Understanding the immunosuppressive microenvironment of glioma: mechanistic insights and clinical perspectives.

Glioblastoma (GBM), the predominant and primary malignant intracranial tumor, poses a formidable challenge due to its immunosuppressive microenvironment, thereby confounding conventional therapeutic interventions. Despite the established treatment regimen comprising surgical intervention, radiotherapy, temozolomide administration, and the exploration of emerging modalities such as immunotherapy and integration of medicine and engineering technology therapy, the efficacy of these approaches remains constrained, resulting in suboptimal prognostic outcomes. In recent years, intensive scrutiny of the inhibitory and immunosuppressive milieu within GBM has underscored the significance of cellular constituents of the GBM microenvironment and their interactions with malignant cells and neurons. Novel immune and targeted therapy strategies have emerged, offering promising avenues for advancing GBM treatment. One pivotal mechanism orchestrating immunosuppression in GBM involves the aggregation of myeloid-derived suppressor cells (MDSCs), glioma-associated macrophage/microglia (GAM), and regulatory T cells (Tregs). Among these, MDSCs, though constituting a minority (4-8%) of CD45+ cells in GBM, play a central component in fostering immune evasion and propelling tumor progression, angiogenesis, invasion, and metastasis. MDSCs deploy intricate immunosuppressive mechanisms that adapt to the dynamic tumor microenvironment (TME). Understanding the interplay between GBM and MDSCs provides a compelling basis for therapeutic interventions. This review seeks to elucidate the immune regulatory mechanisms inherent in the GBM microenvironment, explore existing therapeutic targets, and consolidate recent insights into MDSC induction and their contribution to GBM immunosuppression. Additionally, the review comprehensively surveys ongoing clinical trials and potential treatment strategies, envisioning a future where targeting MDSCs could reshape the immune landscape of GBM. Through the synergistic integration of immunotherapy with other therapeutic modalities, this approach can establish a multidisciplinary, multi-target paradigm, ultimately improving the prognosis and quality of life in patients with GBM.

State of the Art in Low-Grade Glioma Management: Insights From Isocitrate Dehydrogenase and Beyond.

Low-grade gliomas present a formidable challenge in neuro-oncology because of the challenges imposed by the blood-brain barrier, predilection for the young adult population, and propensity for recurrence. In the past two decades, the systematic examination of genomic alterations in adults and children with primary brain tumors has uncovered profound new insights into the pathogenesis of these tumors, resulting in more accurate tumor classification and prognostication. It also identified several common recurrent genomic alterations that now define specific brain tumor subtypes and have provided a new opportunity for molecularly targeted therapeutic intervention. Adult-type diffuse low-grade gliomas are frequently associated with mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), resulting in production of 2-hydroxyglutarate, an oncometabolite important for tumorigenesis. Recent studies of IDH inhibitors have yielded promising results in patients at early stages of disease with prolonged progression-free survival (PFS) and delayed time to radiation and chemotherapy. Pediatric-type gliomas have high rates of alterations in BRAF, including BRAF V600E point mutations or BRAF-KIAA1549 rearrangements. BRAF inhibitors, often combined with MEK inhibitors, have resulted in radiographic response and improved PFS in these patients. This article reviews emerging approaches to the treatment of low-grade gliomas, including a discussion of targeted therapies and how they integrate with the current treatment modalities of surgical resection, chemotherapy, and radiation.

Glioma.

Gliomas are primary brain tumours that are thought to develop from neural stem or progenitor cells that carry tumour-initiating genetic alterations. Based on microscopic appearance and molecular characteristics, they are classified according to the WHO classification of central nervous system (CNS) tumours and graded into CNS WHO grades 1-4 from a low to high grade of malignancy. Diffusely infiltrating gliomas in adults comprise three tumour types with distinct natural course of disease, response to treatment and outcome: isocitrate dehydrogenase (IDH)-mutant and 1p/19q-codeleted oligodendrogliomas with the best prognosis; IDH-mutant astrocytomas with intermediate outcome; and IDH-wild-type glioblastomas with poor prognosis. Pilocytic astrocytoma is the most common glioma in children and is characterized by circumscribed growth, frequent BRAF alterations and favourable prognosis. Diffuse gliomas in children are divided into clinically indolent low-grade tumours and high-grade tumours with aggressive behaviour, with histone 3 K27-altered diffuse midline glioma being the leading cause of glioma-related death in children. Ependymal tumours are subdivided into biologically and prognostically distinct types on the basis of histology, molecular biomarkers and location. Although surgery, radiotherapy and alkylating agent chemotherapy are the mainstay of glioma treatment, individually tailored strategies based on tumour-intrinsic dominant signalling pathways have improved outcome in subsets of patients.

