Dual roles and therapeutic targeting of tumor-associated macrophages in tumor microenvironments

Macrophages are white blood cells located in tissues and are derived from monocytes, which in turn are derived from precursor cells in the bone marrow. Macrophages are involved in both nonspecific and specific immunity and are immune cells with a variety of functions. M1 and M2 cells are two distinct subpopulations of macrophages and are classified according to their activation status and function. M1 macrophages are classically differentiated and activated by interferon-γ²¹ and LPS (cytoplasmic polysaccharides), whereas M2 macrophages are selectively differentiated and activated by Th2 (helper cell 2) cytokines and inflammatory factors such as IL-4 and IL-13.²² In terms of function, M1 macrophages secrete mainly proinflammatory factors and phagocytose pathogens and apoptotic cells, whereas M2 macrophages secrete mainly inhibitory inflammatory factors that suppress inflammatory responses and act on tissue repair and remodeling.²³

M1-like macrophages inhibit tumor cell survival and metastasis in three main ways: first, antibody-dependent cell-mediated cytotoxicity (ADCC) action; second, antibody-dependent cellular phagocytosis (ADCP) action; and third, indirectly modulating immunity through proinflammatory factors; for example, M1-like macrophages can release proinflammatory factors such as IL-1β, IL-6, IL-12, IL-23, C-X-C motif chemokine ligand 9 (CXCL9), CXCL10, TNF-α and major histocompatibility complex (MHC) molecules. Through a constructed mouse model, Elsas et al. demonstrated that late activated M1-type macrophages are critical for effective tumor control. The expression of M1-type macrophage surface proteins (e.g., CD68 and CD80) and the intracellular protein SOCS3 are also upregulated when M1-type macrophages perform antitumor functions. Therefore, the main role of M1-type macrophages is to kill tumor cells and inhibit their growth and metastasis.²⁵ In contrast, M2-type macrophages promote tumor cell proliferation and metastasis by secreting a variety of proliferation-inducing and immunosuppressive cytokines and chemokines, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), TGF-β1, and basic fibroblast growth factor (bFGF).²⁴ Moreover, M2-type macrophages also inhibit CD8+ T cells and promote the growth and proliferation of tumor cells, as well as tumor metastasis, by secreting matrix metalloproteinases (MMPs), serine proteases and histones to destroy the stromal membranes of tumor endothelial cells, as well as by secreting proangiogenic factors and enzymes involved in the regulation of angiogenesis, thus ensuring that tumor tissues have sufficient oxygen and nutrients to promote tumor growth.²⁶

In recent years, M2 macrophages have been classified into four subtypes, M2a, M2b, M2c, and M2d, according to the differences in cytokines and signaling pathways involved in macrophage activation,^27–30 and these four subtypes differ in tumor microenvironments and have their own unique functions, which will be described next. Next, we describe the differences and functions of each of these four macrophage subtypes.

In M2a macrophages, IL-4 and IL-13 are the main cytokines that can polarize macrophages to the M2a phenotype;³¹ M2b macrophages are activated primarily by the expression of high levels of anti-inflammatory cytokines in response to combined exposure to IgG immune complexes and Toll-like receptor (TLR) agonists;^32,33 and M2c macrophages are activated by either glucocorticoid or IL-10-dependent macrophage colony-stimulating factor (M-CSF) signaling, which induces an M2 macrophage subtype.²⁸ Unlike the first three subtypes, M2d macrophages are derived from polarized M1 macrophages²⁸ and are induced by IL-6, TLR, and A2 adenosine receptors.^27,34 In addition, M2b, M2c, and M2d macrophages produce more ATP through anaerobic glycolysis than do M2a macrophages, which are more dependent on the tricarboxylic acid (TCA) cycle and OXPHOS for energy supply.

Each of the four isoforms also has a unique function in the tumor microenvironment. CD206, CD209, Dectin-1, and CCL22 are known to be surface markers of M2a macrophages and are highly expressed on their surface.^28,35 The main roles of M2a macrophages are mainly in tissue reconstruction and remodeling and in the inflammatory response,^28,32 and their functions can be summarized in the following six aspects: 1. M2a macrophages are able to promote tissue reconstruction and remodeling through the release of a variety of cytokines and chemokines by M2a macrophages, and the released factors include IL-10, TGF-β, CCL17, CCL18, etc.²⁸ In addition, some chitinase-like substances play major roles in the mechanism of reorganization;³¹ 2. M2a macrophages can promote the growth of tumor cells. M2a macrophages produce a variety of growth factors and cytokines, such as vascular endothelial growth factor (VEGF) and PDGF, which promote angiogenesis and, in turn, promote the growth of tumors.²⁸ 3. M2a macrophages produce a variety of proteases, such as MMPs and histone proteases, which degrade the ECM and thus promote tumor cell invasion and migration;²⁸ 4. M2a macrophages play a role in anti-inflammatory and antitumor immunity. This role is dependent on the inhibition of T-cell and NK cell activation and proliferation by anti-inflammatory factors and chemokines produced by M2a macrophages;²⁸ 5. M2a macrophages release IL-4, which further promotes the polarization of more M2 macrophages toward M2a, which in turn produces more IL-4, creating a positive feedback pathway;³¹ 6. In response to IgG4, M2a macrophages can also repolarize to M2b macrophages.²⁷

