The Warburg effect is a hallmark metabolic characteristic of tumor cells, wherein they preferentially rely on glycolysis to metabolize glucose and produce substantial amounts of lactate, even in the presence of oxygen. This metabolic shift inhibits the conversion of glucose into acetyl-CoA, thereby preventing its entry into the tricarboxylic acid (TCA) cycle (17, 18). To compensate for this altered metabolism, tumor cells accelerate intracellular glutamine metabolism, ensuring a continuous supply of metabolic intermediates (nitrogen and carbon sources) to sustain the TCA cycle. This phenomenon is referred to as “anaplerosis” (19, 20). Glutamine undergoes metabolism through various enzymatic processes, producing byproducts that integrate into the TCA cycle. It enters the cytoplasm via solute carrier (SLC) proteins (21) and is converted into glutamate by glutaminase (GLS) (22). Glutamate is then converted to α-ketoglutarate (α-KG) by glutamate dehydrogenase (GLUD) or transaminases, facilitating the replenishment of the TCA cycle (22–24). Furthermore, glutamine metabolism supports the production of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH), contributing to the maintenance of cellular redox homeostasis (25, 26).

Glutamine metabolism is pivotal for tumor cell survival and proliferation. Studies indicate that many tumor cells upregulate SLC family transporters to enhance glutamine uptake and stimulate the activity of enzymes involved in glutamine metabolism, thus sustaining proliferative demands (27, 28). Among these, SLC1A5 (also known as ASCT2) serves as the primary transporter for glutamine, with its overexpression strongly linked to the growth, metastasis, and drug resistance of several cancers, including breast, lung, liver, and gastric cancers (29–31). Other transporters, such as SLC7A5, SLC38A2, and SLC7A11, also play contributory roles in glutamine and glutamate transport (32–34).

As a rate-limiting enzyme in the “anaplerosis” pathway, GLS catalyzes the breakdown of glutamine into free ammonia and glutamate (22). Studies have demonstrated that GLS1 is overexpressed in various tumor types and is closely associated with advanced tumor stages, high invasiveness, metastatic potential, and poor clinical prognosis (35, 36). Similarly, GLUD1, a key enzyme in glutamine catabolism, is highly expressed in many tumors (37). Research indicates that GLUD1 converts glutamate into α-KG, releasing substantial amounts of free ammonia in the process (38, 39). Under the influence of GLUD1 and enzymes such as glutamine synthetase (GS), the ammonia is incorporated into amino acid synthesis pathways, thereby promoting tumor cell proliferation and migration (40, 41). Alternatively, glutamate can be converted into α-KG by transaminases, such as glutamate-pyruvate transaminase 2 (GPT2), without producing ammonia. GPT2 catalyzes the reaction between glutamate and pyruvate, generating α-KG and alanine, which accelerates liver cancer cell proliferation (42). GPT2 is highly expressed in various cancers and correlates with patient prognosis (43, 44) ( Figure 1 ).

The oncogene c-Myc is a major regulator of glutamine utilization in tumor cells (45). c-Myc enhances glutamine uptake by binding to the promoter regions of glutamine transporters or repressing the expression of miR-27a, which promotes SLC1A5 upregulation (46, 47). Furthermore, c-Myc upregulates GLS1 expression by transcriptionally inhibiting miR-23a/b, thereby enhancing glutamine metabolism (48). c-Myc also influences glutamine metabolism indirectly by modulating the expression of miR-15a and miR-16 (49, 50). Recent studies have highlighted that c-Myc can also regulate glutamine metabolism through the upregulation of long non-coding RNAs (lncRNAs) such as H19 and MALAT1 (51, 52). Similarly, KRAS mutations in tumor cells promote glutamine metabolism by inducing the expression of glutamate oxaloacetate transaminase (GOT), contributing to the maintenance of tumor cell proliferation and survival (53, 54). Conversely, the loss of tumor suppressor proteins from the Rb family activates E2F transcriptional activity, directly upregulating the expression of SLC1A5 and GLS1, thus facilitating glutamine uptake and utilization (55) ( Figure 1 ).

