Effects of tumor metabolic microenvironment on regulatory T cells

A tumor is a type of polygenic disease in which malignant cell proliferation is caused by a variety of oncogenic factors that promote abnormal gene expression and regulation in the body [1]. It differs from the body’s normal tissue cells, and is mainly characterized by the following ten characteristics: unlimited proliferation, resistance to apoptosis, evasion of growth inhibitory factors, invasion and metastasis, stimulation of angiogenesis, continuous production of proliferative signals, resistance to cellular energy metabolism, genomic instability, evasion of immune suppression, and enhancement of tumor-related inflammatory responses [2‐4]. Acquisition of these characteristics is inseparable from changes in the cells themselves and the influence of the surrounding environment. The internal environment in which tumor cells grow is called the tumor microenvironment, which consists of the extracellular matrix, myofibroblasts, cytokines, fibroblasts, neuroendocrine cells, adipocytes, immune-related cells, and vasculature [5‐8].Tumor cells can affect the physicochemical properties, components, and cytokines of the tumor microenvironment, making it more conducive to their growth, metastasis, and invasion [9‐13]. At the same time, changes in tumor microenvironment play an important role in all stages of tumor development, inhibiting or promoting tumor growth. The interaction between the two affects the whole process of tumor growth, and mutual adaptation maintains the dynamic balance of tumor microenvironment [14‐19].

Owing to changes in their physiological characteristics, tumor cells promote a series of changes in the tumor microenvironment [20‐23]. Rapidly growing tumor cells and mesenchymal cells lead to a decrease in local oxygen content and thus to a microenvironment in an oxygen-starved state, with the most severe hypoxia in the center of the tumor and a gradient decrease from external to internal oxygen content. Tumor growth to 1–2 mm in diameter stimulates local angiogenesis [24], and local hypoxia induces the expression of vascular endothelial growth factor (VEGF), which in turn promotes the formation of blood vessels in tumors [24, 25]. However, owing to the abnormal tumor microenvironment, new blood vessels often have uneven distribution, large capillary distance, short arteriovenous circuit, incomplete endothelial cells, and ruptured basal membranes [26]. Therefore, these localized microvessels cannot improve the state of hypoxia. With changes in the tumor microenvironment, the metabolism of tumor cells is also quite different from that of normal cells. The most significant difference is the Warburg effect, in which, compared with normal cells, tumor cells obtain energy through anaerobic glycolysis even under oxygen-rich conditions [27, 28]. At the same time, hypoxia causes high expression levels of hypoxia-inducible factor (HIF) in cells. This in turn promotes the expression of glycolysis-related proteins [29], and changes in glucose metabolism lead to the massive production of lactic acid metabolites. An oncogene such as c-myc is activated by tumor cells and up-regulates the expression of lactate dehydrogenase A (LDH-A) and promotes the conversion of pyruvate to lactic acid [30]. This leads to an increase in lactate content and low pH in tumor cells. To avoid the effects of intracellular lactic acid on basal metabolic activity, the efficiency of the monocarboxylate transporter (MCT) on the cell membrane is increased, removing excess lactic acid from the cell [31]. This causes the tumor microenvironment to be at a low pH. HIF can induce high expression levels of carbonic anhydrase (CA) in tumor cells, which catalyzes the reaction of CO₂ with H₂O to generate carbonic acid [32]. Stimulation of various types of carcinogens and cytokines leads to disturbances in the acid-base balance in tumor cells. Tumor cells promote an efflux of intracellular hydrogen ions by up-regulating hydrogen ion-related transport proteins in the cell membrane [33]. The above changes will further exacerbate the low pH of the microenvironment. In addition, de novo synthesis of fatty acids in tumor cells is also enhanced [34, 35]. Glutamine metabolism is enhanced, and the metabolism of other amino acids and nucleic acids also changes [36, 37]. These metabolic changes promote tumor cell proliferation, metastasis, and invasion [38‐40] and also cause changes to the microenvironment of the tumor, affecting other cells infiltrating the tumor tissue. Metabolic changes occur not only in tumor cells, but also in immune cells infiltrated in the tumor tissue that undergo metabolic reprogramming to accommodate functional changes.

