Glutamine and cancer: metabolism, immune microenvironment, and therapeutic targets

Abstract

Glutamine is the most abundant amino acid in human serum, and it can provide carbon and nitrogen for biosynthesis, which is crucial for proliferating cells. Moreover, it is widely known that glutamine metabolism is reprogrammed in cancer cells. Many cancer cells undergo metabolic reprogramming targeting glutamine, increasing its uptake to meet their rapid proliferation demands. An increasing amount of study is being done on the particular glutamine metabolic pathways in cancer cells.

Further investigation into the function of glutamine in immune cells is warranted given the critical role these cells play in the fight against cancer. Immune cells use glutamine for a variety of biological purposes, including the growth, differentiation, and destruction of cancer cells. With the encouraging results of cancer immunotherapy in recent years, more investigation into the impact of glutamine metabolism on immune cell function in the cancer microenvironment could lead to the discovery of new targets and therapeutic approaches.

Oral supplementation with glutamine also enhances the immune capabilities of cancer patients, improves the sensitivity to chemotherapy and radiotherapy, and improves prognosis. The unique metabolism of glutamine in cancer cells, its function in various immune cells, the impact of inhibitors of glutamine metabolism, and the therapeutic use of glutamine supplements are all covered in detail in this article.

Introduction

Glutamine is involved in numerous cellular biosynthesis processes, including the creation of proteins, lipids, nucleotides, and amino acids [1]. Though glutamine is a non-essential amino acid (NEAA), transformed cells consume glutamine at a rate faster than the rate at which glutamine is synthesized through endogenous pathways, leading to the conclusion that glutamine is crucial in cancer cells [2, 3].

In cancer cells, even under aerobic conditions, glucose undergoes glycolysis to produce lactate instead of entering the tricarboxylic acid (TCA) cycle. However, glutamine can replenish the TCA cycle by generating the intermediate product α-ketoglutarate (α-KG) [1, 4]. A characteristic of cancer cells is thought to be metabolic reprogramming, which guarantees a sufficient supply of lipids, proteins, and nucleotides to enable processes of rapid growth and proliferation [5]. Although cancer metabolism exhibits significant heterogeneity, increased consumption of glutamine has been found in many types of cancers, such as melanoma, breast cancer and prostate cancer [6,7,8]. Therefore, glutamine has been utilized for clinical imaging of cancer metabolism as a tracer for metabolic analogs. Due to the increased focus on the variations in glutamine metabolism found in malignancies, multiple kinds of glutamine metabolism inhibitors have been developed [9].

Important for a strong anti-cancer immune response, metabolic reprogramming is also another characteristic of immune cell activation. It was discovered lately that glutamine is a nutrient that modulates immunity. In immune cells that divide quickly, such as lymphocytes, glutamine consumption is on par with or even higher than glucose consumption [10].

This paper has been divided into three parts. The first part deals with glutamine and its metabolism, followed by its role in cancers cells and immune cells. Finally, we will talk about the glutamine supplements.

Glutamine and its metabolism

What is Glutamine

One of the most prevalent L-alpha amino acids in the human body is glutamine. Its molecular weight is 146.15 kDa, and the elemental composition is carbon (41.09%), hydrogen (6.90%), oxygen (32.84%), and nitrogen (19.17%). It makes up 40–60% of all the amino acids in plasma and tissues, which is 10–100 times more concentrated than other amino acids. Glutamate is an NEAA that the body can produce on its own and that, when needed, can also be acquired through food [10, 11].

Glutamine plays many biosynthetic roles in cells and is involved in nitrogen transport, acid–base balance regulation, and signal transduction of breakdown metabolism [12]. Glutamine has a five-carbon backbone and two nitrogen atom(α and γ) to donate [13]. In addition to being necessary for the production of other amino acids, proteins, nucleic acids, nucleotides, and hexosamines, it acts as a substrate for gluconeogenesis in specific organs [14]. It is clear that glutamine plays various biosynthetic roles in cells, and has different effects on various types of cells. Further research on its potential effects on cancer patients is crucial.

In 1955, Harry Eagle noted that one major common feature of mammalian cell culture is the requirement for glutamine [15]. Furthermore, immune cells seem to utilize glutamine more than glucose under catabolic conditions, such as sepsis, recovery from burns or surgery [16]. Other normal cells also need glutamine, including neurons, gut mucosa, skeletal muscle, etc. Besides, glutamine is a crucial nutrient for many cancer cells [17]. There is a competition for glutamine uptake in cancer cells and normal cells, especially immune cells. However, the mechanism and regulation of competition between cancer cells and immune cells remains unclear, which may deserve more research in the future.

Additionally, glutamine is a supplement recognized as Generally Recognized as Safe (GRAS) by the FDA [11]. A high-protein diet of 10–20 g per day can provide sufficient glutamine, but if there are injuries and stress, 20–40 g may be required [18]. The efficacy of glutamine supplementation in cancer patients is frequently questioned due to confusion and controversial results.

Cellular metabolism of glutamine

Glutamine is one of the main amino acids for biosynthesis during cell growth and division. While the carbon in glutamine can be utilized to synthesize fatty acids and amino acids, the nitrogen in glutamine is directly engaged in the creation of purines and pyrimidines. Glutamate is transported into the cytoplasm by glutamine transport proteins (SLC1A5, SLC38A1/SLC38A2, and SLC6A14) that are expressed on cell membranes (Fig. 1) [2]. The SLC1A5 variation then carries glutamine into the mitochondrial matrix, where glutaminase (GLS) transforms it into glutamate (Fig. 1) [19]. Subsequently, glutamate dehydrogenase 1 (GLUD1) or multiple mitochondrial transaminases, such as alanine aminotransferase 2 (GPT2) and aspartate aminotransferase 2 (GOT2), convert mitochondrial glutamate to α-KG (Fig. 1) [20, 21]. α-KG generated from glutamine can facilitate oxidative phosphorylation (OXPHOS) or reductive carboxylation pathways, in addition to providing metabolites for the tricarboxylic acid cycle (Fig. 1) [3, 22]. Once guanosine triphosphate (GTP) and adenosine triphosphate (ATP) are synthesized, glutamine metabolism products take part in the oxidative phosphorylation process by helping to generate electron donors like NADH or FADH2 [2, 23].

