Also known as MCP-1, CCL2 plays an important role in tumor growth and macrophage recruitment in the setting of a number of malignancies ( Figure 3A ) (50). For example, breast cancer, gastric cancer, ovarian cancer, etc. (51–53) CCL2 is also present in the central nervous system. A potent chemoattractant, CCL2 is produced by many central nervous system cells such as astrocytes, endothelial cells and microglia (54). Glioma cells also express high levels of CCL2 (17). This was reported to significantly positively correlate with GAM quantity within the glioma TME. However, whether CCL2 directly acts on tumor cells remains unclear. An earlier study of the U87 glioma cell line utilized flow cytometry to confirm that these cells lack CCR2, the CCL2 receptor. Moreover, CCL2 did not appear to markedly influence proliferation or migration of U87 cells (55). A different study of the U251 glioma cell line, however, suggested differently: after CCL2 knockdown, glioma cells exhibited significantly decreased proliferation and migration, and significantly increased apoptotic activity, as compared to control cells (56). Effects of CCL2 on glioma cells thus warrant further clarification.

A number of molecules also influence macrophage infiltration of the central nervous system via interactions with CCL2 and thereby affect glioma pathogenesis. Among these, MEX3A expression was found to be significantly increased in the setting of glioma and significantly positively correlated with the degree of malignancy as well as poor prognosis. A likely downstream target of MEX3A, CCL2 is also regulated by this protein. Possible relevant pathways include PPARα/RXRα activation and AMPK signaling (57). Under CCL2 knockout conditions, the tumor-promoting effect of MEX3A was found to be significantly attenuated (56). Homeobox C10 (HOXC10), known to induce angiogenesis via VEGFR upregulation, is also highly expressed in the setting of glioma and exerts a similar regulatory effect on CCL2 (58). As HOXC10 knockdown was confirmed to inhibit CCL2 expression, it thus likely plays an important role in CCL2-mediated macrophage recruitment (59). Proteins such as ELF-1 and Notch1 also exert upstream regulatory effects on CCL2, thus affecting macrophage recruitment (60–62).

Upon CCL2 interaction with CCR2, release of IL-6 from microglia is promoted in addition to cellular recruitment. Greater IL-6 levels further increase TME microglia, reduce cytotoxic CD8+ T cells within the TME, and effectively promote malignant proliferation and metastasis (63). Interestingly, a number of studies have reported that CCL-7 (MCP-3) plays a more significant role in GAM infiltration in glioma as compared to CCL2 (64). However, a relatively early publication year of this study, as well as a lack of relevant follow-up research, necessitates verification.

Stromal cell-derived factor-1 (SDF-1), also known as chemokine CXC ligand 12 (CXCL12), is expressed and secreted by a variety of cells including myeloid, endothelial, epithelial and tumor cells ( Figure 3B ) (65). CXCR4, expressed primarily by monocytes and neutrophils in the immune microenvironment, is the primary receptor for CXCL12, but there are others (66). For example, CXCR7 and atypical chemokine receptor 3 (ACKR3) also bind CXCL12 and exert corresponding downstream regulatory effects. Signaling pathways such as the mitogen-activated protein kinase ACCRA (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) are activated under the influence of the CXCL12-CXCR4/CXCR7 axis, thus affecting cellular proliferation and metastasis (67, 68). The regulation of angiogenesis by the CXCL12-CXCR4/CXCR7 axis attracts increasing research attention. Studies have reported that the CXCL12/CXCR4 axis effectively upregulates VEGF expression within the TME, thereby promoting angiogenesis. Inhibition of VEGF alone, however, is known to increase CXCR4 expression. Existing experimental treatment methods therefore utilize CXCR4 and VEGF inhibitors in combination have achieved good therapeutic effect (66). The CXCL12-CXCR4/CXCR7 axis also influences resistance to radiotherapy and chemotherapy, immune cell infiltration into the TME and tumor stem cell proliferation. Although the relevant mechanisms of these phenomena are not described in detail in this review, such activity greatly affects malignant pathogenesis (69–73).

