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Review

Immunopathology of Extracellular Vesicles in Macrophage and Glioma Cross-Talk

by
Timothy J. Kopper
,
Xiaoli Yu
and
Michael W. Graner
*
Department of Neurosurgery, University of Colorado Anschutz Medical Campus, 12700 E 19th Ave., Aurora, CO 80045, USA
*
Author to whom correspondence should be addressed.
J. Clin. Med. 2023, 12(10), 3430; https://doi.org/10.3390/jcm12103430
Submission received: 14 March 2023 / Revised: 25 April 2023 / Accepted: 11 May 2023 / Published: 12 May 2023
(This article belongs to the Section Clinical Neurology)
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Abstract

:
Glioblastomas (GBM) are a devastating disease with extremely poor clinical outcomes. Resident (microglia) and infiltrating macrophages are a substantial component of the tumor environment. In GBM and other cancers, tumor-derived extracellular vesicles (EVs) suppress macrophage inflammatory responses, impairing their ability to identify and phagocytose cancerous tissues. Furthermore, these macrophages then begin to produce EVs that support tumor growth and migration. This cross-talk between macrophages/microglia and gliomas is a significant contributor to GBM pathophysiology. Here, we review the mechanisms through which GBM-derived EVs impair macrophage function, how subsequent macrophage-derived EVs support tumor growth, and the current therapeutic approaches to target GBM/macrophage EV crosstalk.

Graphical Abstract

1. Introduction

Glioblastomas (CNS5 WHO Grade 4 astrocytomas, IDH-wildtype, henceforth called GBM) are a devastating disease, with extremely poor clinical outcomes evidenced by the median survival time of 15 months post-diagnosis [1]. New therapies are desperately needed; however, many recent approaches have failed to improve outcomes [2,3,4,5], including immunotherapeutic approaches. Insufficient understanding of the tumor microenvironment is a critical challenge impairing the development of effective therapies. Resident (microglia) and infiltrating macrophages are a substantial component of the tumor environment and the tumor itself, constituting 30–50% of the cellular content [6,7], and are primary mediators of inflammatory responses [8]. Macrophages are a key cellular component of a successful anti-tumor response in which they identify and phagocytose cancerous tissues [9,10]. In GBM and other cancers, this capability is impaired or blocked entirely by tumor-induced suppression of anti-tumor immune responses [11,12,13]. In an increasing number of instances, this is recognized as being induced through extracellular vesicle (EV) intercellular communication [12]. EVs are lipid-enclosed vesicles released from cells that contain proteins, nucleic acids, and other biological mediators, allowing for intercellular communication in both health and disease [14,15]. GBM/macrophage EV crosstalk is a critical component of the tumor microenvironment [11,12]. With improved understanding, these interactions could be harnessed—or discouraged—to engage endogenous anti-tumor responses and develop novel targeted therapies. Here, we review the current state of GBM/macrophage EV crosstalk in the immunopathology of GBM.

2. Macrophage/Microglial Polarization in GBM Pathology

Macrophages are a key component of the innate and adaptive immune response. Circulating monocytes exit the bloodstream into numerous tissues and mature into resident macrophage populations throughout the body [16]. Microglia, the resident macrophage population in the central nervous system, are unique in that this population is established during embryonic development [17]. Functionally, however, monocyte-derived macrophages and microglia retain extensive similarities. In innate immunity, macrophages contain numerous receptors and sensors to identify the presence of infection, cellular damage, or cancer [16]. These macrophages can then induce numerous signaling cascades, inducing localized inflammation (production of reactive oxygen species, lymphocyte and Natural Killer (NK) cell activation, recruitment of neutrophils from the bloodstream, etc.). Similarly, these macrophages can induce systemic responses, such as a fever [16]. Once the activating stimulus is resolved, macrophages also produce factors contributing to cellular repair and the resolution of inflammation [18]. In adaptive immunity, macrophages are an important antigen-presenting cell which identify microbial antigens, or in the case of cancer, antigens from tumors that are either not expressed by healthy cells or antigens that are expressed much more highly in tumor cells [16]. Through this antigen expression, macrophages are able to activate adaptive immune responses, notably T-cells, to specifically target that antigen throughout the body [19]. Unfortunately, in tumors, the cancer often adapts to evade and manipulate the immune response. For example, tumors can begin to produce proteases, clipping off the ligand necessary for the macrophage-activated NK cells to attack the tumor cells [20]. Further, the tumor can signal directly to nearby macrophages to manipulate their activities, as discussed in detail in this review.
Macrophages are an extremely malleable cell type capable of adopting a wide spectrum of activation states [21,22]. These activation states control many aspects of macrophage physiology, including morphology, phagocytic activity, cytokine production, inflammatory profiles, and many other components of the immune response [23,24]. While macrophage activation is complex and multifaceted, for practical purposes this is often simplified to a pro-inflammatory “M1” subset and a second anti-inflammatory “M2” subset. The M1/M2 nomenclature is overly simplistic and does not cleanly apply across most disease states [25]; however, for general scientific discourse, it remains the best option available so long as its limitations are understood. In GBM, it is generally understood that pro-inflammatory “M1”-polarized macrophages promote a robust immune response and anti-tumor effects, whereas anti-inflammatory “M2”-polarized macrophages promote pro-tumor effects and dampen the broader immune response [13,26]. M0 is sometimes used in reference to naïve unstimulated macrophages [27]. Intracellularly, these macrophage activation states are regulated by several canonical and non-canonical signaling pathways. For M1 macrophages, this is typically via the NF-κβ/STAT1 [28,29] signaling axes, often modeled in vitro using lipopolysaccharide and interferon gamma stimulation of TLR-4 and the interferon-gamma receptor [30]. Conversely, M2 macrophages are more typically associated with the STAT3/STAT6 signaling axes [28], modeled with Interleukin 4 and/or 13 stimulation [31]. In the context of GBM, however, countless potential environmental stimuli likely induce complex activation states not fully in line with the M1/M2 nomenclature [32,33]. Further, given that macrophages are highly impressionable by extremely localized environmental factors, there are likely diverse activation states across the GBM macrophage populations [21]. Continued advances in single-cell RNA sequencing technologies will allow for an improved understanding of how the macrophage activation spectrum impacts GBM pathophysiology [34].
Critically, in the context of GBM, macrophage activation status determines whether the macrophage attacks the cancerous tissue or promotes continued growth and broader immune suppression [17,25]. M1 macrophages express high levels of antigen-presenting MHC complexes, through which they can display tumor-derived antigens and induce robust adaptive immune responses against the tumor [35,36]. M1 macrophages also express elevated levels of the costimulatory molecule CD87 (B7-2) allowing for improved activation of naïve T-cells [31]. Similarly, M1 macrophages produce elevated levels of cytotoxic factors such as reactive oxygen species, nitric oxide, and pro-inflammatory cytokines to directly target nearby tumor cells. Conversely, M2 macrophages are generally anti-inflammatory and immunosuppressive [26]. With reduced MHC and costimulatory molecule expression, M2 macrophages have a reduced ability to activate the adaptive immune response [35]. Further, under certain conditions, M2 macrophages can directly promote tumor growth in response to tumor signals, as discussed in this review. GBM macrophages are a heterogenous cell population derived from both tissue-resident microglia and infiltrating myeloid-derived macrophages. While these populations share many similarities and are often referred to collectively as macrophages, some distinctions can occur [25,37]. When examined separately experimentally, specific terminology will be utilized for clarity.

3. EVs as Biological Mediators of Intercellular Communication

EVs are small lipid-enclosed vesicles released from cells into the extracellular space [38,39]. EVs contain a broad mixture of biological materials, including proteins, lipids, nucleic acids, and other metabolites, whose contents can vary depending on the cellular source and EV subtype [40]. Even the lipid composition of the EV membrane can influence biological activity [41]. Historically, EVs were further distinguished into several EV subtypes based on size and cellular source or biogenesis. Exosomes are a subtype of EVs derived from the endosomal/multivesicular body system and range from 30–150 nm in diameter. Microvesicles are a subtype of EVs formed from the plasma membrane through outward budding and range from 100 nm to 1 µm in diameter. Apoptotic bodies form from dying cells. Notably, apoptotic bodies differ in that they contain additional cellular material, such as intact organelles and chromatin [42]. In recent years, the EV research field has updated its guidelines on EV terminology [43]. When specified in the source literature, the original terminology will be used in this review; however, where sufficient method detail is provided, we will also include the current terminology. Notably, these recent guidelines describe EVs in terms of their physical characteristics, molecular composition, and cellular origin via biogenesis [43]. Here, this will support a shift in terminology from the previous term “exosome” towards the modern terminology, “small EVs” (sEVs) for EVs under 200 nm diameter. We note that the biogenesis of “exosomes” versus “microvesicles” is seldom known when collecting EVs from biofluids or conditioned cell/tissue culture media, and thus the term “EVs” refers to both such populations [44].
While EVs were originally thought to be a form of cellular waste, it has since been appreciated that EVs conduct an intricate form of cellular communication [40]. They allow for cellular crosstalk both locally and across great distances [45]. These communications occur both in normal physiological conditions and in disease [15]. GBM tumor cells show an increased production of EVs relative to healthy tissue [46,47]. Critically, in GBM, EVs are thought to be a primary means through which the tumor manipulates its surroundings to invoke a more conducive environment for further growth and anti-cancer drug resistance [44,48,49]. Here, we review the mechanisms of GBM/macrophage EV communications and the implications of these signals in GBM immunopathology. Table 1 provides a brief summary of the key mechanisms through which GBM tissue and macrophages exchange EV crosstalk.