Fig. 1

Metabolism of glutamine between normal cells and cancer cells. The glutamine transporters (SLC1A5, SLC38A1/SLC38A2, and SLC6A14) expressed on the cell membrane facilitate the transport of glutamine into the cytoplasm. Subsequently, SLC1A5 variants transport glutamine into the mitochondrial matrix, where it is converted to glutamate by GLS. Then, mitochondrial glutamate is converted to α-KG by GLUD1 or various mitochondrial transaminases (including GPT2 and GOT2). Glutamine-derived α-KG provides metabolites for the tricarboxylic acid cycle and can also support OXPHOS or reductive carboxylation pathways. Cancer cells uptake a significant amount of glutamine from plasma through protein transporters. The first and rate-limiting step of glutamine breakdown is catalyzed by GLS in the mitochondria, converting glutamine to glutamate. This enzyme exists in two forms: GLS1 and GLS2, both of which convert it into glutamate. Glutamate is further converted to α-KG by mitochondrial GLUD1 and releases ammonia. α-KG enters the TCA cycle in cancer cells and is converted back to glutamate. In cancer cells, the production of NAAG, derived from glutamine, is significantly increased, and high levels of NAAG are observed in advanced cancer. The NAAG storage cycle provides a mechanism for cancer cells to store this important metabolic product by converting glutamate generated from glutamine into NAAG and then back to glutamate. c-Myc promotes glutamine uptake by targeting the expression of glutamine transporters SLC1A5, SLC38A1, and SLC7A5. Glutamine activates mTORC1 through the SLC7A5/SLC3A2 antiporter system, leading to the efflux of glutamine out of the cell in exchange for leucine influx, a potent Rag-dependent mTORC1 activator. GLS, glutaminase; αKG, α-ketoglutarate; GLUD1, glutamate dehydrogenase 1; GOT, glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; GSH, glutathione; ROS, reactive oxygen species; OXPHOS, oxidative phosphorylation; GLS1, kidney-type glutaminase; GLS2, liver-type glutaminase; NAAG, N-Acetyl-aspartyl-glutamate

The primary sources of glutamine include the lungs, liver, brain, adipose tissue, and skeletal muscle due to their highly tissue-specific glutamine synthesis activity, which catalyzes the synthesis of glutamine from glutamate and ammonia in cells and tissues that synthesize glutamine. In contrast, intestinal mucosal cells, renal tubular cells, and leukocytes are the main consumers of glutamine. They hydrolyze glutamine through enzymes such as phosphate-dependent GLS [24].

Glutamine provides essential precursors for the biosynthesis of reduced glutathione [25]. In order to maintain an appropriate supply of glutamine, an essential substance for survival of cells and proliferation, a recent study also discovered that proline production declines in the absence of glutamine [26].

Glutamine metabolism in cancer cells

In cancers, glucose metabolism is primarily aerobic glycolysis, resulting in the secretion of lactate in the extracellular space [2, 27]. The vast majority of cancers, including melanoma and breast cancer, have an elevated glutamine requirement [6, 7, 28]. Furthermore, it has been demonstrated that glutamine is essential to the metabolic reprogramming of advanced prostate cancer (PCa) [8]. It has also been found that glutamine can participate in the metabolism of cancer stem cells (CSCs) [29]. From an other viewpoint, during times of glutamine deprivation strain, the majority of cancer cells significantly depend on autophagy to withstand the existential threat [30]. Glutamate is taken up by cancer cells in significant quantities from the plasma via protein transporters [31, 32]. GLS in the mitochondria catalyzes the initial and rate-limiting step in the breakdown of glutamine, which transforms glutamine into glutamate [33] (Fig. 1). This enzyme converts it to glutamate and comes in two forms: kidney-type glutaminase (GLS1) and liver-type glutaminase (GLS2) (Fig. 1). Ammonia is released when glutamate is further converted to α-KG by mitochondrial GLUD1. As α-KG enters the TCA cycle, it gets oxidized to generate succinate and fumarate, which are cancer metabolites that produce ATP, NADH, and FADH2 [34] (Fig. 1). Glutamine also creates α-KG through redox processes, which serves as a precursor for purines, pyrimidines, and aspartate biosynthesis [35]. However, it's important to remember that Quek et al. found that glutamine can enhance glucose oxidation and counteract α-KG depletion through essential amino acids (EAA) degradation [36]. The enzymes alanine aminotransferase (GPT), aspartate aminotransferase (GOT), and phosphoserine aminotransferase (PSAT1) can also convert glutamate to the amino acids alanine, aspartate, and serine, in that order [37]. Isocitrate dehydrogenase 1 (IDH1) reduces glutamine metabolism in hypoxic conditions, which promotes the lipogenesis and proliferation of cancer cells [38]. Interestingly, in pancreatic ductal adenocarcinoma (PDAC), uncoupling protein 2 (UCP2) can transport the aspartate derived from glutamine metabolism from mitochondria to the cytoplasm to generate NADPH, and silencing UCP2 can significantly inhibit the growth of PDAC. Therefore, UCP2 can be regarded as a critical therapeutic target [39]. However, it is noteworthy that, while upregulation of glutamine metabolism is a phenomenon present in many cancer cells, recent studies have shown a decrease in glutamine metabolism in cholangiocarcinoma, glioblastoma multiforme, liver hepatocellular carcinoma, and thyroid carcinoma, demonstrating tissue heterogeneity in glutamine metabolism [40].

Recently, it has been discovered that N-Acetyl-aspartyl-glutamate (NAAG) serves as a storage reservoir for glutamate in cancer. In cancer cells, NAAG produced from glutamine is significantly increased, and high levels of NAAG are observed in advanced cancer (Fig. 1) [41]. The NAAG storage cycle provides a mechanism for cancer cells to store this important metabolic product by converting glutamate produced from glutamine into NAAG and then back into glutamate, thus providing a mechanism for storing this important metabolic product under stress conditions such as inhibition of glutamine breakdown [37] (Fig. 1).

