1.5.1 Glucose and Glutamine

Glucose and glutamine are the primary nutrients for cancer cells, T cells, and macrophages, which share similar metabolic profiles. As a result, this leads to fierce competition for the limited supply of glucose and glutamine in TME. The capacity to uptake these essential nutrients is vital for the survival and function of different cell subsets in the TME. One recent PET imaging study revealed that intratumor myeloid cells (including F4/80+ TAMs) possess the most significant capacity to uptake glucose within TME, followed by T cells and cancer cells [45]. Consistent with this observation, histological analysis showed that glucose transporter 1 (GLUT1) is highly expressed in TAMs localized in hypoxic regions of both transplanted and autochthonous murine breast tumor tissues [46]. In contrast, cancer cells exhibited the highest uptake of glutamine across a range of cancer models [45]. They used 2-NBDG, a fluorescent glucose analog, to measure glucose uptake in different cell populations. Additionally, the study assessed GLUT1 expression to quantify glucose transport activity. This distinct nutrient utilization profile was programmed in a cell-intrinsic manner through mTORC1 signaling and the overexpression of genes related to glutaminolysis, such as GLS1 and GLUT1 [47]. In addition, tumor cells upregulate glutamine transporters, such as solute carrier family 1 member 5 (SLC1A5) and glutaminase (GLS), to enhance glutamine uptake and utilization [48].

The conflict between nutrient utilization leads to metabolic stress on both tumor and immune cells. However, the metabolic flexibility of cancer allows them to survive under low glucose and glutamine conditions [49]. Furthermore, the production of lactate and CO2 from glycolysis and glutaminolysis results in the acidification of the tumor microenvironment, which favors the development of more aggressive and invasive tumor cells [50, 51]. Distinct from cancer cells, TAMs possess a superior capacity to take up glucose, allowing them to outcompete other cell types within the tumor, promoting their pro-tumor and immunosuppressive function [10, 52, 53]. Supporting these findings, a recent study using an orthotopic murine pancreatic ductal adenocarcinoma (PDAC) tumor model demonstrates that the GLUT1 depletion in TAMs via lysozyme M-cre (LysMcre) improved natural killer and CD8⁺ T cell activity and inhibited tumor progression. In contrast to tumor cells and TAMs, effector T cells are deprived of glucose and glutamine, which are crucial for their effector functions [54, 55] and survival [56-58]. These findings suggest that the metabolic interaction, particularly the competition for limited nutrients in the tumor microenvironment, can profoundly impact antitumor immunity.

1.5.2 Methionine

The catabolism of the essential amino acid methionine has been reported as a biomarker of tumors in various studies [59]. In cancer cells, methionine is primarily converted into S-adenosylmethionine (SAM) and polyamines. SAM serves as a critical methyl donor in the methylation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, and lipids. Aberrant methylation patterns, such as hypermethylation of tumor suppressor genes (like p53) and hypomethylation of oncogenes, are common phenomena in cancer and contribute to tumorigenesis [60]. Increased methionine catabolism in tumor cells resulted in methionine depletion in the extracellular space. T cells are reliant on amino acid transporters, including Slc7a5, for methionine transport [61]. Mechanistically, Slc7a5-null T cells lack effective engagement with antigen as well as have an impaired ability to engage in inflammatory profiles and cellular cycling profiles1. Slc7a5-null T cells are dramatically lower in methionine levels and subsequently have reductions within mTORC1 activation, c-MYC expression, as well as reduced glycolysis and engagement in central carbon metabolism [62]. In addition to this, deprivation of methionine can suppress T effector function by causing lower intracellular levels of methionine and SAM and resulting in loss of demethylation at lysine 79 of histone H3 (H3K79me2) [63]. The loss of H3K79me2 leads to low expression of STAT5 and impaired T-cell immunity, which, in turn, suppresses antitumor-immune responses [63]. Conversely, the conversion of methionine to polyamines is essential for cancer cell proliferation but also plays a broad role in modulating immunity [63]. The extracellular accumulation of polyamines alters the expression of T cell surface receptors, including T cell receptor (TCR) and co-stimulatory molecules like CD28, which directly impact the signaling pathways necessary for T cell proliferation and cytokine production [64]. Polyamines can also influence the expression of immune checkpoint molecules, such as programmed death-ligand 1 (PD-L1) on tumor cells. Increased PD-L1 expression inhibits T cell activity through PD1 engagement, contributing to immune evasion by the tumor [65]. Additionally, polyamines promote the polarization of macrophages toward the immunosuppressive M2 phenotype, which promotes tumor growth and inhibits immune responses [66]. Mechanistically, polyamine biosynthesis regulates the activity of the enzyme eIF5A through a posttranslational modification process referred to as hypusination. The hypusinated form of eIF5A facilitates the efficient expression of numerous mitochondrial proteins that enhance OXPHOS and alternative macrophage activation [67]. The polyamine compound spermidine can stimulate the expression of immunosuppressive factors like arginase and inhibit the pro-inflammatory activities of M1 macrophages by decreasing the production of cytokines and the expression of costimulatory molecules CD80 and CD86 [66]. In summary, methionine depletion and polyamine accumulation cooperatively create an immunosuppressive microenvironment in tumors.

