Exosomes in Glioma

INTRODUCTION

Malignant glioma, cancer of glial cells, has an incidence of 8.62 per 100,000 population in adults [1]. Overall survival (OS) after glioma diagnosis is impacted by histology, grade, and molecular factors [2–4] Median OS ranges from 12 months [5] to 20.5 months [6] for glioblastoma (GBM) and can exceed 15 years for grade 3 oligodendroglioma [3,4], but these tumors are uniformly fatal. Our understanding of glioma pathophysiology has improved drastically in the last few decades due to refined methods of cellular and molecular analysis technologies [3,7–9]. The most recent 2021 World Health Organization classification system of central nervous system (CNS) tumors integrated newly recognized molecular tumor types, as opposed to historically histopathologic-based categorization [4]. Key molecular alterations in glioma include isocitrate dehydrogenase 1 and 2, telomerase reverse transcriptase promoter mutation, epidermal growth factor receptor (EGFR) amplification and mutation, alpha-thalassemia mental retardation X-linked loss, cyclin-dependent kinase inhibitor 2 A/B deletion, and O-6-methylguanine-DNA methyltransferase (MGMT) methylation status [4].

Diagnosis, despite advances in MRI and radiogenomics, still requires tissue for histologic and molecular characterization [10], in addition to the therapeutic benefit of resection [11]. Surgical biopsy carries risks, including anesthetic complications, poor wound healing, neurologic injury, and intracranial hemorrhage. In addition, therapy may need to be delayed for adequate wound healing. The current standard of care for GBM, the most common glioma, involves maximal safe resection followed by radiotherapy with concomitant temozolomide (TMZ) followed by 6 or more cycles of TMZ therapy and tumor treating fields [12,13]. Unfortunately, GBM survival has not significantly improved [14].

Many investigators are exploring the complex glioma tumor microenvironment (TME) pathophysiology to address these dismal outcomes. The glioma TME is characteristically heterogeneous and comprises various cell types, including microglia and other immune cells, astrocytes, neurons, glial stem cells, and endothelial cells [15]. These components utilize various intra- and inter-cellular communication strategies, either through direct cell-to-cell physical contact or secretion of communication mediators. Amongst these mediators, the release of extracellular vesicles (EVs) has sparked interest from both a diagnostic and therapeutic point of view. This review explores biogenesis, the role of exosomes in glioma pathogenesis, and their potential in diagnosing and treating GBM.

BIOGENESIS AND FUNCTION OF THE EXOSOMES

Chargaff and West first described EVs in 1946 during their studies of coagulation physiology. EVs are defined as 30 to 100 nm in diameter, composed of a bilipid membrane, and secreted from the parent cell via an exocytic mechanism [16]. EVs can be further subdivided into 4 general groups: apoptotic vesicles, membrane particles, microvesicles, and exosomes [17]. Exosomes are released from normal and pathologic cells in animal and plant tissues. EVs play an important role in inter-cellular communication and are involved in processes such as tissue regeneration, immunomodulation, and TME regulation [18,19]. Synthesis occurs via an initial invagination of the parent cell plasma membrane, creating a small biphospholipid-membrane vesicle termed an intraluminal vesicle (ILV). Multiple ILVs are then collected within a larger structure called a multivesicular body (MVB). Once an MVB contains a sufficient volume of ILVs, it fuses with the plasma membrane to release the ILVs, which are then defined as exosomes [20]. Formation from the invagination of the parental cell membrane results in similar membrane surface proteins. Their bilipid membrane composition also contains ubiquitous components, such as tetraspanins CD63, CD9, CD81, and CD82, heat shock protein 70 (HSP70), HSP90, tumor susceptibility gene-101 (Tsg101), and ALIX, as well as unique proteins and nucleic acids derived from their cell of origin [21]. The Endosomal Sorting Complexes Required for Transport (ESCRT) pathway determines exosome content. The ESCRT pathway contains 4 main components, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, as well as an assortment of accessory proteins, including ATPase Associated with various cellular Activities (AAA ATPase) and Vacuolar protein sorting-associated protein 4 (Vsp4) [22]. ESCRT-I and ESCRT-II prompt invagination of the plasma membrane, followed by ESCRT-III cleavage to form the new ILVs. Tsg101 is a subunit of ESCRT-I and a universal component of final exosome membranes. Similarly, ALIX binds ESCRT-III during the exosome formation process and is also found within the final exosome membrane [14]. Exosomes contain messenger RNAs, proteins, DNA, micro RNAs (miRNAs), long non-coding RNAs (lncRNAs), lipids, metabolic residues, and fragments of organelles. The profile of exosome contents mimics the parental cell cytosol.

