It has been demonstrated that microglia and BMDM display distinct phenotypic signatures and localisations within GBM indicating complexity and diversity of the myeloid compartment in malignant glioma [7,8].

It is becoming increasingly clear that endothelial cells are a key component in organising the perivascular niche and sustaining the survival and stemness of glioma stem cells GSCs [9]. Recent accumulating evidence indicated that GSCs predominantly reside in a perivascular niche [10,11] and usually surround by tumour-associated macrophages and microglia [12]. A previous study by Yi et al. has further reported that GSCs are better at recruiting microglia than glioma cells as GSCs expressed more Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 5 (CCL5), Chemokine (C-C motif) ligand 7 (CCL7), Vascular endothelial growth factor A (VEGF-A) and neurotensin (NTS) than glioma cells that infiltrate the brain tissue [12]. In addition, tumour associated macrophages and microglia (TAMs) maintain the phenotype of glioma-stem cells by releasing TGF-β1, which in turn promotes glioma growth and invasion [13]. MSR1 (CD204) is a class A macrophage scavenger receptor and pattern recognition molecule associated with a pro-tumourigenic phenotype of brain macrophages [14]. High levels of MSR1+ (CD204) are associated with increased expression of immune checkpoint markers PD-L1 and TIM3 which raise the possibility that MSR1+ may contribute to T cell exhaustion [15,16]. A recent study employing single-cell RNA sequencing (scRNA-seq) reported that while BMDM aggregate perivascularly and within necrotic foci, microglia expression signatures are enriched within the glioma infiltration front (“tumour edge”). Furthermore, the increased presence of blood-derived tumour-associated macrophages correlated with shorter survival times [7]. Sørensen and Zhang et al. have shown that the number of MSR1+ glioma-associated microglia/macrophages increases with malignancy grade [14,17]. A later study performed mRNA transcriptome profiling followed by pathway and connectivity network analysis (STRING, https://www.string-db.org/; accessed on 1 November 2022) revealed that the accumulation of MSR1+ microglia/macrophages in glioblastoma correlates with an interleukin-6-enriched profile and poor survival [18]. Sørensen and Kristensen [18] further demonstrated that MSR1+ microglia/BMDM accumulate perivascularly and around necrotic areas and that they often co-reside with stem-like GBM cells expressing the marker, podoplanin (PDPN) [18]. Thus, MSR1+ microglia/BDMDs may support stem-like GBM cells and the progression of GBM in perivascular and necrotic niches.

Results from recent flow cytometry experiments indicate that P2RY12 and CD49 may be used to distinguish microglia from BMDM in GBM [7,8]. In line with this, a single-cell image analysis study by Woolf et al. [47] reported that P2RY12 and TMEM119 label microglia in GBM and the authors further proposed that the markers can be used to discriminate microglia from BMDM. Of note, patients with high P2RY12 expression survived longer. Moreover, a higher microglia to BMDM ratio in GBM conferred a survival advantage that was independent of O-6-methylguanine-DNA methyltransferase (MGMT) methylation status [47]. Interestingly, activation of P2Y12 receptors causes extension of microglial cell processes [48,49]. It is noteworthy that phagocytic GBM-associated microglia and macrophages have also been observed in the non-necrotic parts of (pseudo)palisading GBM necrosis [48,49]. Importantly, microglia/macrophages in the GBM resection zone have been suggested to function as part of a glioma stem cell niche at the tumour border [50,51] which is important for tumour recurrence. It is suggested that they express a distinct gene signature [52]. The supportive influence of microglial cells on glioma growth is now established beyond doubt and can even be reproduced in a xenograft model [52]. Interestingly, microglia seem to exert a sex-specific influence in the TME [53] which may help to explain why GBM is more common and aggressive in male patients. In fact, the hijacking of sexual immune privilege by GBM has been identified as an immune evasion strategy of the glioma [53,54,55].

