Immune Microenvironment In Glioblastoma Research Articles

Research progress on the role of PTEN deletion or mutation in the immune microenvironment of glioblastoma.

Recent advances in immunotherapy represent a breakthrough in solid tumor treatment but the existing data indicate that immunotherapy is not effective in improving the survival time of patients with glioblastoma. The tumor microenvironment (TME) exerts a series of inhibitory effects on immune effector cells, which limits the clinical application of immunotherapy. Growing evidence shows that phosphate and tension homology deleted on chromosome ten (PTEN) plays an essential role in TME immunosuppression of glioblastoma. Emerging evidence also indicates that targeting PTEN can improve the anti-tumor immunity in TME and enhance the immunotherapy effect, highlighting the potential of PTEN as a promising therapeutic target. This review summarizes the function and specific upstream and downstream targets of PTEN-associated immune cells in glioblastoma TME, providing potential drug targets and therapeutic options for glioblastoma.

Association Between Diverse Cell Death Patterns Related Gene Signature and Prognosis, Drug Sensitivity, and Immune Microenvironment in Glioblastoma

Glioblastoma (GBM) is the most invasive type of glioma and is difficult to treat. Diverse programmed cell death (PCD) patterns have a significant association with tumor initiation and progression. A novel prognostic model based on PCD genes may serve as an effective tool to predict the prognosis of GBM. The study incorporated 11 PCD patterns, namely apoptosis, necroptosis, pyroptosis, ferroptosis, cuproptosis, entotic cell death, netotic cell death, parthanatos, lysosome-dependent cell death, autophagy-dependent cell death, alkaliptosis, and oxeiptosis, to develop the model. To construct and validate the model, both bulk and single-cell transcriptome data, along with corresponding clinical data from GBM cases, were obtained from the TCGA-GBM, REMBRANDT, CGGA, and GSE162631 datasets. A cell death-related signature containing 14 genes was constructed with the TCGA-GBM cohort and validated in the REMBRANDT and CGGA datasets. GBM patients with a higher cell death index (CDI) were significantly associated with poorer survival outcomes. Two separate clusters associated with clinical outcomes emerged from unsupervised analysis. A multivariate Cox regression analysis was conducted to examine the association of CDI with clinical characteristics, and a prognostic nomogram was developed. Drug sensitivity analysis revealed high-CDI GBM patients might be resistant to carmustine while sensitive to 5-fluorouracil. Less abundance of natural killer cells was found in GBM cases with high CDI and bulk transcriptome data. A cell death-related prognostic model that could predict the prognosis of GBM patients with good performance was established, which could discriminate between the prognosis and drug sensitivity of GBM.

Time-resolved single-cell transcriptomics defines immune trajectories in glioblastoma

Deciphering the cell-state transitions underlying immune adaptation across time is fundamental for advancing biology. Empirical invivo genomic technologies that capture cellular dynamics are currently lacking. We present Zman-seq, a single-cell technology recording transcriptomic dynamics across time by introducing time stamps into circulating immune cells, tracking them in tissues for days. Applying Zman-seq resolved cell-state and molecular trajectories of the dysfunctional immune microenvironment in glioblastoma. Within 24hours of tumor infiltration, cytotoxic natural killer cells transitioned to a dysfunctional program regulated by TGFB1 signaling. Infiltrating monocytes differentiated into immunosuppressive macrophages, characterized by the upregulation of suppressive myeloid checkpoints Trem2, Il18bp, and Arg1, over 36 to 48hours. Treatment with an antagonistic anti-TREM2 antibody reshaped the tumor microenvironment by redirecting the monocyte trajectory toward pro-inflammatory macrophages. Zman-seq is a broadly applicable technology, enabling empirical measurements of differentiation trajectories, which can enhance the development of more efficacious immunotherapies.

