Immunometabolic Maladaptations to the Tumor Microenvironment

Abstract

Tumors consist of cancer cells and a wide range of tissue resident and infiltrating cell types. Tumor metabolism, however, has largely been studied on whole tumors or cancer cells and the metabolism of infiltrating immune cells remains poorly understood. It is now clear from a range of analyses and metabolite rescue studies that metabolic adaptations to the tumor microenvironment (TME) directly impede T-cell and macrophage effector functions. The drivers of metabolic adaptation to the TME and metabolic immune suppression include depletion of essential nutrients, accumulation of waste products or immune suppression metabolites, and metabolic signaling through altered posttranslational modifications. Each infiltrating immune cell subset differs, however, with specific metabolic requirements and adaptations that can be maladaptive for antitumor immunity. Here, we review T-cell and macrophage adaptation and metabolic immune suppression in solid tumors. Ultimately, understanding and addressing these challenges will improve cancer immunotherapy and adoptive chimeric antigen receptor T-cell therapies.

Hanahan and Weinberg first described six hallmarks of cancer in 2000. These have since been expanded to 14, and now include hallmarks for an ability to evade the immune system and deregulation of metabolism (Hanahan 2022). It has become increasingly appreciated that these two new cancer hallmarks are closely linked. While the immune system is designed to recognize foreign or new antigens and damaged tissues, transformation-induced changes to cancer cell metabolism can directly impair this process to contribute to tumor immune evasion. In addition to the disruption of normal tissue structures that ordinarily facilitate immune surveillance for new or mutated antigens in growing tumors, the tumor microenvironment (TME) reflects the metabolic impact of cancer cells. Given the need of activated immune cells for increased nutrient uptake and metabolic reprogramming to support proliferation and effector functions, the TME can offer a hostile environment that prevents metabolic events essential for immune-mediated tumor killing (Bader et al. 2020). How T cells and macrophages respond to metabolic challenges in the TME and confront metabolic immune suppression can thus place severe limits to antitumor immunity.

While metabolic adaptations of T cells and macrophages to local conditions in the TME may have some benefits to preserve cell viability and avoid processes including activation or restimulation-induced cell death, these adaptations more generally suppress inflammatory immune function and are maladaptive for immune efforts to eliminate cancers (ElTanbouly and Noelle 2021). Both T cells and antigen presenting cells are impaired (McLane et al. 2019; Yang et al. 2023). Given the association of mutational burden with immunotherapy response (Jardim et al. 2021), understanding the nature of metabolic stresses and adaptations of immune cell types in the TME is now critical to improve immune therapies and fully harness the potential of antitumor immunity. These stresses include chronic antigen exposure in large solid tumors that would induce tolerance in normal tissues, tumor-induced changes in tissue architecture, tumor genetics and neoantigen availability, and poor vasculature that cause key nutrients to be depleted and waste products to accumulate. These changes affect signaling, metabolism, and ultimately the antigen presentation and inflammatory functions of tumor-infiltrating immune cells (Fig. 1). Here we review how key immune cell populations adapt and are shaped by stress in the TME in ways that are maladaptive for antitumor immunity.

Figure 1.

Metabolic stresses in the tumor microenvironment (TME). The TME of solid tumors is characterized by nutrient competition, accumulation of metabolic waste products and immunosuppressive molecules, metabolic feedback by posttranslational modifications (PTMs), and hypoxic regions that contribute to metabolic reprogramming of infiltrating immune cells to both promote tumor progression and suppress antitumor immunity. (ARG1/2) Arginase 1/arginase 2, (IDO) indoleamine 2,3-dioxygenase, (MDSC) myeloid-derived suppressor cell, (Mito) mitochondrial, (mtROS) mitochondrial reactive oxygen species, (PD-1/PD-L1) programmed death 1/programmed death-ligand 1, (PGE2) prostaglandin E2, (TAM) tumor-associated macrophage, (TCR) T-cell receptor, (TIL) tumor-infiltrating lymphocyte, (Treg) regulatory T cell.

METABOLIC STRESSES AND MODIFIERS IN THE TME

Nutrient Availability and Competition

The TME presents a complex milieu of many different metabolically active cell types that may compete for limited resources as tumors grow. One hundred years ago, Warburg (1956) proposed cancer cells consume glucose at high rates due to mitochondrial defects. This glycolytic phenotype has provided the rationale for ¹⁸F-2-deoxyglucose positron emission tomography (PET) for tumor imaging for diagnosis and monitoring. However, Warburg's cancer cell metabolism studies did not consider tumor cell heterogeneity in tumors and PET imaging offers only tissue-level resolution. The contributions of individual cells and various heterogeneous cell types to tumor metabolism have remained largely unclear within the overall TME. Like cancer cells, stimulated immune cells rapidly up-regulate glucose uptake and glycolysis. If vascular exchange in the TME is insufficient to efficiently renew nutrients and remove waste, this may result in an imbalance of glucose consumption and replenishment.

In principle, limited glucose could result in competition between cancer and immune cells for this critical nutrient (Pavlova and Thompson 2016). Indeed, targeted glucose restriction or deletion of the glucose transporter, Glut1, reduces mTORC1 activity and glycolytic capacity in T cells, which blunts interferon γ (IFN-γ) production and effector function (Macintyre et al. 2014; Chang et al. 2015). Decreased glycolysis also reduces availability of the glycolytic metabolite phosphoenolpyruvate (PEP), an important metabolite for sustaining T-cell receptor (TCR)-mediated activation of calcium, nuclear factor of activated T-cell (NFAT) signaling, and ultimately effector function (Ho et al. 2015). In contrast to the Warburg model that presumes cancer cells account for altered metabolism in the tumor as a whole, however, fractionation of cells following infusion of the PET tracer ¹⁸F-2-deoxyglucose revealed that tumor-associated macrophages (TAMs), rather than cancer cells, are the predominant consumers of glucose in the TME (Reinfeld et al. 2021). These findings are consistent with proteomics studies showing high expression of glycolytic enzymes in TAMs) (Liu et al. 2017). Importantly, the ability of immune cells to take up glucose was not limited by glucose availability (Reinfeld et al. 2021). Blockade of glutamine uptake within tumors led to increased glucose uptake in all tumor cell types. These data do not preclude locally limiting glucose levels, such as in hypoxic or necrotic tumor regions, but do demonstrate glucose uptake within tumors is not widely capped by availability to glucose itself. Glucose uptake is instead controlled by cell-intrinsic metabolic programing within the TME.