Secreted phosphoprotein 1 (Spp1), also known as osteopontin (OPN), is a glycophosphoprotein expressed by various cell types, including macrophages, T-cells, osteoblasts, epithelial cells and tumour cells. Moreover, expression of Spp1 can regulate cell-matrix interactions by binding to CD44 and integrin receptors thus mediating cell adhesion, chemotaxis, angiogenesis and resistance to apoptosis [59]. Notably, Spp1 expression which like CD68 [60] is especially high in mesenchymal GBM, correlates with both tumour grade and the extent of macrophage infiltration. GBM and glioblastoma stem cells (GSCs) use Spp1 to recruit macrophages into the TME [19]. Integrin αvβ5 (ITGαvβ5), a key receptor for Spp1, is highly expressed on GBM-infiltrating macrophages [19]. This is in line with previous findings by Ellert-Miklaszewska et al. that Spp1 and lactadherin enable glioma cells to gain an advantage through M2 reprogramming of tumour-infiltrating brain macrophages [61]. Therefore, Spp1 is likely to play a key role in the progression of GBM.

POSTN is a disulfide-linked cell adhesion protein which belongs to the fasciclin (Fas) family [62]. Recent studies have revealed that POSTN contributes to malignant tumour progression by supporting metastatic colonisation of breast cancer stem cells through upregulation of Wnt signalling [63]. A study by Zhou et al. [20] has suggested that BMDM are the main source of macrophages in GBM and that their recruitment from the bloodstream is stimulated by GBM-secreted periostin (POSTN) that interacts with integrin αvβ3 (ITGαvβ3) on BMDMs. As expected, POSTN knockout mice show significantly extended survival times. It is noteworthy that POSTN is preferentially expressed by putative GSCs expressing SOX2 and OLIG2, respectively [20]. This is in keeping with the results of Guo et al. [64] who reported that hypoxia promotes glioma-associated macrophage infiltration via POSTN.

IL-33, a member of the IL-1 cytokine family, is secreted as an alarmin by damaged or necrotic cells [65]. It is now clear that IL-33 plays a pro-tumorigenic role in various cancers including glioma [65]. De Boeck and colleagues [34] recently suggested that both the nuclear and secreted form of IL-33 are present within tumour cells in ~50% of human glioma specimens and GBM murine models. In addition, IL-33 was previously shown to associate with chromatin and be involved in the regulation of gene transcription [66]. Using multiplex cytokine/chemokine analysis, these authors further demonstrated that nuclear IL-33 facilitates tumour growth by triggering glioma-mediated expression of inflammatory cytokines (LIF, IL-6, IL-8, IL-1RN, IL-1β and secreted IL-33 [34]. Strikingly, a subset of microglia in the IL-33+ xenografts also expressed a significant amount of IL-33 and showed enrichment in pro-inflammatory cytokines, Spp1 (osteopontin), and the lipid metabolism gene, Apoe. These microglial cells further displayed a notable upregulation of the monocyte chemoattractant genes (CCL2, CCL3, and CCL12). Activation of monocyte chemoattractant genes fuels further recruitment of immune cells to the glioma microenvironment [34]. Conversely, loss of nuclear IL-33 resulted in significantly smaller IL-33-associated tumour burden and increased overall survival. Furthermore, elevated levels of both nuclear and soluble IL-33 were associated with enhanced activation of AIF1 (Iba1)+ resident microglia and recruitment of CD163+ BMDM [34]. In addition, neutralising IL-33 effects by-means of anti-IL-33 and anti-CCL2 (which is up-regulated by recombinant IL-33) antibodies significantly reduced the recruitment of microglia and BMDM [34].