Glutamine uptake is enhanced in Myc and KRas-activated cells [2, 42]. The glutamine synthetase (GLUL) gene promoter is demethylated and thymine DNA glycosylase expression is elevated as a consequence of Myc-induced thymine DNA glycosylase upregulation [23, 43]. Moreover, by suppressing the expression of the glutamine transporters SLC1A5, SLC38A5, and SLC7A5, c-Myc increases the absorption of glutamine [23, 42] (Fig. 1). It's interesting to note that lung cancers do not express glutamine synthetase (GS), which catalyzes the synthesis of glutamine from glutamate and ammonia [44]. This results in Myc-induced liver tumors consuming glutamine, while lung tumors produce glutamine [45].

The invasion of cancer cells is also influenced by glutamine metabolism. For instance, in colorectal cancer, the amount of GLS1 mRNA expression is linked to lymph node metastases. GLS1 converts glutamine into glutamate [46]. Pharmacological inhibition of glutamine metabolism has been demonstrated in murine cancer models to limit glycolytic metabolism and oxidative phosphorylation in cancer cells, leading to hypoxia, acidosis, and decreased absorption of nutrients [47]. Regarding ASCT2, recently, Du et al. found that RNA binding motif protein 45 (RBM45) enhances its stability by antagonizing non-autophagy lysosomal degradation, which is also associated with the poor prognosis of hepatocellular carcinoma [48].

Cancer cells metabolize glucose through the glycolysis pathway in the presence of oxygen, while glutamine is converted into α-KG and enters the tricarboxylic acid cycle. However, there is also a connection between glucose and glutamine in the tumor microenvironment (TME). Recently, studies have found that in the tumor microenvironment, glutamine uptake is mainly dominated by cancer cells. When glutamine is deficient, TME resident cells will increase their ability to uptake glucose [49]. Another metabolite that is relatively closely related to glutamine is asparagine. Mechanistically, glutamate and oxaloacetic acid are catalyzed by GOT2 to aspartate and α-KG, and then asparagine is generated in the presence of asparagine synthetase. It is worth noting that when glutamine is depleted, asparagine will up-regulate glutamine synthetase and increase the content of glutamine again [50]. Therefore, inhibiting asparagine synthesis is also an important target.

18F-(2S,4R)4-fluoroglutamine has the potential to illustrate the distinct glutamine metabolism of cancer cells, especially those with brain metastases, in therapeutic imaging applications [51]. Thus, 18F-(2S,4R)4-fluoroglutamine PET could be a novel method for identifying glutamine metabolism in cancer patients and directing the use of glutamine therapy that is specifically targeted [52]. Furthermore, the active metabolism of CSCs and the tumor uptake of glutamine can be evaluated by magnetic resonance imaging (MRI) [53].

The role of glutamine in immune cells

T cells

Dietary and endogenously synthesized glutamine can regulate T cell proliferation [54]. When glutamine is deficient in the cell growth environment, T cell activation is completely inhibited [55]. When T cell receptors are stimulated in naïve and memory CD4 subpopulations, the main route for the TCA cycle and OXPHOS is glutamine metabolism [56]. Different types of T cells have different requirements for glutamine and exhibit different functions. For example, initial T cells have the lowest metabolic efficiency for glutamine metabolism because they only require it for survival, while effector T cells (Teff) that require rapid proliferation have a higher rate of glutamine metabolism, providing them with sufficient macromolecular synthesis materials. Different types of T cells also have different ways of immune response against cancers [57]. While activated CD4 + helper T cell 1 (Th1) cells fight cancer by secreting interferon-γ (IFN-γ) to activate macrophages and natural killer (NK) T cells, activated CD8 + T cells have direct cytotoxic effects against tumors [57] (Fig. 2). While regulatory T cells (Treg) and activated CD4 + helper T cell 2 (Th2) cells support immune suppression brought on by cancer, CD4 + helper T cell 17 (Th17) cells, depending on the context, either assist or impede the growth of cancer (Fig. 2). A combination of all cytokines can cause Th1 expression to shift in a glutamine-restricted binding model, where Th2 expression takes over [58] (Fig. 2). It has been discovered that glutamine metabolism plays a crucial regulatory role in the development of γδ T cells that produce interleukin-17 (IL-17) in the skin tissues of individuals with psoriasis [59] (Fig. 2).

Fig. 2

Metabolism of glutamine between different kinds of T cells and cancer cells. Th1 combat tumors by secreting IFN-γ to activate macrophages and NK cells. Th2 and Treg promote cancer-induced immune suppression. A mixture of cytokines that bind to glutamine-restricted cells can lead to a shift from Th1 to Th2 expression. Th17 cells can either support or inhibit cancer progression depending on the context. Glutamine metabolism is also a critical regulator of γδ T cell differentiation and IL-17 production. Loss of SLC7A5 results in reduced mTORC1-dependent Th1 and Th17 effector T cells, while Treg numbers are largely unaffected. Proliferation of antigen-stimulated CD8 + T cells already present in the body is completely inhibited. This mechanism is due to the inability of the mTORC1 signaling pathway to activate in the absence of SLC7A5. Furthermore, the absence of the tumor suppressor Menin can enhance mTORC1 signaling and glutamine breakdown. Th1, CD4 + T helper 1 cells; Th2, CD4 + T helper 2 cells; Treg, regulatory T cells; Th17, CD4 + T helper 17; IL-17, interleukin-17

Glutamine transporter plays a critical role in T cells. When SLC7A5 is absent, CD8 + T cell proliferation is completely inhibited even if the cells receive antigen stimulation in the body. This process results from the mTORC1 signaling pathway's incapacity to activate in the absence of SLC7A5 [60, 61] (Fig. 2). Nabe et al. proposed a novel cancer-specific CD8 + T cell adoptive transfer culture under glutamine-deficient conditions, which may be a promising method for improving the efficacy of cell transfer immunotherapy [62]. Similarly, in a mouse model lacking SLC7A5, mTORC1-dependent Th1 and Th17 effector T cells are reduced, while Treg numbers are almost unaffected(Fig. 2). According to Suzuki et al., through restricting cell metabolism, the cancer suppressor menin stops activated CD8 + T lymphocytes from developing functional abnormalities [63] (Fig. 2). Vallion et al. found that the stability of Treg lacking mTOR decreases [64].

