Neuro-Oncology

CREB-regulated transcription coactivator 1 (CRTC1) regulates neuronal development, plasticity, and activity-dependent transcription, but its role in medulloblastoma (MB) remains poorly understood. We investigated expression of the CRTC1 gene during human cerebellar development and across pediatric brain tumors and examined its molecular and clinical associations in MB using public transcriptomic datasets. CRTC1 was broadly expressed in the developing cerebellum, with cell-state-specific variation within the granule-cell lineage. Among pediatric brain tumors, MB showed the highest median CRTC1 expression, which differed significantly among MB molecular groups and subtypes, with highest levels in Group 4 MB. High CRTC1 expression was associated with longer overall survival specifically in Group 3 {beta} MB and remained significant after correction across the 12 molecular subtypes. Within Group 3 {beta} tumors, CRTC1 expression was inversely associated with enrichment of mitochondrial and bioenergetic processes, including oxidative phosphorylation, mitochondrial organization, and mitochondrial translation, whereas CRTC1-high and CRTC1-low tumors displayed distinct transcriptional profiles. CRTC1 was also associated with selected cell-type-related transcriptional signatures related to the tumor microenvironment. These findings identify CRTC1 as a marker of biologically and clinically relevant transcriptional heterogeneity in Group 3 {beta} MB and support further investigation of its functional and prognostic significance.

Mismatch repair (MMR) deficiency contributes to temozolomide (TMZ) resistance in a subset of isocitrate dehydrogenase 1/2 mutant (IDHmut) gliomas, creating a need for therapies that retain activity despite MMR loss. We evaluated the novel interstrand crosslinking agent KL-50, alone and in combination with the histone deacetylase inhibitor belinostat, in patient-derived IDHmut glioma models. MSH6 knockout or constitutive expression established four matched MMR-proficient and MMR-deficient culture models. Drug responses were assessed using live cell count and cytotoxicity assays, and KL-50 monotherapy was tested in intracranial BT142 orthotopic xenografts. LOEWE synergy analysis, RNA sequencing, and a genome-wide CRISPR knockout screen assessed combination activity and potential mechanisms. MMR deficiency increased TMZ GI values 2.2- to 30.4-fold, whereas KL-50 retained antiproliferative activity and showed enhanced cytotoxicity in selected MMR-deficient models. In TMZ-naive, MMR-proficient BT142 xenografts, KL-50 increased median survival from 107.5 to 194 days. Two of eight treated mice had no histologically detectable residual tumor after 220 days post-engraftment. KL-50 and belinostat exhibited synergistic cytotoxicity in BT142, 905, and TS603 IDHmut glioma cultures. Transcriptomic gene ontology analyses identified downregulation of DNA repair pathways following histone deacetylase inhibition, including terms for homologous recombination and double-strand break repair. The CRISPR screen identified depletion of PKMYT1-targeting gRNAs during KL-50 treatment, implicating this mitotic checkpoint kinase as a candidate determinant of drug sensitivity. PKMYT1 expression was also reduced by histone deacetylase inhibition. These findings support further preclinical development of KL-50, alone and in combination with belinostat, for IDHmut gliomas including tumors with MMR-associated TMZ resistance.

ABSTRACT Background: High grade glioma (HGG), encompassing IDH-wildtype glioblastoma (CNS WHO grade 4), diffuse midline glioma (CNS WHO grade 4), oligodendroglioma (IDH-mutant and 1p/19q-codeleted) CNS WHO grade 3, together with IDH-mutant astrocytoma of CNS grade 3 and grade 4, represents the most lethal group of adult diffuse gliomas. Despite significant efforts, obtaining improvements in patient survival has been limited, in part, by difficulties in comprehensive ex vivo modeling of the complexities of HGG. Although organotypic slice cultures and other platforms have shown promise, existing models often fail to capture the intrinsic cellular heterogeneity, microenvironment, and architecture of HGG, restricting investigations into their fundamental biology as well as design of novel therapeutics. Methods: We developed a patient-derived HGG organotypic slice culture (GOSC) platform demonstrating long-term preservation of intrinsic tumor characteristics. Vibratome-sectioned slices were cultured in defined medium under conditions approximating those in situ: reduced oxygen (4%), low glucose (2.5 mM), and no added growth factors. The characteristics of GOSCs after 28 days in culture, including expression of immunotherapy targets, were compared to parental tumors using histological staining, immunofluorescence, multiplexed PhenoCycler (CODEX) imaging, bulk RNA-seq expression profiling, and CAR T cell killing assays. Results: GOSCs preserved the histopathology, cell heterogeneity, tumor microenvironment, and tissue architecture of their parental tumors throughout at least 28 days in culture. Importantly, GOSCs retained parental patterns of immunotherapy target expression, and CAR T cells co-cultured with GOSCs demonstrated target-specific killing consistent with parental tumors. Conclusions: This ex vivo GOSC platform offers long-term preservation of the cellular and structural properties of parental HGG tumors, thereby providing a robust tool for extended preclinical studies, and facilitating development and evaluation of advanced tumor-targeting strategies.

