The glioma microenvironment and its impact on antitumor immunity

Abstract

Gliomas are a heterogeneous group of intrinsic brain tumors that are among the most difficult cancers to treat. Diffuse invasion into normal brain tissue prevents complete surgical resection; therefore, adjuvant therapy is necessary to curtail tumor progression and recurrence. High-grade, isocitrate dehydrogenase wild-type gliomas, also known as glioblastomas, are particularly resistant to treatment. Despite aggressive therapy with maximal safe resection, radiation, and chemotherapy, the median survival remains less than 2 years and has changed little in the past 2 decades. A major focus of therapeutic development for cancer treatment is immunotherapy, which aims to enhance the immune system’s ability to destroy tumor cells wherever they reside. While cancer immunotherapy has dramatically improved outcomes for patients with advanced melanoma, lung cancer, and many other malignancies, immunotherapies have not yet demonstrated the ability to reliably improve survival for glioblastoma patients. One of the fundamental challenges to developing effective immunotherapy for glioblastoma is the heterogenous and complex tumor microenvironment (TME), where there are multiple anatomic, molecular, and functional barriers to generating and sustaining antitumor immunity. Recent insights into the contributions of specific components of the glioma tumor microenvironment are leading the way from a trial-and-error approach to rationally targeted combination therapies. In this focused review, we discuss specific characteristics of the TME that impede immunotherapy for glioma and approaches in various stages of development aimed at overcoming these barriers.

Gliomas are the most common type of primary brain tumor and are characterized by their infiltrative nature and poor prognosis. These tumors impact tens of thousands of individuals worldwide each year and pose a substantial challenge to treatment due to their resistance to conventional therapies.¹ Gliomas are classified based on genetic and histologic characteristics, with grade 4 lesions known as glioblastoma (GBM) being the most common and aggressive form. The median survival time for glioma patients varies from over 10 years for grade 2 oligodendrogliomas to only 15 months for patients diagnosed with GBM, even with comprehensive treatment involving maximum safe surgical resection, radiation, and chemotherapy.² Gliomas are particularly difficult to treat due to multiple factors including the invasive dispersion of tumor cells, the delicate nature of the surrounding host organ, the unique immune environment of the CNS, the restricted access of the blood-brain barrier, and the inherent cellular resistance to therapy.

Given the diffusely infiltrative nature of this disease into the indispensable and poorly healing surrounding brain tissue, the precise and adaptive nature of immunotherapy is particularly appealing. Immunotherapy has revolutionized care for many cancer types and is now a mainstay of cancer therapy. The concept of arming the patient’s immune system with the tools necessary to defend itself against invasive and recalcitrant tumor cells has reshaped the way we understand and approach cancer.³ Despite recent progress, however, the promise of immunotherapy-based approaches for treating brain tumors remains to be fully realized. One of the chief reasons is that these tumors are profoundly immunosuppressive and employ nonredundant, parallel mechanisms to escape immune surveillance, recognition, and elimination.^4–9

A tumor microenvironment conducive to potent and sustained antitumor immune responses is critical for effective immunotherapy. The glioma TME is a complex interaction of tumor cells, astrocytes, immune cells, fibroblasts, endothelial cells, growth factors, cytokines, and metabolites (Figure 1). This intricate ecosystem of signaling molecules and cellular cross-talk determines the likelihood of generating and sustaining antitumor immune responses. The general term “immune cells” in this instance includes a diverse array of tumor-infiltrating cell types including microglia, macrophages, myeloid-derived suppressor cells (MDSCs), neutrophils, dendritic cells, Tregs, CD4⁺ T cells, CD8⁺ T cells, natural killer (NK) cells, and B cells.^10–16 In this focused review, we will discuss the most potent immunosuppressive mechanisms at work in the TME and explore strategies to shift the TME to an environment conducive to antitumor immune activity.

Figure 1.

Schematic summarizing selected therapeutic strategies targeting the TME. Central to the schematic is the tumor cell which instigates the process of tumor formation and the creation of the tumor microenvironment. Cells that are recruited to the microenvironment include microglia, macrophages, T cells, NK cells, dendritic cells, reactive astrocytes, fibroblasts, and others. These cells secrete factors and components that influence each other and modify the extracellular matrix. Tumor cells also interact with neurons and form structural connections like synapses that facilitate tumor growth. Metabolic derangements result in autocrine and paracrine signaling in the microenvironment through secreted molecules and metabolites. Strategies to target glioma growth may focus on any of these aspects of the microenvironment, only a small sample of which are illustrated here. Created with BioRender.

Structural Components of the TME

Tumor Cells

The foundation of the brain TME is the tumor cells themselves. Glioma, like all cancers, is initiated by the underlying genetic alterations that enable deregulated cell growth and proliferation, transforming normal cells into tumor cells. This process of transformation allows tumor cells to ignore cues from their microenvironment that normally regulate cell growth, such as cellular density, nutrient and oxygen availability, growth factors (or the lack thereof), and extracellular matrix (ECM) support. The mutations and copy number alterations that arise in glioma have been particularly well-characterized, with distinct patterns of mutations specific to each of the glioma subtypes.

