Lactic acid and lactate: revisiting the physiological roles in the tumor microenvironment

Abstract

Lactic acid production has been regarded as a mechanism by which malignant cells escape immunosurveillance. Recent technological advances in mass spectrometry and the use of cell culture media with a physiological nutrient composition have shed new light on the role of lactic acid and its conjugate lactate in the tumor microenvironment. Here, we review novel work identifying lactate as a physiological carbon source for mammalian tumors and immune cells. We highlight evidence that its use as a substrate is distinct from the immunosuppressive acidification of the extracellular milieu by lactic acid protons. Together, data suggest that neutralizing the effects of intratumoral acidity while maintaining physiological lactate metabolism in cytotoxic CD8⁺ T cells should be pursued to boost anti-tumor immunity.

Physiological lactic acid metabolism

Lactic acid is one of the most abundant metabolites in the human circulatory system [1]. It is generated from the end product of glycolysis, pyruvate, by the enzyme lactate dehydrogenase (LDH). Under aerobic conditions, pyruvate can be shuttled into the mitochondria to contribute to biosynthetic pathways and adenosine triphosphate (ATP) production. When oxygen is low, pyruvate is converted to lactate, and this reaction regenerates nicotinamide adenine nucleotide (NAD⁺), which is essential for sustained glycolysis [2]. Even with sufficient oxygen, cells might convert pyruvate into lactic acid (Figure 1) [3]. First described by Otto Warburg for tumor tissues that produced lactic acid and released it extracellularly [3], this process of aerobic glycolysis has since been identified in many other cell types [4], [5], [6], including immune cells [7], [8], [9], [10], [11], [12]. Lactic acid is a weak hydrophilic acid, which in aqueous solution with a physiological pH, converts almost entirely into its conjugate base, lactate, by releasing hydrogen ions (H⁺). The lactate exchange between cells and the extracellular space is facilitated by monocarboxylate transporters (MCT) [13]. Specifically, MCT1–4 mediate the transport of monocarboxylates, such as lactate, simultaneously with protons across the plasma membrane, and this transport can be bidirectional [13] (Figure 1). Systemic lactate concentrations are tightly regulated around 1–2 mM, but conditions such as shock [14], sepsis [15], muscle exercise [16], and cancer [17] can lead to much higher concentrations. Such supraphysiological concentrations of lactic acid and lactate might affect cellular function.

Figure 1. Lactic acid and lactate metabolism.

In mammalian cells, glycolysis converts glucose to two molecules of pyruvate with a net yield of two molecules of adenosine triphosphate (ATP) and two molecules of reduced nicotinamide adenine nucleotide (NADH). Pyruvate can be imported into the mitochondria and converted to acetyl-coenzyme A (acetyl-CoA), which enters the tricarboxylic acid (TCA) cycle. Alternatively, the enzyme lactate dehydrogenase (LDH) can convert pyruvate to lactate. This reaction is bidirectional, so when lactate is high, LDH converts it to pyruvate that can either enter the TCA cycle or initiate the process of gluconeogenesis, i.e., de novo glucose synthesis [2]. Lactate can be exported via the monocarboxylate transporters MCT1–4, which also function bidirectionally [13]. Acquisition of a proton, for instance, from NADH, leads to the conversion of lactate to its conjugate, lactic acid. In aqueous solution with physiological pH, lactic acid dissociates almost entirely to lactate and hydrogen ions (H⁺), thus acidifying the extracellular microenvironment. Lactate can also be used to produce lactyl-coenzyme A (lactyl-CoA) and affect gene transcription via histone lactylation [43]. Figure adapted from “Lactic acid fermentation” by Biorender.com (2022). Retrieved from https://app.biorender.com/biorender-templates, last accessed on October 8, 2022.

Abbreviations: Acetyl-CoA: acetyl-coenzyme A, ADP: adenosine diphosphate, ATP: adenosine triphosphate, H⁺: hydrogen ion, Lactyl-CoA: lactyl-coenzyme A, LDH: lactate dehydrogenase, MCT: monocarboxylate transporter, NAD/NADH: oxidized/reduced nicotinamide adenine nucleotide, TCA: tricarboxylic acid.