Identification of key genes for glioblastoma microenvironment

Glioblastoma(GBM), as a common kind of brain cancer, has a poor prognosis and a recurrence rate of nearly 100 percent. Tumor immune microenvironment is a crucial factor for glioblastoma progression. Some prognostically relevant genes in the immunologic microenvironment of glioblastoma remain to be further explored. This article aims to ascertain genes involved in the immune microenvironment of glioblastoma, and this topic has not been identified before. R version 4.2.1 and the appropriate packages were used for all analyses in this paper. Relevant transcriptomic data for GBMs as well as clinical data were extracted from immune score data in the TCGA database, and stromal score data were acquired from this URL (http://www.cbioportal.org/). Differentially expressed genes were obtained from immune score data and Stromal score data. After intersections of all these genes, the PPI interaction network was applied. According to functional analysis, these genes are intensively distributed in immune-related pathways. Eventually LncRNAs co-expressing with immune-related genes were positively identified. We characterized genes related to the GBM tumor microenvironment not previously identified in similar studies, as well as some lncRNAs that co-expressed with these immune genes

IMMU-13. DEVELOPMENT OF ADAPTIVE IMMUNE RESISTANCE IN A MURINE GLIOMA MODEL TREATED WITH DENDRITIC CELL VACCINATION AND PD-1 CHECKPOINT BLOCKADE

Abstract Immunotherapies such as immune checkpoint inhibitors and active vaccination have shown great potential in unleashing antitumor immunity for cancer treatment. However, their effectiveness in treating glioblastoma (GBM) is limited. In a preclinical glioma model, administration of both autologous tumor lysate-pulsed dendritic cell vaccination (ATL-DC) and anti-PD-1 checkpoint blockade (aPD1) significantly increased survival and enhanced intratumoral T cell activation. We hypothesize that the combination of these two treatments will substantially increase the abundance and activity of tumor-reactive cytotoxic T cells. As such, we recently started a phase I trial administering ATL-DC and neoadjuvant aPD1 to recurrent GBM patients (NCT ID: NCT04201873). To understand the local antitumor immune response in greater depth, we performed single-cell RNA sequencing analysis of the CD45+ immune compartment of explanted gliomas from twelve mice, four of which were treated with ATL-DC and aPD1 while the other eight received no treatment. We found that the combination therapy increased tumor-infiltrating T cells, monocytes and DCs. We also observed the expansion and differentiation of CD8+ effector T cells under the effect of combination therapy. These T cells exhibited a differentiation trajectory from a Tcf7+Il2ra+ early activated phenotype to a terminally exhausted state marked by high expression of Pdcd1, Tox and Tigit. Interestingly, combination therapy specifically increased a macrophage subset that overexpressed Ch25h, an interferon-stimulated gene encoding for the oxysterol lipid 25-hydroxycholesterol (25-HC). Preliminary results from in vitro studies demonstrated that 25-HC restricted T cell proliferation and tumor cell cytolysis, suggesting its immunosuppressive function. In summary, our study reveals the impact of ATL-DC + aPD1 combination therapy on the immune microenvironment of GBM and a potential maladaptive mechanism mediated by 25-HC. Future studies will seek to exploit these targets therapeutically to develop more effective GBM treatment regimens.

CNSC-19. HYPOXIA-INDUCED NEURONAL ACTIVITY IN GLIOMA PATIENTS POLARIZES MICROGLIA BY POTENTIATING RNA M6A DEMETHYLATION

Abstract Neuronal activity in the brain has been reported to promote the malignant progression of glioma cells via nonsynaptic paracrine and electrical synaptic integration mechanisms. However, the interaction between neuronal activity and the immune microenvironment in glioblastoma (GBM) remains largely unclear. In this study, by applying chemogenetic techniques, we enhanced and inhibited neuronal activity in vitro and in a mouse model to study how neuronal activity regulates microglial polarization and affects GBM progression. Here, we demonstrate that hypoxia induces glioma stem cells (GSCs) to produce glutamate, which activates local neurons. The hypoxia-induced neuronal activity promotes GBM progression by facilitating microglial M2 polarization. We found that when microglia were co-cultured with activated neurons, the level of RNA m6A methylation in microglia was impaired, which subsequently induced microglial M2 polarization. In the mouse model, cortical neuronal activation promoted regional microglial M2 polarization while cortical neuronal inhibition decreased regional microglial M2 polarization in GBM xenografts. Together, these findings indicate that hypoxic GSC-induced neuronal activation promotes GBM progression by polarizing microglia via decreasing their m6A methylation levels.

Warburg effect-related risk scoring model to assess clinical significance and immunity characteristics of glioblastoma.