ISOLATION OF EXOSOMES

There are multiple methods available for exosome isolation for clinical and research purposes. Ultracentrifugation (UC), the most widely used technique for exosome isolation across various cancer types, is challenging due to the low density and small size of exosomes [23]. UC involves spinning down larger objects, such as apoptotic bodies, followed by another spin at a higher speed to pellet exosomes and generate a supernatant devoid of EVs [24]. The equipment required (ie, UC) can be a significant barrier to this method. Though widely used, UC produces a lower EV yield than precipitation techniques [25]. Ultrafiltration offers a quicker and more cost-effective alternative to UC. However, it suffers from lower efficacy and specificity due to potential vesicle entrapment and the inability to distinguish similar particles. Multiple commercially available kits can isolate exosomes via precipitation from a small volume of biofluid with a greater yield than UC [26]. Exosome research has significantly advanced with commercially available kits using precipitation techniques, which offer higher yields and efficiency than traditional UC. The ExoQuick ULTRA EV Isolation Kit requires just 250 mL of serum or plasma and 1 hour to isolate exosomes, using a purification column to reduce immunoglobulin G and albumin contaminants [24]. This method simplifies the isolation process, speeding up sample processing and allowing for the easy storage of exosomes in a buffer solution. Other kits such as exoEasy, Exo-spin, and ExoQuick Plus further streamline the process, demonstrating the rapid evolution of exosome isolation technologies [27]. Immunoaffinity isolation is notable for its ability to purify and subtype exosomes through the specific interaction between exosome surface markers and corresponding antibodies. However, it is costly, and antibody production is still being optimized. Additionally, using water-excluding polymers like polyethylene glycol (PEG) modifies exosome solubility and dispersibility, enhancing isolation efficiency [28]. These methods utilize the biochemical and physical properties of exosomes, leveraging advanced techniques such as microfluidics to facilitate rapid, efficient isolation while minimizing sample and reagent use. In Table 1, we have summarized the advantages and disadvantages of exosome isolation methods.

TABLE 1.

Exosome Isolation Methods: Advantages and Limitations

Isolation Technique	Pros	Cons	Ref.
Ultracentrifugation (UC)	Easy and direct to use	Limited discrimination (challenging with low density and small sizes)	[23–25]
Ultrafiltration (UF)	Quick and economical	Reduced efficacy and specificity	[26,27]
Immunoaffinity Isolation	Achieves high purity	Costly and requires specific antibodies	[24,25]
Polyethylene Glycol (PEG) Precipitation	Simple and cost-effective	Inconsistent efficiency and struggles with similar-sized particles	[28]

ROLE OF THE EXOSOMES WITHIN THE PHYSIOLOGIC CENTRAL NERVOUS SYSTEM

Exosome-mediated intercellular communication plays a crucial role across various tissues, including the CNS, where it facilitates interactions among neurons, glial cells, and microglia. Neuron-derived exosomes, isolated from culture mediums, are particularly rich in miRNA and small RNAs, underscoring their importance in neural communication. The role of exosomes in intercellular CNS communication has been demonstrated in both healthy and pathologic conditions. The miRNAs are small (21–23 oligonucleotides long), non-coding RNA molecules that regulate gene expression by binding to the target messenger. Through these regulatory functions, miRNAs play an important function in controlling cellular processes. MiR124-3p, vital for neuronal function and identity, are abundantly present in neurons and can be transmitted to astrocytes and other glial cells via exosomes facilitating intercellular communication in the nervous system. In 2013, Morel and colleagues observed neuron-derived exosome miR-124a-mediated induction of astrocyte glutamate transporter levels [29]. Neuron-derived exosome miRNAs have promoted surrounding vascular integrity (miR-132) and myelination (miR-219) [29,30]. Exosome-mediated intercellular communication is also observed by microglia within the CNS. MiRNA, such as miR-124-3p, has been correlated with the change from the acute to chronic inflammatory phases and, subsequently, the transition from M1 to M2 polarization [14,31]. The CNS uses exosome-mediated intercellular communication in normal homeostasis, inquiry response, and pathologic states.

