Facts

  • TAMs potentially establish the complicated, unique intercellular interactions of the glioma ecosystem. Meanwhile, they also provide a potent alternative for the targeted therapeutics.

  • Exosomes would provide us with promising nanoplatforms for targeted drug delivery with enormous advantages, which would open a new era of eradication of tumors under immunoregulation.

  • TAM-centered strategies can be divided into reducing the recruitment or depletion and reprograming into the M2-like phenotype.

  • Exosomes would significantly assist the TAMs-centered treatment as drug delivery vehicles and liquid biopsy biomarkers.

Question

  • What is the difference between different types of macrophages, such as microglia and bone marrow-derived macrophages?

  • How to realize the clinical applications of exosomes and overcome challenges such as massive production, standard isolation, drug loading, stability, quality control, etc.?

  • How to design the combined modality therapy with higher efficacy?

  • How to make a better transition from pre-clinical trials to clinical applications?

Introduction

Gliomas, accounting for ~25% of primary CNS tumors, have an average mortality rate of 4.43 per 100,000 and 16,606 deaths per year [1, 2]. Standard therapies of surgery resection combined with radiotherapy and alkylating agent chemotherapy showed little curative effect, with a median survival of 16 months [3]. Intensive research and clinical efforts have revealed growing knowledge about glioma [4]. However, there has been no significant breakthrough in therapeutics [5]. Efforts like temozolomide, bevacizumab (a molecule blocking vascular endothelial growth factor-A (VEGF-A)), cilengitide (a molecule blocking αVb3 and αVb5 integrin), lomustine, etc. [6], offered minimal survival benefit during the past decades [6,7,8]. Therefore, more effective therapeutic options are urgently needed. Accordingly, lots of laboratory research and increasing clinical laboratory investigations have been initiated to aid standard therapies [9]. Unfortunately, up to now, the result is still far from satisfactory. Many approaches have achieved encouraging outcomes in preclinical and early clinical stages, like immune checkpoint inhibitors (ICIs), including antibodies against PD-1, its ligand PD-L1, CTLA-4, and CAR T cell therapy, failed to exert their therapeutic effects in glioma [57]. Exosomal STAT3 derived from glioblastoma stem cells traverses the monocyte cytoplasm, causes a molecular change of the actin cytoskeleton, and induces monocytes to polarize toward the tumor-promoting M2 phenotype [58]. Besides, dendritic cell-derived exosomes with TNF superfamily ligands can boost tumor cell apoptosis [59], Natural killer, cell-derived exosomes with miR-186 exert cytotoxic effects on blastoma cells [60, 61]. Neutrophils-derived exosomes, with the different constitutions of a vast repertoire of cytokines, immunosuppressive or stimulatory molecules, can be pro-tumorigenic or antitumorigenic [62]. Also, glioma-derived exosomal EGFRvIII is transferred to neighboring glioma cells lacking EGFRvIII and then activates the MAPK and AKT pathway, horizontally transforming the phenotype among subsets of cancer cells [63].

More importantly, exosomes from specific sources exhibit different content profiles. For instance, the unique content of exosomes derived from tumor cells or immune cells containing a mass of tumor antigens like MHC-I could directly induces anticancer immunotherapy. With the potential to elicit antitumor responses, accumulating investigations are trying to develop exosomes into vital cancer vaccines or vaccine adjuvants [64, 65]. Exosome-based vaccines have presented infusive and hopeful results against various tumors. Exosome-based vaccines already approved have infusive and bright effects against various tumors and are approved by the FDA. TheraCys® is applied to treat early-stage bladder cancer, PROVENGE can be used for metastatic castration-resistant prostate cancer, and IMLYGIC® shows its efficacy in metastatic melanoma [66].

Exosomes as potential glioma biomarkers

In addition to their therapeutic potential, exosomes also present the potential to facilitate disease diagnosis and prognosis as robust biomarkers. Released continuously by all living cells, including glioma cells, exosomes are enriched in body fluids such as blood, urine, saliva, cerebrospinal fluid, sputum, etc [32, 67]. With advantages of minimal invasive characteristic, cost-saving, real-time evaluation of tumor condition, and so on, liquid biopsy attracts increasing attention in cancer. Compared with evaluating circulating tumor cells or other strategies, exosomes offer extraordinary advantages. Exosomes show high biological concentration, continuously released by alive cells, there is a relatively high level of cell-derived exosomes in various biofluid. In addition, containing different cell-derived molecules like DNA, RNA, bioactive protein, chains of glucose, metabolites, exosomes have more abundant information. And cargos contained within exosomes are protected from degradation [38, 68]. Exosome-based liquid biopsies provide information about the actions of tumor cells and present possible applications for tumor diagnosis, therapeutic response monitoring, and prognosis evaluation [55, 69]. Specifically, compared to circulating tumor cells, exosomes give better sensitivity during the initiating process, which is closely linked to carcinogenesis [70].