One of the defining genetic alterations of gliomas is the isocitrate dehydrogenase (IDH1) mutation. As of 2021, the World Health Organization classifies gliomas based on whether or not they have IDH mutations, due to the clear prognostic and diagnostic utility.¹⁷ IDH mutant tumors include astrocytoma, which also have mutations in ATRX and TP53, and oligodendroglioma, which has TERT promoter mutations and loss of chromosome arms 1p and 19q.^17–20 IDH mutant tumors that also have CDKN2A/CDKN2B deletion or histologic characteristics of microvascular proliferation and necrosis behave more aggressively and are given a grade 4 designation. IDH wild-type (WT) tumors, which are the most aggressive, include GBM, and have mutations in the TERT promoter, loss of chromosomal arm 10q (PTEN gene locus), loss of the chromosome 9p21 housing tumor suppressor genes CDKN2A/CDKN2B and MTAP, amplification of chromosome 7, and frequent mutations in EGFR, TP53, PIK3CA, PTEN, AKT, and STAT. Most of these mutations function to deregulate the cell cycle controls, allowing unchecked tumor cell proliferation. Some genetic alterations also play a role in protecting tumor cells from the immune response and suppressing immune function, a requirement for tumor growth. Additional attention will be given to some of these immunosuppressive alterations in later sections.

While in many cancer types, a higher tumor mutational burden correlates with increased antigen recognition and improved immune response, the same association has not been found with GBM.^21,22 In fact, some studies have shown the inverse, with high mutational burden associated with worse survival,²³ although this is confounded by the fact that higher mutation number is also associated with older age and IDH WT status. One study, however, looked at patients that underwent immunotherapy with either immune checkpoint blockade or recombinant polio virotherapy and found that those with a very low tumor mutational burden had improved survival.²⁴

The standard of care treatment for high-grade glioma is surgical resection followed by radiation and temozolomide chemotherapy.² Temozolomide is a DNA alkylating agent that demonstrates single-agent antitumor activity against GBM. The mechanism of temozolomide is directly counted by the action of DNA-repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT), and levels of tumor MGMT expression and promoter methylation have been shown to correlate with patient survival.²⁵ One of the main side effects of temozolomide is lymphopenia,²⁶ with decreased circulating CD8⁺ T cells and NK cells, but increased Tregs.^27,28 Interesting, despite T-cell depletion by temozolomide, patients coadministered temozolomide and an EGFRvIII vaccine was able to develop potent cellular and humoral immune responses that were enhanced by higher temozolomide doses.²⁸ Of note, dendritic cells are relatively unaffected by temozolomide and thus may be able to exert more influence in the TMZ-depleted microenvironment.²⁹

Immune Infiltrates

Macrophages are the most abundant cell type in gliomas, comprising a staggering 30%–50% of the cells in a tumor mass.¹⁶ It has been shown that a combination of tissue-resident microglia and bone marrow–derived macrophages both contribute to the tumor-associated macrophage (TAM) population. Microglia are embryologically yolk sac–derived and reside in normal brain tissue, where they account for 10%–20% of the non-neuronal population in the brain.³⁰ They play an important role in brain development, homeostatic maintenance, angiogenesis, synaptic formation, and protection from pathogens.^31,32 Extra-parenchymal, bone marrow–derived macrophages typically reside in the perivascular spaces and the meninges.³³ Both microglia and macrophages infiltrate gliomas, and the density of infiltration correlates with glioma progression and grade, with more highly infiltrated lesions being more aggressive.^14,15,34 Glioma cells secrete high levels of chemoattractants including CCL2, CCL7, MCP-3, CSF-1, G-CSF, Lox, and Osteopontin.^35–38 These attractants stimulate robust infiltration of macrophages and microglia into the tumor where they are manipulated into a pro-tumoral role. In addition to attractants, tumor cells secrete an abundance of anti-inflammatory cytokines and metabolites, such as IL-6, IL-10, TGF-β, adenosine, glucocorticoids, and prostaglandins.^39–44 The combination of chemoattractants and immunosuppressive signals results in a resident macrophage population that is unable to carry out their expected functions. Ideally, macrophages, along with other antigen-presenting cells (APCs), would function by engulfing tumor cell debris and presenting processed peptides on major histocompatibility complexes (MHC) I and II for T-cell surveillance. Macrophage activation, mediated by IFN-γ involves secretion of TNF-α, chemokines, cytokines, and nitric oxide to augment the pro-inflammatory environment.⁴⁵ Macrophage activation to an inflammatory state (M1-like phenotype) is normally balanced in the body by subsequent transition to an anti-inflammatory (M2-like) state to promote healing.⁴⁶ While the M1 vs M2 polarization model is an oversimplification of macrophage activation states in gliomas, markers of these phenotypes are useful in identifying cell states that are pro or anti-inflammatory. In the classically activated M1 state, macrophages produce inflammatory cytokines and are identified by cell surface markers CD80, CD86, and MHC II. In the alternatively activated M2 state, macrophages secrete factors to dampen the immune system and promote tissue healing and remodeling, including ECM deposition and angiogenesis. This M2 polarization is the predominant macrophage phenotype observed in gliomas and can be detected by expression of markers CD163, CD206, and arginase-1 (ARG-1).^47,48 Alternative (M2) activation is triggered by the abovementioned anti-inflammatory factors (IL-4, IL-10, IL-13, CSF-1, TGF-β) and maintained autocrine signaling by macrophage secretion of IL-10 and expression of the IL-10 receptor.^49–51 As expected, expression of M2 markers correlates with higher glioma grade.⁴⁷ This is because these M2 macrophages in the TME do not recognize tumor cells as intruders, but rather, as healing tissue for which they carry out supportive roles of promoting proliferation, connectivity, and angiogenesis.