EXOSOMES IN GLIOMA: PERSPECTIVES ON DIAGNOSTIC POTENTIAL OF BIOMARKERS

Exosomal Micro Ribonucleic Acids (miRNAs) in GBM

The field of liquid biopsy for glioma diagnosis has rapidly expanded to mitigate the surgical risk and potentially, more importantly, allow for a reliable assessment of response to treatment. The exosome lipid membrane prevents degradation of miRNA content, resulting in a more stable circulating diagnostic target than free miRNAs. Several miRNAs have been explored as diagnostic markers in patient blood, plasma, serum, and cerebrospinal fluid (CSF) (Table 2). The miR-454-3p regulates cell proliferation and growth by targeting key signaling pathways, acting as both an oncogene and a tumor suppressor in various cancers. In glioma, it inhibits cell proliferation by targeting PDK1 and is selectively exported via exosomes, suggesting roles in tumor progression and intercellular communication. In 2019, Shao and colleagues investigated the role of the tumor suppressor miR-454-3p in tissue and serum exosomes of patients with glioma compared to healthy controls [32]. Twenty-four glioma tissue samples were compared to 12 normal brain tissues, revealing a significant down-regulation of miR-454-3p in glioma. Conversely, serum exosomes from the same 24 glioma patients exhibited significantly elevated levels of miR-454-3p compared to 24 age-matched and sex-matched healthy controls. These findings suggest that glioma cells selectively export miR-454-3p via exosomes. Additionally, miR-454-3p was markedly reduced in glioma cell lines U251 and LN229 compared to the normal glial cell line human embryonic brain (HEB), leading to their selection for further experiments. High expression of miR-454-3p in serum exosomes correlated significantly with shorter survival [32]. Exosomal miR-454-3p similarly decreased following surgical resection. Diagnostic sensitivity and specificity were 79% and ~92~%, respectively, a threshold that would not be adequate for replacing the role of surgically obtained tissue specimens for diagnosis.

TABLE 2.

Examples Indicating Diagnostic/Prognostic Value of Exosomal miRNAs in Glioma

Author and ref	mRNA(s)	Sample and Group	Findings & Outcomes
Shao et al, [32] 2019	miR-454-3p	Serum, glioma vs controls	miR-454-3p higher in glioma, correlates with poor survival; 79.17% Sensitivity (SN), 91.67% Specificity (SP)
Lan et al, [33] 2020	miR-210	Serum, glioma grades 1–4	Elevated in higher grades decreases post-surgery; 83.2% SN, 94.3% SP
Santangelo et al, [36] 2018	miR-21, miR-222, miR-124-3p	Serum, glioma vs controls	Increased in GBM, improves diagnostic accuracy; AUC 0.87
Stakaitis et al, [38] 2020	miR-181b, miR-181d	Serum, glioma	Downregulated in higher-grade gliomas
Zeng et al, [40] 2018	miR-151a, miR-93	CSF and serum, TMZ-resistant vs sensitive	Lower miR-151a in resistant GBM correlates with a worse prognosis

GBM characteristically contains hypoxic regions, prompting exploration of miR-210 by Lan and colleagues, which is directly associated with hypoxic conditions in patients with glioma and may serve as a diagnostic, prognostic, and hypoxic biomarker to reflect glioma status and hypoxic signatures [33]. Serum samples from 91 glioma patients and 50 healthy controls were collected. Exosomal miR-210 levels were significantly higher in glioma patients. Overall, and statistically greater in patients with high-grade glioma (HGG) (grades 3 and 4) compared to low-grade glioma (grades 1 and 2), a dichotomization, which may have less relevance in the molecular diagnostic era. miR-210 levels did not correlate with clinical function as measured by Karnofsky Performance Scores. Analysis of serum exosome miR-210 levels in 20 GBM patients found markedly decreased levels after maximal surgical resection, with significantly increased levels at the time of tumor recurrence [33], lending support for the potential of using exosomes for assessing disease control and progression. The sensitivity of serum exosomal miR-210 was ~83%, specificity ~94%, positive predictive value ~94%, and negative predictive value ~70%.