Both cancer cells and immune cells rely on amino acid uptake and metabolism. Cancer cells up-regulate multiple transporters to uptake and utilize amino acids, creating potential competition between immune cells for amino acids within the TME (Lieu et al. 2020). In addition to essential amino acids, which must be obtained through diet and transport into cells, amino acids such as glutamine can be conditionally essential and require uptake if synthesis fails to meet demand. Indeed, observations of increased cellular glucose uptake following inhibition of glutamine uptake suggests glutamine uptake in tumors is limiting in vivo (Reinfeld et al. 2021). Cancer cells avidly consume glutamine to support their bioenergetic and biosynthetic demand, thus potentially depleting available glutamine from the TME (Zhang et al. 2017; Reinfeld et al. 2021). Selectively blocking glutamine metabolism in cancer cells by targeting glutamine uptake or metabolism via glutaminase improved antitumor T-cell function, supporting a model that glutamine accessibility is limiting in the TME (Edwards et al. 2021). Glutamine is a key nutrient for activated effector T cells (Teffs) and insufficient access to glutamine impairs Teff metabolism and function while promoting the development of immune-suppressive regulatory T cells (Tregs) (Klysz et al. 2015). Glutamine depletion can also induce dysfunction in natural killer (NK) cells, as glutamine is essential to maintain cMyc expression and cytotoxic function (Loftus et al. 2018). Additionally, conventional dendritic cells (cDCs) require glutamine for effective antigen cross-presentation to CD8 T cells; however, cancer cells outcompete cDCs for glutamine by overexpression of the transporter SLC38A2 to impair T-cell priming (Guo et al. 2023). Increased tryptophan uptake and catabolism by cancer cells, certain macrophages, and DCs “starves” T cells of tryptophan, thereby inhibiting antitumor Teff while stimulating suppressive Tregs (Lemos et al. 2019). Cancer cells may also outcompete infiltrating T cells for methionine, resulting in methionine deprivation in T cells and subsequent reductions of intracellular methyl donors and STAT5 signaling, which is important in CD8⁺ T effector function and regulation of CD4⁺ T-cell metabolism (Tripathi et al. 2010; Bian et al. 2020; Villarino et al. 2022). Arginine is yet another amino acid important in T-cell activation, effector function, and memory development often depleted in tumors due to high rates of arginine catabolism in tumor cells and myeloid-derived suppressor cells (MDSCs) (Geiger et al. 2016; Lemos et al. 2019).

Hypoxia is a characteristic feature of the solid tumor TME and contributes to an immunosuppressive environment when oxygen is not replenished through the vasculature at the rate it is consumed. Oxygen-dependent prolyl-hydroxylases and the E3 ubiquitin ligase VHL degrade the transcription factor, hypoxia inducible factor 1α (HIF1α) if oxygen is abundant. When limiting HIF1α is stabilized, this leads to transcription of a range of glycolytic genes and immune-suppressive ligands such as programmed death-ligand 1 (PD-L1) (Noman et al. 2014). The combination of hypoxia paired with chronic stimulation of tumor-infiltrating lymphocytes (TILs) inhibits proliferation and drives T-cell exhaustion through repressed mitochondrial biogenesis, function, and heightened reactive oxygen species (ROS) accumulation (Scharping et al. 2016, 2021; Vardhana et al. 2020). Hypoxic conditions can also disturb mitochondrial dynamics and promote the exhausted TIL phenotype through the repression of Myc-regulated, mitofusin 1 (Mfn1)-mediated mitochondrial fusion (Liu et al. 2020). Hypoxia and chronic TCR signaling also coordinate with programmed death 1 (PD-1) signaling via ligation by PD-L1 on tumor cells or infiltrating myeloid cells, elevating T-cell mitochondrial ROS and inhibiting mitophagy, resulting in the accumulation of depolarized, dysfunctional mitochondria that impair TIL effector function (Yu et al. 2020).

Suppressive Metabolites and Molecules

Cancer cells produce and export metabolites and waste products that directly inhibit immunity. Lactate is produced as a byproduct of aerobic glycolysis, leading to accumulation of lactic acid and creation of an acidic TME. Subsequent lactate and proton uptake by T and NK cells, decrease their intracellular pH, interfering with NFAT translocation to the nucleus and impairing IFN-γ production (Brand et al. 2016). Lactate also suppresses glycolysis in CD4 Teff by inhibiting NAD⁺ recycling necessary for GAPDH activity and inhibits Teff function by liberating GAPDH from its enzymatic role and facilitating translational repression of IFN-γ (Chang et al. 2013; Haas et al. 2015). Conversely, lactate accumulation promotes Treg function and can drive an immunosuppressive phenotype of DCs within the tumor (Colegio et al. 2014; Angelin et al. 2017; Watson et al. 2021; Møller et al. 2022). Activation of the lactate receptor GPR81 on DCs can disrupt antigen presentation by suppressing MHC class II expression (Brown et al. 2020). Lactate induces polarization of TAMs toward a protumor phenotype and this polarization increases arginine metabolism, to feed back into the cycle of nutrient competition and highlight the metabolic interplay of cells in the TME (Colegio et al. 2014; Zhang et al. 2019).