In glioma, CXCL12-CXCR4/CXCR7 was reported to be significantly increased. Indeed, increased expression of CXCR4 is considered to be one hallmark of malignant glioblastoma (74). Its ligand CXCL12 was further identified as a potential biomarker of cancer stem cell resistance to radiotherapy, likely via induction of autophagy among malignant cells (75, 76). CXCR4 was preferentially expressed in glioma stem cells. As the differentiation degree of glioma increased, the expression level of CXCR4 decreased gradually, instead, the expression level of CXCR7 increased (77, 78). This axis is involved in malignant processes such as tumor angiogenesis, inflammatory response, immunosuppression and reprogramming (79). Recruitment of monocytes is one of the many tumor-promoting effects exerted by CXCR4.

A study of tumor-associated fibroblast (CAF)-induced monocyte migration revealed that CXCL12-supplemented media significantly increased monocyte chemotaxis (80). In glioma, the CXCL12-CXCR4/CXCR7 axis similarly plays a decisive role in macrophage recruitment (77). CXCR4 and CXCR7 are highly expressed on the surface of both microglia and glioma cells (78). As CXCL12 concentration within the TME increases, microglia (mainly M2 macrophages) are increasingly recruited toward the vicinity of the malignancy.

Hypoxia, an essential characteristic of the TME, results in enhanced resistance to damage by tumor cells due to an adaptive modulatory response (81). In the setting of glioma, hypoxia further promotes malignant pathogenesis as well as drug resistance (82). The hypoxic microenvironment upregulates members of the hypoxia-inducible factor (HIF) family, which influence glioma phenotype via effects on angiogenesis, cellular resistance to therapy and enhanced metastasis (83). Members of the HIF family also affect macrophage recruitment via CXCL12 regulation. An earlier study showed that HIF-1α, as an upstream regulator of CXCL12, effectively influences CXCL12 release (84).While HIF-1α regulates CXCL12, it is also activated by CXCL12 via RAS/ERK1-2 and PI3K/AKT signaling (85). In another study of a mouse model of astrocytoma revealed that SDF-1 (CXCL12) effectively promotes GAM recruitment to the vicinity of the malignancy in a concentration-dependent fashion within the hypoxic TME. Co-localization of HIF-1 and CXCL12 by immunofluorescence subsequently complemented the aforementioned findings (86).

In the setting of the hypoxic TME, HIF members play regulatory roles in the CXCL12-CXCR4/CXCR7 axis along with other cytokines such as VEGF and CD26 (87, 88). A 2006 study of the U251 glioma cell line revealed that VEGF increases expression of both SDF-1 and CXCR4 mRNA, thus effectively improving metastatic capabilities (89). In addition, CXCL12 induces macrophage polarization toward the M2 subtype, thus further promoting malignant process (73).

Colony-stimulating factor 1 (CSF-1) and IL-34 are ubiquitous cytokines of great significance to the regulation of monocyte function. Their common ligand, CSF-1R, is a transmembrane tyrosine kinase receptor widely expressed on the surface of cells such as monocytes and marrow-derived macrophages. Ligand-receptor interaction results in effective promotion of monocyte proliferation and differentiation via downstream signaling pathways such as JAK-STAT and PI3K/AKT ( Figure 3C ) (90–93).

Expression of CSF-1 and IL-34 in various tumors is significantly increased. In breast cancer, significant upregulation of CSF-1 effectively promotes migration of non-resident macrophages to the TME and induces their polarization toward the M2 subtype (94). In melanoma, the ERK pathway-mediated RUNX1 transcription factor promotes CSF-1R expression, increasing tumor cell survival and malignancy (95).

In normal brain tissue, CSF-1 and IL-34 are respectively secreted by resident microglia and neurons (96). Both cytokines significantly affect microglial development, maturation and function (97). However, in glioma, especially glioblastoma, these cytokines influence tumor progression. While CSF-1R, CSF-1 and IL-34 directly support tumor cell growth, CSF-1, highly expressed by glioma cells, effectively promotes macrophage recruitment and indirectly promotes malignant pathogenesis via GAM interactions (98). One study of the SETDB1 enzyme revealed significantly increased expression in glioma and resultant promotion of CSF-1 secretion via AKT/mTOR signaling, thereby enhancing macrophage recruitment and polarization (99). Inhibition of CSF-1R signaling in vivo was similarly reported to reduce macrophage infiltration of the TME, further validating the roles CSF-1 and IL-34 play in immune cell recruitment (100).