4. Impact of GBM-Derived EVs on Macrophage Function

EVs are a critical mechanism utilized by GBM tissue to evade the immune system [59]. Macrophages are the predominant immune cells in the tumor vicinity; however, they are ineffective at targeting tumors [60]. Worse, some macrophages begin to support the tumor, allowing for increased and faster growth [61]. GBM tissue supports this maladaptive macrophage response through numerous EV-dependent mechanisms as detailed below.
In 2015, de Vrij et al. applied GBM-derived EVs (presumably small EVs) onto monocyte-derived macrophages and observed several changes [48] (Table 1). Notably, the application of GBM-derived EVs resulted in an increased expression of CD163, a marker associated with the M2 pro-tumor phenotype, as measured by flow cytometry. The macrophages were found to produce increased levels of VEGF and IL-6. These cytokines have been previously found to support tumor growth (IL-6) [62] and tumor hyper-vascularization (VEGF) [63]. The authors hypothesized that these effects were caused by small RNA molecules enriched within GBM EVs, as had been demonstrated elsewhere with EVs from different tumor types [64]; however, they did not identify an exact candidate. Work by van der Vos et al. (2016) supported this hypothesis by visualizing the extensive uptake of GBM EVs (presumably small EVs) by primary microglia and noting the increase in GBM EV-cargo microRNAs in the microglia and macrophage. These studies suggest the transfer of microRNAs from EVs to the microglia/macrophage [65].
Zhao et al. (2022) also demonstrated an M2 polarization shift in macrophages treated with GBM EVs (presumably small EVs). They found that GBM EVs are enriched in microRNA-27a-3p and have even higher levels under hypoxic conditions (Figure 1, Table 1). Further, they demonstrated that microRNA-27a-3p inhibits enhancer of zeste homologue 1 (EZH1) [50]. Inhibition of EZH1 has been previously demonstrated to promote M2 macrophage polarization [66]. Indeed, the authors observed that microRNA-27a-3p-treated macrophages had elevated levels of Arginase-1 (M2 phenotypic marker) and reduced iNOS (M1 phenotypic marker). Myeloid-cell-derived arginase-1 (via arginine depletion) has a noted ability to suppress T-cell responses [67]. A similar study confirmed the transfer of miR-21 from GBM to microglia in an in vivo mouse model and observed substantial microglial reprogramming [51]. This was an important study in that the host mice were miR-21 null, directly implying the microRNA was transferred from GBM EVs to cells of the tumor microenvironment. This is further evidence that GBM EVs may be preferentially taken up by tumor-associated macrophages to promote a supportive (immune-suppressed) tumor environment. These effects are mediated by GBM EV microRNAs such as microRNA-27a-3p and miR-21, although other EV components could certainly contribute towards this effect (Table 1, Figure 1).
Proteomics analysis has demonstrated a diverse set of proteins enriched in GBM EVs with likely bioactive effects [68]. Indeed, in 2018, Gabrusiewicz et al. also found that GBM stem-cell-derived exosomes (presumably small EVs) applied to monocytes induced a shift in monocyte-derived macrophage polarization state [49]. Specifically, through flow cytometry they found that macrophages exposed to these exosomes had reduced expression of M1 indicators (MHCII and CD80) and increased M2 indicators (CD163 and CD206). The authors found that the exosomes contained the transcription factor STAT3, a transcription factor heavily associated with M2 macrophage polarization [49,69]. This suggested that GBM-derived STAT3 is transferred to the macrophages inducing their phenotypic shift towards a tumor-supportive phenotype. Further, they demonstrated the exosomes used had a propensity to be taken up by macrophages relative to other cell types (Table 1, Figure 1) [49]. Additional work to understand how GBM-derived EVs target macrophage populations could unveil an advantageous therapeutic target.
Additional studies have also investigated the roles of GBM EVs on macrophage physiology. Yang et al. (2019) found that the GMB EV microRNA miR-214-5p was associated with poor clinical prognosis and targeted microglial CXCR5 transcripts, and thus reduced protein expression (Table 1, Figure 1) [52]. CXCR5 has numerous roles in cancer biology, notably through chemoattractant interactions with its ligand CXCL13 [70]. Xu et al. (2021) found that exosomes (presumably small EVs) from hypoxic glioma cells also induce macrophages towards an M2 state through an induction of autophagy pathways (Table 1). These results were greatly subdued in EVs derived from normoxic glioma cells [53].
Collectively, it is clear that GBM EVs are taken up by macrophage populations and subsequently modulate cellular functions towards a tumor-supportive phenotype. While this is frequently addressed as an M2 macrophage or M2-like, it is likely far more complex, with numerous caveats. In 2015, de Vrij et al., for example, saw a macrophage shift towards the M2 phenotype after GBM EV exposure, yet observed increased IL-6 production. While IL-6 has been documented as a tumor-supportive cytokine in this context, it is typically associated with M1 macrophages [31,71]. This highlights the uniqueness of the GBM microglial/macrophage population to consider when developing macrophage-targeted therapies developed in other contexts.

5. Impact of Macrophage-Derived EVs on GBM Immunopathology

While GBM regulation of macrophage physiology is well documented, macrophage-derived EVs also influence tumor health. Under normal physiological conditions, macrophage EVs are a component of homeostatic immune function and cancer surveillance. For example, sEVs taken from healthy microglia displayed anti-cancer protective effects in a mouse model of GBM [54]. Specifically, microglia-derived sEVs modified glioma metabolism, thereby supporting the restoration of glutamate homeostasis [54], a key aspect of glioma pathophysiology [72]. Notably, these sEVs contained miR-124, an onco-suppressor highly downregulated in GBM patients’ tumors relative to healthy control normal brain tissue (Table 1, Figure 2) [54,73,74]. Unfortunately, following exposure to GBM EVs, the sEVs produced by macrophages shift towards a tumor-supportive phenotype, performing tumor-supportive functions [54]. Intriguingly, one mechanism through which macrophages are co-opted to support tumor growth is through their own sEV production. These macrophage sEVs then exert tumor-supportive functions through numerous distinct mechanisms detailed here.
Zhang et al. (2020), for example, demonstrated that tumor-associated macrophages produced sEVs containing a series of microRNAs (miR-27a-3p, miR-22-3p, and miR-221-3p) (Table 1, Figure 2) [55]. These microRNAs induced glioma stem cells to undergo proneural-to-mesenchymal transition through a CHD7-dependent mechanism. Proneural-to-mesenchymal transition during GBM progression is believed to be tumor-supportive, as it leads to increased resistance to various therapeutic modalities, including radiotherapy [55,75,76]. This suggests that GBM-derived EVs induce macrophages to produce EVs that in turn increase resistance to one of the few viable treatments clinically available to treat GBM. Interestingly, it is not yet known whether this process is instigated by mutagenic chance during GBM development or as a direct response to radiotherapy. Curiously, macrophage EVs from cervical cancer patients treated with radiation drove macrophages from M2-like to M1-like phenotypes. The impact of radiation on GBM-associated microglia and macrophages is complex, but usually considered detrimental to tumor treatment effects [77,78]; notably, this is often in the context of infiltrating macrophages following radiation [79]. The effect of microglial/macrophage-derived EVs in GBM therapeutic resistance seems understudied. Further insight into primary vs. recurrent GBM macrophage populations could begin to answer these questions.
Recently Zhao et al. (2022) implicated exosomes (likely small EVs) from M2-like tumor-associated macrophages in the phenotypes of glioblastoma stem cells. Specifically, they isolated glioblastoma stem cells and tumor-associated macrophages through fluorescence-activated cell sorting (FACS) of GMB tissue specimens. EVs isolated from the macrophage population were applied to the GBM stem cells where the EVs were found to maintain some stem cell properties of the glioblastoma stem cells (Table 1, Figure 2) [56]. Through an in vivo xenograft tumor model of GBM, they implicated the microRNA miR-27b-3p as the mediator for the macrophage EV-induced increase in the tumorigenicity of the glioblastoma stem cells. This miR-27b-3p-induced process was found to be mediated through the MLL4/PRDM1/IL-33 cell signaling axis [56].
Another key mechanism through which macrophage EVs support tumor growth is by supporting angiogenesis in the tumor microenvironment, allowing for the delivery of oxygen and metabolites to fuel further growth. One mechanism by which macrophage sEVs accomplish this is through the transfer of circular RNA circKIF18A from macrophage sEVs to brain microvessel endothelial cells, which enhances the activity of the FOXC2 transcription factor (Table 1, Figure 2) [57]. FOXC2 is a key regulator of angiogenesis [80]. Here, the authors attributed the pro-angiogenic effects of circKIF18A to the RNA-aiding nuclear entry of FOXC2, leading to the transcription factor upregulating the expression of ITGB3, CXCR4, and DLL4, and activating the PI3K/AKT signaling axis [57].
Azambuja et al. (2020) demonstrated that GBM EV-reprogramed macrophages produce EVs that promote glioma cell proliferation and migration in vitro [58]. Intriguingly, they attributed these effects, at least in part, to the expression of the anti-inflammatory enzyme Arginase-1 on the surface of these macrophage EVs (size not directly measured but likely sEVs) (Table 1, Figure 2). Arginase-1 is a potent anti-inflammatory mediator and is strongly associated with M2 macrophages [31,81]. Previous studies have identified Arginase-1 as an important mediator of the survival and progression of tumors [67,82]. Arginase-1 catalyzes the conversion of arginine to ornithine and urea. Local and systemic levels of these molecules have substantial impacts on inflammation, T-cell development and ability to recognize tumors, and general progression of some tumors [83]. For example, L-arginine is a key factor in the maturation of the T-cell receptor and is thus critical in the recognition of tumor antigens by T-cells. Arginase-1’s enzymatic function is to convert L-arginine into ornithine and urea; therefore, high Arginase-1 expression can directly deplete L-arginine and impair T-cell responses. This is in addition to Arginase-1’s roles in tumorigenesis and metastasis [84]. This novel observation of Arginase-1 on macrophage EVs, therefore, has significant implications in the tumor microenvironment [58].
Macrophage EVs are not inherently tumor-supportive; however, following exposure to GBM-derived EVs, macrophages adopt a polarization state that promotes tumor growth. While the numerous microRNAs and Arginase-1 described here are significant contributors to the growth-permissive effects of macrophage EVs, there are likely additional mechanisms contributing to the multifaceted macrophage response. With continued investigation, these pathways hold great potential in the development of future therapeutic targets.