Lipids are abundant in the TME of many tumors. In addition to promoting tumor growth and metastasis, lipid uptake and storage modulates the metabolism and function of infiltrating immune cells to create an immunosuppressive TME (Prendeville and Lynch 2022). Elevated lipid uptake in Tregs and M2-like TAMs fuels fatty acid oxidation (FAO) to support their immune suppressive functions (Prendeville and Lynch 2022). Conversely, intracellular accumulation of lipid droplets blunts antigen presentation by DCs by preventing the translocation of peptide:MHC complexes to the cell surface (Veglia et al. 2017). Enhanced FAO in DCs has also been shown to promote tolerance in melanoma (Zhao et al. 2018; Giovanelli et al. 2019). In CD8 TILs, the accumulation of very long chain fatty acids or cholesterol induces exhaustion, inhibits cytotoxicity, and production of IFN-γ and TNF-α (Ma et al. 2019; Xu et al. 2021). Uptake of oxidized lipids in the TME via the scavenger receptor and lipid transporter CD36 can impair CD8 T-cell effector function and blocking CD36 can increase antitumor immunity (Xu et al. 2021). Lipid accumulation also interferes with mTOR-mediated glycolysis in NK cells, inhibiting production of IFN-γ and direction of lytic granules to the target cell synapse (Michelet et al. 2018).

In addition to waste products, suppressive cells also actively produce immune inhibitory metabolites. Cancer cells and infiltrating myeloid cells often up-regulate tryptophan catabolism through increased indoleamine 2,3 dioxygenase (IDO). This depletes tryptophan and produces the metabolite kynurenine (Munn and Mellor 2013; Lemos et al. 2019). Kynurenine is exported into the TME where it can activate aryl hydrocarbon receptors (AHRs) on T cells and subsequently inhibit their effector function (Munn and Mellor 2013; Liu et al. 2018). Kynurenine accumulation also promotes the generation of Tregs and Treg-dependent recruitment and activation of MDSCs (Holmgaard et al. 2015; Campesato et al. 2020). MDSCs produce the glycolytic metabolite methylglyoxal, an α-dicarbonyl that can paralyze TILs by cell–cell transfer and subsequent depletion of intracellular L-arginine (Baumann et al. 2020). Methylglyoxal also inhibits TIL metabolism by glycation-induced dysfunction of L-arginine containing proteins imperative for mitochondrial respiration (Baumann et al. 2020). MDSCs also secrete the TCA cycle metabolite itaconate, which is taken up by CD8 TILs and inhibits their activation, proliferation, and cytotoxic function, at least in part, by disrupting aspartate and serine/glycine biosynthesis (Zhao et al. 2022). The lipid mediator prostaglandin E₂ (PGE₂) is also abundant in the TME. PGE₂ receptor EP1-4 signaling promotes MDSC and TAM recruitment and activation while repressing NK and TIL function, resulting in an immunosuppressive environment (Jin et al. 2023).

An interesting new feature of metabolic suppression may be through the regulation of electron transport and subsequent epigenetic changes. The methylation-controlled J protein (MCJ) can act as an endogenous inhibitor of mitochondrial electron transport complex I and MCJ-deficient T cells had increased respiration, and although proliferation was unchanged secretion of inflammatory cytokines was elevated (Champagne et al. 2016). Ultimately, the greater respiratory capacity through complex I enhanced T-cell survival and memory responses against influenza. This may occur in part because MCJ inhibits complex I, leading to increased glycolysis and cell death through caspase 3 (Secinaro et al. 2019). This pathway has also been shown to affect tumor cells and antigen presentation, with the balance of complex I– and complex II–mediated respiration as key (Mangalhara et al. 2023). In this case, inhibition of complex II–dependent respiration led to elevated succinate and complex I–mediated respiration together with increased expression of MHC-I and antigen presentation. Targeting MCJ in tumor cells had a similar effect and increased both succinate and complex I respiration, leading to greater MHC-I expression and antitumor immunity. Thus, changes that regulate mitochondrial electron transport complex I and II may ultimately shape tumor immunogenicity and the function of antitumor T cells.

Metabolic Signaling and Posttranslational Modifications in the TME

Posttranslational modifications (PTMs) widely influence homeostasis, gene expression, and cell proliferation (Li et al. 2018; Jennings et al. 2022). Importantly, PTMs rely on metabolic intermediates as substrates to modify and influence protein activity, structure, and location (Ducker and Rabinowitz 2017; Li et al. 2018). The availability of these nutrients, therefore, can limit or promote specific PMTs to regulate cell signaling and gene expression. Site specificity and abundance of PTMs is dictated enzymatically through dedicated writer, reader, and eraser enzymes that respond to substrate availability (Jennings et al. 2022). Writer enzymes catalyze the addition of the PTM on amino acid residues, reader enzymes recognize PTMs for downstream signaling, and eraser enzymes catalyze the removal of PTMs. However, not all PTMs require an enzymatic writer and there is growing evidence that eraser enzymes can regulate abundance of nonenzymatic PTMs (Jin et al. 2016; Zheng et al. 2019; Gaffney et al. 2020; Jennings et al. 2021). While listing all PTMs associated with immune cells is beyond the scope here, many reviews have covered PTMs in tumors and the TME (Hitosugi and Chen 2014; Chang and Ding 2018; Chandler et al. 2019; Jennings et al. 2022; Pan and Chen 2022). Less is known, however, on the impact of TME and altered nutrients on PTMs of tumor-infiltrating immune cells (Li et al. 2021; Pan and Chen 2022).