A number of studies have investigated macrophage recruitment by CSF-1. Although administration of anti-CSF-1R therapy to glioma experimental animals exhibited an excellent initial reaction, nearly half of experimental animals eventually developed drug resistance and tumor recurrence. PTEN/PI3K pathway activation and increased levels of IGF-1 were found in the drug-resistant glioma setting. As such, targeting of the above two mechanisms with a combined therapeutic strategy involving CSF-1R inhibition was reported to improve therapeutic outcome (101). Although these findings suggest that CSF-1 and IL-34 are susceptible to regulation, anti-CSF-1R therapy warrants further study for effective use in glioma treatment.

Osteopontin (OPN), also known as secreted phosphoprotein-1(SPP1), is highly expressed in microglia of the early postnatal brain and injured adults (102). It is an exocrine immunoregulatory protein involved in the inflammatory process, and also expressed in fibroblasts, dendritic cells and macrophages. It is involved in both physiological and pathological processes such as bone formation, osteoarthritis, obesity and Alzheimer’s disease, as well as carcinogenesis and metastasis (103–106). In the central nervous system, OPN effectively monitors acute or chronic injuries in the central nervous system, such as inflammation (102). In various pathological settings, OPN plays roles in mediating inflammation, inducing immune cell proliferation and attracting mature macrophage migration to the vicinity of the lesion ( Figure 3D ) (103, 107, 108).

A component of the extracellular matrix, OPN primarily binds three receptors relevant in the TME including α4β1, αvβ3 and CD44. Activation of corresponding pathways promotes macrophage recruitment, angiogenesis and T cell inhibition, thereby promoting malignant progression (109). Increased expression of OPN in glioma was reported to significantly positively correlate with the degree of malignancy (110). It has been reported that the increased expression of OPN in glioma is significantly positively correlated with the degree of malignancy, and its expression is effective in maintaining the survival and angiogenesis of glioma cells. Furthermore, OPN increases secretion of metalloproteinase-2 (MMP-2), thereby promoting glioma metastasis. OPN also effectively reduces glioma cell sensitivity to the immune system (106, 111). Selective inhibition of OPN expression in glioma was noted to significantly reduce malignant cell proliferation (112).

As previously mentioned, in various diseases, the secreted glycoprotein OPN effectively induces macrophage migration to the lesion site in a dose-dependent manner (104). Recruitment of M0 and M2 (but not M1) macrophages to tumor tissue via αvβ5-integrin signaling facilitates a continued increase in OPN secretion and further enhances macrophage recruitment (111). Interestingly, existing literature is inconsistent regarding effects of OPN on macrophage polarization within the TME. Studies focusing on obesity, liver cancer and colitis underscored that apart from promoting macrophage recruitment, OPN effectively induces macrophage polarization to the M2 phenotype via the αvβ3 and CD44 receptors, and activates the downstream STAT3/PPARg signaling pathway (103, 113, 114). However, a glioma study reported that despite regulation of macrophage recruitment via CD44, OPN does not significantly influence macrophage polarization (111). Mechanisms relevant to the function of OPN in malignancy thus require further study.

OPN offers unique immunotherapeutic prospects. After OPN knockout, significantly decreased levels of M2 macrophages and tumor cell expression of PD-L1 were reported. These phenomena are not due to direct effects of OPN on tumor cells, but rather due to indirect regulatory effects that induce secretion of CSF-1 by macrophages via the PI3K/AKT/NF-κB/p65 pathway. After CSF-1R inhibition, anti-PD-L1 treatment in a mouse model of OPN-expressing malignancy was significantly improved (113). However, these findings are limited to liver cancer, and CSF-1R inhibition therapy needs further investigation.