Glioblastoma is among the most lethal human malignancies. Immune-based therapies have failed due to a strong immunosuppressive tumor microenvironment (TME)1. We uncovered that LAIR-12 expressed by tumor cells simultaneously drives TME fibrosis and inhibits migration of immune cells to brain tumors, thus achieving powerful immune exclusion3. We demonstrate that glioma-cell-specific LAIR-1 knockdown (KD), but not LAIR-1 KD from host cells, significantly extends survival in an immune-dependent manner. Glioma-cell-specific LAIR-1 signals through SHP2 to activate JNK -which on one hand sustains high levels of Lysyl Oxidase-Like 1 and collagen I to block immune cells' entry- and on the other hand suppresses STAT3 signaling. In the absence of LAIR-1, gliomas' collagen-dense ECM becomes disassembled and, through STAT3-driven upregulation of ADAM10 and ADAM17, promotes release of CXCL16, and recruitment of NK cells and cytotoxic T cells. Combining LAIR-1 KD with immune-stimulatory gene therapy4 achieved 100% long-term survival with durable immunological memory in immunocompetent mice. Pharmacological SHP2 inhibition in LAIR-1 WT mouse and human glioma cells recapitulated the LAIR-1 KD molecular phenotype and similarly potentiated gene therapy. These findings define LAIR-1 as a tumor-cell-intrinsic pro-fibrotic and immunosuppressive checkpoint and identify the LAIR-1>SHP2>JNK>CXCL16 and/or LOXL1 axis as a therapeutic target for sensitizing glioma to immunotherapy.

Pediatric high-grade gliomas (pHGGs) are diffuse and highly aggressive CNS tumors which remain incurable, with a 5-year overall survival of less than 20%. Within glioma, mutations in the genes encoding the histones H3.1 and H3.3 have been discovered to be age-restricted and specific of pHGGs. This work focuses on the study of pHGGs harboring the H3.3-G34R mutation. H3.3-G34R tumors represent the 9-15% of pHGGs, are restricted to the cerebral hemispheres, and are found predominantly in the adolescent population (median 15.0 years). We have utilized a genetically engineered immunocompetent mouse model for this subtype of pHGG generated via the Sleeping Beauty-transposon system. The analysis of H3.3-G34R genetically engineered brain tumors by RNA-Sequencing and ChIP-Sequencing revealed alterations in the molecular landscape associated to H3.3-G34R expression. In particular, the expression of H3.3-G34R modifies the histone marks deposited at the regulatory elements of genes belonging to the JAK/STAT pathway, leading to an increased activation of this pathway. This histone G34R-mediated epigenetic modifications lead to changes in the tumor immune microenvironment of these tumors, towards an immune-permissive phenotype, making these gliomas susceptible to TK/Flt3L immune-stimulatory gene therapy. The application of this therapeutic approach increased median survival of H3.3-G34R tumor bearing animals, while stimulating the development of anti-tumor immune response and immunological memory. Our data suggests that the proposed immune-mediated gene therapy has potential for clinical translation for the treatment of patients harboring H3.3-G34R high grade gliomas.

Gliomas are aggressive tumors with a poor prognosis. The protocols presented here outline the methods used to study tumor progression, the tumor microenvironment (TME), and the effects of experimental treatments. The Sleeping Beauty (SB) transposase system induces tumors de novo to generate mouse models that recapitulate human gliomas. Plasmids are constructed with oncogenic drivers and other genetic alterations of interest. which are recognized by their unique position in between inverted/direct repeat (IR/DR) sequences. Luciferase enzyme is used to monitor the uptake of the plasmid, tumor growth, and response to experimental therapies. The genes of interest are tracked using fluorescent markers. Tumors will arise in immunocompetent hosts, which provides a relevant preclinical platform for analysis of tumor initiation, progression, survival, immune microenvironment, and histopathological features. Once the tumor grows within the desired brain location, it can be harvested to generate cell cultures of neurospheres for future experimentation. The benefit of implantable models generated from SB tumors is that they provide specific anatomical and genetic context, in which specific genetic characteristics can be tracked, as they are co-expressed with fluorescent markers. Post glioma cell implantation, additional analysis of the TME and tumor growth can be performed through immunohistochemistry (IHC) and flow cytometry. (c) 2025 Wiley Periodicals LLC.Basic Protocol 1: Creation of mouse glioma models by Sleeping-Beauty-mediated transpositionBasic Protocol 2: Generation of orthotopic implantable brain tumors and neurospheresBasic Protocol 3: Hematoxylin and eosin staining of glioma tissue samplesBasic Protocol 4: Immunohistochemistry of glioma tissue samplesBasic Protocol 5: Flow cytometry for immune cell analysis of the tumor microenvironment

