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: Hepatocellular carcinoma (HCC), Ammonia metabolism, Tumor
   microenvironment, Metabolic reprogramming

Introduction

   Hepatocellular carcinoma (HCC) is among the most lethal malignancies
   worldwide, with both high incidence and mortality rates [[36]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 [[37]2]. The burden of HCC is particularly
   severe in China, where approximately 431,383 new cases and 412,216
   deaths were reported in 2022 [[38]3, [39]4]. This concerning situation
   places a significant burden on patients, their families, and healthcare
   systems [[40]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
   [[41]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
   [[42]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 [[43]8]. Understanding the mechanisms that regulate CSC
   behavior is critical for identifying new therapeutic strategies in HCC
   [[44]9]. Recent studies have increasingly focused on the role of
   metabolic byproducts in the tumor microenvironment [[45]10], such as
   lactate and ammonia [[46]11, [47]12], which significantly influence CSC
   properties and tumor progression [[48]13, [49]14]. Lactate, a key
   byproduct of glycolysis, has been shown to promote tumor invasiveness
   and enhance migratory capacity [[50]15, [51]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 [[52]17, [53]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 [[54]19, [55]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 [[56]21, [57]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 [[58]23, [59]24].

   The liver is the primary organ responsible for ammonia detoxification
   through the urea cycle [[60]25]. However, in HCC, due to defects in
   urea cycle function, ammonia levels are elevated within tumor cells and
   the surrounding microenvironment [[61]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 [[62]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 mm^3 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 [[63]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. [64]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. [65]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. [66]1D–F).
   Interestingly, although high ammonia inhibited cell growth, it did not
   induce apoptosis in HCC cells (Fig. [67]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. [68]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. [69]1I). SK-Hep1 cells also exhibited enhanced
   proliferation, though to a lesser extent than HepG2 (Fig. [70]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. [71]1K).

Fig. 1.

   [72]Fig. 1
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   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. [74]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. [75]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. [76]2B). Final tumor weights were also significantly
   higher in the high-ammonia group (Fig. [77]2D, E), consistent with in
   vitro findings. Plasma ammonia concentrations were significantly
   elevated in the high-ammonia group (Fig. [78]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. [79]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. [80]2I).

Fig. 2.

   [81]Fig. 2
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   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. [83]3A). RNA quality control showed high-quality total RNA,
   and sequencing data quality control indicated no significant overall
   differences between groups (Fig. [84]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. [85]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.

   [86]Fig. 3
   [87]Open in a new tab

   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. [88]4A, D). These genes were primarily enriched in pathways
   related to proliferation, migration, adhesion, and metabolism
   (Fig. [89]4B, E). Even after ammonia removal, many metabolism- and
   migration-related pathways remained active, indicating the persistence
   of transcriptional changes (Fig. [90]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. [91]4C, F). Even after
   ammonia removal, some of these pathways remained highly active
   (Fig. [92]4I), suggesting that high-ammonia treatment enhances both
   short-term and long-term tumor cell survival and metastasis.

Fig. 4.

   [93]Fig. 4
   [94]Open in a new tab

   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. [95]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. [96]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.

   [97]Fig. 5
   [98]Open in a new tab

   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. [99]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. [100]6D, E). These findings
   suggest that high ammonia induces complex gene regulation that enhances
   tumor cell growth and metastasis.

Fig. 6.

   [101]Fig. 6
   [102]Open in a new tab

   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. [103]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. [104]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. [105]7D, G).
   Differential abundance scores for these metabolites showed significant
   shifts, indicating that high ammonia induced substantial metabolic
   reprogramming (Fig. [106]7E, H). These changes reflect the metabolic
   adaptability of tumor cells in response to high ammonia.

Fig. 7.

   [107]Fig. 7
   [108]Open in a new tab

   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. [109]8A, D and G). It presents the results of
   differential enrichment analysis, revealing significantly altered
   metabolic pathways under each condition (Fig. [110]8B, E and H). Which
   show violin plots of individual metabolites, suggest that high-ammonia
   treatment did not immediately alter metabolite levels (Fig. [111]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.

   [112]Fig. 8
   [113]Open in a new tab

   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. [114]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.

   [115]Fig. 9
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   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 [[117]29]. Additionally, lactate can activate key signaling
   pathways, such as HIF-1α and NF-κB [[118]30, [119]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
   [[120]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)
   [[121]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 [[122]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 [[123]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 [[124]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.

Acknowledgements