Santangelo and colleagues studied the exosome content of miR-21, miR-222, and miR-124-3p in serum from patients with glioma, brain non-glial metastases, and healthy controls. Previous work established a negative correlation between glioma tissue miR-21 level and OS [34]. miR-221/222 family levels were significantly higher in glioma patients in a study by Zhang and colleagues in 2016, wherein they also correlated with poor survival [35]. Based on these results, Santangelo and colleagues prospectively followed 44 glioma patients prior to the first surgery and throughout their disease course. Serum exosomes were isolated and evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The miR-21, miR-222, and miR-124-3p were significantly greater in patients with GBM than healthy controls. Interestingly, a combination of miR-21, miR-222, and miR-124-3p slightly improves diagnostic accuracy with an area under the curve from 0.84 (miR-21–0.84; mir-222–0.80; mir-124-3p – 0.78) to 0.87 (95% CI 0.7885–0.9524, P<.0001) [36].

The relationship between the miR-181 family (a-d) and glioma pathology has been under investigation due to its importance in several other cancers. miR-181a, miR-181b, and miR-181c were all downregulated in primary GBM tissues [37]. In 2020, Stakaitis and colleagues examined the miR-181b and miR-181d expression levels in 92 tumor tissues and serum exosomes of 63 glioma patients (with 64 match control serum samples) [38]. miR-181b and miR-181d were both downregulated in tissue and serum exosomes of patients with grade 3 gliomas, though only miR-181b distinguished between grade 2 and grade 3 gliomas. The clinical value of this may be somewhat limited in the current molecular era of neuropathology.

Exosomes are emerging as pivotal factors in GBM therapy, serving as diagnostic biomarkers, as well as actively contributing to TMZ resistance. They have the potential to predict TMZ resistance, similar to MGMT methylation status, and can be utilized to monitor tumor progression during and after TMZ treatment [39,40]. In 2018 Zeng and colleagues isolated exosomes from 2 GBM cell lines (U251 and N3) after TMZ treatment [40]. After isolation of TMZ-resistant and TMZ-sensitive cells, exosomes from TMZ-resistant cells displayed lower expression of miR-151a and miR-93 compared to TMZ-sensitive cells, with miR-151a displaying the most significant decrease. In vitro, overexpression of miR-151a in GBM cell lines resulted in decreased cell viability and increased apoptosis. Notably, intracellular expression of miR-151a in recipient cells significantly decreased after treatment with TMZ-resistant cell-derived exosomes. CSF and serum samples were collected from 14 patients with GBM, and exosomes were isolated. A worse prognosis was correlated with a significant decrease in CSF exosome miR-151a expression, though no difference was appreciated in serum exosome miR-151a levels. CSF exosome miR-151a levels were positively correlated with GBM tissue expression [40]. This highlights that the location (CSF vs serum) of exosome obtainment may influence their sensitivity and specificity for diagnostic, prognostic, and predictive purposes.

Exosomal long non-coding Ribonucleic Acids in GBM

lncRNAs are defined as non-protein-coding RNA sequences larger than 200 nucleotides. Functional roles of lncRNAs include cell survival, proliferation, cell death, and various other cell biologic functions [41]. In 2018, Bian and colleagues investigated the role of lncRNA activated by TNF-C (lncRNA-activated by transforming growth factor β [ATB]) in glioma-derived exosomes [42]. Prior studies implicated lncRNA-ATB in the migration and invasion of glioma cells via inhibition of miRNA-200a [43]. Using in vitro models of human glioma cell lines A172 and U251 and normal human astrocytes (NHA), Bian and colleagues demonstrated effective uptake of glioma cell-derived exosomes by astrocytes, as well as a significant activation of astrocytes after treatment with glioma-derived exosomes. Subsequent RT-qPCR analysis revealed a significant elevation in lncRNA-ATB levels within glioma-derived exosomes. Astrocytes transfected with lncRNA-ATB displayed a significant reduction in miR-204-3p and miR-200a [42]. A172 and U251 cell lines were co-cultured with lncRNA-ATB-transfected astrocytes, and a significant increase in glioma cell migration and invasion was observed.

Given the characteristic hypoxic environment within GBM, Dai and colleagues explored the lncRNA AHIF, the antisense transcript of hypoxia-inducible factor-1α [44]. In vitro studies utilizing the human GBM cell lines U87, U251, A172, and T98G revealed significant upregulation of AHIF within T98G and A172 lines compared to U87 and U251. Expression increased in U87 and U251 lines after irradiation. Overexpression of AHIF significantly increased cell survival and invasion. Notably, when cell lines were treated with exosomes derived from AHIF-overexpressing cells, viability, invasion, and radioresistance increased. These data indicate the transfer of AHIF via exosomes [44].