Immune cells are essential components of the TME, playing pivotal roles in tumor initiation, progression, immune evasion, and metastasis (56, 57). Increasing evidence suggests that while immune cells are tasked with mounting anti-tumor responses, they also contribute to tumor progression through various mechanisms (57, 58). Within the TME, tumor cells competitively consume glutamine, depriving immune cells of this vital nutrient. This results in restricted glutamine metabolism in immune cells, diminishing their anti-tumor efficacy and promoting immune evasion (57) ( Figure 2 ). The metabolic profiles of immune cells significantly vary based on their differentiation and functional states.

In addition to tumor cells and T cells, the TME also contains various other cell types that play important roles in tumor initiation, progression, immune evasion, and response to therapy.

As an indispensable component of the TME, tumor-associated macrophages (TAMs) also exhibit a high dependence on glutamine metabolism (58). Research has demonstrated that glutamine not only supports the TCA cycle in TAMs but also influences their polarization (76, 77). M2-like TAMs, compared to their M1-like counterparts, exhibit elevated expression of glutamine transporters and metabolic enzymes. Inhibition of glutamine synthetase can trigger the conversion of M2-like TAMs to the more inflammatory M1-like phenotype, thus impeding tumor metastasis (77, 78). TAMs, particularly M2 macrophages, rely on glutamine metabolism to sustain cell proliferation and immune suppression (79). Recent findings indicate that chemotherapy-responsive macrophages secrete interleukin (IL)-18, which upregulates the expression of L-type amino acid transporter 2 (LAT2) in osteosarcoma cells. This enhances leucine and glutamine uptake, activating mTORC1 and promoting c-Myc-mediated transcription of CD47, which inhibits macrophage phagocytosis and facilitates immune evasion (80). Another study highlights that competitive uptake of extracellular glutamine by clear cell renal carcinoma activates hypoxia-inducible factor 1α (HIF-1α) in tumor-infiltrating macrophages, inducing IL-23 secretion (81). IL-23 subsequently activates Treg proliferation and stimulates the release of immunosuppressive cytokines, including IL-10 and transforming growth factor (TGF)-β, thus suppressing cytotoxic lymphocyte effector functions (81).

Myeloid-derived suppressor cells (MDSCs) are a diverse subset of bone marrow-derived cells, typically generated in response to stress stimuli such as tissue injury and inflammation (82, 83). MDSCs play a pivotal role in maintaining immune tolerance and preventing excessive immune responses (82). In the TME, competition for glutamine between tumor cells and myeloid cell SLC1A5 transporters significantly promotes MDSC generation and recruitment, while inhibiting the formation of anti-tumor inflammatory TAMs, thus impairing anti-tumor immunity (79). This occurs as glutamine scarcity in the TME induces endoplasmic reticulum stress in myeloid cells, activating the IRE1α/XBP1 signaling pathway, which upregulates the expression of GPR109A, driving their immunosuppressive polarization (79). Additionally, glutamine metabolism regulates MDSC suppressive function via the glutamate-NMDA receptor axis (84).

Dendritic cells (DCs) are essential to the immune system, particularly in antigen presentation (85). Recent studies have emphasized the central role of glutamine metabolism in DC function, particularly in type 1 conventional dendritic cells (cDC1s) (33). In the TME, tumor cells and cDC1s compete for glutamine uptake through the SLC38A2 transporter (33, 86). This glutamine deprivation in cDC1s diminishes their antigen presentation capacity via the FLCN-Tfeb signaling pathway, ultimately impairing anti-tumor T cell responses (33).

Natural killer (NK) cells, key effectors in the immune defense against viruses and tumors, rely on glutamine for proper function (30, 87). Research shows that SLC7A5 serves as the primary glutamine transporter in activated NK cells. Glutamine uptake activates mTORC1, increasing c-Myc expression and promoting NK cell proliferation (30). However, when tumor cells competitively deplete glutamine, activated NK cells undergo glutamine scarcity, leading to reduced glycolysis and oxidative phosphorylation, which suppresses IFN-γ production (30, 88). Although glutamine is crucial for maintaining c-Myc expression, it is not a primary metabolic fuel for NK cells. Studies suggest that while glutamine metabolism inhibitors can significantly impact tumor cells, they do not suppress NK cell metabolism or functional responses (30).