Maintaining a balance in the concentration of glutamine is critical to achieving its beneficial cancer-suppressive effects. In triple negative breast cancer (TNBC), cancer cells develop an addiction to glutamine, competing with T cells for its availability [57, 65]. Compared to effector CD8 + T cells, memory CD8 + T cells have higher levels of CD8 protein expression. According to Madi et al., pretreating memory CD8 + T cells with an agonistic anti-CD8 antibody can increase the effectiveness of memory T cell-based cancer immunotherapy by activating memory T cells' glutamine metabolism [66]. According to Chatterjee et al., adoptive T cell therapy for cancer treatment can be more effective when the CD38-NAD + axis is targeted [67]. Consequently, the challenge in the development of anti-cancer therapies is to produce pharmaceuticals that selectively target cancer cells while maintaining the ability of vital immune cells, like T cells, to function. Our goal is to find targets that specifically limit the glutamine metabolism of tumor cells while leaving T cells unaffected.

Macrophages

As the body's initial line of defense in the innate immune response, macrophages are crucial for controlling host inflammation and preserving tissue homeostasis [68]. Macrophages can polarize into two different states, M1 and M2. M2 macrophages have weakened antigen presentation capacities and downregulate immune responses [57, 69] (Fig. 3).

Fig. 3

The role of glutamine in Macrophages. Macrophages can polarize into two distinct states, M1 and M2. They can be induced by various stimuli, most notably polarizing towards M1 macrophages under LPS/IFN-c stimulation, and towards M2 macrophages under IL-4/IL-13 stimulation. M1 and M2 macrophages have different biological functions. The ratio of succinate to α-ketoglutarate regulates M1 polarization through PHD-dependent proline hydroxylation of IKKβ, which is crucial for NF-κB signaling activation. High levels of succinate in the early stages of M1 polarization favor a strong inflammatory response. Compared to M1 macrophages, M2 macrophages exhibit intact TAC and enhanced OXPHOS. Glutamine itself can influence macrophage phenotype through unknown mechanisms. Another important factor in alternative activation is PPAR-γ signaling. PPAR-γ is not only involved in the conversion of glutamine metabolism but also participates in the upregulation of OXPHOS. TAMs are a special type of macrophage with functional plasticity. MSO regulates TAMs' glutamine metabolism, inducing them towards an M1 phenotype. LPS, lipopolysaccharide; IFN-c, interferon-c; IL-4, interleukin-4; IL-13, interleukin-13; TAC, tricarboxylic acid cycle; OXPHOS, oxidative phosphorylation; TAMs, tumor-associated macrophages; MSO, L-glutamine sulfoximine

A unique subset of macrophages with versatile functions are known as tumor-associated macrophages (TAMs). Although they are usually characterized as M2-like, there may be M1-like TAMs as well, according to some data. TAMs have an inflammatory phenotype in the early stages of cancer formation and an immunosuppressive phenotype in the later stages of cancer progression [57]. According to Du et al., l-methionine sulfoximine (MSO) controls TAMs' glutamine metabolism and causes them to polarize toward the M1 phenotype [70] (Fig. 3). The consumption of glutamine by the cancer cells in clear cell renal cell carcinoma (ccRCC) results in the local deprivation of extracellular glutamine This, in turn, triggers the release of IL-23 by cancer-infiltrating macrophages via the activation of hypoxia-inducible factor 1α (HIF1α) [71].

M1 and M2 macrophage polarization is related to glutamine metabolism. It is unknown whether suppressing the anti-cancer immune response generated by M2 macrophage polarization in the TME is more effective than boosting the anti-cancer immune response generated by M1 macrophage polarization because M2 macrophages consume more glutamine than M1 macrophages (Fig. 3). According to Liu et al., dimethyl α-KG suppresses M1 polarization by blocking IKKβ activation, which in turn prevents the nuclear factor-κB (NF-κB) signaling pathway from activating. The hydroxylation of IKKβ serine residues in a prolyl hydroxylase (PHD)-dependent manner may regulate this mechanism [72].

An essential metabolic regulator for M2 polarization is glutamine. Increased glutamine metabolism may be the fundamental mechanism behind EPHB2's ability to regulate M2-like polarization of macrophages in lung cancer [73]. Furthermore, Nelson Liu et al. discovered that glutamine-mediated alternate activation of macrophages requires the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) [74]. Macrophages rely on large amounts of glutamine utilization to provide energy for OXPHOS, while macrophages lacking PPARγ express a higher level of proinflammatory transcriptome [74]. It is worth noticing that glutamine produced through GS may also play a key role in selective macrophage activation [68]. According to research by Palmieri et al., pharmacological inhibition of GS causes M2-like macrophages to change into M1-like cells, which have higher succinate and less intracellular glutamine [75]. Therefore, glutamine itself may influence macrophage phenotype through unknown mechanisms.

Moreover, effector T cells and M2 macrophages compete for arginine and glutamine. Fatty acid oxidation (FAO) is used by M2 macrophages to support many tasks, such as proliferation, by supplying energy for oxidative phosphorylation and electron transfer via the TCA cycle [76]. Overall, glutamine is crucial for controlling the polarization of macrophages and the activity of macrophages linked to cancer. Additional investigation into how glutamine metabolism affects macrophage activity may lead to the discovery of novel targets and therapeutic approaches for cancer.

Other immune cells

Only an appropriate concentration of glutamine content can promote the secretion of cytokines such as IFN-γ and TNF-α by CD8 + T cells and NK cells to kill tumor cells [57, 77]. In the cancer microenvironment, glutamine is mainly concentrated in cancer cells, while the glutamine content in other cells is lower, which leads to a decrease in the level of GSH in NK cells [78] (Fig. 4). The reduction of GSH levels affects the ability of NK cells to protect cells and promote immune function, which is crucial for cancer patients (Fig. 4).

