Glioma Microenvironment Research Articles

PATH-63. M6A REGULATOR-BASED MOLECULAR CLASSIFICATION AND HUB GENES ASSOCIATED WITH IMMUNE INFILTRATION CHARACTERISTICS AND CLINICAL OUTCOMES IN DIFFUSE GLIOMAS

Abstract BACKGROUND m6A methylation modification is a new regulatory mechanism involved in tumorigenesis and tumor-immunity interaction. However, its impact on glioma immune microenvironment and clinical outcome remains unclear. METHODS Comprehensive expression profiles of 18 m6A regulators were used to identify molecular subtypes exhibiting distinct m6A modification patterns in 1673 glioma samples sourced from public datasets. A multi-genes signature was constructed for predicting clinical outcome and response to immunotherapy in glioma patients. Immunohistochemistry and cellular experiments were performed for validation. RESULTS Two m6A subtypes of gliomas were identified. The m6A-low-risk subtype was characterized by paucity of immune infiltrates; Whereas the m6A-high-risk subtype had higher abundances of multiple immune cells including lymphocyte and macrophage as well as increased expression of PD-L1, corresponding to an immunosuppressive phenotype. The m6A-high-risk subtype had poorer survival than the m6A-low-risk subtype in both the glioblastoma and lower grade gliomas cohorts. Eight m6A-related hub genes of high prognostic significances were identified and selected for developing a scoring signature termed as m6Ascore. Elevated m6Ascore indicated worse survival for glioma patients under standard care, but showed enhanced response to immunotherapy. Moreover, we demonstrated that overexpression of FTO, a m6A demethylase, inhibited the expressions of m6A-related hub genes (PTX3, SPAG4), impaired glioma cell viability and reduced macrophage chemotaxis. CONCLUSION This work develops an immune- and clinical-relevant m6A subtyping and a scoring model, which enhances our understanding of the role of m6A modification in regulating immune infiltration microenvironment in gliomas and helps to identify patients who are more likely to benefit from immunotherapy.

EXTH-79. BREAKING THE BARRIER: ADOPTIVE CELLULAR THERAPY HALTS GLIOMA IMMUNOSUPPRESSION BY TARGETING MYELOID-DERIVED SUPPRESSOR CELL MIGRATION AND CELL CYCLE MECHANISMS

Abstract INTRODUCTION Our group previously demonstrated that adoptive cellular therapy (ACT) significantly increased overall survival in mice across several brain cancer models. ACT remodels the tumor microenvironment (TME) in a pleiotropic manner, and specifically depletes immunosuppressive cells like myeloid-derived suppressor cells (MDSCs) which are a significant barrier to efficacious immunotherapy responses. This led us to hypothesize that ACT depletes MDSCs from the glioma microenvironment by inhibiting MDSC proliferation and migration, thereby overcoming glioma immunosuppression. METHODS We quantified MDSC cell cycle, apoptosis, and localization in the TME and peripheral lymphoid tissues using flow cytometry as well as chemokines in the TME from glioma-bearing mice treated with ACT. C57BL/6J mice intracranially received KR158b-luciferase glioma and were treated with ACT. After treatment, MDSC cell cycle was quantified using bromodeoxyuridine and 7-aminoactinomycin to define cell cycle phases. MDSC apoptosis and cell death states were quantified by measuring annexin-V and propidium iodide. Anatomic localization of MDSCs was measured by comparing host (CD45.2) and transferred HSC-derived cell markers (CD45.1) in the TME and spleen. A multiplex array was performed using tumor lysates to quantify cytokine and chemokine levels within the TME. RESULTS We observed that MDSCs in the TME were significantly less proliferative and exhibited significantly increased levels of apoptosis and cell death after ACT. HSC-derived MDSCs were significantly enriched in the spleen of ACT-treated mice, but both transferred HSC-derived MDSCs and host MDSCs were depleted in the TME. Finally, ACT significantly decreased several chemokines in the TME, including interleukin-16 and monocyte chemoattractant protein-5. CONCLUSION These results indicate that ACT shifts MDSC cell cycle states away from proliferation towards cell death, and inhibits peripheral recruitment to the TME. Our investigation revealed novel, myeloproliferative chemokines that are decreased by ACT, representing a novel mechanism underlying disrupted MDSC migration to the glioma microenvironment and ultimately to overcoming glioma-mediated immunosuppression.

