Inhibition or promotion, the potential role of arginine metabolism in immunotherapy for colorectal cancer

Abstract

Colorectal cancer (CRC) is the third most common cancer in the world, with increasing morbidity and mortality. The current diagnosis and treatment plan rely on early detection and comprehensive treatment based on surgery. Still, most patients are in the middle and advanced stage at the time of diagnosis, and the therapeutic effect is very limited. Immunotherapy is a new cancer treatment method. The most commonly used are immune checkpoint inhibitors, specifically anti PD-1/PD-L1 interaction. However, immunotherapy has limitations, with its therapeutic effect closely related to the immune response in the tumor microenvironment. In recent years, the effect of arginine metabolism disorder on the occurrence and development of cancer and tumor immune regulation has attracted wide attention. Studies have confirmed that cancer cells cannot survive without arginine and arginine metabolites. In addition, arginine is essential for the proliferation, differentiation, and survival of tumor-infiltrating lymphocytes. High arginine levels can promote the anti-tumor immune response, thus increasing the effect of tumor immunotherapy. In this paper, the molecular mechanisms of arginine metabolism in the development and immune regulation of CRC are reviewed, with the authors hoping to provide potential evidence for the development of immunotherapy for CRC.

Abbreviation

CRC = Colorectal cancer, dMMR = Mismatch repair deficient, MSI-H = Microsatellite highly Instability, MSS = Microsatellite stability, TME = Tumor microenvironment, MHC = Major histocompatibility complex, pMMR = Mismatch repair proficient, NOS = Nitric Oxide synthase, NO = Nitric oxide, ASS-1 = Arginine succinate synthetase 1, ASL = Argininosuccinate lyase, ARG = arginase, CAT = Cationic amino acid transporter, AGAT = Arginine-glycine amidinotransferase, ADC = Arginine decarboxylase, ADI = Arginine deiminase, ODC = Ornithine decarboxylase, GCN2 = General control non-depressible 2, mTOR = Mammalian target of rapamycin, HIF-1 = Hypoxia-inducible factor 1, VEGF = Vascular endothelial growth factor, ICI = Immune checkpoint inhibitor, ORR = Objective response rate, DCR = Disease control rate, OS = Overall survival, PFS = Progress free survival, TIL = Tumor-infiltrating lymphocyte, TCM = Central memory T cell

KEYWORDS:

• Colorectal cancer
• tumor immunotherapy
• immune checkpoint
• PD-1
• arginine metabolism

Introduction

Colorectal cancer (CRC) is a group of malignant tumors occurring in the colon and the rectum. Among malignancies, Colorectal cancer ranks third in morbidity and second in mortality globally. In 2020, there were more than 1.9 million new cases and 935,000 deaths worldwide (Sung et al. 2021). Men tend to have higher rates of morbidity and mortality than women (Hang and Shen 2021). The number of new cases of CRC worldwide is expected to rise to 2.5 million by 2035 (Dekker et al. 2019). In China, the incidence and mortality of CRC have been on the rise. According to the 2018 China Cancer Statistics Report, there were 376,000 new cases of CRC and 191,000 deaths in China, ranking third and fifth among all malignant tumors respectively, with urban areas far higher than rural areas (Hospital Authority of National Health Commission of the People's Republic of China, Chinese Society of Oncology, Chinese Medical Association 2020; National Cancer Center, China, Expert Group of the Development of China Guideline for the Screening, Early Detection and Early Treatment of Colorectal Cancer 2021). The etiology and pathogenesis of CRC were not clear, and more and more research evidence showed that its occurrence and development were the results of multiple factors such as genetics, environment and lifestyle (Dekker et al. 2019; Boland et al. 2018; Perrod et al. 2021; Kossenas and Constantinou 2021). Most CRCs followed the ‘adenoma-cancer’ sequence and developed slowly from adenomatous polyps or adenomas. Due to atypical early symptoms, most patients were in the middle and advanced stages at the time of diagnosis (Hospital Authority of National Health Commission of the People's Republic of China, Chinese Society of Oncology, Chinese Medical Association 2020; Aran et al. 2016; Hong et al. 2021). At present, the main treatment method for CRC is the surgery-based comprehensive treatment, but the therapeutic effect for some patients with unresectable or metastatic CRC is very limited. Therefore, alternative effective treatments need to be found, one of which is immunotherapy (Johdi and Sukor 2020; Modest et al. 2019; Kishore and Bhadra 2021; Wrobel and Ahmed 2019).

