Journal of Neuro-Oncology

Background. Glioma surgery requires balancing maximal tumor resection against preservation of neurological and cognitive function. Intraoperative direct electrical stimulation guides this balance but is invasive and not always feasible. Preoperative magnetoencephalography (MEG) can identify tumor regions with high functional connectivity (HFC) to the rest of the brain. These HFC areas contain more malignant glioma cells and relate to poorer short-term outcome when resected. We aimed to determine whether HFC voxel resection relates to postoperative neurological and cognitive outcomes. Methods. In this preregistered analysis, 54 adults with diffuse glioma underwent resting-state MEG before resection. Imaginary coherence identified voxels with significantly higher connectivity against their contralateral controls, and these HFC voxels within the resection cavity were counted. Neurological status was assessed at short-term (~1 week) and long-term (~1 year) follow-up, and neuropsychological assessment took place at baseline and long-term follow-up. Logistic regressions related resected HFC voxels to decline at each timepoint, adjusting for Karnofsky performance status, sex and resectability index. Results. Resected HFC voxels were not associated with neurological decline at short-term (48% declined; odds ratio [OR] 0.98, P = .50) or long-term follow-up (27% declined; OR 0.99). For cognition, more resected HFC voxels were associated with less long-term decline (48% declined; OR 0.85, P = .02); a binarized analysis was consistent (OR 0.076, P = .01), indicating that tumor volume did not drive this result. Conclusions. Resecting more HFC voxels associates with preserved long-term cognition, without apparent neurological cost. Non-invasive MEG connectivity mapping may help optimize the onco-functional balance in glioma surgery.

Background and Objectives: Recurrence following burr-hole drainage of chronic subdural haematoma (cSDH) occurs in 10-25% of cases, sustained by neovascularisation of the subdural neomembrane supplied by the middle meningeal artery (MMA). MMA embolisation reduces recurrence; whether incidental burr-hole intersection of MMA branches during drainage confers similar benefit is unknown. Methods: We performed a multicentre retrospective cohort study of consecutive adults undergoing burr-hole drainage for cSDH at two UK tertiary neurosurgical centres. Postoperative thin-slice CT was used to classify burr-hole intersection of the underlying MMA groove (no hit, distal-branch hit or main-branch hit) and measure perpendicular burr-hole-to-MMA-groove distance. Co-primary outcomes were radiological recurrence and recurrence requiring intervention. Patient-clustered multivariable logistic regression adjusted for prespecified clinical covariates and treating site. Results: 227 patients (284 operated hemispheres) were included. Radiological recurrence decreased from 34.4% with no branch hit to 22.9% with main-branch intersection, with the gradient confined predominantly to unilateral cSDH. Main-branch intersection was associated with lower adjusted odds of radiological recurrence in unilateral cSDH (adjusted OR 0.30, 95% CI 0.11- 0.81; P = .018), with a similar but non-significant association in the overall cohort (adjusted OR 0.53, 95% CI 0.26-1.07; P = .075). Burr-hole-to-MMA-groove distance demonstrated a more consistent association: in the overall cohort, each 5-mm increase independently increased the odds of radiological recurrence (adjusted OR 1.38, 95% CI 1.04-1.82; P = .025). In unilateral cSDH, each 5-mm increase was independently associated with both radiological recurrence (adjusted OR 1.45, 95% CI 1.03-2.04; P = .034) and recurrence requiring intervention (adjusted OR 1.52, 95% CI 1.05-2.20; P = .027). Conclusion: Main-branch intersection of the middle meningeal artery during routine burr-hole surgery is associated with lower recurrence of unilateral cSDH, while the accompanying burr-hole-to-MMA-groove distance gradient provides biologically plausible support for a dose-response relationship. Together, these findings provide mechanistic rationale for prospective evaluation of intentional neuronavigation-guided MMA targeting (BURR-MMA; NCT07549893).

Background. Hematoporphyrin derivative (HpD, Hiporfin) is used for photodynamic therapy (PDT) of solid tumors but had never been evaluated for fluorescence guided resection (FGR) in glioma. We assessed Hiporfin fluorescence accuracy against histopathology in recurrent glioblastoma (rGBM) and the feasibility of adding cavity PDT to FGR. Methods. In this prospective single arm series, adults with rGBM received a single preoperative intravenous Hiporfin dose of 5 mg/kg before FGR under blue light excitation and 630 nm cavity PDT. Intraoperative fluorescence assessment was the index test and histopathology the reference standard. Specimen level diagnostic accuracy was pooled with bivariate random-effects modeling. Results. Of 63 specimens, 35 were true positive, 7 false positive, 8 false negative and 13 true negative. Pooled sensitivity was 0.751 (95% confidence interval 0.600 to 0.858) and specificity 0.606 (0.407 to 0.775); the crude positive predictive value was 0.833 (0.694 to 0.917) and the negative predictive value 0.619 (0.409 to 0.792). All seven false positive specimens were peritumoral tissue and the eight false negatives were non fluorescent tumor. FGR combined with cavity PDT was completed in all six patients without new neurological deficits. Conclusions. Hiporfin fluorescence identified rGBM with moderate accuracy: fluorescent regions were likely tumor, while non fluorescent tissue often still contained tumor. The main source of error was peritumoral tissue of the treated cavity, and not the drug or the optics. Hiporfin is a new fluorescence agent for glioma. One drug provides both fluorescence guidance during resection and immediate cavity PDT.

