Notch Signaling in Macrophages in the Context of Cancer Immunity

Macrophages play both tumor-suppressing and tumor-promoting roles depending on the microenvironment. Tumor-associated macrophages (TAMs) are often associated with poor prognosis in most, but not all cancer. Understanding how macrophages become TAMs and how TAMs interact with tumor cells and shape the outcome of cancer is one of the key areas of interest in cancer therapy research. Notch signaling is involved in macrophage activation and its effector functions. Notch signaling has been indicated to play roles in the regulation of macrophage activation in pro-inflammatory and wound-healing processes. Recent evidence points to the involvement of canonical Notch signaling in the differentiation of TAMs in a breast cancer model. On the other hand, hyperactivation of Notch signaling specifically in macrophages in tumors mass has been shown to suppress tumor growth in an animal model of cancer. Investigations into how Notch signaling is regulated in TAMs and translates into pro- or anti-tumor functions are still largely in their infancy. Therefore, in this review, we summarize the current understanding of the conflicting roles of Notch signaling in regulating the effector function of macrophages and the involvement of Notch signaling in TAM differentiation and function. Furthermore, how Notch signaling in TAMs affects the tumor microenvironment is reviewed. Finally, the direct or indirect cross-talk among TAMs, tumor cells and other cells in the tumor microenvironment via Notch signaling is discussed along with the possibility of its clinical application. Investigations into Notch signaling in macrophages may lead to a more effective way for immune intervention in the treatment of cancer in the future.

Macrophages play both tumor-suppressing and tumor-promoting roles depending on the microenvironment. Tumor-associated macrophages (TAMs) are often associated with poor prognosis in most, but not all cancer. Understanding how macrophages become TAMs and how TAMs interact with tumor cells and shape the outcome of cancer is one of the key areas of interest in cancer therapy research. Notch signaling is involved in macrophage activation and its effector functions. Notch signaling has been indicated to play roles in the regulation of macrophage activation in pro-inflammatory and wound-healing processes. Recent evidence points to the involvement of canonical Notch signaling in the differentiation of TAMs in a breast cancer model. On the other hand, hyperactivation of Notch signaling specifically in macrophages in tumors mass has been shown to suppress tumor growth in an animal model of cancer. Investigations into how Notch signaling is regulated in TAMs and translates into pro-or anti-tumor functions are still largely in their infancy. Therefore, in this review, we summarize the current understanding of the conflicting roles of Notch signaling in regulating the effector function of macrophages and the involvement of Notch signaling in TAM differentiation and function. Furthermore, how Notch signaling in TAMs affects the tumor microenvironment is reviewed. Finally, the direct or indirect cross-talk among TAMs, tumor cells and other cells in the tumor microenvironment via Notch signaling is discussed along with the possibility of its clinical application. Investigations into Notch signaling in macrophages may lead to a more effective way for immune intervention in the treatment of cancer in the future.
Keywords: Notch signaling, macrophages, tumor-associated macrophages, metastasis, tumor immunity iNTRODUCTiON The biological functions of macrophages are diverse and not only limited to their role as the first line of defense during innate immune response. In addition to their protective role against infections, the known roles of macrophages have expanded in recent years, and their involvement in organ development, tissue homeostasis, and metabolic dysfunctions, such as diabetes and obesity, are increasingly appreciated. Cancer is another area in which macrophages have emerged as a crucial player in the creation of a tumor microenvironment that supports tumor growth and metastasis, in opposition to their traditional role as an innate immune cell, whose function is to eliminate cancer cells (1). Therefore, understanding the signaling pathway(s) governing the development, differentiation, activation, deactivation, proliferation, and cell death of macrophages in the context of tumorigenesis is expected to reveal novel strategies for tar geting cancer growth more effectively.