More recently, in 2024, Tian and colleagues studied the role of intercellular communication of glioma cells and myeloid-derived suppression cells (MDSCs) in TME regulation. Specifically, they investigated the relationship between glioma-derived exosome levels of the lncRNA AGAP2 antisense RNA 1 (AGAP2-AS1) and MDSC immunosuppressive transforming growth factor-β1 (TGF-β1) secretion [45]. AGAP2-AS1 displayed significantly increased expression in 48 GBM tissue samples compared to 33 normal brain tissue samples. These results were demonstrated in vitro via glioma cell lines compared to NHA. In addition, silencing of TGF-β correlated with decreased glioma cell migration, invasion, and cell viability. Exosomes isolated from U87 and U251 cells had elevated expression of AGAP2-AS1. After the co-culture of MDSCs with exosomes from glioma cell lines, MDSCs had significant elevations in AGAP2-AS1 and TGF-β1 levels. Further investigation implicated AGAP2-AS1 inhibition of miR-468-3p expression as an important mechanism in this process [45].

Exosomal proteins in GBM

Exosomal proteins have been shown to provide higher specificity than soluble serum proteins as cancer biomarkers, enhancing their effectiveness in diagnosis and surveillance. Over recent decades, significant strides have been made to pinpoint potential protein biomarkers for gliomas. These biomarkers can be detected in various bodily fluids such as urine, serum/plasma, or CSF, aiding in diagnosis, detecting recurrences, or monitoring tumor activity post-therapy, as detailed by Kros and colleagues [46]. Among these proteins, glypican-1 found in tumor-derived exosomes is significantly more specific than its soluble form and the serum marker CA-19.9 for distinguishing pancreatic tumor tissue from normal tissue. Similarly, Chen and colleagues identified markedly higher levels of nuclear transcription factor X-box-binding protein 1 and cGMP-dependent protein kinase in serum exosomes from breast cancer patients compared to healthy controls. Unlike serum-free proteins, which degrade quickly, proteins encapsulated within exosomes remain stable and are less likely to degrade or get diluted, reducing the risk of false negatives. In glioma research, exosomes from the T98G GBM cell line showed increased surface levels of the L1 domain of the L1CAM protein, involved in neuronal differentiation and migration. Additionally, Shao and colleagues detected elevated levels of the EGFR variant EGFRvIII, a pro-tumorigenic protein, in exosomes from serum and CSF of GBM patients, highlighting their potential in glioma diagnostics [47]. EGFRvIII, a variant of the EGFR growth factor receptor, is one of the most extensively studied oncogenic proteins specific to glioma. This mutant receptor is found in EVs, including exosomes, secreted by glioma cells and cell lines engineered with the EGFRvIII construct. Further studies have confirmed the presence of this mutant receptor on the surface of exosomes derived from receptor-expressing cells and in the sera of GBM patients. Additionally, HSPs like HSP60, HSP70, and HSP90 are prevalent in glioma cell lines and have been linked to promoting cell proliferation, survival, invasiveness, and migration. These HSPs are consistently found in exosomes and other EVs, positioning glioma cell-derived exosomes, rich in HSPs, as promising candidates for diagnostic applications. One limitation is that a substantial subset of gliomas, including GBM, do not harbor the presence of EGFRvIII.

Current approaches in detecting exosomal biomarkers

Recent advancements highlight the effectiveness of surface-enhanced Raman spectroscopy (SERS) in differentiating cancerous from normal cells, positioning it as a robust tool to detect many cancer exosomes, including gliomas [48–50]. A custom-made platinum-black (Pt-black) SERS template, developed via an economical electroplating technique, specifically targets cancer-associated exosomes, achieving an 83.3% sensitivity and 95.8% specificity [51]. Research by Agarwal and colleagues utilized next-generation sequencing and quantitative real-time PCR (Q-RT-PCR) to identify and validate Cfa-miR-9 as a potential universal tumor marker, found consistently elevated in cancer cell-derived exosomes [52]. This underscores the potential of molecular techniques in enhancing cancer diagnostics. Additionally, surface proteins on glioma-derived exosomes have been further explored as diagnostic biomarkers with advanced detection techniques like atomic force microscopy (AFM) and localized surface plasmon resonance (LSPR). AFM offers precise imaging for delicate samples like exosomes, while LSPR is recognized for its capability to detect diverse tumor biomarkers, enhancing the diagnostic landscape in oncology [15,32,53].

