High-ammonia microenvironment promotes stemness and metastatic potential in hepatocellular carcinoma through metabolic reprogramming

Abstract

Background: Hepatocellular carcinoma (HCC) is a prevalent and aggressive form of liver cancer, characterized by frequent recurrence and metastasis, which remain significant obstacles to effective treatment. Ammonia accumulates in the tumor microenvironment of HCC due to dysfunction in the urea cycle, but the detailed impact of ammonia on HCC cells remains insufficiently understood.

Methods: We exposed HCC cell lines to high concentrations of ammonium chloride to evaluate alterations in proliferation, stemness, and migratory potential. After ammonia removal, changes in cellular behavior were assessed using colony formation, and spheroid assays. Transcriptomic and metabolomic analyses were conducted to investigate ammonia-induced metabolic reprogramming and alterations in gene expression. Additionally, animal models were employed to validate the impact of ammonia on tumor growth and metastasis.

Results: Exposure to high-ammonia conditions transiently suppressed HCC cell proliferation without inducing apoptosis. However, following ammonia removal, cells demonstrated increased proliferation, enhanced spheroid formation, and elevated migratory capacity. Transcriptomic analysis revealed the upregulation of genes associated with cell adhesion, migration, and glycolysis. Concurrently, metabolomic profiling indicated increased lactate production, facilitating the aggressive behavior of HCC cells after ammonia withdrawal. Animal experiments confirmed that high-ammonia exposure accelerated tumor growth and metastasis.

Conclusion: Ammonia exerts a dual effect on HCC progression: it initially suppresses cell growth but later promotes stemness, proliferation, and metastasis through metabolic reprogramming. Targeting ammonia metabolism or glycolysis in the tumor microenvironment may represent a promising therapeutic strategy for mitigating HCC recurrence and metastasis. Future studies utilizing clinical samples are required to validate these findings and identify potential therapeutic strategies targeting ammonia metabolism.

Keywords: Ammonia metabolism; Hepatocellular carcinoma (HCC); Metabolic reprogramming; Tumor microenvironment.

Fig. 1

Effects of Ammonium Chloride on HCC Cell Growth and Recovery (A) Experimental design showing the timeline for ammonium chloride addition, cell seeding, and CCK8 assay. B, C Cell proliferation of HepG2 and SK-SK-Hep1 cells treated with 0 mM, 0.1 mM, 1 mM, and 10 mM ammonium chloride at different time points (Day 0, 1, and 3), measured by optical density. D Colony formation assay in HepG2 and SK-Hep1 cells treated with different concentrations of ammonium chloride. E,G Images of HepG2 and SK-Hep1 cells under various ammonia concentrations, showing morphological changes. F Quantification of colony formation rates in HepG2 and SK-Hep1 cells. H Schematic representation of the experimental timeline for ammonia removal and subsequent analysis. I, J Proliferation recovery in HepG2 and SK-Hep1 cells after ammonia removal (Day 3 to Day 5), measured by CCK8 assay. K Representative spheroid formation in HepG2 cells treated with 0 mM and 10 mM ammonium chloride

Fig. 2

In Vivo Effects of Ammonium Chloride on Tumor Growth and Metastasis (A) Schematic of the experimental design: mice were injected with Hep1-6 cells pre-treated with ammonium chloride, monitored for tumor growth, and tumors harvested. B Body weight changes in mice treated with 0 mM and 10 mM ammonium chloride over 16 days. C Tumor volume over time in both treatment groups, with significant differences observed after Day 12. D Images of tumors from mice treated with 0 mM and 10 mM ammonium chloride. E Final tumor weight comparison between groups. F Ammonia concentration in blood at the time of tumor harvesting, showing increased levels in the 10 mM group. G,H,I Representative H&E, Ki67, and CD133 staining of tumor sections from both groups showed the proliferation activity and stemness characteristics of tumor cells