Glioblastoma (GBM), the most common primary malignant brain tumor, has a poor prognosis, with a median survival of only 14.6 months. The Warburg effect is an abnormal energy metabolism, which is the main cause of the acidic tumor microenvironment. This study explored the role of the Warburg effect in the prognosis and immune microenvironment of GBM. A prognostic risk score model of Warburg effect-related genes (Warburg effect signature) was constructed using GBM cohort data from The Cancer Genome Atlas. Cox analysis was performed to identify independent prognostic factors. Next, the nomogram was built to predict the prognosis for GBM patients. Finally, the drug sensitivity analysis was utilized to find the drugs that specifically target Warburg effect-related genes. Age, radiotherapy, chemotherapy, and WRGs score were confirmed as independent prognostic factors for GBM by Cox analyses. The C-index (0.633 for the training set and 0.696 for the validation set) and area under curve (>0.7) indicated that the nomogram exhibited excellent performance. The calibration curve also indicates excellent consistency of the nomogram between predictions and actual observations. In addition, immune microenvironment analysis revealed that patients with high WRGs scores had high immunosuppressive scores, a high abundance of immunosuppressive cells, and a low response to immunotherapy. The Cell Counting Kit-8 assays showed that the drugs targeting Warburg effect-related genes could inhibit the GBM cells growth invitro. Our research showed that the Warburg effect is connected with the prognosis and immune microenvironment of GBM. Therefore, targeting Warburg effect-related genes may provide novel therapeutic options.

Hypoxia-Induced Neuronal Activity in Glioma Patients Polarizes Microglia by Potentiating RNA m6A Demethylation.

Neuronal activity in the brain has been reported to promote the malignant progression of glioma cells via nonsynaptic paracrine and electrical synaptic integration mechanisms. However, the interaction between neuronal activity and the immune microenvironment in glioblastoma (GBM) remains largely unclear. By applying chemogenetic techniques, we enhanced and inhibited neuronal activity in vitro and in a mouse model to study how neuronal activity regulates microglial polarization and affects GBM progression. We demonstrate that hypoxia drove glioma stem cells (GSC) to produce higher levels of glutamate, which activated local neurons. Neuronal activity promoted GBM progression by facilitating microglial M2 polarization through enriching miR-200c-3p in neuron-derived exosomes, which decreased the expression of the m6A writer zinc finger CCCH-type containing 13 (ZC3H13) in microglia, impairing methylation of dual specificity phosphatase 9 (DUSP9) mRNA. Downregulation of DUSP9 promoted ERK pathway activation, which subsequently induced microglial M2 polarization. In the mouse model, cortical neuronal activation promoted microglial M2 polarization whereas cortical neuronal inhibition decreased microglial M2 polarization in GBM xenografts. miR-200c-3p knockdown in cortical neurons impaired microglial M2 polarization and GBM xenograft growth, even when cortical neurons were activated. Treatment with the anti-seizure medication levetiracetam impaired neuronal activation and subsequently reduced neuron-mediated microglial M2 polarization. These findings indicated that hypoxic GSC-induced neuron activation promotes GBM progression by polarizing microglia via the exosomal miR-200c-3p/ZC3H13/DUSP9/p-ERK pathway. Levetiracetam, an antiepileptic drug, blocks the abnormal activation of neurons in GBM and impairs activity-dependent GBM progression. See related commentary by Cui et al., p. 1073.

Development and validation of an immunogenic cell death-related prognostic model, and landscape of the tumor immune microenvironment in glioblastoma

Development and validation of an immunogenic cell death-related prognostic model, and landscape of the tumor immune microenvironment in glioblastoma

Histone acetylation gene-based biomarkers as novel markers of the immune microenvironment in glioblastoma.