Ammonia metabolism in the TME has garnered significant attention in recent years, emerging as a critical area of study in both immunology and oncology (14). As a major metabolic byproduct, ammonia plays multifaceted roles in the TME, influencing tumor progression, immune evasion, and therapeutic responses (89, 90). Beyond serving as a nitrogen source for nucleic acid and protein synthesis, ammonia accumulates in the TME through various metabolic pathways (14). Rapid tumor cell growth and proliferation in the TME often coincide with shifts in metabolic pathways, with ammonia accumulation tightly linked to cellular metabolic reprogramming, protein degradation, and the urea cycle (14, 91). Ammonia concentrations in the TME are elevated compared to normal tissues, with distinct regional distribution patterns (14).

Both tumor and immune cells exhibit increased amino acid demands to sustain their proliferation and survival (92, 93). The primary source of ammonia within cells is the deamination of amino acids (94). Glutamine, a key nitrogen and energy donor for tumor cells, is imported into the cytoplasm via the transporters SLC1A5, SLC38A1, and SLC38A2. It is subsequently transferred to the mitochondrial inner membrane via a variant of SLC1A5 (29, 95). Within the mitochondria, glutamine is converted into glutamate by glutaminases (GLS1, GLS2, GAC), releasing free ammonia (22, 96). The glutamate produced can either return to the cytoplasm via SLC25A18 and SLC25A22 transporters for the biosynthesis of GSH and non-essential amino acids (e.g., aspartate, alanine, arginine) or undergo complete oxidation or conversion into α-KG by mitochondrial transaminases (e.g., GPT2, GOT2) (97, 98). Pathway variations in the conversion of glutamine to α-KG occur under different pathological conditions. In liver and kidney diseases, such as hepatitis, cirrhosis, and renal failure, glutamate primarily converts to α-KG through transaminase pathways (99). In contrast, within the TME, factors like hypoxia and oxidative stress activate the GLUD pathway as a result of metabolic reprogramming. The oxidative metabolism of glutamate through GLUD activity generates substantial adenosine triphosphate (ATP), with ammonia and carbon dioxide as byproducts (93), significantly contributing to ammonia buildup in the TME ( Figure 3 ). Isotope tracing studies have confirmed that the majority of ammonia in the TME is derived from the amino group of glutamines (15), further underscoring that ammonia accumulation is predominantly driven by glutamine metabolism.

Ammonia clearance is essential for maintaining cellular homeostasis and supporting growth and proliferation. Under normal conditions, the urea cycle (ornithine cycle) serves as the primary mechanism for ammonia detoxification, particularly in hepatocytes (100). In liver cells, urease-related enzymes in the mitochondria—such as carbamoyl-phosphate synthetase I, ornithine transcarbamylase, and argininosuccinate synthetase—convert ammonia into urea, which is then transported to the kidneys via the bloodstream and excreted in urine (100). Extracellular tissues can also incorporate ammonia into glutamine via transaminases, with glutamine then transported through the circulatory system to the liver to participate in the urea cycle (101). While the urea cycle is not commonly present in most tissue cells, many cells retain some capacity to clear ammonia (101). Non-hepatic cells can utilize alternative synthetic pathways for nitrogen-containing compounds, such as purines and pyrimidines, to clear ammonia, although these mechanisms are less efficient and cannot completely eliminate ammonia (102). In the TME, despite various ammonia clearance mechanisms, rapidly proliferating cells, particularly tumor and immune effector cells, have a heightened demand for α-KG derived from glutamine breakdown to fuel the TCA cycle, leading to substantial free ammonia production (14). Research indicates that tumor cells downregulate the expression of urea cycle enzymes (e.g., ornithine aminotransferase), reducing ammonia conversion into urea (103). This disruption in ammonia production and clearance balance contributes to the accumulation of ammonia. Furthermore, although lysosomes, as crucial organelles for degrading and recycling cellular metabolic waste, can transport ammonia into the lumen via the Rhesus glycoprotein C (RHCG) to reduce the concentration of free ammonia in cells, this clearance pathway is biologically limited. Prolonged and sustained ammonia stimulation may instead impair lysosomal degradative function (15, 104). Consequently, ammonia accumulation is not a transient phenomenon; rather, ammonia-induced cellular damage is more closely associated with chronic conditions, such as cancer ( Figure 3 ).

