Comprehensive single-cell transcriptomic and proteomic analysis reveals NK cell exhaustion and unique tumor cell evolutionary trajectory in non-keratinizing nasopharyngeal carcinoma

Abstract

Background Nonkeratinizing nasopharyngeal carcinoma (NK-NPC) has a strong association with Epstein-Barr virus (EBV) infection. The role of NK cells and the tumor cell evolutionary trajectory in NK-NPC remain unclear. In this study, we aim to investigate the function of NK cell and the evolutionary trajectory of tumor cells in NK-NPC by single-cell transcriptomic analysis, proteomics and immunohistochemistry. Methods NK-NPC (n = 3) and normal nasopharyngeal mucosa cases (n = 3) were collected for proteomic analysis. Single-cell transcriptomic data of NK-NPC (n = 10) and nasopharyngeal lymphatic hyperplasia (NLH, n = 3) were obtained from Gene Expression Omnibus (GSE162025 and GSE150825). Quality control, dimension reduction and clustering were based on Seurat software (v4.0.2) process and batch effects were removed by harmony (v0.1.1) software. Normal cells of nasopharyngeal mucosa and tumor cells of NK-NPC were identified using copykat software (v1.0.8). Cell-cell interactions were explored using CellChat software (v1.4.0). Tumor cell evolutionary trajectory analysis was performed using SCORPIUS software (v1.0.8). Protein and gene function enrichment analyses were performed using clusterProfiler software (v4.2.2). Results A total of 161 differentially expressed proteins were obtained between NK-NPC (n = 3) and normal nasopharyngeal mucosa (n = 3) by proteomics (log2 fold change > 0.5 and P value < 0.05). Most of proteins associated with the nature killer cell mediated cytotoxicity pathway were downregulated in the NK-NPC group. In single cell transcriptomics, we identified three NK cell subsets (NK1-3), among which NK cell exhaustion was identified in the NK3 subset with high ZNF683 expression (a signature of tissue-resident NK cell) in NK-NPC. We demonstrated the presence of this ZNF683 + NK cell subset in NK-NPC but not in NLH. We also performed immunohistochemical experiments with TIGIT and LAG3 to confirm NK cell exhaustion in NK-NPC. Moreover, the trajectory analysis revealed that the evolutionary trajectory of NK-NPC tumor cells was associated with the status of EBV infection (active or latent). The analysis of cell-cell interactions uncovered a complex network of cellular interactions in NK-NPC. Conclusions This study revealed that the NK cell exhaustion might be induced by upregulation of inhibitory receptors on the surface of NK cells in NK-NPC. Treatments for the reversal of NK cell exhaustion may be a promising strategy for NK-NPC. Meanwhile, we identified a unique evolutionary trajectory of tumor cells with active status of EBV-infection in NK-NPC for the first time. Our study may provide new immunotherapeutic targets and new sight of evolutionary trajectory involving tumor genesis, development and metastasis in NK-NPC.

Revealing the role of SPP1+ macrophages in glioma prognosis and therapeutic targeting by investigating tumor-associated macrophage landscape in grade 2 and 3 gliomas

“…For instance, the interaction between the glycoprotein SPP1 and the cell surface receptor CD44 has been found to inhibit T cell activation and proliferation, as well as promote tumor immune evasion [ 55 , 56 ]. Additionally, Galectin-9 (encoded by LGALS9 ), a recognized suppressive immunomodulator, has been reported to confer immune tolerance by exhausting natural killer (NK) cells and stimulating regulatory T cell (Treg) differentiation through its interaction with CD44 [ 57 , 58 ].…”

The Role of Natural Killer Cells in the Tumor Immune Microenvironment of EBV-Associated Nasopharyngeal Carcinoma

“…TIGIT PVR TIGIT-PVR interaction was detected between NK and tumor cells and between NK and macrophages/dendritic cells in NPC; TIGIT expression was upregulated on exhausted NK cells in NPC. [48,64] CD96-PVR interaction was detected between NK and tumor cells in NPC CD96 TIGIT and CD96 were positively correlated with FCER2 and KHDRBS2 and negatively correlated with IGSF9 in NPC *. [63] LAG3 Gal-3…”

“…According to single-cell transcriptomic and proteomic analysis research, infiltrated NK cells within EBV + NPC biopsies could be divided into three sub-groups, which showed cytotoxic, inhibited, and exhausted phenotypes, respectively. Notably, these exhausted NK cells showed an upregulation of both lymphocyte-activation gene 3 (LAG3) and T cell immunoreceptor with Ig and ITIM domain (TIGIT) expression, both known as immune checkpoint receptors [64]. Also, comparing the EBV + NPC and EBV − NPC biopsies, the percentage of activated CD56 + CD3 − granzyme B + NK cells was found to be lower for EBV + NPC [65].…”

“…Unfortunately, these studies have not indicated NK cell subtypes. On the contrary, when detected with immunohistochemical staining (IHC), CD56 + NK cells were reduced in nonkeratinizing NPC tissue compared with normal nasopharyngeal mucosa [64]. Peng and colleagues demonstrated that CD56 dim CD16 + NK cells, the cytotoxic subtype, accounted for the majority of NK cells in both treatment naïve and recurrent EBV + NPC tissues.…”

Single-cell technologies in multiple myeloma: new insights into disease pathogenesis and translational implications

“…Mechanistically, ZNF683 can directly suppress SH2D1B (EAT-2) expression, thus disrupting EAT-2/SLAMF7-mediated activating signals and eventually promoting NK cell exhaustion. Likewise, a ZNF683 + NK cell subset was also found in nonkeratinizing nasopharyngeal carcinoma exhibiting similar exhausted phenotypes with upregulated LAG3 and TIGIT expressions [ 201 ]. These consistent findings lead to a confirmation of ZNF683 + NK cell present in cancer context and highlight potential roles of ZNF683 in regulating NK cell function.…”