Fig. 4

The role of glutamine in NK cells, dendritic cells, MDSCs and CAFs. A In the TME glutamine is primarily concentrated in cancer cells, leading to a decrease in glutamine levels in NK cells. This reduction in glutamine results in a decrease in GSH levels in NK cells, reducing their cytotoxic function and allowing cancer cells to evade immune attack. B Elevated glutamine levels in cancer patients affect Th17 cell responses by activating the GluR4 receptor on DCs, leading to cancer cell proliferation and tumor growth. Both cDC1s and cancer cells express SLC38A2, with higher expression levels in cancer cells. Mechanistically, in cDC1s, glutamine induces the assembly of the FLCN-FNIP2 complex, inhibiting the activity of the TFEB transcription factor and promoting the antigen-presenting ability of cDC1s. Therefore, glutamine-dependent signaling in cDC1s enhances the generation of cytotoxic effector CD8 + T cells in the TME. C Inhibiting the glutamine metabolism of MDSCs can lead to cell death induced by MDSC activation and their transformation into inflammatory macrophages. mGluR2/3 are expressed on MDSCs, and the mGluR2/3 antagonist LY341495 can weaken the immunosuppressive activity of MDSCs. D CAFs upregulate both glutamine synthesis and secretion, increasing the concentration of glutamine in the TME. TME, tumor microenvironment; NK cells, Natural killer cells; GSH, glutathione; DCs, dendritic cells; cDC1s, type-1 conventional DCs; Th17,CD4 + T helper 17; MDSCs, myeloid-derived suppressor cells;, mGluR2/3, metabotropic glutamate receptors; CAFs, cancer-associated fibroblasts

In conclusion, the TME's glutamine content influences NK cells' ability to perform their regular immunological activity. Thus, NK cells and tumor cells have different glutamine metabolisms, and researching these differences may help discover novel targets for tumor cell-targeting medications.

The control of glutamine levels outside of the cell can help B cells differentiate into lymphocytes and plasma cells. Mechanically, a distinct group of microRNAs, known as the let-7 family, exhibits the capability to restrict the metabolic rate of glutamine, consequently repressing the activation of B lymphocytes [79]. Elevated glutamine levels in cancer patients affect Th17 cell responses by activating GluR4 receptors on dendritic cells (DC), leading to cancer cell proliferation and cancer growth [78] (Fig. 4). Both conventional type 1 dendritic cells (cDC1s) and cancer cells express SLC38A2, with higher expression levels in cancer cells [80] (Fig. 4). Mechanistically, glutamine enhances the ability of cDC1s to deliver antigens by inducing the FLCN-FNIP2 complex to assemble and suppressing the TFEB transcription factor's activity (Fig. 4).

Compared to normal fibroblasts, cancer-associated fibroblasts (CAFs) show increased glutamine production and release, which can raise the glutamine content in the TME [81] (Fig. 4). Furthermore, research has demonstrated that glutamine deprivation stimulates the migration and invasion of CAFs, contributing to the leading edge of migration through mechanisms involving the regulation of TRAF6 and p62-dependent glutamine activity, along with a polarized distribution of Akt2 [82]. Another way that glutamine can be produced in stromal fibroblasts is by macropinocytosis, a mechanism that has been demonstrated to be controlled by RAS, PTEN, and PI3K [83]. Myeloid-derived suppressor cells (MDSCs), a population of immature myeloid cells produced from bone marrow, are also present in the suppressive cancer microenvironment. It is possible to cause activation-induced cell death in MDSCs and their transformation into inflammatory macrophages by blocking their glutamine metabolism [84] (Fig. 4). According to Morikawa et al., MDSCs express the metabotropic glutamate receptor 2/3 (mGluR2/3), and the immunosuppressive effect of MDSCs can be reduced by the mGluR2/3 antagonist LY341495 [85] (Fig. 4).

In summary, glutamine plays a crucial role in regulating B cell immune response, dendritic cell antigen presentation, and neutrophil immune function. The complexity of glutamine metabolism in the tumor microenvironment warrants further research.

Glutamine is an indispensable substance for the growth of tumor cells. In preclinical studies, cancer cells were dependent on glutamine, and glutaminase inhibition led to reduced cell growth and induced apoptosis [86,87,88,89]. Glutaminase inhibitor CB-839 is now under clinical trials to evaluate safety and efficacy in combination with other anticancer drugs in cancers (Table 1). Glutaminase inhibitor CB-839 were reported to show therapeutic value in combination therapy with other anticancer drugs in advanced renal cell carcinoma, renal cell carcinoma,advanced myelodysplastic syndrome, etc. [90,91,92]. Glutaminase inhibitor alone in cancer treatment remains unknown. Therefore, Glutaminase inhibitor is a new potential target for cancer therapy and more clinical trials are needed.

Table 1 Clinical trials of glutamine and glutamine inhibitors

On the other hand, glutamine is selected as a dietary supplement for patients undergoing anti-tumor treatment in clinical practice, and at an appropriate dose, it does play an important role in reducing weight loss and radiation toxic side effects [98]. However, these clinical trials did not report whether glutamine will promote cancer progress. Besides, glutamine also plays a significant role in the differentiation and activity of immune cells. However, recent studies have shown that in the tumor microenvironment, cancer cells are the dominant players in glutamine metabolism [99]. Cancer cells may competitively uptake glutamine more than immune cells, further leading to the loss of immune activity of immune cells. The competition of glutamine between immune cells and cancer cells requires more studies.

Therefore, more glutamine uptake is not recommendation in advance or metastatic cancers. In early-stage cancer patients after curative treatment, appropriate glutamine supplementation might be considered to reduce the side effects of treatment.

Glutamine antimetabolites

L-DON

Antibiotics secreted by Streptomyces bacteria: 6-nitro-5-oxo-L-norleucine (L-DON), nitroacetylserine, and glutamine analogs are irreversible competitive inhibitors of glutamine transferase and glutaminase [87, 88, 98]. For instance, L-DON prevents the induction of PPAT and PAICS by glutamine and the reduction of pyruvate kinase activity [87] (Fig. 5). The pro-drug form of L-DON, sirpiglenastat (DRP-104), attempts to avoid the toxicity that is often associated with L-DON, and when used in combination with gemcitabine, it has been observed to dramatically increase the survival rate of PDAC [89]. Dependent on CD8 + T cells, DRP-104 produces a strong immunological memory. Furthermore, in lung cancer with KEAP1/NRF2 mutations, DRP-104 combined with checkpoint inhibitors can enhance anti-tumor immunity [99]. Furthermore, L-DON can also act on asparagine synthetase (ASNS) [100]. Studies have found that in PDAC, in the absence of glutamine, it can inhibit the synthesis of Asn to exert tumor-suppressive effects [101].