Fig. 3

RNA Sequencing and Quality Control Analysis (A) Workflow for RNA extraction, cDNA synthesis, sequencing, and quality control (QC). B Agarose gel electrophoresis of RNA samples showing intact total RNA from three experimental groups. C Box plot displaying the distribution of gene expression levels (log10FPKM) across the three groups, indicating consistent data quality. D Heatmap showing sample-to-sample correlation based on RNA-seq data, illustrating high correlation between biological replicates within each group. A: control group B: high-ammonia group C: ammonia removal group

Fig. 4

Differential Gene Expression and Pathway Enrichment Analysis in High-Ammonia Conditions A, D, G Volcano plots showing differentially expressed genes (DEGs) in three comparisons: high-ammonia vs. control, ammonia removal vs. control, and ammonia removal vs. high-ammonia. Red points represent significantly upregulated genes, and blue points represent downregulated genes. B, E, H Bar plots showing the number of DEGs involved in KEGG pathways for the three comparisons, categorized by upregulated and downregulated genes. C, F, I KEGG pathway enrichment analysis illustrating the most significantly affected pathways in each condition, with pathway significance indicated by bubble size and color. A: control group B: high-ammonia group C: ammonia removal group

Fig. 5

Gene Set Enrichment and KEGG Pathway Analysis in High-Ammonia Conditions (A–C) Bubble plots showing KEGG pathway enrichment analysis for differentially expressed genes in three comparisons: high-ammonia vs. control, ammonia removal vs. control, and ammonia removal vs. high-ammonia. Pathway significance is represented by bubble size and color. D, E Gene set enrichment analysis (GSEA) of key transport pathways, including calcium ion transport and protein transmembrane transport, under high-ammonia conditions. F–H KEGG pathway enrichment results for the top enriched metabolic and signaling pathways across the three comparisons, high-ammonia vs. control, ammonia removal vs. control, and ammonia removal vs. high-ammoniawith, bubble plots illustrating pathway significance and gene counts

Fig. 6

Protein–Protein Interaction Networks and Venn Diagram Analysis of Differential Gene Expression (A–C) Protein–protein interaction (PPI) networks showing interactions between significantly upregulated and downregulated genes in three conditions: high-ammonia vs. control, ammonia removal vs. control, and ammonia removal vs. high-ammonia. Nodes represent proteins, and edges represent interactions. D Venn diagrams showing overlap in differentially expressed genes (DEGs) between the three comparisons, divided into upregulated and downregulated categories. E Venn diagrams depicting common and unique DEGs among the different groups, indicating shared and distinct gene expression patterns across conditions. A: control group B: high-ammonia group C: ammonia removal group

Fig. 7

Metabolomic Profiling and Pathway Enrichment Analysis Under High-Ammonia Conditions (A) Workflow for sample preparation and LC–MS/MS-based metabolomic analysis. B, C, F Heatmaps of differentially expressed metabolites across experimental groups, showing metabolic changes induced by high-ammonia conditions and following ammonia removal. D, G Bar charts displaying significantly altered metabolic pathways identified by KEGG pathway enrichment analysis in response to high-ammonia conditions and subsequent removal. E, H Bubble plots of enriched metabolic pathways, with bubble size representing the number of metabolites involved and color indicating statistical significance. D: control group E: high-ammonia group G:ammonia removal group

Fig. 8

Detailed Metabolic Pathway Analysis Following Ammonia Exposure (A, D, G) Bar plots showing altered metabolic pathways based on fold change for different experimental conditions: high-ammonia exposure VS control, ammonia removal VS control, and ammonia removal VS high-ammonia exposure. B, E, H Bubble plots displaying enriched metabolic pathways, with bubble size corresponding to the number of metabolites involved and color representing the significance (p-value). C Violin plot comparing metabolite abundance between high-ammonia and control conditions. F, I Box plots of individual metabolites across different pathways, illustrating the distribution of metabolite levels under various experimental conditions, ammonia removal VS control, and ammonia removal VS high-ammonia exposure

Fig. 9

Lactate Levels in Response to Ammonium Chloride Treatment (A) Violin plot showing lactate levels in cells indicating increased lactate production upon higher ammonia removal. B Graph of intracellular lactate concentrations across different ammonia treatments. C Graph of lactate concentration in culture medium, showing a similar trend of increasing levels with higher ammonium chloride concentrations