Ammonia is generally regarded as a cytotoxin, and elevated blood levels lead to hyperammonemia, impairing cellular function (105, 106). In the TME, excessive ammonia accumulation triggers endoplasmic reticulum (ER) stress, activating ER stress sensors such as IRE1 and PERK. This activation promotes reactive oxygen species (ROS) production and induces oxidative stress, resulting in significant cellular and tissue toxicity (107).

Ammonia accumulation is closely associated with protein synthesis and degradation processes (90, 106, 108). Excess ammonia disrupts cellular protein metabolism. The mTOR pathway, a key regulator of cell growth, proliferation, and metabolism (90), is modulated by ammonia levels. Under normal conditions, ammonia activates amino acid sensors, enhancing mTORC1 activity and promoting protein synthesis. However, at high concentrations, ammonia and increased oxidative stress initiate negative feedback on mTORC1. Through modulation of upstream factors like AMPK, ammonia inhibits mTOR activity, thereby reducing protein synthesis (106). Regarding protein degradation, ammonia primarily influences this process via the ubiquitin-proteasome system (UPS) and autophagy (108). Excess ammonia enhances the activity of ubiquitin ligases, such as E3 ubiquitin ligase, facilitating protein degradation. It can also activate cellular stress responses, including plasma membrane and ER stress (e.g., UPR), accelerating the removal of damaged proteins (108).

Ammonia accumulation has a dual effect on autophagy (15, 109, 110). At low to moderate levels, ammonia activates the AMPK signaling pathway, enhancing autophagy to clear ROS-induced damage and dysfunctional proteins, helping cells adapt and survive (110). However, at high levels of ammonia, it may excessively activate the mTORC1 pathway or interfere with the autophagy initiation complex, inhibiting autophagic function. In this scenario, the failure to effectively clear damaged proteins and organelles leads to cellular dysfunction and eventual cell death (110).

In 2024, Professor Huang’s team uncovered a novel form of T cell death, termed “Ammonia death” (15, 16). This study, for the first time, explored ammonia-induced cell death from a metabolic perspective, offering a new explanation for the rapid exhaustion of effector T cells (Teffs) following anti-tumor activity. Ammonia death is a distinct form of cell death driven by lysosomal and mitochondrial damage due to ammonia accumulation, characterized by lysosomal alkalization and mitochondrial swelling (15, 16). In contrast to traditional cell death mechanisms such as apoptosis and T cell exhaustion, the accumulation of ammonia from glutamine metabolism is identified as the primary trigger for “Ammonia death” (15, 16). During tumor immune responses, rapidly proliferating T cells become highly dependent on glutamine metabolism, leading to the release of large quantities of ammonia in the mitochondria (15, 16). However, when ammonia accumulates excessively in lysosomes, it raises the lysosomal pH, which not only damages the lysosomal membrane but also halts the storage of ammonia in the lysosome, resulting in its release back into the cytoplasm (15). As lysosomes lose their capacity to store ammonia, it accumulates around the mitochondria, triggering mitochondrial dysfunction and initiating oxidative stress (15, 104). This dual damage to lysosomes and mitochondria due to excessive ammonia accumulation ultimately promotes cell death (15). Furthermore, autophagy is one of the crucial mechanisms for maintaining intracellular homeostasis, preserving normal cellular function by clearing damaged or unnecessary cellular components (15). However, due to lysosomal impairment, excess ammonia accumulates in mitochondria, leading to increased mitochondrial mass. Although autophagosomes can form in the cytoplasm, autophagic flux appears to be interrupted. The blockade of autophagy prevents the efficient clearance of damaged mitochondria, which further exacerbates cellular dysfunction and ultimately accelerates cell death (15, 16) ( Figure 4 ).

While Ammonia death differs in morphology and mechanism from other cell death forms, excessive ammonia promotes the generation of ROS, inducing oxidative stress (15, 16). This suggests that ammonia-induced Ammonia death is not an isolated mode of cell death but may be interconnected with other well-established cell death processes, such as apoptosis, autophagic cell death, and necrosis.