The critical functions of the evolutionarily wellconserved Notch signaling pathway in myeloid lineage cell development and, in particular, monocyte/macrophage development are well recognized and have been reviewed extensively elsewhere (2,3). Recent evidence, using state of the art technologies, revealed better defined subsets of circulating monocytes and the unique ness and the origin of tissueresident macrophages (TRMs). This new insight reignited the excitement in the field of macrophage biology. In addition, these studies cast new light and contro versy over the origin of macrophages found in tumors, called tumorassociated macrophages (TAMs), and the involvement of TAMs in cancer progression and suppression (4,5). Within tumors of various origins, macrophages have been observed to accumulate in large numbers and exhibit unique combinations of activated phenotypes (6). In general, TAMs in large quantities are associated with poor disease prognosis, partly by promoting tumor growth, dampening immune responses, and inducing angiogenesis and metastasis (7,8). Together with the recent advances in the understanding of the roles, Notch signaling plays in the activation and regulation of the immune effector functions of macrophages and in TAMs, these observations have led to the conclusion that Notch signaling is one of the candidate pathways to be manipulated to enhance the host antitumor response. In this review, we summarize the current knowledge of the involvement of Notch signaling in macrophage activation, with an emphasis on its role(s) in TAMs. We also discuss the cross talk among macrophages, tumor cells, and other cells associated with the tumor microenvironment and the potential utility and challenges in manipulating Notch signaling in TAMs for tumor suppression in ways that are beneficial to the host.

Notch Signaling in Macrophage Activation and Function
The biological functions of macrophages are multifaceted depending on the external microenvironment, and some func tions may be contradictory or opposing to others. For example, during infection or tissue injury, macrophages sense danger via various receptors, actively eliminate the source of danger by phagocytosis and chemical mediators, and trigger inflamma tion by producing inflammatory cytokines to alert other immune cells. After the elimination phase, wounds are healed mainly by antiinflammatory woundhealing macrophages (9). The contradictory inflammatory and antiinflammatory microenvi ronments are conducive to driving macrophage activation into two opposite functional spectra. The most simplistic view of macrophage effector functions divides activated macrophages into proinflammatory macrophages, in which macrophages are activated by pathogenassociated molecular patterns (PAMPs) and/or inflammatory cytokines. In contrast, antiinflammatory macrophages, activated by IL4/IL13, represent a woundhealing and immunosuppressive phenotype (10). However, more detailed characterization and studies in various in vivo models have revealed a more complicated view of macrophage effector pheno types that are often observed in an in vivo setting (11). Thus, the narrow concept of pro vs. antiinflammatory macrophages may be oversimplified, and the presence of various hybrid phenotypes of macrophages has been described (11). Some of the genes uniquely expressed in pro or antiinflammatory macrophages are summarized in Table 1 (12,13).
To avoid oversimplification and confusion over macrophage effector phenotypes, this review will adopt the macrophage nomenclatures proposed by Murray et al. to describe specific macrophage subsets based on the stimuli and effector functions described in each referred study (19). In some instances, where the stimuli were not identified, the microenvironments in which macrophages were described will be used.
Initial reports generally found that Notch signaling primarily operates in macrophages that are activated toward inflamma tory functions such as in lipopolysaccharide (LPS)activated macrophages M(LPS) or LPS in combination with IFNγ M(LPS + IFNγ) (15,20,21). Subsequent findings in various pathophysiological conditions also indicated the involvement of Notch signaling in activation and effector functions of pro infla mmatory macrophages (3). Notch signaling, therefore, favors inflammatory macrophages, and when the Notch signaling path way is pharmacologically or genetically blocked, some of the key proinflammatory functions are compromised, including the decrease in the production of proinflammatory cytokines, such as IL6, and the reduction in nitric oxide production (15,22). To this end, Notch signaling is reported to directly or indirectly influence proinflammatory effector functions. Notch signaling can directly regulate transcription of some of the inflammation induced signature genes, such as il6, il12b, and nos2 (23)(24)(25). Using Rbpjdeficient mice, Xu et al. demonstrated that canonical Notch signaling tips the effector phenotypes toward inflammatory ones by directly influencing the transcription of a transcription factor IRF8 (22). In addition, Notch signaling also indirectly regulates proinflammatory phenotypes through a crosstalk with other signaling pathways, such as NFκB and mitogenactivated pro tein kinases (15,20). Interestingly, metabolic analysis found that Notch signaling supports inflammatory macrophage phenotypes by reprograming mitochondrial metabolism toward oxidative phosphorylation (25). Abrogating Notch signaling in myeloid lin eage cells attenuated inflammation in a mouse model of alcoholic steatohepatitis and reduced the severity of endotoxininduced hepatitis (25). All evidence, therefore, points to a critical role of Notch signaling in macrophage activation toward proinflam matory phenotypes in a canonical Notch signalingdependent (intracellular Notch and CSL/RBPJκdependent) manner. The question remains whether inhibition of Notch signaling under an inflammatory microenvironment can switch macrophages toward the opposite phenotype, such as antiinflammatory functions, or whether a lack of Notch signaling only dampens the inflammatory response without directing the macrophages toward other effector phenotypes.