6. Approaches to Target GBM/Macrophage EV Crosstalk

GMB/macrophage EV crosstalk has substantial impacts on GBM disease progression and therefore represents a potent therapeutic target. Given the complexities of GBM EV release, transfer to macrophages, and the subsequent production of EVs by macrophages, there are numerous aspects that could be therapeutically targeted, including EV neutralization, macrophage depletion, macrophage stimulants, exogenous EV therapies, and numerous other emerging approaches.
We have previously targeted the toxic effects of GMB sEVs on neurons using customized peptides targeting GBM sEVs, which we isolated with peptide phage display technology [85]. A similar approach could block the tumor-supportive effects of GBM sEVs on macrophage physiology and/or allow for additional anti-tumor cellular activity. This approach could be further enhanced through the use of multiple peptides in combination to maximize effects. Clearly, the delivery of such peptide blockers requires novel approaches, but one could envision application during surgery into the resection cavity [86], or perhaps even using healthy macrophage-derived EVs [87].
Macrophages perform predominantly detrimental functions in the GBM tumor environment as a result of macrophage manipulation by GBM sEVs [8,13,77]. While microglia and macrophages are capable of performing anti-tumor functions, in this case, it may be beneficial to remove them from the tumor microenvironment. One well-developed approach to deplete macrophages is through the use of clodronate-enclosed liposomes. These small artificial lipid vesicles are selectively taken by macrophages, minimizing off-target effects on other cells and tissues. When loaded with clodronate, the liposomes can deplete macrophages across numerous disease models, including a clinical trial in canines with soft-tissue sarcomas [88].
GBM-derived sEVs are a primary mediator suppressing macrophage anti-tumor activity [12]. One approach to rectify this could be macrophage-targeted therapies to shift macrophage polarization states towards anti-tumor activities. This could be performed through a systemic drug or a targeted approach, such as drug-enclosed liposomes. Stimulating macrophages with bacterial components (Mycobacterium bovis bacillus Calmette–Guérin), for example, have been found to induce increased anti-cancer macrophage activity [89]. Similarly, Banerjee et al. (2015) utilized Mycobacterium indicus pranii to repolarize macrophages towards an anti-tumor M1 phenotype [90]. Numerous other therapeutics are available with proven capabilities to modulate macrophage phenotype and anti-tumor activity [91]. With continued development, these therapeutics could be utilized to counter the detrimental effects of GBM-derived sEVs on macrophage physiology in the context of the GBM microenvironment.
Grimaldi et al. (2019) used an interesting approach to rescue microglia manipulated by GBM EVs. Microglia were exposed to the standard M1 stimulants lipopolysaccharide and interferon-gamma (LPS/IFN-γ), with microvesicles (EVs identified as 100–1000 nm, see [92]) isolated from the resulting culture. They then injected these treated EVs into the brains of tumor-bearing mice, resulting in a reduction in tumor size. Intriguingly, the M1 microglial-derived EVs resulted in a phenotypic shift of tumor macrophages towards an anti-cancer phenotype. Molecular analysis of the M1 microglia EV cargo identified a wide variety of inflammation-associated transcripts. While GBM patient microglia would be difficult to implement as a therapeutic approach, patient macrophages would be simple to culture from blood monocytes. Further work is needed to determine if this approach would be feasible or safe to develop as a treatment for GBM in humans. In a multi-layered approach, Wang et al. (2022) isolated sEVs from M1 macrophages that had been treated with AQ4N, a pro-drug that is activated in hypoxic conditions. AQ4N was sequestered in the released sEVs. These sEVs were further membrane-modified with two agents: a chemical excitation source, CPPO [hydrophobic bis(2,4,5-trichloro-6-carbopentoxyphenyl) oxalate]; and a photosensitizer, Ce6 (chlorin e6) [93]. Across multiple GBM models, the modified sEVs were able to induce M1 phenotypes in macrophages, leading to in vivo settings with intravenous injection of the modified sEVs. In addition to the anti-tumor effects of M1 macrophages, the increased H2O2 produced by M1 macrophages triggered a chemical reaction between CPPO and Ce6 creating cytotoxic reactive oxygen species within the tumor [93]. Further, the chemically driven induction of additional hypoxia released the activated anti-cancer agent AQ4. This unique approach, blending tumor microenvironment manipulation with localized chemical ablation of the tumor, holds great potential, but likely requires considerable development before reaching its clinical potential.
Given that GBM/macrophage EV crosstalk predominately supports pro-tumor functions, a blockade of these lines of communication may provide therapeutic benefits. Numerous compounds, chemicals, and peptides have been utilized to interfere with EV uptake [94]; however, the diverse nature of EV physiology presents a major challenge. The numerous subsets of EVs can each be taken up by several district cellular pathways, such as endocytosis, micropinocytosis, phagocytosis, membrane fusion, clathrin-dependent endocytosis, caveolin-mediated endocytosis, and lipid raft-mediated endocytosis [94]. To address this, further understanding of which EV populations are detrimental in GBM/macrophage EV crosstalk is needed. A targeted therapy, such as liposomal delivery to macrophages, may be able to bypass some of these challenges through combinatorial approaches targeting multiple uptake pathways.
Numerous publications have detailed the roles of sEV-derived microRNAs in pathological GBM/macrophage crosstalk [95,96], while another group has instead developed microRNA-loaded EVs as a potential GBM therapeutic in vitro. Specifically, Hong et al. (2021) loaded miRNA-124 into HEK293T-derived EVs (likely small EVs). In GBM cells (U373MG cells), these EVs reduced mRNA expression levels indicative of tumor progression, proliferation, migration, and epithelial–mesenchymal transition (STAT3, c-Myc, Mcl-1, ITG-β1, Vimentin, and Slug). In human microglia (SV40 microglial cell line), miRNA-124-loaded EVs (presumably small EVs) reduced expression of the M2 macrophage markers TGF-β and Arginase-1 and upregulated the M1 marker IL-6 [97]. It was noted that miR-504 appears downregulated in GBM and GBM stem cells compared to normal brain, particularly in those stem cells of the mesenchymal GBM subtype [98]. The miR targets the expression of Grb10, the latter acting as an oncogene. Forced over-expression of miR-504 in GBM stem cells reduced both their mesenchymal phenotype and their tumorigenicity. The miR could be passed from the GBM cells to microglia through EV secretion inducing a shift towards M1 polarization of the microglia, also likely reducing the pro-tumor capacities of the microglia (Bier et al., 2020). Similarly, Li et al. (2021) loaded long non-coding RNAs into exosomes (size not specified; protocol would suggest sEVs) as a means to demonstrate a mechanism in which GBM chemotherapy resistance develops through microglial complement production [99]. These studies highlight the adaptability of EVs as potential carriers of GBM therapeutics.
Extensive work across cancer disciplines has developed numerous approaches to target tumor-associated macrophages, many of which could be adapted to target GBM/macrophage EV crosstalk. Grégoire et al. (2020) performed a comprehensive review of clinical trials targeting macrophages as a therapeutic strategy to fight cancer [77]. These trials identified dozens of monoclonal antibodies and small molecule drug candidates aiming to block the recruitment of macrophages to the tumor, deplete macrophages from the tumor microenvironment, or reprogram macrophage polarization state towards anti-cancer phenotypes with Toll-like receptor (TLR) antagonists [77]. These studies emphasize the immense impact macrophages have on the tumor microenvironment and their impact as a therapeutic target. Similarly, Wang et al. (2022) reviewed recent clinical trials targeting macrophages, specifically for the treatment of GBM [17]. While these clinical trials were not performed with the explicit intent to target macrophage/GBM EV crosstalk, any approach that significantly depletes macrophages or blocks their recruitment would inevitably impact EV crosstalk. Similarly, any attempt to reprogram macrophages could also alter EV cargo that is impacted by macrophage activation state [100]. Intriguingly, many of these clinical trials aim to deplete macrophages or macrophage recruitment [77]; however, these approaches would not likely impact GBM-derived EVs. It is currently unknown whether the absence of macrophages could alter or enhance the GBM-derived EV impacts on the remaining cells in the tumor environment [101,102].
These diverse approaches to target GBM/macrophage EV crosstalk highlight the immense potential for the development of GBM therapeutics targeting these pathways; however, continued development is needed to push these approaches towards clinical applications.

