Data_Sheet_1_Immunocyte Profiling Using Single-Cell Mass Cytometry Reveals EpCAM+ CD4+ T Cells Abnormal in Colon Cancer.docx

Colon cancer (CC) is one of the leading causes of cancer related mortality. Research over past decades have profoundly enhanced our understanding of immunotherapy, a major clinical accomplishment, and its potential role toward treating CC. However, studies investigating the expression of these immune checkpoints, such as epithelial cell adhesion molecule (EpCAM), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1), by peripheral blood mononuclear cells (PBMCs) is lacking. Here, high-dimensional mass cytometry (CyTOF) is used to investigate immune alterations and promising immunotherapeutic targets expression by PBMCs of CC patients. Results reveal that expression of EpCAM and PD-L1 on CD4+ T cells significantly increased in patients with CC, compared with age- and sex- matching healthy controls and patients with colonic polyps. These differences are also validated in an independent patient cohort using flow cytometry. Further analysis revealed that EpCAM+ CD4+ T cells are PD-L1+ CCR5+ CCR6+. Immunofluorescence staining results demonstrate that the increase of EpCAM+ CD4+ T cells is also observed in tumor tissues, rather than para-cancerous tissues. To ascertain the functional disorders of the identified cell subset, phosphorylated signaling protein levels are assessed using imaging mass cytometry. Increases in pp38 MAPK and pMAPKAPK2 are observable, indicating abnormal activation of pp38 MAPK-pMAPKAPK2 signaling pathway. Results in this study indicate that EpCAM+ CD4+ T cells may play a role in CC development. Detailed knowledge on the functionality of EpCAM+ CD4+ T cells is of high translational relevance.

结肠癌（CC）是导致癌症相关死亡率的主要原因之一。数十年的研究极大地深化了我们对免疫疗法的理解，这一重大临床成就及其在治疗结肠癌中的潜在作用。然而，关于外周血单个核细胞（PBMCs）中这些免疫检查点（如上皮细胞粘附分子（EpCAM）、程序性死亡-1（PD-1）和程序性死亡配体-1（PD-L1））表达的研究尚显不足。在本研究中，通过高维质谱流式细胞术（CyTOF）对结肠癌患者PBMCs的免疫改变和有希望的免疫治疗靶标表达进行了调查。研究结果揭示，与年龄和性别匹配的健康对照者和结肠息肉患者相比，结肠癌患者CD4+ T细胞上EpCAM和PD-L1的表达显著增加。这些差异在独立患者队列中通过流式细胞术也得到了验证。进一步分析显示，EpCAM+ CD4+ T细胞同时表达PD-L1、CCR5和CCR6。免疫荧光染色结果显示，EpCAM+ CD4+ T细胞的增加仅在肿瘤组织中观察到，而非癌旁组织中。为了确定所识别的细胞亚群的生理功能障碍，使用成像质谱流式细胞术评估了磷酸化信号蛋白水平。观察到pp38 MAPK和pMAPKAPK2水平的增加，这表明pp38 MAPK-pMAPKAPK2信号通路异常激活。本研究的结果表明，EpCAM+ CD4+ T细胞可能在结肠癌的发生发展中发挥作用。对EpCAM+ CD4+ T细胞功能的深入了解具有高度的临床转化意义。