Is Notch signaling dispensable for other types of macrophage effector functions? In macrophages treated with IL4/IL13 M(IL4/IL13), which normally induces antiinflammatory mac rophages. Notch signaling was long considered to be irrelevant; however, an indicator that Notch signaling is activated in the form of cleaved Notch1 was observed in this condition, albeit Notch receptors Notch1 Notch ligands Jagged1 Italiani and Boraschi (12) provide reviews on murine blood monocyte subsets based on Ly6C expression and their functions in inflammation and tissue repair. b Franklin et al. (14) propose TAM markers found in a breast cancer mouse model. c  with different kinetics than those reported in M(LPS + IFNγ) (26). More importantly, in macrophages with targeted deletion of Rbpj, CSL/RBPJκ, possibly through canonical Notch signaling, was found to be required for activation of M(IL4) or M(chitin), including the expression of the gene signature associated with M(IL4), such as Arg1 expression (27). This involvement was independent of STAT6, C/EBPβ, and IRF8. In addition, our obser vation revealed that Notch signaling functions in macrophages activated by PAMPs in the presence of immune complexes and LPS M(LPS + Ic), which predominantly produce high amounts of IL10 and low levels of IL12 to function in dampening the immune response (28,29). Together, these data indicate the need for rethinking the roles that Notch signaling plays in macrophage activation. Notch signaling may be involved in various types of macrophage activation in a contextdependent manner. Whether Notch signaling functions as an instructor or a signal amplifier during macrophage activation remains to be determined, but this feature is similar to what has been postulated for the involvement of Notch signaling in the polarization of helper T cells (30).

Notch Receptors and ligands During Macrophage Activation
Four Notch receptors and five Notch ligands have been identi fied thus far. Differences in signals sent via different combina tions of ligand-receptor interactions have long been suspected.
For example, two ligands, Dll1 and Dll4, send different signals through the same receptor, Notch1, that are either pulsatile or sustained, thereby inducing different cell fates (31). During macrophage activation, various Notch receptors and ligands have been detected ( Table 1). All Notch receptors, except for Notch4, are expressed in proinflammatory M(LPS) or M(LPS + IFNγ) (15). Notch3 is selectively upregulated in proinflammatory macrophages, such as in M(LPS) and M(LDL) (21). Notch1 and Notch2 are required for differentiation of CD11c + CX3CR1 + macrophage subset in the small intestine (16). Similarly, Jagged1, Dll1, and Dll4 are detected in proinflammatory macrophages (18). In M(LPS), Foldi et al. reported that Jagged1 is the ligand responsible for autoamplification of Notch signaling in pro inflammatory macrophages (32). The importance of the Notch Dll4 axis in proinflammatory macrophages was highlighted in a study using blocking antibodies against Dll4. The results revealed that the antiDll4 antibody reduced proinflammatory macrophage accumulation in inflammatory lesions and attenu ated atherosclerosis and metabolic disease (33). Furthermore, during influenza infection, Dll1 expression on macrophages is crucial for dictating the effective antiviral responses of CD4 and CD8 T cells (34). Nevertheless, knowledge of the effect of specific combinations of Notch receptors and ligands on macrophage activation is still limited, and requires each receptor and ligand to be specifically blocked to evaluate the relevance of different interaction pairs.   (5). In a breast cancer model, newly recruited monocytes differentiated to become TAMs, while in brain tumors, both bloodderived monocytes and resident microglia cells contributed to the TAM population (14,36). When TAMs arise from monocytes recruited from circulation, tumor cells need to secrete factor(s) that trigger the migration of monocytes to the tumor sites (Figure 1).
