[J]. 实用肿瘤学杂志, 2020, 34(3): 266-270.
[10]	梁永琴, 赵宏伟. 抗血管生成药物在铂耐药卵巢癌中的应用进展[J]. 实用肿瘤学杂志, 2020, 34(1): 60-63.
[11]	黄梨, 龙佑梅, 付义霞, 夏良斌. 紫檀芪对卵巢癌SKOV3细胞凋亡和糖酵解的影响及机制研究[J]. 实用肿瘤学杂志, 2019, 33(6): 502-507.
[12]	邓明新, 张颖. 环状RNA与卵巢癌研究新进展[J]. 实用肿瘤学杂志, 2019, 33(6): 549-552.
[13]	张慧婷, 娄阁. 卵巢癌肿瘤微环境的三维细胞培养模型研究进展[J]. 实用肿瘤学杂志, 2019, 33(6): 557-560.
[14]	宋玲, 付琼. 紫云英苷通过上调PHD2抑制缺氧诱导的卵巢癌细胞增殖和侵袭[J]. 实用肿瘤学杂志, 2019, 33(5): 407-414.
[15]	张蓓蕾, 李怡, 吴涛, 姜锋, 赵宏喜, 李艳红. 血根碱通过诱导细胞凋亡抑制卵巢癌肿瘤生长的机制研究[J]. 实用肿瘤学杂志, 2019, 33(4): 305-309.

单细胞转录组数据揭示PHLDA1是卵巢癌T细胞耗竭的关键分子

Single cell sequencing data reveal PHLDA1 as a critical molecule responsible for T cell exhaustion in ovarian cancer

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价