Pediatric posterior fossa group A (PFA) ependymoma is an aggressive, chemo-resistant brain tumor with a poor 10-year overall survival and no therapeutic options at relapse. Although conventionally classified as immune-cold, the spatial organization and therapeutic relevance of its rare immune infiltrates remain poorly characterized. Here, we present a spatially resolved immune analysis of 14 PFA ependymoma sections using published Visium spatial transcriptomics data analyzed through a multi-stage immune-focused computational framework combining reference-guided deconvolution, functional lymphocyte state scoring, ligand-receptor inference, and {beta}/{gamma}{delta} T cell immunotherapy opportunity scoring. Lymphocyte-associated hotspots were identified in 6 of 14 sections and, among confidently classified hotspots, were dominated by inflammatory-associated and retention-associated transcriptional states rather than cytotoxic or exhausted programs. These hotspots were preferentially confined to myeloid- and mesenchymal-rich tumor zones with near-complete exclusion from epithelial regions; ligand-receptor analysis identified four candidate restriction axes (SPP1-CD44, FN1-integrin, collagen VI-integrin, and APP-CD74). Immunotherapy opportunity scoring revealed heterogeneous and spatially compartmentalized {beta} and {gamma}{delta} T cell recognition landscapes across sections: {beta}T ligand availability and immunosuppressive tone both rose in the mesenchymal compartment, while the two components of {gamma}{delta} phosphoantigen-mediated recognition were spatially and transcriptionally dissociated from one another. Together, these findings reframe PFA ependymoma not as a monolithic immune desert but as a tumor type with spatially compartmentalized, mechanistically tractable immune niches, and provide a generalizable computational strategy for mapping immune architecture and T cell therapy susceptibility in low-infiltrated pediatric CNS tumors.

Background: Brain-penetrant histone deacetylase inhibitors (HDACi) are promising radiosensitizing agents for glioblastoma (GBM). HDAC inhibitors can impair DNA damage response (DDR) pathways and promote persistent DNA damage. Here, we evaluated whether combined inhibition of HDAC and the DNA repair kinase, ataxia-telangiectasia mutated (ATM), could synergize with radiation (IR) and improve therapeutic efficacy in GBM. Methods: In vitro dose-response assays were performed in patient-derived xenograft (PDX)-derived glioma stem cells (GSCs) with both inhibitors of HDAC (quisinostat, QST) and ATM (AZD1390) with or without IR treatment to assess synergy. Pharmacodynamic responses were evaluated by immunoblotting for histone acetylation, DDR signaling, and DNA damage markers. Transcriptomic analyses were performed to evaluate treatment-associated changes in DNA repair pathways. The combination of QST, AZD1390, and IR was further evaluated in an orthotopic GBM survival study. Results: Combined treatment with QST and AZD1390 produced synergistic radiosensitization in multiple GSC models in vitro. Furthermore, QST combined with IR suppressed transcription of homologous recombination (HR) pathway genes in GSCs, impairing double-strand break repair capacity through an epigenetic mechanism that does not directly inhibit ATM kinase activity. Pharmacodynamic analyses confirmed that QST increased histone acetylation while AZD1390 suppressed ATM signaling, together impairing complementary mechanisms to increase radiosensitivity of GSCs. In an orthotopic GBM model, QST combined with IR significantly prolonged survival compared with IR alone. However, the addition of AZD1390 did not provide a significant survival benefit beyond QST+IR, and AZD1390 combined with IR did not significantly improve survival relative to IR alone. Conclusions: Combined HDAC and ATM inhibition with QST and AZD1390 enhances radiosensitivity in patient-derived GBM cells, mechanistically underpinned by QST-mediated increase in DNA damage and dual disruption of DDR through complementary, non-overlapping mechanisms. However, this in vitro synergy did not confer additional survival benefit beyond QST+IR in an orthotopic GBM model under the dosing conditions tested, suggesting pharmacological limitations with the tumor microenvironment or suboptimal dosing schedules may have constrained in vivo efficacy.