Macrophage phenotypes, in general, are considered highly plastic and can change depending on the microenvironment, and this may also be true for the phenotypes of TAMs in the tumor microenvironment (10). In one study, human breast cancer cells skewed TAMs toward an antiinflammatory pheno type partly by secretion of MCSF (39). In an in vivo model of BALB/c 4T1 mammary carcinoma, the tumor micro environment condition encouraged monocyte precursors to differentiate into diverse TAM subsets with either pro or anti inflammatory phenotypes (40). Furthermore, studies in renal cell carcinoma have shown mixed pro and antiinflammatory phenotypes of TAMs (41). These observations indicate that there are variations in TAM phenotype that depend on the type of tumors and that the activation of TAMs is highly complex and contextdependent.

Notch Signaling and TAMs
In TAMs, Notch1 and 2 have been detected in breast cancer model, while Dll1 and Dll4 have been detected in a lung cancer model (Table 1) (14,17). Jagged1 expression in a breast cancer cell line was shown to modulate TAM differentiation result ing in antiinflammatory and IL10producing TAMs (42). In human cancer, evidence is still lacking regarding the expression profiles of Notch receptors and ligands in TAMs associated with different types of cancer. Recent study of head and neck head and neck squamous cell carcinoma, increasing Notch1 level is associated with CD68 + /CD163 + TAMs, indirectly suggest the link between Notch signaling and TAMs (43). Knowing the expression profiles of Notch receptors and ligands in TAMs and the importance of the signals that they send will provide better targets for intervention.

Notch Signaling and Migrations of Monocytes and Differentiation into TAMs
For monocytederived TAMs, the presence of TAMs begins with the recruitment of blood monocytes/macrophages to the tumor microenvironment through newly formed blood vessels around the solid tumor (14,44). Diverse chemokines, i.e., CCL2 (MCP1), CCL5 (RANTES), CCL7 (MCP3), CXCL8 (IL8), and CXCL12 (SDF1), released by tumor cells induce migration, differentiation, and survival of tumorinfiltrating myeloid cells (45,46). The chemokine receptor CCR2 has been a subject of intense study as a key molecule of monocyte recruitment into tumors. An in vitro study revealed that GMCSFinduced mac rophages M(GC) showed higher CCR2 expression than their MCSFinduced counterparts M(MC). After CCL2 stimulation, M(GC) exhibited enhanced LPSmediated IL10 production, indicating an antiinflammatory role. These phenomena were confirmed by an in vivo study in which Ccr2deficient bone marrowderived macrophages displayed profiles indicative of inflammatory macrophages (47). In the MMTVPyMT mam mary tumor model, a decrease in the number of TAMs in the tumor site was observed in Ccr2null background animals, sug gesting the importance of CCR2/CCL2 signaling in the recruit ment of TAMs to tumor sites (14). Further investigation revealed that the deletion of Rbpj in macrophages results in loss of CCR2 and TAM markers, suggesting a crosstalk between canonical Notch signaling and the CCR2/CCL2 signaling pathway in TAMs in the tumor microenvironment. One can speculate that in the early phase, monocytes are recruited to the tumor site in a CCR2dependent manner and perhaps begin to encourage activation toward an inflammatory phenotype, but tumor cells educate these cells by creating a tumor microenvironment that redirects them toward a tumorfriendly phenotype in a later phase of tumor growth (Figure 1). In fact, a gradual increase in M(IL4)associated markers such as a high level of CD206 expression and low or no MHC Class II molecule expression has been reported in TAMs in a mouse colon cancer model and in human cancer samples (37). Interestingly, expression of the immune checkpoint receptor, programmed cell death protein 1 (PD1), was significantly increased in CD206 + TAMs compared to the expression in TAMs negative for CD206.