NIMG-44. ELEVATED TRANSLOCATOR PROTEIN 18KDA BINDING IN LOW MEAN DIFFUSIVITY CLUSTERS IS A FEATURE OF THE TRANSFORMING GLIOMA AND IDH MUTANT GLIOMA MICROENVIRONMENT

Abstract Around 95% of low-grade gliomas undergo anaplastic de-differentiation into high-grade gliomas, a process only partially understood from in vivo clinical imaging. 11 patients with suspected transforming gliomas underwent a combined magnetic resonance imaging (MRI) and positron emission tomography (PET) protocol, incorporating post-contrast T1-weighted/FLAIR acquisition for tumour delineation and diffusion tensor imaging. The PET radiotracer [11C]-(R)-PK11195 assessed translocator protein 18kDa (TSPO) distribution, whose upregulation is associated with inflammation/glioma progression. [11C]-(R)-PK11195 binding potential (BPND) maps were generated using the simplified reference tissue model, with the grey-matter cerebellar time-activity curve serving as the reference tissue input function. KMeans clustering of mean diffusivity (MD) intensity values defined spatially distinct “habitats” of high/low MD voxels within the glioma microenvironment. BPND values within high/low MD clustering habitats were evaluated, across glioma grades and isocitrate dehydrogenase (IDH) mutational status. Across all grade 2 and 3 gliomas (n= 6), mean TSPO BPND values in low MD habitats (0.0383 ±0.102) were significantly greater than high MD habitats (-0.045 ±0.142) (p= 0.003). In grade 4 gliomas (n= 5), no significant difference was observed in mean BPND values between low (0.514 ±0.176) and high (0.465 ±0.296) MD habitats (p= 0.543). IDH mutant gliomas (n= 7) exhibited significantly higher mean BPND in low MD habitats (0.116 ±0.225), relative to high habitats (0.052 ±0.287) (p= 0.021). IDH wildtype gliomas (n= 4) showed no significant differences between BPND values in low (0.498 ±0.199) and high MD habitats (0.424 ±0.325) (p= 0.467). Our novel findings demonstrate spatial heterogeneity in TSPO expression within 2/3/IDH mutant gliomas, a characteristic absent from grade 4/IDH wildtype varieties. Regions of restricted diffusion with high TSPO signal in grade 2/3/IDH mutant tumours indicate the heterogenous distribution of active TSPO-expressing immune/neoplastic cell populations, contrasting the homogenous distribution in grade 4/IDH wildtype gliomas, highlighting differences between their inflammatory profile and pathological features.

TMOD-05. GLIOMA-ASSOCIATED OLIGODENDROCYTE PROGENITOR CELLS’ DYNAMIC PATTERNS SUGGEST FUNCTIONAL ADAPTATIONS DURING GLIOMA PROGRESSION IN A UNIQUE MOUSE MODEL

Abstract Gliomas account for more than one-third of brain tumors in children, with high fatality rate despite aggressive adjuvant treatment. Two-thirds of survivors suffer from health conditions due to the toxicity of conventional therapies. Notably, gliomas with the BRAFV600E mutation, occurring in ~20% of all glioma cases, are difficult to treat and have a high risk for progression, especially when combined with CDKN2A deletion. The mechanisms for glioma progression remain unclear and unraveling them could lead to therapies that inhibit glioma progression, preventing high-grade gliomas. Objectives: To gain mechanistic insights into glioma progression and exploit therapeutically for preventing progression. We developed two novel mouse models for BRAFV600E mutant progressive gliomas – one at high genetic risk for progression and one at intermediate genetic risk for progression. Expression of genetic alterations were induced by injecting compound transgenic mice with adenovirus-Cre. Our new mouse models therefore more faithfully recapitulated the genetic changes accompanying glioma formation and progression from low- to high-grade than earlier mouse models which used the RCAS system to overexpress the oncogene (PMCID: PMC11112369 ). Glioma formation was followed non-invasively by magnetic resonance imaging. Single cell RNA sequencing and spatial transcriptomics were performed and immunofluorescence was used for validation (PMCID: PMC10619673 ). We present new data showing that animal subject survival is significantly shorter in the high risk model than in the low risk model. Our two models with distinct latency can be used for preclinical studies and studies of the changes associated with progression, including in the glioma microenvironment. Analyses revealed temporal, functional, and spatial changes in glioma-associated oligodendrocyte progenitor cells (GA-OPCs) at distinct stages (initiation, expansion, progression) and grades. Our findings that GA-OPCs closely interact with glioma cells and their morphological transformation as gliomas progress, suggest changing functions, including potential roles in phagocytosis and immunomodulation. Understanding the unclear role of glioma microenvironment OPCs in promoting glioma progression could reveal an Achilles heel for targeted therapies.