As shown above, exosomes act as a critical mediator of intercellular communication. Exosomes are significantly associated with tumor progression and microenvironment modulation. The exosome-based gene test to predict malignancies was the earliest available for prostate cancer in 2016 [71]. For CNS tumors, where sample collection is a chief obstacle, exosomes unfold a new vista for clinical evaluation. The structural and functional properties of exosomes make exosomes to be potent diagnostic/prognostic makers. With the potential to efficiently cross the intact BBB [72], Exosomes provide a valuable alternative that is less invasive than cerebrospinal fluid sampling. Exosomes derived from malignant glioma cells have been investigated for searching for robust biomarkers for glioma evaluation. For instance, exosomes containing significantly high MCT1 and CD147 indicate malignant glioma progression [73]. What’s more, exosome-derived miRNAs, the ample and fundamental biomolecules mediating intercellular communication, play essential roles in immunosuppression, induction, intrusion, metastasis, and treatment unresponsiveness of tumors [74]. The serum exosomal EGFRvIII mRNA of glioblastoma patients could be used to provide sufficient diagnostic information [55]. In short, many circulating exosomes and exosomal cargos that may play significant roles in the intricate cross-talk systems in glioma initiation, development, and dissemination, also provide us potent opportunities for glioma diagnosis and prognosis evaluation. To monitor drug response in GBM, Shao et al. have developed a microfluidic chip to evaluate the levels of unique exosomal mRNA (MGMT and APNG) [75]. In addition, for detecting prognostic biomarkers in glioma-derived exosomes, the TiO2-CTFE-AuNIs real-time label‐free plasmonic biosensor demonstrates its promising application in liquid glioma biopsy [76].

The vital role of TAMs in the GME and potential strategies underlying

The tumor microenvironment is widely recognized as critical in tumor development and treatment response. TAMs, the effective innate immune system, constitute a diminishing population among the complex components of the tumor microenvironment. In addition, they are the vital mononuclear phagocytes that bridge the natural immune response with the adaptive immune response by presenting relevant antigens to the T cells following the phagocytosis of apoptotic cells. It is not surprising that TAMs are increasingly recognized as crucial in normal processes like neural development, homeostasis, and central nervous system diseases. At the intersection of neuroscience and immunology, investigations on macrophage biology are gaining increasing attention. Meanwhile, knowledge about TAMs is fast-growing [77, 78]. (Fig. 2).

Fig. 2: Recruitment and polarization of TAMs.
figure 2

TAMs consist of residual microglia and bone marrow-derived macrophages (BMDMs). Various molecules are associated with the recruitment and polarization of TAMs. Under different stimuli, macrophages show distinct phenotypes, which can be roughly divided into immunosuppressive M2 phenotype (There’s significant upregulation of STAT3, PD-L1, Arg-1, CECR1, Romo1, etc.) and immune-stimulating M1 phenotype. (By Figdraw).

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A brief classification of TAMs

From the origin perspective, glioma TAMs consist of microglia and bone marrow-derived macrophages. Macrophages reside in most significant tissues and contribute to tissue homeostasis and disease. Following the expression of unique sets of transcriptional regulators, the progenitor cells differentiate into tissue-specific macrophages during organogenesis. Most adult macrophages, resident or infiltrating, are classical hematopoietic stem cells (HSC)-derived, except microglia. Nevertheless, the mechanisms responsible for the development of macrophage diversity remain unclear. The exact origins are still under debate [79,80,81]. Microglia, a population of resident macrophages derived from erythro-myeloid progenitors of the yolk sac [81], is the type of macrophages in the central nervous system under normal conditions. As a whole, microglia turn over slowly, and individual cells can potentially be decades old [82]. Besides serving as professional phagocytes, microglia significantly regulate neuronal activity at the synaptic level, ultimately supporting circuit plasticity [83]. Main classifications and more detailed information about microglia have been extensively reviewed [84, 92].