Many immune cells in the TME experience elevated glycolytic flux that can provide glycolytic intermediates for anabolic metabolism and as precursors and substrates for PTMs. Glucose supports a plethora of glycation modifications formed from the nonenzymatic covalent binding of reducing sugars to Arg, Lys, and Cys amino acid residues (Jennings et al. 2022). Fructose-6-phosphate fuels the hexosamine biosynthetic pathway, ultimately leading to the highly studied enzymatic glycosylation PTMs (Palaniappan and Bertozzi 2016; Smith and Bertozzi 2021). 1,3-Bisphosphoglycerate can nonenzymatically modify Lys residues by phosphoglycerylation to regulate glycolytic flux (Moellering and Cravatt 2013). Glycolytic intermediates also support epigenetic modifications on histones. Serine generated by 3-phosphoglycerate through de novo serine metabolism can go through one-carbon metabolism to produce S-adenosylmethionine (SAM), ultimately generating enzymatically driven Lys and Arg methylation of histones (Mentch et al. 2015). While methylation is most known for transcriptional regulation through epigenetic modifications on histones, methylation is pervasive throughout the cell-regulating cell signaling, cell cycle, and nutrient sensing (Di Blasi et al. 2021). Conversely, TCA intermediates α-ketoglutarate and succinate regulate demethylation reactions.

The TCA cycle is an important supplier of substrates for PTMs. Acetylation is a critical PTM for both epigenetic and nonhistone modifications that is based on cytosolic acetyl-CoA. There are multiple pathways to generate cytosolic acetyl-CoA, including by ATP citrate lyase from mitochondrially derived citrate, by ACSS2 after uptake of acetate, and by nuclear pyruvate dehydrogenase from pyruvate (Sutendra et al. 2014; Zhao et al. 2016; Ali et al. 2018; Guertin and Wellen 2023). In addition to enzymatically generated histone acetylation, acetyl-CoA modifies nonhistone proteins throughout the cell both enzymatically and nonenzymatically on Lys and Cys residues (James et al. 2017). Further, many of the acyl-CoAs generated through the TCA cycle and FAO can enzymatically or nonenzymatically modify proteins throughout the cell, regulating enzyme activity and localization in addition to epigenetic transcriptional regulation (Jennings et al. 2022). Under inflammatory conditions, some cells produce itaconate from cis-aconitate after the induction of IRG1. Itaconate is an α,β-unsaturated electrophile that can nonenzymatically modify proteins (i.e., LDH, ALDOA, KEAP1) through a Michael addition and remodels cells, specifically macrophages, toward an anti-inflammatory phenotype (Mills et al. 2018; Qin et al. 2019, 2020).

Cells within the TME can also experience elevated oxidative stress that leads to protein and lipid modifications (Aboelella et al. 2021). Electron leakage from the mitochondrial electron transport chain is the biggest contributor of oxidative stress and renal carcinoma TILs have increased mitochondrial ROS due in part to reduced mitochondrial superoxide dismutase (Siska et al. 2017). Both ROS and reactive nitrogen species (RNS) can nonenzymatically modify protein Cys residues (Jennings et al. 2022). One such RNS, nitric oxide (NO), can be found within tumor-associated immune cells as a byproduct of elevated arginine metabolism. NO can accumulate and nonenzymatically oxidize Cys residues, generating nitrosylation PTMs (Smith and Marletta 2012). Although NO is a signaling molecule, it can be further metabolized into reactive peroxynitrite and S-nitrosoglutathione in addition to protein S-nitrosylation (Smith and Marletta 2012). Oxidative stress can also lead to secondary metabolites capable of nonenzymatically modifying Cys residues. For example, ROS-generated lipid peroxidation produces α,β-unsaturated alkenals (e.g., 4-hydroxy-2-nonenal, 4-oxo2-nonenal) prone to modifying Cys, His, and Lys residues, resulting in carbonylation PTMs (Yin et al. 2011; Galligan et al. 2014; Chen et al. 2017). Carbonylation modifications can damage proteins and result in protein degradation; additionally, the presence of 4-hydroxy-2-nonenal (HNE) in DCs has been associated with immune suppression in ovarian cancer models (Yin et al. 2011; Galligan et al. 2014; Chen et al. 2017). Last, lipid peroxidation can lead to iron-dependent cell death via ferroptosis (Jiang et al. 2021). Each of these processes are counterbalanced by ROS-modification and KEAP1 to release and stabilize the master redox regulatory transcription factor NRF2, which induces redox neutralization and protective mechanisms (Bellezza et al. 2018).

METABOLISM OF TUMOR-INFILTRATING IMMUNE CELL POPULATIONS

Tumor-Associated Macrophages

Macrophages exist in a wide range of phenotypes and use a metabolic continuum from highly glycolytic to highly lipid oxidative that play roles to promote inflammation or to stimulate tissue repair and inflammatory resolution, respectively (Fig. 2). At the ends of these spectra are proinflammatory, classically activated, M1-like macrophages or anti-inflammatory, alternatively activated, M2-like macrophages. Classically activated macrophages are stimulated by IFN-γ, lipopolysaccharide (LPS) from infection or microbiome, or a combination of both to stimulate IFN-γ receptor (IFNGR) and Toll-like receptors (TLRs) and activate STAT1, NF-κB, and PI3K/AKT/mTORC1 signaling pathways. These signals induce the proinflammatory M1-associated enzyme, inducible nitric oxide synthase (iNOS) (Liu et al. 2017; Van den Bossche et al. 2017; Kolliniati et al. 2022). In contrast, alternatively activated macrophages are stimulated by interleukin 4, 10, and 13 (IL-4, IL-10, and IL-13), glucocorticoids, and macrophage colony-stimulating factor (M-CSF) (Van den Bossche et al. 2017). The binding of both IL-4 to the IL-4 receptor a (IL-4Ra) and IL-13 to the IL-13 receptor subunit a1 (IL-13Ra1) triggers a dimerization of these two receptors to form a type II cytokine receptor complex (McCormick and Heller 2015). This dimerization leads to downstream anti-inflammatory signaling pathways, including the induction of the M2 anti-inflammatory enzyme arginase-1 (ARG1) (Van den Bossche et al. 2017; Lundahl et al. 2022). Metabolites and danger- or damage-associated molecular patterns (DAMPs) in the TME thus lead to metabolically heterogeneous TAMs (Kolliniati et al. 2022). While TAMs are M2-like for many metabolic pathways, mTORC1-regulated glucose uptake and glycolytic flux are elevated compared to in vitro studied M2 macrophages (Reinfeld et al. 2021; Kolliniati et al. 2022). Despite high metabolic activity, these M2-like TAMs are often regarded as protumorigenic due to their immunosuppressive phenotypes.