TMIC-40. TARGETING GLIOMA TUMOR MICROENVIRONMENT BY SPECIFIC NEUTRALIZATION OF EXTRACELLULAR NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (ENAMPT) USING ALT-100, A NOVEL HUMANIZED MONOCLONAL ANTIBODY

Abstract BACKGROUND Intracellular nicotinamide phosphoribosyltransferase (iNAMPT is the rate-limiting enzyme of NAD salvage pathway and is preferentially used by glioma cells for regulating energy metabolism and homeostasis. When secreted, extracellular NAMPT (eNAMPT) exerts cytokine-like functions and is associated with poor prognosis in cancer patients. We have previously reported an essential role of iNAMPT for glioma cell proliferation and survival. Here, we examined whether eNAMPT influences glioma biology and tumor microenvironment by neutralizing eNAMPT using ALT-100, a novel humanized monoclonal antibody. METHODS Native levels of eNAMPT in blood samples of patients with primary/recurrent gliomas were determined using ELISA under an IRB approved protocol. Next, the effects of ALT-100 on glioma microenvironment were determined by treating viable human glioma tissue slices with ALT-100 and assessing downstream effects on RNA and protein using RNASeq, reverse phase protein array and spatial transcriptomics. Lastly, the in vivo effect of ALT-100-mediated eNAMPT neutralization on survival was determined in a mouse intracranial glioma model. RESULTS Measurable levels of eNAMPT were noted in all patient samples with a range of (3.7-71.2 ng/ml). Preliminary results showed a correlation of eNAMPT levels with grade of tumor. Human organotypic glioma slice cultures treated with ALT-100, showed a profound alteration of gene and protein expression particularly related to key energy metabolism and proliferation pathways and the PD-1/PD-L1 axis. Additionally, results of spatial profiling of ALT-100 treated human organotypic glioma slices will be presented. Survival and response data from ALT-100 treatment in ongoing in vivo experiment will also be reported. CONCLUSIONS This is the first report of ALT-100-mediated eNAMPT neutralization in gliomas and demonstrates profound effects on the tumor and its microenvironment. Given that ALT-100 is in phase 2 trials (NCT05938036) against ARDS, data from our studies can potentially accelerate translating ALT-100-mediated eNAMPT neutralization to treat gliomas.

TMIC-26. IMMUNE CELL DYNAMICS WITHIN GLIOMAS: THE INFLUENCE OF GRADE AND IDH STATUS

Abstract Mutations in isocitrate dehydrogenase (IDH) are known to significantly impact the prognosis and biology of diffuse gliomas. A number of studies have compared the immune microenvironment of IDH-wildtype glioma versus IDH-mutant glioma, and have also characterized the impact of mutant-IDH on lymphocytes. However, few studies have explored how mutant-IDH may affect tumors as they progress and increase in grade, or have delved into the myeloid immune cell compartment. Thus, we sought to understand whether tumor progression affected intratumoral immune cell composition within IDH-mutant glioma, and aimed to compare IDH-mutant versus IDH-wildtype glioma in both humans and mice. Using bulk RNA sequencing, we compared paired samples from patients with IDH-mutant glioma before and after progression, focusing on differentially-expressed immunological genes and relative immune cell proportions. We also compared grade 4 IDH-mutant astrocytoma cases to IDH-wildtype glioblastoma, performing both transcriptomic and cellular analyses. Finally, we engineered murine glioma cells to test the effect of mutant-IDH alone in the tumor microenvironment. We found that multiple immune-related genes distinguish low- and high-grade IDH-mutant glioma, and that high-grade tumors contain greater proportions of suppressive myeloid cells. When comparing IDH-wildtype to IDH-mutant glioma, we found enrichment of suppressive macrophages and myeloid-derived suppressor cells (MDSC) in IDH-wildtype tumors. Finally, our murine model of IDH-wildtype versus IDH-mutant glioma mirrored many of the myeloid cell shifts observed in human tumors. Our data show that progression of IDH-mutant tumors is a significant immunological phenomenon, associated with marked alterations in the tumor immune microenvironment. We also show that IDH-mutant glioma harbors greater inflammatory signatures and fewer suppressive myeloid cells than IDH-wildtype glioma, and that engineering mutant IDH into murine tumors is sufficient to model these differences. These findings improve our understanding of key intratumoral cell populations that may be harnessed by future immunotherapeutic strategies.