Simply put, TAMs are recruited to the GME and then release a considerable amount of growth factors and cytokines that strongly contribute to tumor proliferation, vascularization, invasion, metabolism, treatment resistance, and the immunosuppressive microenvironment. Intriguingly, TAMs can exert duplex influences, either to orchestrate a tumor-promoting response or enhance the anti-tumor effect, which presents us with a new vista of macrophage-centered therapies [93]. As the complicated interactions establish a unique tumor ecosystem, they also offer promising opportunities for therapeutic targets with attractive prospects [94].

Recruitment and polarization of TAMs

Under physiological conditions, exclusively via the IL-1α-dependent pathway, microglial populations are replenished from brain-resident microglia [95]. While under pathological conditions like trauma, infection, and brain tumor, microglia undergo substantial phenotypic changes, bone marrow-derived macrophages cross the impaired BBB and colonize the microglial niche. Distinct from other solid tumors, it is well-accepted that TAMs dominate the GME [96]. Among them, bone marrow-derived macrophages recruited peripherally constitute the significant population [94, 97]. Factors mediating TAMs chemoattraction mainly include various chemokines, surficial ligands, and other essential factors like neurotransmitters, ATP, etc [98].

TAMs are vital cells with different molecular profiles potentially regulated by microenvironment factors in sophisticated ways. Briefly, TNF-α, IFN-γ, and LPS (Toll-like receptor four ligands) drive polarization toward the M1 phenotype. While under stimulation of IL-4, IL-10, and IL-13, macrophages typically shift to the M2 phenotype [99]. At different states of differentiation, activation, and polarization, TAMs show different phenotypes and distinct functions. While regarding the separation of M1/M2 macrophages, little exclusivity was observed [93]. What’s more, as for TAMs from different sources, functions of resident microglia and recruited macrophages appear to differ in ways that remain unknown [100].

Monocyte chemoattractant protein-2 (CCL2, or MCP-1), the first identified chemoattractant molecule, is a critical molecule that recruits resident microglial cells to glioma and promotes its progression [101]. Glioblastoma cells produce kynurenine, which plays a role by stimulating aryl hydrocarbon receptor (AHR) in TAMs, which then contributes to the recruitment of TAMs via upregulated CCR2 expression [102]. Similarly, the up-released IL1β by TAMs activates the p38 MAPK signaling pathway and expression of CCL2 by tumor cells [103]. later, researchers confirmed a stronger correlation between the expression of CCL7 (also known as MCP-3) and the level of infiltrated microglia and macrophages [104].

CXCL12 (stroma-derived factor-1, SDF-1) is another critical chemoattraction for TAMs recruiting via the CXCL12/CXCR4 pathway, especially for infiltrating areas of hypoxia and tumor invasiveness [105]. Glial cell line-derived Neurotrophic Factor (GDNF) strongly induces recruiting of microglia. Accordingly, shRNA knockdown GDNF showed reduced glioma expansion and prolonged survival in mice [106].

Periostin released by glioma stem cells accumulates in the perivascular areas. It acts as a chemoattractant by stimulating the integrin receptor αvβ3 of the peripheral monocytes and M2-like TAMs [107]. Osteopontin (OPN/SPP1) also plays a vital role in recruiting macrophages to glioblastoma, regulating cellular communication between tumor cells and the TAMs via integrin αvβ5 on the glioblastoma-infiltrating macrophages [108]. IL-33 is associated with the recruitment and invasion of TAMs via platelet-derived growth factor (PDGF)–BB–PDGF receptor beta (PDGFRβ)–Sox7 signaling [109]. In PTEN-null glioma, YAP1 is activated, upregulating lysyl oxidase expression (LOX) in glioma cells. Then LOX acts as a significant macrophage chemoattractant by activating the β1 integrin-PYK2 signaling of macrophages [110].

Macrophage colony-stimulating factor (M-CSF, encoded by the CSF1 gene) significantly controls the recruitment and polarization of TAMs [111]. Tumor-derived M-CSF induces polarization of TAMs toward the pro-tumor M2 phenotype [112]. Granulocyte-macrophage colony-stimulating factor (GM-CSF, encoded by the CSF2 gene) is another essential molecule that attracts, supports survival, and induces M2 polarization of TAMs [113].