The function of M2b macrophages is reflected mainly in their inhibitory effect on immune responses,²⁸ and they are referred to as regulatory macrophages with immunomodulatory activity.³⁶ M2b macrophages play an immunomodulatory role through the release of many anti-inflammatory and proinflammatory cytokines, such as IL-6, IL-10, and IL-12,³⁷ and they are able to activate Th2 cells,^27,35 promoting naive T-cell differentiation into Treg cells. In addition, a marker of M2b cells is the high expression of CCL1, which is important for the maintenance of the M2b cell subtype.^28,31,38 CCL1 is a chemokine that recruits NK cells, DCs, and other cells by interacting with chemokine receptor 8 (CCR8) on the cell surface²⁸ and attracts CCR8-expressing Th2 and Treg cells,³⁵ which promotes an immunosuppressive microenvironment that promotes tumor cell proliferation, migration, and metastasis.^35,39 M2b macrophages have an autocrine proliferative capacity, which allows the cells to survive without the need for exogenous growth factors; this capacity is mediated by CCL1 signaling. This ability also allows M2b macrophages to survive longer in the tumor microenvironment and thus be able to sustain the suppression of antitumor immune responses.^28,38

M2c macrophages are a subtype of anti-inflammatory M2 macrophages induced by glucocorticoid- or IL-10-dependent M-CSF signaling,^28,40 with CD206, CD163, and Mer tyrosine kinase (MerTK) as the main markers and highly expressed on the surface of M2c macrophages.^35,41 M2c macrophages are capable of releasing large amounts of IL-10 and TGF-β, resulting in their anti-inflammatory activity. In addition, M2c macrophages are able to produce a sustained anti-inflammatory response, which is due to the ability of M2c macrophages to produce GAS6, which is a ligand for MerTK, and when the two combine, they amplify the production of IL-10, and the large amount of IL-10 production causes M2c macrophages to exhibit anti-inflammatory activity; in addition to its anti-inflammatory activity, the release of IL-10 and the overexpression of MerTK promote its phagocytosis, removal of apoptotic cells and cell debris, etc.^28,42 The removal of apoptotic cells not only promotes the release of anti-inflammatory factors, e.g., IL-10, from macrophages but can also trigger an anti-inflammatory response, which enhances the anti-inflammatory response; M2c macrophages also play a proangiogenic role, which is realized by M2c macrophages through the upregulation of genes, such as SPPX2 and VCAN;^27,42,43 and M2c macrophage-released IL-10 released by M2c macrophages exerts a series of matrix remodeling, phagocytosis, and other effects by participating in signal transducer and activator of transcription 3 (STAT3)-mediated signaling, mitogen-activated protein kinase (MAPK), and other pathways.^31,43,44 In terms of tumorigenicity, M2c macrophages play similar roles as M2a macrophages do, e.g., both exert anti-inflammatory effects, promote angiogenesis and thus tumor metastasis and invasion, promote stromal remodeling, etc.^27,31

Unlike other subtypes, M2d-type macrophages are derived from polarized M1 macrophages, and a key factor in the polarization of M2d macrophages is an increase in extracellular adenosine levels, in addition to M2d macrophages being induced by IL-6 and TLRs.^27,29 Both apoptotic and necrotic cells can secrete adenosine, resulting in increased extracellular adenosine levels, which induce the polarization of M2d macrophages.²⁸ On the surface of M2d macrophages, phenotypes such as CD14, CD163, and CD86 are highly expressed, and CCL17 and CCL22 are expressed at lower levels.^31,45 The protumorigenic effects of M2d macrophages are manifested in two main aspects: anti-inflammatory effects and proangiogenic effects. The anti-inflammatory effect is realized mainly through the IL-10 signaling pathway, which is activated to promote mucosal and epithelial cell healing and inflammation. In addition, IL-10 can inhibit the excessive proinflammatory response through the MAPK pathway; IL-10 also inhibits the synthesis of proinflammatory mediators, such as IL-1, IL-6, and IL-8.^27,34,46 In terms of proangiogenic effects, M2d macrophages secrete growth factors (e.g., VEGF and PDGF) and matrix metalloproteinases (e.g., MMP2 and MMP9), which promote angiogenesis and extracellular matrix elaboration and promote tumor metastasis and growth.^28,31,45 There is also potential communication between M2d macrophages and M1 macrophages through the VEGF and CCL3-CCR1 signaling pathways, and the polarization of M1 macrophages to M2d macrophages is facilitated through this pathway.^27,46

In summary, the four subtypes of M2 macrophages, although differing in polarization mode and surface markers, promote tumor growth and metastasis mainly by exerting similar effects, such as anti-inflammatory and angiogenesis-promoting effects.