Dendritic cells (DCs) constitute about 5% of the cell population in IDH WT glioma and 10% of the population in IDH mutant glioma.⁵² Dendritic cells are antigen-processing and -presenting cells and interact with both the innate and adaptive immune system including T cells, B cells, mast cells, and NK cells. They are not found in the quiescent brain parenchyma but reside in vascular-rich compartments such as the meninges and choroid plexus. In gliomas, the infiltrative dendritic cell population consists of different subtypes, such as conventional DC type 1 (cDC1), conventional DC type 2 (cDC2), premature DCs (pre-DC), migratory DCs (CD2), and plasmacytoid DCs, though they display limited function compared to their peripheral blood counterparts.^52,53 Suppressive components of the TME, such as macrophage-derived IL-10, limit full maturation and antigen-presenting capability.^52,54 Recent work using single-cell RNA-seq analysis has identified additional DC subsets in blood and GBM tissue,^53,55 though further investigation is needed to understand the role each of these subsets plays in the microenvironment.

MDSCs are a group of myeloid progenitor and precursor cells consisting of macrophages, dendritic cells, and granulocytes in different stages of differentiation. Under the chronic inflammatory conditions of the TME, immature myeloid cells do not fully differentiate into a mature myeloid cell type, causing accumulation of an immunosuppressive population of cells known as MDSCs.⁵⁶ There are different subtypes of MDSCs based on which differentiation markers they express, namely granulocytic (G-MDSC), mononuclear (M-MDSC), and early-stage (E-MDSC), while all lacking mature markers of myeloid cells such as MHCII-HLA DR.⁵⁷ The MDSC cell population is particularly immunosuppressive, with ability to block T cell, NK cell, macrophage, and dendritic cell function. They utilize reactive oxygen species and reactive nitrogen species, generated through NADPH oxidase or ARG-1 to induce oxidative stress that blocks T-cell or NK cell function. MDSCs also secrete soluble factors such as IL-10, TGF-β, and IFN-γ that are able to polarize macrophages to M2 phenotype, inhibit DC cell stimulation of T cells, and influence T reg differentiation.⁵⁶

Tumor-infiltrating lymphocytes (TILs) are an essential component of the host immune response and the body’s most effective weapon against tumor growth. The number of infiltrating T cells in glioma tissue is associated with improved survival independent of age, MGMT status, or treatment.¹¹ There have been observations that the number of CD8⁺ TILs in glioma is inversely correlated with tumor grade⁵⁸ and with EGFR amplification and homozygous PTEN loss.⁵⁹ The overall density of lymphocytes in glioma tissue is low, comprising only 1% of all cell types.⁶⁰ In addition, the glioma microenvironment is particularly adept at inducing T-cell dysfunction through immunosuppressive cytokines, metabolites, immune checkpoints, antigen suppression, and regulatory T cells.^4,5 Together, with low TIL density, these microenvironmental factors render T-cell responses ineffective.

Virtually every cell in the body displays internally processed peptide fragments on their cell surface in MHC I receptors for T-cell inspection. APCs that engulf pathogens, debris, or apoptotic cells present peptide fragments on MHC II receptors. When T cells interact with MHC I and II receptors that contain irregular sequences (ie, tumor mutations), this can trigger a cascade of T-cell activation and proliferation, releasing an army of CD4⁺ T helper cells and CD8⁺ cytotoxic T cells to scan the body for the aberrant sequence. When a T helper cell locates mutant peptides on a tumor cell, they secrete pro-inflammatory cytokines to recruit cytotoxic T cells and other immune cells to the target. Cytotoxic T cells are then able to kill tumor cells through secretion of perforin and granzymes to induce apoptosis. T regulatory cells (Tregs) serve to temper the inflammatory response once the threat has been neutralized. While this seems like a straightforward program to eliminate any cell that is going awry, tumor cells employ multiple evasive maneuvers to avoid detection. For example, tumor cells downregulate MHC I expression, so that antigens are not presented on the cell surface for T-cell detection. Even if functional APCs are able to present tumor peptides on MHC II for T-cell activation, the T cells are unable to locate the mutant tumor cells that lack MHC I. Loss of MHC I expression is correlated with worse clinical outcomes, and resistance to immune checkpoint blockade and adoptive immunotherapy.⁶¹