Mutant isocitrate dehydrogenase 1 (mIDH1) catalyzes 2-hydroxyglutarate production which leads to epigenetic reprogramming. RNA-seq, scRNA-seq, and ChIP-seq analysis revealed that human and mouse mIDH1 gliomas exhibit downregulated gene ontologies (GOs) related to mitochondrial metabolism and upregulated autophagy-related GOs. Decreased mitochondrial metabolism was accompanied by decreased glycolysis, rendering autophagy a source of energy in mIDH1 gliomas. Human and mouse mIDH1 glioma cells exhibited increased expression of autophagy-related proteins and enhanced LC3 I/II conversion, indicating augmented autophagy. Inhibiting autophagy in vivo by administration of synthetic protein nanoparticles encapsulating siRNA targeting Atg7 sensitized mIDH1 glioma cells to radiation, resulting in tumor regression, long-term survival, and immunological memory. This work uncovered autophagy as a critical pathway for survival in mIDH1 gliomas and its inhibition elicits radiosensitivity in vitro in human and mouse mIDH1 glioma cells, and in vivo in mIDH1 mouse models. Thus, autophagy inhibition emerges as an attractive therapeutic target for mIDH1 gliomas.

Mutant isocitrate dehydrogenase 1 (mIDH1) exhibits a gain of function mutation enabling 2-hydroxyglutarate (2HG) production and epigenetic reprogramming. This leads to enhanced DNA-damage response and radioresistance in mIDH1 gliomas. RNA-seq and ChIP-seq data revealed that human and mouse mIDH1 glioma neurospheres have downregulated gene ontologies (GOs) related to mitochondrial metabolism and upregulated GOs related to autophagy. Decreased mitochondrial metabolism was accompanied by decreased glycolysis, rendering autophagy a source of energy in mIDH1 gliomas. Human and mouse mutant IDH1 glioma cells exhibited increased expression of pULK1-S555 and enhanced LC3 I/II conversion, indicating augmented autophagy. Additionally, scRNA-seq data from human mIDH1 astrocytoma patients' samples showed decreased mitochondrial metabolism and increased autophagy. We further demonstrate that inhibiting autophagy in vivo by systemic administration of synthetic protein nanoparticles encapsulating siRNA targeting Atg7 sensitized mIDH1 glioma cells to radiation-induced cell death, resulting in tumor regression, long-term survival, and immunological memory. In summary, our work uncovered that autophagy is a critical pathway for survival in mIDH1 gliomas and by blocking this pathway we can elicit radiosensitivity in vitro in human and mouse mIDH1 glioma cells, and in vivo in genetically engineered mouse models. Our data also highlights that blocking autophagy has significant potential for clinical translation. Abstract Figure:

High-grade gliomas are a major health challenge with poor prognosis and high morbidity. Immune-checkpoint inhibitors (ICI) have emerged as promising therapeutic options for several malignancies yet show little efficacy against central nervous system (CNS) tumors. CD200 is a newly recognized immune checkpoint that modulates immune homeostasis. CD200 protein is expressed by a variety of cells, including immune cells and stromal cells, and is overexpressed by many tumors. The shedding of CD200 from tumor cells can create an immunosuppressive environment that dampens anti-tumor immunity by modulating cytolytic activity and cytokine expression both within and outside the tumor microenvironment (TME). While it is well-accepted that CD200 induces a pro-tumorigenic environment through its ability to suppress the immune response, we sought to determine the role of glioma-specific expression of CD200. We show that CD200 is expressed across glioma types, is shed from tumor cells, and increases over time in the serum of patients undergoing immunotherapy. Using CD200 knockout (KO) glioma models, we demonstrated that glioma cell-derived CD200 promotes tumor growth in vivo and in vitro. Notably, CD200 KO gliomas are spontaneously rejected by their host, a process that required a fully functional immune system, including NK and T-cells. Moreover, we report that glioma-derived or brain-injected soluble CD200 contributes to the suppression of antigen-specific CD8 T-cells in the draining lymph nodes (dLNs). Our work provides new mechanistic insights regarding CD200-mediated immunosuppression by gliomas. Statement of significance:We demonstrate mechanisms of the druggable glioma-derived CD200 checkpoint on tumor growth and immune suppression.