Apoptosis, a form of programmed cell death, is typically executed through an intrinsic self-destruction pathway that involves caspase family activation (mainly caspases 3 and 7), alterations in the cell membrane, and DNA fragmentation (111). Mitochondrial dysfunction caused by ammonia accumulation creates an oxidative stress environment, a critical condition for activating the caspase pathway (112). At low ammonia concentrations, cells typically undergo a sub-lethal apoptotic response, a mechanism designed to eliminate mildly damaged cells (113). However, at higher concentrations, ammonia induces significant mitochondrial damage, compromising mitochondrial membrane integrity and promoting the release of cytochrome c and other pro-apoptotic molecules (such as SMAC/DIABLO) into the cytoplasm. This activates the apoptosome, leading to caspase-9 and caspase-3 activation, which triggers the apoptotic program (112).

Autophagy is a process in which cells degrade and recycle damaged organelles and macromolecules, typically activated in response to nutrient deprivation or stress (114). The relationship between Ammonia death and autophagy is complex. As mentioned earlier, varying ammonia concentrations yield different effects on autophagy (109, 115). In the early stages of autophagy, ammonia triggers oxidative stress and mitochondrial damage, stimulating the autophagic machinery to clear damaged mitochondria and oxidative damage. However, as ammonia accumulates to higher concentrations, it may disrupt the initiation and degradation stages of autophagy (e.g., inhibiting autophagosome-lysosome fusion), impairing autophagic flux and promoting autophagic cell death (109, 115).

Cell necrosis is an acute, non-programmed form of cell death typically triggered by membrane rupture, leading to the release of cellular contents and subsequent inflammation (116). The link between ammonia-induced cell death and necrosis is likely due to the local accumulation of ammonia at high concentrations, which disrupts cell membrane integrity and initiates necrotic responses such as cell swelling, membrane rupture, and the leakage of intracellular components into the extracellular space (117, 118). This leakage contributes to local inflammation and tissue damage.

Pyroptosis, a programmed cell death process driven by iron ions and lipid peroxidation, has been implicated in various pathological conditions (119). While direct evidence connecting ammonia-induced cell death with pyroptosis is currently lacking, several studies suggest that ammonia accumulation exacerbates oxidative stress, potentially activating inflammasomes and thereby promoting pyroptotic cell death (119, 120).

In conclusion, ammonia-induced cell death represents a distinct mode of cellular demise caused by ammonia accumulation, overlapping with and interacting with apoptosis, autophagy, necrosis, and other forms of cell death. Its unique characteristic is the metabolic reprogramming of the cell and the specialized response to ammonia buildup, positioning ammonia death as a promising target for future immunotherapeutic strategies.

Upon antigen stimulation, CD8+ naïve T cells differentiate into effector cells, with over 95% undergoing rapid cell death following antigen clearance in a process known as T cell contraction (121). This death of effector T cells is essential for maintaining immune homeostasis and preventing autoimmune responses. However, the precise mechanisms underlying effector T cell death have remained unclear. Professor Huang Bo’s team demonstrated that the ketone-body-gluconeogenesis-glycogen metabolic pathway activates the pentose phosphate pathway to reduce intracellular ROS, promoting the formation and maintenance of CD8+ T cell memory (122, 123). Furthermore, they showed that the urea cycle plays an active role in reducing intracellular ammonia levels, which supports the long-term survival of memory T cells (104).

Recent studies have highlighted ammonia’s critical role in effector CD8+ T cell death (15). While glutamine metabolism provides essential energy for CD8+ T cells during immune responses, contributing carbon and nitrogen for biosynthesis, its metabolic byproduct, ammonia, gradually accumulates within the cells, ultimately leading to their demise (15, 16). To prevent ammonia-induced cell death and extend the survival of CD8+ T cells, at least three strategies have been proposed. First, since glutamine metabolism is the precursor to ammonia accumulation, inhibitors such as 6-diazo-5-oxo-L-norleucine (DON) or CB-839, a non-competitive allosteric inhibitor of GLS1, can significantly reduce intracellular ammonia levels (35, 124). Both DON and CB839 have been shown to promote Teff survival, upregulate CD25, IFN-γ, and TNF-α, and enhance the formation of memory precursor effector cells (KLRG1-CD127+) (125–127). Second, removing excess ammonia from the cell provides an alternative approach to mitigate ammonia-induced cell death. Ammonia scavengers, such as phenylbutyrate (a prodrug of phenylacetic acid), reduce ammonia levels by promoting its conversion into phenylacetyl glutamine, which is excreted in urine, thus mitigating its toxic effects (128). Additionally, upregulating the key urea cycle enzyme, carbamoyl-phosphate synthetase 1 (CPS1), which is expressed at low levels in CD8+ T cells, can significantly lower intracellular ammonia concentrations (15, 129). Third, because ammonia-induced damage to lysosomes and mitochondria directly triggers cell death, accelerating the clearance of damaged organelles or enhancing autophagy offers a promising strategy to rescue T cells. Restoring lysosomal function, for example, by activating the transcription factor TFEB using compounds like Torin1 or Metformin to upregulate lysosomal gene expression, promotes lysosome biogenesis (130–132). When lysosomal function is severely impaired, lysosome-independent degradation pathways, such as those activated by Bortezomib, a proteasome modulator, can enhance ubiquitination and proteasomal activity, compensating for defective lysosomal function and facilitating the removal of damaged proteins and organelles, thus improving immunotherapy efficacy (133) ( Table 1 ). In summary, targeting ammonia-induced cell death can significantly prolong the survival of CD8+ T cells in the TME, enhancing their anti-tumor activity. This approach offers a promising direction for future cancer immunotherapy.