Immunotherapy, a new type of cancer therapy via the patient's own immune system to fight the tumor, has the characteristics of specific recognition compared with standard therapy (Hegde and Chen 2020). Immunotherapy targets tumor antigens on tumor cells, including tumor-specific antigens and tumor-associated antigens. When the immune system is activated, it can mediate the effect of killing tumor cells through the cascade regulation effect of various immune signals (Abbott and Ustoyev 2019; Pulendran and Davis 2020). Cancer immunotherapy can be divided into two categories according to the immune mechanisms involved. Passive immunotherapy, including tumor-targeted monoclonal antibodies, adoptive cellular immunity and oncolytic virus therapy. Active immunotherapy includes immunomodulatory monoclonal antibodies, anticancer vaccines, immunostimulatory cytokines, immunosuppressants, pattern recognition receptor agonists, immunogenic cell death inducers, etc. (Johdi and Sukor 2020). Some reports showed that cancer immunotherapy was very effective for many types of cancer, and some rare malignancies even disappeared completely after immunotherapy (Kumar et al. 2021). Studies have found that mismatch repair deficient (dMMR) / microsatellite highly Instability (MSI-H) in CRC patients can increase tumor-related gene mutations and produce neoantigens, stimulating the body's tumor immune response, which will benefit more from immunotherapy (Le et al. 2017; Zhao et al. 2019; Picard et al. 2020). However, with the progression of cancer, tumors can also evade immune surveillance by modifying their antigens or changing the tumor microenvironment (TME), such as the expression of tumor antigens or major histocompatibility complex (MHC) molecules reduced, the release of immunosuppressive factors, and interference with immune responses through regulatory T cells, tumor-associated macrophages, and myeloid-derived suppressor cells, ultimately leading to immunosuppression (Jin et al. 2020). Therefore, it is necessary to study the immunomodulatory pathway in the microenvironment of CRC (Khalyfa et al. 2021).

In recent years, the dual roles of arginine metabolism in tumorigenesis and antitumor immune response have attracted much attention. Studies have shown that arginine is essential for T cell proliferation and T cell receptor maturation in the TME, and arginine deficiency leads to the loss of the ability of T cells to interact with tumor antigens (Geiger et al. 2016; Sikalidis 2015). However, abnormal arginine metabolism is also a feature of tumor cell metabolism, and various enzymes and metabolites (polyamines, nitric oxide, etc.) in the arginine metabolism pathway are involved in the occurrence and development of tumors, including CRC (Szefel et al. 2019). This suggests that arginine metabolism plays a dual role in tumor progression and antitumor immunity (Zou et al. 2019; Selvi et al. 2019). In recent years, although there have been some studies on arginine metabolism in CRC, for example, appropriate supplementation of arginine can inhibit the excessive proliferation of crypt cells, thus decreasing CRC production, and nitric oxide synthase (NOS) inhibitors can reduce the proliferation of CRC cells, etc. (Karimian et al. 2019). In addition, the role of arginine metabolism in the immunotherapy of CRC is still unclear, and a systematic review is lacking. To better understand the role of arginine metabolism in CRC, this paper reviews the molecular mechanisms of arginine metabolism in the development and tumor immune regulation of CRC. The authors hope that this review will provide new ideas for the treatment of clinical CRC, especially for the development of immunotherapy, and search for potential treatment methods.