Fig. 5

Three main types of drugs that inhibit glutamine metabolism. 1. Glutamine antimetabolites: L-DON can block glutamine-mediated induction of PPAT and PAICS as well as decrease the activity of pyruvate kinase, but it lacks selectivity for glutamine-dependent cells. JHU083 is the prodrug form of L-DON, which can deplete glutamine metabolism in cancer cells, but it can stimulate T-cell proliferation. 2. Glutaminase inhibitors: BPTES is a selective inhibitor of GLS1; CB-839 (telaglenastat) is a selective inhibitor of GLS1 currently undergoing comprehensive clinical trials; 968 is a pan-glutaminase inhibitor, with fourfold selectivity for GLS2 over GLS. 3. Glutamine uptake inhibitors: GPNA can inhibit the growth of colorectal cancer cells by suppressing the overexpressed glutamine transporter SLC1A5 on the cell surface. A GPNA derivative, V-9302, has been developed to effectively inhibit cellular glutamine uptake, but its mechanism of action requires further investigation. MeAIB targeting SLC38A1 and/or SLC38A2, has shown anticancer effects in various cancer cells. α-MT inhibits the growth of SLC6A14-positive breast cancer, PDAC, and CRC cells. L-DON, 6-diazo-5-oxo-L-norleucine; JHU083, ethyl 2-(2-Amino-4-methylpentanamido)DON; BPTES, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide; GPNA, L-γ-glutamyl-p-nitroanilide; CB-839, telaglenastat; V-9302, 2-amino-4-bis (aryloxy benzyl) aminobutanoic acid; MeAIB, n-methyl-aminoisobutyric acid; α-MT, α-methyltryptophan; PDAC, pancreatic ductal adenocarcinoma; CRC, colorectal cancer

JHU083

JHU083 is a prodrug form of L-DON designed to be cleaved by tissue proteases and other enzymes in the cancer, thereby limiting its effects on other organs [87, 102] (Fig. 5).

JUH083 produces different metabolic effects on cancer cells and T cells in the TME [87, 102]. This drug can deplete glutamine metabolism in cancer cells but can stimulate T cell proliferation, enhance cell survival, improve effector function, and even induce persistent anti-cancer memory [87, 102]. Furthermore, Huang et al. discovered that JUH083 plus EGFR peptide vaccination (EVax) combination therapy dramatically slowed the growth of lung cancer in tumor-bearing animals. Mechanistically, JUH083 decreased immunosuppressive cells such as monocytes and myeloid-derived suppressor cells, regulatory T cells, pro-tumorigenic CD4 + Th17 cells, and boosted the infiltration of CD8 + T cells and CD4 + Th1 cells. On the other hand, EVax induced a robust Th1 cell-mediated immune response [103]. In conclusion, the cytotoxic effect of JUH083 on immune cells against tumors aligns with expectations, and its potential for combination therapy with vaccines or immune checkpoint inhibitors holds great promise for further research.

Glutaminase inhibitors

BPTES

GLS-specific inhibitors do not affect other functions of glutamine but selectively target the first enzymatic step of glutamine hydrolysis, glutaminase [55]. In highly proliferative cancer cells, GLS1 is often overexpressed in order to meet the increased glutamine demand [104]. For instance, GLS1 is the specific target of bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)disulfide (BPTES), but GLS2 exhibits cancer-inhibitory action [55, 98, 105]. Preclinical research on it is presently being conducted. It also suppresses the growth of ovarian and colorectal cancer cells that are resistant to platinum [55, 98, 105]. However, further research is needed to understand the in vivo effects of this drug more thoroughly.

968

968 has been found to be a pan-glutaminase inhibitor with fourfold selectivity for GLS2 over GLS [9] (Fig. 5). In non-small cell lung cancer (NSCLC), combining 968 with erlotinib reverses erlotinib resistance and decreases both glycolysis and glutamine metabolism [105]. When combined with mTOR inhibition, 968 treatment exhibits synergistic anti-tumor efficacy in glioblastoma, inhibiting the neoplastic transformation mediated by Rho GTPases in fibroblasts and breast cancer [106].

CB-839

Recently, another GLS1 selective inhibitor, CB-839 (telaglenastat), is currently undergoing comprehensive clinical trials (Fig. 5, Table 1). Cancer cells that express high levels of SLC7A11 and are glutamine-dependent may benefit from treatment with the glutaminase inhibitor CB-839 [107]. In addition to restricting mitochondrial metabolism, GLS inhibition also leads to a decrease in glutamate and ammonia concentrations [108].

There are currently numerous studies investigating the combination of CB-839 with other drugs. For instance, in ovarian clear cell carcinoma xenograft mice, it was discovered that immune checkpoint blockage using anti-PD-L1 antibody and glutaminase inhibitor CB-839 work in concert when ARID1A inactivation is present [109]. And it is worth noting that many KRas-dependent tumors develop resistance to mTORC1 inhibition by upregulating glutamine metabolism, but the combination of mTORC1 inhibition with CB-839 can overcome this resistance [110].

R162

Targeting GLUD1 has been demonstrated in prior research to reduce the migration and proliferation of cancer cells, suggesting that it may be a useful therapeutic target for the treatment of cancer [34]. Recent research has shown that the GLUD1 inhibitor purpurogallin analog R162 may have inhibitory effects on the growth of glioblastoma, non-small cell lung cancer, and breast cancer cells in vitro and in animal models [111]. Additionally, it has been demonstrated that the combination of docetaxel and R162 inhibits the development and metastasis of non-small cell lung cancer cells resistant to docetaxel, hence bolstering the effectiveness of combination therapy including anticancer medications and GLUD1 inhibitors as a cancer treatment approach [111].