The polarization of TAMs in tumors correlates with the downregulation of the activity of the transcription agent signal transducer and activator of transcription 3 (STAT3). The enhanced levels of sialic acid of the GME, induced by the hypoxia condition, mediate the disruption of CD45 protein dimerization, upregulation of CD45 phosphatase, and the downregulation of STAT3 signaling in recruited monocytes [114]. Lactic acid expressed by tumor cells with aerobic or anaerobic glycolysis significantly promotes the expression of VEGF and the shift toward M2-like TAMs. HIF1α mediates the mechanism and the effect of lactic acid. The lactate-facilitated expression of arginase 1 by macrophages significantly promotes glioma growth [115]. Carbonic anhydrase IX contributes to the polarization of M2-like TAM through the EGFR/STAT3/HIF-1α axis under hypoxic conditions [116]. Reactive oxygen species modulator 1 (Romo1) is significantly upregulated in macrophages. The overexpression of Romo1 in macrophages induces the shift of bone marrow-derived macrophages toward M2 macrophages via stimulating mTORC1 signaling [117]. In addition, experiments indicate that relatively lower concentrations of S100B attenuate microglia polarization via inducting STAT3 [118]. Recent investigations reveal that miR-155-3p and IL-6 drive shifts toward M2 macrophages through the positive feedback loop of IL-6-pSTAT3-miR-155-3p-autophagy-pSTAT3 signaling [119].

Glioma stem cells (GSCs) release Wnt-induced signaling protein 1 (WISP1) by the signal integrin α6β1-Akt to maintain M2 TAMs. The Wnt/β-catenin-WISP1 signaling axis also presents us with a promising target [120]. GSC-derived exosomes containing various essential components, like members of the STAT3 pathway, can traverse the monocyte cytoplasm and cause the polarization of monocytes toward the M2 phenotype with upregulated expression of PD-L1 [58].

Exosomal circNEIL3 can be transmitted to infiltrated TAMs, enabling them to polarize to the immunosuppressive phenotype by stabilizing IGF2BP3 [78]. ERp57/PDIA3 is another potential factor modulating microglial pro-tumor polarization toward the M2 phenotype [121]. RSK1, a downstream target of Ras/extracellular signal-regulated kinase signaling, also strongly correlates with the infiltration of M2 macrophages [122]. Also, arsenite-resistance protein-2 plays a critical role in M2-like TAM polarization via ARS2/MAGL signaling [123]. (Fig. 3).

Fig. 3: Roles of TAMs in the TME and the molecules concerned.
figure 3

TAMs play a significant role in glioma progression by contributing to glioma proliferation, vascularization, and invasion, promoting immunosuppression in the GME, generating treatment resistance, and influencing glioma metabolism. Acting through specific signaling pathways, various molecules are closely involved. Detailed mechanisms are discussed as follows. (By Figdraw).

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TAMs contribute to glioma proliferation

The polarized microglial cells release high levels of O2 radicals to contribute to the genomic mutations and enhance the expression of IL-6 and TNF-α to support tumor survival. TAMs play a significant role in promoting the stem-like properties of Brain tumor-initiating cells (BTICs), which possess the capacity for self-renewal and accurate recapitulation of the initial tumor and contribute to the genesis and recurrence of gliomas [124].

Microglia increase the expression of LPA1 and ATX in GBM, further contributing to GBM proliferation and migration [125]. Microglia also synthesizes and releases stress-inducible protein 1 (STI1), a cellular ligand that contributes to glioblastoma proliferation and migration.

M2 TAMs-derived exosomal miR-27b-3p raise the activity of BTICs through the MLL4/PRDM1/IL-33 signaling [126]. WISP1 also plays an indispensable role in maintaining TAMs and GSCs through the Wnt/β-catenin-WISP1 signaling [120]. TAMs secrete a large amount of pleiotrophin (PTN) to control GSCs via the PTN-PTPRZ1 paracrine signaling [127]. The highly expressed CCL8 on TAMs contributes to the invasive activities and stem-like characteristics of GBM cells via signaling on the CCR1 and CCR5 and activating the ERK1/2 phosphorylation [107]. Disruption of these signaling pathways may give us an encouraging outcome. Cilengitide, which inhibits signaling of the αvβ3 and αvβ5, is under investigation in clinical trials. While as for the recurrent GBM, it’s worthwhile to search for more therapeutic strategies like integrating cilengitide into combinatorial regimens [181]. Utilization of exosomes may give us a different result.