Tumors also utilize innate immune regulatory programs to quell antitumor immune responses. For example, T cells express surface inhibitory proteins, called checkpoints, such as programmed-death 1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), and cytotoxic T lymphocyte Antigen 4 (CTLA-4). One of the best-known, and successfully modulated, mechanisms of immune evasion is upregulation of checkpoint ligands on tumor cells, such as the PD-1 ligand, PDL-1, which can bind to PD-1 on surveilling T cells and prevent activation even when a tumor antigen is detected.⁶² Chronic antigen exposure in the setting of anti-inflammatory signals, such as occurs in the glioma microenvironment, results in hyporesponsive T-cell states that share many similarities with the exhaustion observed in chronic viral infections. This hypofunction is marked by even further upregulation of multiple checkpoint receptors and a state of severe T-cell dysfunction.⁵ In addition to these active immunosuppressive processes, at baseline the TME is an uninviting place for T cells. Nutrient deficiency, hypoxia, high lactate levels, acidic pH, and high levels of released ions and metabolites such as potassium (K⁺) and adenosine all limit function of infiltrating T cells. A major line of tumor defense is inactivation of the APCs as described above, limiting antigen presentation and co-stimulation of T cells and secreting anti-inflammatory signals.

Stromal Cells

Gliomas are diffuse tumors that spread throughout the brain parenchyma. As such, the normal parenchymal cells become involved in the tumor mass and part of the TME. Like the immune cells we have discussed, stromal cells are not merely trapped in the TME but play an active role in tumor growth. Astrocytes are the most common glial cell in the brain and are best-known for their role in structural support, blood-brain barrier maintenance, synapse structural and functional maintenance, nutrient dispersion, and other homeostatic roles.^63,64 Reactive astrocytes have been found in close association with glioma cells and have been found to promote tumor proliferation and invasion through secretion of stromal cell-derived factor 1 (SDF-1) and metalloproteinases.⁶⁵ Tumor-associated astrocytes are predominately found in the tumor niche, along with fibroblasts, pericytes, and endothelial cells.⁶⁶ This is also the primary location of the glioma stem-like cell (GSC) population.⁶⁷ Similar to immune cells, reactive astrocytes are initially recruited to aid in brain healing, but quickly become hijacked to help with tumor progression. GBM cells produce RANKL, which acts on the astrocyte RANK receptor, to activate the NF-κB pathway. These activated astrocytes in turn secrete high levels of TGF-β, TNF-α, IGF-1, and GDF-15 which support glioma cell proliferation.^68,69 Astrocytes have also been shown to facilitate glioma cell resistance to hypoxia through CCL20 secretion, and resistance to chemotherapy/apoptosis through GAP junctions and helping glioma cells clear calcium ions.⁷⁰

Recent work demonstrated that neurons directly participate and support glioma development through aberrant synapses and secretion of growth stimuli. Gliomas integrate into neuronal circuitry where they form synapses with neurons and receive depolarizing currents which promote glioma growth and proliferation through voltage-sensitive signaling pathways.⁷¹ Glioma cells secrete the synopatogenic molecule thrombospondin-1 (TSP-1), promoting functional connectivity with neighboring neurons. Neurons also secrete synaptic molecule neuroligin-3 at neuronal-glioma synapses, which promotes glioma cell proliferation through the PI3K-MTOR pathway and results in feed-forward neuroligin-3 expression by glioma cells.^72,73 Of note, these areas of high functional connectivity between neurons and glioma cells are inversely correlated with areas of immune activity.⁷⁴ Animal models show that TSP-1 knockout not only abrogates neuronal connectivity but also restores pro-inflammatory responses, prolonging survival in immunocompetent but not immunodeficient models.⁷⁴ This suggests that glioma cells utilize functional connectivity with neurons not only for neuron-derived growth signals but also to manipulate the microenvironment to evade immune cells by mimicking neuronal mechanisms.

Fibroblasts are additional stromal cells that play a role in tumor cell invasion and growth. These cells play roles in processing tumor-secreted metabolites and waste products to optimize tumor growth and immune evasion.⁷⁵ Glioma-associated fibroblasts have also been shown to play an important role in matrix metalloprotease production for ECM restructuring.^76,77 Recent work has also shown that cancer-associated fibroblasts produce extra domain A fibronectin which binds to macrophage TLR4 receptor to stimulate M2 polarization. Spatial transcriptomics also demonstrates close association between fibroblasts and GSCs in the perivascular niche. GSCs stimulate fibroblasts with PDGF and TGF-β and fibroblasts in turn secrete HGF and osteopontin.⁷⁵

Endothelial cells play a critical role in tumor vascularization as well as carry an immunomodulatory role through cross-talk with immune cells.⁷⁸ The proliferation, migration, and morphogenesis of endothelial cells is essential to angiogenesis, and angiogenesis is essential to glioma growth and progression.^78,79 In fact, one of the defining histologic features of high-grade glioma is vascular proliferation. Vascular endothelial growth factor (VEGF) is a secreted signaling molecule that promotes angiogenesis. VEGF is secreted by tumor cells and TAMs to promote neovascularization. The main signals driving VEGF production are either cytokine cross-talk as described above, or hypoxia and HIF-1α signaling. Endothelial cells also play a role in promoting immunosuppression in the TME by upregulating PD-L1 expression on their cell surface in response to VEGF signaling and hypoxia.⁷⁸