The role of arginine metabolism in the human body

Synthesis and degradation of arginine

Arginine is a kind of non-essential or conditionally essential amino acid, which plays an important role in cell proliferation, protein synthesis, endocrine, immune regulation and other biological functions (Böger 2014; Wu et al. 2021). There are three sources of arginine: endogenous synthesis, exogenous intake and protein turnover. In humans and most other mammals (including pigs, sheep, and rats), endogenous arginine synthesis is mainly accomplished through the small – intestine renal metabolic axis. Namely, citrulline is synthesized from glutamine, glutamate and proline in the mitochondria of enterocytes, released from the small intestine, and then ingested primarily by kidneys for arginine production that is catalyzed by arginine succinate synthetase 1 (ASS-1) and argininosuccinate lyase (ASL) (Wu et al. 2009). Although arginine can be synthesized in the liver, the net synthesis amount of arginine in the liver is very low due to the high activity of arginase (ARG) in the liver, which can rapidly decompose arginine into urea and ornithine and participate in the urea cycle (Zou et al. 2019). Exogenous arginine is dependent on the cationic amino acid transporter (CAT) system, which transfers arginine from the extracellular to the intracellular to perform its functions (Szefel et al. 2019). The degradation of arginine requires the involvement of a variety of enzymes and transporters, the main enzymes include arginases (ARGs), nitric oxide synthases (NOSs), arginine-glycine amidinotransferase (AGAT), arginine decarboxylase (ADC), arginine deiminase (ADI), ornithine decarboxylase (ODC), etc. These pathways produce polyamines, nitric oxide, agmatine, glutamine, proline and other metabolic products, which are of great significance in cell proliferation and immune regulation (Zou et al. 2019; Kim et al. 2018). Once arginine metabolism disorder, it is easy to cause the occurrence of tumor.

The role of arginine metabolites in the development of CRC

Arginine metabolism plays an important role in the development of CRC. It has been reported that colon cancer cell lines could not survive in vitro in the arginine-free medium. Studies have found that DNA replication of cancer cells is blocked, and the translation process and cyclin expression can be inhibited by activating the protein kinase General control non-depressible 2 (GCN2) pathway and inhibiting the mammalian target of rapamycin (mTOR) signaling pathway, which can be corrected after the addition of exogenous arginine (Alexandrou et al. 2018; Vynnytska-Myronovska et al. 2016). In addition, enzymes and transporters related to arginine metabolism also participate in the development of CRC. For example, the overexpression of ASS1 and ASL is negatively correlated with the prognosis of CRC; NOS, ARG and ODC are up-regulated and promote the occurrence of colon cancer in CRC; CAT-1, a transporter, is highly specifically expressed in CRC, and is negatively correlated with the pathological grade of the tumor (Du and Han 2021; Graboń et al. 2009). Arginine produces a variety of metabolites through a variety of metabolic pathways, some metabolites are important for the development of tumors.

Polyamines are important products of arginine metabolism. ODC plays a key role in the metabolism of polyamines. Arginine is first converted to ornithine, and then further decomposed into polyamines by ODC, including putrescine, spermine and spermidine. Polyamines can bind with RNA, miRNA, protein and other substances, participate in the transcriptional regulation of genes, and affect the transcription and translation of downstream oncogenes and tumor suppressor genes (Paz et al. 2014). In addition, studies have shown that polyamines are up-regulated in CRC and can promote the proliferation and metastasis of tumor cells, which are often associated with increased ODC activity and expression (Dai et al. 2017). At the same time, ODC inhibitors can reduce the occurrence of colon polyps and adenomas (Battaglia et al. 2014). This suggests that ODC inhibitors could be one of the potential treatments for CRC.