Diffuse midline glioma is an epigenetically driven disease defined by alterations targeting the histone post-translational modification H3K27me3 resulting in epigenetic rewiring and imposing unique metabolic dependencies. ACVR1-mutations arise in ~25% of DMG H3K27-altered patients and impart a selective dependency on the kinase it encodes (ALK2), however ALK2 inhibitors show modest single-agent efficacy in vivo. In an attempt to better understand the cellular consequences of ALK2 inhibition to identify mechanistically-driven drug combinations, integrated multi-omics analysis was performed and revealed a novel role for ALK2 in cholesterol homeostasis while CRISPR and high-throughput drug screens identified hits targeting cholesterol metabolism as sensitisers to ALK2i. In vivo assessment of ALK2i plus clinically well-tolerated statins revealed a significant increase in the median survival compared to vehicle. Forced differentiation of DMG cells from an oligodendrocyte precursor-like to an astrocyte-like cell-state and led to a significant decrease in ALK2i sensitivity and synergy with statins, which was phenocopied when DMG cells were co-cultured with normal astrocytes. We identify a previously unappreciated role for ALK2 signalling in cholesterol homeostasis, showing cell-state dependency, and identify a rational combinatorial strategy for clinical translation.

Glioma is a fatal tumor of the central nervous system (CNS) with limited therapeutic options. While immune checkpoint inhibitors (ICI) are gaining traction as a novel alternative treatment for human gliomas (HG), clinical trials have yielded mixed results. A proposed major barrier to ICI therapy in cancer is epithelial-mesenchymal plasticity (EMP)-driven intratumoral immunosuppression. However, studying this mechanism is difficult in ex vivo and murine models, which fail to adequately replicate cancer cell-immune cell interactions. Formalin-fixed, paraffin-embedded (FFPE) patient-derived canine glioma (CG) samples represent an attractive alternative approach. Although mechanistic perturbation is unachievable in archival FFPE tissues, CG is notoriously 'cold' immunologically and is actively being investigated as a comparative model of HG. Moreover, the immune microenvironment in CG advantageously develops under exposure to environmental conditions directly shared with humans. Yet, it remains unknown whether CGs harbor heterogeneous niches combining varied plastic phenotypes and immunosuppressive populations. Defining the immune landscape of CG in relation to EMP is therefore critical to determining its potential as a comparative model for immunotherapy response in HG. By combining standard histology, immunohistochemistry, and tissue microarray-based spatial transcriptomics, we analyzed 30 low-grade (LGr) and high-grade (HGr) FFPE CG cases (n=10 cases/subtype): astrocytomas (4xLGr, 6xHGr), oligodendrogliomas (1xLGr, 9xHGr), and undefined gliomas (3LxGr, 7xHGr). All CG subtypes harbored heterogeneous immunosuppressive niches associated with varied EMP phenotypes. Astrocytomas exhibited a more-mesenchymal identity correlated with recruitment of tumor-associated macrophages (TAMs). Oligodendrogliomas displayed a strong stem-like signature and uniquely upregulated a FOXP3/LAG3/SNAI1 triad, positioned directly at the interface of immunosuppression and EMP. Across all CG subtypes, a more-mesenchymal phenotype co-occurred with TAMs, and intratumoral T-cell dysfunction and exhaustion. We conclude that CG replicates the EMP-immunosuppression heterogeneity seen in humans. Importantly, our results emphasize the existence of subtype-specific mechanisms of EMP and immune evasion, underscoring the major relevance of subtyping for translational applications.