Although current experimental research on ammonia-induced cell death primarily focuses on CD8+ T cells, the immune system operates as a complex network. As discussed in the previous section, glutamine metabolism, which leads to ammonia accumulation, is active in a variety of immune cells. This metabolic pathway facilitates ammonia buildup in these cells (68, 69, 76, 77, 79). Furthermore, research indicates that CPS1, the key enzyme in the urea cycle, is predominantly expressed in hepatocytes and certain renal cells, with low or absent expression in most immune cells due to the urea cycle’s inactivity in these cells (134, 135). This limitation further contributes to ammonia accumulation in other immune cells. Notably, effector CD4+ T cells, including Th1, Th2, and Th17 cells, play vital roles in short-term immune responses. Following activation and execution of their effector functions, the majority of effector CD4+ T cells undergo programmed cell death (apoptosis), making them unsuitable for long-term immunity (136, 137). Therefore, it is reasonable to speculate that ammonia-induced cell death may not be a form of cell death unique to CD8⁺ T cells. Although CD8⁺ T cells may be more susceptible to ammonia under certain tumor microenvironments or immunosuppressive conditions, other immune cells (such as CD4⁺ T cells and macrophages)may also undergo cell death due to ammonia overload.

Research on ammonia-induced cell death has primarily focused on immune cells, particularly CD8+ T cells (15, 16). Inhibiting ammonia-induced cell death appears to significantly extend the lifespan of CD8+ T cells, thereby enhancing long-term immune efficacy (15). However, whether tumor cells undergo ammonia-induced cell death has yet to be investigated. Current evidence suggests that the primary mechanism triggering ammonia-induced cell death involves the excessive accumulation of ammonia, a byproduct of glutamine metabolism, which leads to lysosomal and mitochondrial damage (15, 16). As a metabolic byproduct, ammonia plays a critical role not only in immune cells but also in tumor cells (138, 139).

In the TME, the rapid proliferation of tumor cells is often associated with increased metabolic activity and competition for nutrients, including glutamine, from the surrounding environment. Consequently, tumor cells typically exhibit higher glutamine metabolic activity than immune cells (140). Studies indicate that, under normal conditions, ammonia produced by glutamine metabolism can promote tumor progression. For instance, ammonia serves as a key activator of lipogenesis, which is a prominent marker of cancer progression and metastasis (141). Lipogenesis is regulated by Sterol Regulatory Element-Binding Proteins (SREBPs) (141, 142). Cheng et al. demonstrated that glutamine is essential for SREBP activation and lipogenesis. While glucose is required for the N-glycosylation-mediated stabilization of SCAP, glutamine is necessary for the nuclear translocation of SREBP (90). Further studies revealed that ammonia, rather than glutamate or α-KG, is the primary activator of SREBP cleavage (90). Interestingly, gene inhibition and CB-839, an inhibitor of GLS activity, can eliminate SREBP activation, suggesting that ammonia derived from GLS is a critical mediator of lipogenesis (90, 142). Additionally, studies have shown that in certain tumors, blocking glutamine uptake leads to cell death, a phenomenon known as “glutamine addiction” (143, 144). Consequently, recent studies have increasingly focused on inhibiting glutamine metabolism in tumor cells as a strategy to suppress tumor progression.

