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.