PDCD10 is an evolutionarily conserved protein expressed by neurons, astrocytes, endothelial and cancer cells. Zhang et al. [37] recently reported that overexpression of PDCD10 by GBM cells promotes tumour progression via recruitment of microglia and BMDM. Furthermore, PDCD10 up-regulation is followed by an increase in CXC motif chemokine ligand 2 (CXCL2) and resulting activation of CXCR2 in microglia. In sum, CXCL2-CXCR2 signalling stimulated by PDCD10 appears to be a key mechanism in the crosstalk between GBM cells and microglia/macrophages that promotes tumour progression.

SLITs are evolutionarily conserved polypeptides that bind to cells expressing ROBO receptors. SLIT-ROBO binding activates the recruitment of adaptor protein to the cytoplasmic domain of ROBO receptors which in turn regulates cell motility by modulating the actin and microtubule cytoskeleton [84]. Recently, SLIT2 has been found to promote microglia/macrophage chemotaxis (via ROBO1/2 induced PI3Kγ activation) and polarization, and its expression to increase with malignant progression and correlate with poor survival and immunosuppression [21]. It is worth noting that SLIT2 knockdown in tumour cells inhibits mouse macrophage invasion [21].

The family of let-7 microRNAs, which share an evolutionarily conserved sequence, are highly expressed in the brain [85]. Recent studies have indicated that let-7 microRNAs are involved in cancer initiation and brain tumour progression [85]. Moreover, it is known that Toll-like receptors are pattern recognition receptors found on microglia that detect pathogen- and host-derived factors such as miRNAs [85]. Let-7 miRNA, specifically ones carrying the core sequence motif UUGU, can activate microglial and BMDM Toll-like receptor (TLR) 7 and induce TNF-α production which might lead to suppression of glioma growth [68]. It is worth noting that selective groups of let-7 miRNAs regulate the expression of antigen-presenting molecules in the CNS. These includes let-7b and let-7e miRNAs which stimulate up-regulation of MHC I and ICAM1 (CD54) through TLR7 signalling [68]. Of note, MHC I and ICAM1 are critically important for the communication between innate and adaptive immune cells and activation of T cell-mediated cytotoxic responses [68]. Interestingly, let-7 miRNA oligoribonucleotides that lack the GU-rich core motif may act as a chemoattractant for microglial cells in glioma [68]. Furthermore, the let-7 miRNAs induced TLR-7 activation is observed in both neonatal and adult microglia/BMDMs [68]. Thus, it is hypothesised that let-7 miRNAs can potentially shift microglia towards an “anti-tumour” phenotype and enhance the efficacy of immunotherapies.

Numerous studies have demonstrated that GBMs are infiltrated by immune cells, and by microglia and monocyte-derived macrophages in particular [86]. It has been shown that an increased number of microglia and brain macrophages is associated with a higher WHO grade in gliomas [87]. The WHO classification is essentially a malignancy scale that helps clinicians predict a patient’s disease course (prognostication). Tumors are graded benign (grade 1) or malignant, and there are different grades of malignancy expressed as “grade 2–4” with 4 being worst (shortest expected survival time) [88]. In order to avoid confusion, a grade 2 glioma will progress and cannot be considered benign although it is not fully malignant yet. Gabrusiewicz et al. recently reported that the number of CD14+ monocytes is increased in the blood of GBM patients [35]. Using whole-genome expression profiling, the authors also observed that GBM-associated myeloid cells do not exist in distinct polarized M1 and M2 states. Moreover, gene set enrichment analysis revealed MYC and E2F transcriptional regulation in CD14+ cells. Interestingly, Gabrusiewicz et al. also pointed out that miRNA-146 may play a role in pro-inflammatory macrophages; its expression was significantly suppressed in GBM-infiltrating CD14+ cells. Strikingly, a previouly mentioned glioma-derived molecule, Spp1 was also highly expressed in GBM-infiltrating CD14+ BMDM [35].