Early studies observed that human and mouse tumor cell lines released lactic acid in vitro [18], [19]. Elevated lactic acid in the cell culture media impaired T cell survival and cytokine production, while anti-inflammatory gene expression in macrophages increased [18], [19]. The authors hypothesized that lactic acid secretion by tumors is a mechanism which fosters an immunosuppressive TME [18], [19]. However, many early studies were performed in in vitro systems, where the metabolic milieu differed substantially from the physiological setting. Several technological advances have brought exciting insights into the role of lactic acid and lactate in the TME. First, in vivo and in vitro isotope tracing by mass spectrometry, both in mice and humans, has allowed researchers to focus on the physiological contributions of lactic acid and lactate to cellular metabolism. Second, the development of culture media resembling the metabolic composition of human serum has provided more accurate conditions for in vitro studies. Here, we revisit our understanding of the role of lactic acid and lactate metabolism in tumor cells, cytotoxic CD8⁺ T cells, regulatory T cells (T_reg), and macrophages. We discuss evidence that lactate metabolism is integral to energy homeostasis in these cell types. Based on the findings to date, we propose that lactate ion-related metabolism and the acidic pH caused by an accumulation of (lactic acid) protons in the TME can and should be targeted with separate putative therapeutic approaches as novel avenues in cancer treatment.

Lactate is a metabolic fuel for cancer cells

Patients with cervical cancer and head-and-neck squamous cell carcinoma (HNSCC), whose tissue specimens presented elevated lactate concentrations, had high metastatic spread and a significantly shorter disease-free survival compared to patients with low lactate concentration tumors (n=34 patients for each entity, high- versus low-lactate groups defined based on the median lactate concentration in the cohort) [17], [20]. Furthermore, high expression of LDHA, which encodes a subunit of LDH, correlated with low expression of the T cell receptor (TCR) complex subunit CD3d and shorter overall survival in a gene expression analysis of metastatic melanoma tissue from 38 patients, indicating a potential negative impact of lactic acid production on T cell infiltration [21], [22]. These observations suggest two non-mutually exclusive hypotheses. First, high lactic acid production might benefit tumor cells by allowing them to withstand conditions of metabolic stress, such as during metastasis. Second, elevated local lactic acid concentrations might create an immune environment beneficial to tumor growth. Both of these mechanisms would be relevant for tumor persistence and metastasis.

Many studies have documented lactic acid secretion by tumor cells in culture [3], [23]. Serum analysis of 140 patients with different malignancies showed significantly higher lactate concentrations in those with high tumor burden than with low tumor burden or patients in complete remission, suggesting that at least some tumors produce lactic acid and release it into the circulation [18] (Figure 2A). Still, only recent advances in isotope tracing technologies have allowed researchers to study whether lactate is also utilized as a metabolic fuel by tumor cells under physiological conditions. For instance, a landmark study of in vivo infusion of [U-¹³C]-glucose in patients with non-small cell lung cancer (NSCLC) undergoing surgery made several vital observations [24]. First, many tumors exhibited heterogeneous enrichment patterns of ¹³C-labeled metabolites compared to adjacent healthy tissue. High labeling of lactate from [U-¹³C]-glucose – indicating aerobic glycolysis – did not correlate with invasion or lymph node spread at the time of infusion but was associated with tumor progression and metastatic behavior in the years of follow-up. Second, concurrent in vivo isotope tracing with ¹³C-labeled glucose and lactate was performed in mice injected with HCC15 NSCLC xenografts. Enrichment of lactate-derived ¹³C in the tricarboxylic acid (TCA) cycle intermediates citrate, glutamate, and malate was two-fold higher compared to glucose-derived ¹³C [24]. Moreover, CRISPR-Cas9-mediated deletion in HCC15 cells of solute carrier family 16 member 1 (Slc16a1) encoding MCT1, resulted in nearly abolished labeling of tumor metabolites from lactate in vivo in mice, suggesting that in this setting, MCT1 acted as the primary transporter that mediated lactate uptake [24]. Another study performed ¹³C-labeled substrate tracing in mouse healthy tissues and genetically-engineered mouse models of lung (Kras^G12D/+Stk11^−/−) and pancreatic tumors (Kras^LSL-G12D/+ Trp53^−/−, Kras^LSL-G12D/+Trp53^−/−Ptf1a^CRE/+), obtaining similar conclusions [25]. In particular, ¹³C-lactate extensively labeled TCA intermediates in vivo in all healthy tissues, and its contribution to TCA metabolites in tumors of fasted mice exceeded that of ¹³C-glucose. These studies suggest that even in the presence of glucose, lactate is an essential metabolic fuel of the TCA cycle in tumor cells in vivo – a process that is central to energy generation in the cell (Figure 2A). Moreover, melanoma xenografts with high potential of metastasis incorporated more lactate in TCA metabolites via MCT1-mediated uptake than xenografts with low metastatic potential, as shown by in vivo isotope tracing [26]. Thus, using lactate as a primary TCA cycle substrate to generate most ATP in the mitochondria might potentially uncouple energy production from aerobic glycolysis; this might allow glucose to serve as an essential substrate for other processes that contribute to cell proliferation and metastasis, although this remains to be further investigated [25], [26]. Taken together, these novel studies identify lactate as a physiological metabolic fuel for mouse and human tumor cells in vivo and suggest a link between lactate uptake and metastatic potential [24], [25], [26].