1.5.3 Serine

Extracellular serine is essential for optimal T cell expansion and effector function. T cells require serine to fuel one-carbon metabolism to support their proliferation. T cells rely on serine to maintain effector functionality. Activated T cells increase the expression of amino acid transporters such as ACT2 to increase the extracellular uptake of serine [68, 69]. When deprived, T cells can generate serine de novo from glycine and other one-carbon metabolites to rescue deprivation. Notably, low levels of extracellular serine compromise de novo purine nucleotide synthesis and cellular division [69]. While T and tumor cells can produce serine, the de novo serine synthesis capacity is often suppressed in the TME. Consequently, the competition between tumor and T cells for limited extracellular serine can lead to the suppressed T cell proliferation. In contrast, TME upregulates the phosphoserine aminotransferase 1 (PSAT1) and promotes serine biosynthesis in TAMs by activating the protein kinase RNA-like ER kinase (PERK) signal pathway [70, 71]. This enhanced serine metabolism supports mitochondrial fitness and provides ketoglutarate, which enables jumonji domain-containing protein 3 demethylase-dependent epigenetic modification that amplifies the immunosuppressive function in intratumoral macrophages. These activated TAMs can lead to further mechanistic suppression of T cells by releasing reactive oxygen species and other intermediates, which promote lipid oxidation, further creating a harsh metabolic environment [72]. Collectively, these studies indicate the importance of serine metabolism in the interplay between tumor cells, T cells, and TAMs, thereby influencing the balance of antitumor immunity and tumor progression.

1.5.4 Tryptophan

Tryptophan is an essential amino acid reported as a tumor biomarker in various studies [73]. In tumor cells, tryptophan is converted to kynurenine by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO). High expression of IDO and TDO in various cancers facilitates tumor cells in evading immune surveillance [74, 75]. Upregulated tryptophan catabolism in tumor tissue resulted in the depletion of tryptophan in the extracellular space, which may cause dysfunction and apoptosis of effector T cells [76, 77]. The accumulation of kynurenine and its downstream metabolites, including 3-hydroxykynurenine, can induce T cell apoptosis and promote the expansion of regulatory T cells (Tregs), thereby further suppressing the antitumor immunity [73, 78]. Moreover, kynurenine and potentially other catabolic metabolites of tryptophan are identified as natural ligands of aryl hydrocarbon receptor (AhR), which plays a broad role in modulating immunity [78]. The AhR activity is elevated in TAMs and plays a vital role in macrophage polarization [79]. Deletion of AhR in TAMs or pharmacologic inhibition of AhR reduced PDAC tumor growth, improved efficacy of immune checkpoint blockade, and increased intratumoral frequencies of IFN-γ⁺ CD8⁺ T cells [39]. Consistent with this observation, high AhR expression is associated with rapid disease progression and mortality, as well as with an immunosuppressive TAM phenotype in patients with PDAC [39]. Thus, tryptophan depletion and its catabolite accumulation cooperatively foster an immunosuppressive microenvironment in tumors.