PERSPECTIVES ON THE THERAPEUTIC POTENTIAL FOR TARGETING GLIOMAS

The structure and ability of exosomes to deliver contents in a cell-specific manner provide an exciting avenue for treatment delivery. Glioma has traditionally challenged treatment delivery due to the selective natural blood-brain barrier (BBB). Exosomes can cross the BBB via endothelial cell endocytosis, accumulation within endosomes, and subsequent MVBs, followed by exocytosis [54]. In addition, exosome surface markers and receptors can be modified for desired tissue delivery [55]. Cargo loading within exosomes has been accomplished by incubation, electroporation, sonication, freeze and thaw, biosynthesis, extrusion, chemical permeabilization, and transfection [55]. Due to its high molecular weight and hydrophilic nature, the BBB drastically limits monoclonal antibody therapy delivery. In July 2024, Chu and colleagues developed an innovative strategy to optimize the monoclonal antibody cetuximab (CTX) delivery in combination with doxorubicin (DOX) to glioma tissue using exosomes [56]. Exosomes were collected from rat glioma C6 cell lines, CTX-PEG lipids were fused to their surface, then incubated with DOX, resulting in CTX-Exo-DOX. In vitro studies demonstrated a significant reduction in C6 cell invasiveness after treatment with CTX-Exo-DOX. In vivo, evaluation was completed using a rat in situ glioma model and tail vein injection of CTX-Exo-DIX every 2 days for 5 doses total. CTX-Exo-DOX effectively crossed the BBB and significantly increased GBM tissue apoptosis [56]. Wang and colleagues developed an oligopeptide-modified exosome strategy to optimize GBM DOX delivery [57]. Exosomes were isolated from the mouse microglial cell line BV2 (BV2-Exos) due to favorable BBB penetration. The functional oligopeptide (Pep2) contained consecutive lysine residues within the hydrophilic exosome region and cysteine residues in the hydrophobic area. The cysteine residues could form crosslinks by disulfide bonds and subsequently lock in DOX. The high glutathione environment within GBM cells leads to the breakage of these disulfide bonds and the release of DOX. In vivo, studies with the treatment of DOX-loaded Pep2 modified exosomes (Pep2-Exos-DOX) in a mouse U87 cell orthotopic model revealed no apparent toxicity, favorable brain-targeting capacity, and a significant reduction in tumor volume compared to DOX alone, Exos-DOX, and PBS (control) [57].

In 2019, Salarpour et al. demonstrated effective loading and delivery of the microtubule-stabilizing agent paclitaxel (PTX) by exosomes in vitro [58]. PTX historically has faced a significant challenge crossing the BBB, limiting its efficacy in GBM. Human glioma cells U87 were cultured, and exosomes were isolated from the media. The exosome-isolated pellet was resuspended in PTX solution and incubated or sonicated. Sonication demonstrated superior efficiency (0.92%) compared to incubation (0.74%). U87 cell viability significantly decreased after treatment with PTX-loaded exosomes compared to free PTX and empty exosomes [58]. Zhu and colleagues also explored using exosomes to deliver PTX to GBM cells. However, they used exosomes isolated from embryonic stem cells (ESC) due to their abundant self-renewal capabilities [59]. Prior studies have suggested ECS-exosomes (ESC-exos) carry innate anti-tumor properties [60] The ECS-exos surface was modified to express the Cyclo (Arg-Gly-Asp-D-Tyr-Lys) peptide (c(RGDyK)) to target actively proliferating endothelium of GBM (cRDG-Exo). ECS-exos alone demonstrated tumor suppressive activity in vitro within U87 and U251 human GBM cell lines and a significant decrease in tumor growth curves in vivo in a U87 GBM xenograft model. cRGD-Exo-PTX treatment in vitro resulted in significant inhibition of cell viability and an increase in apoptosis. They further observed a significantly decreased tumor volume in vivo following tail vein injection treatment of cRDG-Exo-PTX every other day for 2 weeks compared to Exo-PTX, PTX, and PBS (control). Exosome treatment was well-tolerated with no difference in body weight between groups [59]. cRDG-Exo-PTX treatment in an orthotopic GBM mouse model significantly increased survival compared to ESC-exos, PTX, and Exo-PTX treatment groups.