Key figure, Figure 2. Effects of lactic acid and lactate on mammalian cell metabolism and function.

(A) Tumor cells with high aerobic glycolysis generate lactate via lactate dehydrogenase (LDH) [3], [21]. In mouse breast cancer and multiple myeloma cell lines, lactate can be exported via the monocarboxylate transporter 1 or 4 (MCT1, 4) simultaneously with protons and acidify the extracellular space in vitro [50], [51]. Lung and pancreatic cancer cells in mice and humans in vivo acquire lactate via MCT1, which is then converted to pyruvate to fuel the tricarboxylic acid (TCA) [24], [25].

(B) Lactic acid can enter the cytosol of cytotoxic CD8⁺ T lymphocytes and decrease the intracellular pH. Acidification leads to the inhibition of glycolysis, proliferation, and cytokine production in mice and humans [18], [21], [23]. Lactate can also be taken up via MCT1 and fuel the TCA cycle in mouse CD8⁺ T cells in vitro [30]. At higher concentrations (40 mM), lactate acts as a histone deacetylase (HDAC) inhibitor in vitro, which enables the increased transcription of Tcf7 and drives a stem cell-like phenotype in mice [36].

(C) For intratumoral regulatory CD4⁺ T cells, lactate uptake via MCT1 is an essential fuel for the TCA cycle [38]. Furthermore, it can be used for gluconeogenesis and reduces the requirement for extracellular glucose. Lactate and lactic acid exposure are tightly linked to T_reg proliferation and suppressive function in mice and humans [38], [39], [40].

(D) Extracellular lactic acid regulates the transcription of genes such as Vegf, Arg1, and Retnla in mouse macrophages, influencing their anti-inflammatory differentiation [19], [42], [43]. Figure created with Biorender.com.

Abbreviations: Acetyl-CoA: acetyl-coenzyme A, ATP: adenosine triphosphate, H⁺: hydrogen ion, HDAC: histone deacetylase, MCT: monocarboxylate transporter, OXPHOS: oxidative phosphorylation, PEP: phosphoenolpyruvate, TCA: tricarboxylic acid, Treg: regulatory T cells.

Lactic acid and lactate – immunosuppressive metabolites or fuel for tumor-infiltrating CD8⁺ T lymphocytes?

Certain metabolic traits, such as a low rate of aerobic glycolysis and high mitochondrial respiration, are central characteristics of CD8⁺ memory T cells that can suppress tumor growth following adoptive cell transfer in mice, as in models of lymphoma [27] and melanoma [28]. However, tumor-infiltrating CD8⁺ T cells have exhibited decreased mitochondrial mass compared to their lymph node or circulating counterparts in mouse models of implantable melanoma, colon adenocarcinoma, lung carcinoma, as well as in samples from patients with HNSCC [29]. These changes were found to be specific to CD8⁺ T cells isolated from tumors, because they were not observed in a mouse model of Vaccinia virus infection, suggesting that conditions in the TME might impact CD8⁺ T cell metabolic reprogramming [29]. The effects of lactic acid and lactate in this context have been extensively explored. Early studies showed that lactic acid reduced the proliferation, cytokine production, and cytotoxic function of human antigen-specific CD8⁺ cytotoxic T lymphocytes (CTL) and mouse CD8⁺ T cells in vitro [18], [21] (Figure 2B). In a subcutaneous mouse B16.SYI melanoma model, small hairpin ribonucleotide acid (shRNA)-mediated knockdown of Ldha in tumor cells resulted in higher infiltration of NK1.1⁺ natural killer (NK) cells and CD3⁺ CD8⁺ T cells compared to control tumors [21]. Within these populations, increased percentages of interferon γ (IFN-γ)⁺ and granzyme-B⁺ cells were detected by flow cytometry [21]. While lactic acid significantly reduced intracellular IFN-γ and interleukin 2 (IL-2) in mouse and human CD8⁺ T cells in vitro, an equal concentration of lactate did not affect cytokine production [18], [21]. Acidification of the media with hydrochloric acid (HCl) to a similar pH as with 15 mM lactic acid (~6.4) partially decreased IFN-γ and IL-2 production [18], [21]. These results suggested that TME acidification by lactic acid protons might be responsible for suppressing CD8⁺ T cell function and, indeed, buffering the pH of cell culture media containing lactic acid to 7.4 restored cytokine production in human CTL in vitro [18]. Tumor cell-derived lactic acid inhibited glycolysis, proliferation, and cell cycle progression of CD8⁺ T cells during acute myeloid leukemia (AML), in a mouse model of myeloid leukemia and in patients with relapse after allogeneic hematopoietic cell transplantation [23] (Figure 2B). These studies suggest that lactic acid production by tumor cells may facilitate escape from immunosurveillance by blunting CD8⁺ T cell function [18], [21], [23].