More radicle strategies for depleting TAMs are also under investigation. Several molecules and mAbs targeting the CSF-1/CSF-1R signaling axis are in clinical development as monotherapy and combined therapy. Inhibitors of the (CSF-1R) (anti-CSF1R antibody-like Cabiralizumab, SNDX-6352, BLZ945, PLX3397, etc.) to diminish the TAMs population in mouse GBM model significantly increases the survival and shrinks the established tumors [182]. While further preclinical and clinical trials failed to show their effectiveness [183]. Glioma may acquire resistance driven by elevating levels of high IGF-1R and macrophage-derived IGF-1, which enhance survival and invasion of glioma cells [158]. Rational combination therapies are currently promising strategies under investigation. Incorporating these therapeutic molecules with exosomes would be a direction worthy of trying.

MiR-142-3p is revealed to play a unique part in modulating M2 TAMs through the TGF-β signaling. M2 macrophages have a lower level of miR-142-3p expression compared with M1 macrophages. Therapeutic administration of miR-142-3p coherently induces M2- apoptosis and results in glioma growth inhibition [184]. Intracerebral administration of liposome-encapsulated clodronate shows promising efficacy by inducing microglial apoptosis once phagocytosed by macrophages. At the same time, a lack of specificity for TAMs causes lesions of other brain cells and damages blood vessel integrity [185]. In this respect, an exosome-based specific delivery system may present a promising prospect for TAMs-targeted therapies.

Yuan Qian et al. have developed M2-like TAM dual-targeting nanoparticles (M2NPs), the lipid nanoparticles modified with a fusion peptide composed of α-peptide (a scavenger receptor B type 1 targeting peptide) and linked with M2pep (an M2 macrophage binding peptide). Loaded with siRNA targeting anti-CSF-1R to inhibit the survival of M2 TAMs, these nanoparticles could lead to selective depletion, tumor regression, and prolong the survival of B16 melanoma-bearing mice [186]. The dual-targeting capacity of M2NPs, combined with RNA interference, provides new insight into designing TAMs-targeted therapeutics via exosomes.

Similarly, targeting the overexpressed folate receptors on TAMs, Tingting Luo et al. synthesized the oxygen/paclitaxel-loaded microbubbles. Combined with ultrasound-mediated delivery, these microbubbles act as immunomodulatory agents, efficiently depleting the TAMs [219].

Recent research indicates that circNEIL3 is related to YAP1 signaling activation and CCL2 and LOX secretion, thus driving the infiltration of macrophages. Exosomal circNEIL3 could be transmitted to infiltrating TAMs, thereby enabling them to acquire pro-tumor functionality by stabilizing IGF2BP3 [78]. Kristan E van der Vos et al. revealed that high levels of exosomal miR-451/miR-21 were transferred from glioma cells to microglia, which increased microglia proliferation and shifting of cytokine profile toward glioma promotion [220]. Mingyu Qian et al. discovered that by targeting TERF2IP to activate the STAT3 signaling pathway and inhibit the NF-κB signaling pathway, miR-1246 mediated M2 macrophage polarization [221]. MiR-1246 in the CSF might present us a novel biomarker for glioma diagnosis. Moreover, therapeutics targeting microRNA-1246 may assist the anti-glioma immunotherapy. Erik R. Abels et al. confirmed that glioma cells could reprogram microglia in part by transferring exosomal miR-21 [222], which also opens up opportunities for therapeutics aiming at disrupting this form of communication between glioma cells and TAMs. A recent study indicated that exosomal the long noncoding RNA (lncRNA) TMZ-associated lncRNA in GBM recurrence (lnc-TALC) could be delivered to TAMs and then promote M2 polarization of the microglia [223]. Liangyi Zhu et al. lately demonstrated that downregulated exosomal let-7i-5p and miR-221-3p could trigger M2 polarization of TAMs-through upregulating peroxisome proliferator-activated receptor gamma [224].

Nevertheless, the specific mechanisms by which exosome signals regulate TAMs in gliomas still need to be defined. Understanding the intricate interaction between gliomas and TAMs could open a new vista for the therapy. The utilization of exosomes as clinical biomarkers still demands further evaluation and investigation. The main requirements are validation in larger patient cohorts, more standardized methodologies for identifying exosomal biomarkers, better-isolating exosomes, more accurate quantifying of miRNAs or proteins, etc. With increasing research efforts on these applications of exosomes and continuous technological advances, diagnostic applications of exosomes in glioma will be promising shortly.