Chen et al. investigated the effect of M2 macrophages on cancer cell metastasis. They cocultured M1 and M2 macrophages with gastric cancer cells, breast cancer cells and melanoma cells and reported that the number of migrated cancer cells cocultured with M2 macrophages increased significantly and that coculture of M1 macrophages with cancer cells did not affect the number of migrated cancer cells, suggesting that M2 macrophages play an important role in the migration of gastric cancer and breast cancer cells. Migration of gastric cancer and breast cancer. In addition, this research team experimentally reported that the CHI3L1 protein secreted by M2 macrophages may play an important role in promoting cancer cell metastasis.⁴⁷

Macrophages can differentiate into two types, M1 and M2, due to the influence of different cytokines and metabolites in the TME. This classification was initially proposed on the basis of the stimulatory response of macrophages to type 1 or type 2 cytokines in in vitro experiments,^48,49 but with the continuous development of technology and the deepening understanding of macrophages, we have shown that macrophages have a high degree of plasticity and heterogeneity and that they can reregulate their phenotype in response to different bits of environmental stimuli. Therefore, it is overly simplistic to categorize them as M1 and M2 only.^50,51

Single-cell sequencing is a technology that sequences and analyzes genomes, transcriptomes and epigenomes at the single-cell level. Single-cell sequencing technology has been widely utilized because it is a good solution to the problem of loss of information on cellular heterogeneity caused by the traditional technique of sequencing on a multicellular basis. In recent years, many researchers have discovered new tumor-associated macrophage subtypes via single-cell sequencing technology, and these subtypes play important roles in tumor growth and proliferation, which affects the prognosis of patients. Q. Zhang, Y. He et al. collected six macrophage clusters in hepatocellular carcinoma and reported that among them, THBS1+ macrophages (myeloid-derived suppressor cell (MDSC)-like macrophages) and C1QA+ macrophages (TAM-like macrophages) were enriched in tumor tissues; these features are similar to those of TAMs but more complex than those of the classical M1/M2 model is, thus identifying these two types of macrophages as new subtypes.⁵² Zhang et al. also used single-cell sequencing to identify a new macrophage subtype, FABP4+C1q+, in which two genes, fatty acid binding protein 4 (FABP4) and the complement C1qA chain, are highly expressed and serve as marker genes. In addition, the team reported that FABP4+C1q+ macrophages focus on positive regulation of cytokine production, the inflammatory response, chemokine production, neutrophil activation, leukocyte chemotaxis and migration and have increased proinflammatory cytokine secretion, phagocytosis, and antiapoptotic functions. These effects may constitute one of the main mechanisms by which FABP4+C1q+ macrophages exert antitumor effects. The antitumor capacity of FABP4+C1q+ macrophages was also verified by a team that used tumor tissues from non-small cell lung cancer (NSCLC) patients and reported that there was a better prognosis with FABP4+C1q+ macrophage enrichment.⁵³ In glioma cells, Xu et al. identified a macrophage subtype with high FN1 gene expression, defined it as FN1+ TAMs, and reported that FN1+ TAMs play a key role in glioma recurrence.⁴⁸ In addition, in melanoma, Wu et al. identified and obtained a subpopulation of MerTK+ macrophages and reported that this subpopulation has potent immunosuppressive activity that promotes tumor growth.⁵⁴

Using single-cell sequencing technology, multiple novel macrophage subpopulations can be identified, and they influence tumor progression through different signaling pathways; for example, aryl hydrocarbon receptor (AHR)-ALKAL1 signaling is a key regulator of the MerTK+ macrophage subpopulation,⁵⁴ and in-depth studies of these novel macrophages may be able to identify new targets for tumor therapy.

The macrophage phenotype is plastic and can change in response to cytokines, cell‒cell interactions and tissue-specific signals. Immunosuppressive molecules and inhibitory pathways, including mechanistic targets of the NF-κB and rapamycin (mTOR) signaling pathways, are involved in macrophage differentiation. TAMs respond to sufficient upstream activation signals to produce abundant reactive oxygen species (ROS), which subsequently mediate immunosuppressive activity via the NF-κB pathway. It is hypothesized that the NF-κB pathway manipulates signal activation in cancer cells and tumor-infiltrating leukocytes to promote inflammatory responses in the TME. For example, Clec4e molecules can activate the NF-κB signaling pathway via Syk kinase, which in turn promotes the protumorigenic effects of TAMs.⁶⁸ Therefore, blocking TNF-α with anti-TNF-α antibodies may be therapeutically useful.⁶⁹ IFN-γ is an anti-inflammatory factor that inhibits the production of TAMs, thereby reversing the immunosuppressive and tumorigenic properties of TAMs,⁷⁰ whereas the NF-κB signaling pathway is involved in the regulation of macrophage activation, which mediates cytotoxicity against tumor cells.⁷¹

Another pathway that promotes inflammation and tumor cell proliferation is the STAT pathway. TAMs have multiple effects on the STAT signaling pathway, and they can activate or inhibit the STAT signaling pathway by secreting cytokines and growth factors, thereby affecting tumor growth and progression. TAMs can secrete a variety of cytokines, such as IL-6 and IL-10, which can activate Janus kinase (JAK), which phosphorylates STAT3, causing it to form a dimer and translocate to the nucleus, initiating the transcription of downstream genes. The activation of STAT3 promotes the expression of a variety of genes, including VEGF, MMPs, and cyclin D1, which are associated with tumor angiogenesis, invasion, and progression. tumor angiogenesis, invasion and proliferation. Studies have shown that STAT3 can also promote the expression of programmed death-ligand 1 (PD-L1), which inhibits the antitumor activity of T cells. One study revealed that in ovarian cancer, STAT3-activated TAMs can express PD-L1 and bind to programmed cell death protein 1 (PD-1) receptors on T cells, leading to T-cell inactivation.⁷² The activation of STAT5 can promote the expression of various genes, including Bcl-xL and Mcl-1, which are related to cell survival and proliferation.

