Exploring the role of innate immunity in Cholangiocarcinoma: implications for prognosis, immune infiltration, and tumor metastasis

Abstract

The innate immune system serves as the body's primary physiological defense against the intrusion of pathogenic microorganisms, playing a pivotal role in restricting viral infections. However, current research on the interplay between innate immune pathways and cancer is limited, with reported effects often inconsistent. Therefore, we aimed to elucidate the relationship between innate immune pathways and tumors through an amalgamation of bioinformatics and extensive data analysis. Conducting a pan-cancer analysis encompassing expression, genomic alterations, and clinical prognosis, we identified a close association between the innate immune pathway and cholangiocarcinoma. Subsequently, our focus shifted to unraveling the role of innate immune pathway proteins in cholangiocarcinoma. TIMER database analysis showed that the innate immune pathway predominantly influences the infiltration of macrophages and B cells in cholangiocarcinoma. Additionally, gene ontology (GO) and pathway analyses were performed for significantly differentially expressed genes correlated with the innate immune pathway in cholangiocarcinoma. Single-cell transcriptome analysis in cholangiocarcinoma demonstrated that genes in the innate immune pathway are primarily expressed in malignant cells, endothelial cells, monocytes and macrophages. To further validate the expression of proteins in the innate immune pathway in the tumor tissues of patients with cholangiocarcinoma, tumor tissue slices from patients with liver intrahepatic cholangiocarcinoma and normal tissue slices from the HPA database were analyzed. These results indicated pronounced activation of the innate immune pathway in the tumor tissues of patients with cholangiocarcinoma. Finally, proteomic data from patients with or without intrahepatic cholangiocarcinoma metastasis were analyzed. The results revealed a significant correlation between the expression and phosphorylation of IKKε and the occurrence of intrahepatic cholangiocarcinoma metastasis. These findings not only demonstrate the significance of the innate immune pathway in cholangiocarcinoma but also its potential as a prospective prognostic biomarker and therapeutic target for this malignancy.

Keywords: cholangiocarcinoma; immune cell infiltration; innate immune pathway; metastasis; prognosis.

Figure 1

The schematic workflow representing the study's design and the data collection. Primarily, a comprehensive analysis of the pan-cancer expression, genomic alterations and survival prognosis of key proteins within the innate immune pathway. Subsequently, functional enrichment analysis of relevant genes, immune infiltration analysis, single-cell transcriptomic analysis, immunohistochemical analysis, promoter methylation analysis and correlation analysis with tumor metastasis, all focused on the key proteins within the innate immune pathway in cholangiocarcinoma.

Figure 2

Innate immune pathway expression levels in different types of human cancers. Key genes of innate immune pathway expression levels in different tumor types from TCGA were determined by TIMER (A-G) (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

Expression levels of the Type I interferons and interferon-stimulated genes (ISG). For the types of BRCA, CHOL, COAD, ESCA, LIHC, LUSC and STAD in the TCGA project, the corresponding normal tissues in the GTEx database were included as controls. The box plot data were supplied. (*P<0.01).

Figure 4

Innate immune pathway alternation analysis in different tumors. (A) The proportions of various innate immune pathway alternations in different subclasses of tumors. (B) The genetic alternations of innate immune pathway in tumors.

Figure 5

Mutation features of innate immune pathway in different tumors of TCGA. Mutation sites and the mutation site in the 3D structure of proteins are displayed (A-G).

Figure 6

Prognostic significance of innate immune pathway in various cancer patients. Heatmap showing the impact of innate immune pathway on OS (A) and DFS (B). The survival curves for OS of TBK1 in KICH patients (C), OS of TBK1 in READ patients (D), DFS of IKBKE in UVM patients (E), DFS of TMEM173 in UVM patients (F), DFS of C6orf150 in CHOL patients (G), and DFS of TMEM173 in CHOL patients (H) in TCGA data.

Figure 7

The correlation between differentially expressed innate immune pathway and immune cell infiltration in cholangiocarcinoma. The stacked histogram showing the composition of immune cells infiltrating in cholangiocarcinoma tissues (A). The correlations between the transcriptional levels of TMEM173 (B) and C6orf150 (C) with the infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells in cholangiocarcinoma were shown. Scatterplots of correlations between TMEM173 expression and gene markers of B cell (D) or dendritic cell (E).

Figure 8

Analysis of differentially expressed genes in correlation with DDX58 in cholangiocarcinoma. (A) Volcano plot showing the up-regulated and down-regulated genes correlated with DDX58 expression (Pearson test). The significantly positively correlated (B) and negatively correlated (C) genes were shown in heatmaps. (D) Kyoto Encyclopedia Genes and Genomes (KEGG) pathway analysis of the significantly differentially expressed genes in correlation with DDX58. (E) Gene ontology (GO) analysis of the significantly differentially expressed genes in correlation with DDX58.

Figure 9

Analysis of differential expression of the innate immune pathway across distinct cellular subtypes in cholangiocarcinoma (single-cell transcriptomic dataset GSE138709). TISCH2 analysis reveals the correlation between innate immune pathway expression and immune cell populations in cholangiocarcinoma.

Figure 10

Expression of RIG-I, MAVS, cGAS and IRF3 in normal and intrahepatic cholangiocarcinoma tissues. IHC images demonstrating RIG-I, MAVS, cGAS and IRF3 expression in normal liver tissue (left) or intrahepatic cholangiocarcinoma liver tissue (right).

Figure 11

The promoter methylation levels of innate immune pathway in normal and intrahepatic cholangiocarcinoma tissues. The promoter methylation levels of DDX58, MAVS, TMEM173, IKBKE, TBK1, and IRF3 in normal and primary tumor tissues (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 12

Correlation analysis between the expression and phosphorylation of key proteins within the innate immune pathway and the metastasis of intrahepatic cholangiocarcinoma. (A) The relationship between protein expression levels and the metastasis of intrahepatic cholangiocarcinoma. (B) The correlation between protein phosphorylation levels and the metastasis of intrahepatic cholangiocarcinoma (*P < 0.05, **P < 0.01, ***P < 0.001).