Research progress of Tim-3 and M2 macrophages in immunotherapy in colorectal cancer

Wang Zhujiang, Shang Xueqin, Lu Zhengmin

summary

The aim of this review was to summarize the research progress of Tim-3 and M2 macrophages in the immunotherapy of colorectal cancer (CRC). As an important immune checkpoint molecule, Tim-3 (T cell immunoglobulin mucin-3) plays an important role in regulating T cell function and macrophage polarization, and has attracted extensive attention in the research of tumor immunity and autoimmune diseases in recent years. M2 macrophages, as a polarized state of macrophages, are also closely related to immune escape and progression of tumors. This article will explore how Tim-3 affects the polarization of M2 macrophages and how this interaction affects immunotherapy for colorectal cancer.

Keywords: colorectal cancer; TIM-3; immune evasion; M2 macrophages; immunotherapy;

Keywords: colorectal cancer; TIM-3; immune evasion; M2 macrophage; Immunotherapy;

I. Introduction

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, with high morbidity and mortality. Although surgery is the preferred treatment for early-stage tumors, the side effects of surgery and chemoradiotherapy are greater for patients with recurrence and metastasis. In recent years, immunotherapy, as an emerging treatment method, can kill tumors by blocking immune checkpoint inhibitors (ICIs) to activate immune cells in the tumor microenvironment. However, there are currently no ICI drugs that can effectively reduce the mortality rate of patients with microsatellite stable colorectal cancer. In this context, the role of Tim-3 as an emerging immune checkpoint molecule and M2 macrophages in the immunotherapy of colorectal cancer (CRC) has attracted a lot of attention in recent studies, highlighting their complex interactions in the tumor microenvironment and their implications for therapeutic strategies1.

2. Structure, ligand and biological characteristics of Tim-3

TIM-3Tim-3 (T cell immunoglobulin and mucin domain 3) is a type I transmembrane protein containing an immunoglobulin and a mucin-like domain that is expressed on activated Th1 cells but not on Th2 cellsIt is also expressed on Th1, Th17, and CD8+ T cells of mouse bone marrow lines2. The structure of TIM-3 consists of an N-terminal IgV domain (an amino-terminal immunoglobulin variable domain with five atypical cysteines, the V-domain). ), a mucin-like region containing O-linked glycosylation, an N-chain glycosylation site, a single transmembrane domain, and a c-terminal cytoplasmic domain, The IgV domain is a key region where Tim-3 binds to ligands, and is able to recognize and bind a variety of ligands, such as galectin 9, carcinoembryonic antigen cell adhesion molecule 1, etc. Its gene, named Havcr2, belongs to the TIM family and was first identified in CD4+T helper 1 (Th1) and CD8+ T cytotoxic 1 (Tc1) cells, this family has been shown to have eight members (TIM-1 to TIM-8) in mice, while only three genes (HAVCR1, HAVCR2, and TIMD4) are encoded in humans), which can be expressed in a variety of innate and adaptive immune and non-immune cells3,4. Studies have shown that Tim-3 is expressed in a variety of immune cells, including regulatory T cells (Tregs), B cells, dendritic cells (DCs), macrophages, and NK cells, in addition to CD4+ and CD8+ T cells5.