7. Conclusions

GBM is a devastating disease in great need of new therapies to improve outcomes. GBM EV-mediated manipulation of macrophage immune defenses and reciprocal EV crosstalk from macrophages is a critical component impairing recovery; therefore, we have provided a brief description of these key mechanisms mediating direct GBM/macrophage/microglia EV crosstalk (Figure 1 and Figure 2, Table 1). To address the challenges produced through GBM EV crosstalk with macrophages and microglia, further insight is needed into the mechanisms through which GBM manipulates macrophage physiology to the tumor’s benefit. With this knowledge, we can better develop therapies to impair these detrimental processes and improve patient outcomes for this disease.
It is well established that GBM EVs are a primary mechanism through which macrophages are targeted [48,50,65]; however, much remains uncertain. EVs are incredibly diverse extracellular entities derived from several distinct populations and taken up through numerous cellular pathways [38]. Further understanding of which exact pathway(s) is/are mediating GBM control of macrophage populations is key for the development of future macrophage-directed therapeutics. Similarly, macrophages are a diverse heterogenous population of cells. Distinct roles and spatiotemporal contributions of both microglia and monocyte-derived macrophages likely have differential roles throughout the pathogenesis of GBM progression. Some of the above-mentioned studies do note the potential for recruitment or conversion of macrophages towards a tumor-attacking phenotype, suggesting that cells already within the tumor microenvironment may be therapeutically malleable. With continued understanding, therapeutics can be utilized to their greatest potential while minimizing unintended consequences.
Given the complexity of GBM/macrophage EV crosstalk, there are countless therapeutic targets and areas for proposed therapies. Unfortunately, most of these approaches are early in development and will require considerable time and investment before any potential clinical application. Given the dire need for new therapies for GBM patients, strong considerations should be made in the selection of therapeutic agents. Often, existing drugs can be repurposed, greatly improving the speed at which new therapies can be implemented. To date, numerous clinical trials across cancer disciplines have developed several distinct approaches to target tumor-associated macrophages [77]. Adapting successful approaches from other cancer disciplines into GBM therapies may allow for faster clinical implementation.
GBM/macrophage EV crosstalk is an important component of the GBM microenvironment that is relatively early in development. Given this, there remain many uncertainties and challenges to overcome. With continued development, however, these pathways hold great promise in the development of novel treatments for the GBM patient population for which there are currently so few currently available.