During proliferation and metastasis, tumor cells need to obtain enough energy and reaction substrates to satisfy their own metabolism [41‐44], and it is necessary to escape the surveillance and elimination of abnormal tissue cells by the immune system [45]. The immune evasion of tumor cells occurs in two ways: covering the self-antigens to hide or remove the target that can be recognized by the host immune system, also known as antigen coverage; or secretion of immunosuppressive cytokines to inhibit the immune effector cells or induction of suppressive immune cells to exert immunosuppressive effects [46]. Tregs belong to a T cell subgroup with regulatory immune functions. These cells participate in the immune escape of tumor cells mainly through the induction of immune incompetence and immunosuppression [47]. Clinical studies have found a large infiltration of Tregs in tumor tissues of patients, and their amount is closely related to the prognosis of patients [48, 49]. Some tumors can be treated by reversing local Tregs levels in tumor tissue [50, 51]. Animal experiments showed that tumor growth is positively correlated with Tregs local content in tumors, and removal of Tregs can effectively enhance the body’s resistance to tumors [52, 53]. Tregs have a strong ability to infiltrate and accumulate in tumor tissues. The chemokines in the tumor microenvironment can bind to the corresponding receptors on the surface of Tregs, recruit Tregs to the tumor tissues, and exert immunosuppressive effects [54]. Antigens in tumor tissues can promote the production of Tregs after dendritic cell processing and extraction [55]. Tumor cells secrete cytokines to directly induce the transformation of T cells into Tregs [56], or indirectly promote the production of Tregs by inducing the maturation of myeloid dendritic cells that secrete immunosuppressive factors [57].

Tregs are abundantly present in tumor tissues and mainly mediate immunosuppressive effects. An increasing number of studies has focused on the metabolic reprogramming of Tregs in tumor tissues and whether tumors and Tregs can influence each other, and on how to modulate Tregs or the biologically active substances or cells that affect these T cells in the tumor microenvironment to improve the patient’s prognosis. Therefore, this review focuses on the metabolic reprogramming of Tregs in tumor tissues and the effects of the physicochemical properties of the tumor microenvironment on the generation, proliferation, and infiltration of Tregs and its mechanisms of action. Advances in understanding of the complex regulatory networks of tumor immune evasion provide a theoretical basis and new biological targets for clinical treatment.

Tregs are a subset of T cells that express the interleukin (IL)-2 receptor CD25 and the forkhead/flanking helix nuclear transcription factor (Foxp3), and were first discovered in 1995 [58]. Tregs in the body are divided into two groups: natural Tregs (nTregs), which mature after positive and negative selection of the thymus and exert immunosuppressive effects in peripheral blood and lymphoid tissues; and inducible Tregs (iTregs), which originate after T cells receive antigen stimulation and are transformed by inhibitory cytokines [59], having differentiated into different subtypes by different cytokines and playing different biological roles [60] (Fig. 1/Table. 1).

Foxp3 is a key transcription factor of Tregs, mainly related to the development and function of these cells. Foxp3 regulates the immunosuppressive function of Tregs mainly through transcriptional co-regulatory proteins, including transcription factors, co-repressors, co-activators, histones, and chromatin remodeling factors, which combine to form protein complexes to dynamically regulate the expression of related genes [61‐65].

Tregs exert immunosuppressive effects by inhibiting effector T cells and dendritic cells mainly through the following pathways: (a) secretion of perforin and granzyme B that act directly on effector cells promoting apoptosis [66]; (b) secretion of inhibitory cytokines such as transforming growth factor (TGF)-β, IL-10, and IL-35 that bind to immune cells and result in immunosuppressive effects [67]; (c) by interacting with target cells via its surface receptors, such as cytotoxic T lymphocyte-associated antigen (CTLA)-4, to inhibit immune function by binding to CD80/CD86 on the surface of effector cells [68], thereby inducing effector cells to secrete indoleamine 2,3 dioxygenase (IDO), which catalyzes the conversion of tryptophan in tissues to kynurenine. The loss of tryptophan inhibits the activation of effector cells and induces apoptosis [69] . The increased kynurenine not only inhibits the immune function of effector cells, but also regulates Treg proliferation and differentiation by acting on the aromatic hydrocarbon receptor (AhR) [70]. Specifically, invasive Tregs in pancreatic cancer can bind to CD80/CD86 on the surface of dendritic cells via surface CTLA-4 to inhibit the function of dendritic cells in tissues, which, in turn, affects the function of cytotoxic CD8+ T cells to suppress immune responses in pancreatic cancer [71]. Tregs surface also expresses costimulatory molecules belonging to the CD28 family----induced costimulatory molecules (ICOS) [72]. ICOS binds to ICOSL, a receptor on the surface of effector cells, and promotes the secretion of inhibitory cytokines, especially IL-10 [73]; and (d) conversion of intercellular ATP to adenine through the cell surface receptors CD73 and CD39, with adenine binding to the adenosine receptor A2AR on the surface of effector cells and inhibiting its proliferation and immune function [74]. (Fig. 2).