Extracellular Matrix

The ECM is a network of macromolecules such as chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, hyaluronic acid, laminins, tenascins, and glycoprotein-fibronectin that provide the scaffold for the extracellular tissue microenvironment.⁸⁰ The normal brain ECM differs from other tissues in that it lacks collagen (except in basal lamina of blood vessels) and carries out important roles in neuronal migration, differentiation, and maturation.⁸¹ The ECM of gliomas relative to normal brain has upregulated hyaluronic acid, tenascins, and fibronectin, with higher levels of these molecules being correlated with higher tumor grade and aggressiveness. Upregulation of these ECM components has been shown to increase the mobility and invasiveness of tumor cells. The ECM in gliomas is a dynamic structure that is actively remodeled through MMPs to facilitate tumor cell invasion, with new ECM deposition being a constant process.⁸¹

Demographic Factors

Age

There is a strong association between age at the time of glioma diagnosis and outcomes.⁸² One factor is the genetic composition of the tumor, as IDH mutant gliomas occur predominately in younger adults while IDH WT gliomas occur in a bimodal distribution in children and elderly adults.¹⁷ The incidence of IDH WT GBM progressively increases throughout adulthood and peaks in the eighth decade of life.⁸² Within the same glioma subtype classification, advanced age is associated with poorer outcomes in general and has been shown to have decreased survival benefit with immunotherapy treatment.⁸³ General population studies show that circulating CD8⁺ T-cell numbers progressively decrease in peripheral circulation with age, while regulatory T-cell numbers increase.⁸⁴ Advanced age is also associated with immunosuppressive gene expression profile in the brain including increased PD-L1 and IDO1 expression.⁸⁴ While outcomes are certainly multifactorial, it is clear that the immune system is less capable of an antitumor response in the elderly population.

Gender

Men have higher incidence of glioma and worse survival outcomes compared to women.^82,85,86 Investigations into molecular differences between gliomas in males and females have revealed differences in mutations and copy number alterations, with males having amplification of AKT1 on chromosome 17 and females having amplification of MYC on chromosome 8. Males also have enrichment of PIK3R1 and NF1 mutations while females are more likely to have PIK3CA mutations.⁸⁶ Overall, females have a higher aneuploidy fraction and tumor mutational burden. Women also are more likely to have methylation of the MGMT promoter. Analysis of tumor gene expression patterns shows differences in transcriptional signatures with increased neuronal transcriptional signatures in males with IDH WT tumors compared to females.⁸⁷ Aside from molecular differences in tumor cells, there are also broader TME differences that have been reported. Males have increased monocytic MDSCs in the TME.⁸⁸ There are also intrinsic T-cell differences between sexes, with males having decreased T-cell infiltration into gliomas and increased rates of T-cell exhaustion associated with hormonal and sex gene differences.⁸⁹

Metabolism and the TME

The brain TME is an area of high energy demand and insufficient vascular supply. One of the first noted characteristics of cancer cells is the increased rate of glycolysis, known as the Warburg Effect, discovered by Otto Warburg in 1927.⁹⁰ The brain normally utilizes alternate energy sources like lactate and ketone bodies during periods of high energy demand. Tumor cells prioritize metabolic pathways that facilitate high rates of cell turnover and provide the energy and building components necessary for cell proliferation. Oncogenic pathways altered in cancer that regulate cell proliferation naturally also regulate cellular metabolism, increasing rates of glycolysis and secretion of lactic acid in the microenvironment.⁹¹ Glioma cells have been shown to upregulate the rate-limiting glycolytic enzyme hexokinase2.^92,93 Lactic acid accumulation leads to an acidic microenvironment with a pH around 6.5 in glioma, compared to 7.2 in normal brain.⁹⁴ Mass spectrometry imaging confirms elevated lactate levels in glioma tissue compared to surrounding tissue,⁹⁵ and higher lactate levels in IDH WT vs IDH mutant gliomas.⁹⁶ Serum lactate levels are also elevated in glioma patients, particularly with higher grade lesions.⁹⁷ Lactic acid promotes angiogenesis and cancer cell migration and inhibits immune cell function.⁹⁸ High lactate levels are associated with decreased CD8 T-cell infiltration in gliomas.⁹⁹ When TAMs are exposed to lactate, HIF-1α is stabilized and VEGF production increases.¹⁰⁰ In addition to glycolysis, gliomas also have upregulated activity through the pentose phosphate pathway, glutamine metabolism, and fatty acid oxidation. These all represent energy sources and pathways essential to generating biosynthesis components for cellular division that are necessary in GBM and provide the tumor with metabolic plasticity to deal with a dynamic nutrient microenvironment.¹⁰¹

Control of immune cell metabolism in the TME is not fully understood. Identifying targetable pathways to drive specific metabolic programs has the potential to provide the metabolic support necessary for immune cells to perform antitumor functions under duress. We recently identified a novel mechanism by which tumors inhibit immune cell function is through the metabolically active cytokine Meteorin-like (METRNL).¹⁰² T cells have limited resources in the TME and are susceptible to metabolic insufficiency.¹⁰³ T cells that undergo repeated stimulation in this context enter a hypofunctional state characterized by upregulation of checkpoint molecules as well as secretion of METRNL. METRNL acts via autocrine and paracrine signaling to induce T-cell mitochondrial dysfunction and shift metabolic activity to glycolysis, temporarily conserving energy but decreasing T-cell function.¹⁰² Ablating METRNL improves T-cell function and supports tumor control. Future studies will determine if METRNL blockade or ablation alone is adequate metabolic support for endogenous and/or genetically modified antitumor T cells to remain active in the TME long-term.