Angiogenesis is an important process in tumor biology and plays a key role in promoting tumor nutrition and oxygen supply; metabolism and growth; invasion and metastasis; and remodeling of the tumor microenvironment. Among them, the VEGF pathway and hypoxia-inducible factor 1α (HIF-1α) pathway are strongly influenced by TAMs, which can secrete VEGF, activate the VEGF pathway, and promote tumor angiogenesis. Moreover, HIF-1α is upregulated in TAMs, which can increase VEGF expression and further promote angiogenesis. Therefore, inhibiting the angiogenic process has become an important strategy in tumor therapy, by which angiogenesis can be inhibited to slow or stop tumor growth and metastasis. For example, antiangiogenic drugs such as Avastin and Sorafenib have been used to treat certain types of cancer.

MMPs are a group of protein hydrolases capable of degrading the ECM; they play a key role in tumor invasion, metastasis, and angiogenesis, and the action of TAMs on the MMP pathway involves a variety of mechanisms. TAMs can activate the production and secretion of MMPs by secreting a variety of cytokines, such as TNF-α, IL-1β, and TGF-β, to activate the production and secretion of MMPs. One study revealed that TAMs can induce the production of MMP-2 and MMP-9 via IL-1β.⁷³ Moreover, certain proteases secreted by TAMs, such as histone B (Cathepsin B), can activate inactive pre-MMPs (e.g., pre-MMP-2 and pre-MMP-9) to become active forms. Active MMPs can degrade the ECM, providing a pathway for tumor cell invasion and metastasis. TAM-mediated activation of the MMP pathway also promotes the invasion and metastasis of breast cancer tumor cells.

First, TAMs can express PD-L1, one of the ligands for PD-1. PD-L1 binds to the PD-1 receptor on T cells and inhibits T-cell activation and function. One study revealed that in ovarian cancer, TAMs express PD-L1 and bind to the PD-1 receptor on T cells, leading to T-cell inactivation.⁷⁴ TAMs also express the PD-1 receptor. The activation of PD-1 can inhibit the phagocytosis and clearance of TAMs, which can affect the immunosurveillance role of TAMs. A previous study revealed that the inhibition of PD-1 enhances the phagocytosis of TAMs, thereby prolonging survival time in a mouse model of colon cancer.⁷⁵ In addition, TAMs can interact with T cells and inhibit the antitumor immune response of T cells through the PD-1/PD-L1 signaling pathway. This interaction can lead to T-cell depletion and dysfunction, thereby promoting tumor growth and metastasis.

Second, the action of TAMs on the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) pathway involves multiple mechanisms. CTLA-4 is an immune checkpoint molecule that is expressed on tumor cells and immune cells and inhibits the activation and proliferation of T cells. TAMs can express CTLA-4 and bind to the CTLA-4 ligands B7.1 and B7.2 on T cells, which inhibits T-cell activation and proliferation.⁷⁶ This inhibition reduces the killing of tumor cells by T cells, thereby promoting tumor growth. TAMs can also secrete immunosuppressive molecules, such as IL-10 and TGF-β, which can act synergistically with the CTLA-4 pathway to further enhance the immunosuppressive effect.

In terms of cell survival and proliferation, TAMs promote cell survival and proliferation by activating the PI3K/Akt pathway while inhibiting apoptosis.⁷⁷ TAMs also activate the Ras/ERK pathway, which is involved in cell proliferation and differentiation.⁷⁸ In terms of the cell‒cell interaction signaling pathway, TAMs interact with the extracellular matrix via integrins to influence cell adhesion and migration. In addition, TAMs have intrinsic signaling pathways, i.e., the CSF-1/CSF-1R pathway. CSF-1 binds to its receptor (CSF-1R) and is a key regulator of TAM survival and function.⁷⁹

The activation and regulation of these signaling pathways in TAMs are very complex, and they are intertwined with each other, not only affecting tumor growth and metastasis but also participating in the development of the tumor microenvironment, which plays an important role in immune escape and therapeutic resistance. Therefore, an in-depth understanding of these signaling pathways can help in the development of new tumor therapeutic strategies to improve the efficacy of treatment by regulating the function of TAMs.

During tumor progression, acute and chronic inflammation, wound healing, and the female reproductive cycle, the original vascular system teeth out epithelial cells to form new blood vessels via a process called angiogenesis. During angiogenesis, factors such as VEGFs and placental growth factor (PIGF) stimulate quiescent endothelial cells to release proteases such as MMP-9, thereby reducing intercellular adhesion and degrading the basement membrane. Disruption of intercellular adhesion and the continuous basement membrane results in blood vessels that are not in their normal state, become distorted or inflated, and are more prone to invade the tumor microenvironment and become locally hypoxic. Tumors require a vascular bed formed by endothelial cells to provide nutrients and oxygen and carry away waste and carbon dioxide.⁸⁰

Tumor-associated macrophages are involved mainly in tumor angiogenesis through the following three mechanisms: (1) the hypoxic tumor microenvironment stimulates macrophages to overexpress HIF, which acts as a transcription initiation factor that binds to the promoters of target genes, such as VEGF-A, and induces the expression of VEGF-A;⁸¹ macrophages express factors such as IL-1β, TGF-β, and TNF-α, which stimulate fibroblasts and adenocarcinoma cells to express VEGF;⁸² (2) macrophages are capable of secreting proteases such as MMP-7,⁸³ MMP-9,⁸⁴ and MMP-12;⁸⁵ and (3) macrophages may also differentiate into endothelial-like cells (expressing Tie2), which reside at sites of intense angiogenesis and promote angiogenesis through the expression of VEGF-A, MMP-9, and cyclooxygenase-2 (COX2).^86,87