Glioblastoma (GBM) is a primary malignant tumour with high intracranial morbidity, high malignancy and poor prognosis. Abnormal changes in histone acetylation are closely related to the occurrence and development of cancer. However, there is still a lack of systematic research on histone acetylation in GBM. Whole-transcriptome sequencing data and clinical data of GBM patients were obtained through the TCGA database. Single-cell RNA-sequencing (scRNA-seq) data from GBM patients were obtained from GSE146711 in the Gene Expression Omnibus database. Cell descending fractionation was first performed for scRNA-seq on GBM. The CellChat and PROGENy scores explore the impact of the histone acetylation pathway in GBM on intercellular chat and tumour pathways. The AddModuleScore function evaluates the enrichment score of histone acetylation in cells and divides them into high-histone acetylation and low-histone acetylation groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on the differential genes between different histone acetylation states, and the biological processes and pathways that may be affected by histone acetylation were evaluated. Based on this, a prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) analysis, and survival analysis was performed to evaluate its prognostic performance. Finally, we also analysed the main effects of the constructed histone acetylation-related model on GBM immune infiltration by multiple methods, and analysed the main mutation data of its different subgroups. GBM samples mainly include seven large cell populations: oligodendrocyte precursor cells (OPCs), myeloid, neoplastic, oligodendrocytes, astrocytes, vascular and neurons. Cellchat and ProgenY scores revealed that in GBM tumours, histone acetylation interacts closely with multiple immune cells and tumour pathways. GO and KEGG analyses revealed the main impact proteins and pathway correlates of histone acetylation. Five histone acetylation genes were screened using LASSO analysis and a prognostic model was constructed. The results revealed that prognostic models were significant in the prognostic stratification of patients in both the training and validation groups of GBM patients. Immune infiltration analysis revealed that the mechanism of histone acetylation in GBM may be related to the immune infiltration of multiple effector immune cells. Our histone acetylation-based biomarkers are closely associated with immune microenvironmental infiltration and functional mutations in multiple tumour pathways in GBM. This suggests that histone acetylation may reveal microscopic alterations in the tumour microenvironment, and may provide potential evidence and a research basis for the development of novel therapeutic targets for GBM. On this basis, a novel perspective on the spatial biology and immunological understanding of GBM is provided.

Abstract LB097: Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma

Abstract Introduction: We have completed two CAR T cell clinical trials for glioblastoma (GBM) and have identified several key challenges to therapeutic efficacy, including the inherently heterogenous genomic landscape and the immunosuppressive tumor microenvironment (TME) found in GBM. Our previous study showed that EGFR variant III (EGFRvIII)-targeting monovalent CAR T cells reduced target-positive tumor cell populations, but tumor recurrence resulted from target-negative tumor cells, highlighting the limitation of single-target approaches in heterogenous tumors. With regards to the highly immunosuppressive TME in GBM, we found that transforming growth factor-β (TGFβ) was present in the GBM TME as a major driver of suppression of the anti-GBM response in clinical samples. TGFb is consistently highly expressed in both GBM tumor cell lines and patient tumor tissues. Methods: We used two parallel scFv constructs, independently targeting both IL13Rα2 and EGFRvIII, in combination with a truncated dominant negative (dn) TGFβ receptor II. This trivalent construct was designed to explore possible additive effects in both in vitro and in vivo GBM model systems to limit tumor escape and overcome the immunosuppressive GBM TME. The CART-EGFR-IL13Rα2-dnTGFb construct broadened the targeted tumor cell repertoire, blocked TGFβ signaling, and served as a sink for free TGFβ in the GBM TME to overcome the suppressive function of TGFβ. Results: The tri-modular CAR T construct had an enhanced proliferative response when compared with the CART-EGFR-IL13Rα2 construct, in vitro. In co-culture assays, this construct led to reduced PD-1 expression and increased central memory phenotype, when compared to the bicistronic CAR T construct, which suggested a lower fraction of exhausted T cells. Tri-modular CAR T cells blocked the suppressive pSmad2/3 signaling pathway, leading to the increased tumor killing activity in co-culture experiments with both adherent and suspension GBM cell lines. In an immunodeficient mouse model, tri-modular CAR T cells eradicated tumor cells efficiently and mice had a longer median survival when compared those treated with the bicistronic CART-EGFR-IL13Rα2 cells, lacking the dnTGFb receptor II. Conclusion: Overcoming the adaptive changes in the local TME and addressing antigen heterogeneity will be required to improve the clinical efficacy of CAR T-directed strategies. Our combination work showed that bicistronic CART constructs cooperate with truncated TGFβ receptor II efficiently. In summary, the dominant-negative TGFβ RII CART-EGFR-IL13Rα2 structure is a promising strategy to address the clinical challenges of antigenic heterogeneity and the immunosuppressive TME in GBM we have observed in our two GBM CART cell trials at UPenn. Citation Format: Nannan Li, Jesse Rodriguez, Zev Binder, Donald O’Rourke. Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB097.