Ligands for TIM-3 In the tumor microenvironment, the consistently highly expressed TIM-3 molecule can bind to its ligands and interact, and four different ligands have been discovered: galectin-9 (Gal-9) and phosphatidylserine (PtdSer)., high mobility group protein B1 (HMGB1), and carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM-1). Gal-9, the first ligand to be discovered, binds to TIM-3 after glycosylation with IgV, inducing calcium flux into cells and thereby inducing apoptosis6,7. PtdSer is a phospholipid molecule exposed to the surface of apoptotic cells that binds to the Mertk and Axl receptors, which are commonly expressed in tumor cells, Tim-3Binding to PtdSer with a lower affinity than the other three receptors, PtdCell binding to a pocket of the N-terminal IgV domain in the human TIM family stimulates the clearance and engulfment of apoptotic cells, due toPtdCellis susceptible to a variety of factors (intracellular calcium level8, radiotherapy, chemotherapy) in the tumor microenvironment, and the mechanism is still unknown. CEACAM1 as the third ligand of TIM-3, it can also be used in T cells, macrophages, tumor cells (e.g., melanoma), DCs (dendritic cells) Carcinoembryonic antigen-associated adhesion factor-1 can form a heterodimer with the N-terminal domain of TIM-3 in both intracellular and extracellular homeopathic or trans forms, mediate immune tolerance of T cells, promote the maturation and expression of TIM-3 in intracellular or cis-way interactions, and bind TIM-3 extracellularly to release HLA-B-related transcripts3(Bat-3), which inhibits TIM-3-mediated TCR (T cell receptor) signaling. Therefore, TIM-3-CEACAM1 plays a crucial role in autoimmune and anti-tumor immunity9. In the tumor microenvironment, Tim-3 on tumor-infiltrating DCs binds to the HMGB1 molecule released by tumor cells. HMGB1 is a damage-associated molecular pattern protein that is able to interact with DNA to release from dying cells and facilitate the sensing of nucleic acids through Toll-like receptors (TLRs), thereby promoting tumor immune evasion. In the Nature Immunology study, Chiba et al. found that myeloid cells of Tim-3+ act as molecular sinks for HMGB1 in the tumor microenvironment, interfering with its function in innate immune activation. This discovery provides a potential target for the development of new immunotherapy strategies10.

Biological characteristics Tim-3 plays a negative regulatory role in immune regulation, and its interaction with ligands can induce T cell apoptosis, inhibit immune cell activity, and enable tumor cells to evade the attack of the immune system. There are many views on the transcriptional control of TIM-3 in various acute and chronic inflammation and tumors, and it is known that the expression of Tim-3 is regulated by at least three transcription factors: activated T cell nuclear factor (NFIL3), T-bet and STAT311, and there was a positive correlation between the three and IL-27-induced expression of TIM-3 and IL-10, and in IL-27-stimulated T cells, NFIL3 induced the expression of Tim-3 and IL-10 was more effective than T-bet, and T-bet and STAT3 could synergize with each otherNFIL3 promotes overexpression of TIM-3 and IL-10. 3,12,13IL-10 is a key agonist of M2 macrophage polarization, which indirectly indicates that there is a link between TIM-3 and macrophages to promote tumor cell progression.

3. The role of TAMs in colorectal cancer

Characteristics and Polarization Mechanism of TAMs TAMs, also known as tumor-associated macrophages, are mainly derived from peripheral blood mononuclear cells (MDMS/PBMCs). ), with the blood circulation to various tissues differentiation, or by tumor cells to produce a variety of cytokines and chemokines from the integration mediation recruitment into tumor differentiation, a small part from the yolk sac, colonization in specific tissues. TAMs are mainly divided into two types: M1 macrophages (classical activated cells, pro-inflammatory) and M2 macrophages (alternative activated cells, tumori-prototypical), M2 macrophages are divided into M2a, M2b, M2c three subtypes according to different stimuli, the stimulators of M2a are IL-4 and IL-13, however, IL-4 and IL-13 are the key factors that induce the production of M2 type TAM, and the stimulators of M2b are immune complexes and TLR ligands, The stimulants of M2c are IL-10 and glucocorticoids14. It is worth mentioning that the interaction between macrophages and tumors is not only related to the number of TAMs, but also to the different functional subtypes of TAMs. M1 macrophages promote the type 1 T helper cell (Th1) response, and M2 enhances the type 2 T helper cell (Th2) response, and the products of the two reactions can inhibit the activity of M1 and M2, thereby regulating the immune response. Cell surface markers such as histocompatibility complex II. (MHC-II.), CD68 markers, and CD80 and CD86 costimulatory molecules have been shown to recognize M1, while arginase-1 (Arg-1) is secreted by M2 (M1 and M2 Macrophages: Oracles of Health and). Disease) (enhancer-mediated control of macrophage-specific arginase I expression). Tumor necrosis factor (TNF-α), interferon γ (IFNγ), macrophage colony-stimulating factor (M-CSF), and lipopolysaccharide (LPS) Activates immature monocyte precursors (M0) to differentiate into M1 type TAM, thereby producing a variety of inflammatory factors (such as IL-1, IL-6, IL-12, IL-18, IL-23, reactive oxygen species and TNF), which exert anti-tumor and promote immune response15。