Given the established mechanisms of ammonia-induced cell death in CD8+ T cells, inducing ammonia-induced cell death in tumor cells through excessive ammonia accumulation presents a promising avenue for cancer immunotherapy research. CPS1 catalyzes the reaction between ammonia and carbon dioxide to form carbamoyl phosphate, the first step in urea synthesis (134, 145). This process helps maintain metabolic balance by clearing ammonia from cells and mitigating oxidative damage (134, 145). Research indicates that CPS1 is predominantly expressed in liver, kidney, and a small number of intestinal epithelial cells (134, 135, 146). In the TME, CPS1 expression varies across cancer types and is highly expressed in liver, pancreatic, kidney, colorectal, breast cancers, and non-small cell lung cancer (135, 146–148). Several small-molecule CPS1 inhibitors have been identified, showing potential in inhibiting CPS1 activity and inducing tumor cell death under experimental conditions (135, 146, 149–151). However, these inhibitors remain in the early stages of research and have yet to be extensively validated in clinical cancer treatments. Despite this, the common outcome observed across these studies is that CPS1 inhibition leads to a significant increase in intracellular ammonia levels. Therefore, inhibiting CPS1 expression in tumor cells may cause excessive ammonia accumulation, but whether this accumulation can induce ammonia-induced cell death in tumor cells requires further validation through comprehensive experimental studies.

Based on current research progress, anti-tumor strategies targeting glutamine metabolism have established a multi-level intervention system, primarily encompassing three major directions: transport inhibition, key enzyme blockade, and combination therapies. At the level of glutamine transport, SLC1A5, as a key transporter, has emerged as an important target. Preclinical studies have confirmed that IMD-0354 significantly suppresses the growth of melanoma and hepatocellular carcinoma (HCC) through competitive inhibition of SLC1A5 (152, 153). Similarly, V-9302, which also targets this transporter, has demonstrated anti-tumor activity in models of triple-negative breast cancer (TNBC), HCC, and melanoma. Its mechanism of action involves blocking glutamine uptake by tumor cells, thereby disrupting metabolic homeostasis (60, 154, 155). JHU083, a novel SLC1A5 inhibitor, has been shown to be effective against various solid tumors in animal experiments, particularly exhibiting potential in regulating the metabolic reprogramming of TAMs (76, 156).

Multiple inhibitors targeting glutaminase (GLS), a key enzyme in glutaminolysis, are currently in various stages of development. BPTES, a selective GLS inhibitor, has been shown to suppress the growth of multiple solid tumors in preclinical studies (157, 158). CB-839 (also known as Telaglenastat), one of the more advanced candidates, has entered clinical trials for the treatment of various solid tumors (156, 159). Other GLS inhibitors, such as IACS-6274 in head and neck squamous cell carcinoma (HNSCC) and IPN60090 in non-small cell lung cancer (NSCLC) and ovarian cancer, have demonstrated efficacy in preclinical models (160) (161). These agents inhibit the conversion of glutamine to glutamate, thereby blocking α-KG production, disrupting TCA cycle replenishment and biosynthesis, and ultimately leading to metabolic collapse in tumor cells.

Combination therapy strategies aim to overcome the limitations of monotherapy and enhance therapeutic efficacy. Preclinical studies have demonstrated that DRP-104, a glutamine mimetic prodrug, in combination with the MEK inhibitor trametinib, effectively suppresses pancreatic ductal adenocarcinoma by simultaneously inhibiting glutamine metabolism and the ERK signaling pathway (162, 163). Furthermore, progress has been made in targeting compensatory mechanisms employed by tumor cells under glutamine deprivation. For instance, IFRD1 knockout combined with CB-839 treatment effectively inhibited the “low-cost” survival of tumor cells under nutrient stress in HCC animal models by blocking the autophagic degradation pathway of histone H1.0 (164). Other strategies, such as targeting the RAS signaling pathway to suppress its mediated dysfunction in macrophage phagocytosis, remain at the theoretical exploration stage (165, 166). These multi-pathway, multi-target intervention strategies provide new research directions and therapeutic approaches for overcoming tumor metabolic heterogeneity and microenvironment-mediated drug resistance. ( Table 2 ).