Figure 2.

Metabolic reprogramming of macrophages. Naive macrophages undergo context-dependent activation that dictates metabolic programming. M1 macrophages primarily rely on glycolysis for bioenergetic needs and induction of proinflammatory genes, while M2 macrophages use oxidative metabolism and are anti-inflammatory. TAMs are considered M2-like with high mitochondrial metabolism, but also exhibit elevated glycolytic flux. (Acetyl-CoA) Acetyl-coenzyme A, (ADP) adenosine diphosphate, (αKG) α-ketoglutarate, (ARG1) arginase 1, (ATP) adenosine triphosphate, (DAMPs) damage-associated molecular patterns, (HIF1α) hypoxia-inducible factor 1α, (iNOS) inducible nitric oxide synthase, (IFN-γ) interferon γ, (IFNGR) interferon γ receptor, (IL-4) interleukin 4, (IL-10) interleukin 10, (IL-13) interleukin 13, (LPS) lipopolysaccharide, (M-CSF) macrophage colony-stimulating factor, (mTORC1) mammalian target of rapamycin complex 1, (MYC) MYC proto-oncogene, (OXPHOS) oxidative phosphorylation, (succinyl-CoA) succinyl-coenzyme A, (TCA cycle) tricarboxylic acid cycle, (TLRs) Toll-like receptors, (TME) tumor microenvironment.

HIF1α stabilization induces the expression of Glut1 and glycolytic enzymes that promote elevated glycolytic flux in TAMs. In conjunction with tumor-associated DAMPs, inflammatory cues activate mTORC1 signaling, and increased expression of HIF1α in hypoxic regions of tumors stabilize HIF1α and promote glycolytic flux (Huang et al. 2016; Kolliniati et al. 2022). Exogenous lactate secreted from tumor cells and taken up by TAMs can stabilize HIF1α and induce VEGF expression to drive angiogenesis (Zhang and Li 2020; Kolliniati et al. 2022). In addition to lactate abundance, elevated succinate and low pH in the TME can stabilize HIF1α (Liu et al. 2017; Mehla and Singh 2019). Many TCA intermediates can signal to modify TAM phenotypes and hypoxic responses. For example, itaconate, generated from TCA-derived aconitate after LPS or IFN-γ activation and induction of IRG1, regulates the metabolic switch from proinflammatory to anti-inflammatory phenotypes. Itaconate drives PTMs to reprogram macrophages for OXPHOS and enhanced lactate production through aerobic glycolysis (Lampropoulou et al. 2016; Kolliniati et al. 2022). Inhibition of SDH by itaconate results in succinate accumulation (Lampropoulou et al. 2016; Seim et al. 2019), leading to increased mitochondrial ROS and inhibition of prolyl hydrolases that promote HIF1α degradation and demethylation reactions (Wu et al. 2020). In contrast, α-ketoglutarate provides a substrate for prolyl hydrolases that promote HIF1α degradation, with a high α-ketoglutarate/succinate ratio supporting the anti-inflammatory and protumorigenic phenotype of TAMs (Liu et al. 2017). Glutamine uptake and glutaminolysis can promote this phenotype as a source of α-ketoglutarate for both the replenishment of the TCA cycle and to support α-ketoglutarate-dependent demethylation of histone H3 in promoter regions of anti-inflammatory M2 genes (Mehla and Singh 2019). In hypoxic conditions, or after HIF1α or MYC activation, glutamine supports the TCA cycle through anaplerotic flux and reductive carboxylation (Dubey et al. 2023). MYC-induced glutaminolysis results in the shunting of citrate to cytosolic acetyl-CoA for lipid biogenesis and protein PTMs (Yang et al. 2014). Indeed, blocking glutamine metabolism can enhance the antitumor activity of TAM (Liu et al. 2017; Oh et al. 2020) and regulate antigen presentation by DCs (Guo et al. 2023). Thus, hypoxic TAMs repurpose glutamine for a variety of biosynthetic pathways and immune-suppressive processes rather than for ATP production (Li et al. 2022).

Metabolic changes in M2-like TAMs also augment TME mechanisms that suppress other immune cell types. TAM inhibition of T-cell activity occurs by secreting NOS or ARG1 into the TME that depletes the TME of arginine required for the expression of TCR, proliferation, and memory establishment of T cells (Li et al. 2022). ARG1 is regarded as a universal marker for the M2, anti-inflammatory phenotype. Induction of ARG1 may be perpetuated by IL-4 and acidic pH in the TME (Li et al. 2022). Production of iNOS is supported by T-cell-associated IFN-γ and CD40 (Marigo et al. 2016). While ARG1 and iNOS induction are indicative of distinct polarization states, in vitro evidence has shown iNOS expression in hypoxic states, suggesting the potential for dual expression of iNOS and ARG1 in TAMs of hypoxic TME regions (Carmona-Fontaine et al. 2017; Li et al. 2022). Juxtaposing the M1 antitumor phenotype of iNOS-producing macrophages, inhibition of iNOS in TAMs blocks TAM suppression of T-cell function while inhibition of ARG1 reduces tumor growth (Li et al. 2022). Thus, iNOS expression may regulate the protumorigenic phenotype of TAMs with a higher induction in hypoxic TME regions where ARG1 and iNOS may be coexpressed (Li et al. 2022).