In addition, TAMs can help tumors evade immune surveillance by suppressing adaptive immune responses. Organisms can inhibit tumorigenesis and development through natural and acquired immunity, while tumor cells can evade recognition and attack by the organism's immune system through a variety of mechanisms. The inhibition of antitumor immunity by TAMs involves the following mechanisms: (1) tumor cells produce IL-10, which induces the expression of PD-L1 on the surface of TAMs,⁸⁸ binds to PD-1 on the surface of T cells in the TME, and inhibits cytotoxic T-cell function; (2) TAMs produce CCL22, which recruits regulatory T cells into the TME and inhibits the activation and function of effector T cells;⁸⁹ and (3) TAMs produce Arg-1, which catalyzes the hydrolysis of L-arginine to urea and L-ornithine, inhibits the upregulation of cytokinin D3 and cytokinin-dependent kinase 4, and prevents the T-cell cycle from proliferating by arresting in the G0/G1 phase.⁹⁰ Overall, TAMs play dual roles as "immune suppressors" and "tumor promoters" because they can promote tumorigenesis and act as central drivers of the immunosuppressive TME.

The process of specific recognition and clearance of tumor cells by immune cells is complex, and macrophages are among the most important members of this process. TAMs are key components of leukocyte infiltration and are widely observed in a variety of tumors. In most studies, the density of TAMs has been found to be associated with poor prognosis in cancer patients,⁹¹ whereas very few studies have shown that the density of TAMs in the TME is associated with good prognosis.⁹² This duality has been found in a variety of cancers, including prostate, lung, and brain cancers.⁹³ Other researchers have reported that TAMs inhibit the growth and metastasis of osteosarcoma and are associated with a favorable prognosis. Both the M1 and M2 isoforms of TAM inhibit the growth of osteosarcoma cells under certain conditions.⁹⁴ The dual nature of the TME may be due to the influence of other cell types present within the TME.

First, macrophage-mediated programmed cell removal (PrCR) plays an important role in tumor elimination and monitoring. The activation of the TLR pathway in macrophages induces the activation of Bruton's tyrosine kinase (Btk) signaling pathway,⁹⁵ which dissociates from endoplasmic reticulum cell surface calreticulin (CRT) phosphorylation. The dissociated CRT is expressed on the surface of macrophages, which then forms the CRT/CD91/C1q compound, which targets cancer cells for phagocytosis.⁹⁶ Second, activated macrophages can also defend against tumors by directing tumor cytotoxicity and secreting cytokines. For example, M-CSF and muramyl dipeptide (MDP) are added to macrophages in in vitro culture to enhance macrophage cytotoxicity, or immunomodulators are loaded by intravenous injection of liposomes to increase macrophage toxicity. Molecules of microbial agents and pathogens can also stimulate antitumor cytotoxicity in macrophages, as in the case of the use of Bacillus Calmette-Guerin (BCG) in the treatment of bladder cancer, which increases macrophage cytotoxicity against certain bladder cancer cell lines by stimulating macrophages.⁹⁷ Moreover, many studies have shown that TAMs have the ability to phagocytose and remove damaged cells. In the early stage of tumorigenesis, TAMs can phagocytose and remove abnormal or damaged cells, preventing them from developing into tumor cells.⁹⁸ Moreover, TAMs can degrade the extracellular matrix by secreting MMPs, a process that is usually promoted in tumorigenesis. However, studies have demonstrated that under appropriate conditions, MMPs can promote the clearance of tumor cells. Moreover, TAMs may inhibit the self-renewal ability of tumor stem cells by secreting antitumor factors, regulating metabolic pathways, suppressing stemness gene expression, and modulating immunosuppressive cells, thereby reducing tumorigenesis.⁹⁹

In the early stages of tumor development, since free radicals produced by macrophages often lead to DNA damage, which in turn causes mutations and host cell transformation, it is widely believed that in the early stages of cancer development, macrophages exist in a form similar to classically activated macrophages with an inflammatory phenotype, contributing to the early eradication of transformed cells.¹²⁵ However, as the tumor progresses and grows, the tumor microenvironment significantly influences the onset/development of TAMs, and these macrophages take on a phenotype that more closely resembles that of regulatory macrophages.¹²⁶ Regardless of the stimulus, this new class of macrophages produces high levels of IL-10 to inhibit immune responses to neoantigens expressed by tumor cells and can inactivate neighboring macrophages.^127,128 It has also been demonstrated that regulatory macrophages promote angiogenesis, which in turn promotes tumor growth.¹²⁹

Some studies have classified mouse Th lymphocytes into Th1 and Th2 cells on the basis of their respective cytokine production (IFN-γ and IL-4).¹³⁰ These cytokines have cross-regulatory properties that are critical for triggering IFN-γ production and Th1 cell development or IL4/IL-13 secretion and Th2 cell proliferation.^81,131 IL-12 or IL-10 production likewise sets the stage for M1/M2-type cell polarization.^62,132–134