MIF is a molecule that is highly conserved across species suggesting it has a role in fundamental biological processes [89]. Accumulating evidence suggests that MIF is expressed by immune cells in various cancers including breast and lung cancer [89]. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of bone marrow-derived progenitor cells that consist of monocytic (M-MDSC) and granulocytic (G-MDSC) subsets which exhibit potent immunosuppressive activity. They interfere with the cytotoxic functions of natural killer (NK) cells and T lymphocytes in tumours including GBM [90,91]. A recent experimental study by Alban and colleagues [92] has found that the monocytic subset of myeloid-derived suppressor cells (M-MDSCs) expresses high levels of CD74 in the presence of glioma-derived macrophage migration inhibitory factor (MIF) and glioma cells. Using a syngeneic murine model, the authors further described that disruption of the MIF-CD74 pathway using Ibudilast minimises downstream activation of CCL2 (MCP-1). CCL2 was previously shown to have a critical role in driving recruitment of monocytes and expansion of MDSCs [93,94]. Of note, MIF is also capable of mediating signalling via non-cognate receptors such as CXCR2, CXCR4, and CXCR7 [92]. Importantly, activation of microglial CD74 weakens the microglial defense against glioma cells [23]. Furthermore, MIF expression is significantly increased in malignant glioma and interferon (IFN)-γ secretion by microglia is inhibited by MIF-CD74 signalling [23].

SELP is a well-known adhesion molecule involved in leukocyte rolling and recruitment [95]. It is now evident that SELP and its ligand PSGL-1 are involved in the metastatic spread of melanoma and colon cancer [96]. Recently, a mechanism has been proposed by which SELP-PSGL1 mediate GBM progression influencing microglia/macrophage phenotype [22]. The authors found that recombinant SELP reduced the phagocytic activity of microglia/BMDM, decreased their expression of inducible nitric oxide synthase (iNOS) and release of nitric oxide (NO) while increasing expression of IL-10 and TGF-β. It is worth noting that following exposure to soluble SELP (sSELP), a positive feedback loop causes overexpression of SELP and PSGL-1 by GBM and microglia cells [22]. On the flip side, expression of actin nucleation promoting factor wasla by microglial cells was found to revive microglial phagocytotic activity and slow down GBM progression in zebrafish [97].

A recent experimental study by Hutter and co-workers has indicated that tumour-associated microglia are capable of tumour cell phagocytosis in vivo if the immune evasion of tumour cells is blocked by a humanized anti-CD47 monoclonal antibody [29]. Notably, Li et al. have shown that CD47 is expressed by human and mouse glioma cell lines and that positive cells have many characteristics of cancer stem cells [98]. The view is also supported by Hu et al. [99] who report that overexpression of the LRIG2 (Leucine Rich Repeats And Immunoglobulin Like Domains 2) gene in GBM cells induces upregulation of CD47 and activation of the CD47-SIRPα anti-phagocytic axis [30]. Further experiments revealed that soluble LRIG (sLRIG) induces recruitment of BMDM that exhibit an immunosuppressive phenotype and also express high levels of CD47 receptor, SIRPα [99]. As expected, knockdown of LRIG2/sLRIG2 in GL261 (murine GBM) cells interferes with the activation of the CD47–SIRPα anti-phagocytic axis and enhances BMDM-mediated phagocytosis of GBM cells and suppresses GBM progression [99]. This is in keeping with the results of a xenograft study by Gholamin et al. showing ubiquitous expression of CD47 in paediatric GBM and diffuse midline glioma [30]. Blockage of the anti-phagocytic CD47-SIRPα axis using an anti-CD47 antibody, Hu5F9-G4, strongly induced BMDM-mediated phagocytosis of glioma cells, and mice treated with Hu5F9-G4 demonstrated significant longer survival [30]. It is worth noting that the “don’t eat me” signal mediated by the CD47-SIRPα axis [100] also protects synapses from non-specific pruning during development and disease. Accordingly, CD47 deficiency in mice leads to reduced synaptic density resulting from excessive pruning by microglia [31]. Loss of microglial SIRPα has a similar effect in preclinical models of neurodegeneration [101]. Thus, the CD47-SIRPα axis may deserve special attention in the context of cognitive deficits of brain tumour patients even though Li et al. were unable to detect damage to neurons and astrocytes in a treatment model [98].