1.5.5 pH and ion

Lactate produced by tumor cells causes acidification of the TME [80]. This acidification helps tumor cells invade surrounding tissues by promoting the activity of proteases, such as matrix metalloproteinases (MMPs), which degrade the extracellular matrix [81]. The acidic environment can also lead to the selection of more aggressive cancer cell phenotypes and promote drug resistance [82]. The acidic microenvironment can impede the activity of immune cells such as T cells and natural killer (NK) cells. In addition, low pH conditions can impair the function of dendritic cells, which are crucial for antigen presentation and the activation of T cells. Proton pumps, such as V-ATPases, and proton exchangers, like sodium-hydrogen exchanger 1 (NHE1), are often upregulated in tumors and are potential therapeutic targets. Inhibitors of these proton pumps can disrupt the acidic microenvironment and inhibit tumor growth. Similarly, proton exchange is regulated by other cell membrane channels and transporters, such as Na⁺/H⁺ exchangers (NHE), Na⁺/H⁺ ATPase, and epithelial sodium channels (ENaC), which are often dysregulated in cancer cells. For example, increased intracellular sodium levels can promote cancer cell proliferation and migration. The Na⁺/H⁺ exchanger (NHE1) plays a role in regulating intracellular pH and cell volume. Necrotic tumor cells can release intracellular potassium [K⁺] ions into the extracellular fluid and cause profound suppression of T cell effector function. Potassium-mediated immune suppression is mediated through the serine/threonine phosphatase protein phosphatase 2-dependent suppression of Akt-mTOR signaling [83]. Accordingly, augmenting potassium efflux in tumor-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K⁺], improves effector functions in vitro and in vivo, and enhances tumor clearance and survival in melanoma-bearing mice [83]. In addition, the acidic pH of 6.1 can improve the expression of protumor molecules such as arginase 1 (ARG1) in macrophages [84]. The acidification of TME can be sensed by macrophages by a G protein-coupled receptor (GPCR), such as GPCR64 and GPCR64 [85, 86]. Activating this signaling pathway enhances the production of cyclic AMP (cAMP) to upregulate the expression of Inducible cAMP Early Repressor (ICER) and other genes associated with immunosuppressive macrophages, like Arg1, Clec10a, and Vegfa [86]. Neutralization of intratumoral acidity can delay tumor progression by inhibiting protumor differentiation of TAMs [87].

1.5.6 Taurine

Taurine, an amino acid derivative, is primarily synthesized in the liver through cysteine and methionine metabolic pathways. In tumors, taurine is mainly transported by solute carrier family 6 member 6 (SLC6A6), and its level correlates with aggressiveness and outcomes in multiple cancers [88]. Tumor cells outcompete CD8⁺ T cells for taurine by overexpressing SLC6A6 [29, 88], which consequently induces endoplasmic reticulum (ER) stress in CD8⁺ T cells through a PERK-ATF4-dependent mechanism [88]. Activating transcription factor 4 (ATF4) transactivates multiple immune inhibitory genes and induces T cell death, malfunction, and exhaustion [88]. In contrast, culturing CD8⁺ T cells with taurine in vitro enhances their secretion of cytotoxic cytokines, PLCγ1-mediated calcium production, and MAPK signaling, which determine the functional state of CD8⁺ T cells and the therapeutic efficacy of programmed cell death protein 1 (PD-1) blockade antibodies in tumors [89]. Consistent with the preclinical studies, high serum taurine levels in lung cancer patients correlate with high levels of cytotoxic cytokines (IFN-γ and TNF-α) and better outcomes in responding to PD-1 blockade therapy [89]. Furthermore, TAMs can produce taurine to support the growth of prostate cancer cells by signaling through the Liver X receptor alpha/stearoyl-coenzyme A desaturase 1 (LXR-α/SCD1) pathway to protect them from ferroptosis [90]. This tumor cell-macrophage interaction can be abolished by inhibiting the taurine transporter, TauT, thereby sensitizing the cancer cells to ferroptosis [90].

1.5.7 Nucleotide and Nucleoside Metabolism

Nucleotides, the building blocks of DNA and RNA, are intrinsically required for driving cancer growth [91]. In addition, their catabolic products (nucleosides) can directly serve as alternative carbon sources to support the proliferation of cancer and immune cells under nutrient-scarce conditions. The ribose moiety in inosine, uridine, and thymidine has been reported to provide carbon to sustain cell proliferation [91-94]. Within the TME, cancer cells can actively excrete nucleotides through exocytosis and transporters [95-97]. Furthermore, hypoxia and cytotoxic reagents can induce nucleotide release by damaging cancer cells [97-99]. Apart from tumor cells, immune cells, such as macrophages and T cells, can also release nucleotides. For TAMs, release pyrimidines such as deoxycytidine, modulating chemotherapy resistance [100]. Additionally, macrophages undergoing necrotic cell death passively release UDP activating P2Y6 receptors and inducing MCP-3 expression. Effector CD8⁺ T cells, under hypoxia or cytotoxic stress, can undergo activation-induced cell death, releasing ATP via P2×7 receptors, while necrotic T cells passively release nucleotide [101].