T-Cell Metabolism and Adaptation to Tumor Microenvironments

T cells undergo metabolic reprogramming from catabolism to anabolism upon antigen encounter with appropriate costimulation (Fig. 3). This transition enables T cells to rapidly double cell mass for proliferation while supporting diverse effector functions. While anabolic pathways are broadly elevated, glucose uptake and glycolysis are among the pathways most strikingly increased (Jacobs et al. 2008; Wang et al. 2011; Macintyre et al. 2014). This is initiated in part when the TCR is stimulated by antigen as a first signal and activation of pyruvate dehydrogenase kinase 1 (PDK1), which promotes pyruvate conversion to lactate by preventing pyruvate import into the mitochondria (Menk et al. 2018). Signaling downstream of TCR activation is enhanced by costimulatory signals through CD28 ligation, allowing the T cells to maintain high glycolytic activity (Frauwirth et al. 2002; Jacobs et al. 2008). CD28 stimulation also activates the PI3K/Akt/mTORC1 signaling pathway, leading to Myc-dependent remodeling of the metabolic transcriptome including an increase in genes that regulate glucose uptake and glycolysis (Frauwirth et al. 2002; Jacobs et al. 2008; Wang et al. 2011; Chi 2012). Aerobic glycolysis, while yielding less ATP per cycle than OXPHOS, is important to maintain intracellular redox balance and generate metabolic intermediates for biosynthesis to support rapid cell growth and production of ATP (Buck et al. 2015; Hosios and Vander Heiden 2018). Glycolytic intermediates enable the shunting of glucose-6-phosphate to the pentose phosphate pathway (PPP) for lipid synthesis, redox balance (Wang et al. 2011; Patra and Hay 2014), and to support the one carbon metabolism pathway that generates nucleotide precursors and SAM for methylation reactions (Ron-Harel et al. 2016; Roy et al. 2020; Sugiura et al. 2022).

Figure 3.

T-cell metabolic programming. Naive T cells are characterized by catabolic metabolism that rapidly switches to anabolism upon activation and costimulation to promote proliferation and effector function. Following antigen clearance, T-cell populations contract to generate a pool of long-lived memory T cells that rely primarily on oxidative metabolism to enable a rapid recall response upon antigen re-encounter. Conversely, antigen persistence promotes differentiation into an exhausted T-cell state characterized by metabolic dysfunction and progressive loss of effector function. (IL-7) Interleukin 7, (IL-7R) interleukin 7 receptor, (S1P) sphingosine-1-phosphate, (S1PRTCR) sphingosine-1-phosphate receptor T-cell receptor.

Costimulation via the CD28 family plays a central role as a signal two to promote or suppress a productive T-cell response upon TCR stimulation as a signal one. In the absence of CD28-mediated costimulatory activation of the PI3K/Akt/mTORC1 pathway, TCR-stimulated T cells enter an unresponsive state of anergy in which glucose uptake and metabolism are suppressed (Jacobs et al. 2008; Zheng et al. 2009; ElTanbouly and Noelle 2021). Conversely, the inhibitory receptors cytotoxic-T-lymphocyte-associated protein 4 (CTLA4) or PD-1 recruit phosphatases that prevent activation of the PI3K/Akt/mTORC1 pathway and antagonize CD28 costimulation to reduce anabolic metabolism (Frauwirth et al. 2002; Parry et al. 2005). A key mechanism of CTLA4 and PD-1 checkpoint immunotherapies, therefore, is to release this break on T-cell metabolism and enable T-cell anabolism (Bengsch et al. 2016). Indeed, transgenic expression of constitutively active Akt or Glut1 could partially restore T-cell function in models of B-cell leukemia (Siska et al. 2016).

While efficient antigen clearance leads to differentiation of long-lived, protective memory T cells (Tmem), chronic antigen exposure, such as in large solid tumors, promotes restimulation-induced T-cell death or differentiation of dysfunctional, exhausted T cells (Tex) (McLane et al. 2019; ElTanbouly and Noelle 2021). Tex are characterized by loss of effector function and memory recall, sustained expression of inhibitory receptors, and metabolic insufficiency (McLane et al. 2019). Sustained expression and signaling through PD-1 via ligation by PD-L1 expressed on tumor and suppressive myeloid cells reduces glycolysis and mitochondrial function in Tex, partially through inhibition of the master mitochondrial biogenesis and regulatory transcription factor PGC-1α (Bengsch et al. 2016). Additionally, persistent but suboptimal mTORC1 activation during chronic antigen exposure can induce the accumulation of large but dysfunctional, depolarized mitochondria and mtROS production (Bengsch et al. 2016). Accumulation of depolarized mitochondria results in transcriptional and epigenetic changes characteristic of Tex, supporting a link between mitochondrial function and the altered transcriptomic and epigenomic state of exhaustion (Yu et al. 2020).