The PTEN-associated immune prognostic signature reveals the landscape of the tumor microenvironment in glioblastoma

Glioblastoma (GBM) is a common brain tumor with a complex and diverse tumor microenvironment (TME). As PTEN mutation is the most common mutation in GBM, we aimed to investigate how PTEN mutation regulates the immune response in GBM TME and thus affects the prognosis of GBM patients. In this study, we conducted a comprehensive analysis of multiple levels of data, including whole-exome sequencing (WES), transcriptome RNA sequencing, patient survival and immune signatures, to study the relationship between PTEN mutation and TME in GBM. We developed an immune-related prognostic signature (IPS) based on the PTEN-associated immune-related genes (IRGs), and the IPS exhibited a powerful prognosis prediction capacity in different GBM cohorts. A scoring nomogram based on the IPS was also established for clinical application. In addition, the correlations of the IPS with tumor immune cell infiltration and immune checkpoints were systematically analyzed. This study illustrates the influence of PTEN mutation on the immune microenvironment of GBM. Our IPS, which is sensitive to PTEN mutation status, can enhance the prognosis prediction ability for GBM patients and provides potential targets for immunotherapy.

Integrative analysis of single-cell transcriptomics reveals age-associated immune landscape of glioblastoma.

Glioblastoma (GBM) is the most malignant tumor in center nervous system. Clinical statistics revealed that senior GBM patients had a worse overall survival (OS) comparing with that of patients in other ages, which is mainly related with tumor microenvironment including tumor-associated immune cells in particular. However, the immune heterogeneity and age-related prognosis in GBM are under studied. Here we developed a machine learning-based method to integrate public large-scale single-cell RNA sequencing (scRNA-seq) datasets to establish a comprehensive atlas of immune cells infiltrating in cross-age GBM. We found that the compositions of the immune cells are remarkably different across ages. Brain-resident microglia constitute the majority of glioblastoma-associated macrophages (GAMs) in patients, whereas dramatic elevation of extracranial monocyte-derived macrophages (MDMs) is observed in GAMs of senior patients, which contributes to the worse prognosis of aged patients. Further analysis suggests that the increased MDMs arisen from excessive recruitment and proliferation of peripheral monocytes not only lead to the T cell function inhibition in GBM, but also stimulate tumor cells proliferation via VEGFA secretion. In summary, our work provides new cues for the correlational relationship between the immune microenvironment of GBM and aging, which might be insightful for precise and effective therapeutic interventions for senior GBM patients.

TMET-11. QUINOLINATE PROMOTES ALTERNATIVELY ACTIVATED MACROPHAGE-INDUCED IMMUNE TOLERANCE IN GLIOBLASTOMA THROUGH THE NMDA/PPARG SIGNALING AXIS

Abstract There has been considerable interest in understanding the biological consequence and therapeutic implications of aberrant tryptophan metabolism in brain tumors and neurodegenerative diseases. An overwhelming majority of this work has focused on the first-step of tryptophan metabolism (kynurenine); however, this has yet to result in clinical application. Using global metabolomic profiling on >100 patient-derived brain tumors, we identified a 64-fold accumulation of quinolinate (QA), a downstream metabolic intermediate of the tryptophan pathway, in glioblastoma when compared to low-grade glioma. As several metabolites in the tryptophan pathway have been implicated in immune modulation, we sought to determine the impact of QA on the immune microenvironment. We identified the capacity of QA to strongly skew macrophage polarization towards the “pro-tumorigenic” M2-phenotype with suppressive properties, which recent studies suggest play a dominant role in the immune microenvironment in glioblastoma. Intriguingly, QA conferred an “M2-like” phenotype to M1 macrophages and microglia, attenuating their phagocytosis efficiency. We went on to systematically delineate a novel mechanism of macrophage polarization through QA-induced NMDA receptor activation and Foxo1/PPARg signaling. We then determined that tumor cells and host macrophages/microglia work in concert to complete both upstream and downstream metabolism of tryptophan, respectively, resulting in the accumulation of QA. We discovered a very strong positive feedback loop involving the expression of kynureninase (KYNU) in macrophages, an enzyme involved in the downstream metabolism of tryptophan and QA production, making this a lead candidate for targeting this pathway. As this represents a novel target and agents designed to inhibit this enzyme are not commercially available, we generated a Kynu-/- knockout mouse model. Intriguingly, consistent with in vitro data, tumors grown in Kynu-/- mice had a ~50% reduction in M2 macrophages, increased immune activation, decreased growth, and improved overall survival in two orthotopic glioblastoma models, supporting the therapeutic potential of targeting this pathway.