As the tumor microenvironment undergoes a series of changes in physical and chemical properties, Tregs infiltrating tumor tissues are also metabolically reprogrammed to meet the needs of differentiation, proliferation, and function. Metabolic reprogramming is primarily involved in glycolysis, fatty acid oxidation (FAO), and oxidative phosphorylation (OXPHOS). Glycolysis is a process in which glucose is degraded in the cytoplasm to produce pyruvic acid and then reduced to lactic acid in an oxygen-deficient state. It can quickly provide the required energy to the cells, and the metabolites can participate in the growth and function of the cells as substrates for other reactions. FAO is a process in which intracellular fatty acids are oxidatively decomposed into CO₂ and H₂O by the action of related enzymes and release a large amount of energy. OXPHOS mainly occurs in mitochondria, the process by which the metabolically produced active substance is electronically coupled through the respiratory chain to ultimately couple to form ATP. These metabolic changes interact to affect the immune function of Tregs.

Hypoxia in the microenvironment leads to high expression levels of the transcriptional regulatory factor, HIF, in tissue cells, which are positively correlated with the degree of hypoxia [88]. HIF is a transcriptionally active heterodimer consisting of an α-subunit and a β- subunit. HIF-α has three subtypes: HIF-1α, HIF-2α, and HIF-3α [89]. HIF binds to hypoxia response elements (HREs) to promote the transcription of related downstream genes and to express the encoded protein to respond to the effects of hypoxia on cells [90].

The acidic microenvironment not only promotes the release of active substances such as immunosuppressive cytokines by affecting the intracellular metabolic environment, but also affects the biological and physical properties of tumor cell membranes to promote exosome release [91]. Exosomes are small vesicles containing complex RNA and proteins for information exchange between cells [92].Caveolin-1 can also promote the release of tumor-derived exosomes under acidic conditions [91]. Similarly, microenvironment hypoxia may promote the secretion of exosomes by tumor cells by activating the HIF signaling pathway [93].

Owing to their rapid proliferation and invasion and metastasis characteristics, tumor cells have large differences in their metabolic activities from normal cells. There are many cell types that infiltrate the microenvironment in which the tumor cells are located, including immune cells and mesenchymal cells [118]. Changes in metabolic activity and an increase in infiltrating cells lead to a change in the physicochemical properties of the tumor microenvironment, the most notable of which are hypoxia and pH reduction. Metabolic reprogramming and microenvironmental changes will cause changes in cell-associated transcription factors and secretory substances, which will affect the occurrence and development of tumors.

Tregs are T cells that suppress immunity and heavily infiltrate tumor tissues, regulating tumor proliferation, invasion, and metastasis. Metabolic reprogramming and microenvironmental changes generally play a role in promoting the function of Tregs: i) invasion of Tregs into tumor tissue by cytoskeletal rearrangement or secretion of chemokines; ii) induce of transcription factors or suppressive cytokines that promote the differentiation of T cells into Tregs; and iii) stimulation of the immunosuppressive function by regulating metabolism or promoting Treg secretion of functional cytokines. There are probably still chemokines, cytokines, and relevant signaling pathways to be discovered with the above modes of action. The effects of metabolism on Tregs are two-sided. Glycolysis can promote the proliferation of Tregs but it inhibits its immunosuppressive function. The metabolites of glycolysis can participate in other metabolic activities that promote Treg function. The effects of metabolic reprogramming on Tregs are not limited to the role of a single signaling pathway or a single type of metabolism. Instead, they are affected by changes in the microenvironment to regulate the cell function. The key transcription factor, HIF-1α, in the tumor microenvironment is also bidirectional in regulating Tregs. The expression of HIF-1α is increased under hypoxia. On one hand, it can inhibit the differentiation of Tregs by promoting glycolysis [119, 120], and on the other hand, it can promote the expression of Foxp3 and promote the differentiation of Tregs. The above phenomenon may be due to the different means of regulating the expression level of HIF-1α in cells. Different cytokines stimulates HIF-1α to activate different downstream signaling pathways, which leads to different functions of regulatory T cells [120]. Intracellular feedback regulation also affects the function of HIF-1α. Tregs affects its function according to the stimulation and regulation inside and outside the cell. In addition to traditional methods of regulation, non-coding RNAs can also affect the function of Tregs. miRs can be secreted by cells in exosomes, and then modulate the Tregs to act on the corresponding target regulatory cells. There are many kinds of non-coding RNAs, and their mode of action is complex. Further research is necessary to explore these regulatory mechanisms of Treg function. Many of the above experiments were performed in vitro, failing to completely simulate the complex dynamic balance of the tumor microenvironment, and Tregs are susceptible to this microenvironment. The experimental results may be inconsistent with the in vivo experiments; therefore, it is necessary to continue the exploration with rigorous experiments.