Abstract Gliomas are primary brain tumors with a poor prognosis. IDH1R132H exhibits a gain of function mutation leading to 2-hydroxyglutarate (2HG) production. We previously demonstrated, 2-HG mediated epigenetic rewiring is associated with better prognosis and its presence impacts several cellular functions including immune responses and enhanced DNA-damage response in IDH1R132H gliomas harboring p53 and ATRX loss-of-function mutations. Also, IDH1R132H elicits metabolic reprogramming which implies that IDH1R132H could modify the energetic state of gliomas with consequences in tumor biology and therapeutic responses. In this study, RNA-seq and ChIP-seq data revealed human and mouse IDH1R132H gliomas have downmodulated gene ontologies related to mitochondrial metabolism. This was paralleled by a decrease in glycolysis, rendering autophagy as a source of energy for IDH1R132H glioma cells. We also found that IDH1R132H-neurospheres have decreased levels of oxygen consumption and extracellular acidification rates, characteristic of an autophagic/quiescent state when compared to IDH1WT-neurospheres. Analysis of the autophagy pathway revealed that both human and mouse IDH1R132H gliomas have increased expression of pULK1-S555, pATG4b-S383, enhanced conversion of LC3I to LC3II, and decreased expression of pULK1-S757, p62 indicating that IDH1R132H cells have augmented autophagy activity compared to IDH1WT cells. Blocking autophagy selectively impairs the growth of IDH1R132H glioma cells in vitro. Targeting autophagy by systemic administration of synthetic protein nanoparticles packaged with siRNA targeting Atg7 sensitized IDH1R132H glioma cells to radiation-induced cell death, resulting in tumor regression, long-term survival, and immunological memory when used in combination with irradiation. Our results thus indicate that the metabolic changes in IDH1R132H glioma cells lead to compromised mitochondrial functions and increased autophagic activity which contributes to their radioresistance, representing a novel therapeutic target for IDH1R132H cells. *Corresponding author: Maria G. Castro: mariacas@med.umich.edu Equal contributions: KB-FN-AM-AM-CT Funding: This work was supported by National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) Grants: [R37-NS094804, R01-NS122165, R21-NS123879-01 awarded to MGC]; [R01-NS122378, R01-NS122234 awarded to PRL], Ian’s Friends & Chad Tough Foundation.

Epigenetic remodeling is a molecular hallmark of gliomas, and it has been identified as a key mediator of glioma progression. Epigenetic dysregulation contributes to gliomagenesis, tumor progression, and responses to immunotherapies, as well as determining clinical features. This epigenetic remodeling includes changes in histone modifications, chromatin structure, and DNA methylation, all of which are driven by mutations in genes such as histone 3 genes (H3C1 and H3F3A), isocitrate dehydrogenase 1/2 (IDH1/2), α-thalassemia/mental retardation, X-linked (ATRX), and additional chromatin remodelers. Although much of the initial research primarily identified how the epigenetic aberrations impacted glioma progression by solely examining the glioma cells, recent studies have aimed at establishing the role of epigenetic alterations in shaping the tumor microenvironment (TME). In this review, we discuss the mechanisms by which these epigenetic phenomena in glioma remodel the TME and how current therapies targeting epigenetic dysregulation affect the glioma immune response and therapeutic outcomes. Understanding the link between epigenetic remodeling and the glioma TME provides insights into the implementation of epigenetic-targeting therapies to improve the antitumor immune response.

The search for reliable protein biomarker candidates is critical for early disease detection and treatment. However, current immunoassay technologies are failing to meet increasing demands for sensitivity and multiplexing. Here, the authors have created a highly sensitive protein microarray using the principle of single-molecule counting for signal amplification, capable of simultaneously detecting a panel of cancer biomarkers at sub-pg/mL levels. To enable this amplification strategy, the authors introduce a novel method of protein patterning using photolithography to subdivide addressable arrays of capture antibody spots into hundreds of thousands of individual microwells. This allows for the total sensor area to be miniaturized, increasing the total possible multiplex capacity. With the immunoassay realized on a standard 75x25 mm form factor glass substrate, sample volume consumption is minimized to <10 μL, making the technology highly efficient and cost-effective. Additionally, the authors demonstrate the power of their technology by measuring six secretory factors related to glioma tumor progression in a cohort of mice. This highly sensitive, sample-sparing multiplex immunoassay paves the way for researchers to track changes in protein profiles over time, leading to earlier disease detection and discovery of more effective treatment using animal models.