Nevertheless, it is essential to note that the effects of lactic acid and lactate on CD8⁺ T cells are highly complex and appear to be intensely dependent on the concentration of these metabolites and their impact on intracellular pH. In contrast to the reported immunosuppressive role of lactic acid, a recent study identified lactate as a physiological carbon source for activated CD8⁺ T cells by in vitro and ex vivo mass spectrometry-based isotope tracing using customized cell culture media [30]. Classical cell culture media composition can vary significantly from the serum metabolome [31]. Recent advances in understanding physiological cell metabolism have been achieved by generating media that mimic the natural environment in the human body [31], [32], [33]. To study the physiological metabolic flux in mouse CD8⁺ T cells, researchers utilized cell culture media in which the concentrations of crucial nutrients (e.g., glucose, glutamine, glycine, alanine, aspartate, methionine, threonine, and serine) were adapted to mouse serum concentrations, unlike their higher abundance in standard T cell culture media (IMDM, Iscove’s modified Dulbecco’s medium). In addition, the media was supplemented with physiological carbon sources that are present at >100 µM in mouse serum but absent in most cell culture media (acetate, citrate, lactate, and β-hydroxybutyrate) [30]. Under these conditions, competitive ex vivo tracing of ¹³C-glucose and ¹³C-lactate in CD8⁺ T cells isolated from mice after Listeria monocytogenes infection showed higher labeling of TCA intermediates citrate, malate, and aspartate from lactate, than from glucose [30] (Figure 2B). Furtehrmore, supplementation of cell culture media with physiological lactate concentrations (0.5 – 2 mM) slightly increased IFN-γ expression in activated mouse CD8⁺ T cells in vitro relative to media devoid of lactate. These data suggested that lactate is a physiological carbon source tied to optimal T cell function, even in the presence of glucose [30]. Importantly, shRNA-mediated knockdown of Ldha in mouse CD8⁺ T cells resulted in reduced antigen-specific T cell expansion and IFN-γ production in an in vivo model of a Listeria monocytogenes infection [30]. These data are in line with another recent study, in which the authors observed that specific deletion of Ldha in mouse effector T cells (Tbx21^CreLdha^fl/fl) diminished antigen-specific CD8⁺ T cells expansion in mouse models of Listeria monocytogenes and lymphocytic choriomeningitis virus (LCMV) infections [34]. Additionally, the oncometabolite D-2-Hydroxyglutarate (D-2HG) inhibited LDH in mouse CD8⁺ T cells, and treatment with either D-2HG or LDH inhibitors oxamate and GSK decreased IFN-γ and granzyme B production; it also reduced the killing of B16 melanoma cells in vitro [35]. Collectively, these novel studies suggest that lactate is a physiological carbon source for mouse CD8⁺ T cells and that the intact function of LDH is essential for their cytotoxic function [30], [34], [35].