The role of TAMs in promoting tumor growth, angiogenesis, and immune evasionTAMs play a key role in CRC, TAMs are the cells with the highest proportion of tumor microenvironment (TEM), and the interconversion of their phenotypic subtypes stimulates tumor-associated angiogenesis, promotes tumor cell proliferation, invasion, and metastasis. Inhibition of anti-tumor immune responses and other ways promote tumor initiation and malignant progression16. In colorectal cancer, M2 macrophages are closely related to tumor progression and prognosis. In TEM, in terms of tumor cell proliferation, in vitro studies have confirmed that colon cancer cells upregulate the expression of macrophage's complement 32 response gene (RGC-32) through TGF-β secretion. RGC-32 not only promotes M2 macrophage polarization, but also macrophage migration17. TAMs drive oxidative stress, regulate reactive oxygen species (ROS) by changing the activity of NADPH oxidase, and maintain redox status and angiogenesis in the tumor microenvironmentC26 Effect of colon cancer cell proliferation18。 In terms of metastasis and invasion, the VEGFC/VEGFR3 pathway is the cause of lymphatic vessel (LV) production to promote metastasis, because VEGF receptor 3 (VEGFR3) ligand VEGF C (VEGFC) induces lymphatic vessel (LV) expansion, and the VEGFC/VEGFR3 axis is highly expressed on lymphatic vessels and TAMs in primary colorectal cancer, which is essential for the modification of lymphatic vessels and TAMs, and studies have shown that lymphatic endothelial cells (LECs) stimulate the upregulation of PD-L1, which causes T cells to exhaust and thus exert immunosuppressive effects19. Lan, J., et al. have demonstrated that M2 macrophage-derived exosomes (MDEs) are highly expressed in miR-21-5p and miR-155-5p, dependent on migration and invasion of colorectal cancer cellsThese two miRNAs, which are metastasized by MDE, are transferred to colorectal cancer cells, bind to the BRG1 coding sequence and reduce its expression, promoting tumor cell metastasis20。 In terms of angiogenesis, the vascular network and vascular density act as the channels for macrophages to enter and exit, providing high nutrient correlation for tumor cells, TAM can activate VEGF receptor 2 (VEGFR2) to bind to its ligand, and can secrete a variety of angiogenesis to regulate the process of vascular formation and remodeling growth, and can also secrete a variety of inflammatory cytokines (such as: IL-1, IL-6, TNF, etc.). ) promotes the activation of the transcription factor NF-kappaB in colon cancer cells and increases the release of VEGF21,22. In macrophages, Gpr35 stimulates vascular growth by stimulating MMP activity by proton pump Na/K-ATPase23.In addition, M2-polarized TAMs enhanced METTL3-mediated m6A modification to confer oxaliplatin (OX) resistance in cells, suggestedM2-TAMs are an important mediator for the acquisition of oxaliplatin resistance in colorectal cancer patients24.