Curcumin (Cur), a plant polyphenolic compound, has shown promise in glioma tumor suppression via inhibition of SHH/GLI1 signaling pathway [61]. Despite promising results, Cur has difficulty crossing the BBB and does not have tumor-targeting ability. Jia and colleagues explored the possibility of exosome-mediated Cur delivery in glioma [62]. In addition to loading exosomes with Cur, they included SPIONs. SPIONs have been used in intracellular hyperthermia therapy to achieve tumor heating with minimal side effects [63]. Exosomes were modified to express RGERPPR peptide (RGE) on their surface, a specific ligand of the glioma-specific transmembrane glycoprotein neuropilin-1. Studies in an orthotopic mouse model of glioma demonstrated strong tumor tissue specificity. MRI evaluation of orthotopic tumors revealed that RGE-Exo-SPION/Cur and RGE-Exo-SPION treatment led to clearer, more distinct tumor borders. In vitro studies using the human glioma U251 cell line showed REG-Exo-SPION exosome treatment significantly increased the inhibitory effect of magnetic flow hyperthermia on cell viability compared to free-SPION and exosomes without SPION. Additional co-treatment with RGE-Exo-Cur exosomes further inhibited cell viability, supporting a synergistic effect. These results were also seen within the orthotopic mouse model, with a significant decrease in tumor volume in both RGE-Exo-SPION and RGE-Exo-Cur treatment groups, with a drastic further reduction in tumor volume in the RGE-Exo-Cur/SPION treatment group [61–63]. Importantly, REG-Exo-Cur/SPION therapy was well-tolerated in vivo. Table 3 lists exosome-based treatment strategies for glioma.

TABLE 3.

Exosome-Based Treatment Strategies for Glioma

Study and ref	Exosome Source	Drug/Modification	Brief of Key Findings
Chu et al, [56] 2024	Rat glioma C6 cell lines	CTX-PEG lipids, Doxorubicin (DOX)	Enhanced apoptosis, BBB penetration
Yutong et al, [57] 2024	Mouse microglial BV2 cell line	Oligopeptide-modified, DOX	Reduced tumor volume, better BBB targeting
Salarpour et al, [58] 2019	Human glioma U87 cells	Paclitaxel (PTX)	Increased delivery efficiency, reduced cell viability
Zhu et al, [59] 2019	Embryonic stem cells	PTX, c(RGDyK) peptide	Inhibited cell viability, decreased tumor size
Jia et al, [62] 2018; Kobayashi et al, [63] 2011	N/A	Curcumin (Cur), SPIONs	Targeted delivery, enhanced hyperthermia

DISCUSSION

Given that exosomes enable complex intercellular communication within the TME, exosomes are essential to the pathophysiology of gliomas. They have a critical role in mediating processes like immunologic tolerance, angiogenesis, proliferation, invasion, migration, stemness, and malignant transformation, frequently resulting in increased resistance to traditional treatments. Importantly, exosomes convey and transfer biochemical cues that stimulate tumor development and survival between glioma cells, as well as other cellular entities in the TME. Over the past decades, the prognosis for patients with GBM and other HGGs has remained virtually unaltered. This stagnation underscores the need for a deeper understanding of the glioma TME, which could drive breakthroughs in diagnosis, treatment monitoring, and surveillance. While traditional imaging and surgical biopsies continue to be necessary for glioma management, the emergence of liquid biopsies offers a promising complement to these methods, enhancing clinical decision-making. The use of such techniques has the potential for superiority over traditional imaging for assessment of response, stability, and progression in patients with gliomas receiving treatment or in clinical follow-up.

Exosomes have emerged as powerful tools in the glioma landscape, carrying specific biomarkers reflective of their cells of origin. Able to facilitate intricate intercellular communication within healthy and malignant microenvironments, they provide previously inaccessible insights into tumor physiology and pathology. Research has successfully isolated miRNAs, proteins, lncRNAs, and other entities from glioma-derived exosomes, linking them to crucial pathologic features such as tumor progression, invasiveness, and angiogenesis. This growing body of evidence suggests that glioma-specific exosomes could play pivotal roles in understanding and manipulating the TME, demanding further exploration of their interactions with other glioma markers. Recent advancements highlight the potential of engineered exosomes as innovative tools in cancer therapy. These exosomes are designed to improve drug delivery systems, notably through their inherent ability to traverse the BBB. This property is attributed to their small size, flexible nature, and specific adhesive surface proteins, facilitating brain tissue penetration. Moreover, exosomes’ encapsulation in lipid bilayers significantly reduces their immunogenicity and toxicity, enhancing their biocompatibility and stability in the bloodstream. However, the clinical application of exosome-based strategies faces several challenges. One major hurdle is the efficient and specific isolation of exosomes from bodily fluids. While effective, current techniques, such as UC and immunoaffinity capture, require further refinement to improve yield, purity, and scalability, the lack of standardized protocols for their production, purification, and storage hinders widespread clinical adoption.