Background: Immune checkpoint inhibitors (ICI) are efficacious in many solid tumors, but response in glioma is restricted to a small subgroup. The determinants of response and resistance to ICI remain poorly understood. Methods: Here we exploit a syngeneic hypermutated high-grade glioma model with dichotomous response to combined PD-1 and CTLA-4 inhibition to unravel determinants of tumor-infiltrating T-cells driving response. Tumor-infiltrating T-cells from ICI-responsive and non-responsive tumors were analyzed by single-cell RNA and T-cell receptor sequencing and tumor-reactive T-cell receptor clonotypes were functionally validated to characterize their transcriptional phenotypes. We verify our findings in IDH1 wildtype glioblastoma patients treated with neoadjuvant pembrolizumab. Results: ICI response was associated with intratumoral clonal expansion of tumor-reactive cytotoxic T-cells and increased infiltration of CXCR6+ CD4+ tissue resident memory T-cells (Trm). CD4 stem-like memory T-cells in responding tumors demonstrated elevated interferon responses, following trajectories toward clonally expanded Trm, versus trajectories toward exhaustion in non-responsive tumors. In responsive tumors, CD4+ Trm interacted with infiltrating CXCR3+ tumor-reactive and clonally expanded, yet transcriptionally versatile cytotoxic T-cells. Probing the post neoadjuvant ICI high-grade glioma patient tissue dataset, we confirmed increased CXCR6 expression in CD4+ T cells and the association of CD4+ Trm with prolonged overall survival. Conclusion: These findings identify CD4 tissue-resident memory T-cells as determinants of ICI response in IDH1 wildtype high-grade glioma and warrant their further investigation to improve immunotherapy outcomes.

Neuronal-cancer cell interactions are a key component of brain tumor pathophysiology, yet many fundamental questions remain unanswered, with even more remaining unasked. Here, we present a comprehensive brain tumor reference map generated from bulk RNA-seq data of ~4,300 adult and pediatric brain tumors, including various glioma subtypes, medulloblastomas, ependymomas, and meningiomas, alongside ~1,400 healthy brain samples. By analyzing synaptic processes and synaptic gene expression across tumor types, we reveal the complexity of neuron-tumor crosstalk and its potential impact on patient survival. Our findings identify key synaptic signaling pathways and dysregulated patterns in brain tumors, including HNRNPH2 upregulation, a key factor in RNA processing and metabolism, in gliomas and medulloblastomas. Additionally, we highlight the role of cholinergic signaling, particularly CHRNA9, as a driver of glioma progression, which is also upregulated in WNT and Group 4 medulloblastomas, ependymomas, and meningiomas. Furthermore, P2 purinergic signaling emerges as a key player in glioma progression. These insights lay the groundwork for future studies exploring whether targeting these pathways could reprogram the tumor microenvironment toward an anti-tumor state, ultimately leading to novel therapeutic strategies and improved patient outcomes.

Background: Glioblastoma (GBM) adapts to therapy through coordinated malignant-cell and microenvironmental responses, but the mechanisms linking treatment-associated inflammation to tumor-cell phenotypic plasticity remain poorly defined. Here, we investigated whether preoperative temozolomide exposure is associated with inflammatory-repair remodeling and a ROCK-linked therapeutic vulnerability in recurrent GBM using a window-of-opportunity clinical trial cohort. Methods: We integrated patient-resolved single-cell transcriptomics, spatial RNA profiling and multiplex protein imaging with experimental perturbations and orthotopic glioblastoma models. In the presurgical window-of-opportunity subgroup of NCT05236036, four patients with recurrent glioblastoma received additional preoperative temozolomide (TMZ; 150 mg/m2/day for 5 days) before resection and were compared with three recurrent comparators who did not receive additional preoperative TMZ. Results: Across seven tumors, malignant-cell inflammatory activity covaried with integrin-binding and wound-healing programs (median partial {rho} = 0.45 and 0.42, respectively). Tumors exposed to preoperative TMZ showed enrichment of inflammatory, chemokine, adhesion and wound-healing programs (GSEA q < 0.05), with the direction of enrichment preserved in all leave-one-patient-out analyses. In two tumors exposed to preoperative TMZ, CXCL12 localized to vascular territories, whereas chemokine and wound-repair programs increased near injury-reference regions. Across seven specimens analyzed by multiplex protein imaging, MYL9 abundance correlated with local CXCL12 ({rho} = 0.61), phosphorylated MYPT1 ({rho} = 0.48) and nuclear phosphorylated STAT3 ({rho} = 0.45), with positive associations in every specimen. Experimentally, TMZ increased CXCL8 and reactive-state markers and enhanced subsequent scratch closure, while CXCL8 and CXCL12 increased MLC2 phosphorylation. Fasudil attenuated TNF-NF-{kappa}B, inflammatory-response and IFN-{gamma}-response programs in TMZ-treated cells and reduced scratch closure and p-MLC2 in complementary assays. Across three orthotopic models, fasudil plus TMZ prolonged survival versus TMZ alone (model-stratified HR, 0.21; 95% CI, 0.07-0.61; P = 0.001). Conclusions: These findings link treatment-associated inflammatory-repair programs with cytoskeletal remodeling and support further translational evaluation of fasudil plus TMZ in glioblastoma.