Nitric oxide (NO) is a gaseous signaling molecule, which is the main metabolite of arginine under the catalysis of NOSs, and plays an important role in the physiological and pathological processes of the organism (Böger 2014). NOS can be divided into three types. Among them, calcium ion-dependent NOS subtypes nNOS (NOS-1, expressed in nervous tissues) and eNOS (NOS-3, endothelial type) produce a small amount of NO under physiological stimulation, which is respectively involved in the regulation of blood flow and intestinal dynamics. The expression of non-calcium-dependent NOS (NOS-2, induced type) is stimulated by inflammation, oxidative stress, cytokines or some pathogenic substances, and its overexpression can generate a large amount of toxic peroxynitrite, which will further damage DNA and other macromolecules and destabilize the genome, promoting the occurrence of CRC. Magorzata Krzystek-Korpacka et al. analyzed 55 colon cancer tissues and matched non-cancer tissues, and the results showed that NOS-2 transcription was significantly increased (Brankovic et al. 2017; Bednarz-Misa et al. 2020). In addition, appropriate enhancement of NO can inhibit proline hydroxylase activity and lead to increased expression of hypoxia-inducible factor 1 (HIF-1), which can regulate multiple target genes and thus regulate important biological processes required for tumor survival and development, such as glucose metabolism, cell proliferation, migration and angiogenesis (Zou et al. 2019; Masoud and Li 2015; Robrahn et al. 2020). Otherwise, NO also regulates the tumor immune response in tumor microenvironment, linking innate and adaptive immunity (Sahebnasagh et al. 2022; Vedenko et al. 2020). Some researchers found it can activate innate immune cells, such as macrophages and dendritic cells, and differentiate Th0 cells into Th1 cells, to produce the anti-tumor effect in the early stage of tumorigenesis. However, with the progression of tumor, it proved that NO from NOS-expressing tumoral cells, tumor-associated macrophages and myeloid-derived suppressor cells potentially suppresses T cell-mediated antitumoral responses (Garcia-Ortiz and Serrador 2018; Thwe and Amiel 2018; Weigert et al. 2018). Besides, different concentrations of NO play different roles. A low concentration of NO (<100 nmol/L) promotes tumor progression, while the high concentration of NO (>500 nmol/L) produces cytotoxic effects and has anti-tumor effects (Vannini et al. 2015). Some studies have shown that NO promotes angiogenesis in tumors, such as CRC, and thus contributes to tumor proliferation and migration (Brankovic et al. 2017; Hulin et al. 2019). A photoactive inhibitor of NOS named ‘NS1’ inhibits vascular endothelial growth factor (VEGF) and angiogenesis by reducing NO levels in tumors (Yarlagadda et al. 2017). Other studies have found that in xenotransplantation models of human colon cancer in nude mice, arginine supplementation can increase the concentration of NO to play an anti-tumor angiogenesis effect (Yeh et al. 2010). It has also been found in other animal models that high concentrations of NO reduce tumor angiogenesis and induce cell death by inhibiting the epithelial–mesenchymal transformation process and the PI3K-Akt signaling pathway. In addition, NO sensitizes tumor cells to chemotherapy by mediating the inhibition of the activity of nuclear transcription factor NF-κB (Bonavida and Garban 2015; Baritaki et al. 2010). Altogether, the evidence suggests that NO exerts dichotomous effects within the multistage model of tumors.

Agmatine, also known as decarboxylarginine, is produced by arginine catalyzed by ADC. Studies have shown that agmatine inhibits the expression of ODC and the biosynthesis of polyamines, thus inducing caspase-dependent apoptosis and inhibiting the proliferation of CRC cells (Wolf et al. 2007). Soo Kyung Ahn et al. found that agmatine inhibited the production of NO mainly by inhibiting the activity of NOS (Ahn et al. 2011). In addition, agmatine can reduce the production of polyamines in several ways, such as inhibiting ODC can reduce the source of polyamines, improve the activity of the polyamine-degrading enzyme (polyamine-N-acetyltransferase), promote the metabolism of polyamines, etc. However, agmatine also has cytotoxic effects on normal cells, and its clinical application still needs further exploration (Hesterberg et al. 2018).

Potential application of arginine metabolism in immunotherapy for CRC

Immunotherapy for CRC

At present, immunotherapy as a new effective treatment is gradually appearing in people's sight (Dekker et al. 2019; Modest et al. 2019). Immune checkpoint inhibitor (ICI) is a common immunomodulatory monoclonal antibody, which has made great breakthroughs in the field of tumor immunotherapy in recent years. In malignant tumors such as colon cancer, high activation and overexpression of immune checkpoints can inhibit the anti-tumor immune response and promote the proliferation and proliferation of tumor cells (Seidel et al. 2018). ICI can specifically bind or block the immune checkpoint ligands on the surface of T cells or other immune cell subsets to restore the body's anti-tumor immune function. Among them, the most widely studied immune checkpoint targets are programmed cell death receptor 1 and ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4). The former is more widely used (Wu et al. 2019; Makaremi et al. 2021).