Abstract Glioma is one of the most aggressive cancers and represents approximately 80% of malignant brain tumors. The majority of CNS World Health Organization (WHO) grades 2-3 gliomas and a subset of high-grade gliomas (CNS WHO grade 4) harbor mutated isocitrate dehydrogenase 1 (IDH1R132H; mIDH1). Mutant IDH1 results in the accumulation of 2-hydroxyglutarate (2HG) which elicits profound changes in the glioma transcriptome/biology. Consequently, mIDH1 glioma patients have a significantly increased median survival compared to wildtype IDH1 patients. Recent data from our lab showed that mIDH1 gliomas exhibit increased immune-reactivity of tumor-infiltrating immune cells. A critical pathway that mediates reduction of anti-tumor immune cell reactivity is the adenosinergic pathway (AP), which converts ATP into adenosine via the enzymes CD39 and CD73. Adenosine binds to adenosine receptors, A2AR and A2BR, on immune cells resulting in a decreased immunoreactivity. Although mIDH1 has been implicated in altering the AP, the extent to which the AP is affected by mIDH1 in gliomas is largely unknown. Here we used human and mouse mIDH1 glioma cells in vitro along with in vivo mIDH1 glioma mouse models to examine changes in the AP. Our results show reduced levels of the enzyme, CD73, on the surface of mIDH1 glioma cells, and the ATP concentration in the mIDH1 glioma cell growth media is lower than from wildtype IDH1 glioma cells. Additionally, we discovered that mIDH1 glioma-infiltrating macrophages have decreased expression of CD39 and A2AR compared to their wildtype IDH1 glioma counterparts. Taken together, these data suggest a reduction of the AP mediated-immune suppression in mIDH1 gliomas. Uncovering these differences in the AP provides novel insights on the use of AP inhibitors for glioma treatment, revealing whether the AP is a preferred target in specific glioma types based on the IDH1 mutation status.

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4+ monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

The preclinical and clinical development of novel immunotherapies for the treatment of central nervous system (CNS) tumors is advancing at a rapid pace. High-grade gliomas (HGG) are aggressive tumors with poor prognoses in both adult and pediatric patients, and innovative and effective therapies are greatly needed. The use of cytotoxic chemotherapies has marginally improved survival in some HGG patient populations. Although several challenges exist for the successful development of immunotherapies for CNS tumors, recent insights into the genetic alterations that define the pathogenesis of HGG and their direct effects on the tumor microenvironment (TME) may allow for a more refined and targeted therapeutic approach. This review will focus on the TME in HGG, the genetic drivers frequently found in these tumors and their effect on the TME, the development of immunotherapy for HGG, and the practical challenges in clinical trials employing immunotherapy for HGG. Herein, we will discuss broadly the TME and immunotherapy development in HGG, with a specific focus on glioblastoma multiforme (GBM) as well as additional discussion in the context of the pediatric HGG diagnoses of diffuse midline glioma (DMG) and diffuse hemispheric glioma (DHG).

Here, we present a mass cytometry protocol optimized to examine the phenotype of immune cells within the mouse glioma microenvironment, using a Sleeping Beauty transposon-mediated mouse glioma model. We describe antibody conjugation and titrations for analysis of immune cells. We then detail mouse brain tumor tissue collection and processing, staining, followed by data acquisition, analysis, and gating strategy. This protocol can be applied to any brain tumor-harboring mouse model. For complete details on the use and execution of this protocol, please refer to Alghamri et al. (2021).