Both tumor and immune cells express ecto-nucleotidases, which are critical in controlling nucleotides and nucleoside turnover in the TME [102]. It is well-established that purine nucleosides can engage purinergic receptors in autocrine or paracrine manners, fostering an immune suppressive microenvironment [103]. Recent studies have also shown that pyrimidine nucleosides are critically involved in mediating communications between macrophages and tumor cells through engaging cytidine deaminase (CDA) and P2Y6 signaling [100]. Moreover, CDA expression could predict PDAC patient outcomes in response to immune check point (ICB) therapy [104]. Thus, nucleotide metabolism in TAMs significantly influences their function and promotes tumor progression.

1.5.8 Ammonia

Tumor cells, due to increased amino acid catabolism, produce a large amount of ammonia (NH₃). Ammonia is a weak base that generates ammonium ions (NH4⁺) when combined with hydrogen ions (H⁺). This allows ammonia to buffer acidic changes in the microenvironment and regulate pH, helping tumor cells adapt to harsh conditions and improve their survival and proliferation. Recent studies have shown that imbalances in intracellular ammonia in the TME can suppress TCR signaling and inhibit T cell activation, thereby reducing T cell proliferation and cytokine secretion, such as IL-2 and IFN-γ [105, 106]. Interestingly, memory T cells have less ammonium present compared with effector CD8⁺ T cells and are more resistant to NH4Cl-induced cell death [107]. Further investigation into memory CD8⁺ T cells revealed enhanced utilization of Carbamoyl phosphate synthetase 1 (CPS1) to form arginine in the cytosol [107], which can translocate to the mitochondria, catalyzed by arginase 2 and NO synthase, and produce urea, ornithine, NO, or citrulline. CPS1 ablation compromised the memory cell differentiation, implying a role in the balance of ammonia and utilization of the urea cycle in altering CD8⁺ T responses [107]. Other pathways, including the malate shuttle enzyme glutamic-oxaloacetic transaminase 1 (GOT1), can also scavenge ammonia by converting 2-KG to glutamate [108]. Deletion of GOT1 compromised effector function, such as IFN-γ and TNF cytokine production, in CD8⁺ T cells, and accelerated their dysfunctional process [108]. Therefore, targeting and balancing ammonia concentration could dictate the short-term and sustained response of effector T cells in the TME. In metastatic mouse models of colon cancer, tumors impair CD8⁺ T cell functionality through ammonia production and accumulation in the TME [105]. Therapeutically, an imbalance of ammonia can hinder the efficacy of PD-L1 therapy and decrease tumor-associated ammonia-enhanced immune checkpoint efficacy and long-term durability of CD8⁺ memory responses.

1.5.9 Lactate

Tumor cells actively engage in aerobic or anaerobic glycolysis to convert glucose-derived carbon into lactate. Recent studies have demonstrated that both tumor and immune cells can utilize and catabolize lactate [109-112]. For instance, isotope tracing of carbon-13 (13C) labeled lactate in non-small-cell lung cancer (NSCLC) patients revealed extensive 13C labeling of TCA cycle intermediate metabolites within surgically removed tumor tissues [113]. This suggests lactate is an in vivo fuel for the TCA cycle and OXPHOS in cancer cells. Outside of the cancer cell-intrinsic fates of lactate, lactate can also be excreted into the TME as an immunosuppressive acidic factor. Lactic acid is a weak acid that dissociates in the TME, reducing the pH and possibly suppressing immune functionality simply due to pH changes [110, 111, 114].

Studies have shown that acidification of the medium by physiologically relevant lactate levels partially decreased IFN-γ and IL-2 production in T cells [110, 111]. Buffering the culture media back to physiological pH (7.4) restores cytokine production in human CTLs in vitro [110, 115, 116]. In addition to the pH-dependent changes in functionality, tumor-derived lactate can significantly disrupt T-cell metabolism [117]. Tumor-derived lactate can induce a pyruvate utilization switch in CD8⁺ T cells, enhancing pyruvate carboxylase (PC) utilization instead of pyruvate dehydrogenase (PDH). Eventually, this results in a negative feedback loop that impairs the TCA cycle and reduces substantial levels of intracellular succinate within T cells [115]. Lack of succinate production failed to induce specific G protein-coupled receptor succinate receptor 1 (SUCNR1) signaling, thus compromising T cell responses [115]. SUNCR1 activation is essential for CD8⁺ killing capacity as well as IFN-γ production. Accordingly, inhibiting PDH synergized with T cell responses in a SUNCR1-dependent manner in a high lactate setting since SUNCR1 KO T cells failed to mount antitumor responses when PDH was inhibited [115, 117].

