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.

Introduction

Hepatocellular carcinoma (HCC) is among the most lethal malignancies worldwide, with both high incidence and mortality rates [1]. Data from the International Agency for Research on Cancer (IARC) indicate that HCC is the sixth most common cancer and the third leading cause of cancer-related deaths globally, contributing to 7.8% of all cancer-related fatalities [2]. The burden of HCC is particularly severe in China, where approximately 431,383 new cases and 412,216 deaths were reported in 2022 [3, 4]. This concerning situation places a significant burden on patients, their families, and healthcare systems [5]. Despite the availability of treatment options, including surgical resection, liver transplantation, radiotherapy, and chemotherapy, the prognosis for HCC remains poor, primarily due to the high rates of recurrence and metastasis. Even among early-stage patients receiving curative treatments, the five-year recurrence rate exceeds 50%, underscoring the challenges in managing HCC effectively [6].

The frequent recurrence and high metastatic potential of HCC are largely attributed to the presence of cancer stem cells (CSCs), a subpopulation of tumor cells with distinct biological characteristics [7]. CSCs possess the capacity to self-renew, differentiate, and migrate, enabling them to survive under harsh microenvironmental conditions and driving tumor relapse, metastasis, and therapeutic resistance [8]. Understanding the mechanisms that regulate CSC behavior is critical for identifying new therapeutic strategies in HCC [9]. Recent studies have increasingly focused on the role of metabolic byproducts in the tumor microenvironment [10], such as lactate and ammonia [11, 12], which significantly influence CSC properties and tumor progression [13, 14]. Lactate, a key byproduct of glycolysis, has been shown to promote tumor invasiveness and enhance migratory capacity [15, 16]. However, compared to lactate, the role of ammonia in the tumor microenvironment remains less well understood.

Ammonia, a byproduct of protein and amino acid metabolism, is generally considered toxic and is primarily detoxified by the liver via the urea cycle [17, 18]. However, recent studies have revealed that ammonia also plays an important role in tumor metabolism and contributes to shaping the tumor microenvironment. Ammonia can act as a nitrogen source for cells, supporting energy metabolism through the glutamate dehydrogenase (GDH) pathway, and can also modulate immune responses within the tumor microenvironment [19, 20]. For example, in breast and colorectal cancers, ammonia has been shown to enhance tumor growth and metastasis by regulating nitrogen metabolism and recycling pathways [21, 22]. Additionally, ammonia can induce metabolic reprogramming in T cells, leading to functional exhaustion and impairing the efficacy of immune checkpoint inhibitors, such as anti-PD-1 therapies [23, 24].

The liver is the primary organ responsible for ammonia detoxification through the urea cycle [25]. However, in HCC, due to defects in urea cycle function, ammonia levels are elevated within tumor cells and the surrounding microenvironment [26]. Research has demonstrated that ammonia can accumulate intracellularly and is also actively exported through ion channels, further altering the tumor microenvironment. Elevated ammonia levels have been associated with enhanced tumor cell proliferation, increased stemness, and a greater metastatic potential. Although ammonia’s role in promoting tumor growth has been studied in other cancers, such as breast and colorectal malignancies, its specific effects on HCC cells remain insufficiently characterized.

In this study, we aim to explore the impact of high-ammonia conditions on the proliferation, stemness, and metastatic potential of HCC cells. Our hypothesis is that ammonia, beyond its conventional toxic effects, serves as a regulatory factor in HCC progression by driving metabolic reprogramming. Specifically, we investigate whether high-ammonia conditions induce quiescence in HCC cells, followed by an increase in proliferation and metastatic potential upon ammonia removal. Through transcriptomic and metabolomic analyses, we seek to elucidate the underlying molecular mechanisms and metabolic pathways altered by ammonia exposure. Understanding these processes may provide new insights into the role of ammonia in HCC progression and identify potential therapeutic targets for managing recurrence and metastasis.

Methods

Cell lines and culture conditions

HepG2, SK-Hep1, and Hep1-6 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified incubator with 5% CO₂. Ammonium chloride (NH₄Cl) stock solution (1 M) was prepared by dissolving 0.053 g of ammonium chloride in 1 mL of DMEM, followed by filtration through a 0.2 µm membrane to prevent contamination. Fresh working solutions were prepared before each experiment to avoid ammonia volatilization, and the concentration was adjusted according to experimental needs [27].

