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

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.

Rainbow Kaposi's Sarcoma-Associated Herpesvirus Revealed Heterogenic Replication with Dynamic Gene Expression

“…Next-generation sequencing (NGS) enables researchers to routinely obtain millions of sequence reads that reveal genomic mutations, transcriptome variations, and epigenetic differences at the single-cell level (3)(4)(5). New developments in mass cytom-etry have also enabled analysis of cell surface protein expression and metabolites at single-cell resolution (6)(7)(8)(9). Thus, advancing single-cell technologies are revolutionizing experimental approaches and allowing researchers to investigate cell-to-cell heterogeneity with unprecedented depth and understand biological outcomes as a sum of individual effects.…”

Recent advances in mass spectrometry based clinical proteomics: applications to cancer research

“…This is accomplished by coupling antibody probes to unique stable heavy metal isotopes rather than fluorophores [125]. The cells are nebulized by inductively-coupled plasma (ICP) and the metal ions provide the mass spectrometer a quantitative readout of a marker's distribution within a sample, such as immune cell infiltration [126]. For example, single-cell mass cytometry was used to quantify a panel of 73 proteins related to tumourimmune cell signalling in 144 breast tumours and 50 healthy tissue samples.…”

Progress and applications of mass cytometry in sketching immune landscapes

“…With relative high cellular throughput and high dimensions, CyTOF is an ideal and potent technique that reveals intratumor heterogeneity in a variety of tumors, including breast, [70][71][72][73] lung, [74][75][76][77] oropharyngeal, 78 brain, [79][80][81][82][83] colon, 7,11,84,85 ovarian, 86,87 kidney, 88,89 gastric cancer, 90,91 leukemia and lymphoma, 19,[92][93][94][95] and melanoma. 96,97 A summary of single-cell CyTOF studies on primary tumors of various human cancers is presented in Table 3.…”