So, can lactate metabolism be exploited to boost anti-tumor immunity? Intratumoral application of lactate, but not glucose, has reduced tumor volume in a subcutaneous MC38 mouse model of colon cancer [36]. This beneficial effect was observed when tumors were induced in immunocompetent animals but not in Rag1^−/− mice lacking B and T cells, or when CD8⁺ T cells were depleted by in vivo administration of a monoclonal antibody [36]. Lactate supplementation synergized with antibodies directed against programmed death receptor 1 (PD-1) or via a cancer vaccine; this was demonstrated by the reduced tumor size in mouse models of MC38 colon cancer, TC-1 human papilloma virus (HPV)-positive tumors, and B16F10 melanoma compared to untreated animals [36]. The augmented in vivo anti-tumor immune response of CD8⁺ T cells upon lactate treatment was attributed to the induction of T cell stemness through epigenetic regulation of T cell factor 7 (Tcf7) expression – the gene encoding T cell factor 1 (Tcf1), a key transcriptional regulator of T cell fate. The response was also accompanied by reduced apoptosis of CD8⁺ T cells during expansion in cell culture media containing lactate compared to traditional media [36] (Figure 2B). In summary, these novel studies challenge the classical view that lactic acid compromises CD8⁺ T cell function. Specifically, while an acidic pH blunts CD8⁺ T cell proliferation, cytokine production, and cytotoxicity [18], [21], [23], [37], lactate appears to be a crucial physiological carbon source contributing to T cell activity [30]. At higher concentrations, lactate also regulates epigenetic processes related to CD8⁺ T cell stemness [36]. Thus, these data suggest that the effects of environmental acidity and lactate itself should be studied separately and in physiological environments.

Lactic acid can support tumor-promoting immune cell populations

The effects of lactic acid and lactate on immune cells are strongly cell-type-specific. For example, mouse CD8⁺ T cells appear more sensitive to lactic acid than CD4⁺ T cells [21], and mouse regulatory T cells (T_reg) are more resistant to increased lactic acid concentrations than conventional CD4⁺ T cells (T_conv) [38]. Indeed, T_reg showed high uptake and utilization of lactic acid in the TCA cycle (Figure 2C) [38]. Tamoxifen-inducible T_reg-specific deletion of Slc16a1 (Slc16a1^f/fFoxp3^cre-ERT2) resulted in attenuated growth of syngeneic B16 melanoma tumors, MC38 colon tumors, and MEER head-and-neck squamous cell carcinoma relative to tamoxifen-treated Foxp3^cre-ERT2 mice [38]. T_reg isolated from B16 tumors exhibited an impaired capacity to suppress T_conv proliferation ex vivo if they were deficient in Slc16a1 relative to controls [38]. Addition of pH-neutral lactate to a culture of mouse T_conv under polarizing conditions to form T_reg led to an increase in induced T_reg formation, which was detected by the elevated expression of the transcription factor forkhead box protein 3 (Foxp3) relative to non-polarizing conditions [39]. Moreover, exposure of human T_reg to lactic acid induced PD-1 expression and improved the ability of these cells to suppress human T cell proliferation in vitro [40]. These results link lactic acid/lactate uptake and utilization by T_reg directly to their formation and immunosuppressive properties and imply that lactic acid and lactate are essential substrates for intratumoral T_reg function (Figure 2C) [38], [39], [40].

Lactic acid can also foster an immunosuppressive environment by modulating the differentiation of myeloid cells, such as macrophages. Specifically, lactic acid induced the gene expression of vascular endothelial growth factor, Vegf, and arginase 1, Arg1, via stabilization of hypoxia-inducible factor 1 alpha, Hif1a; it also promoted their differentiation towards an anti-inflammatory “M2-like” phenotype [19] (Figure 2D). In addition, exposure of mouse bone marrow-derived macrophages (BMDM) to an acidic pH (6.8) in vitro increased the expression of anti-inflammatory genes such as Arg1, Cd206, and resistin-like alpha (Retnla) [41]. A newer study showed that similar to other immune cells, mouse IL-4-induced anti-inflammatory BMDM could acquire lactate and catabolize it via pyruvate in the TCA cycle to support their metabolic reprogramming [42] (Figure 2D). Furthermore, supplementation of lactate to glucose-starved mouse BMDM increased histone H3 lysine 9 (H3K9) acetylation in the promoter regions of Arg1 and Retnla, suggesting that lactate can affect macrophage gene expression via epigenetic modulations [42]. Recently, lactate-derived lactylation of histone lysine residues was identified as a novel epigenetic modification that directly stimulated gene transcription in mouse and human cells; among other functions, histone lactylation was identified as being a driver of Arg1 expression in mouse BMDM (via chromatin immunoprecipitation sequencing and RNA sequencing) (Figure 2D) [43]. Together, these studies suggest that anti-inflammatory immune cells such as T_reg and macrophages utilize lactic acid and lactate efficiently to fuel their metabolic needs. This process might be central to acquiring a suppressive function in the TME and promoting tumor persistence.