Case report: Temozolomide induced hypermutation indicates an unfavorable response to immunotherapy in patient with gliomas.

Background: Temozolomide (TMZ) is a key component in the treatment of gliomas. Hypermutation induced by TMZ can be encountered in routine clinical practice, and its significance is progressively gaining recognition. However, the relationship between TMZ-induced hypermutation and the immunologic response remains controversial. Case presentation: We present the case of a 38-year-old male patient who underwent five surgeries for glioma. Initially diagnosed with IDH-mutant astrocytoma (WHO grade 2) during the first two surgeries, the disease progressed to grade 4 in subsequent interventions. Prior to the fourth surgery, the patient received 3 cycles of standard TMZ chemotherapy and 9 cycles of dose-dense TMZ regimens. Genomic and immunologic analyses of the tumor tissue obtained during the fourth surgery revealed a relatively favorable immune microenvironment, as indicated by an immunophenoscore of 5, suggesting potential benefits from immunotherapy. Consequently, the patient underwent low-dose irradiation combined with immunoadjuvant treatment. After completing 4 cycles of immunotherapy, the tumor significantly shrank, resulting in a partial response. However, after a 6-month duration of response, the patient experienced disease progression. Subsequent analysis of the tumor tissue obtained during the fifth surgery revealed the occurrence of hypermutation, with mutation signature analysis attributing TMZ treatment as the primary cause. Unfortunately, the patient succumbed shortly thereafter, with a survival period of 126 months. Conclusion: Patients subjected to a prolonged regimen of TMZ treatment may exhibit heightened vulnerability to hypermutation. This hypermutation induced by TMZ holds the potential to function as an indicator associated with unfavorable response to immunotherapy in gliomas.

Clinical characteristics and prognostic factors of adult brainstem gliomas: A retrospective analysis of histologically-proven 40 cases.

To illustrate the clinical characteristics and prognostic factors of adult patients pathologically confirmed with brainstem gliomas (BSGs). Clinical data of 40 adult patients pathologically diagnosed with BSGs admitted to Beijing Shijitan Hospital from 2009 to 2022 were recorded and retrospectively analyzed. The primary parameters included relevant symptoms, duration of symptoms, Karnofsky performance status (KPS), tumor location, type of surgical resection, diagnosis, treatment, and survival. Univariate and multivariate analyses were evaluated by Cox regression models. The gliomas were located in the midbrain of 9 patients, in the pons of 14 cases, in the medulla of 5 cases, in the midbrain and pons of 6 cases and invading the medulla and pons of 6 cases, respectively. The proportion of patients with low-grade BSGs was 42.5%. Relevant symptoms consisted of visual disturbance, facial paralysis, dizziness, extremity weakness, ataxia, paresthesia, headache, bucking, dysphagia, dysacousia, nausea, dysphasia, dysosmia, hypomnesia and nystagmus. 23 (57.5%) patients accepted stereotactic biopsy, 17 (42.5%) patients underwent surgical resection. 39 patients received radiotherapy and 34 cases were treated with temozolomide. The median overall survival (OS) of all patients was 26.2 months and 21.5 months for the median progression-free survival (PFS). Both duration of symptoms (P = .007) and tumor grading (P = .002) were the influencing factors for OS, and tumor grading was significantly associated with PFS (P = .001). Duration of symptoms for more than 2 months and low-grade are favorable prognostic factors for adult patients with BSGs.

Neutrophils in glioma microenvironment: from immune function to immunotherapy.

Glioma is a malignant tumor of the central nervous system (CNS). Currently, effective treatment options for gliomas are still lacking. Neutrophils, as an important member of the tumor microenvironment (TME), are widely distributed in circulation. Recently, the discovery of cranial-meningeal channels and intracranial lymphatic vessels has provided new insights into the origins of neutrophils in the CNS. Neutrophils in the brain may originate more from the skull and adjacent vertebral bone marrow. They cross the blood-brain barrier (BBB) under the action of chemokines and enter the brain parenchyma, subsequently migrating to the glioma TME and undergoing phenotypic changes upon contact with tumor cells. Under glycolytic metabolism model, neutrophils show complex and dual functions in different stages of cancer progression, including participation in the malignant progression, immune suppression, and anti-tumor effects of gliomas. Additionally, neutrophils in the TME interact with other immune cells, playing a crucial role in cancer immunotherapy. Targeting neutrophils may be a novel generation of immunotherapy and improve the efficacy of cancer treatments. This article reviews the molecular mechanisms of neutrophils infiltrating the central nervous system from the external environment, detailing the origin, functions, classifications, and targeted therapies of neutrophils in the context of glioma.