TMIC-06. TUMOR ASSOCIATED MACROPHAGES DRIVE TUMOR PROGRESSION AND ARE TRANSCRIPTIONALLY SHAPED BY HISTONE MUTATIONS IN DIFFUSE MIDLINE GLIOMA

Abstract Little progress has been made to delineate the inflammatory microenvironment in pediatric high-grade glioma (pHGG) and diffuse midline glioma (DMG). We utilize molecularly defined human samples and immunocompetent mouse models to study how tumor location and genetic driver mutations influence the tumor microenvironment (TME). We first induced tumors of each pHGG/DMG molecular subtype (H3WT, H3G34R, H3.1K27M, H3.3K27M) into both the cortical hemisphere and midline of separate mice and performed survival studies and flow cytometry. The main driving factor shaping the immune landscape and survival is the driver mutation, not tumor location. Next, we performed single cell RNA sequencing on murine H3WT cortical pHGG, H3WT DMG, H3.1 and H3.3K27M DMGs, paired with multiplex flow cytometry. H3K27M DMGs have sparse T cell infiltration and limited T cell recruiting chemokine production. The main non-neoplastic cell type in all tumors was infiltrating myeloid cells and microglia. H3WT DMGs had the greatest myeloid cell and monocyte-derived macrophage (MDM) infiltration, while H3.3K27M DMGs were enriched for microglia. Disease associated gene signatures were found in tumor-associated macrophages (TAMs) that are found in other neurodegenerative diseases, marked by down-regulation of inflammatory signaling, interferon responses, and up-regulation of metabolism-related pathways. H3.3K27M DMGs were enriched for disease-associated TAMs. These gene signatures were then identified in human pHGG/DMG scRNA sequencing data. CD16 monocytes and microglia were enriched for diseased signatures, while CD14 monocytes were pro-inflammatory. We demonstrate that reprogramming this diseased signature in mice results in increased T cell infiltration and extension of survival in H3K27M DMG. Last, through pharmacologic inhibition of CCR1 and CCR5, we demonstrate preventing TAM infiltration in DMG results in enhanced survival, comparable to radiation therapy. Together, this work provides the foundation for developing immunotherapies targeted at specific subgroups of pHGG and DMGs, such as CAR-T-cell, oncolytic viral therapy, and checkpoint blockade.

TMIC-71. ELUCIDATING IMMUNE MICROENVIRONMENTAL DISTINCTIONS BETWEEN IDH-MUTANT ASTROCYTOMA AND OLIGODENDROGLIOMA USING SINGLE-CELL TRANSCRIPTIONAL PROFILING

Abstract Despite exhibiting longer overall survival that IDH-wt counterparts, IDH-mutant gliomas remain uniformly deadly and impact patients in the prime of adult life. The immune microenvironment of IDH-mutant gliomas remains incompletely characterized, particularly with regard to distinctions between IDH-mutant astrocytoma and IDH-mutant, 1p/19q codeleted oligodendroglioma. To address this knowledge gap, we subjected dissociated tumor suspensions derived from 7 astrocytoma and 7 oligodendroglioma clinical samples to flow cytometry-based sorting for the pan-immune marker, CD45, and the microglial marker, P2RY12. This process yielded both microglial and non-microglial immune cell populations, which were then analyzed by scRNA sequencing. This workflow revealed a single large cluster of microglia, characterized by expression of Galectin-1 and lysosomal Lipase A, which was present at a significantly higher level in oligodendroglioma. While the biological significance of this cluster is unclear, gene set enrichment analysis exploring phenotypic differences between microglia in astrocytoma and oligodendroglioma revealed significant increases in expression of gene sets related to antigen presentation in astrocytoma. Parallel analysis of the non-microglial immune fraction showed an increase in the expression of pro-inflammatory gene sets, such as genes related to cytokine secretion, within the CD8 T-cell compartment of astrocytomas. We also observed upregulation of the expression of inflammatory gene sets within the dendritic cells of astrocytomas. Taken together, these changes suggest that the immune compartment of astrocytoma is more active and inflamed, and targeting the immune system in these tumors may represent a potential therapeutic avenue.