IFN-γ is the only type II IFN that is recognized by a receptor consisting of two ligand-binding IFN-γ receptor 1 (IFNGR1) chains and two signaling IFNGR2 chains.¹³⁵ It is now well established that IFN-γ is involved in macrophage "initiation" by increasing macrophage responses to inflammatory molecules such as Toll-like receptor ligands and tumor necrosis factor.¹³⁶ Muller et al. demonstrated that IFN-γ synergized with Toll-like receptor ligands to induce tumor-killing activity in preconditioned macrophages and enhanced the expression of TNF-α and IL-12 family cytokines.¹³⁷ Furthermore, in the TME, IFN-γ produced by cytotoxic immune cells increases the number of iNOS(+)CD206(-) M1-type macrophages, thereby inhibiting tumor growth.¹³⁸ iNOS(+)CD206(-) M1-type macrophages have been shown to be correlated with a favorable prognosis in a variety of tumors, such as breast, lung, ovarian, and gastric cancers.^139–142 On the other hand, M1-type TAMs in the TME secrete CXCL9, CXCL10, and CD86, which stimulate CTL recruitment to the TME and its activation, and recruit CTLs to produce IFN-γ, which is equally important for maintaining M1-type TAM activity and tumor suppression.¹⁴³ However, IFN-γ also induces the apoptosis of CD4+ T cells and reduces secondary antitumor immune responses.¹⁴⁴

While the immunity of Th2 cells to parasites and the pathogenic role of allergic diseases are well established, the regulation and function of Th2 cells in the TME remain largely neglected and controversial. In general, Th2 cell immunity to tumors is mediated by Th2 cytokines and acts synergistically with macrophages through secondary recruitment of tumor-killing myeloid cells, such as eosinophils.^145,146 It has been demonstrated that mice deficient in the Th2 cytokines IL-2 and IL-4 show reduced tumor clearance.¹⁴⁷ Neutralization of IL-4 by monoclonal antibodies may also help restore tumor growth.^148–152 The possible mechanism involves the secretion of the cytokines IL-4 and IL-13 by Th2 cells, which prompts the transformation of TAMs into M2-type macrophages, which in turn promotes tumor growth.

Th2 cytokine production and type 2 immunity are also mediated by type 2 intrinsic lymphocytes (ILC2s). ILC2s also secrete Th2 cytokine and are dependent on the Th2 cell transcription factor GATA binding protein 3 (GATA3) but lack TCR expression.^153–155 Notably, Th2 cells in the TME correlate with the progression of breast and cervical cancers.^156–158 In addition, type 2 immunity, largely driven by Th2 cells, has been shown to promote tumor metastasis in breast, colorectal, and lung cancers.^159–162

Th17 cell development is distinct from that of Th1, Th2, and Treg cells, and a number of mouse models have shown that Th17 cells can promote CD8+ T-cell-mediated antitumor immune responses.¹⁶³ In addition, the polarization of CD8+ T cells to Tc17 cells also increases antitumor immunity.¹⁶⁴

CD8+ T cells are cytotoxic T lymphocytes whose main role is to kill tumor cells directly. As mentioned previously, TAMs release immunosuppressive cytokines such as IL-10 and TGF-β, which directly inhibit the function of CD8+ T cells. Studies of the immunomodulatory function of TAMs have shown that they can inhibit CTLs and reduce their tumor-killing capacity in a number of ways.¹⁶⁵ For example, the expression of PD-L1 and B7-1 (or CD80), which are ligands for the inhibitory checkpoint receptors PD-1 and CTLA4 expressed by activated T cells, leads to T-cell functionality by binding to PD-1 on CD8+ T cells "exhaustion". Additionally, TAMs can reduce the bioavailability of L-arginine through the production of arginase 1 (ARG1), which is critical for T-cell function.^165–168

CCL17 (also known as thymic and activation-regulated chemokine TARC) and CCL22 (also known as macrophage-derived chemokine MDC) are ligands for CCR4.^169–171 The expression of CCL17 and CCL22 is elevated in breast cancer tumors^,172 and the expression of CCL22 is increased in colorectal adenocarcinomas.¹⁷³ CCL22 secreted by M2-type TAMs and other chemotactic factors can attract Tregs into the tumor microenvironment by attracting CCR4, the receptor for CCL22, to be highly expressed on Tregs; cytokines secreted by TAMs, such as IL-10 and TGF-β, can promote the proliferation and activation of Tregs; and at the same time, Tregs can in turn counteract macrophages, which tend to differentiate into M2-type macrophages, thus further supporting tumor growth. Recent findings have emphasized the integration of M2-polarized macrophages with immunostimulatory pathways to induce the differentiation of Treg cells.¹⁷⁴

Natural killer T cells are a heterogeneous lymphoid population that may have both immune-enhancing and immune-suppressive effects, and in tumor immunity, the two NKT subpopulations (type I and type II) play opposing roles, which not only cross-modulate each other but also influence the innate immune cell population. In liver–lung metastasis models, type I NKT cells can rapidly release large amounts of Th1-type cytokines, such as IFN-γ, upon recognition of specific lipid antigens on the surface of tumor cells or macrophages.^175–178 As mentioned previously, IFN-γ can induce polarization of TAMs toward the M1 type (antitumor phenotype) to enhance tumor immunity; CXCL16 is classified into a chemokine α subfamily and is induced by a variety of cells.¹⁷⁹ CXCL16 is important for monocyte polarization to become macrophages in the tumor microenvironment, and when CXCL16 attracts monocytes, it is involved in recruiting them into the tumor ecotone, which then differentiates them into TAMs.^180,181 Studies have shown that soluble CXCL16 (sCXCL16) may also be a macrophage-polarizing factor. sCXCL16 may also be a macrophage polarizing factor, and such polarized macrophages display characteristics of the M2 macrophage subpopulation: increased expression of CD163 and decreased expression of CD80, CD86, and HLA-DR. In addition, the secretion of large amounts of IL-10 and IL-15 by these macrophages also inhibits normal NK cell function.¹⁸²