H3K27M diffuse midline gliomas (DMGs) are lethal and lack treatment options. These tumors exhibit tumor heterogeneity with aggressive oligodendrocyte precursor (OPC)- and mesenchymal (MES)-like cell states. Because all tumor cells bear H3K27M, tumor heterogeneity has been attributed to microenvironmental cues that remain obscure. H3K27M DMGs exhibit increased glycolysis and the Warburg effect to produce lactate. Lactate can directly modify histone lysines including H3K27Lac, and we hypothesized that lactate epigenetically regulates tumor cell states in H3K27M DMGs. We undertook a comprehensive and integrated analysis of H3K27M DMG cell lines, animal models, tumor tissues, and magnetic resonance spectroscopy in patients to decipher the epigenetic roles of lactate. Here, we report that H3K27M cells uniquely exhibit elevation of the activating mark H3K27Lac. We observed significant heterogeneity in lactate levels within tumors, and the addition of exogenous lactate resulted in marked increases in H3K27Lac and the expression of critical OPC- and MES- like gene signature genes, which correlates with poorer patient outcomes. Our therapeutic approach leverages the inhibition of CBP/p300, which deposits both H3K27Lac and H3K27ac. Targeting CBP/p300 effectively reduces both H3K27Lac and H3K27ac levels, suppresses key OPC- and MES-like signatures, and shows efficacy in preclinical models. Collectively, our findings identify lactate as a key player in the epigenetic regulation of tumor cell state heterogeneity and suggest that targeting this pathway offers promise for addressing the heterogeneous complexities of H3K27M DMGs.

Neuroblastoma is the most common extracranial solid tumor of childhood. Its clinical behavior ranges from spontaneous regression to lethal, treatment-refractory disease. Aberrant 2,6-sialylation contributes to aggressive phenotypes in many cancers, but the role of ST6Gal2, a neural-enriched 2,6-sialyltransferase, in neuroblastoma is largely unexplored. Here, we examine the clinical and functional significance of ST6Gal2 in neuroblastoma. In two independent public cohorts (SEQC, n=498; Kocak, n=649), high ST6GAL2 expression was associated with significantly worse overall and event-free survival. In the SEQC cohort, ST6GAL2 expression was higher in high-risk and MYCN-amplified tumors, varied across International Neuroblastoma Staging System stages, and correlated positively with a mesenchymal transcriptional signature (Spearman {rho}=0.181). The mesenchymal correlation was reproduced in the Kocak cohort ({rho}=0.204). Stable shRNA-mediated knockdown of ST6GAL2 in SK-N-AS and SK-N-BE(2) cells reduced proliferation and viability, impaired wound closure, and decreased migration and invasion. In preliminary experiments in SK-N-AS cells, ST6GAL2 knockdown reduced binding of Sambucus nigra agglutinin, consistent with a role for ST6Gal2 in 2,6-sialylation. Together, these findings link ST6Gal2 expression to aggressive clinical and transcriptional features and pro-tumorigenic phenotypes in neuroblastoma and nominate ST6Gal2-mediated sialylation as a candidate pathway for mechanistic study.