Mutations in IDH1/2 alter the enzymatic active site and disrupt the normal metabolic function of converting isocitrate to α-ketoglutarate, and instead confer the neomorphic function of converting α-ketoglutarate to 2-hydroxyglutarate (2-HG). 2-HG cannot be utilized by the cell causing it to accumulate within the cell and within the TME. IDH mutation is believed to be one of the earliest mutations that occurs in low-grade glioma formation and has been shown to heavily impact tumor cell epigenetics and gene expression patterns. Investigations of the impact of 2-HG on the microenvironment have shown that 2-HG impairs T-cell chemotaxis, accumulation, and function through interference with STAT signaling pathways and decreased NFAT and NF-κB-mediated transcription.^104–106 2-HG has also been shown to directly impair dendritic cell maturation, downregulating MHC II and IL-6 and upregulating immunosuppressive mediators CD206 and PD-L1, resulting in decreased T-cell activation and IFN-γ production.⁵²

In 1975, it was first demonstrated that extracellular adenosine suppresses the activity of cytotoxic T cells.¹⁰⁷ Subsequently, the effects of adenosine on the immune system have been further elucidated but translation to therapeutic strategies has lagged. There are 4 adenosine G protein-coupled receptors, A1, A2_A, A2_B, and A3, which are expressed to varying degrees on the surface of lymphocytes, macrophages, and other cells in the TME. Activation of T-cell receptors results in upregulation of A2_A expression on T cells, and activation of toll-like receptors on macrophages upregulates expression of both A2_A and A2_B receptors.¹⁰⁸ Adenosine stimulation of A2_A on T cells results in intracellular cAMP production and downstream impairment of T-cell activation and proliferation, and causes upregulation of co-inhibitory molecules PD-1, TIM-3, LAG-3, and CTLA-4 as well as differentiation of CD4⁺ T cells into Tregs.^40,109–111 Stimulation of A2_A and A2_B receptors on microglia and macrophages results in M2 polarization and increased expression of IL-10, VEGFA, and TGF-β.^42,112 Gliomas manipulate this signaling mechanism in the TME through upregulation of adenosine levels. At baseline, adenosine levels are increased in the TME due to high cellular turnover. Following cell death, ATP is rapidly released into the extracellular space, shifting concentrations from the nanomolar to micromolar range.¹¹³ ATP is a pro-inflammatory molecule in the extracellular space; however, in the TME it is quickly hydrolyzed to adenosine, which is potently immunosuppressive. Tumor cells, and other cells in the TME, such as fibroblasts, express high levels of ectonucleotidases CD39 and CD73. CD39 is responsible for hydrolyzing extracellular ATP to ADP and AMP, and CD73 subsequently converts AMP to adenosine.^114,115 Adenosine can then be catabolized by membrane-bound adenosine deaminase into inosine, or transported into cells.¹⁰⁸

One of the most frequent genetic alterations in GBM is loss of the 9p21 locus including tumor suppressor genes CDKN2A, CDKN2B, and MTAP. MTAP deletion has been shown to have prognostic significance, correlating with shortened disease-free survival in GBM patients.¹¹⁶

Loss of 9p21 has been associated with an immunosuppressive microenvironment across several cancer types and resistance to immune checkpoint therapy.⁹ Further studies demonstrated that this immunosuppressive effect of 9p21 loss was specific to MTAP deletion and not CDKN2A or CDKN2B deletion.¹¹⁷ When MTAP enzyme function is lost, its substrate methylthioadenosine (MTA), a purine metabolite, is left to accumulate to high levels within and around the tumor cells, in a manner similar to 2-HG in IDH mutant gliomas. MTA inhibits several methyltransferase enzymes intracellularly, resulting in epigenetic changes to tumor cells,^116,118,119 and has also been shown to have an impact on its microenvironment, likely by acting on purinergic receptors as well as affecting methyltransferase activity within other cells of the TME.^6,117,120 Exposure of macrophages to MTA has been shown to downregulate TNF-α production and to shift macrophages to an M2-polarized phenotype with elevated expression of VEGFA and IL-10,^116,121 and MTA has been shown to directly suppress the proliferation and function of T lymphocytes.¹²² As 9p21 loss is one of the cancer-initiating events in the process of tumorigenesis, it is likely that the resultant metabolic alteration of the TME is one of the first immune escape mechanisms employed by tumor cells and further cements the importance of purinergic signaling in immune modulation.