CCK-8 assay for cell proliferation

HepG2, SK-Hep1, and Hep1-6 cells were seeded into 96-well plates at a density of 3000–5000 cells per well in 100 µL of culture medium. Cell viability and proliferation were measured using the CCK-8 reagent (CK04; Dojindo Laboratories, Kumamoto, Japan) at 0, 24, 48, and 72 h after ammonium chloride treatment. At each time point, 10 µL of CCK-8 reagent was added to each well, followed by a 2 h incubation at 37 °C. Optical density (OD) was measured at 450 nm using a microplate reader (Multiskan SkyHigh; Thermo Fisher Scientific, USA).

Colony formation assay

HepG2 and SK-Hep1 cells were seeded in 6-well plates at a density of 500–1000 cells per well. After adherence, the cells were treated with various concentrations of ammonium chloride or left untreated as controls. After 14–20 days, the medium was removed, and the cells were washed twice with PBS. Colonies were fixed with 1 mL of 4% paraformaldehyde for 15 min at room temperature and stained with 0.1% crystal violet for 30 min. The stained plates were rinsed gently with running water, air-dried, and photographed. Colony numbers were counted using a microscope, and statistical analysis was performed.

Spheroid formation assay

Single-cell suspensions of HepG2 cells, treated with ammonium chloride for 3 days or cultured under normal conditions, were prepared. A total of 3000 cells were seeded into low-attachment 6-well plates containing 4 mL of spheroid formation medium, consisting of DMEM/F12 supplemented with 20 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), 1:50 B27 supplement, and 4 µg/mL heparin. The cells were incubated at 37 °C with 5% CO₂ for 7–10 days. Spheroid formation was monitored, photographed, and recorded for subsequent analysis.

Plasma ammonia concentration analysis

Plasma ammonia levels were measured using an ammonia assay kit (Abcam, #ab83360). Whole blood samples were collected from mice into heparinized tubes and stored at 4 °C. Plasma was separated by centrifugation at 1000 × g for 10 min and transferred to clean tubes. Ammonia concentrations were determined using a standard curve prepared according to the kit instructions. Absorbance was measured at 570 nm with a microplate reader, and results were expressed as mean ± SEM.

Animal experiments

C57BL/6 mice (6–8 weeks old) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and housed under specific pathogen-free (SPF) conditions at 21–23 °C with 40–60% humidity. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Zhengzhou University. Hep1-6 cells, either treated with ammonium chloride or left untreated, were harvested, washed, and resuspended to a final concentration of 1 × 10⁷ cells/mL. A total of 100 µL of cell suspension was subcutaneously injected into the right axilla of each mouse to establish xenograft tumors. Tumor volume and body weight were measured every 3 days, and growth curves were plotted.

Tumor size was monitored throughout the study using calipers. Tumors were considered to have reached their maximum permissible size if the volume exceeded 1000 mm³ or the maximum diameter exceeded 2 cm, in accordance with the ethics committee’s approved protocol. Tumors were measured per three day to ensure accurate monitoring. This study strictly adhered to the ethical standards approved by the IACUC, ensuring the tumor size and burden were closely monitored to ensure they remained within the ethical limits.

After 2 weeks, the mice were euthanized using cervical dislocation. The mice were first anesthetized with isoflurane by placing them in an anesthesia chamber and exposing them to 5% isoflurane in 1.5 L/min oxygen until signs of deep anesthesia were observed. After confirming deep anesthesia, the neck was swiftly and carefully dislocated to ensure humane euthanasia. Death was immediately confirmed by the cessation of heart and respiratory activity. After euthanasia, tumors were excised, weighed, photographed, and fixed in 4% paraformaldehyde for histological analysis.

Histology and immunohistochemistry (IHC)

Tumor tissues were embedded in paraffin and sectioned. The sections were deparaffinized, rehydrated, and treated with 5% hydrogen peroxide to block endogenous peroxidase activity. Antigen retrieval was performed using sodium citrate buffer under microwave heating. Sections were blocked with 10% normal goat serum for 1 h, followed by overnight incubation at 4 °C with anti-Ki67 antibody (1:5000; Servicebio, China). After incubation with HRP-conjugated secondary antibodies, DAB was used for chromogenic detection, and hematoxylin was applied for nuclear staining. Images were captured using an Olympus microscope.