Background: While immunotherapies have transformed outcomes in several adult cancers, more than half of pediatric patients with high-risk neuroblastoma fail to achieve sustained responses, resulting in poor outcomes. Therefore, understanding the phenotypic and spatial organization of immune cells within the neuroblastoma tumor microenvironment is essential to identify mechanisms driving immunotherapy failure. Methods: To identify spatial determinants of the neuroblastoma immune landscape that may contribute to treatment failure, we dissected immunoregulatory mechanisms at single-cell and protein levels in neuroblastoma samples from 28 patients, using imaging mass cytometry (IMC, n=14) and high-dimensional spectral flow cytometry (n=18). Results: We characterized four distinct cellular neighborhoods, ranging from tumor-dominant to stromal and immune niches. Macrophages were broadly distributed across these niches. In contrast, T and B cells were largely confined to immune niches, forming immature tertiary lymphoid structure-like aggregates within the tumor and stromal cores. T cells displayed a CD69+ tissue-resident memory phenotype, with an overall reduced cytotoxic profile (in CD8+ T cells) and increased expression of inhibitory receptors (predominantly in CD4+ T cells), indicative of dysfunctional features. Intriguingly, T cell functional states differed between spatial niches, with a TCF7+/Ki-67+ self-renewing profile within immune niches, and increased granzyme B expression in CD8+ T cells in tumor cores. Interaction analysis pinpointed CD163+ macrophages as the dominant immunosuppressive interaction partner of T cells, particularly within the tumor edge and immune cores. Analysis of inhibitory ligand-receptor interactions identified the TIGIT/PVR/Nectin-2 axis as the main engaged axis between CD163+ macrophages and T cells. Nectin-2 expression on macrophages was particularly high within immune cores, and elevated in macrophages with direct T cell contact, suggesting a high probability of in situ engagement. Consistent with this immunosuppressive role, both CD163 and Nectin-2 expression on macrophages correlated with poor patient survival, substantiating the clinical relevance of inhibitory macrophage-T cell interactions. Conclusions: This study reveals T cell-excluded tumor niches and targetable inhibitory interactions between CD163+ macrophages and T cells. These insights provide a rationale for combinatorial immunotherapeutic strategies that both enhance T cell infiltration into tumor-rich areas and disrupt inhibitory signaling to improve immunotherapy efficacy in neuroblastoma.

Supratentorial ependymomas (ST-EPNs) are aggressive pediatric brain tumors predominantly driven by the recurrent ZFTA-RELA fusion, an oncogenic chromatin regulator that establishes an aberrant transcriptional program. Although metabolic pathways modulate epigenetic regulation by controlling the availability of metabolic cofactors, how ZFTA-RELA coordinates metabolic reprogramming with epigenetic regulation is not well understood. Here, we show that ZFTA-RELA-positive ependymomas exhibit enhanced methionine dependency, elevated adenosyl homocysteinase (AHCY) expression, and epigenetically control aspartate metabolism for tumor growth. Mechanistically, ZFTA-RELA upregulates AHCY, increasing cellular methylation potential which promotes H3K4me3-dependent expression of CAD, the first enzyme complex of de novo pyrimidine biosynthesis, while simultaneously enforcing H3K27me3 mediated repression of ASNS, which encodes asparagine synthetase. Through this coordinated epigenetic program, aspartate utilization is directed toward pyrimidine biosynthesis while endogenous asparagine production is limited. Restricting S-adenosylmethionine availability or genetically or pharmacologically inhibiting AHCY reverses these epigenetic states, leading to CAD downregulation, ASNS upregulation, impaired nucleotide synthesis, and suppression of tumor growth in vitro and in vivo. Similarly, EZH2 inhibition also restores ASNS expression and suppresses tumor proliferation. These findings suggest that ZFTA-RELA EPN maintain ASNS-low phenotype which conditionally make them dependent on extracellular asparagine. We further show that enhanced de novo pyrimidine synthesis not only supports nucleotide production but also provides UDP-sugars required for N-glycosylation and stabilization of the asparagine transporter SLC1A5, thereby promoting extracellular asparagine uptake and tumor proliferation. Collectively, our findings identify AHCY as a critical metabolic regulator of epigenetic and metabolic reprogramming that drives pyrimidine synthesis and establishes extracellular asparagine dependency, revealing actionable metabolic vulnerabilities in ZFTA-RELA-positive ST-EPNs.

Background: The Central Brain Tumor Registry of the United States (CBTRUS) uses a histology grouping model based on the World Health Organization (WHO) to classify cancer registry records for clinically relevant statistical reporting. Methods: CBTRUS, together with its consulting neuropathologists, reviewed and aligned its histology groupings with the 2021 WHO Classification update. Obsolete histology nomenclature along with their International Classification of Diseases for Oncology 3rd edition (ICD-O-3) codes were identified, currently included histologies were reclassified as appropriate, and new codes found in 2021 WHO were incorporated. An evaluation was conducted of the frequency of histology codes affected in the reclassification process, as well as incidence and survival pre- and post- reclassification of CBTRUS Tumor Type Groupings. Results: After review of the CBTRUS grouping scheme, 205 codes were reclassified and 2 new codes were incorporated. Tumor type groups most affected were those classified as glioma. Reclassification resulted in 167,038 of 491,804 (34%) cases having a grouping reclassification or reporting change, indicating that the 2021 revision of the WHO Classification has significantly impacted the collection and reporting of primary b CNS tumors. Conclusion: This work demonstrates the need to be responsive to changes in classification and coding for statistics to reflect real-world clinical use. This will require collaboration from all parties to have the ability to reconcile clinical practices and surveillance requirements.