The relationship between imaging features, therapeutic response, and overall survival in pediatric diffuse intrinsic pontine glioma.

We aimed to evaluate the relationship between imaging features, therapeutic responses (comparative cross-product and volumetric measurements), and overall survival (OS) in pediatric diffuse intrinsic pontine glioma (DIPG). A total of 134 patients (≤ 18 years) diagnosed with DIPG were included. Univariate and multivariate analyses were performed to evaluate correlations of clinical and imaging features and therapeutic responses with OS. The correlation between cross-product (CP) and volume thresholds in partial response (PR) was evaluated by linear regression. The log-rank test was used to compare OS patients with discordant therapeutic response classifications and those with concordant classifications. In univariate analysis, characteristics related to worse OS included lower Karnofsky, larger extrapontine extension, ring-enhancement, necrosis, non-PR, and increased ring enhancement post-radiotherapy. In the multivariate analysis, Karnofsky, necrosis, extrapontine extension, and therapeutic response can predict OS. A 25% CP reduction (PR) correlated with a 32% volume reduction (R2 = 0.888). Eight patients had discordant therapeutic response classifications according to CP (25%) and volume (32%). This eight patients' median survival time was 13.0 months, significantly higher than that in the non-PR group (8.9 months), in which responses were consistently classified as non-PR based on CP (25%) and volume (32%). We identified correlations between imaging features, therapeutic responses, and OS; this information is crucial for future clinical trials. Tumor volume may represent the DIPG growth pattern more accurately than CP measurement and can be used to evaluate therapeutic response.

Contemporary Management of Pediatric Brainstem Tumors.

Brain tumors are the second most common malignancy in childhood. Around 15-20% of pediatric brain tumors occur in the brainstem. The most common type of brainstem tumor are diffuse tumors in the ventral pons, whereas focal tumors tend to arise from the midbrain, medulla, and dorsal pons. Glioma is the most common pathological entity. Contemporary management consists of surgery, radiotherapy, chemotherapy, and other adjuvant treatment. Surgical options range from biopsy to radical excision. Biopsy can be performed for diagnostic and prognostic purposes, or in the setting of clinical trials, mainly for diffuse intrinsic pontine gliomas. For focal tumors, surgeons need to carefully balance clinical outcomes against possible neurological sequelae in order to achieve maximal safe resection. Radiotherapy is essential for control of high-grade tumors and may be applied to residual or recurrent low-grade tumors. Proton therapy may provide similar efficacy and less neurotoxicity in comparison to conventional photon therapy. Oncological treatment continues to evolve from conventional chemotherapy to targeted therapy, immunotherapy, and other novel treatment methods and holds great potential as adjuvant therapy for pediatric brainstem tumors.

Understanding supported self-management for people living with a lower-grade glioma: Implementation considerations through the lens of normalisation process theory.

BACKGROUND: Supported self-management can improve clinical and psychosocial outcomes in people with cancer; the considerations required to implement self-management support (SMS) for people living with a lower-grade glioma (LGG)-who often have complex support needs-are not known. We aimed to identify and understand these implementation considerations through the lens of normalisation process theory (NPT), from the perspectives of healthcare professionals (HCP) and people with LGG. METHODS: We conducted semistructured interviews with HCPs who support adults with brain tumours (n = 25; 12 different healthcare professions), and people with LGG who had completed primary treatment (n = 28; male n = 16, mean age 54.6 years, mean time since diagnosis 8.7 years), from across the United Kingdom. Interviews were transcribed and inductive open coding conducted, before deductively mapping to constructs of NPT. We first mapped HCP data, then integrated data from people with LGG to explore alignment in experiences and perspectives. RESULTS: We generated supporting evidence for all four NPT constructs and related subconstructs, namely: 'Coherence', 'Cognitive participation', 'Collective action' and 'Reflexive monitoring'. Data from HCPs and people with LGG clearly demonstrated that effective SMS constitutes a collective activity. Key implementation considerations included: ensuring awareness of, and access to, support; building strong HCP-support recipient relationships; and careful inclusion of close family and friends. We identified pertinent challenges, such as identifying support needs (influenced by the extent to which those with LGG engage in help-seeking), resistance to support (e.g., technology literacy), training for HCPs and HCP cooperation. CONCLUSIONS: This study demonstrates the collective nature of, and provides insight into the individual roles within, supported self-management. We outline considerations to operationalise, sustain and appraise the implementation of SMS for people with LGG. PATIENT OR PUBLIC CONTRIBUTION: People with brain tumours, and informal caregivers, were involved in the development of information materials and topic guides to ensure accessibility and pertinence. They also had opportunities to comment on interview findings.