In the TME, both CD8⁺ T cells and Tregs utilize monocarboxylate transporters (MCTs) to regulate lactate metabolism but in distinct ways. MCT1, a bidirectional lactate transporter, is primarily involved in lactate uptake in both cell types, whereas MCT4 mainly facilitates lactate export. Effector CD8⁺ T cells predominantly express MCT1, particularly under glucose-deprived conditions, enabling them to absorb extracellular lactate to sustain metabolic activity [118]. However, excessive lactate uptake via MCT1 can impair CD8⁺ T cell function, leading to mitochondrial dysfunction, metabolic reprogramming, and reduced IFN-γ and IL-2 production [110]. Notably, PD-1⁺CD8⁺ T cells exhibit higher MCT1 expression, making them more susceptible to lactate-induced metabolic suppression [119]. While CD8⁺ T cells can upregulate MCT4 upon activation or hypoxia to expel intracellular lactate, MCT4 plays a relatively minor role compared with MCT1-mediated lactate uptake. In addition, new studies have found that exhausted T cells depend on lactic acid uptake through monocarboxylate transporter 11 (MCT11) to reinforce an exhausted phenotype [120]. Genetic ablation and pharmacological blockade of MCT11 or Slc16a11 lead to dramatic reductions in an exhausted population and improvements in ICB responsiveness in preclinical models [120]. Regarding exhaustion, lactic acid uptake through MCT11 leads to reduced stem-like signatures of T cells and enhanced genes associated with terminal exhaustion, including Havcr2 (TIM3), Eomes, and Id2, and disrupted mitochondrial biogenesis [120].

In contrast, Tregs exhibit a metabolic profile that favors lactate utilization rather than export. Intratumoral Tregs show high MCT1 expression, allowing efficient lactate uptake, which supports Foxp3-mediated metabolic reprogramming and enhances their suppressive function [112]. MCT1-deficient Tregs exhibit impaired lactate uptake, resulting in reduced suppressive function and enhanced antitumor immunity. Unlike CD8⁺ T cells, Tregs express relatively low levels of MCT4, suggesting that lactate import rather than export is crucial for their function in the TME. Furthermore, MCT1-mediated lactate uptake has been linked to PD-1 upregulation in Tregs via NFAT1 nuclear translocation, reinforcing their immunosuppressive activity. Notably, intratumoral Tregs exhibit higher PD-1 expression in highly glycolytic tumors, such as MYC-amplified and liver tumors, and PD-1 blockade paradoxically enhances Treg function, contributing to immune checkpoint therapy resistance [119]. Additionally, Wagner et al. demonstrate that lactate can induce PD-1 expression on innate lymphoid cells (ILCs), further contributing to immunosuppression in the TME [121]. Beyond T cells, lactate also plays a role in modulating other immune cell populations in the TME. This suggests that lactate-driven PD-1 upregulation is not exclusive to CD8⁺ T cells and Tregs but extends to other immune subsets, highlighting lactate as a central regulator of immune suppression in the tumor context. Collectively, these findings underscore the critical role of MCT1 in Treg-mediated immune suppression and suggest that targeting MCT1 may represent a promising strategy to selectively disrupt Treg function and enhance antitumor immunity in the TME.

In contrast to the above findings, activating CD8⁺ T cells with high amounts of lactate is also reported to enhance immunotherapy efficacy. CD8⁺ T cells primed with high amounts of lactate before infusion displayed several markers associated with a stem-like memory fate, including higher amounts of TCF1 and enhanced tumor control [122]. Lactate supplementation was also found to improve accessibility to TCF1 within CD8⁺ T cells and the efficacy of immune checkpoint therapy [122]. Taken together, the effects of intratumoral lactate are substantial, but the context dictates the impact of CD8⁺ T cell functionality.

Elevated lactate levels in the TME significantly enhance the ability of macrophages to promote tumor growth and suppress antitumor immunity. Specifically, lactate induces the upregulation of hypoxia-inducible factor 1α (HIF1α), which leads to increased expression of vascular endothelial growth factor (VEGF) and arginase 1 (Arg1) in TAMs [123]. Furthermore, lactate also modulates the interplay between metabolism and epigenetics, favoring an M2-like macrophage polarization within the tumor [124]. Isotope tracing studies have demonstrated that macrophages can metabolize extracellular lactate to fuel the TCA cycle and produce citrate [124]. This lactate-derived citrate is then cleaved by ATP citrate lyase, generating acetyl-coenzyme A, which serves as a critical donor for histone acetyltransferases, thereby increasing histone acetylation. Functional assays suggest that this ACLY-dependent histone acetylation is enriched at the promoter regions of M2-specific immunosuppressive genes, ultimately leading to an attenuated T-cell response and accelerated tumor progression.