TMIC-64. EXPLORING ULTRASTRUCTURAL PATHOLOGY IN GLIOMAS: INSIGHTS FROM RECENT STUDIES

Abstract Glioma tumors arise from the glial cells making up to 40% of the brain tumors, and contributing up to 85% of the malignant tumors of the brain. Gliomas are a heterogeneous group of tumors classified based on various factors such as genetics, molecular profiles, etc. Glioblastoma, an aggressive form of gliomas, has incredibly low survival of only 15 months suggesting that thorough research, improved diagnostics, and therapeutics development is needed to improve the outcomes for patients with gliomas. These advancements require a critical analysis of the microenvironment of gliomas to understand the pathophysiology at play. Ultrastructural analysis (UA) using high-resolution electron microscopy offers crucial insights into the cellular and subcellular characteristics of gliomas, revealing organelle abnormalities and confirming molecular alterations. In this review, we focus on the recent developments exploring the ultrastructural pathology of gliomas. UA shows a detailed microenvironment of gliomas to reveal the abnormal presence of lipids in the intra- and extracellular environment of gliomas accompanied by the disrupted structure of the cellular membranes and organelles. The disruptions were noted in the blood vessels of the tumors displaying intriguing details regarding the formation of neovasculature and the absence of typical contents of vessel walls like endothelial cells. A comparative survey of the mitochondrial ultrastructure of gliomas with or without IDH1 mutations also reveals the role of UA in deciphering the impact of different mutations on the microenvironment level. Here, we also discuss the major implications of these findings regarding the research direction, diagnostic modalities, and therapeutic options.

CNSC-19. ISCHEMIC STROKE DRIVES GLIOMA PROGRESSION BY REMODELING TUMOR-ASSOCIATED ASTROCYTES

Abstract Ischemic stroke and glioma are two deadly neurological disorders worldwide. Recent epidemiological studies reveal an increased risk of brain tumors, notably glioma, in patients with a history of stroke, resulting in a 10% co-occurrence. Comparative transcriptome analyses of brain samples collected from patients with common neurological disorders show that glioma is particularly enriched with stroke-associated gene signatures, especially in pathways related to their shared pathological features. These findings suggest a stroke-glioma interrelationship; however, it remains unknown how stroke alters the brain microenvironment to drive tumor progression. Here, we induced photothrombotic stroke in three mouse glioma models recapitulating multiple aspects of human glioma. Across all glioma models, we consistently observed that stroke increases tumor proliferation and infiltration towards stroke-affected regions, accompanied by reduced survival compared to sham control groups. Further analyses using single-cell RNA sequencing and spatial transcriptomic profiling in our stroke-glioma models reveal pronounced stroke-induced glioma microenvironment changes, among which tumor-associated astrocytes (TAAs) are most dramatically remodeled. Remarkably, we identified a unique stroke-induced astrocyte (SAA) at the tumor leading edge of the stroke-glioma group, characterized by diminished physiological astrocyte signatures and increased chromosomal and metabolic reprogramming. Among the top differentially expressed genes in SAA compared with TAA, the sodium-bicarbonate cotransporter Slc4a4 is interesting due to its enriched expression in TAA in both human and mouse gliomas, and it is significantly down-regulated at the infiltrating tumor edge. Consequently, we examine whether restoring astrocytic Slc4a4 expression rescues stroke-induced glioma progression. We found that astrocytic Slc4a4 overexpression reduces glioma growth and infiltration, accompanied by extended astroglial scar formation. Collectively, our study demonstrates that a subset of TAAs induced by stroke exacerbates glioma pathology, highlighting an indispensable astrocytic contribution to the stroke-glioma interaction.