The interactions between tumour-associated microglia/brain macrophages and T cells may lead to T cell malfunction and diminished T-cell mediated anti-tumour responses [102]. By analysing RNAs found in extracellular vesicles (EVs) derived from microglia that had interacted with GBM, Maas et al. [103] recently found that GBM-interacting microglia down-regulate genes involved in the detection of tumour cells (sialic acid-binding immunoglobulin-like lectin-H (Siglec-H), CD33 and GPR34) and tumour-derived metabolic by-products (Gpr183, Adora3, Il6Ra, Cx3cr1, P2ry12, P2ry13, Csf1r, and Csf3r) [103]. In contrast, levels of CD274 (PD-L1) and PD-L2 transcripts are elevated in GBM-interacting microglia, suggesting that genes involved in immunologic tolerance are up-regulated in microglia by GBM contact, resulting in indirect inhibition of anti-tumour functions of T cells [104]. Interestingly, the authors further demonstrated that GBM-interacting microglia show up-regulated expression of phagocytic receptors (Cd93, Msr1, Cd36, Olr1, Megf10, Clec7a, Scarf1) and extracellular matrix (ECM) degrading enzymes such as Mmp14 [103]. A recent study supports the invasion-facilitating role of microglial cells by describing that glial cell line-derived neurotrophic factor (GDNF), a chemoattractant of microglia [105], stimulates the production of microglia-derived MMP9 and MMP14 in neonatal mice [106]. Huang et al. further indicated that GDNF induced up-regulation of microglial TLR1 and TLR2 and that the activation of TLR2 can increase expression of microglial MMP9 and MMP14 [106]. In addition to stimulating expression of ECM degrading enzymes, the activation of TLR2 also inhibited expression of MHC class II by microglial cells via loss of histone H3 acetylation at the master regulator of MHC class II molecule transcription, Ciita (Class II Major Histocompatibility Complex Transactivator). Accordingly, inhibited MHC II expression impedes CD4+ T cell activation and proliferation which weakens T-cell dependent anti-tumour responses [107]. Expression of the GBM-associated microglial phenotype appears to be mediated by EVs, a view that is supported by animal experiments demonstrating that intracranial injection of glioma-derived EVs in healthy mice results in similarly modified transcription [103]. Recently, Mirzai and Wong [5] have reviewed microglia-T cell communication and pointed out that the synthesis of immunosuppressive cytokines such as TGF-β and IL-10 is increased as a consequence of their interaction. In addition to the release of immunosuppressive cytokines, Acod1 (aconitate decarboxylase 1) has been identified as a gene that is involved in the regulation and subsequent adjustment of the microglia/macrophage phenotype during GBM progression [108]. These findings are in line with the view that GBM alters gene transcription in microglia, supporting tumour invasion and migration while microglia remove necrotic debris and digested ECM in the TME. It is also worth mentioning that CXCL14 has been proposed as an important determinant of the glioma immune microenvironment where it is thought to promote activated CD8+ T cell chemotaxis which appears to prolong survival [109]. Interestingly, pleomorphic xanthoastrocytoma (PXA) shows increased CXCL14 secretion and contains a higher number of activated cytotoxic CD8+ T cells, increased expression of MHC class I and other genes associated with antigen presentation and processing as well as a higher number of AIF1 (Iba1)+ immunoreactive microglia/macrophages when compared to IDH-mutant astrocytoma [109]. Blockage of Spp1 not only reduces recruitment of macrophages but also renders GBM cells more sensitive to direct CD8+ T cell cytotoxicity [19]. Inhibition of MIF-CD74 interaction also leads to the expansion and activation of CD8+ T cells [92].