Concluding Remarks

Future directions and therapeutic perspectives

With modern technologies for isotope tracing in vivo in humans and animal models, the field has come to appreciate lactate as more than a byproduct of glycolysis. Lactate is now established as a physiological carbon source for healthy tissues, immune cells, and tumors [24], [25], [30] [38], [42]. Lactate concentrations in the tumor might potentially be used as a candidate biomarker to help assess the aggressive course of disease, including pro-metastatic cell behavior and resistance to anti-tumor immunity [17], [18], [20], [21], [23], [24], [38]. Current evidence suggests that the immunosuppressive effects of lactic acid on tumor-fighting T and NK cells are mediated via acidification, particularly of intracellular pH, which is likely associated with the inhibition of essential metabolic pathways, and subsequent loss of functionality [18], [21], [23]. The lactate ion itself is not immunosuppressive [18], [21], [30], [36]. On the contrary, as discussed, lactate might fuel the TCA in CD8⁺ T cells [30] and epigenetically promote their stemness [36] which may contribute to an improved anti-tumor effect following adoptive T cell transfer.

How can the detrimental accumulation of protons be uncoupled from the potentially beneficial effects of the lactate ion? Inhibition of LDHA with oxamic acid or deletion of the Ldha gene from tumor cells in mouse melanoma spheroids or B16 tumors reduced the total concentrations of lactic acid. It resulted in increased infiltration by NK and CD8⁺ T cells, thus suggesting that LDH might be targeted for melanoma treatment [18], [21]. However, recent studies have demonstrated that intact LDH function is essential for antigen-specific T cell expansion and cytotoxic function in mouse models of infection and cancer [30], [34], [35]. Thus, compounds targeting LDH should be assessed concerning their direct effects on cytotoxic T cells in vivo. A second potential intervention is buffering the acidic pH with sodium bicarbonate, by which the intracellular pH and metabolic activity of mouse CD8⁺ T cells might be restored even at high concentrations of lactic acid in vitro [23]. Accordingly, sodium bicarbonate administration to patients with AML relapse after allogeneic transplantation has resulted in higher mitochondrial respiration and IFN-γ production in circulating CD8⁺ T cells [23]. Further evidence for the efficacy of sodium bicarbonate, either as a monotherapy or in combination with other treatments mainly from mouse models, has recently been reviewed [44]. It remains unclear whether systemic administration of sodium bicarbonate actually neutralizes the acidic pH in human tumors, and no clinical studies have shown a benefit in cancer patients so far. Nevertheless, more specific approaches targeted at making T cells less susceptible to an acidic environment might improve antitumor immunity. Furthermore, MCT1 inhibitors might potentially prove to be effective when considering several mechanisms [45]. First, they might abrogate lactate uptake by T_reg, thus suppressing their pro-tumorigenic functions [38]. Also, diminished suppressive activity of T_reg might synergize with immune checkpoint inhibition and result in better tumor control [38]. Second, inhibition of MCT1 might also impair metabolic processes (such as the TCA cycle) directly in tumor cells and reduce metastatic rates [26]. Accordingly, the MCT1 inhibitor AZD3965 has been tested in a non-randomized, single group assignment, Phase 1 clinical trial for patients with advanced solid tumors (53 participants; ClinicalTrials.gov Identifier: NCT01791595) and the results should be informative.

From another angle, expanding CD8⁺ T cells for adoptive cell transfer under high-lactate culture conditions has improved their anti-tumor efficacy in a mouse model of colon cancer, presumably through enhanced expression of Tcf7, which conveyed stem cell-like properties [36]. However, this recent finding requires further investigation with human cells and using more physiological tumor models. These studies only begin to uncover the potential of lactic acid and lactate metabolism as putative targets for improving anti-tumor immunity (see outstanding questions). However, we posit that antagonizing the effects of intratumoral acidification while preserving physiological lactate metabolism by CD8⁺ T cells might be a successful approach for maintaining their anti-tumor activity. In addition, tumor cell and T_reg-directed inhibition of lactate transport and metabolism should be further explored. Targeting these metabolites might even be considered beyond cancer for diseases such as ischemic tissue injury and wound healing in which lactic acid is thought to accumulate in the local inflammatory environment, thus modulating immune responses and tissue remodeling [46].