PD-1 (also known as CD279) is a type I transmembrane protein with a molecular weight of approximately 50–55 kDa, consisting of the extracellular IgV domain, the transmembrane region, and the cell tail containing two phosphorylation sites containing the inhibitory motif based on immune receptor tyrosine and the switching motif based on immune receptor tyrosine, first identified in the screening of mouse T cell apoptosis-related genes (Wu et al. 2019; Pascolutti et al. 2016). PD-1 belongs to the CD28/CTLA-4 co-receptor family and is only 21%−33% consistent with the amino acid sequence of other receptors in the family. It can be expressed in CD4, CD8T cells, B cells, macrophages, dendritic cells and other immune cells, mainly T cells. Researchers have found that two ligands of PD-1. PD-L1 is expressed in both hematopoietic and non-hematopoietic cells, PD-L2 is induced and expressed on dendritic cells, macrophages, mast cells, and certain B cell populations (Zak et al. 2017). Under normal circumstances, PD-1 functions as an immune checkpoint to maintain the body's peripheral immune tolerance response, acting as the ‘brake button’ of the immune response (Kumar et al. 2021). The T cells of the organism are activated by antigen recognition to express PD-1 on the cell membrane and produce interferon to induce the normal tissue to express the ligand PD-L1. PD-1 binds to its ligand to inhibit T cell activity and to achieve immune tolerance to autoantigens by negatively regulating the immune response. However, malignant tumors such as CRC also take advantage of this by expressing a large number of PD-L1 molecules to impede the proliferation and function of T effector cells, thereby inhibiting the tumor immune response (Payandeh et al. 2020). These characteristics provide a theoretical basis for immunotherapy targeting the immune checkpoint target PD-1/PD-L1.

The U.S. Food and Drug Administration has approved three ICI agents for CRC: nivolumab, pembrolizumab, and ipilimumab, the former two of which target PD-1 (Johdi and Sukor 2020). Due to dMMR/MSI-H can lead to high tumor mutational burden, increased tumor immunogenicity, and greater benefit from immunotherapy, ICIs are mainly used in CRC patients with dMMR/MSI-H and have achieved significant results (Cohen et al. 2020). Checkmat-142, a phase II clinical trial study in immunotherapy for CRC, showed that compared with the treatment of patients with microsatellite stability (MSS), The objective response rate (ORR), disease control rate (DCR), and overall survival (OS) rate at 12 months were significantly higher with Nivolumab and pembrolizumab than with dMMR/MSI-H at 55% (95% CI, 452-63.8), 80% (95% CI, 71.5–86.6), and 85% (95% CI, 77.0–90.2) (Overman et al. 2018). Another recent phase III clinical trial of immunotherapy for CRC, KEYNOTE 177-Designed to evaluate the efficacy and safety of pembrolizumab versus standard first-line treatment (FOLFOX or FOLFIRI chemotherapy ± targeted bevacizumab or cetuximab) in patients with dMMR/MSI-H metastatic CRC (André et al. 2020). The results showed that the median progress free survival (PFS) in the pembrolizumab group and the control group were 16.5 and 8.2 months (HR = 0.59, 95%CI, 0.45-0.79, P = 0.0002) after the median follow-up of 28.4 and 27.2 months respectively. The PFS time of the pembrolizumab group doubled 60% of patients in the control group who received PD-1/PD-L1 immune checkpoint inhibitor treatment after disease progression. The PFS2 of the pembrolizumab group was superior to that of the chemotherapy group, of which the former group was not achieved, and the control group was 23.5 months (HR = 0.63, 95%CI, 0.45–0.88). Results of the final OS data analysis of KEYNOTE-177 were officially announced at the annual meeting of the American Society of Clinical Oncology (ASCO) in 2021. At the median follow-up of 44.5 and 44.4 months, the median OS of the pembrolizumab group was not reached yet, and the median OS of the control group was 36.7 months, and the risk of death was reduced by 26% (HR = 0.74, 95%CI, 0.53–1.03, P = 0.0359). The pembrolizumab group showed a trend of overall survival benefit, with an overall survival rate of 61% at 36 months and 50% in the control group. The ORR of the pembrolizumab group was 45.1% (20 complete response, 49 partial response), and that of the control group was 33.1% (6 complete response, 45 partial response). The median duration of response (DOR) of the pembrolizumab group was not reached, and that of the control group was 10.6 months. Together, these two studies establish the gold standard status of ICIs, particularly pembrolizumab, in first-line treatment for patients with unresectable or metastatic dMMR/MSI-H CRC. Unfortunately, neither CheckMate-142 nor Keynote-177 published translational surgery rates or R0 resection rates after ICI treatment, so further research is needed to determine whether they can be used in translational therapy (Long et al. 2021).