In addition to surgical treatment, chemotherapy, and radiotherapy, immunotherapy has gained increasing attention, including treatment of PD-L1 and its related signaling pathways [121], inhibitors of CTLA-4 [122], and CAR-T treatment [123]. Tregs as major immunosuppressive cells have an important influence on the occurrence and development of tumors. Although large numbers of Tregs are usually associated with poor clinical outcomes, the role of Tregs in colorectal cancer [124], head and neck squamous cell carcinoma [125], and esophageal cancer [49] remains controversial. This may be a result of the presence of chronic inflammation in tumor tissue that promotes abnormal cell proliferation, angiogenesis, and distant metastasis [126], whereas Tregs act as immunosuppressive cells that inhibit tumor development by reducing chronic inflammation. In addition, Tregs can also be divided into different subtypes according to the expression levels of Foxp3 and CD45RA, and the prognosis of patients with colorectal cancer is closely related to the infiltrating Treg subtype [127]. The complex role of Tregs in tumor progression may also be affected by the tumor microenvironment. Changes in tumor microenvironment and metabolic reprogramming can affect not only the differentiation of primary T cells into Tregs, but also its immune suppression function, and therefore also the balance of Tregs and Th17 by affecting the expression of Foxp3 and RORgt [128, 129]. The above factors comprehensively affect tumor progression. Therapeutic methods targeting Tregs are also receiving increasing attention. However, directly killing the Tregs in the tumor tissue does not improve the patient’s condition, because the adenosine produced by apoptotic Tregs will continue to inhibit the function of the effector cells [130]. Therefore, inhibiting the migration of Tregs to tumor tissues may have an effect on the treatment of cancer. Clinical trials have been conducted for treatment with chemokines, such as the use of monoclonal antibodies against CCR4 in the treatment of lymphoma, and a significant anti-tumor effect has been observed [131]. Regarding tumor infiltrating Tregs, Tregs can be depleted in tumor tissues by targeting the Treg surface-specific receptor, CD25, to inhibit tumor growth. Improving anti-CD25 antibodies and using them together with PD-1 antibodies may lead to a better therapeutic effect [132]. The fusion protein formed by IL-2 and diphtheria toxin can recognize IL-2R on the surface of Tregs to clear the cells. Treatment of renal cancer can effectively reduce the number of Tregs in the body, thereby achieving the purpose tumors treatments [133]. In addition to the expression of the transcription factor Foxp3, Tregs also expresses the transcription factor, Helios, to maintain stability. Deleting Helios will lead to the conversion of Tregs into effector T cells, which significantly delays tumor growth. Therefore, Helios targeting can be used as a feasible treatment strategy [134]. Similarly, targeting of Foxp3 can also help to regulate the expression of Tregs [135]. Moreover the treatment based on metabolic reprogramming has also been used in some immune diseases [136].

Based on the current status of immunotherapy, research still needs to be performed to improve our understanding of the mechanism of interaction between the tumor, Tregs and its subtypes, and other immune cells. Finding key cytokines, signaling pathways, non-coding RNAs that are relevant to Treg’ function is needed, and these will serve as potential targets for immunotherapy. The present review, which highlights the changes in the tumor microenvironment that result in changes in metabolites and thus affect the function of Tregs, enriches the theoretical basis of tumor immunosuppression, and may provide new directions for cancer immunotherapy.