Outstanding questions:

• Do CD8⁺ T cells in the TME directly acquire lactate? How does lactate utilization by CD8⁺ T cells compare to tumor cells in vivo? – A novel study showed that lactate is used as a physiological carbon source to sustain TCA activity in in vitro and ex vivo mouse CD8⁺ T cells. It is unclear whether this also occurs in the TME in vivo and how lactate uptake and utilization rates of CD8⁺ T cells compare to cancer cells.

• What are the lactate concentrations and the pH in the TME in vivo? – Multiple studies have documented high lactate concentrations within tumor cells and lactic acid secretion in vitro. Still, there is little direct evidence about the concentration of lactate in the tumor interstitial fluid, to which immune cells would be directly exposed.

• Does LDH inhibition reduce tumor growth and metastasis in vivo? – Deletion of Ldha in mouse tumor cell lines resulted in increased infiltration by T cells and NK cells. However, intact LDH function is also required for mouse CD8⁺ T cell function.

• How can the susceptibility of CD8⁺ T cells to an acidic pH be reduced? – Several studies, mostly from mouse tumor models, suggest that neutralizing the environmental acidic pH might improve CD8⁺ T cell anti-tumor cytotoxicity. The specific molecular mechanisms by which T cells might withstand a low pH in the TME and maintain their function remain to be determined.

• Can exposure to high lactate concentrations be a successful approach for boosting the anti-tumor efficacy of adoptively transferred T cells? – Recently, expansion of mouse CD8⁺ T cells in media containing a high concentration of lactate induced a stem cell-like phenotype by epigenetic regulation of transcription factor expression. This resulted in better control in one mouse model of colon cancer. Further studies are warranted to assess the efficacy of such an approach for inducing a stem cell-like phenotype in clinically relevant agents, such as human chimeric antigen receptor (CAR) T cells, as well as in physiological tumor models.

Limitations and caveats

The effects of lactic acid and lactate have been extensively studied in vitro using concentrations of 15 – 40 mM. While elevated lactate concentrations within tumor cells might reach these concentrations [17], it remains unclear whether the lactate concentration in the extracellular tumor environment is equally high. As reviewed elsewhere [47], there is only limited evidence of elevated lactate in the tumor interstitial fluid. In patients with HNSCC, tumor tissue showed about 2-fold higher lactate concentrations than healthy mucosa [48]. High-grade astrocytoma in humans had a 1.6-fold increase of lactate compared to peri-tumoral brain tissue [49]. Thus, further studies must assess the lactate concentrations that immune cells can encounter in the TME.

Highlights.

• Many tumors and activated immune cells have high glycolytic activity and accumulate intracellular and potentially extracellular lactic acid. Besides acidifying the intratumoral pH, this might lead to an increased lactate concentration in the tumor microenvironment.

• An acidic pH and high lactate concentrations affect tumor and immune cell function. A low pH impairs metabolic activity, proliferation, and cytokine production of human and mouse CD8⁺ T cells, while it can promote melanoma metastasis formation. Neutralizing intratumoral acidification might improve antitumor immunity and attenuate cancer metastasis.

• Novel findings from in vivo and in vitro isotope tracing establish lactate as a physiological carbon source fueling the tricarboxylic acid cycle in human and mouse cancer cells, mouse CD8⁺ T cells, regulatory T cells, and antiinflammatory bone marrow-derived macrophages.

• The intact function of lactate dehydrogenase in mouse CD8⁺ T cells is required for their antigen-specific expansion, cytokine production, and cytotoxicity in vivo.

• We posit that future efforts to improve the anti-tumor immune response should overcome the harmful effects of an acidic pH on CD8⁺ T cells while sustaining their physiological use of lactate as a metabolic fuel.

Significance.

Recent studies establish that the metabolic byproduct of glycolysis, lactate, is a physiological carbon source for both cancer and immune cells, including CD8⁺ T cells. Maintaining physiological lactate metabolism in anti-tumor CD8⁺ T cells is essential for their cytotoxic activity, while improving their resistance to acidic pH in the tumor microenvironment might be a beneficial candidate approach to cancer immunotherapy.