RNA sequencing and data analysis

Total RNA was extracted using Trizol reagent (Invitrogen, USA), and RNA quality was assessed using a NanoDrop spectrophotometer and Qubit 4.0 fluorometer. RNA integrity was confirmed with an Agilent 2100 Bioanalyzer. Libraries were constructed using 3 µg of RNA, followed by cDNA synthesis, fragment selection, and PCR amplification. Sequencing was performed on a DNBSEQ-T7 platform, generating 150 bp paired-end reads. Differentially expressed genes (DEGs) were identified using DESeq2 with |log₂FoldChange|> 1 and adjusted p-value ≤ 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the ClusterProfiler package, with significant pathways identified at p < 0.05.

Mass spectrometry-based metabolomic analysis

Samples were thawed on ice and extracted in 80% methanol, followed by three cycles of vortexing and freezing in liquid nitrogen. After centrifugation at 12,000 rpm for 10 min, supernatants were collected and analyzed using a Waters ACQUITY UPLC system coupled with a QTRAP 6500 + mass spectrometer. A gradient elution was applied, and ionization was performed under both positive and negative modes. Metabolites were identified based on VIP scores and KEGG pathway analysis, with pathway enrichment determined by Log₂FC values [28].

Statistical analysis

All experiments were performed at least three times independently. Data are expressed as mean ± standard error of the mean (SEM). Statistical comparisons between two groups were made using Student’s t-test, while multiple group comparisons were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A P-value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 8.0 software.

Results

High concentration of ammonium chloride inhibits hepatocellular carcinoma cell growth but promotes stemness

To investigate the effects of high ammonia on hepatocellular carcinoma (HCC) cell growth, we treated HepG2 and SK-Hep1 cells with different concentrations of ammonium chloride and monitored cell proliferation at various time points (Fig. 1A). The results showed that 0.1 mM and 1 mM ammonium chloride had minimal impact on cell growth, with no significant inhibition observed even after three days of treatment. However, treatment with 10 mM ammonium chloride led to a marked reduction in cell proliferation after one day, and this inhibition became more pronounced by day 3 (Fig. 1B, C). Colony formation assays further confirmed that while low concentrations of ammonium chloride slightly inhibited colony formation, high concentrations significantly reduced the number of colonies (Fig. 1D–F). Interestingly, although high ammonia inhibited cell growth, it did not induce apoptosis in HCC cells (Fig. 1G). Given previous studies showing that HCC patients with urea cycle deficiencies often exhibit cellular quiescence, we hypothesized that high ammonia may induce quiescence in HCC cells, temporarily inhibiting their growth. To test this, we removed ammonium chloride from the medium after high-ammonia treatment and cultured the cells in fresh, ammonia-free medium (Fig. 1H). After ammonia removal, HepG2 cells showed significantly higher proliferation rates than the control, even cells treated with low concentrations of ammonium chloride exhibited increased proliferation (Fig. 1I). SK-Hep1 cells also exhibited enhanced proliferation, though to a lesser extent than HepG2 (Fig. 1J). This difference could be attributed to the differentiation status of the cells, with HepG2 cells being less differentiated and tending to form clusters, while SK-Hep1 cells are more differentiated and grow in a dispersed manner. These results suggest that high ammonia induces quiescence in HCC cells without causing cell death. Once ammonia is removed, the cells resume growth with enhanced proliferative capacity. Spheroid formation assays further confirmed that HepG2 cells treated with high ammonia had increased spheroid-forming ability, indicating enhanced stemness (Fig. 1K).