In basallike breast cancer, tumor cells secrete both CCL2 and IL1β in a Notchdependent manner, and the secreted cytokine/ chemokines, in turn, recruit monocytes to the tumor site (48). In this case, canonical Notch signaling directly regulates the expression of CCL2 and IL1β, leading to the adhesion of monocytes to blood vessel and extravasation to migrate toward tumor tissue. CCL2 can be produced by bone marrowderived stromal cells or tumor cells, while tumor cells produce IL1β (49). Once monocytes are recruited, tumor microenvironments train/ educate monocytes to differentiate to become TAMs with a pro tumor phenotype that can function to support tumor growth and metastasis (5). In this breast cancer model, TAMs interact with cancer cells via TGFβ to potentiate the expression of Jagged1, one of the Notch ligands (48). The Notch/Jagged1 positive feedback loop amplifies cytokine/chemokine secretion leading to more TAM recruitment. In an animal model of breast cancer using MMTVPyMT mice, Franklin et al. showed conclusively that TAMs are recruited from blood inflammatory monocytes and exhibit phenotypes and functions that are distinct from mam mary TRMs. Importantly, the terminal differentiation of these TAMs from monocytes is CSL/RBPjκdependent, indicating that the canonical Notch signaling pathway plays a vital role in TAM differentiation (14). Therefore, at least for TAMs in this breast cancer model, Notch signaling plays both an extrinsic role, i.e., regulating the production of recruiting factors by tumor cells, and an intrinsic role, i.e., regulating the differentiation of TAMs. Whether TAMs associated with other tumor types also require CSL/RBPjκ for their differentiation or function is still an open question.

Notch Signaling in Anti-Tumor Responses of TAMs
Forced activation of the Notch receptor in TAMs in a Lewis lung carcinoma cell (LCC) model of cancer was shown to repress tumorpromoting activity by enhancing the antitumor pheno type and suppressing the protumor phenotype. The mechanism of antitumor activity is reported to be mediated in part by microRNAs (miRNAs) (50). miRNAs are small regulatory non coding RNAs of 21-22 nt that play important roles in regulating gene expression through posttranscriptional silencing of targets mRNAs. miRNAs play important roles in the activation and effector function of macrophages in TAMs by regulating their target genes and signaling pathway (51). In the LCC model, miR152a, which is under regulation by Notch signaling, targets factorinhibiting hypoxia 1 and IRF4, a transcription factor involved in M(IL4) activation, to enhance the antitumor phe notype (52). In addition, another miRNA downstream of Notch signaling, miR148a3p, also helps to skew the activation of macrophages toward the antitumor phenotype by targeting the PTEN/Akt pathway and activation of the NFκB pathway (53). This observation is consistent with the role of Notch signaling in favoring antitumor macrophage activation, and by forced activation of the Notch signaling pathway, these processes can result in the suppression of tumor growth.
Targeted deletion of Rbpj in macrophages resulted in reduced activity of CD8 + T cells by diminishing the cytotoxic activity against tumor cells in a B16 cell melanoma model (17), suggesting that the crosstalk between TAMs and CTLs is crucial for the anti tumor immune response, and Notch signaling plays an important role in eliciting the antitumor activity of CTL. Moreover, activa tion of Notch signaling in macrophages was demonstrated to increase the CD8 + T cell population infiltrating the tumor site in the LCC model (50). These data indicate the ability of Notch signaling in TAMs to increase antitumor activity directly as pro inflammatory macrophages or indirectly via cytotoxic T cells.