Glutamine uptake inhibitors

GPNA

The frequent upregulation of SLC1A5, a glutamine transporter protein, as a transcriptional target of c-Myc in cancers suggests its specific role, and its widespread overexpression is associated with poor prognosis [105]. By employing L-γ-glutamyl-p-nitroanilide (GPNA) to pharmacologically inhibit SLC1A5, it is possible to reduce the proliferation of stomach and colon cancer cell lines and improve the effectiveness of cetuximab (Fig. 5) [105, 112, 113]. For instance, Polet et al. discovered that SLC38A2, a glutamine compensatory transporter, is markedly increased in leukemia cells when SLC1A5 is absent or inhibited [114].

V-9302

The GPNA derivative V-9302 has been developed, which enhances the ability to inhibit cellular glutamine uptake by 100-fold [34, 87]. However, there is doubt about whether V-9302 is a specific inhibitor of SLC1A5. Some have suggested that the aforementioned drug V-9302 targets SLC38A2 and SLC7A5 instead of SLC1A5 [34, 87] (Fig. 5).

Research has demonstrated that V-9302 can boost anti-cancer immune responses. In patients with breast cancer who are not responsive to anti-PD-1 therapy, combined therapy involving both V-9302 and anti-PD-1 may have a synergistic impact [115]. V-9302 boosts the uptake of glucose by all cancer cell groups in the TME while significantly reducing the uptake of glutamine by TME cells [116]. Thus, more research is needed to determine how different glutamine inhibitors affect immune cells in the TME.

MeAIB and α-MT

SLC38A1 is overexpressed in melanoma, breast cancer, gastric cancer, osteosarcoma, and endometrial cancer cells, while SLC38A2 is overexpressed in prostate cancer, hepatocellular carcinoma (HCC), and TNBC cells [34]. The n-methyl-aminoisobutyric acid (MeAIB) targeting SLC38A1 and/or SLC38A2 has been used to elucidate their functions in various cells, and MeAIB has been shown to exert anti-cancer effects in multiple cancer cells [117, 118]. α-methyltryptophan (α-MT) can act as an inhibitor on SLC6A14, which is overexpressed in cells of pancreatic cancer, ER-positive breast cancer, colon cancer, and cervical cancer [34].

While the majority of drugs targeting the inhibition of glutamine metabolism have been described above, in practical application, we still need to consider the issue of resistance. For example, cancer cells frequently experience glutamine famine in the body as a result of their rapid glutamine consumption. In response to glutamine shortage, arginine can operate as an effector of mTORC1 activation, which may be linked to resistance to inhibitors of glutamine metabolism [119]. Furthermore, the above-mentioned drugs may face the challenge of insufficiently effective in vivo tumor treatment. A new strategy utilizing nanomaterial-mediated silencing of mutant genes Kras and glutaminase 1 (GLS1) with siKras and siGLS1 has shown great potential in inhibiting cancer [120].

Glutamine supplements

Oral administration of GLN significantly enhances the level of apoptosis in cancer cells [121]. Oral supplementation with glutamine has multiple effects, including supporting host glutamine storage and glutathione production, enhancing NK cell activity, and inhibiting cancer growth [121]. Mechanistically, it may be related to the GSH-mediated inhibition of PGE2 synthesis [122].

Oral GLN dramatically increases the expression of the pro-apoptotic factor Bad and suppresses the expression of Bcl-2, according to the detection of the apoptosis pathway. In addition, GLN dramatically raises PARP, caspase-3, caspase-8, and caspase-9 activity [121].

Numerous studies have demonstrated that taking glutamine orally can enhance surgical outcomes and lessen side effects from chemotherapy and radiation for cancer patients. L-glutamine supplementation shields the rat penis's structural integrity from radiation-induced alterations, which frequently happen during radiation therapy for pelvic cancers [123].The deficiency of amino acid metabolism in gastric cancer patients after chemotherapy can be reduced by the administration of Jianpi Yangzheng Xiaoshi Decoction, which corrects metabolic abnormalities such as glutamine and improves the quality of life of patients [124].

Compared with the control group, adding immune nutrition to the perioperative diet can improve the postoperative immune function of patients, reduce postoperative infections, and shorten hospital stays, which is safe and effective [125].Compared with standard enteral nutrition, immune-enhanced enteral nutrition (EN + Gln) can improve the defense mechanisms and regulate inflammatory responses in patients after elective major surgery for gastric cancer [126]. It can also improve the nutritional status and immune function of patients after total gastrectomy for advanced gastric cancer [127].

Oral supplementation with glutamine can prevent bladder wall damage caused by radiotherapy [128] (Fig. 6). At appropriate doses, oral supplementation with arginine and glutamine can improve serum protein levels in non-bedridden patients with head and neck cancer after surgery and reduce the incidence of oral mucositis in the radiotherapy subgroup [129, 130] (Fig. 6). For esophageal cancer patients, oral supplementation with glutamine can protect lymphocytes and reduce intestinal permeability during radiotherapy and chemotherapy [131] (Fig. 6). L-alanyl-L-glutamine dipeptide can be used as an adjuvant treatment for chemotherapy in colon cancer patients, improving their quality of life and overall survival [132] (Fig. 6). Supplementation of L-glutamine can reduce the size of intrahepatic cholangiocarcinoma (ICC) and cancer-related cachexia in Walker-256 cancer rats, supporting its beneficial therapeutic effect against cancer [133] (Fig. 6). And supplementation of L-glutamine has a protective effect on the enteric nerve innervation of cancer-related cachexia, possibly through mechanisms associated with reducing oxidative stress [134]. Receiving parenteral nutrition (Gln-PN) containing L-Gln may support the recovery of lymphocytes after allogeneic bone marrow transplantation (BMT) [135]. And also Immunomodulatory enteral nutrition can improve the cellular and humoral immune function of postoperative patients with colorectal cancer [136] (Fig. 6). However, there are also studies that suggest that supplementation with glutamine has no significant effect on the growth of solid cancers, cannot prevent the occurrence of enteritis during radiotherapy, and does not support routine supplementation of glutamine in children with solid cancers to enhance lymphocyte function [137, 138]. However, it may be beneficial for the immune response to disease recurrence, metastasis, treatment, or secondary infection. Even though multiple studies have indicated the beneficial effects of glutamine supplementation for cancer patients, the underlying mechanisms remain unclear and require further exploration. Perhaps serum levels of glutamine could be measured, and different appropriate values could be set for patients with different types of cancer undergoing different treatments, thus enhancing personalized therapy.