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

High ammonium chloride initially inhibits, then promotes HCC cell growth and metastasis

To confirm the phenomenon of initial inhibition followed by enhanced growth under high ammonia conditions, we treated murine HCC cells (Hep1-6) with ammonium chloride and subcutaneously implanted 1 × 10⁶ cells into C57BL/6 mice. Tumor growth and body weight were monitored over time (Fig. 2A). Tumors began to form around day 9 with no significant difference between groups. However, by day 14, tumor volumes in the high-ammonia group were significantly larger than in the control group (Fig. 2C). Throughout the experiment, body weights remained similar between groups, though by the end of the study, the high-ammonia group showed a slight increase in body weight compared to controls (Fig. 2B). Final tumor weights were also significantly higher in the high-ammonia group (Fig. 2D, E), consistent with in vitro findings. Plasma ammonia concentrations were significantly elevated in the high-ammonia group (Fig. 2F), possibly due to increased glutamine consumption or ammonia release from tumor cells. Surprisingly, liver metastasis was observed in the high-ammonia-treated mice (Fig. 2G, H), indicating that high-ammonia treatment not only enhanced tumor cell proliferation but also significantly increased metastatic potential. Additionally, CD133 staining revealed a substantial increase in the proportion of CD133-positive cells in the high-ammonia-treated tumors, suggesting that high-ammonia conditions may promote tumor cell stemness (Fig. 2I).

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

Transcriptomic and metabolomic analyses reveal persistent gene expression changes induced by high ammonia treatment

To explore the mechanisms underlying high ammonia's effects on tumor cell growth, we performed transcriptomic analysis of HCC cells under various conditions: untreated cells refer to A, high-ammonia-treated cells refer to B, and cells from which ammonia had been removed refer to C (Fig. 3A). RNA quality control showed high-quality total RNA, and sequencing data quality control indicated no significant overall differences between groups (Fig. 3B, C). However, correlation analysis revealed that gene expression profiles of the high-ammonia and ammonia-removal groups were nearly identical, with a high correlation (Fig. 3D). Differential gene expression analysis revealed that high-ammonia treatment induced significant transcriptional reprogramming, and these changes persisted even after ammonia removal. Genes involved in cell proliferation, migration, and metabolism were significantly upregulated, suggesting that high-ammonia treatment induces long-lasting changes that may contribute to enhanced growth and metastasis once ammonia is removed.

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

High ammonia activates key pathways in HCC cells

Differential gene expression (DEG) analysis combined with Gene Ontology (GO) and KEGG pathway analyses revealed significant upregulation and downregulation of numerous genes following high-ammonia treatment (Fig. 4A, D). These genes were primarily enriched in pathways related to proliferation, migration, adhesion, and metabolism (Fig. 4B, E). Even after ammonia removal, many metabolism- and migration-related pathways remained active, indicating the persistence of transcriptional changes (Fig. 4G, H). KEGG pathway analysis revealed that high-ammonia treatment activated key pathways, including glycolysis and extracellular matrix remodeling, which are closely linked to tumor metabolism and invasion (Fig. 4C, F). Even after ammonia removal, some of these pathways remained highly active (Fig. 4I), suggesting that high-ammonia treatment enhances both short-term and long-term tumor cell survival and metastasis.

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

Metabolic pathway changes induced by high ammonia

Gene Set Enrichment Analysis (GSEA) and KEGG pathway enrichment analysis of differentially expressed genes revealed that key pathways related to calcium ion and glucose transport were significantly activated by high ammonia (Fig. 5A–E). KEGG analysis showed that glycolysis and amino acid metabolism pathways were particularly affected, with significant upregulation of genes involved in these processes (Fig. 5F–H). These findings indicate that high-ammonia treatment leads to metabolic reprogramming that enhances tumor cell adaptability, and many of these pathways remained active even after ammonia removal.

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

Protein–protein interaction networks and differential gene analysis

Protein–protein interaction (PPI) network analysis revealed significantly enhanced gene interactions in the high-ammonia group compared to untreated cells (Fig. 6A–C). After ammonia removal, some interactions persisted, particularly in metabolism and migration pathways. Venn diagram analysis of upregulated and downregulated genes showed substantial overlap, indicating persistent gene expression changes across different conditions (Fig. 6D, E). These findings suggest that high ammonia induces complex gene regulation that enhances tumor cell growth and metastasis.

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

Metabolic profiling of tumor cells after high ammonia treatment

Metabolomic analysis using LC–MS/MS revealed significant changes in metabolite levels following high-ammonia treatment (Fig. 7A). The abundance of several metabolites involved in energy metabolism, amino acid metabolism, and lipid metabolism changed significantly, with many remaining altered even after ammonia removal (Fig. 7B, C, F). KEGG classification analysis confirmed that high-ammonia treatment primarily affected energy metabolism pathways, such as glycolysis and amino acid synthesis, with significant enrichment observed (Fig. 7D, G). Differential abundance scores for these metabolites showed significant shifts, indicating that high ammonia induced substantial metabolic reprogramming (Fig. 7E, H). These changes reflect the metabolic adaptability of tumor cells in response to high ammonia.