Author Contributions

Conceptualization, T.J.K. and M.W.G. Formal analysis, T.J.K., X.Y. and M.W.G. Investigation, T.J.K. Resources, T.J.K., X.Y. and M.W.G. Writing—original draft, T.J.K. and M.W.G. Writing—reviewing and editing, T.J.K., X.Y. and M.W.G. Visualization, T.J.K. Supervision, M.W.G. Project Administration, X.Y. and M.W.G. Funding acquisition, X.Y. and M.W.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work is supported by National Institutes of Health 5R33MH118174-05 grant support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Thakkar, J.P.; Dolecek, T.A.; Horbinski, C.; Ostrom, Q.T.; Lightner, D.D.; Barnholtz-Sloan, J.S.; Villano, J.L. Epidemiologic and molecular prognostic review of glioblastoma. Cancer Epidemiol. Biomark. Prev. 2014, 23, 1985–1996. [Google Scholar] [CrossRef] [PubMed]
  2. Burster, T.; Traut, R.; Yermekkyzy, Z.; Mayer, K.; Westhoff, M.-A.; Bischof, J.; Knippschild, U. Critical View of Novel Treatment Strategies for Glioblastoma: Failure and Success of Resistance Mechanisms by Glioblastoma Cells. Front. Cell Dev. Biol. 2021, 9, 695325. [Google Scholar] [CrossRef] [PubMed]
  3. Sun, R.; Kim, A.H. The multifaceted mechanisms of malignant glioblastoma progression and clinical implications. Cancer Metastasis Rev. 2022, 41, 871–898. [Google Scholar] [CrossRef] [PubMed]
  4. Vilar, J.B.; Christmann, M.; Tomicic, M.T. Alterations in Molecular Profiles Affecting Glioblastoma Resistance to Radiochemotherapy: Where Does the Good Go? Cancers 2022, 14, 2416. [Google Scholar] [CrossRef]
  5. Fabro, F.; Lamfers, M.L.M.; Leenstra, S. Advancements, Challenges, and Future Directions in Tackling Glioblastoma Resistance to Small Kinase Inhibitors. Cancers 2022, 14, 600. [Google Scholar] [CrossRef]
  6. Charles, N.A.; Holland, E.C.; Gilbertson, R.; Glass, R.; Kettenmann, H. The brain tumor microenvironment. Glia 2011, 59, 1169–1180. [Google Scholar] [CrossRef]
  7. Badie, B.; Schartner, J.M. Flow cytometric characterization of tumor-associated macrophages in experimental gliomas. Neurosurgery 2000, 46, 957–962. [Google Scholar] [PubMed]
  8. Basheer, A.S.; Abas, F.; Othman, I.; Naidu, R. Role of Inflammatory Mediators, Macrophages, and Neutrophils in Glioma Maintenance and Progression: Mechanistic Understanding and Potential Therapeutic Applications. Cancers 2021, 13, 4226. [Google Scholar] [CrossRef]
  9. Annovazzi, L.; Mellai, M.; Bovio, E.; Mazzetti, S.; Pollo, B.; Schiffer, D. Microglia immunophenotyping in gliomas. Oncol. Lett. 2018, 15, 998–1006. [Google Scholar] [CrossRef]
  10. Wu, S.-Y.; Watabe, K. The roles of microglia/macrophages in tumor progression of brain cancer and metastatic disease. Front. Biosci. 2017, 22, 1805–1829. [Google Scholar] [CrossRef]
  11. Andersen, J.K.; Miletic, H.; Hossain, J.A. Tumor-Associated Macrophages in Gliomas-Basic Insights and Treatment Opportunities. Cancers 2022, 14, 1319. [Google Scholar] [CrossRef] [PubMed]
  12. Yekula, A.; Yekula, A.; Muralidharan, K.; Kang, K.; Carter, B.S.; Balaj, L. Extracellular Vesicles in Glioblastoma Tumor Microenvironment. Front. Immunol. 2019, 10, 3137. [Google Scholar] [CrossRef] [PubMed]
  13. Ma, J.; Chen, C.C.; Li, M. Macrophages/Microglia in the Glioblastoma Tumor Microenvironment. Int. J. Mol. Sci. 2021, 22, 5775. [Google Scholar] [CrossRef] [PubMed]
  14. Buzas, E.I. The roles of extracellular vesicles in the immune system. Nat. Rev. Immunol. 2022, 23, 236–250. [Google Scholar] [CrossRef]
  15. Shetty, A.K.; Upadhya, R. Extracellular Vesicles in Health and Disease. Aging Dis. 2021, 12, 1358–1362. [Google Scholar] [CrossRef] [PubMed]
  16. Parham, P. The Immune System; Garland Science: New York, NY, USA, 2009. [Google Scholar]
  17. Wang, G.; Zhong, K.; Wang, Z.; Zhang, Z.; Tang, X.; Tong, A.; Zhou, L. Tumor-associated microglia and macrophages in glioblastoma: From basic insights to therapeutic opportunities. Front. Immunol. 2022, 13, 964898. [Google Scholar] [CrossRef]
  18. Kotwal, G.J.; Chien, S. Macrophage Differentiation in Normal and Accelerated Wound Healing. Results Probl. Cell Differ. 2017, 62, 353–364. [Google Scholar]
  19. Chang, R.B.; Beatty, G.L. The interplay between innate and adaptive immunity in cancer shapes the productivity of cancer immunosurveillance. J. Leukoc. Biol. 2020, 108, 363–376. [Google Scholar] [CrossRef]
  20. Chitadze, G.; Kabelitz, D. Immune surveillance in glioblastoma: Role of the NKG2D system and novel cell-based therapeutic approaches. Scand. J. Immunol. 2022, 96, e13201. [Google Scholar] [CrossRef]
  21. Mosser, D.M.; Edwards, J.P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 2008, 8, 958–969. [Google Scholar] [CrossRef]
  22. Xue, J.; Schmidt, S.V.; Sander, J.; Draffehn, A.; Krebs, W.; Quester, I.; De Nardo, D.; Gohel, T.D.; Emde, M.; Schmidleithner, L.; et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 2014, 40, 274–288. [Google Scholar] [CrossRef] [PubMed]
  23. Arango Duque, G.; Descoteaux, A. Macrophage Cytokines: Involvement in Immunity and Infectious Diseases. Front. Immunol. 2014, 5, 491. [Google Scholar] [CrossRef]
  24. Lichtnekert, J.; Kawakami, T.; Parks, W.C.; Duffield, J.S. Changes in macrophage phenotype as the immune response evolves. Curr. Opin. Pharmacol. 2013, 13, 555–564. [Google Scholar] [CrossRef]
  25. Buonfiglioli, A.; Hambardzumyan, D. Macrophages and microglia: The cerberus of glioblastoma. Acta Neuropathol. Commun. 2021, 9, 54. [Google Scholar] [CrossRef]
  26. Liu, J.; Geng, X.; Hou, J.; Wu, G. New insights into M1/M2 macrophages: Key modulators in cancer progression. Cancer Cell Int. 2021, 21, 389. [Google Scholar] [CrossRef]
  27. Zhao, Y.-L.; Tian, P.-X.; Han, F.; Zheng, J.; Xia, X.-X.; Xue, W.-J.; Ding, X.-M.; Ding, C.-G. Comparison of the characteristics of macrophages derived from murine spleen, peritoneal cavity, and bone marrow. J. Zhejiang Univ. Sci. B 2017, 18, 1055–1063. [Google Scholar] [CrossRef]
  28. Wang, N.; Liang, H.; Zen, K. Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front. Immunol. 2014, 5, 614. [Google Scholar] [CrossRef]
  29. Haydar, D.; Cory, T.J.; Birket, S.E.; Murphy, B.S.; Pennypacker, K.R.; Sinai, A.P.; Feola, D.J. Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-κB Signaling Pathways. J. Immunol. 2019, 203, 1021–1030. [Google Scholar] [CrossRef]
  30. Kopper, T.J.; Zhang, B.; Bailey, W.M.; Bethel, K.E.; Gensel, J.C. The effects of myelin on macrophage activation are phenotypic specific via cPLA 2 in the context of spinal cord injury inflammation. Sci. Rep. 2021, 11, 6341. [Google Scholar] [CrossRef]
  31. Gensel, J.C.; Kopper, T.J.; Zhang, B.; Orr, M.B.; Bailey, W.M. Predictive screening of M1 and M2 macrophages reveals the immunomodulatory effectiveness of post spinal cord injury azithromycin treatment. Sci. Rep. 2017, 7, 40144. [Google Scholar] [CrossRef]
  32. Kopper, T.J.; Gensel, J.C. Myelin as an inflammatory mediator: Myelin interactions with complement, macrophages, and microglia in spinal cord injury. J. Neurosci. Res. 2017, 21, 1831–1839. [Google Scholar] [CrossRef] [PubMed]
  33. Garofalo, S.; Porzia, A.; Mainiero, F.; Di Angelantonio, S.; Cortese, B.; Basilico, B.; Pagani, F.; Cignitti, G.; Chece, G.; Maggio, R.; et al. Environmental stimuli shape microglial plasticity in glioma. Elife 2017, 6, e33415. [Google Scholar] [CrossRef] [PubMed]
  34. Khan, F.; Pang, L.; Dunterman, M.; Lesniak, M.S.; Heimberger, A.B.; Chen, P. Macrophages and microglia in glioblastoma: Heterogeneity, plasticity, and therapy. J. Clin. Investig. 2023, 133, e163446. [Google Scholar] [CrossRef] [PubMed]
  35. DeNardo, D.G.; Ruffell, B. Macrophages as regulators of tumour immunity and immunotherapy. Nat. Rev. Immunol. 2019, 19, 369–382. [Google Scholar] [CrossRef]
  36. Duan, Z.; Luo, Y. Targeting macrophages in cancer immunotherapy. Signal Transduct. Target. Ther. 2021, 6, 127. [Google Scholar] [CrossRef]