In China, the results of the KEYNOTE-177 study were also recommended by experts in the GUIDELINES OF CHINESE SOCIETY OF CLINICAL ONCOLOGY (CSCO) COLORECTAL CANCER (2021) (referred to as the guidelines). In the guidelines, patients with dMMR/MSI-H are listed separately. No matter for first-line, second-line or third-line treatment, PD-1 monoclonal antibody immunotherapy is firstly recommended for these patients. The guidelines recommend that pembrolizumab (Class 1A evidence, Class I recommendation) be used for first-line treatment in patients with dMMR/MSI-H. For palliative second and third-line treatment in dMMR/MSI-H patients, PD-1 inhibitors (Class 2A evidence, Class II recommendation) can be used instead of limiting PD-1 inhibitor types (Chinese Society of Clinical Oncology Guidelines Working Committee 2021). This can be said to be a milestone of precision immunotherapy for CRC in China.

However, at present, the treatment effect of ICIs such as PD – 1/PD-L1 inhibitors on dMMR/MSI-H is good in CRC patients, but poor in patients with mismatch repair proficient (pMMR) / microsatellite stability (MSS) (Fan et al. 2021). Some studies have shown that this is related to the significant decrease of T cells (such as CD8 + T cells) in the latter tumor microenvironment (Chen et al. 2021; Breakstone 2021). Therefore, how to improve the number and efficacy of T cells in the tumor microenvironment, enhance the anti-tumor effect of PD-1/PD-L1 inhibitors, and benefit more patient groups have become the key to current research, one of which is the mechanism of arginine metabolism in immune regulation.

Arginine increases the efficacy of immune checkpoint PD-1/PDL-1 inhibitor by promoting tumor immune response

1. Arginine enhances tumor immune response by promoting T cell proliferation, differentiation and survival

Flow cytometry analysis of rectal cancer tissues showed that tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment were mainly CD + 4 and CD + 8 T cells, which were also the main force of anti-tumor immunity. In most cases, however, the function of TILs is inhibited, which contributes to tumor progression (Hadrup et al. 2013; Meraviglia et al. 2017). In recent years, a large number of studies have shown that arginine plays an important role in the proliferation, differentiation and survival of T cells. The deletion of arginine not only hindered the DNA replication of T cells but also down-regulated the CD3 protein chain, affected the signal transduction of the T cell receptor complex, led to the proliferation of T cells, decreased cytokine production or impaired cell function, which could be recovered after the supplementation of arginine or citrulline (Geiger et al. 2016; Sosnowska et al. 2021; Albaugh et al. 2017; Rodriguez et al. 2007; Bansal et al. 2004). Studies in mice and a variety of cancer patients have shown that myeloid-derived suppressor cells inhibit T cell proliferation by upregulation of ARG-1, increasing arginine metabolism in the TME (Raber et al. 2012). Susanne M. Steggerda et al. reported that an ARG-1 small molecule inhibitor named ‘CCB-1158’ could block the proliferation inhibition of T cells mediated by myeloid-derived suppressor cells in vitro (Steggerda et al. 2017). In the mouse model, CCB-1158 decreased tumor growth, and tumor microenvironment analysis showed that CB-1158 increased the expression of tumor-infiltrating CD8 + T cells, NK cells, inflammatory cytokines and interferon-induced genes. Other researchers have found that mitochondrial ARG-2 is a negative regulator of CD8 + T cell activity (Marti et al. 2019). In a mouse tumor model of 17 cases, ARG-2 deletion mice significantly reduced tumor growth and nearly 60% remained tumor-free, which is related to the increased number, activity, and duration of TILs in the tumor microenvironment. Serum arginine concentration in ARG-2 deficient mice was about 1.5 times higher than that in normal mice. In vitro studies have confirmed that increased arginine concentration can significantly improve the survival rate of human and mouse CD4 + T and CD8 + T cells, independent of the mTOR signaling pathway. In addition, arginine induces immature T cells to differentiate into central memory T cells (TCMs), which have long-term memory and can be homed to lymph nodes to receive antigen restimulation (Geiger et al. 2016). When stimulated by antigens, TCMs can produce a large number of clonal Effective Memory T cells carrying the same antigens. Studies have confirmed that TCMs and their derived cloned T cells have the strong anti-tumor ability (Gattinoni et al. 2011; Klebanoff et al. 2005). All these indicate that the increase of arginine level can improve the tumor immune response-ability in the tumor microenvironment.

