Alterations of gut mycobiota profiles in intrahepatic cholangiocarcinoma

Abstract

Objective: Intrahepatic cholangiocarcinoma (ICC) is a silent liver malignancy with an increasing incidence. Gut mycobiota plays a crucial role in benign liver diseases; however, its correlation with ICC remains elusive. This study aimed to elucidate fungal differences in patients with ICC compared to healthy controls.

Methods: The 40 fecal samples from 23 ICC patients and 17 healthy controls were collected and analyzed using ITS2 rDNA sequencing. Obtaining the OTUs and combining effective grouping, we carried out the biodiversity and composition of the fungi, as well as FUNGuild functional annotation.

Results: Our results revealed the presence of intestinal fungal dysbiosis with significant enrichment of opportunistic pathogenic fungi such as Candida and C. albicans, and significant depletion of the beneficial fungus Saccharomyces cerevisiae in ICC patients compared with healthy controls. Alpha-diversity analysis demonstrated that patients with ICC showed decreased fungal diversity compared to healthy controls. Beta diversity analysis indicated that the two groups exhibited significant segregated clustering. Besides, C. albicans was found to be significantly more abundant in the ICC patients with TNM stage III-IV than those with stage I-II. The FUNGuild functional classification predicted that pathotrophs were the most abundant taxon in the ICC group, well above their abundance in healthy controls.

Conclusion: This study indicates that dysbiosis of the fecal mycobiome might be involved in ICC development. Further research into gut fungi may contribute to new therapeutic options for ICC patients.

Keywords: Candida albicans; ITS2 rDNA sequencing; dysbiosis; gut mycobiome; intrahepatic cholangiocarcinoma.

Figure 1

Diversity analysis in the ICC and NC groups. (A) Rank abundance curves. (B) Venn diagram displaying the overlap of OTUs identified between the two groups. Alpha diversity was estimated by the ACE index (C), Chao1 index (D), Simpson index (E), and Shannon index (F). The distributional difference of gut mycobiota profiles was assessed using PCoA (G) and NMDS (H) based on a weighted_unifrac matrix. ICC, intrahepatic cholangiocarcinoma; NC, healthy controls.

Figure 2

Differential analysis of fungal communities in the ICC and NC groups. Stacked bar plot of the predominant intestinal fungi at the phylum (A), genus (B), and species (C) levels. The three most abundant of the differential taxonomic units at the phylum level (D) between ICC patients and healthy controls. The ten most abundant of the differential taxonomic units at the genus level (E) and species level (F) between ICC patients and healthy controls. The Wilcoxon rank-sum test was used. ICC, intrahepatic cholangiocarcinoma; NC, healthy controls.

Figure 3

The differential taxa in the ICC and NC groups using the linear discriminant analysis effect size (LEfSe) analysis. (A) Taxonomic cladogram from LEfSe showing differences in fecal taxa of ICC patients and healthy controls. (B) LDA scores were computed for differentially abundant taxa in the gut fungi of ICC patients and healthy controls. Length indicates the effect size associated with a taxon. p = 0.05 for the Kruskal-Wallis sum-rank test; LDA score > 4; NC, healthy controls; ICC, intrahepatic cholangiocarcinoma; LDA, linear discriminant analysis.

Figure 4

Correlation of gut mycobiota with TNM stage in ICC patients. The distributional difference of gut mycobiota profiles was assessed using PCoA (A) and NMDS (B) based on a weighted_unifrac matrix. (C) Taxonomic cladogram from LEfSe showing differences in fecal taxa of ICC patients with stage III-IV and stage I-II. (D) LDA scores were computed for differentially abundant taxa in the gut fungi of ICC patients with stage III-IV and stage I-II. Length indicates the effect size associated with a taxon. p = 0.05 for the Kruskal-Wallis sum-rank test; LDA score > 4; ICC, intrahepatic cholangiocarcinoma; LDA, linear discriminant analysis;

Figure 5

Functional predictive analysis between the ICC and NC groups. Stacked bar plot of the predominant functional classification at the trophic modes (A). Wilcox rank-sum test was applied to the guild annotated results (B). Control, healthy controls; ICC, intrahepatic cholangiocarcinoma.