Single-cell RNA sequencing of immune cells in gastric cancer patients

Abstract

Cancer immunotherapy has achieved positive clinical responses in the treatment of various cancers, including gastric cancer (GC). In this study, we characterized the heterogeneity of T cells isolated from GC patients at the single-cell level using single-cell RNA sequencing. We identified different immune cell subtypes and their heterogeneous transcription factors and depicted their developmental trajectories. In particular, we focused on exhausted CD8⁺ cells and Tregs and discovered that, as compared to control, the IRF8 transcription factor was downregulated in CD8⁺ tumour-infiltrating lymphocytes (TILs) from GC tissues, and that GC patients with lower IRF8 levels in blood CD8⁺ T cells tended to be a at a more advanced disease stage. These findings provide a theoretical basis for targeted immune therapy in GC.

Keywords: exhausted; gastric cancer; immunotherapy; single-cell RNA sequencing.

Figure 1

Overview of the study design. (A) ScRNA-seq was performed on immune cells isolated from GC preoperational peripheral blood samples and GC tissues and corresponding adjacent non-tumor tissues. 10 cell clusters in tissues and 9 cell clusters in peripheral blood were identified based on CD45 isolation. (B) Each immune cell subtype, their heterogeneous transcription factors, and their developmental trajectories. (C) Correlation between the expression of specific genes and clinical significance.

Figure 2

The transcription factor IRF8 was associated with CD8⁺ T cells in GC. (A) Heat map displaying the top 50 genes differentially expressed in CD8⁺ exhausted T cells from tissues. (B and C) Pathway analysis for CD8⁺ exhausted T cells. (D) Trajectory analysis for CD8⁺ T cells in tissues. (E). Trajectory analysis for CD8⁺ T cells in blood. (F) Expression of IRF8 in CD8⁺TILs from GC tissues and normal tissues. (G) Expression of IRF8 in peripheral blood CD8⁺ T cells from GC patients. (H) TGCA analysis of IRF8 in GC prognosis. (I). Pathway and disease analysis of IRF8.

Figure 3

Identification of genes uniquely associated with Treg function in GC. (A) Heat map displaying the top 50 genes differentially expressed in Tregs from tissues. (B and C) Pathway analysis for different genes in Tregs. (D) Trajectory analysis for Tregs in tissues. (E) Expression of various molecules in Tregs. (F) STRING analysis of RBPJ. (G) Single-cell analysis using CancerSEA. (H) Top 20 differentially expressed TFs in cancers as shown by Cistrome DB Toolkit for RBPJ. (I) GEPIA analyses showing the association between RBPJ and LAG3.

Figure 4

Gene signature of B cells and pathway analysis. (A) The expression analysis of functional molecules in B cell cluster in T vs N. (B) The expression analysis of functional molecules in B cell cluster in PB vs HB. (C) Pathway analysis of in B cell cluster in T vs N. (D) Pathway analysis of in B cell cluster in PB vs HB. (E) The expression analysis of functional molecules in B cell cluster in T vs N. (F) The expression analysis of functional molecules in B cell cluster in PB vs HB.

Figure 5

More inhibitory receptors and fewer activated receptors are secreted by NK cells in response to GC. (A). Expression analysis of functional molecules in the NK cell cluster in T vs N. (B). Expression analysis of functional molecules in the NK cell cluster in PB vs HB. (C). Pathway analysis of functional molecules in the NK cell cluster in T vs N. (D). Pathway analysis of functional molecules in the NK cell cluster in PB vs HB. (E). Expression analysis of functional molecules in the NK cell cluster in T vs N. (F). Expression analysis of functional molecules in the NK cell cluster in PB vs HB.

Figure 6

Different DC subtypes and their interactions in GC. (A) Expression analysis of functional molecules in the DC cell cluster in T vs N. (B) Expression analysis of functional molecules in the DC cell cluster in PB vs HB. (C) Pathway analysis of functional molecules in the DC cell cluster in T vs N. (D) Pathway analysis of functional molecules in the DCB cell cluster in PB vs HB. (E) Expression analysis of functional molecules in the DC cell cluster in T vs N. (F) Expression analysis of functional molecules in the DC cell cluster in PB vs HB.