Interaction of Tim-3 with M2 macrophages

TIM-3 plays an important role as an inhibitor in the activation of macrophages, and the Frisancho-Kiss25 study found that down-regulation of TIM-3 signaling in virus-infected mice led to the expression of CD80 costimulatory molecules on macrophages, thereby promoting the inflammatory response. Administration of anti-tim-3 antibodies in vivo such as Monney26 increases macrophage numbers and activation levels. In Gencheng Han et al. 27, it was also found that down-regulating the TIM-3 signaling pathway on macrophages can enhance the activation of macrophages, or up-regulating the TIM-3 signaling pathway on macrophages can inhibit macrophage activity. These findings suggest that TIM- is an activation marker on macrophages and exerts an inhibitory effect on macrophages. In addition, in colorectal cancer, Tim-3 is associated with macrophage polarization towards the M2 phenotype, which is characterized by anti-inflammatory properties and supporting tumor growth, but the mechanism by which TIM-3 specifically regulates macrophage polarization is not well understoodEnhancing Tim-3 signaling can inhibit the release of inflammatory cytokines from macrophages and promote their M2-type polarization, while attenuating Tim-3 signaling can promote the secretion of inflammatory cytokines in macrophages and enhance their M1-type polarization. On the one hand, Tim-3 plays a negative regulatory role in the secretion of inflammatory factors after macrophage activation. The polarization of macrophages reflects the activation state of TIM-3 to a certain extent, and M1 polarization often reflects its "pro-inflammatory and killer" function by the expression levels of various molecules such as NOS2, CDl6/32, CCR7, MHC-II. and IL-12. M2 polarization, on the other hand, often reflects its "anti-inflammatory and repair" ability by the expression levels of various molecules such as Arg-1, Dectin-1, IL-4Ra, IL-10 and TGFβ. These results suggest that TIM-3 supports the development of tumors by promoting the M2-type polarization of macrophages and inhibiting the production of M1-type polarization-related molecules, resulting in an imbalance in macrophage polarization28,29. In immune-related diseases such as sepsis, inflammatory bowel disease, and gastrointestinal tumors, the expression of Tim-3 and the polarization of macrophages are unbalanced There is also a certain latent relationship with the occurrence and development of diseases. On the other hand, transforming growth factor β in macrophages, both at the protein level and at the gene level, has a positive relationship with TIM-3, and TGFβ can promote the expression of Tim-3 on macrophages. TGFβ down-regulated the expression of ADAM10 and ADAM17 at the gene level, while the shedding process of Tim-3 required the participation of the shedding enzymes metalloproteinases ADAM10 and ADAM17. Secondly, LSP down-regulated the expression of TIM-3 in macrophages and up-regulated the expression of ADAM10 and ADAM17 at the gene level. Once again, TIM-3 has been shown to be a process that drives macrophages to actively exhibit M2 and inhibits their polarization to M1, resulting in an imbalance in macrophage polarization30,31. In the study of Yan 32, blocking TGFβ significantly inhibited the transformation of TIM-3+ macrophages to M2 type, and significantly inhibited the growth and migration of HCC. These findings reveal the role of Tim-3 in regulating macrophage polarization, and how this regulation affects the progression of colorectal cancer and the effect of immunotherapy, providing new research directions and ideas for future immunotherapy combination therapy.

5. Research progress of Tim-3 in the immunotherapy of colorectal cancer

TIM-3 has been evaluated in multiple clinical trials as an important target for immunotherapy. These studies focused on the development of monoclonal antibodies (mAbs) against Tim-3, and after BIS2/LB4330 treatment, tumor infiltrating CD8+ T cells were significantly increased, IFNγ secretion was enhanced, and, The drug administered in combination with chemotherapy and anti-PD-1 antibodies can further enhance the anti-tumor effect and explore the potential of Tim-3 in combination with PD-1/PD-L1 blockade strategy (bispecific antibody (BsAb)), BIS5/ LB1410 is the first PD-1/TIM-3 bispecific antibody in China with the highest clinical progress, and has also shown good anti-tumor and delayed survival effects in clinical practice At present, there is also some progress in the combination of TIM-3 and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) blocking strategies, such as ipalolimab/tovolelimab. Sabatolimab (MBG453) is a monoclonal antibody to the T cell immunoglobulin domain and mucin domain-3 (TIM-3), and spartalizumab is programmed death-1 (PD-1) monoclonal antibody, Giuseppe33 et al., found that the combination of the two was safer and more effective than sabatolimab alone. In glioma treatment34, blockade of Gal-9/TIM-3 signaling inhibits macrophage M2 polarization and inhibits M2-type macrophages to promote plaque angiogenesis, leakage, inflammation, and progressionThe main mode is the CD163/HIF1α/VEGF-A pathway. In breast cancer35, integrins β 3 promote macrophage polarization to replace macrophages, and the M2 regulator peroxisome proliferator-activated receptor γ (PPAR). γ) Participating in integrin-mediated M2 polarization, blocking or reducing its expression can inhibit the increase of specific M2-like phenotype and inhibit tumor growth during tumor progression. In summary, the targeting strategy against macrophages may be a novel therapeutic anti-vascularization approach, first, to promote the repolarization of M2 to M1, and second, to target Tim-3 with bispecific antibodies, The combination of the two may be a promising approach to anti-tumor.