Biomimetic Dual-Target Theranostic Nanovaccine Enables Magnetic Resonance Imaging and Chemo/Chemodynamic/Immune Therapy of Glioma.

Development of theranostic nanomedicines to tackle glioma remains to be challenging. Here, we present an advanced blood-brain barrier (BBB)-crossing nanovaccine based on cancer cell membrane-camouflaged poly(N-vinylcaprolactam) (PVCL) nanogels (NGs) incorporated with MnO2 and doxorubicin (DOX). We show that the disulfide bond-cross-linked redox-responsive PVCL NGs can be functionalized with dermorphin and imiquimod R837 through cell membrane functionalization. The formed functionalized PVCL NGs having a size of 220 nm are stable, can deplete glutathione, and responsively release both Mn2+ and DOX under the simulated tumor microenvironment to exert the chemo/chemodynamic therapy mediated by DOX and Mn2+, respectively. The combined therapy induces tumor immunogenic cell death to maturate dendritic cells (DCs) and activate tumor-killing T cells. Further, the nanovaccine composed of cancer cell membranes as tumor antigens, R837 as an adjuvant with abilities of DC maturation and macrophages M1 repolarization, and MnO2 with Mn2+-mediated stimulator of interferon gene activation of tumor cells can effectively act on both targets of tumor cells and immune cells. With the dermorphin-mediated BBB crossing, cell membrane-mediated homologous tumor targeting, and Mn2+-facilitated magnetic resonance (MR) imaging property, the designed NG-based theranostic nanovaccine enables MR imaging and combination chemo-, chemodynamic-, and imnune therapy of orthotopic glioma with a significantly decreased recurrence rate.

RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model.

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

TLR agonists polarize interferon responses in conjunction with dendritic cell vaccination in malignant glioma: a randomized phase II Trial.

In this randomized phase II clinical trial, we evaluated the effectiveness of adding the TLR agonists, poly-ICLC or resiquimod, to autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination in patients with newly-diagnosed or recurrent WHO Grade III-IV malignant gliomas. The primary endpoints were to assess the most effective combination of vaccine and adjuvant in order to enhance the immune potency, along with safety. The combination of ATL-DC vaccination and TLR agonist was safe and found to enhance systemic immune responses, as indicated by increased interferon gene expression and changes in immune cell activation. Specifically, PD-1 expression increases on CD4+ T-cells, while CD38 and CD39 expression are reduced on CD8+ T cells, alongside an increase in monocytes. Poly-ICLC treatment amplifies the induction of interferon-induced genes in monocytes and T lymphocytes. Patients that exhibit higher interferon response gene expression demonstrate prolonged survival and delayed disease progression. These findings suggest that combining ATL-DC with poly-ICLC can induce a polarized interferon response in circulating monocytes and CD8+ T cells, which may represent an important blood biomarker for immunotherapy in this patient population.Trial Registration: ClinicalTrials.gov Identifier: NCT01204684.

Targeted Ferroptosis-Immunotherapy Synergy: Enhanced Antiglioma Efficacy with Hybrid Nanovesicles Comprising NK Cell-Derived Exosomes and RSL3-Loaded Liposomes.

Ferroptosis therapy and immunotherapy have been widely used in cancer treatment. However, nonselective induction of ferroptosis in tumors is prone to immunosuppression, limiting the therapeutic effect of ferroptosis cancer treatment. To address this issue, this study reports a customized hybrid nanovesicle composed of NK cell-derived extracellular versicles and RSL3-loaded liposomes (hNRVs), aiming to establish a positive cycle between ferroptosis therapy and immunotherapy. Thanks to the enhanced permeability and retention effect and the tumor homing characteristics of NK exosomes, our data indicate that hNRVs can actively accumulate in tumors and enhance cellular uptake. FASL, IFN-γ, and RSL3 are released into the tumor microenvironment, where FASL derived from NK cells effectively lyses tumor cells. RSL3 downregulates the expression of GPX4 in the tumor, leading to the accumulation of LPO and ROS, and promotes ferroptosis in tumor cells. The accumulation of IFN-γ and TNF-α stimulates the maturation of dendritic cells and effectively induces the inactivation of GPX4, promoting lipid peroxidation, making them sensitive to ferroptosis and indirectly promoting the occurrence of ferroptosis. This study highlights the role of the customized hNRV platform in enhancing the effectiveness of synergistic treatment with selective delivery of ferroptosis inducers and immune activation against glioma without causing additional side effects on healthy organs.