With the use of the opposite approach, manipulating canoni cal Notch signaling in TAMs in a mouse model of cancer was clearly demonstrated to be able to control tumor growth. Targeted deletion of Rbpj in macrophages resulted in antiinflammatory phenotypes under proinflammatory inducers (such as LPS), and these macrophages lost the ability to control tumor growth (17). Therefore, if the Notch signaling pathway is dampened in TAMs, this dampening probably results in TAMs shifting toward an anti inflammatorylike phenotype and helping tumor growth. One caveat is that this study employed in vitro-activated macrophages mixed with a tumor cell line that was administered to mice. Whether switching the Notch signaling on or off in TAMs after differentiation in the tumor influences the antitumor immunity remains an open question.
Contradictory to the studies described above, several reports have indicated that activation of Notch signaling supports anti inflammatory phenotypes of macrophages and possibly favors TAMs (27,54). A study in breast cancer patients who exhibited resistance to aromatase inhibitor treatment showed higher expres sion of Jagged1 in the tumor and an increasing density of anti inflammatory TAM infiltration in breast cancer tissue compared to that in control (42). This study indirectly suggests that Jagged1 on cancer cells may drive TAMs into protumor phenotype by activating Notch signaling in TAMs. These contradictory reports on Notch signaling in TAMs imply that the difference in TAM phenotype possibly depends on the tumor microenvironment and types of tumor, and this need to be taken into consideration. In addition, different Notch ligands may activate Notch signaling in different ways, and this may impact the phenotypes of TAMs.

TAMs, Tumor Angiogenesis, and Notch Signaling
Angiogenesis requires contact between macrophages and endo thelial cells together with cytokines and angiogenic molecules. Inflam matory macrophages, including TAMs, are involved in angiogenesis based on the expression of cytokines, such as TNF α and IL6, and angiogenic factors, such as vascular endo thelial growth factor (VEGF) (5). Because Notch signaling, directly or indirectly, regulates the expression of genes involved in angiogen esis, such as VEGFR and EphrinB2 (55), Notch signaling in TAMs may regulate tumor angiogenesis. In retinal choroidal neovascu larization (CNV), the deletion of Rbpj in myeloid cells results in the inhibition of the inflammatory response in the retina and choroid after injury. This inhibited inflammatory response is accompanied by suppression of VEGF and TNFα production and CNV devel opment in the choroid (56). Moreover, Notch1expressing mac rophages interact with two Dll4expressing sprouts of endothelial cells, leading to the activation of Notch signaling in macrophages. This interaction regulates the function of macrophages during vessel anastomosis in retina angiogenesis (57). Loss of Notch1 in myeloid lineage cells reduces microglia recruitment and results in abnormal angiogenesis (58).
Vascular cell adhesion molecule (VCAM) 1 is highly expressed in TAMs, whereas loss of VCAM1 in macrophages reduces the number of hematopoietic stem cells in the spleen and the inflam mation in atherosclerosis due to an inability of macrophages to attach to vascular endothelial cells (59). Although little is known about the role of Notch signaling in the regulation of VCAM1 expression in macrophages, lung endothelial cells express high levels of VCAM1, and increased numbers of TAMs have been observed in lung cancer tissue compared to that in control. Endothelial cells were reported to undergo cellular senescence after implantation of tumor cells expressing Notch ligands (Dll4 and Jagged1), suggesting that VCAM1 expression in endothelial cells is under the regulation by Notch signaling and, together with Notch activation, required for TAM localization (60). VCAM1 expression in endothelial cells is under regulation of the Notch signaling pathway even in the absence of inflammatory cytokines. However, in the presence of IL1β, VCAM1 expression in endothelial cells is greatly enhanced in a Notchdependent manner (61). These studies suggest that endothelial VCAM1 is important for the survival of TAMs in the tumor microenvi ronment. However, this interaction through VCAM1 may be bidirectional because VCAM1 is also highly expressed in TAMs, suggesting that it may play an important role in the survival of endothelial cells as well. Blood vessel endothelial cells have also been found to play a role in TAM differentiation. A recent study demonstrated that Dll1 