  37. Yin, J.; Valin, K.L.; Dixon, M.L.; Leavenworth, J.W. The Role of Microglia and Macrophages in CNS Homeostasis, Autoimmunity, and Cancer. J. Immunol. Res. 2017, 2017, 5150678. [Google Scholar] [CrossRef]
  38. Doyle, L.M.; Wang, M.Z. Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis. Cells 2019, 8, 727. [Google Scholar] [CrossRef]
  39. Di Bella, M.A. Overview and Update on Extracellular Vesicles: Considerations on Exosomes and Their Application in Modern Medicine. Biology 2022, 11, 804. [Google Scholar] [CrossRef]
  40. Bazzan, E.; Tinè, M.; Casara, A.; Biondini, D.; Semenzato, U.; Cocconcelli, E.; Balestro, E.; Damin, M.; Radu, C.M.; Turato, G.; et al. Critical Review of the Evolution of Extracellular Vesicles’ Knowledge: From 1946 to Today. Int. J. Mol. Sci. 2021, 22, 6417. [Google Scholar] [CrossRef]
  41. Cauvi, D.M.; Hawisher, D.; Dores-Silva, P.R.; Lizardo, R.E.; De Maio, A. Macrophage reprogramming by negatively charged membrane phospholipids controls infection. FASEB J. 2019, 33, 2995–3009. [Google Scholar] [CrossRef]
  42. Battistelli, M.; Falcieri, E. Apoptotic Bodies: Particular Extracellular Vesicles Involved in Intercellular Communication. Biology 2020, 9, 21. [Google Scholar] [CrossRef]
  43. Théry, C.; Witwer, K.W.; Aikawa, E.; Alcaraz, M.J.; Anderson, J.D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G.K.; et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 2018, 7, 1535750. [Google Scholar] [CrossRef]
  44. Graner, M.W. Roles of Extracellular Vesicles in High-Grade Gliomas: Tiny Particles with Outsized Influence. Annu. Rev. Genom. Hum. Genet. 2019, 20, 331–357. [Google Scholar] [CrossRef]
  45. van Niel, G.; D’Angelo, G.; Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 2018, 19, 213–228. [Google Scholar] [CrossRef]
  46. Hallal, S.; Mallawaaratchy, D.M.; Wei, H.; Ebrahimkhani, S.; Stringer, B.W.; Day, B.W.; Boyd, A.W.; Guillemin, G.J.; Buckland, M.E.; Kaufman, K.L. Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways. Mol. Neurobiol. 2019, 56, 4566–4581. [Google Scholar] [CrossRef]
  47. Andre, F.; Schartz, N.E.C.; Movassagh, M.; Flament, C.; Pautier, P.; Morice, P.; Pomel, C.; Lhomme, C.; Escudier, B.; Le Chevalier, T.; et al. Malignant effusions and immunogenic tumour-derived exosomes. Lancet 2002, 360, 295–305. [Google Scholar] [CrossRef]
  48. de Vrij, J.; Maas, S.L.N.; Kwappenberg, K.M.C.; Schnoor, R.; Kleijn, A.; Dekker, L.; Luider, T.M.; de Witte, L.D.; Litjens, M.; van Strien, M.E.; et al. Glioblastoma-derived extracellular vesicles modify the phenotype of monocytic cells. Int. J. Cancer 2015, 137, 1630–1642. [Google Scholar] [CrossRef]
  49. Gabrusiewicz, K.; Li, X.; Wei, J.; Hashimoto, Y.; Marisetty, A.L.; Ott, M.; Wang, F.; Hawke, D.; Yu, J.; Healy, L.M.; et al. Glioblastoma stem cell-derived exosomes induce M2 macrophages and PD-L1 expression on human monocytes. Oncoimmunology 2018, 7, e1412909. [Google Scholar] [CrossRef]
  50. Zhao, G.; Yu, H.; Ding, L.; Wang, W.; Wang, H.; Hu, Y.; Qin, L.; Deng, G.; Xie, B.; Li, G.; et al. microRNA-27a-3p delivered by extracellular vesicles from glioblastoma cells induces M2 macrophage polarization via the EZH1/KDM3A/CTGF axis. Cell. Death Discov. 2022, 8, 260. [Google Scholar] [CrossRef]
  51. Abels, E.R.; Maas, S.L.N.; Nieland, L.; Wei, Z.; Cheah, P.S.; Tai, E.; Kolsteeg, C.-J.; Dusoswa, S.A.; Ting, D.T.; Hickman, S.; et al. Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21. Cell. Rep. 2019, 28, 3105–3119.e7. [Google Scholar] [CrossRef]
  52. Yang, J.-K.; Liu, H.-J.; Wang, Y.; Li, C.; Yang, J.-P.; Yang, L.; Qi, X.-J.; Zhao, Y.-L.; Shi, X.-F.; Li, J.-C.; et al. Exosomal miR-214-5p Released from Glioblastoma Cells Modulates Inflammatory Response of Microglia after Lipopolysaccharide Stimulation through Targeting CXCR5. CNS Neurol. Disord. Drug. Targets 2019, 18, 78–87. [Google Scholar] [CrossRef] [PubMed]
  53. Xu, J.; Zhang, J.; Zhang, Z.; Gao, Z.; Qi, Y.; Qiu, W.; Pan, Z.; Guo, Q.; Li, B.; Zhao, S.; et al. Hypoxic glioma-derived exosomes promote M2-like macrophage polarization by enhancing autophagy induction. Cell. Death Dis. 2021, 12, 373. [Google Scholar] [CrossRef] [PubMed]
  54. Serpe, C.; Monaco, L.; Relucenti, M.; Iovino, L.; Familiari, P.; Scavizzi, F.; Raspa, M.; Familiari, G.; Civiero, L.; D’Agnano, I.; et al. Microglia-Derived Small Extracellular Vesicles Reduce Glioma Growth by Modifying Tumor Cell Metabolism and Enhancing Glutamate Clearance through miR-124. Cells 2021, 10, 2066. [Google Scholar] [CrossRef]
  55. Zhang, Z.; Xu, J.; Chen, Z.; Wang, H.; Xue, H.; Yang, C.; Guo, Q.; Qi, Y.; Guo, X.; Qian, M.; et al. Transfer of MicroRNA via Macrophage-Derived Extracellular Vesicles Promotes Proneural-to-Mesenchymal Transition in Glioma Stem Cells. Cancer Immunol. Res. 2020, 8, 966–981. [Google Scholar] [CrossRef] [PubMed]
  56. Zhao, G.; Ding, L.; Yu, H.; Wang, W.; Wang, H.; Hu, Y.; Qin, L.; Deng, G.; Xie, B.; Li, G.; et al. M2-like tumor-associated macrophages transmit exosomal miR-27b-3p and maintain glioblastoma stem-like cell properties. Cell Death Discov. 2022, 8, 350. [Google Scholar] [CrossRef]
  57. Jiang, Y.; Zhao, J.; Xu, J.; Zhang, H.; Zhou, J.; Li, H.; Zhang, G.; Xu, K.; Jing, Z. Glioblastoma-associated microglia-derived exosomal circKIF18A promotes angiogenesis by targeting FOXC2. Oncogene 2022, 41, 3461–3473. [Google Scholar] [CrossRef] [PubMed]
  58. Azambuja, J.H.; Ludwig, N.; Yerneni, S.S.; Braganhol, E.; Whiteside, T.L. Arginase-1+ Exosomes from Reprogrammed Macrophages Promote Glioblastoma Progression. Int. J. Mol. Sci. 2020, 21, 3990. [Google Scholar] [CrossRef]
  59. Low, J.J.W.; Sulaiman, S.A.; Johdi, N.A.; Abu, N. Immunomodulatory effects of extracellular vesicles in glioblastoma. Front. Cell Dev. Biol. 2022, 10, 996805. [Google Scholar] [CrossRef]
  60. De Leo, A.; Ugolini, A.; Veglia, F. Myeloid Cells in Glioblastoma Microenvironment. Cells 2020, 10, 18. [Google Scholar] [CrossRef]
  61. Geribaldi-Doldán, N.; Fernández-Ponce, C.; Quiroz, R.N.; Sánchez-Gomar, I.; Escorcia, L.G.; Velásquez, E.P.; Quiroz, E.N. The Role of Microglia in Glioblastoma. Front. Oncol. 2020, 10, 603495. [Google Scholar] [CrossRef]
  62. Sasaki, A.; Ishiuchi, S.; Kanda, T.; Hasegawa, M.; Nakazato, Y. Analysis of interleukin-6 gene expression in primary human gliomas, glioblastoma xenografts, and glioblastoma cell lines. Brain Tumor Pathol. 2001, 18, 13–21. [Google Scholar] [CrossRef] [PubMed]
  63. Wei, Q.; Singh, O.; Ekinci, C.; Gill, J.; Li, M.; Mamatjan, Y.; Karimi, S.; Bunda, S.; Mansouri, S.; Aldape, K.; et al. TNFα secreted by glioma associated macrophages promotes endothelial activation and resistance against anti-angiogenic therapy. Acta Neuropathol. Commun. 2021, 9, 67. [Google Scholar] [CrossRef] [PubMed]
  64. Fabbri, M.; Paone, A.; Calore, F.; Galli, R.; Gaudio, E.; Santhanam, R.; Lovat, F.; Fadda, P.; Mao, C.; Nuovo, G.J.; et al. MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response. Proc. Natl. Acad. Sci. USA 2012, 109, E2110–E2116. [Google Scholar] [CrossRef] [PubMed]
  65. van der Vos, K.E.; Abels, E.R.; Zhang, X.; Lai, C.; Carrizosa, E.; Oakley, D.; Prabhakar, S.; Mardini, O.; Crommentuijn, M.H.W.; Skog, J.; et al. Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain. Neuro. Oncol. 2016, 18, 58–69. [Google Scholar] [CrossRef] [PubMed]
  66. Jia, X.; Xu, H.; Miron, R.J.; Yin, C.; Zhang, X.; Wu, M.; Zhang, Y. EZH1 Is Associated with TCP-Induced Bone Regeneration through Macrophage Polarization. Stem Cells Int. 2018, 2018, 6310560. [Google Scholar] [CrossRef]
  67. Munder, M. Arginase: An emerging key player in the mammalian immune system. Br. J. Pharmacol. 2009, 158, 638–651. [Google Scholar] [CrossRef]
  68. Mallawaaratchy, D.M.; Hallal, S.; Russell, B.; Ly, L.; Ebrahimkhani, S.; Wei, H.; Christopherson, R.I.; Buckland, M.E.; Kaufman, K.L. Comprehensive proteome profiling of glioblastoma-derived extracellular vesicles identifies markers for more aggressive disease. J. Neurooncol. 2017, 131, 233–244. [Google Scholar] [CrossRef]