The Recombinant Oncolytic Virus VV-GMCSF-Lact and Chemotherapy Drugs against Human Glioma.

Virotherapy is one of the perspective technologies in the treatment of malignant neoplasms. Previously, we have developed oncolytic vaccinia virus VV-GMCSF-Lact and its high cytotoxic activity and antitumor efficacy against glioma was shown. In this work, using immortalized and patient-derived cells with different sensitivity to VV-GMCSF-Lact, we evaluated the cytotoxic effect of chemotherapy agents. Additionally, we studied the combination of VV-GMCSF-Lact with temozolomide which is the most preferred drug for glioma treatment. Experimental results indicate that first adding temozolomide and then the virus to the cells is inherently more efficient than dosing it in the reverse order. Testing these regimens in the U87 MG xenograft glioblastoma model confirmed this effect, as assessed by tumor growth inhibition index and histological analysis. Moreover, VV-GMCSF-Lact as monotherapy is more effective against U87 MG glioblastoma xenografts comparing temozolomide.

Association of Clinical, Tumor, and Treatment Characteristics With Seizure Control in Patients With IDH1/2-Mutant Lower-Grade Glioma.

BACKGROUND AND OBJECTIVES: Patients with IDH1/2-mutant lower-grade glioma have a high frequency of seizures. We aimed to investigate the correlations between seizures and tumor/patient characteristics and the impact of surgery and adjuvant treatments (AT) on seizure control along the disease trajectory. METHODS: We retrospectively included patients with IDH1/2-mutant lower-grade glioma who underwent surgery at the neurosurgery divisions of the University of Turin and Milan and were treated at the Division of Neuro-Oncology of Turin. Inclusion criteria were a diagnosis according to the 2021 WHO Classification and presentation with seizures; exclusion criteria were presence of CDKN2A/B homozygous deletion, intense/ring contrast enhancement on MRI at presentation, and small tissue biopsy. We evaluated seizure freedom for 2 months after surgery, 6 months from starting observation or AT, at recurrence, and for 6 months after treatments of recurrence. RESULTS: We included 150 patients. There were 77 (51%) and 31 (21%) patients with IDH-mutant/1p19q-codeleted grade 2 and 3 oligodendroglioma and 30 (20%) and 12 (8%) with IDH-mutant grade 2 and 3 astrocytoma, respectively. Total resection was accomplished in 68 (45%). Seventy-five patients (50%) received AT while the remaining 75 were observed with MRI. After 6 months after AT, 28 of 29 patients (96.5%) displayed seizure reduction, 5 of 28 (18%) being seizure-free. 66 of 124 patients (53%) had seizures at recurrence. After 6 months after second-line treatments, 60 of 66 patients (91%) had seizure reduction, 11 (17%) being seizure-free. In multivariable analyses, grade 3 histology positively correlated with seizure freedom at 2 months after surgery (OR 3.5, 1.4-8.9, p = 0.008), 6 months after AT (OR 9.0, 1.5-54.9, p = 0.017), and 6 months after treatment of recurrence (OR 4.9, 1.5-16.5, p = 0.009). Adjuvant radiotherapy reduced seizures at recurrence in a univariate analysis (OR 0.14, 0.03-0.7, p = 0.020). Patients with seizure freedom after surgery and AT displayed longer progression-free survival (PFS) (65, 24.5-105, vs 48 months, 32-63.5, p = 0.037). DISCUSSION: This study analyzed seizure control in patients with IDH1/2-mutant lower-grade glioma across multiple time points. Grade 3 correlated with better seizure control throughout the entire disease trajectory, and seizure freedom after surgery and AT correlated with a longer PFS regardless of tumor grade. These results could serve as an external control arm in clinical trials evaluating the efficacy on seizures of antitumor agents in patients with IDH-mutant lower-grade glioma.

The ins and outs of microglial cells in brain health and disease.