Among other challenges in tumors, T cells may simultaneously experience chronic TCR stimuli, insufficient costimulation, sustained PD-1 signaling, hypoxia, and altered nutrients. These stresses lead to both functional and metabolic impairments. CD8 T cells isolated from primary patient renal cell carcinoma tumors had accumulated dysfunctional, fragmented mitochondria with elevated production of ROS (Siska et al. 2017). Mitochondrial dysfunction in CD8 T cells is caused by chronic antigen stimulation in hypoxic environments that are frequent in the TME (Scharping et al. 2021). Uptake of oxidized lipids through CD36 in the TME also led to lipid peroxidation and stress response in these CD8 T cells (Xu et al. 2021). While glucose may not be broadly limiting in the TME, TILs also accumulated multiple defects in glycolysis that nonetheless can restrain glucose metabolism. These include deficient Glut1 cell surface trafficking, reduced GAPDH expression, suppression of Enolase 1, and insufficient PEP, which has been reported to signal to drive T-cell calcium responses (Ho et al. 2015; Siska et al. 2017; Gemta et al. 2019). Depletion of amino acids, such as glutamine, in the TME can directly affect T cells and, surprisingly, may promote Teff differentiation and antitumor immunity. Genetically targeting glutaminase (Gls) or broadly inhibiting glutamine metabolism was shown to promote increased glucose metabolism and increased CD4 Th1 and CD8 effector functions (Johnson et al. 2018; Leone et al. 2019; Varghese et al. 2021). However, targeting glutamine metabolism as a whole can also inhibit several essential enzymes (Madden et al. 2023) and promote T-cell exhaustion that may ultimately limit T-cell function (Johnson et al. 2018). In contrast to glutamine depletion, lactate accumulation in the TME may suppress effector functions and both promote Tmem and Treg phenotypes (Angelin et al. 2017; Quinn et al. 2020; Watson et al. 2021; Elia et al. 2022; Feng et al. 2022). Oxidized lipids can also accumulate in the TME, causing stress and impairing T-cell functions, which can be prevented by targeting the lipid transporter CD36 (Xu et al. 2021).

The role of cell metabolism to directly impair or repair T-cell function is evident if metabolic pathways are rescued to restore inflammatory potential, respectively. Consistent with immune checkpoints PD-1 and CTLA4 inhibition of costimulatory signals, T-cell function was partially restored in a glycolysis-dependent manner if human kidney tumor T cells were activated with both TCR and CD28-mediated costimulation (Beckermann et al. 2020). Additionally, mitochondria can be directly rescued by provision of exogenous pyruvate, by neutralizing mitochondrial ROS (Beckermann et al. 2020), or by overexpression of the mitochondrial biogenesis factor PGC1α (Bengsch et al. 2016; Scharping et al. 2021; Lontos et al. 2023). Each of these metabolic rescue treatments reversed metabolic immune suppression, resulting in improved Teff antitumor function. Metabolic conditioning to enhance mitochondrial quantity and quality prior to T-cell adoptive therapy can also improve antitumor immunity. Stimulation of T cells in low doses of 2-deoxyglucose to suppress glycolysis and effector differentiation increased mitochondrial capacity in vitro, leading to improved longevity and function upon subsequent transfer in vivo (Sukumar et al. 2013). Similar selection for T cells with highly coupled and efficient mitochondria by selecting T cells with low mitochondrial membrane potential also showed enhanced antitumor activity relative to bulk populations (Sukumar et al. 2016). Other approaches, such as increased temperatures that result in mitochondrial biogenesis, also increased antitumor immunity (O'Sullivan et al. 2021). These findings show that metabolic maladaptations of TIL, and mitochondria in particular, directly contribute to T-cell functional impairments in the TME.

Rewiring T-Cell Metabolism to Improve CAR-T Therapy for Solid Tumors

Adoptive transfer of chimeric antigen receptor–modified T (CAR-T) cells has proven an effective treatment strategy for a variety of hematologic malignancies after FDA approval in 2017 for B-cell acute lymphoblastic leukemia (Schultz and Mackall 2019). Despite success targeting other advanced leukemias, lymphomas, and multiple myeloma, similar clinical efficacy in the solid tumor setting remains elusive. Early clinical trials have demonstrated the feasibility and safety of CAR-T therapy in a variety of solid tumors, but durable responses comparable to those observed in hematologic malignancies have not yet been achieved in solid tumors (Brown et al. 2016; Tchou et al. 2017; Feng et al. 2018; Wang et al. 2018; Adusumilli et al. 2021; Narayan et al. 2022; Qi et al. 2022). A key difference between solid and liquid tumors is the establishment of the TME and the potential for metabolic immune suppression of CAR-T-cell signaling and metabolism (Fig. 4). Challenges faced in developing CAR-T cells for solid tumors include choice of tumor antigen and heterogeneity, tumor bed trafficking, T-cell-intrinsic regulatory programs, and the broadly immunosuppressive TME that limit CAR-T proliferation, function, and persistence (Wagner et al. 2020). It has become increasingly apparent that the metabolic fitness of T cells is a critical determinant of their antitumor function and survival. Components engineered into the CAR can shape CAR-T-cell metabolism. CD3ζ together with CD28 signaling domains yield CAR-T cells with high rates of glycolysis and anabolic metabolism while CD3ζ with 4-1BB signaling domains promote mitochondrial metabolism and CAR-T-cell longevity (Kawalekar et al. 2016; van Bruggen et al. 2019). Thus, there has recently been an intersection between the fields of immunometabolism and CAR-T engineering.

Figure 4.

Metabolic reprogramming of chimeric antigen receptor–modified T (CAR-T) cells. CAR design impacts metabolism based on the costimulatory domain used. During manufacturing, CAR-T cells can be induced to overexpress metabolic genes to enable adaptation or resistance to the suppressive tumor microenvironment (TME). Additionally, CAR-T cells may be supplemented with cytokines, nutrients, or metabolic pathway inhibitors to modulate metabolism. Such “metabolically fit” CAR-T cells have improved mitochondrial metabolism, antitumor function, and persistence in vivo.