Bacteria-mediated metformin-loaded peptide hydrogel reprograms the tumor immune microenvironment in glioblastoma

The tumor immune microenvironment (TIME) is one of the significant hallmarks of cancer and has the important role of largely determining the malignancy level of tumors. As an approach to break through this bottleneck of tumor treatment methods, the TIME can be reprogrammed by certain nanomaterials. Here, we coated C-novyi-spores with melittin-RADA32 nanofiber hybrid peptide and loaded the immunomodulator metformin to obtain MRM-coated spores as a powerful antitumor nanodrug against glioblastoma (GBM), which is based on the activation of the TIME. MRM-coated spores exhibit extended-release profiles and an enhanced killing effect on GBM both in vitro and in vivo. Furthermore, MRM-coated spores can educate the innate and adaptive immune system by inducing sustainable CD8+ T cell responses, promoting M1 macrophage polarization, and regulating the expression of HIF1-α, PDL1, and CXCL9 in TIME. In intracranial applications, MRM-coated spores showed excellent biosafety and a strong therapeutic effect. In summary, peptide hydrogels provide a promising strategy in which advantages of different treatment methods can be incorporated to synthesize potent antitumor drugs with mild side effects from bacteria-mediated nanomaterials.

Magnetic Resonance Imaging Correlates of Immune Microenvironment in Glioblastoma.

BackgroundGlioblastoma (GBM) is the most commonly occurring primary malignant brain tumor, and it carries a dismal prognosis. Focusing on the tumor microenvironment may provide new insights into pathogenesis, but no clinical tools are available to do this. We hypothesized that the infiltration of different leukocyte populations in the tumoral and peritumoral brain tissues may be measured by magnetic resonance imaging (MRI).MethodsPre-operative MRI was combined with immune phenotyping of intraoperative tumor tissue based on flow cytometry of myeloid cell populations that are associated with immune suppression, namely, microglia and bone marrow-derived macrophages (BMDM). These cell populations were measured from the central and marginal areas of the lesion identified intraoperatively with 5-aminolevulinic acid-guided surgery. MRI features (volume, mean and standard deviation of signal intensity, and fractality) were derived from all MR sequences (T1w, Gd+ T1w, T2w, FLAIR) and ADC MR maps and from different tumor areas (contrast- and non-contrast-enhancing tumor, necrosis, and edema). The principal components of MRI features were correlated with different myeloid cell populations by Pearson’s correlation.ResultsWe analyzed 126 samples from 62 GBM patients. The ratio between BMDM and microglia decreases significantly from the central core to the periphery. Several MRI-derived principal components were significantly correlated (p <0.05, r range: [−0.29, −0.41]) with the BMDM/microglia ratio collected in the central part of the tumor.ConclusionsWe report a significant correlation between structural MRI clinical imaging and the ratio of recruited vs. resident macrophages with different immunomodulatory activities. MRI features may represent a novel tool for investigating the microenvironment of GBM.