  69. Tugal, D.; Liao, X.; Jain, M.K. Transcriptional control of macrophage polarization. Arterioscler. Thromb. Vasc. Biol. 2013, 33, 1135–1144. [Google Scholar] [CrossRef]
  70. Kazanietz, M.G.; Durando, M.; Cooke, M. CXCL13 and Its Receptor CXCR5 in Cancer: Inflammation, Immune Response, and Beyond. Front. Endocrinol. (Lausanne) 2019, 10, 471. [Google Scholar] [CrossRef]
  71. Hu, H.M.; Baer, M.; Williams, S.C.; Johnson, P.F.; Schwartz, R.C. Redundancy of C/EBP alpha, -beta, and -delta in supporting the lipopolysaccharide-induced transcription of IL-6 and monocyte chemoattractant protein-1. J. Immunol. 1998, 160, 2334–2342. [Google Scholar] [CrossRef]
  72. de Groot, J.; Sontheimer, H. Glutamate and the biology of gliomas. Glia 2011, 59, 1181–1189. [Google Scholar] [CrossRef] [PubMed]
  73. Bhaskaran, V.; Nowicki, M.O.; Idriss, M.; Jimenez, M.A.; Lugli, G.; Hayes, J.L.; Mahmoud, A.B.; Zane, R.E.; Passaro, C.; Ligon, K.L.; et al. The functional synergism of microRNA clustering provides therapeutically relevant epigenetic interference in glioblastoma. Nat. Commun. 2019, 10, 442. [Google Scholar] [CrossRef] [PubMed]
  74. Silber, J.; Lim, D.A.; Petritsch, C.; Persson, A.I.; Maunakea, A.K.; Yu, M.; Vandenberg, S.R.; Ginzinger, D.G.; James, C.D.; Costello, J.F.; et al. miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells. BMC Med. 2008, 6, 14–17. [Google Scholar] [CrossRef] [PubMed]
  75. Fedele, M.; Cerchia, L.; Pegoraro, S.; Sgarra, R.; Manfioletti, G. Proneural-Mesenchymal Transition: Phenotypic Plasticity to Acquire Multitherapy Resistance in Glioblastoma. Int. J. Mol. Sci. 2019, 20, 2746. [Google Scholar] [CrossRef]
  76. Bhat, K.P.L.; Balasubramaniyan, V.; Vaillant, B.; Ezhilarasan, R.; Hummelink, K.; Hollingsworth, F.; Wani, K.; Heathcock, L.; James, J.D.; Goodman, L.D.; et al. Mesenchymal differentiation mediated by NF-κB promotes radiation resistance in glioblastoma. Cancer Cell. 2013, 24, 331–346. [Google Scholar] [CrossRef]
  77. Grégoire, H.; Roncali, L.; Rousseau, A.; Chérel, M.; Delneste, Y.; Jeannin, P.; Hindré, F.; Garcion, E. Targeting Tumor Associated Macrophages to Overcome Conventional Treatment Resistance in Glioblastoma. Front. Pharmacol. 2020, 11, 368. [Google Scholar] [CrossRef]
  78. Gutmann, D.H.; Kettenmann, H. Microglia/Brain Macrophages as Central Drivers of Brain Tumor Pathobiology. Neuron 2019, 104, 442–449. [Google Scholar] [CrossRef]
  79. Andersen, R.S.; Anand, A.; Harwood, D.S.L.; Kristensen, B.W. Tumor-Associated Microglia and Macrophages in the Glioblastoma Microenvironment and Their Implications for Therapy. Cancers 2021, 13, 4255. [Google Scholar] [CrossRef]
  80. Hayashi, H.; Kume, T. Foxc2 transcription factor as a regulator of angiogenesis via induction of integrin beta3 expression. Cell Adhes. Migr. 2009, 3, 24–26. [Google Scholar] [CrossRef]
  81. Yang, Z.; Ming, X.-F. Functions of arginase isoforms in macrophage inflammatory responses: Impact on cardiovascular diseases and metabolic disorders. Front. Immunol. 2014, 5, 533. [Google Scholar] [CrossRef]
  82. Szefel, J.; Danielak, A.; Kruszewski, W.J. Metabolic pathways of L-arginine and therapeutic consequences in tumors. Adv. Med. Sci. 2019, 64, 104–110. [Google Scholar] [CrossRef] [PubMed]
  83. Grzywa, T.M.; Sosnowska, A.; Matryba, P.; Rydzynska, Z.; Jasinski, M.; Nowis, D.; Golab, J. Myeloid Cell-Derived Arginase in Cancer Immune Response. Front. Immunol. 2020, 11, 938. [Google Scholar] [CrossRef] [PubMed]
  84. Niu, F.; Yu, Y.; Li, Z.; Ren, Y.; Li, Z.; Ye, Q.; Liu, P.; Ji, C.; Qian, L.; Xiong, Y. Arginase: An emerging and promising therapeutic target for cancer treatment. Biomed. Pharm. 2022, 149, 112840. [Google Scholar] [CrossRef] [PubMed]
  85. Zhou, W.; Craft, J.; Ojemann, A.; Bergen, L.; Graner, A.; Gonzales, A.; He, Q.; Kopper, T.; Smith, M.; Graner, M.W.; et al. Glioblastoma Extracellular Vesicle-Specific Peptides Inhibit EV-Induced Neuronal Cytotoxicity. Int. J. Mol. Sci. 2022, 23, 7200. [Google Scholar] [CrossRef] [PubMed]
  86. Chiocca, E.A.; Yu, J.S.; Lukas, R.V.; Solomon, I.H.; Ligon, K.L.; Nakashima, H.; Triggs, D.A.; Reardon, D.A.; Wen, P.; Stopa, B.M.; et al. Regulatable interleukin-12 gene therapy in patients with recurrent high-grade glioma: Results of a phase 1 trial. Sci. Transl. Med. 2019, 11, eaaw5680. [Google Scholar] [CrossRef]
  87. Bai, L.; Liu, Y.; Guo, K.; Zhang, K.; Liu, Q.; Wang, P.; Wang, X. Ultrasound Facilitates Naturally Equipped Exosomes Derived from Macrophages and Blood Serum for Orthotopic Glioma Treatment. ACS Appl. Mater. Interfaces 2019, 11, 14576–14587. [Google Scholar] [CrossRef]
  88. Guth, A.M.; Hafeman, S.D.; Elmslie, R.E.; Dow, S.W. Liposomal clodronate treatment for tumour macrophage depletion in dogs with soft-tissue sarcoma. Vet. Comp. Oncol. 2013, 11, 296–305. [Google Scholar] [CrossRef]
  89. Luo, Y.; Han, R.; Evanoff, D.P.; Chen, X. Interleukin-10 inhibits Mycobacterium bovis bacillus Calmette-Guérin (BCG)-induced macrophage cytotoxicity against bladder cancer cells. Clin. Exp. Immunol. 2010, 160, 359–368. [Google Scholar] [CrossRef]
  90. Banerjee, S.; Halder, K.; Ghosh, S.; Bose, A.; Majumdar, S. The combination of a novel immunomodulator with a regulatory T cell suppressing antibody (DTA-1) regress advanced stage B16F10 solid tumor by repolarizing tumor associated macrophages in situ. Oncoimmunology 2015, 4, e995559. [Google Scholar] [CrossRef]
  91. Zhou, J.; Tang, Z.; Gao, S.; Li, C.; Feng, Y.; Zhou, X. Tumor-Associated Macrophages: Recent Insights and Therapies. Front. Oncol. 2020, 10, 188. [Google Scholar] [CrossRef]
  92. Grimaldi, A.; Serpe, C.; Chece, G.; Nigro, V.; Sarra, A.; Ruzicka, B.; Relucenti, M.; Familiari, G.; Ruocco, G.; Pascucci, G.R.; et al. Microglia-Derived Microvesicles Affect Microglia Phenotype in Glioma. Front. Cell. Neurosci. 2019, 13, 41. [Google Scholar] [CrossRef] [PubMed]
  93. Wang, X.; Ding, H.; Li, Z.; Peng, Y.; Tan, H.; Wang, C.; Huang, G.; Li, W.; Ma, G.; Wei, W. Exploration and functionalization of M1-macrophage extracellular vesicles for effective accumulation in glioblastoma and strong synergistic therapeutic effects. Signal Transduct. Target. Ther. 2022, 7, 74. [Google Scholar] [CrossRef] [PubMed]
  94. Mulcahy, L.A.; Pink, R.C.; Carter, D.R.F. Routes and mechanisms of extracellular vesicle uptake. J. Extracell. Vesicles 2014, 3, 24641. [Google Scholar] [CrossRef] [PubMed]
  95. Matias, D.; Balça-Silva, J.; da Graça, G.C.; Wanjiru, C.M.; Macharia, L.W.; Nascimento, C.P.; Roque, N.R.; Coelho-Aguiar, J.M.; Pereira, C.M.; Dos Santos, M.F.; et al. Microglia/Astrocytes-Glioblastoma Crosstalk: Crucial Molecular Mechanisms and Microenvironmental Factors. Front. Cell. Neurosci. 2018, 12, 235. [Google Scholar] [CrossRef]
  96. Pancholi, S.; Tripathi, A.; Bhan, A.; Acharya, M.M.; Pillai, P. Emerging Concepts on the Role of Extracellular Vesicles and Its Cargo Contents in Glioblastoma-Microglial Crosstalk. Mol. Neurobiol. 2022, 59, 2822–2837. [Google Scholar] [CrossRef]
  97. Hong, S.; You, J.Y.; Paek, K.; Park, J.; Kang, S.J.; Han, E.H.; Choi, N.; Chung, S.; Rhee, W.J.; Kim, J.A. Inhibition of tumor progression and M2 microglial polarization by extracellular vesicle-mediated microRNA-124 in a 3D microfluidic glioblastoma microenvironment. Theranostics 2021, 11, 9687–9704. [Google Scholar] [CrossRef]
  98. Bier, A.; Hong, X.; Cazacu, S.; Goldstein, H.; Rand, D.; Xiang, C.; Jiang, W.; Ben-Asher, H.W.; Attia, M.; Brodie, A.; et al. miR-504 modulates the stemness and mesenchymal transition of glioma stem cells and their interaction with microglia via delivery by extracellular vesicles. Cell Death Dis. 2020, 11, 899. [Google Scholar] [CrossRef]