Clinical significance of Tim-3 versus M2 macrophages in colorectal cancer

Expression levels of Tim-3 and M2 macrophages may serve as biomarkers to predict response to immunotherapy in colorectal cancer patients. In addition, their value in prognostic assessment is also being explored. High expression of Tim-3 is associated with poor prognosis in colorectal cancer patients, while polarization status of M2 macrophages is associated with immune escape and progression of tumors. M2 macrophages are known to promote tumor progression through a variety of mechanisms, including secretion of immunosuppressive cytokines and enhanced angiogenesis, thereby promoting tumor vascularization and metastasis36. The presence of M2 macrophages in the tumor microenvironment has been shown to be associated with poor prognosis in patients with CRC. Specifically, the expression of Tim-3 on M2 macrophages has been shown to inhibit T cell activation and proliferation, thereby contributing to the formation of an immunosuppressive environment that allows tumors to evade immune surveillance37. Identification of specific biomarkers associated with TIM-3 and M2 macrophage activity may aid in patient stratification for immunotherapy. For example, high levels of TIM-3 expression on tumor-infiltrating lymphocytes and M2 macrophages may serve as indicators of poor prognosis and resistance to conventional therapies, guiding the selection of patients who may benefit from TIM-3-targeted therapies38. In conclusion, the interaction between TIM-3 and M2 macrophages in colorectal cancer immunotherapy is a key area of research. The dual role of TIM-3 as a checkpoint inhibitor and marker of M2 macrophage polarization highlights the need for further investigation of targeted therapies that can modulate these pathways. By understanding and manipulating the dynamics of Tim-3 and M2 macrophages, the investigators aim to improve the effectiveness of immunotherapy for colorectal cancer and ultimately improve patient outcomes.

Conclusions and prospects

The above-mentioned up-regulation of Tim-3 expression was associated with the enhancement of macrophage M2 polarization, which aggravated the polarization imbalance of macrophages and further aggravated the disease of colorectal cancer. The research progress of Tim-3 and M2 macrophages in the immunotherapy of colorectal cancer shows that they play an important role in regulating the tumor microenvironment and influencing the response to immunotherapy. Future research needs to further explore new therapeutic strategies, and VEGFR3-directed therapy can be used to treat primary colorectal cancer. Cross-communication between colorectal cancer cells and TAMs also provides new opportunities for the treatment of metastatic colorectal cancer. and the design of new clinical trials to evaluate the therapeutic efficacy of these targets, whether we can start from inhibiting macrophage recruitment, maintaining tumor microenvironment stability, depleting M2 macrophages, and promoting the transformation of M2 to M1, A new combination adjuvant therapy strategy for Tim-3 and M2 macrophages was developed, and the tolerability, efficacy and safety of therapeutic drugs were considered from multiple perspectives, so as to provide more effective treatment options for colorectal cancer patients.

References

Research progress of TIM-3 in the field of colorectal cancer immunotherapy DOI: 10.3969/j.issn.1671-3141.2022.62.002）（Pinto ML,Rios E,Durães C, et al. The Two Faces of Tumor-Associated Macrophages and Their Clinical Significance in Colorectal Cancer. Front Immunol. 2019; 10:1875. doi:10.3389/fimmu.2019.01875

Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease

Immune regulation by Tim-3 [version 1; peer review: 2 approved]

TIM‑3 as a promising target for cancer immunotherapy in a wide range of tumors

Research progress of T-cell immunoglobulin mucin 3 in colorectal cancer, Zhengmin Lu, Xueqin Shang

TIM‑3 as a promising target for cancer immunotherapy in a wide range of tumors

Cross-regulation of TLR4 and T cell Ig mucin-3 determines the severity of liver injury in CCl4-induced mouse models