EXTH-08. STAT3 AT THE CROSSROADS OF INNATE IMMUNE ACTIVATION IN GLIOBLASTOMA

Abstract BACKGROUND Innate immune cells comprise the majority of the immune microenvironment in glioblastoma (GBM) tumors where they are chiefly thought to foster a hospitable environment for cancer cells by regulating immune suppression and driving resistance to immunotherapy. Of these, myeloid-derived suppressor cells (MDSCs) are regarded as one of the most potent contributors to immune suppression in GBM and thus have become a focus of targeted therapy. Signal transducer and activator of transcription 3 (STAT3) is a key phenotypic regulator of MDSCs. Therefore, we sought to examine if targeted STAT3 inhibition may augment the immunogenicity of these tumors. HYPOTHESIS: Targeted STAT3 inhibition reduces GBM tumor infiltration by MDSCs. METHODS Using syngeneic murine models of GBM, we performed pharmacological inhibition analyses using a specific small molecule inhibitor of STAT3, LLL12B. Circulating numbers of immune cells were assessed in tumor bearing animals with or without concomitant focal radiation. Treated tumors were examined for immune infiltrates, and additional phenotyping analyses were performed. Therapeutic responses to LLL12B alone and in combination with immune checkpoint blockade were evaluated. RESULTS STAT3 is activated in the bone marrow of tumor-bearing animals, preferentially by Gr-1 positive granulocytic myeloid cells. Increased circulating numbers of these cells were also detected. These observations were markedly enhanced in tumor-bearing animals following cranial irradiation. Therapeutic inhibition with LLL12B could mitigate these effects, indicating a dependency on STAT3. Within the tumor compartment, granulocytic myeloid cells that successfully infiltrated following treatment demonstrated a pro-inflammatory phenotype denoted by interferon-gamma expression. Improved survival was also observed following combination treatment with LLL12B and radiation or immune checkpoint blockade. CONCLUSIONS These findings advocate a critically important role for STAT3 in regulating granulocytic myeloid cell mobilization and trafficking to GBM tumors. It further illustrates the plasticity of these cells within these tumors, which may be useful in designing successful immunotherapeutic strategies.

Exploiting Radiation Therapy to Restore Immune Reactivity of Glioblastoma.

Glioblastoma (GBM) is among the most aggressive of brain tumors and confers a dismal prognosis despite advances in surgical technique, radiation delivery methods, chemotherapy, and tumor-treating fields. While immunotherapy (IT) has improved the care of several adult cancers with previously dismal prognoses, monotherapy with IT in GBM has shown minimal response in first recurrence. Recent discoveries in lymphatics and evaluation of blood brain barrier offer insight to improve the use of ITs and determine the best combinations of therapies, including radiation. We highlight important features of the tumor immune microenvironment in GBM and potential for combining radiation and immunotherapy to improve prognosis in this devastating disease.

Extrinsic factors associated with the response to immunotherapy in glioblastoma.

Glioblastoma (GBM) is a heterogeneous and lethal brain tumor. Despite the success of immune checkpoint inhibitors against various malignancies, GBM remains largely refractory to treatment. The immune microenvironment of GBM is highly immunosuppressive, which poses a major hurdle for the success of immunotherapy. Obviously, except for the GBM cells itself, there are also extrinsic reasons for the lack of efficacy of immunotherapy. Accumulated evidence indicates that factors other than GBM cells determine the efficacy of immunotherapy. In this review, we first described the unique immune microenvironment of the brain, which must be considered when using immunotherapy in patients with GBM. Second, we also described the mechanisms by which different immune and non-immune cells in the GBM microenvironment affect the efficacy of immunotherapy. Furthermore, the impact of standard therapies on the response to immunotherapy was delineated. Finally, we briefly discussed strategies for resolving these problems and improving the efficacy of immunotherapy.

Mitochondrial Dysfunction, Macrophage, and Microglia in Brain Cancer.

Glioblastoma (GBM) is the most common malignant brain cancer. Increasing evidence suggests that mitochondrial dysfunction plays a key role in GBM progression as mitochondria is essential in regulating cell metabolism, oxidative stress, and cell death. Meanwhile, the immune microenvironment in GBM is predominated by tumor-associated macrophages and microglia (TAM), which is a heterogenous population of myeloid cells that, in general, create an immunosuppressive milieu to support tumor growth. However, subsets of TAMs can be pro-inflammatory and thereby antitumor. Therapeutic strategies targeting TAMs are increasingly explored as novel treatment strategies for GBM. The connection between mitochondrial dysfunction and TAMs phenotype in the tumor microenvironment is unclear. This review aims to provide perspectives and discuss possible molecular mechanisms mediating the interplay between glioma mitochondrial dysfunction and TAMs phenotype in shaping tumor immune microenvironment.