Therapeutic Targets in the TME

While the number and variety of immune evasion mechanisms in the TME are daunting, each offers a potential opportunity for therapeutic intervention (Figure 1). The most studied immunotherapeutic approach to date has been immune checkpoint blockade using antibodies directed toward surface inhibitory checkpoint proteins, such as anti-PD-1 or anti-CTLA-4. While this approach has been revolutionary in treating other cancer types, it has failed to show the same efficacy against GBM.^123–125 Although there is some evidence that neoadjuvant administration has benefit,^126,127 further work must be done to identify patient subsets that are most likely to benefit from checkpoint inhibitor treatment and trial novel combination therapies.¹²⁸ A summary of phase III immunotherapy trials for glioma treatment is listed in Table 1.

Table 1.

Phase III Glioma Immunotherapy Trials (as of November 16, 2024 in clinicaltrials.gov)

Clinical Trial ID	Study Name	Start Date	Status	Agent	Primary Outcome
NCT00045968	Study of a Drug [DCVax®-L] to Treat Newly Diagnosed GBM Brain Cancer (GBM)	December 2006	Completed	Autologous DC exposed to tumor antigen	Increased OS in Newly Diagnosed GBMa
NCT03149003	A Study of DSP-7888 Dosing Emulsion in Combination with Bevacizumab in Patients With Recurrent or Progressive Glioblastoma Following Initial Therapy	December 2017	Completed	Ombipepimut (Synthetic epitopes of WT1) + bevacizumab	No difference in OSa
NCT01480479	Phase III Study of Rindopepimut/GM-CSF in Patients With Newly Diagnosed Glioblastoma (ACT IV)	November 2011	Completed	Rindopepimut EGFRVIII peptide Vaccine	No difference in OSa
NCT00088400	Comparison of TransMID (Transferrin-mediated delivery of diphtheria toxin) vs Standard Treatment of Cancerous Brain Tumors	July 2004	Completed	Tf-CRM107 Transferrin-Diphtheria toxin conjugate	No difference in OSa
NCT01759810	Proteome-based Personalized Immunotherapy of Glioblastoma	December 2012	Active	Antigen-loaded DCs, co-cultured T cells, hematopoietic stem cells	All-cause mortality
NCT04396860	Testing the Use of the Immunotherapy Drugs Ipilimumab and Nivolumab Plus Radiation Therapy Compared to the Usual Treatment (Temozolomide and Radiation Therapy) for Newly Diagnosed MGMT Unmethylated Glioblastoma	September 2020	Active, Not Recruiting	Ipilimumab Nivolumab	OS
NCT04277221	Autologous Dendritic Cell/Tumor antigen for Adjuvant Immunotherapy in Standard Treatment of Recurrent Glioblastoma Multiforme (GBM)	September 2019	Recruiting	Antigen-loaded autologous DCs	OS
NCT03548571	Dendritic Cell Immunotherapy Against Cancer Stem Cells in Glioblastoma Patients Receiving Standard Therapy	April 2018	Recruiting	DCs transfected w/ survivin, hTERT, and GBM stem cell mRNA	PFS
NCT05235737	The Assessment of Immune Response in Newly Diagnosed Glioblastoma Patients Treated with Pembrolizumab (PIRG)	March 2022	Recruiting	Pembrolizumab	OS PFS
NCT06556563	EF-41/KEYNOTE D58: Phase 3 Study of Optune Concomitant With Temozolomide Plus Pembrolizumab in Newly Diagnosed Glioblastoma	November 2024	Recruiting	Optune + Pembrolizumab	OS
NCT05685004	Study of Neoantigen-Specific Adoptive T cell Therapy for Newly Diagnosed MGMT Negative Glioblastoma Multiforme (GBM)	September 2023	Recruiting	Tumor antigen-stimulated autologous T cells	OS
NCT05100641	AV-GBM-1 vs Control as Adjunctive Therapy Following Surgery and RT/TMZ in Newly Diagnosed GBM	January 2024	Recruiting	Antigen-loaded autologous DCs	OS

Abbreviations: OS, overall survival; PFS, progression-free survival.

^aFinal study result.

Another focus of investigation has been targeting the immunosuppressive effects of infiltrative macrophages.¹²⁹ This includes treatment with IL-12 and inhibitors of CSF-1R to inhibit TAM function,^130,131 inhibition of SDF-1 to inhibit chemotaxis,¹³² antibiotic treatment to inhibit microglial function,¹³³ or promotion of M1 polarization through inhibition of STAT3 signaling.¹³⁴

Altering the cellular composition of the TME through exogenous introduction of autologous immune cells through adoptive cell transfer has been another major effort. In these approaches immune cells (NK cells, dendritic cells, and T cells) are harvested and then modified through cytokine and antigen stimulation or genetic engineering and then reintroduced to the patient. A recent phase III trial using lysate-loaded autologous dendritic cell vaccine infusion showed significant survival improvement in both newly diagnosed and recurrent GBM (Table 1).¹³⁵ Another highly investigated approach is the chimeric antigen receptor (CAR) T cell, which involves engineering the patients T cells to express a receptor that targets a tumor-specific antigen.¹³⁶ This allows the T cell to bypass MHC-dependent antigen recognition. Recent work at 2 institutions demonstrates rapid radiographic responses in recurrent GBM after a single infusion of CAR T cells. In one study, the CAR was engineered to both target the epidermal growth factor receptor (EGFR) variant III tumor-specific antigen, as well as locally secrete t-cell-engaging antibody molecules against the WT EGFR protein.¹³⁷ In the other study, the CAR was bivalent targeting an epitope of EGFR and IL13Rα2.¹³⁸ While the results were mostly transient, these early results demonstrate the incredible potential of this line of therapy.