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

Detailed metabolism change and pathway analysis following ammonia exposure

To gain deeper insights into the metabolic reprogramming induced by ammonia exposure, we conducted a comprehensive analysis of metabolic pathways and changes in metabolites under different experimental conditions. It shows the distribution of various metabolites across the different experimental groups, highlighting the shifts in metabolic profiles between high-ammonia treatment, ammonia removal, and control conditions (Fig. 8A, D and G). It presents the results of differential enrichment analysis, revealing significantly altered metabolic pathways under each condition (Fig. 8B, E and H). Which show violin plots of individual metabolites, suggest that high-ammonia treatment did not immediately alter metabolite levels (Fig. 8C, F and I). However, upon ammonia removal, a marked change in metabolite abundance was observed, particularly in pathways related to metabolism. These results suggest that high-ammonia exposure initially induces gene expression changes, which then lead to subsequent alterations in metabolic profiles. Importantly, the metabolite shifts observed after ammonia removal were largely consistent with the gene expression changes identified in our transcriptomic analysis, indicating a strong interplay between transcriptional reprogramming and metabolic adaptation.

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

Changes in lactate levels following high ammonia treatment

High-ammonia treatment significantly increased lactate levels in tumor cells, indicating activation of the glycolytic pathway (Fig. 9A–C). Intracellure lactate levels rose to 0.6 mmol/L after treatment with 10 mM ammonia, and further increased to 0.8 mmol/L at 1% ammonium chloride, compared to control (P < 0.05). Similarly, entracellure lactate levels increased to 6 mmol/L and 9 mmol/L under 10 mM and 1% ammonium chloride, respectively (P < 0.01). These results demonstrate that high-ammonia conditions enhance glycolysis, leading to the accumulation of key metabolites and promoting tumor cell survival and metabolic adaptability.

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

Discussion

This study provides an in-depth investigation into the effects of a high-ammonia environment on the proliferation, stemness, and metastatic potential of hepatocellular carcinoma (HCC) cells, revealing the complex role of ammonia as a metabolic factor in HCC progression. Our results demonstrate that high-ammonia conditions initially inhibit the proliferation of HCC cells without inducing apoptosis, leading to a quiescent state. Upon ammonia removal, the cells exhibit significantly enhanced proliferative and metastatic capacities. These findings indicate that ammonia participates in metabolic reprogramming, promoting cellular stemness and conferring greater survival and dissemination abilities to tumor cells under stress conditions. This highlights the role of ammonia as a crucial metabolic factor that drives tumor progression.

Our study shows that high-ammonia conditions temporarily suppress cell proliferation through metabolic reprogramming while activating genes associated with cell adhesion and migration. These transcriptional changes may serve as a key mechanism by which tumor cells gain survival advantages under stressful conditions. After ammonia withdrawal, HCC cells display a marked increase in both proliferation and migration, with the upregulation of adhesion-related genes significantly enhancing their invasiveness. This suggests that tumor cells respond to metabolic stress by enhancing stemness and adhesion, laying the foundation for cellular adaptations that facilitate tumor cell survival and invasion.

Metabolomic analyses reveal that lactate accumulation increases substantially after ammonia withdrawal, suggesting that high-ammonia conditions enhance glycolytic pathways to support rapid cell growth and dissemination. Lactate, a primary product of glycolysis, is known to promote tumor cell invasiveness and metastatic potential by acidifying the microenvironment, which in turn enhances the migratory ability of tumor cells [29]. Additionally, lactate can activate key signaling pathways, such as HIF-1α and NF-κB [30, 31], that regulate the expression of genes associated with stemness, thereby supporting the maintenance of tumor stem cell properties. Pyruvate, another key metabolite, is involved in metabolic reprogramming and can affect cellular energy balance by modulating pathways like mTOR and AMPK [32], further promoting tumor cell proliferation and migration. These findings underscore how ammonia-induced metabolic reprogramming shifts energy supply patterns, enhancing both tumor cell proliferation and metastatic potential upon stressor removal.