Interestingly, arginine does not seem to increase immune function by increasing the total number of immune cells. A variety of animal experiments showed that there was no statistically significant difference in the weight of immune organs between the arginine-supplemented animals and the control group (Sun et al. 2020; Lee et al. 2018; Ruan et al. 2020).

1. Arginine combined with immune checkpoint PD-1/PD-L1 inhibitor has a synergistic anti-tumor effect

Increasing arginine levels can improve the efficacy of tumor immunotherapy by increasing the number of TILs and the ability of immune response (Satoh et al. 2020). Some researchers established mouse osteosarcoma models in four groups: the control group, the arginine supplement group, the PD-L1 inhibitor group, and the PD-L1 inhibitor plus the arginine group (the combination group). The results showed that compared with the control group, the number of TILs in the tumor microenvironment of the arginine supplement group was significantly increased, in which the number of CD8 + T cells was increased by about 3 times and the activity was also stronger (He et al. 2017). The researchers also found that arginine supplement alone did not prolong the survival time of mice compared with the control group, but the survival time of mice in the combination group was significantly higher than that of the other three groups, with statistically significant differences (P < 0.0001). In addition, the combination group significantly inhibited the proliferation of myeloid-derived suppressor cells and further enhanced the tumor immune response. In addition to direct arginine supplementation, arginine levels can also be increased by inhibiting arginine metabolism. In another mouse model study of melanoma and colorectal adenocarcinoma, the researchers treated mice with an Arg1-targeted inhibitor in combination with a PD-1 inhibitor and obtained similar results. The combination group was significantly superior to the groups of PD-1 inhibitor alone and arg1-targeted drug alone in improving tumor immune response and prolonging the survival time of mice. The study used mouse models of different tumors, and the results were consistent, suggesting that the combination of drugs may apply to multiple tumors (Aaboe Jørgensen et al. 2021). Fernando P. Canale et al. developed an engineered probiotic named ‘Escherichia coli Nissle 1917’, which can colonize in the tumor microenvironment and convert the ammonia generated in the microenvironment into arginine, increasing the level of arginine and thereby promoting the tumor immune response. Using a mouse model of colonic adenocarcinoma, the engineered bacteria combined with PD-L1 inhibitors improved the antitumor immune response of PD – L1 inhibitors (Canale et al. 2021). These animal model experiments all showed that the combination of arginine elevation and PD-1/PD-L1 inhibitors had a synergistic anti-tumor effect. This provides a potential basis for the further development of immunotherapy in clinical practice.

Discussion

CRC is a group of highly malignant tumors. Arginine metabolism plays a dual role in tumor progression and immune regulation. On the one hand, arginine and its metabolites, such as polyamines and NO, are indispensable in the development of tumors. On the other hand, arginine is essential for the proliferation, differentiation, and survival of TILs. This phenomenon leads to different applications of arginine metabolism in tumor therapy. On the one hand, supplementation of arginine content and inhibition of arginine metabolism can inhibit tumor progression by enhancing tumor immune response. On the other hand, for some tumors with deficient expression of ASS1, such as liver cancer, pancreatic cancer, melanoma, etc., arginine deprivation therapy is very effective due to insufficient synthesis of endogenous arginine. However, for tumors with overexpression of ASS1, such as gastric cancer and CRC, the effect of that is poor (Zou et al. 2019; Feun et al. 2015). Therefore, patients with CRC may benefit more from increased arginine levels. In addition, the mouse CRC model showed that high arginine levels had a synergistic anti-tumor effect combined with PD-1/PD-L1 inhibitors by increasing the number, activity and duration of TILs infiltration in the tumor microenvironment. To sum up, arginine enhancement combined with PD-1/PD-L1 inhibitors is an emerging immunotherapy method with great development potential, but it needs to be further studied in clinical practice. The authors hope that this treatment will benefit more CRC patients in the future.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contribution statement

Literature research: C.C.; manuscript preparation and editing: C.C.; Concept design, manuscript revision/ review: Z.Z. and X.J.; Interpretation, final approval of the manuscript: Z.Z. and X.J.

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.