FGL2172-220 peptides improve the antitumor effect of HCMV-IE1mut vaccine against glioblastoma by modulating immunosuppressive cells in the tumor microenvironment

ABSTRACT Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor characterized by poor prognosis and lack of effective treatments. In recent years, peptide vaccines that use sequences based on tumor-specific or tumor-associated antigens to activate immune responses against tumor cells have emerged as a new therapeutic strategy. In this study, we developed a novel therapeutic polypeptide vaccine targeting the tumor-associated antigen Fibrinogen-Like Protein 2 (FGL2), whose dominant epitope peptide was tandemly linked to the C-terminus of HCMV-IE1mut via a linker. We used this vaccine to compare the therapeutic efficacy of HCMV-IE1mut alone versus HCMV-IE1mut-FGL2172-220 and investigate the potential mechanism of action of HCMV-IE1mut-FGL2172-220 in glioma treatment. An in situ GBM model (GL261-IE1-luc cells) was used to determine the efficacy of the vaccine. Treatment with HCMV-IE1mut-FGL2172-220 exerted antitumor effects and extended the survival of the GL261 animal model. We observed reduced proportions of microglia, regulatory T cells (Treg), and myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME) by immunofluorescence. Flow cytometry showed that compared to HCMV-IE1mut alone, treatment with HCMV-IE1mut-FGL2172-220 increased the proportion of CD8+ T cells and tissue-resident memory T cells (TRM). ELISA analysis showed that it improved the secretion of tumor-specific IFN-γ and TNF-α by these cells and downregulated the expression of IL-6 and IL-10. Our study demonstrates that the long-peptide FGL2172-220 improves the antitumor efficacy of HCMV-IE1mut, possibly by reshaping immune cells in the glioma microenvironment. These findings lay the groundwork for the development of therapeutic antigenic peptide vaccines to improve antitumor effects for cancer.

Single-cell transcriptomics reveals IRF7 regulation of the tumor microenvironment in isocitrate dehydrogenase wild-type glioma.

Mutations in isocitrate dehydrogenase (IDH) are important markers of glioma prognosis. However, few studies have examined the gene expression regulatory network (GRN) in IDH-mutant and wild-type gliomas. In this study, single-cell RNA sequencing and spatial transcriptome sequencing were used to analyze the GRN of cell subsets in patients with IDH-mutant and wild-type gliomas. Through gene transcriptional regulation analysis, we identified the M4 module, whose transcription factor activity is highly expressed in IDH wild-type gliomas compared to IDH-mutants. Enrichment analysis revealed that these genes were predominantly expressed in microglia and macrophages, with significant enrichment in interferon-related signaling pathways. Interferon regulatory factor 7 (IRF7), a transcription factor within this pathway, showed the highest percentage of enrichment and was primarily localized in the core region of wild-type IDH tumors. A machine-learning prognostic model identified novel subgroups within the wild-type IDH population. Additionally, IRF7 was shown to promote the proliferation and migration of T98G and U251 cells in vitro, and its knockdown affected glioma cell proliferation in vivo. This study systematically established the regulatory mechanism of IDH transcriptional activity in gliomas at the single-cell level and drew a corresponding cell map. The study presents a transcriptional regulatory activity map for IDH wild-type gliomas, involving single-cell RNA sequencing and spatial transcriptomics to identify gene regulatory networks, machine learning models for IDH subtyping, and experimental validation, highlighting the role of IRF7 in glioma progression.

Glioma immune microenvironment composition calculator (GIMiCC): a method of estimating the proportions of eighteen cell types from DNA methylation microarray data

A scalable platform for cell typing in the glioma microenvironment can improve tumor subtyping and immune landscape detection as successful immunotherapy strategies continue to be sought and evaluated. DNA methylation (DNAm) biomarkers for molecular classification of tumor subtypes have been developed for clinical use. However, tools that predict the cellular landscape of the tumor are not well-defined or readily available. We developed the Glioma Immune Microenvironment Composition Calculator (GIMiCC), an approach for deconvolution of cell types in gliomas using DNAm data. Using data from 17 isolated cell types, we describe the derivation of the deconvolution libraries in the biological context of selected genomic regions and validate deconvolution results using independent datasets. We utilize GIMiCC to illustrate that DNAm-based estimates of immune composition are clinically relevant and scalable for potential clinical implementation. In addition, we utilize GIMiCC to identify composition-independent DNAm alterations that are associated with high immune infiltration. Our future work aims to optimize GIMiCC and advance the clinical evaluation of glioma.