Importantly, exosomes serve not only as unique biomarkers for monitoring disease progression and treatment efficacy but also as vehicles for targeted therapeutic delivery. This dual capability emphasizes the importance of further studying the molecular characteristics of glioma-derived exosomes. Notably, integrating microfluidic technology and nanotechnology could revolutionize exosome research, offering more precise and scalable methods for isolating and manipulating these vesicles. As research progresses, these technologies may provide the necessary breakthroughs to overcome current limitations, paving the way for exosome-based diagnostics and therapeutics to become a standard part of managing gliomas and potentially other cancers. While the prospects of exosome use in clinical settings are promising, significant scientific and logistical hurdles remain. Addressing these effectively will require a concerted effort to translate this research into viable clinical applications. The biogenesis, isolation, and clinical utility of exosomes are summarized in Fig. 1.

FIG. 1.

Role of exosomes in GBM diagnosis and targeted therapy.

SUMMARY

Exosomes demonstrate significant potential for advancing glioma detection and treatment. Through liquid biopsies, they offer a minimally invasive way to track tumor growth and therapeutic response while also providing additional insights into the dynamics of the TME. However, due to complex and costly isolation techniques and the lack of methodical standardization, further work is needed to realize their full potential. To incorporate exosome-based technologies to improve glioma management in standard clinical practice, it will be imperative to address these obstacles. The ability of exosomes to transport cellular components, antibodies, and drugs across the BBB demands further efforts to understand and refine this powerful diagnostic and therapeutic tool.

KEY POINTS.

• Tumor exosomes play a key role in tumor-microenvironment interactions, angiogenesis, proliferation, and stemness.

• Exosomes may be exploited as a noninvasive diagnostic tool, as they stabilize and protect cellular components from immune surveillance.

• The blood-brain barrier significantly impacts the delivery of drugs to the brain.

• Exosomes serve as carriers to transport drugs, nucleic acids, proteins of interest, and superparamagnetic iron oxide nanoparticles across the blood-brain barrier.

ABBREVIATIONS

AAA ATPase

ATPase associated with various cellular activities

AFM

atomic force microscopy

AHIFATRX

antisense transcript of hypoxia-inducible factor alpha-thalassemia mental retardation X-linked

ALIX

apoptosis-linked gene 2 interacting protein X

BBBLGG

blood-brain barrier low-grade gliomas

CADM1

cell adhesion molecule 1

CNS

central nervous system

CSF

cerebrospinal fluid

CTXIDH1/2

cetuximabIsocitrate dehydrogenase 1 and 2

DNA

deoxyribonucleic acid

DOXKPS

Doxorubicin Karnofsky performance score

ECS

embryonic stem cells

EGFR

epidermal growth factor receptor

ESCRT

endosomal sorting complexes required for transport

EVs

extracelular vesicles

GBM

glioblastoma

GLT1

glutamate transporter

HBMVEC

human brain micro-vessels endothelial cells

HGG

high-grade gliomas

HSP70

heat shock protein 70

HSP90

heat shock protein 90

ILV

intraluminal vesicle

LncRNA

long non-coding ribonucleic acids

LSPR

longitudinal surface plasmon resonance

MDSCs

myeloid-derived suppressor cells

MGMT

O-6-methylguanine-DNA methyltransferase

MiRNAs

microribonucleic acids

MVB

multivesicular body (MVB)

NHAmRNAs

normal human astrocytes messenger ribonucleic acids

OSTERT

overall survival telomerase reverse transcriptase

PEGMRI

polyethylene glycol magnetic resonance imaging

PTXCDKN2A/B

paclitaxel Cyclin-dependent kinase inhibitor 2 A/B

SERS

Surface-enhanced Raman spectroscopy

SPION

superparamagnetic iron oxide nanoparticles

TME

tumor microenvironment

TMZWHO

Temozolomide World Health Organization

Tsg101

tumor susceptibility gene-101

Vsp4

vacuolar protein sorting-associated protein 4