Fig. 6

The benefits of glutamine supplements in patients with cancer. Supplementation of glutamine has multiple benefits for cancer patients. It can prevent bladder wall damage caused by radiotherapy, possibly through the regulation of extracellular matrix density and collagen expression by glutamine. It can also reduce the incidence of oral mucositis in the subgroup of head and neck cancer patients undergoing radiotherapy. Additionally, it protects lymphocytes in esophageal cancer patients during radiation and chemotherapy and reduces intestinal permeability. It can decrease tumor size and cancer-related cachexia in ICC patients. Furthermore, it improves cellular and humoral immune function in colorectal cancer patients after surgery, while supplementation with L-prolyl-L-glutamine dipeptide can enhance the quality of life and overall survival in colon cancer chemotherapy patients

At present, numerous clinical trials on glutamine have been conducted (Table 1). A large proportion of clinical trials involve oral supplementation of glutamine as a supplement for cancer patients who have undergone surgery, chemotherapy, or radiotherapy. Some clinical studies have found that oral supplementation of glutamine can reduce acute radiation toxicity and weight loss, and can also reduce the demand for analgesic drugs in patients with chest and upper respiratory and digestive tract malignancies (NCT05054517) [93], and is helpful in improving vincristine-induced peripheral neuropathy in children with tumors (NCT00365768) [96]. However, some clinical trials have discovered that the severity of oral mucositis in patients with head and neck tumors after radiotherapy is not related to the use of glutamine (NCT03015077) [94], and oral glutamine has no significant improvement effect on acute radiation esophagitis in patients with advanced thoracic malignancies receiving esophageal radiotherapy (NCT01952847) [95]. Therefore, the methods of glutamine supplementation, the amount of supplementation, and the targeted patients population all deserve further research and exploration.

On the other hand, clinical trials also carried out to investigate the combination therapy of glutamine inhibitor CB-839 and other anticancer drugs. LBA54-ENTRATA is a randomized, double-blind, phase II study of CB-839 + everolimus vs placebo + everolimus in patients with advanced/metastatic renal cell carcinoma (mRCC) [91]. LBA54-ENTRATA reported the addition of CB-839 improved PFS over control group. LBA54-ENTRATA reveal that glutaminase inhibitor CB-839 is a new therapeutic approach in RCC [91].

And an interim analysis of a phase II Study of the glutaminase inhibitor CB-839 in combination with Azacitidine in advanced myelodysplastic syndrome (MDS) showed CB-839 at a dose of 600 mg BID orally continuously daily is safe and well tolerated in combination with azacitidine in patients with advanced MDS with an acceptable safety profile [92]. This study reported that encouraging response rates include complete molecular remission (mCR) / hematology improvement (HI) of 62.5% [92]. CANTATA is a randomized, international, double-blind study of CB-839 plus cabozantinib versus cabozantinib plus placebo in patients with metastatic renal cell carcinoma. The results remains unreported [92]. The clinical trial of glutamine antimetabolites DRP-104 is under way (NCT06027086). The clinical trials of other glutamine antimetabolites, glutaminase inhibitors, glutamine uptake inhibitors have not been reported. More clinical trials in the future is necessary for the clinical therapy in cancer patients.

Conclusions and future perspectives

The metabolism of glutamine exhibits distinct characteristics in normal tissue cells, cancer cells, and various immune cells. Glutamine is a non-essential amino acid in normal tissues, but some cancer cells develop a dependency on glutamine. In different immune cells, glutamine plays a crucial role and is indispensable for the functioning of immune cells. On one hand, the identification of differential glutamine metabolism in normal and cancer tissues holds potential for effective intervention in cancer glutamine metabolism. On the other hand, understanding the glutamine characteristics in immune cells is essential, as enhancing the capabilities of immune cells can specifically inhibit cancer development.

Currently, a variety of glutamine metabolism inhibitors have been developed, targeting either the inhibition of glutamine metabolism, key enzymes in the glutamine metabolism process, or critical glutamine transport proteins. And the combined application of glutamine metabolism inhibitors with immune checkpoint inhibitorss has demonstrated enhanced efficacy in inhibiting cancer development. In clinical practice, oral administration of glutamine in various cancer patients has been shown to enhance immunity, reduce adverse reactions in patients undergoing chemotherapy or radiotherapy, and improve treatment outcomes.

However, the mechanism of action of glutamine in the immune cells of cancer patients remains unclear, and further research is needed on the inhibitors of glutamine metabolism and their mechanisms of action, as well as the potential broader application of combined immune checkpoint inhibitors. Further research is also required to understand the mechanism by which oral glutamine enhances immunity in cancer patients and to determine whether glutamine should be administered to all patients undergoing chemotherapy or radiotherapy, as well as the potential use of glutamine metabolism inhibitors.

Glutaminase inhibitor CB-839 are reported to promote anticancer therapy effect combinate with other anticancer drugs in cancers. However, glutamine reducing toxic side effects in cancer patients. It remains unknown about whether glutaminase inhibitor will increase toxic side effects. Besides, glutamine is also necessary in immune cells. It remains unknown about whether glutaminase inhibitor will also inhibit immune cells. In addition, when glutamine is limiting via glutaminase inhibitor, asparagine can modulate glutamine synthesis by up-regulating glutamine synthetase, thereby participate in the regulation of the TCA cycle. Besides, no Phase III Study of glutaminase inhibitor was reported. More clinical trials of glutaminase inhibitors especially Phase III Study are required in future.

Considerably more work will need to be done to determine the role of glutamine between cancer cells, the cancer microenvironment, and different immune cells, combined with clinical practice, is critical for the further adoption of effective targeted therapies.