Variation in the external phosphatidylserine of human cancer cells is regulated by flippase activity and intracellular calcium

CEACAM1 modulates TIM-3-mediated tolerance and depletion

Tumor-infiltrating DCs inhibit nucleic acid-mediated innate immune responses through the interaction between the receptor TIM-3 and the alarm protein HMGB1

T-bet is a Th1 transcription factor that regulates the expression of Tim-3

The IL-27/NFIL3 signaling axis drives Tim-3 and IL-10 expression and T cell dysfunction

Targeting STAT3 abolishes Tim-3 upregulation of adaptive resistance to melanoma regulatory T cell PD-1 blockade

Research advances in tumor-associated macrophages in colorectal cancer

Research progress on the role of tumor-associated macrophages in tumors

Tumor-associated macrophages (TAMs) in colorectal cancer (CRC): from mechanisms to treatment and prognosis

Response genes expressing complement 32 in macrophages increase paracrine stimuli-mediated colon cancer progression

Tumor-associated macrophages promote colon cancer cell proliferation in a c26 mouse in an oxidative stress-dependent manner

VEGFC/VEGFR3 pathway activation induces tumor immune escape in colorectal cancer

M2 macrophage-derived exosomes promote cell migration and invasion in colon cancer.

Macrophages: the inflammatory link between angiogenesis and lymphangiogenesis

Activated macrophages induce metastatic behavior in colon cancer cells

Activation of the GPR35 pathway drives angiogenesis in the tumor microenvironment

Tumor-associated macrophages promote oxaliplatin resistance through METTL3-mediated TRAF5 m6A and necrotizing apoptosis in colorectal cancer

Frisancho-Kiss S, Nyland JF, Davis SE, Barrett MA, Gatewood SJ, NjokuDB, et al. Frontier: T-cell Ig mucin-3 reduces inflammatory heart disease by increasing CTLA-4 during innate immunity. J Immunol (2006) 176: 6411-5.

Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, et al. Th1-specific cell surface protein Tim-3 regulates macrophage activation and the severity of autoimmune diseases. Nature (2002) 415(6871): 536-41. doi：10.1038/415536a

Tim-3: Activation marker and activation limiter of innate immune cells Gencheng Han*, Guojiang Chen, Beifen Shen, and Yan Li

Study on the regulatory mechanism of TIM-3 on macrophage polarization

TIM3 is a well-established inhibitory receptor

The effects of LPS and TGF-B on the expression of Tim-3 in macrophages and their mechanisms were preliminarily explored

A-integrin and metalloproteinases (ADAM) 10 and ADAM17 are the major shedding enzymes of T cell immunoglobulin and mucin domain 3 (Tim-3).

The role and mechanism of TIM-3 in tumor-associated macrophage polarization and hepatocellular carcinoma progression

A Phase I/Ib clinical trial of the anti-TIM-3 antibody Sabatolimab alone and in combination with the anti-PD-1 antibody Spartalizumab in advanced solid tumors

CD163+ macrophages promote angiogenesis and vascular permeability, with inflammation of atherosclerosis

M2 polarization of tumor-associated macrophages relies on receptor activation by integrin β 3 via peroxisome proliferators γ upregulated in breast cancer

Pinto ML, Rios E, Durães C, et al. Two faces of tumor-associated macrophages and their clinical significance in colorectal cancer. Preimmunization. 2019; 10:1875.doi：10.3389/fimmu.2019.01875

Brauneck F, Fischer B, Witt M, et al. TIGIT blockade repolarizes AML-associated TIGIT + M2 macrophages to the M1 phenotype and increases CD47-mediated phagocytosis. J Immune Carcinoma. 2022; 10 (12):.doi：10.1136/jitc-2022-004794

Wei C，Yang C，Wang S， et al. M2 macrophages confer resistance to 5-fluorouracil in colorectal cancer by activating CCL22/PI3K/AKT signaling. Onco Aims Ther.2019; 12:3051-3063.doi：10.2147/OTT. S198126