Integrated analysis of bulk and single-cell RNA sequencing reveals the impact of nicotinamide and tryptophan metabolism on glioma prognosis and immunotherapy sensitivity

BackgroundNicotinamide and tryptophan metabolism play important roles in regulating tumor synthesis metabolism and signal transduction functions. However, their comprehensive impact on the prognosis and the tumor immune microenvironment of glioma is still unclear. The purpose of this study was to investigate the association of nicotinamide and tryptophan metabolism with prognosis and immune status of gliomas and to develop relevant models for predicting prognosis and sensitivity to immunotherapy in gliomas.MethodsBulk and single-cell transcriptome data from TCGA, CGGA and GSE159416 were obtained for this study. Gliomas were classified based on nicotinamide and tryptophan metabolism, and PPI network associated with differentially expressed genes was established. The core genes were identified and the risk model was established by machine learning techniques, including univariate Cox regression and LASSO regression. Then the risk model was validated with data from the CGGA. Finally, the effects of genes in the risk model on the biological behavior of gliomas were verified by in vitro experiments.ResultsThe high nicotinamide and tryptophan metabolism is associated with poor prognosis and high levels of immune cell infiltration in glioma. Seven of the core genes related to nicotinamide and tryptophan metabolism were used to construct a risk model, and the model has good predictive ability for prognosis, immune microenvironment, and response to immune checkpoint therapy of glioma. We also confirmed that high expression of TGFBI can lead to an increased level of migration, invasion, and EMT of glioma cells, and the aforementioned effect of TGFBI can be reduced by FAK inhibitor PF-573,228.ConclusionsOur study evaluated the effects of nicotinamide and tryptophan metabolism on the prognosis and tumor immune microenvironment of glioma, which can help predict the prognosis and sensitivity to immunotherapy of glioma.

Role of T Lymphocytes in Glioma Immune Microenvironment: Two Sides of a Coin.

Glioma is known for its immunosuppressive microenvironment, which makes it challenging to target through immunotherapies. Immune cells like macrophages, microglia, myeloid-derived suppressor cells, and T lymphocytes are known to infiltrate the glioma tumor microenvironment and regulate immune response distinctively. Among the variety of immune cells, T lymphocytes have highly complex and multifaceted roles in the glioma immune landscape. T lymphocytes, which include CD4+ helper and CD8+ cytotoxic T cells, are known for their pivotal roles in anti-tumor responses. However, these cells may behave differently in the highly dynamic glioma microenvironment, for example, via an immune invasion mechanism enforced by tumor cells. Therefore, T lymphocytes play dual roles in glioma immunity, firstly by their anti-tumor responses, and secondly by exploiting gliomas to promote immune invasion. As an immunosuppression strategy, glioma induces T-cell exhaustion and suppression of effector T cells by regulatory T cells (Tregs) or by altering their signaling pathways. Further, the expression of immune checkpoint inhibitors on the glioma cell surface leads to T cell anergy and dysfunction. Overall, this dynamic interplay between T lymphocytes and glioma is crucial for designing more effective immunotherapies. The current review provides detailed knowledge on the roles of T lymphocytes in the glioma immune microenvironment and helps to explore novel therapeutic approaches to reinvigorate T lymphocytes.

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow.

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

Pyrimidine metabolism reshapes immune microenvironment and implies poor prognosis in glioma.

The metabolic environment of glioma is extremely complex. Pyrimidine metabolism can significantly influence malignant progression of multiple kinds of cancer cells. In this study, we intend to explore the relationship between pyrimidine metabolism and malignant progression of glioma. We analyzed two glioma RNA-sequencing databases to construct a pyrimidine metabolism-related risk signature. An individualized prognosis prediction model based on this risk signature was established. Functional analysis and in vitro experiments were conducted to assess the role of pyrimidine metabolism in the tumor-immune microenvironment and malignant progress of gliomas. The high-risk group, as predicted by the pyrimidine metabolism-related risk score, showed a tendency toward more malignant entities and poorer survival outcomes. Functional analysis revealed that pyrimidine metabolism significantly regulates the tumor-immune microenvironment. In vitro experiments confirmed that targeting pyrimidine metabolism-related genes can inhibit malignancy of glioma cell. In short, the pyrimidine metabolism-related signature we established could serve as an independent prognostic biomarker in diffuse gliomas and has a close association with regulation of the tumor-immune microenvironment.