The escape of tumor cells from immune surveillance is one of the key events regulating tumor growth, survival and metastasis. TAMs in an M2 macrophage-like state have poor antigen presentation capacity and suppress the immune response of T cells by releasing the immunosuppressive factors IL-10 and TGF-β, which include the inhibition of the cytotoxic function of NK cells.¹⁸³ Kuang and colleagues reported that TNF-α and IL-10 secretion by activated monocytes strongly induced PD-L1 expression in an autocrine manner and that PD-L1-positive monocytes induced T-cell dysfunction, which was defined as low cytotoxicity against tumor cells and reduced T-cell proliferation.¹⁸⁴ TAMs can bind to inhibitory receptors on NK cells, such as PD-1, through the expression of such inhibitory receptor ligands, which can in turn inhibit NK cell function.

Clinical experiments have confirmed that TAMs can help tumor cells form an inflammatory environment after tumorigenesis, which is conducive to the growth of tumor cells, while their ability to promote angiogenesis enhances the migration and invasion ability of tumor cells.²⁴⁷ Tumor cells are able to secrete some cytokines to recruit TAMs through their cellular pathways of action, further constructing a tumor microenvironment that is conducive to the growth of tumor cells and contributes to tumor development.²⁴⁸ Therefore, termination of macrophage recruitment in cancer tissues can block macrophage growth and signaling pathways of differentiation, thus reducing the ability of TAMs to promote tumor development and spread.

The expression of cytokines within tumors promotes the proliferation of TAMs. For example, the signaling pathway hosted by CSF-1/CSF1R plays an essential role in the recruitment of TAMs and the promotion of polarization of TAMs toward the M2 type.²⁶⁰ CSF1R inhibitors promote the depletion of M2-type TAMs in a tissue-specific manner, revealing the potential of CSF1R as a therapeutic target for cancer treatment. Strachan et al.²⁶¹ investigated a small molecule inhibitor of CSF1R, BLZ945, and found that tumor volume was reduced in the model treated with BLZ945. Further experiments revealed that BLZ945 exhibits tissue specificity in the clearance of TAMs, with incomplete clearance of macrophages from neoplastic lesions and the surrounding cervical stroma observed in a cervical cancer model after treatment with BLZ945. Similar results were obtained in the breast. Notably, in a mouse model of microglia,²⁶² instead of removing TAMs, BLZ945 inhibited the polarization of TAMs toward the M2 phenotype, thereby affecting tumor progression. These findings suggest that the effect of BLZ945 on TAMs is tissue specific. Another CSF1R inhibitor, pexidartinib (PLX3397), also has a depleting effect on the removal of TAMs upon acceptance and dose-dependently downregulates the expression of genes related to polarization toward M2 and upregulates the expression of genes related to polarization toward M1, which is similar to the action of BLZ94.^263,264

In addition, both BLZ945 and PLX3397 were able to modulate the immune cell infiltration profile. For example, increasing the immune infiltration of CD8+ T cells into tumor sites. In combination therapy with monoclonal antibodies, this can further promote the polarization of TAMs toward the M1 type.²⁶⁵ These findings suggest that CSF1/CSF1R is a viable therapeutic target and may be used in combination with other therapies. These discoveries provide promising therapeutic approaches and treatment ideas for immunotherapy.

Cytokine intervention can influence TAM polarization and offer new immunotherapy possibilities.²⁶⁰. Sun et al.²⁷⁶ found that the combination of monophosphoryl lipid A (MPLA) and interferon-gamma increased the expression of M1 markers (iNOS and CD40) and decreased the expression of an M2 marker (CD206), indicating TAM polarization toward the M1 phenotype. This therapy alters the immune microenvironment by secreting chemokines, increasing oxidative stress in tumor cells, and stimulating MHC class II gene transcription, thus killing tumor cells. Ahirwar et al.²⁷⁷ found that Slit2, a tumor suppressor, could regulate TAMs in the breast cancer tumor microenvironment, including increasing the recruitment of M1-type TAMs and increasing their phagocytosis of tumor cells, which demonstrated that Slit2, a tumor suppressor, could effectively play an antitumor role and revealed the potential of Slit2 as an immunotherapeutic agent. Metformin was reported to be able to reeducate the M2 type to the M1 type. Wei et al.²⁷⁸ used mannose-modified macrophage-derived microparticles (Man-MPs), which exhibit a stronger repolarization ability than free metformin, to load metformin. This is achieved through the AMPK‒NF-κB signaling pathway, which regulates the expression of M1- and M2-type cytokines.²⁷⁹