Conclusions, challenges, and perspectives

For glioma, conventional therapies provide unsatisfactory efficacy. Most therapeutic attempts to incorporate immune therapeutics have been futile, mainly due to BBB, the complex GME, the heterogeneity of glioma tissues, off-target effects, and low immunogenicity. It is relatively challenging to “awaken” the immune activities in the complicated GME. To overcome these obstacles and achieve better efficacy, some potential directions are worth investigating: (1) develop a drug delivery platform that could efficiently cross BBB, (2) identify the complicated signaling intracellular or intercellular for develo** new drugs, (3) find convincing biomarkers for more timely and precise glioma diagnosis and monitoring.

TAMs, the significant component of GME that exert nonnegligible effects on glioma proliferation, vascularization, invasion, metabolism, and treatment resistance and significantly contribute to immunosuppressive GME, is closely associated with the genetic phenotype of glioma and primarily influence the treatment response, prognosis of glioma. As TAMs potentially establish the complicated, unique intercellular interactions of the glioma ecosystem, they also provide a potent alternative for the targeted therapeutics. Indeed, various potential targets have already been identified and evaluated by further trials. Increasing attention has been paid to disrupting the specific signaling pathway using diverse interventions—nucleic acids, gene therapy, small molecule inhibitors, antagonistic or agonistic antibodies, synthetic molecules, etc. Nanoimmunotherapy targeting TAMs, the ideal weapons for precision and personalized medicine, opens a new era of eradication of tumors under immunoregulation. At the same time, the targeted delivery of therapeutic agents is still challenging to achieve and represents a significant obstacle that limits cancer treatment results. Currently, exosomes are studied as promising nanoplatforms for drug delivery with enormous advantages, including low toxicity, enhanced bioavailability, good permeability, and specific tissue tropism. More than nanosized extracellular vehicles, exosomes also act as essential regulators of intercellular communication, indicating the vast potential of exosomes to assist the diagnosis and treatment of glioma as less-invasive, real-time liquid biopsy biomarkers, which could provide a promising application for diagnosis and simultaneous monitoring. Advances in the exosomes field and a more profound understanding of the underlying function of exosomes would significantly lead to breakthroughs in clinical applications to benefit patients. Engineering the novel therapeutic exosomes, combined with TAM-targeted immunotherapy, may herald a new era of cancer immunotherapy that potentially brings opportunities to overcome existing limitations.

Deeper investigations and further understanding of the intercellular interactions between TAMs and various tumor cells and other cells in glioma would conclusively yield new glioma treatment strategies. Given that TAMs might be the potent target to facilitate immunotherapeutic efficacy, multiple directions have been investigated for qualitatively repolarizing macrophages toward the anti-tumor subtype. Besides, reducing the amounts of TAMs by inhibiting the recruitment of glioma or radically depleting TAMs also provided us with alternative methods. Microglia and bone marrow-derived macrophages show different potentials in various aspects of glioma development. Knowledge about distinctions between microglia and bone marrow-derived macrophages still needs to be improved. Further investigations into different types of macrophages are required.

The tumor microenvironment restrains the diffusion of nano-drugs, thus decreasing the therapeutic effectiveness. Martin et al. propose that nanomedicines should integrate anti-tumor agents and factors that “normalize” the diverse composition of the tumor microenvironment, inducing increased cancer perfusion and decreased levels of hypoxia. These efforts may ease drug delivery and transform the microenvironment from immunosuppressive to immunostimulating [225]. In another aspect, further investigation to realize a stimuli-responsive release of immunomodulatory therapeutic agents may present us with better efficacy.

In conclusion, TAMs significantly contribute to glioma progression via various signaling molecules, providing new insight into therapeutic strategies. The TAMs-centered strategies can be mainly divided into reducing recruitment or depleting TAMs and reprograming the M1-like phenotype into M2-like. Exosomes would significantly assist the TAMs-centered treatment as drug delivery vehicles and liquid biopsy biomarkers. And much work remains to be done to push forward the hopeful progress established in preliminary work. This would be crucial for develo** different therapies that re-modulate the tumor microenvironment to benefit patients with glioma. With more profound knowledge of TAMs and exosomes, exosome-mediated nanoimmunotherapy may transform glioma immunotherapy in the future. We hope this review contributes to deeper investigations to advance the current understanding and continue challenging the status quo of standard glioma therapy to improve its clinical efficacy.