  99. Li, Z.; Meng, X.; Wu, P.; Zha, C.; Han, B.; Li, L.; Sun, N.; Qi, T.; Qin, J.; Zhang, Y.; et al. Glioblastoma Cell-Derived lncRNA-Containing Exosomes Induce Microglia to Produce Complement C5, Promoting Chemotherapy Resistance. Cancer Immunol. Res. 2021, 9, 1383–1399. [Google Scholar] [CrossRef]
  100. Reed, T.; Schorey, J.; D’Souza-Schorey, C. Tumor-Derived Extracellular Vesicles: A Means of Co-opting Macrophage Polarization in the Tumor Microenvironment. Front. Cell Dev. Biol. 2021, 9, 746432. [Google Scholar] [CrossRef]
  101. Graner, M.W.; Schnell, S.; Olin, M.R. Tumor-derived exosomes, microRNAs, and cancer immune suppression. Semin. Immunopathol. 2018, 40, 505–515. [Google Scholar] [CrossRef]
  102. Graner, M.W. Extracellular vesicles in cancer immune responses: Roles of purinergic receptors. Semin. Immunopathol. 2018, 40, 465–475. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. GBM-derived EV cargo manipulates macrophages and microglia physiology towards tumor-supportive functions. EVs are released from GBM tissue and have immunosuppressive effects on nearby macrophages and microglia, promoting their conversion towards an M2-like tumor-supportive phenotype. This effect on macrophages is mediated through EV cargo, including STAT3 [49], microRNA-214-5p [52], microRNA-27a-3p [50], and potential anti-inflammatory cytokines [49]. Depicted microRNA structures are not representative of actual structure.
Figure 1. GBM-derived EV cargo manipulates macrophages and microglia physiology towards tumor-supportive functions. EVs are released from GBM tissue and have immunosuppressive effects on nearby macrophages and microglia, promoting their conversion towards an M2-like tumor-supportive phenotype. This effect on macrophages is mediated through EV cargo, including STAT3 [49], microRNA-214-5p [52], microRNA-27a-3p [50], and potential anti-inflammatory cytokines [49]. Depicted microRNA structures are not representative of actual structure.
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Figure 2. Macrophages and microglia exposed to GBM-derived EVs produce tumor-supportive EV cargo. These macrophages and microglia, modified by GBM EV-cargo, then release EVs that support tumor growth, migration, and chemotherapy resistance. This is mediated through EV cargo, including microRNA-124 [54,74], microRNA-27b-3p [56], microRNA-27a-3p [55], microRNA-22-3p and 221-3p [55], circularRNA-KIP18A [57], and Arginase-1 [58]. Collectively, this crosstalk suppresses the immune response to the tumor and supports further tumor growth. Depicted microRNA structures are not representative of actual structure.
Figure 2. Macrophages and microglia exposed to GBM-derived EVs produce tumor-supportive EV cargo. These macrophages and microglia, modified by GBM EV-cargo, then release EVs that support tumor growth, migration, and chemotherapy resistance. This is mediated through EV cargo, including microRNA-124 [54,74], microRNA-27b-3p [56], microRNA-27a-3p [55], microRNA-22-3p and 221-3p [55], circularRNA-KIP18A [57], and Arginase-1 [58]. Collectively, this crosstalk suppresses the immune response to the tumor and supports further tumor growth. Depicted microRNA structures are not representative of actual structure.
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Table
Table 1. Key publications detailing direct GBM/macrophage/microglia EV crosstalk.
Table 1. Key publications detailing direct GBM/macrophage/microglia EV crosstalk.
AuthorBiological Mediator in EVsKey Findings
de Vrij et al., 2015 [48]Small RNA moleculesGBM-derived sEVs induced monocyte-derived macrophages towards an M2 pro-tumor phenotype. The macrophages produced increased levels of VEGF and IL-6.
Zhao et al., 2022 [50]microRNA-27a-3pHere, the authors demonstrated that microRNA-27a-3p inhibits enhancer of zeste homologue 1 (EZH1), thereby promoting M2 macrophage polarization. microRNA-27a-3p-treated macrophages had elevated levels of Arginase-1 (M2 phenotypic marker) and reduced iNOS (M1 phenotypic marker).
Abels et al., 2019 [51]microRNA-21The authors found that tumor-derived EVs deliver miR-21 to microglia. This resulted in a shift in numerous gene targets, notably the downregulation of Pdcd4 and Btg2, causing increased microglial proliferation.
Gabrusiewicz et al., 2018 [49]Stat3GBM stem-cell-derived sEVs applied to monocytes induced a shift in monocyte-derived macrophage polarization state towards a tumor-supportive M2 phenotype. This was evidenced through flow cytometry analysis, indicating reduced expression of M1 indicators (MHCII and CD80) and increased M2 indicators (CD163 and CD206). The sEVs contained the transcription factor STAT3, which is associated with M2 polarization.
Yang et al., 2019 [52]microRNA-214-5pGMB EV microRNA miR-214-5p was associated with poor clinical prognosis and targeted microglial CXCR5 transcripts, and thus reduced CXCR5 protein expression.
Xu et al., 2021 [53]Stat3, IL-6, and microRNA-155-3pGBM-derived sEVs were isolated under hypoxic conditions, common in the GBM microenvironment. These EVs induce macrophages towards an M2 state through an induction of autophagy pathways. These results were greatly subdued in EVs derived from normoxic glioma cells.
Serpe et al., 2021 [54]microRNA-124Cultured microglia were stimulated with LPS and IFNγ (M1 activation). sEVs isolated from these microglia were applied intracranially via a cannula infusion into tumor-bearing mice. The application of these microglia-derived EVs resulted in prolonged survival and reduced tumor mass. These effects were attributed to microRNA-124-induced modulation of tumor metabolism.
Zhang et al., 2020 [55]microRNA-27a-3p, microRNA-22-3p, and microRNA-221-3pThe authors found that tumor-associated macrophages produce sEVs that trigger a proneural-to-mesenchymal transition in glioma stem cells. This is believed to be tumor-supportive as it leads to increased resistance to various therapeutic modalities, including radiotherapy. This was attributed to EV cargo, particularly microRNA-27a-3p, microRNA-22-3p, and microRNA-221-3p.
Zhao, G et al., 2022 [56]microRNA-27b-3pThe authors isolated glioblastoma stem cells and tumor-associated macrophages from GMB tissue specimens. Macrophage sEVs were applied to the GBM stem cells where they maintained some of the stem cell properties of the GBM stem cells. They implicated microRNA-27b-3p for the increase in the tumorigenicity of the GBM stem cells. This process was found to be mediated through the MLL4/PRDM1/IL-33 cell signaling axis.
Jiang e al. 2022 [57]Circular RNA circKIF18AHere, a human microglial cell line was treated with GBM-conditioned media to induce an M2 phenotype. sEVs from these treated microglia then transferred circular RNA circKIF18A from microglia. circKIF18A enhances the FOXC2 transcription factor activity, leading to increased angiogenesis in the tumor microenvironment.
Azambuja et al., 2020 [58]Arginase-1Using an in vitro model of GBM/macrophage crosstalk, the authors found that macrophage EVs promote glioma cell proliferation and migration in vitro. Using chemical inhibitors, they identified Arginase-1 on the surface of these EVs as a primary mediator. Arginase-1 is strongly associated with M2 macrophages.
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Kopper, T.J.; Yu, X.; Graner, M.W. Immunopathology of Extracellular Vesicles in Macrophage and Glioma Cross-Talk. J. Clin. Med. 2023, 12, 3430. https://doi.org/10.3390/jcm12103430

AMA Style

Kopper TJ, Yu X, Graner MW. Immunopathology of Extracellular Vesicles in Macrophage and Glioma Cross-Talk. Journal of Clinical Medicine. 2023; 12(10):3430. https://doi.org/10.3390/jcm12103430

Chicago/Turabian Style

Kopper, Timothy J., Xiaoli Yu, and Michael W. Graner. 2023. "Immunopathology of Extracellular Vesicles in Macrophage and Glioma Cross-Talk" Journal of Clinical Medicine 12, no. 10: 3430. https://doi.org/10.3390/jcm12103430

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