Glioblastoma (GBM) is characterized by near-universal recurrence and resistance to therapy. Although tumor-intrinsic mechanisms of radiation resistance have been extensively studied, how the extracellular matrix influences signaling in radiation-resistant GBM remains poorly understood. Here, we investigated discoidin domain receptor 1 (DDR1), a collagen-activated receptor tyrosine kinase, using matched radiation-sensitive parental and radiation-resistant patient-derived GBM models generated through serial in vivo irradiation. Within a three-dimensional collagen I matrix, radiation-resistant GBM cells exhibited distinct temporal patterns of Ki-67 positivity and ERK1/2 phosphorylation compared with parental cells. Despite increased DDR1 expression, radiation-resistant cells exhibited markedly reduced DDR1 phosphorylation and enhanced ectodomain shedding, characterized by accumulation of the membrane-associated C-terminal fragment and release of the N-terminal fragment. DDR1 depletion reduced ERK1/2 phosphorylation, supporting its contribution to ERK1/2 signaling under these conditions. ADAMTS1 was markedly upregulated in radiation-resistant cells, and its knockdown reduced DDR1 ectodomain shedding while increasing full-length DDR1 abundance, ERK1/2 and RB phosphorylation, cyclin D1 expression, and Ki-67 positivity. Together, these findings reveal a distinct DDR1 regulatory state in acquired radiation-resistant GBM, characterized by enhanced ADAMTS1-associated ectodomain shedding, reduced receptor phosphorylation, and attenuated proliferative signaling within a three-dimensional collagen I matrix.

Background Gliomas are primary brain tumors that integrate into the surrounding brain via neuron-glioma synapses and disrupt normal neuronal functioning. Recent work links tumor-brain connectivity to clinical measures such as patients' functional status. However, few experimental systems allow the visualization of tumor and resident brain cell architecture together in human tissue at the scale needed to connect cellular organization to clinical outcomes. To address this gap, we developed an experimental framework for investigating the neuron-glioma network in organotypic human cortex from glioma patients, designed to support future correlation of cellular architecture to clinical outcomes such as functional status and survival. Methods Brain tissue samples containing cortex from seven glioma patients were collected during tumor resection and cultured for seven days as organotypic slices. We established several multiplexed immunohistochemistry panels to stain for glioma cells, various types of resident brain cells including neurons and cells from the oligodendrocyte lineage, as well as axonal networks and myelination patterns. Results We demonstrated the presence of tumor cells in all samples using SOX2, a tumor cell marker. NeuN+ neuronal cells were also successfully identified, as were cells from the oligodendrocyte lineage using SOX10 (pan-lineage marker), BCAS1 (pre-myelinating oligodendrocyte marker), and ASPA (mature oligodendrocyte marker). The neuronal network and myelin were successfully visualized using MAP2 (neurons and dendrites), SMI312 (axons), and PLP1 (myelin). Conclusions High-quality visualization of tumor cells, resident brain cells and the (myelinated) neuronal network in organotypic human cortex from glioma patients is possible after seven days of culturing. Future studies could further optimize culture conditions and should study stability of all cellular components in longer term slice cultures. Preliminary analyses indicate that cellular measures derived from human organotypic slices can be linked to patients' functional status, demonstrating the capacity to generate data suitable for large-scale investigation of the effects of glioma-brain network architecture on clinical outcomes.