Microglia are the brain's resident macrophages that play pivotal roles in immune surveillance and maintaining homeostasis of the Central Nervous System (CNS). Microglia are functionally implicated in various cerebrovascular diseases, including stroke, aneurysm, and tumorigenesis as they regulate neuroinflammatory responses and tissue repair processes. Here, we review the manifold functions of microglia in the brain under physiological and pathological conditions, primarily focusing on the implication of microglia in glioma propagation and progression. We further review the current status of therapies targeting microglial cells, including their re-education, depletion, and re-population approaches as therapeutic options to improve patient outcomes for various neurological and neuroinflammatory disorders, including cancer.

Analysis of the sodium pump subunit ATP1A3 in glioma patients: Potential value in prognostic prediction and immunotherapy.

The ATP1A3 gene is associated with the development and progression of neurological diseases. However, the pathological function and therapeutic value of ATP1A3 in glioblastoma (GBM) remains unknown. In this study, we tried to explore the correlation between the ATP1A3 gene expression and immune features in GBM samples. We found that ATP1A3 gene expression levels showed significant negative correlation with immune checkpoints such as PD-L1, CTLA-4 and IDO1. Next, ATP1A3 gene expression levels showed significant negative correlation with the anti-cancer immune cell process, the immune score and stromal score. By grouping ATP1A3 expression levels, we found that that immunomodulator-related genes and tumor-associated immune cell effector gene expression levels were associated with lower ATP1A3 expression. In addition, immunotherapy prediction pathway activity and a majority of the anti-cancer immune cell process activity levels were also showed to be correlated with lower ATP1A3 gene expression. Further, nine prognostic factors were identified by prognostic analysis, and a GBM prognostic model (risk score) was established. We applied the model to the TCGA GBM training set sample and the GSE4412 validation set sample and found that patients in the high risk score subgroup had significantly shorter survival time, demonstrating the prognostic value and prognostic efficacy of the risk score. Furthermore, ATP1A3 overexpression has also been found to sensitize cancer cells to anti-PD-1 therapy. In conclusion, we showed that ATP1A3 is a highly promising treatment target in GBM and the risk score is an independent prognostic factor for cancer and can be used to help guide the prediction of survival time in patients with GBM.

Identification and characterisation of novel CAR-T cells to target IL13Rα2 positive human glioma in vitro and in vivo.

BACKGROUND: Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13Rα2 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB. METHODS: We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13Rα2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells. RESULTS: The binding of 14-1 clone is to IL-13Rα2Fc-chimeric protein is ∼5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13Rα2Fc and cell free supernatants only from IL-13Rα2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13Rα2-CAR-T cells specifically killed IL-13Rα2+ve but not IL-13Rα2-ve tumour cells in vitro and selectively caused significant release of IFN-γ only from IL-13Rα2+ve co-cultures. These CAR-T cells regressed IL-13Rα2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13Rα2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy. CONCLUSION: Taken together, we conclude that the novel scFv-IL-13Rα2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.

Extensive Shrinkage and Long-term Stable Disease in a Teenage Female Patient With High-grade Glioma Treated With Temozolomide and Radiation in Combination With Oral Recombinant Methioninase and a Low-methionine Diet.

BACKGROUND/AIM: Gliomas are the most common and recalcitrant malignant primary brain tumors. All cancer types are addicted to methionine, which is a fundamental and general hallmark of cancer known as the Hoffman effect. Particularly glioma cells exhibit methionine addiction. Because of methionine addiction, [11C]-methionine positron emission tomography (MET-PET) is widely used for glioma imaging in clinical practice, which can monitor the extent of methionine addiction. Methionine restriction including recombinant methioninase (rMETase) and a low-methionine diet, has shown high efficacy in preclinical models of gliomas, especially in combination with chemotherapy. The aim of the present study was to determine the efficacy of methionine restriction with oral rMETase (o-rMETase) and a low-methionine diet, combined with radiation and temozolomide (TMZ), on a teenage female patient with high-grade glioma. CASE REPORT: A 16-year-old girl was diagnosed with high-grade glioma. Magnetic resonance imaging (MRI) showed a left temporal-lobe tumor with compression to the left lateral ventricle and narrowing of sulci in the left temporal lobe. After the start of methionine restriction with o-rMETase and a low-methionine diet, along with TMZ combined with radiotherapy, the tumor size shrunk at least 60%, with improvement in the left lateral ventricle and sulci. The patient's condition remains stable for 19 months without severe adverse effects. CONCLUSION: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with radiation and TMZ as first-line chemotherapy, were highly effective in a patient with high-grade glioma.