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

Hui Jiang,Hao Li,Yiyang Li,Xianting Ding,Ting Zhang,Ziyang Tan,Antony R Warden,Junwei Lv,Boqian Wang

doi:10.3389/fimmu.2019.01571

Abstract

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.

Highlights

Colon cancer (CC) is one of the main causes of cancer death worldwide [1, 2], with high incidence and mortality rates for all genders
In order to build a comprehensive landscape of immune blockade marker expressions, we employ the spanning-tree progression analysis of density-normalized events (SPADE) plot to sketch the profiles (Figure 1B).The results show that Programmed death1 (PD-1) is mainly expressed by memory T cells for both Tcm and Tem, irrespective of CD4+, and CD8+ (Figure 1C, left panel)
To verify whether the identified population epithelial cellular adhesion molecule (EpCAM)+ CD4+ T cells increased in patients with benign polyps, we examine a cohort of patients with colonic polyps (CP) and determine their T cell subset landscape using the same mass cytometry staining panel above (Supplementary Table 2). viSNE maps in Figures 5A,B show similar subpopulation deficiency pattern comparing CP and CC patients with the identified subset marked in red dotted circle

Summary

Introduction

Colon cancer (CC) is one of the main causes of cancer death worldwide [1, 2], with high incidence and mortality rates for all genders. EpCAM+ CD4+ T Cells in Colon Cancer rates. Programmed death (PD-1), an inhibitory receptor expressed on activated T cells, is regarded as a promising target protein for cancer therapy. Immunotherapeutic drugs targeting EpCAM have been developed by utilizing monoclonal antibodies [7, 8], bispecific antibodies [9], or conjugates with other agents, such as toxins [10]. Data from clinical trials of EpCAM blockade suggest limited anti-tumor effects and low immune-activating efficacy. Reports of the therapeutic effects of PD-1 blockade in melanomas, non-small cell lung cancer, and renal-cell cancer patients are promising, exhibiting only a 3% treatment response rate with CC patients [11]. Examining the role of EpCAM and PD-1 in carcinogenesis and malignant progression would aid the development of more efficacious immunotherapeutic schemes

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