Tissue immune measurements can guide immunotherapy only if one biopsy represents the rest of the tumor. We quantified this in a secondary analysis of public data from 75 magnetic resonance imaging (MRI)-localized glioblastoma (GBM) biopsies from 19 patients. Fourteen transcriptomic modules scored supportive, inhibitory, and checkpoint programs. The intraclass correlation coefficient (ICC) gave the share of variance between patients rather than regions, before and after removing the component explained by a 12-gene leukocyte composite. An ICC of 1 indicates no variation in module scores among biopsies within each patient, whereas an ICC of 0 indicates that biopsies from the same patient are no more similar, on average, than biopsies from different patients. Two hepatocellular carcinoma (HCC) cohorts and a microdissected GBM atlas served as comparisons. CD8 had the lowest unadjusted ICC among abundance scores (0.42; 95% patient-bootstrap interval, 0.10 to 0.68; leukocytes, 0.65). Within patients, CD8 correlated with supportive and inhibitory modules alike (unadjusted mean r, 0.37 and 0.36) but not with major histocompatibility complex class I (MHC-I; r = 0.06). Leukocyte adjustment lowered the median module ICC from 0.56 to 0.34 but interferon (IFN) only from 0.74 to 0.70. IFN had the highest adjusted ICC and ranked first in 86% of patient-bootstrap resamples (post hoc). MHC-I was second (0.47) and not separable from IFN (difference 0.23; interval, -0.04 to 0.43); after purity residualization in 67 biopsies, IFN and MHC-I were similar (0.66 and 0.62). Median adjusted ICCs were 0.34 in bulk GBM, 0.62 and 0.74 in HCC, and 0.08 in microdissected GBM. In bulk GBM, only IFN, and MHC-I after purity residualization, had adjusted ICC point estimates above 0.5. The data support multiregion sampling with CD8, IFN, MHC-I, and checkpoint readouts from the same regions.

Isocitrate dehydrogenase (IDH) is the most frequently mutated metabolic enzyme in human cancers. Mutant IDH produces the oncometabolite D-2-hydroxyglutarate (D-2HG), which promotes tumorigenesis in part through epigenetic alterations. Beyond its cell-autonomous effects, tumor-derived D-2HG acts as a potent immunosuppressant that establishes an immune-cold tumor microenvironment. However, the mechanism by which D-2HG is released from tumors remains largely unknown. Here, we identify the SWELL1 (LRRC8A)/LRRC8C-containing volume-regulated anion channel (VRAC) as a principal pathway for D-2HG efflux from IDH-mutant cells. Genetic deletion of SWELL1, the essential VRAC subunit, markedly reduces D-2HG release and reverses associated immunosuppression in an orthotopic mouse model of IDH-mutant glioma. Strikingly, loss of VRAC causes intracellular accumulation of D-2HG, which paradoxically limits tumor cell proliferation by driving epigenetic remodeling and mitochondrial metabolic stress. Pharmacological inhibition of VRAC with dicumarol suppresses IDH-mutant glioma growth, enhances intratumoral T cell activation, synergizes with immune checkpoint blockade, and prolongs survival in tumor-bearing mice. In human IDH-mutant lower-grade gliomas, elevated LRRC8C expression correlates with DNA hypermethylation and an immunosuppressive tumor microenvironment (TME), and predicts poor overall survival. Together, these findings establish VRAC-mediated D-2HG export as a central mechanism regulating both immune evasion and tumor cell fitness, uncovering new therapeutic opportunities across IDH-mutant cancers.

Glioblastoma (GBM) exhibits extensive loss of circular RNAs (circRNAs), yet the mechanisms driving this depletion and its functional consequences remain unclear. Here, we identify the splicing co-factor SON as a previously unrecognized suppressor of circRNA biogenesis in GBM. CircRNA sequencing revealed widespread increases in circRNA abundance following SON knockdown. Conversely, elevated SON expression in GBM, with further increases in radiation-resistant models, was associated with widespread circRNA loss, including reduced expression of circUSP1 and circSUCO. SON knockdown increased circRNA abundance and attenuated activation of the dsRNA-responsive kinase PKR and downstream NF{kappa}B signaling, whereas radiation-resistant GBM cells exhibited enhanced PKR/NF{kappa}B activation that was attenuated by SON depletion. Importantly, overexpression of the SON-suppressed circRNAs circUSP1 and circSUCO reduced PKR activation and selectively decreased clonogenic survival following irradiation in radiation-resistant GBM cells, with little effect on parental cells. Together, these findings identify a previously unrecognized SON/circRNA/PKR regulatory pathway linking RNA processing to acquired radiation resistance in GBM and highlight specific PKR-inhibitory circRNAs as potential therapeutic tools for resensitizing radiation-resistant GBM to radiotherapy.