Ammonia has long been recognized as a toxic byproduct of protein metabolism, but emerging evidence indicates its significant role in the tumor microenvironment, particularly in hepatocellular carcinoma (HCC) [23]. Elevated ammonia levels in HCC are not merely a consequence of metabolic dysregulation; they actively drive metabolic reprogramming that supports aggressive tumor behavior. Our findings are in line with other studies that demonstrate ammonia's ability to enhance glycolysis, increase lactate production, and induce an acidic microenvironment that promotes tumor invasiveness. Such effects are comparable to observations in other malignancies, such as breast and colorectal cancers, where ammonia is implicated in tumor growth and metastasis.Ammonia-induced metabolic shifts are profound. By promoting enhanced glycolysis, ammonia directly contributes to increased lactate production, which then accumulates in the tumor microenvironment. This lactate not only serves to acidify the microenvironment, making it more conducive for tumor invasion and metastasis [33], but also plays a critical role in maintaining the stemness of tumor cells. The acidic conditions fostered by lactate accumulation enhance the plasticity of cancer stem cells [34], promoting traits associated with invasiveness and therapeutic resistance. Moreover, the Warburg effect, or aerobic glycolysis, facilitated by ammonia, underscores a pivotal shift in energy production from oxidative phosphorylation to glycolysis, which supports rapid tumor cell proliferation and survival under hypoxic conditions often present in solid tumors [35].Our study expands on this concept by specifically linking ammonia-induced glycolysis to the maintenance of tumor stemness and metastasis in HCC. By demonstrating how ammonia exacerbates glycolytic activity and lactate accumulation, we propose that strategies to regulate ammonia levels, such as drugs that clear ammonia or inhibit ammonia metabolism, could serve as promising therapies to attenuate both the stemness and metastatic potential of HCC cells. Moreover, the inhibition of glycolysis or lactate production could provide an additional layer of therapeutic intervention to suppress tumor invasiveness, a strategy that has been shown to enhance the effectiveness of conventional treatments. In summary, our work contributes novel insights into the complex role of ammonia in the tumor microenvironment, particularly its influence on metabolic pathways such as glycolysis.

Although this study validated the effects of ammonia on HCC cells using in vitro experiments and animal models, these findings may not fully capture the complex biological characteristics of human HCC. The HCC microenvironment involves interactions among immune cells, extracellular matrix components, and multiple metabolic pathways. Future studies should incorporate clinical samples, particularly those from patients with hyperammonemia, to further validate the role of ammonia in a clinical setting. Additionally, the long-term biological effects of ammonia and its potential synergistic interactions with other metabolic stressors require further investigation. Future research should explore the relationships between ammonia, lipid metabolism, and oxidative stress to comprehensively understand the metabolic reprogramming mechanisms in HCC.

In conclusion, this study reveals the critical role of ammonia in the HCC microenvironment, particularly in promoting tumor stemness and metastatic potential. By elucidating the regulatory effects of ammonia on metabolic reprogramming and gene expression, we provide novel therapeutic targets for HCC treatment. Future studies should explore strategies for modulating ammonia metabolism to improve patient outcomes, particularly in preventing tumor recurrence and metastasis. Positioning ammonia as a central regulator of metabolic adaptation offers a promising direction for developing new therapeutic approaches, ultimately enhancing the efficacy of HCC treatments.

Author contributions

Renchao Zou and Sicong Jiang contributed equally to the conception and design of the study, and were responsible for conducting the experiments. Jiaqi Mei performed the data analysis and contributed to the writing of the manuscript. Chen Chen, Jia Yu, and Yanqiu Fu were involved in conducting experiments and statistical analysis. Siyu Chen contributed to the initial conception of the study and provided critical revisions to the manuscript.

Data availability

The data supporting the findings of this study are available within the article and its supplementary materials. Additional data can be provided upon reasonable request to the corresponding authors.

Declarations

Ethics approval and consent to participate

This study was conducted in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Zhengzhou University, with the approval number 2024-KY-1721. All animal experiments were performed in compliance with the relevant guidelines and regulations.

Competing interests

The authors declare no competing interests.