expressed by endothelial cells lining the blood vessels in mice induced conversion of Ly6C hi to Ly6C lo monocytes in a Notch2dependent manner (62). This study was the first to demonstrate that the Notch ligand Dll1 in the blood vessel can induce phenotypic changes in monocytes through the Notch2 receptor under steadystate conditions. The Role of Notch-Dependent TAMs in Supporting Tumor growth and immune Suppression As described above, TAMs can directly support tumor growth by secreting factors, such as TGFβ (48). TAMs also affect the overall antitumor immunity mounted by other immune cells, such as T lymphocytes, in tumor sites by dampening the immune functions. Arginase 1, an argininedegrading enzyme produced by M(IL4), can suppress CTL activity (63). Recently, antiinflam matory macrophagelike (CD206 + MHC II low or negative ), but not proinflammatory macrophagelike (CD206 − MHCII hi ) TAMs have been reported to express PD1 in both a mouse model and in human cancers over time with disease progression (37). The socalled immune checkpoint inhibitor is used to block this PD1 PDL1 interaction and trigger a vigorous host immune response against the tumor. Interestingly, blocking this interaction results in increasing phagocytosis by macrophages and a reduction in tumor growth in mouse models of cancer (37). Although there is no evidence linking Notch signaling and PD1 in TAMs, there is a report indicating that canonical Notch signaling regulates the expression of PD1 in activated CD8 + T cells (64). Cancer associated fibroblasts (CAFs) are indicated as accomplices in malignant cancers (38). Because CAFs and TAMs are reported to collaborate via cell-cell interaction in promoting tumor progres sion (65), it is possible that Notch signaling may contribute in the crosstalk between the two cell types. Taken together, these observations suggest that Notch signaling may be involved in regulating this immune suppression mechanism in TAMs via an immune checkpoint inhibitor.

Challenges and Potential for Manipulating Notch Signaling in TAMs for Therapy
Notch signaling clearly plays important roles in TAMs, either to promote or suppress tumor growth. Therefore, Notch signaling in TAMs can be a drug target for manipulating host anticancer immunity. If Notch signaling in TAMs is protumoral, sup pressing it would benefit the host. In contrast, if TAMs require Notch signaling to become more inflammatory antitumor macrophages, it needs to be stimulated. Various types of gamma secretase inhibitor that is a panNotch signaling inhibitor are often used to suppress Notch signaling in cancer clinical trials (66). Unfortunately, this inhibitor has offtarget effect and is highly toxic if applied systemically. Therefore, designing a method that specifically inhibits Notch signaling in TAMs is desirable. One approach is to use a stapled peptide derived from part of mastermindlike protein that interferes with canonical Notch signaling. If coupled with a TAMspecific delivery sys tem, this peptide could specifically inhibit Notch signaling in TAMs (67,68). Antibodybased specific antibody blocking has also been investigated for targeting the ligandbinding domain or the negative regulatory region of Notch receptors (69). To activate Notch signaling to favor inflammatory macrophages, an activating antibody that mimics ligand binding may be used. In any case, an intelligent method that targets TAMs is required to minimize the side effects.

Remaining Unresolved Questions and Future Directions
Notch signaling in macrophages clearly affects their biological functions both directly and indirectly. Notch signaling also affects TAMs and functions in monocyte recruitment, tumormediated training, and angiogenesis. Notch signaling in TAMs is, there fore, an attractive signal to manipulate to promote antitumor immunity. Macrophages have been reported to be epigenetically modified by stimuli that contribute to "trained immunity" and "tolerance, " at least in vitro (70). If the manipulation of mac rophage polarization of TAMs through Notch signaling is to be considered as an alternative for cancer treatment, we must ask whether the epigenetic marks on TAMs imprinted by the tumor microenvironment, created by cancer cells, can be reversed or erased so that TAMs could act to benefit the host.

AUTHOR CONTRibUTiONS
TP is responsible for designing the article concept and scope, reviewing 50% of the content, and conceptualizing the figure. WW is responsible for reviewing 20% of the content. PK is responsible for reviewing 30% of the content and designing the table and part of the scope of the article.