The metabolic influence of the TME on T cells is another recent focus in cancer treatment. Inhibition of adenosine production through ectonucleotidases (anti-CD39, anti-CD73) or direct inhibition of adenosine receptors A2_A and A2_B are being explored in clinical trials for various cancer types.¹³⁹ Drugs that inhibit glycolysis/hexokinase 2,^140,141 block glutamine synthesis and metabolism,¹⁴² or inhibit glucose and/or amino acid uptake¹⁴³ are all approaches that are being investigated to normalize the metabolism of the TME in a way that can impair tumor cell growth and improve immune function. Similarly, targeting the effect of lactic acid buildup through inhibition of lactate dehydrogenase is gaining traction,^139,144,145 as well as targeting hypoxia signaling pathways.^146–148 While direct angiogenesis inhibition with anti-VEGF-A inhibitors has so far not shown to significantly improve survival in GBM patients, multi-modal antiangiogenic therapies and combination with other therapies are being investigated.^149–151 Inhibitors of mutant IDH (Vorasidenib), which block the production of 2-HG, normalizing TCA cycle flow, have recently been shown to significantly improve progression-free survival in IDH mutant gliomas.¹⁵² With the discovery that functional connectivity between neurons and glioma cells promotes tumor growth, efforts to interfere with tumor-neuron synapses revealed that Gabapentin, a common antiepileptic and pain modulation drug that targets synaptogenic thrombospondin signaling, reduces glioma-neuron synapse formation and significantly reduces GBM growth.¹⁵³

Targeting the ECM has been recognized as a high-yield area of therapeutic investigation. Modifying fibronectins, tenascins, fibulin-3, and hyaluronic acid deposition with blocking antibodies has been shown to decrease tumor growth, invasion, and neovascularization in preclinical models.^154–157 Additionally, enzymatic breakdown of ECM components using hyaluronidase, matrix metalloproteinases, or chondroitinase has been shown to improve distribution of immunotherapeutic agents through the thick tumor ECM. Preclinical models utilizing oncolytic adenovirus armed with soluble hyaluronidase or oncolytic herpes simplex virus armed with matrix metalloproteinases or chondroitinase ABC each demonstrated improved viral distribution through the tumor and greater antitumor efficacy.^158–160

Conclusions

The intricate and hostile glioma microenvironment presents a formidable challenge to the body’s natural immune functions and to therapeutic interventions. The TME is an active and dynamic participant in tumor progression and expands the challenge of treating glioma beyond merely targeting the tumor cells themselves. The interplay between glioma cells, immune infiltrates, stromal cells, and the ECM creates a complex ecosystem that fosters tumor growth, immune evasion, and therapeutic resistance.

Despite significant advances in our understanding of glioma biology, the immunosuppressive landscape of the TME remains a major barrier to effective treatment. Glioma cells exploit various mechanisms to suppress immune function, including the recruitment and reprogramming of TAMs, the expansion of MDSCs, and the inhibition of T-cell activity through immune checkpoints and metabolic reprogramming. Metabolic adaptations of glioma cells further render the microenvironment inhospitable to immune cell function. Additionally, the interactions between glioma cells and the surrounding stromal components, such as astrocytes and endothelial cells, promote a pro-tumoral environment that supports tumor cell survival and expansion.

To overcome these challenges, any therapeutic approach aimed at eradicating glioma must include strategies to modulate the TME, overcome immune cell dysfunction, and target the metabolic pathways that contribute to immunosuppression. While the complexity of the glioma microenvironment poses significant hurdles, it also offers numerous therapeutic targets. Continued research into the cellular and molecular mechanisms that drive immunosuppression and tumor progression in gliomas is crucial for the development of novel, more effective treatments. By unraveling the complexities of the glioma microenvironment, we can lay the foundation for innovative therapeutic strategies that may one day offer hope to patients with this devastating disease.

Supplement sponsorship

This article appears as part of the supplement “Immunotherapy for Brain Tumors,” sponsored by the Wilkins Family Chair in Neurosurgical Brain Tumor Research.

Conflict of interest statement

C.M.J. is a consultant and shareholder of Egret Therapeutics. C.M.J. holds Amgen stock. C.M.J. has received research support from Biohaven, InCephalo, and Grifols. Johns Hopkins University has filed a provisional patent on METRNL blockade for cancer treatment on which he is an inventor. Johns Hopkins University has filed a provisional patent on PD-1 agonists for brain ischemia on which he is an inventor. Stanford University has filed a provisional on using PD-1 agonists for myocardial infarction on which he is an inventor.