As mentioned above, evidence demonstrating the potent effects of small metabolic molecules on the CCL2/CCR2 pathways highlights their ability to modulate TAM recruitment, underscoring the intricate crosstalk between metabolic pathways and TAMs. The polarization state of TAMs can reprogram metabolic processes, thereby influencing disease progression.^280,281 Conversely, metabolic changes within the body can also affect TAM polarization.^282,283 This concept is supported by evidence across various biological processes. 3-Oxoacid CoA-transferase 1 (OXCT1), a key enzyme in ketolysis, has been identified as a regulator of TAM polarization. Elevated OXCT1 expression in TAMs promotes the production of succinate, a byproduct with TAM-reprogramming capabilities, which functions via the H3K4me3-Arg1 axis. Notably, in OXCT1mKO mice, OXCT1 deficiency in TAMs does not affect their proliferation, indicating that OXCT1 has no significant effect on the overall TAM population. However, inhibition or deletion of OXCT1 activity, for example, via the use of pimozide (PZ), can reprogram M2-type TAMs into the M1 phenotype, thereby enhancing antitumor immunity.^283,284

Targeted gene silencing technology mediated by siRNA or shRNA is able to inhibit or reduce the expression of target genes, thus altering the phenotype or function of TAMs and regulating them. Targeting the CSF-1/CSF-1R signaling pathway to disrupt CSF-1R expression has demonstrated significant therapeutic potential across various cancers. In prostate cancer (PCa), a study utilizing M2 macrophage-targeting peptides for the delivery of siRNA specifically to M2 TAMs reported an increase in M1 TAM expression, accompanied by a decrease in the expression of M2-specific markers (CD68+/CD206+). These findings indicate the successful reprogramming of M2 TAMs into the M1 phenotype via siRNA-mediated modulation.²⁸⁷ Similarly, in pancreatic cancer (PC), siRNA-mediated interference targeting the same pathway in combination with the PI3K-γ inhibitor BEZ235 yielded comparable outcomes. Notably, BEZ235 alone demonstrated the ability to modulate TAM phenotypes through the suppression of the phosphorylated AKT (pAKT) pathway. Moreover, the combination therapy exhibited synergistic effects, significantly enhancing TAM phenotype reprogramming compared with single agent treatments, with markedly reduced production of IL-6, a signature cytokine of M2 macrophages that is also closely associated with pancreatic cancer progression. In experiments where IL4RPep-1 was modified on the surface for targeted transfection of NF-κBp50 siRNA as well as miR-511-3p into M1-type exosomes, significant downregulation of the expression of M2 markers and cytokines (Arg1, TFG-β, IL-10, and IL-4) was observed, whereas M1 cytokines (IL-12p40 and IFN-γ) were upregulated, suggesting the ability to reprogram the M2 type to the M1 type.²⁸⁸ Researchers have designed nanoparticles (NPs), which include trimethyl chitosan (PEG = MT) and citraconic anhydride-grafted poly(allylamine hydrochloride) (PC) (PEG = MT/PC NPs). The NPs were then double modified with polyethylene glycol (PEG) and mannose (MT) and internally wrapped with VEGF siRNA (siVEGF)/PIGF siRNA (siPIGF) for targeted delivery to M2-type TAMs. These NPs then aggregated at high concentrations in the tumor cells and effectively silenced the target genes after their contents were released. This ultimately repolarizes the M2 type to the M1 type and achieves the regulation of TAMs.²⁸⁹ Selective silencing of genes related to M2-type TAMs can effectively reduce the aggregation of M2-type TAMs. However, to achieve this goal, the targeting ability and effectiveness of siRNA transfer into target cells need to be improved, and the combination of nanotechnology may be a promising solution.

As inhibiting CSF-1R could block the CSF-1/CSF-1R signaling pathway, which is responsible for macrophage differentiation and proliferation, re-education of TAMs could lead to therapeutic therapy for advanced tumors.^308,309 The FDA approved PLX3397 (pexidartinib), a CSF-1R inhibitor, in 2019 as the first and only drug to target CSF-1R for the treatment of malignant fibrous histiocytoma (TGCT), with more drugs being in clinical trials. PLX3397 inhibition of CSF-1R selectively impacts the viability and polarization processes of M2 macrophages, which also enhances cytotoxic CD8+ T-cell infiltration, thereby achieving a tumor-suppressive microenvironment by halting the tumorigenic effects of crosstalk between tumors and M2 TAMs without interfering with the antitumor effects of M1 macrophages.^310,311 Preclinical evidence has shown that this preferentiality could enhance the therapeutic effects of other treatments, supporting the promising role of CSF-1/CSF-1R in cooperation with other therapeutic drugs and its ability as a prognostic factor.^310,312 However, the hepatic toxicity of PLX3397 cannot be ignored and may be induced by the depletion of resident macrophage Kupffer cells in the liver, resulting in impaired physiological enzymatic clearance.^313,314 Strategies targeting CSF-1/CSF-1R have focused mainly on CSF-1R inhibitors, including small molecules and antibodies, while the latter seem to be more likely to have immune-related adverse effects.³¹⁵

The current systematic administration of PLX3397 may cause off-target adverse effects, which introduces the need for targeted delivery. Nanotechnology enables precise drug delivery, enhances therapeutic outcomes and reduces off-target effects. Codelivery of CSF-1R inhibitors with other agents or cytokines via nanocarriers can further reshape the immune microenvironment and strengthen antitumor immunity. Hu et al.³¹⁶ used nanotechnology to co-deliver IL-12-expressing genes and the CSF-1R inhibitor PLX3397, effectively activating T-cell-mediated immunity and reducing the number of M2-type TAMs more significantly than single-drug delivery. This approach induced substantial changes in the tumor microenvironment, demonstrating superior efficacy in impeding M2 polarization.