Overcoming challenges of clinical application of exosomes

Exosomes could transmit therapeutic factors to the targeted cells for therapeutic applications. While there is a necessary condition that exosomes need be able to find their target and release the cargo specifically. At present, the molecular mechanisms of the related exosomes remain undefined. More basic research to better understand these unknowns would aid diagnosis and therapy of glioma shortly.

Besides, clinical applications of exosomes face several challenges, such as massive production, standard isolation, drug loading, stability, and quality control. During the past decade, efforts on basic exosome research, while challenges still exist for the therapeutic delivery of drugs via exosomes [226]. Ivano Luigi Colao et al. reviewed the present manufacturing technologies and strategies that may aid the clinical production of exosomes [227]. Important technologies available for exosome isolation have been precisely discussed by Sergio Ayala-Mar et al. [228]. For the preservation of exosomes, present data indicate that −80 °C remains the promising mode. While considering cost and poses challenges in transportation, alternatives such as lyophilization and the incorporation of additives may be needed [229]. The heterogeneity of exosomes poses a big challenge for the isolation, suitable methods to profile exosome heterogeneity have previously been reviewed [230, 231].

Artificial nanocarriers are the novel direction of drug delivery systems in nanomedicine [54]. Efforts and advances in exosome-related technologies are simultaneous and of significant value.

Designing rational combined modality therapy

Considering the complex intercellular communication in the GBM, employing one effective single drug seems unrealistic. The high loading capacity and rich surface modification characteristics of exosomes provide us with possibilities for improving therapeutic agents’ biocompatibility and targeting capacity. Potently to load multiple drugs simultaneously, exosomes can enhance the efficacy of drugs. Engineering multifunctional exosomes is a step toward efficient immunotherapy and may even replace the current and traditional therapeutic strategies. At the same time, a deeper understanding of the complex intercellular communication between the immune system and cancer cells is required to further increase therapeutic agents’ efficacy. Targeting molecular signatures identified in the glioma context and combining several levels of TAM functionality, from genetic to epigenetic to metabolic molecules, would be a promising direction for TAM-targeted therapeutics.

Transition from pre-clinical trials to clinical applications

Although some strategies have shown considerable therapeutic efficacy in preclinical trials, no single agent has succeeded in clinical trials. The transition from pre-clinical trials to clinical applications is still difficult. The heterogeneity and complexity of human glioma, which induces the discrepancy between patients’ responses under immunotherapies, cannot be entirely simulated by animal models or cell lines. Some may result in unexpected human side effects that animal models cannot affect. In short, more laboratory and clinical efforts are still required.

Therapeutic approaches of targeting, more specifically, the disease-associated microglia and TAM subsets while preserving the homeostatic ones provide us with a grand vista for glioma [232]. TAMs, including microglia and these bone marrow-derived macrophages, reveal diversified phenotypic identity and function, thus resulting in a continuum of states responding to environmental signals. Future studies may focus on sex differences, how bone marrow-derived macrophage subsets interact with resident microglia to regulate their phenotypic state, and whether this cross-talk can be re-directed to control the expression of pro- and anti-tumor responses. Surprisingly, the host microbiome produces some crucial signals that influence microglia. Identifying those signals and to exploiting them therapeutically is prospective [233]. In addition, identifying the pro-resolving TAMs subsets that are long-lived in the brain under disease conditions [91] may provide us with a novel direction for glioma.

Anna Gieryng et al. reviewed the glioma-derived signals and mechanisms driving bone marrow-derived macrophage accumulation and reprogramming [234]. Several glioma-derived factors are revealed to trigger the migration, proliferation, and reprogramming of TAMs. Many preclinical analyses focus on the interaction of TAMs and glioma cells, while the communication between TAMs and other immune cells is still poorly understood [87]. Much remains unclear about the complex cell-cell interactions in the glioma microenvironment. In a clinical setting, it is crucial to figure out the cellular and extracellular matrix-dependent relationships unique to the glioma microenvironment, identify potential targets involved in the intricate intracellular communication, and then find the exceptional opportunities of therapeutic approaches, hopefully contributing in the future to a better outcome for glioma [234].