The CAR-T-cell manufacturing process involves cell culture and genetic modification to introduce the CAR, which provides an opportunity to enforce expression of other genes that may improve the metabolism and function of the cells in vivo. The proline metabolism enzyme PRODH2 was identified in a gain-of-function CRISPR screen as conferring improved effector function and proliferation of CD8 T cells (Ye et al. 2022). Overexpression of this enzyme in CAR-T cells reshaped metabolism to a more memory-like phenotype, including an increase in proline metabolism, mitochondrial mass, and respiration, translating to improved tumor control in both blood and solid tumor models (Ye et al. 2022). Overexpression of the arginine synthesis enzymes argininosuccinate synthase (ASS) or ornithine transcarbamylase (OTC) in CAR-T cells improved proliferation in the arginine-restricted TME, conferring superior tumor control in both acute myeloid leukemia (AML) and solid tumor models (Fultang et al. 2020). Tumor-infiltrating T cells often have dysfunctional mitochondria, but expressing mitochondrial biogenesis transcription factor PGC-1α helped tumor-specific T cells resist mitochondrial deficiency in the TME (Scharping et al. 2016). PGC-1α overexpressing T cells in adoptive cell therapy exhibit increased mitochondrial biogenesis and OXPHOS that enhanced antitumor efficacy in a model of aggressive melanoma (Scharping et al. 2016) and this translates to CAR-T cells in which PGC-1α has also been reported to have increased antitumor activity (Lontos et al. 2023).

The in vitro CAR-T manufacturing process provides yet another opportunity to modulate metabolism by adjusting culture conditions. This might include the use of pharmacological inhibitors to transiently block certain metabolic pathways. For example, inhibition of glycolysis or Akt signaling can promote a more memory-like phenotype and improves tumor control by tumor-specific T cells and CAR-T cells in models of solid and blood tumors, respectively (Sukumar et al. 2013; Ryan et al. 2017). Additionally, inhibition of the mitochondrial pyruvate carrier (MPC) rewired CAR-T cells to increase glutamine- and lipid-derived acetyl-CoA pools leading to epigenetic reprogramming toward a memory-like CAR-T. This metabolic reprogramming conferred greater antitumor potency and persistence in solid tumor and leukemia models (Wenes et al. 2022). A combination treatment with a mitochondrial fusion promoter (M1) and fission inhibitor (Mdivi-1) generates T cells with memory-like bioenergetic profiles and improved tumor control (Buck et al. 2016). Supplementation of IL-15, a cytokine critical for development and survival of memory T cells, reduces glycolysis and increases FAO in CAR-T cells, conferring superior proliferative capacity, persistence, and tumor control in both lymphoma and glioblastoma models (Alizadeh et al. 2019). L-arginine addition to T-cell cultures promotes a switch to OXPHOS and supplementing TCR transgenic, tumor-specific T cells with L-arginine during in vitro activation leads to improved tumor control in vivo (Geiger et al. 2016).

BARRIERS AND OPPORTUNITIES IN METABOLIC IMMUNOTHERAPY

The TME alters immune cell metabolism in ways maladaptive to antitumor immunity. Models must therefore be refined to better fit the environment of the TME and consider both metabolic and cellular heterogeneity. In addition to altering immune cell metabolism, tumors are spatially heterogeneous, leading to diverse metabolism and methods of regulation across regions of the TME or at different metastatic sites. The tumor is also comprised of a variety of cell types, beyond cancer cells, which all contribute to the metabolic regulation of the TME. Thus, the phenotype of the “tumor” is comprised of many cell types along with a heterogenous metabolic environment. Classically, many cancer studies used bulk tumor processing to provide overarching information on the TME. However, this approach does not provide any nuanced information on what cell types drive the phenotype and differential nutrient uptake and usage between cell subsets (Arner and Rathmell 2023). An additional challenge to model the TME is that in vitro differentiation of immune cells can generate immune cell subtype with metabolic profiles that inherently differ from those seen in vivo (Ma et al. 2019). Another challenge of researching cell types within the TME is that sorting of the tumor into its various cell populations is limited to the abundance of the cell type within the tumor under study.

Although the heterogeneous metabolic character of the TME poses challenges to understand the dynamics of tumor immunobiology, it also offers opportunities to improve tumor immunotherapy. The distinct patterns of nutrient uptake and use by different cell types may be exploited in improved cellular-targeted imaging approaches. This same feature of differential nutrient use may then allow selective molecular targeting of specific cell populations to impair suppressive features of the TME or promote inflammatory functions. The metabolism of tumor-infiltrating immune cells is already being exploited in existing immunotherapies, as PD-1 and CTLA4 blockades appear to act in part by reshaping T-cell metabolism to remove a break on glycolysis and anabolism. Similarly, CAR-T cells with CD28 or 4-1BB costimulatory signaling domains have widely different inflammatory potentials and longevity affecting the tumors being targeted. Many pieces of evidence now point to mitochondrial dysfunction as a weak link in T-cell antitumor immune function (Rostamian et al. 2021). Improved understanding of the maladaptive response of T cells to the TME and how this leads to mitochondrial and general metabolic dysfunction will better identify patients most likely to benefit from immunotherapy and will point to new approaches to increase T-cell longevity and antitumor immunity.

COMPETING INTEREST STATEMENT

Dr. Jeffrey Rathmell is an employee of Vanderbilt University Medical Center and appointed to the Vanderbilt University School of Medicine. He is a founder, scientific advisory board member, and stockholder of Sitryx Therapeutics, a scientific advisory board member and stockholder of Caribou Biosciences and holds stock options for Nirogy Therapeutics. He has consulted and received speaker fees from Merck, Pfizer, and Abbie. He has received research support from Incyte Corp. within the past 3 years.