Hypoxic glioma-derived exosomal miR-25-3p promotes macrophage M2 polarization by activating the PI3K-AKT-mTOR signaling pathway

BackgroundExosomes (EXO) play crucial roles in intercellular communication and glioma microenvironment modulation. Tumor-associated macrophages are more likely to become M2-like type macrophages in the immunosuppressive microenvironment. Here, we aimed to investigate the effects and molecular mechanisms of hypoxic glioma-derived exosomes mediated M2-like macrophage polarization.MethodsHighly expressed miRNAs in exosomes derived from glioma cells cultured under hypoxia condition compared to normoxic condition were identified through microRNA sequencing. The polarization status of macrophages was determined using qRT-PCR, Western blotting, flow cytometry, and immunohistochemistry. By using RNA-seq, we aimed to identify the downstream target genes regulated by miR-25-3p in macrophages and investigate the mechanistic pathways through which it exerts its effects. The proliferation and migration capabilities of glioma cells were assessed through EdU, Transwell assays, and in vivo experiments.ResultsWe found that miR-25-3p was upregulated in the exosomes derived from hypoxic glioma cells and can be transferred to the macrophage. In macrophages, miR-25-3p downregulates the expression of PHLPP2, thereby activating the PI3K-AKT-mTOR signaling pathway, ultimately leading to macrophage M2 polarization. As part of a feedback loop, M2-polarized macrophages can, in turn, promote malignant glioma progression.ConclusionOur study reveals that miR-25-3p from hypoxic glioma cells is delivered to macrophages via exosomes as a mediator, promoting M2 polarization of macrophages through the miR-25-3p/PHLPP2/PI3K-AKT signaling pathway. This study suggests that targeted interventions to modulate miR-25-3p expression, transmission, or inhibition of PI3K-AKT pathway activation can disrupt the immune-suppressive microenvironment, providing a novel approach for immunotherapy in gliomas.Graphical

Complement factor H is an ICOS ligand modulating Tregs in the glioma microenvironment.

The survival rate of glioma patients has not significantly increased in recent years despite aggressive treatment and advances in immunotherapy. The limited response to treatments is partially attributed to the immunosuppressive tumor microenvironment, where regulatory T cells (Tregs) play a pivotal role in immunological tolerance. In this study, we investigated the impact of complement factor H (FH) on Tregs within the glioma microenvironment and found that FH is an ICOS ligand. The binding of FH to this immune checkpoint molecule promoted the survival and function of Tregs and induced the secretion of TGF-beta (TGF-β) and IL-10, while also suppressing T-cell proliferation. We further demonstrated that cancer cells in human and mouse gliomas directly produce FH. Database investigations revealed that upregulation of FH expression was associated with the presence of Tregs and correlated with worse prognosis for glioma patients. We confirmed the effect of FH on glioma development in a mouse model, where FH knockdown was associated with decrease in number of ICOS+ Tregs and demonstrated a tendency of prolonged survival (p=0.064). Since the accumulation of Tregs represents a promising prognostic and therapeutic target, evaluating FH expression should be considered when assessing the effectiveness of and resistance to immunotherapies against glioma.

Integrative multi-omics analysis unveils the connection between transcriptomic characteristics associated with mitochondria and the tumor immune microenvironment in lower-grade gliomas

Lower-grade gliomas (LGGs) exhibit diverse clinical behaviors and varying immune infiltration levels. Mitochondria have been implicated in numerous cancer pathogenesis and development, including LGGs. However, the precise biological functions of mitochondrial genes in shaping the immune landscape and the prognostic significance of LGGs remain elusive. Utilizing the Mito-Carta3.0 database, we curated a total of 1136 genes implicated in mitochondrial functions. By leveraging the expression profiles of 1136 genes related to mitochondria, we successfully categorized LGGs into four distinctive mitochondria-related transcriptome (MRT) subtypes. Our thorough analysis conclusively demonstrated that these subtypes exhibited marked disparities. To enable a personalized and integrated evaluation of LGG patients, we developed a prognostic signature known as MRT-related prognostic signature (MTRS). MTRS demonstrated correlation with mitochondria-related transcriptome (MRT) subtypes, allowing the assessment of patients’ prognosis and immune microenvironment. We conducted a detailed exploration of the single-cell distribution of MTRS in lower-grade gliomas and verified the core genes of MTRS within the spatial transcriptome of these tumors. Furthermore, our study pinpointed MGME1 as the pivotal gene in the model, functioning as an oncogene that exerts influence on cell proliferation and migration capabilities. Our research highlights the importance of mitochondrial transcriptomic features in LGGs, offering paths for tailored therapies.