Characterization of Immunogenicity of Malignant Cells with Stemness in Intrahepatic Cholangiocarcinoma by Single-Cell RNA Sequencing

Abstract

Cancer stem cells (CSCs) are responsible for long-term maintenance of tumors and thought to play a role in treatment resistance. The interaction between stemness and immunogenicity of CSCs in the intrahepatic cholangiocarcinoma (iCCA) is largely unknown. Here, we used single-cell transcriptomic data to study immunogenicity of malignant cells in human iCCA. Using an established computerized method CytoTRACE, we found significant heterogeneity in stemness/differentiation states among malignant cells. We demonstrated that the high stemness malignant cells express much lower levels of major histocompatibility complex II molecules when compared to low stemness malignant cells, suggesting a role of immune evasion in high stemness malignant cells. In addition, high stemness malignant iCCA cells exhibited significant expression of certain cytokine members, including CCL2, CCL20, CXCL1, CXCL2, CXCL6, CXCL8, TNFRSF12A, and IL6ST, indicating communication with surrounding immune cells. These results indicate that high stemness malignant cells retain their intrinsic immunological feature that facilitate the escape of immune surveillance.

Figure 1

Differentiation heterogeneity of malignant cells in iCCA. (a) tSNE plots for malignant cells from 5 tumor samples (upper panel). CytoTRACE analysis of malignant cells (lower panel). CytoTRACE scores projected with tSNE plots are colored red to indicate high stemness/low differentiation and blue for low stemness/high differentiation. (b) GSEA enrichment and leading-edge plots. (Top) Differentially expressed genes found in CytoTRACE classified high stemness malignant cells and low stemness malignant cells. Genes were ranked by Log2FC. (Bottom) Genes contributing the most to the enrichment score. The top 10 genes that are predicted to be specifically associated with high stemness malignant cells are indicated on the right box. (c) tSNE plots showing the expression of CSC marker genes. (d) Violin plots showing the expression of CSC marker genes. ∗ indicates P < 0.05.

Figure 2

Differential expressed genes between high stemness and low stemness iCCA cells. (a) Heatmap showing top 10 DEGs (upregulated and downregulated) between high stemness and low stemness malignant cells. (b) Differences in pathway activity (scored per cell by l2p) between high and low stemness malignant cells (upper panel: upregulated genes; lower panel: downregulated genes).

Figure 3

Comparison of MHC pathway profile between high stemness and low stemness iCCA cells. (a) Violin plot of MHC I pathway-related genes. (b) Violin plot of MHC II pathway-related genes. ∗ indicates P < 0.05.

Figure 4

Comparison of C-C and C-X-C cytokine profile between high stemness and low stemness iCCA cells. (a) Violin plot of C-C chemokines. (b) Violin plot of C-X-C chemokines. ∗ indicates P < 0.05.

Figure 5

Comparison of other inflammatory factor profile between high stemness and low stemness iCCA cells. (a) Violin plot of interleukin family. (b) Violin plot of TNF family and other inflammatory factors. ∗ indicates P < 0.05.

Figure 6

Communication between malignant cells and immune cells. Dot plot showing top predicted ligand-receptor interactions of malignant cells and T cells (upper panel) and malignant cells and NK/myeloid/dendritic cells (lower panel). Column represents ligand and receptor pairs. Red, ligand from malignant cells; blue, receptor from immune cells. Row represents pair of malignant cells (red) and immune cell subtype (blue).