CCL5 is an inflammatory cytokine secreted by multiple cell types, including endothelial cells, monocytes, macrophages and NK cells [110]. Moreover, CCL5 is involved in tumour growth and cell migration in various cancers including glioma [110]. Glioma cells that have been stimulated with CCL5 show increased intracellular calcium levels and elevated Akt (p-Akt) and Ca²⁺/calmodulin-dependent protein kinase II phosphorylation (p-CaMKII) in a time- and dose-dependent manner. Increased intracellular calcium levels and p-CaMKII lead to upregulated expression of calcium-dependent MMP2 in glioma cells [81]; MMP2 has been previously associated with GBM cell migration and invasion [111]. In addition to the invasion-promoting role of CCL5, Wu et al. also demonstrated that glioma cells exhibit a strong affinity for glioma-associated microglia/macrophages (GAMs), specifically GAMs that have been activated by Granulocyte-macrophage colony-stimulating factor (GM-CSF). The authors even use the term “homing”. This observation fits with findings showing that conditioned media derived from GM-CSF activated GAMs contain significantly higher concentrations of CCL5 [81]. Moreover, a more recent study has shown that expression of CD11a by microglia may play an important role in the production of glioma derived CCL5 [112]. On the flip side, small interfering RNA silencing of CaMKII resulted in inhibition of CCL5-mediated glioma invasion [81]. Furthermore, downregulated expression of microglial CCL5 and CCR2 in athymic mice showed impaired engraftment of Nf1 optic low grade glioma stem cells [113]. Another study has shown that Na+/H+ exchanger isoform 1 (NHE1), a major interaction partner of calmodulin, stimulates microglial release of soluble factors leading to enhanced glioma proliferation and invasion [114] whereas blockage of NHE1 improves glioma tumour immunity by restoring mitochondrial OXPHOS (oxidative phosphorylation) function in myeloid cells [115]. Furthermore, Venkataramani et al. [116] have found that neural stimulation induces higher intracellular calcium level in GBM cells resulting in de novo formation of GBM microtubes and increased tumour invasiveness.

The co-chaperone STIP1 (STI1), a ligand of the cellular prion protein [117], has been demonstrated to participate in the survival and differentiation of neuronal cells [118]. STIP1 is highly expressed in glioma cells [46]. Strikingly, increased levels of STIP1 are also noted in microglia/macrophages as glioma progresses [46]. Furthermore, a significant upregulation of STIP1 expression is observed in glioma-infiltrating macrophages [46]. Therefore, STIP1 falls into the category of “Janus” genes. They represent promising therapeutic targets.

6. Isocitrate Dehydrogenase (IDH) Mutation Status Influences Glioblastoma Microglia/Macrophage Tissue Phenotype

6.1. Microglia/Macrophages in IDH-Mutant Astrocytoma (Grade 4) and IDH-Wildtype GBM

6.2. SET Domain Containing 2, Histone Lysine Methyltransferase (SETD2)

6.3. Intercellular Adhesion Molecule 1 (ICAM1), Lysosomal Associated Membrane Protein 1 (LAMP1) and Transmembrane Protein 119 (TMEM119)

6.4. C-C Motif Chemokine Ligand 18 (CCL18)/Chemokine (C-C Motif) Receptor 8 (CCR8)/Acid Phosphatase 5 (ACP5)/AKT1 Substrate 1 (AKT1S1/PRAS40)/Akt Pathway

6.5. ATP Binding Cassette Subfamily A Member 1 (ABCA1)

7. Exosomes, Extracellular Vesicles and MicroRNAs in Glioma Progression

7.1. Interleukin-6 (IL-6) and MicroRNA-155-3p (miR-155-3p)

7.2. MicroRNA-1246 (miR-1246)

7.3. Circ_0012381, Arginine Deprivation

7.4. MicroRNA as a Potential Therapeutic Tool for Targeting Glioma