Immunity against malaria depends on germinal center (GC)-derived antibody responses that are orchestrated by T follicular helper (TFH) cells. Emerging data show that the regulatory cytokine IL-10 plays an essential role in promoting GC B cell responses during both experimental malaria and virus infections. Here we investigated the cellular source and temporal role of IL-10, and whether IL-10 additionally signals to CD4 T-cells to support anti-Plasmodium humoral immunity. Distinct from reports of virus infection, we found that IL-10 was expressed by conventional, Foxp3-negative effector CD4 T cells and functioned in a B cell-intrinsic manner only during the first 96 hours of Plasmodium infection to support humoral immunity. The critical functions of IL-10 manifested only before the orchestration of GC responses and were primarily localized outside of B cell follicles. Mechanistically, our studies showed that the rapid and transient provision of IL-10 promoted B cell expression of anti-apoptotic factors, MHC class II, CD83, and cell-cell adhesion proteins that are essential for B cell survival and interaction with CD4 T cells. Together, our data reveal temporal features and mechanisms by which IL-10 critically supports humoral immunity during blood-stage Plasmodium infection, information that may be useful for developing new strategies designed to lessen the burden of malaria.

Mutant isocitrate-dehydrogenase 1 ( mIDH1 ) synthesizes the oncometabolite 2-hydroxyglutarate (2HG), which elicits epigenetic reprogramming of the glioma cells’ transcriptome by inhibiting DNA and histone demethylases. We show that the efficacy of immune-stimulatory gene therapy (TK/Flt3L) is enhanced in mIDH1 gliomas, due to the reprogramming of the myeloid cells’ compartment infiltrating the tumor microenvironment (TME). We uncovered that the immature myeloid cells infiltrating the mIDH1 TME are mainly nonsuppressive neutrophils and preneutrophils. Myeloid cell reprogramming was triggered by granulocyte colony-stimulating factor (G-CSF) secreted by mIDH1 glioma stem/progenitor-like cells. Blocking G-CSF in mIDH1 glioma–bearing mice restores the inhibitory potential of the tumor-infiltrating myeloid cells, accelerating tumor progression. We demonstrate that G-CSF reprograms bone marrow granulopoiesis, resulting in noninhibitory myeloid cells within mIDH1 glioma TME and enhancing the efficacy of immune-stimulatory gene therapy.

Gliomas are one of the most lethal types of cancers accounting for ∼80% of all central nervous system (CNS) primary malignancies. Among gliomas, glioblastomas (GBM) are the most aggressive, characterized by a median patient survival of fewer than 15 months. Recent molecular characterization studies uncovered the genetic signatures and methylation status of gliomas and correlate these with clinical prognosis. The most relevant molecular characteristics for the new glioma classification are IDH mutation, chromosome 1p/19q deletion, histone mutations, and other genetic parameters such as ATRX loss, TP53, and TERT mutations, as well as DNA methylation levels. Similar to other solid tumors, glioma progression is impacted by the complex interactions between the tumor cells and immune cells within the tumor microenvironment. The immune system’s response to cancer can impact the glioma’s survival, proliferation, and invasiveness. Salient characteristics of gliomas include enhanced vascularization, stimulation of a hypoxic tumor microenvironment, increased oxidative stress, and an immune suppressive milieu. These processes promote the neuro-inflammatory tumor microenvironment which can lead to the loss of blood-brain barrier (BBB) integrity. The consequences of a compromised BBB are deleteriously exposing the brain to potentially harmful concentrations of substances from the peripheral circulation, adversely affecting neuronal signaling, and abnormal immune cell infiltration; all of which can lead to disruption of brain homeostasis. In this review, we first describe the unique features of inflammation in CNS tumors. We then discuss the mechanisms of tumor-initiating neuro-inflammatory microenvironment and its impact on tumor invasion and progression. Finally, we also discuss potential pharmacological interventions that can be used to target neuro-inflammation in gliomas.

High grade gliomas are malignant brain tumors that arise in the central nervous system, in patients of all ages. Currently, the standard of care, entailing surgery and chemo radiation, exhibits a survival rate of 14-17 months. Thus, there is an urgent need to develop new therapeutic strategies for these malignant brain tumors. Currently, immunotherapies represent an appealing approach to treat malignant gliomas, as the pre-clinical data has been encouraging. However, the translation of the discoveries from the bench to the bedside has not been as successful as with other types of cancer, and no long-lasting clinical benefits have been observed for glioma patients treated with immune-mediated therapies so far. This review aims to discuss our current knowledge about gliomas, their molecular particularities and the impact on the tumor immune microenvironment. Also, we discuss several murine models used to study these therapies pre-clinically and how the model selection can impact the outcomes of the approaches to be tested. Finally, we present different immunotherapy strategies being employed in clinical trials for glioma and the newest developments intended to harness the immune system against these incurable brain tumors.