Glioblastoma (GBM) is increasingly recognized as a disease that perturbs and interacts with distributed brain networks, yet its imaging-based prognostic assessment remains largely focused on local tumor extent. Here, we ask whether brain-wide tumor involvement provides a more robust representation of prognostic burden than conventional tumor volume. We analyze preoperative imaging from 999 patients with newly diagnosed GBM across multiple institutions, comparing volumetric measures with the lesion-tract density index (L-TDI), a tractography-derived measure of tumor involvement of white-matter pathways. Systematic survival analyses across tumor compartments and stratification thresholds reveal that the prognostic value of tumor volume is strongly dependent on analytical design and is largely confined to the contrast-enhancing compartment. In contrast, L-TDI shows robust associations with overall survival across thresholds and tumor compartments, outperforming volumetric measures and retaining prognostic value after adjustment for established clinical variables. These findings identify brain-wide tumor involvement as a robust dimension of GBM prognosis and delineate the conditions under which conventional volumetric measurements provide reliable survival information. These results highlight the prognostic relevance of considering GBM as a brain-wide disease, beyond its local anatomical extent.

Increasing evidence indicates that neurotransmitter signaling and neuronal interactions are important determinants of glioma biology. However, the clinical and biological significance of serotonin (5-hydroxytryptamine; 5-HT) receptor expression in lower-grade glioma (LGG) remains poorly understood. Here, we investigated G protein-coupled 5-HT receptor genes in LGG using transcriptomic and clinical data from The Cancer Genome Atlas (TCGA-LGG) and Chinese Glioma Genome Atlas (CGGA) cohorts. Initial survival screening identified HTR1A, HTR2A, HTR2C, and HTR6 as the genes most consistently associated with longer overall survival (OS). Multivariable Cox regression further identified HTR2A and HTR6 as independently associated with longer OS after adjustment for age, sex, tumor grade, and IDH/1p19q molecular subtype. Expression of the four genes was preferentially associated with molecular features of less aggressive gliomas, particularly IDH-mutant tumors. Single-cell RNA-sequencing (scRNA-seq) data supported malignant glioma cells as a major source of their expression, while cell-type deconvolution revealed strong positive associations with neuronal enrichment and inverse associations with stromal and immune signatures. Transcriptome-wide co-expression and Gene Ontology analyses showed that all four receptor genes were associated with neuronal and synaptic programs involving neurotransmitter release, synaptic vesicle function, ion channels, and synaptic signaling. These transcriptional programs were particularly coherent in IDH-mutant gliomas and more heterogeneous in IDH-wildtype tumors. Together, these findings identify a subset of 5-HT receptor genes associated with favorable clinical and molecular features in LGG and suggest that their expression may mark a neuronal/synaptic differentiation state, particularly within IDH-mutant gliomas.

Background: Glioblastoma, isocitrate dehydrogenase wildtype, central nervous system World Health Organization grade 4, is the most common primary malignant brain tumor in adults and remains associated with poor prognosis despite aggressive multimodal treatment. The gut-brain axis has been proposed as a modulator of glioblastoma biology, but human data are scarce and the gut mycobiome has not been characterized in these patients. We characterized both bacteria and fungi at diagnosis as well as the intratumoral myeloid compartment and survival. Methods: In a prospective case-control study, we sequenced bacterial (16S rRNA gene) and fungal (internal transcribed spacer, ITS) communities from rectal swabs of 23 patients with newly diagnosed glioblastoma and 23 healthy controls. Additionally, we quantified the intratumoral myeloid compartment by immunofluorescence and performed survival analysis. Results: Both bacterial and fungal communities were more diverse in patients than in controls (Shannon, 16S p < 0.001; ITS p = 0.004), and even more diverse in patients with shorter survival. Community structure itself was unchanged, locating the difference at the level of individual taxa. Fourteen of 218 bacterial and archaeal taxa and four of 102 fungal species differed at nominal, uncorrected p [&le;] 0.05. Members of the Lachnospiraceae and Ruminococcaceae were reduced in glioblastoma patients, while the methanogenic archaeon Methanobrevibacter and Nakaseomyces glabratus (formerly Candida glabrata) were enriched. Survival was also addressed at the tissue level. In the tumor, a higher density of ionized calcium-binding adapter molecule 1-positive myeloid cells was associated with longer survival, independently of age, performance status and extent of resection (hazard ratio 0.82 per percentage point, 95% confidence interval 0.67-0.99). Conclusions: This study characterizes the gut bacteriome and mycobiome of newly diagnosed glioblastoma patients and identifies candidate taxa, among them several butyrate- and propionate producers within the depleted Lachnospiraceae and Ruminococcaceae. Combining these observations with the established role of short-chain fatty acids in microglial maturation, we hypothesize that the myeloid compartment is the interface at which the gut-level and tissue-level findings converge.

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)-mutant gliomas represent most lower-grade diffuse gliomas in young adults. Although IDH-mutant gliomas initially behave indolently relative to IDH-wildtype counterparts, they undergo inevitable malignant transformation. The mechanisms driving this progression remain poorly defined. We analyzed a unique longitudinal cohort of two patients with IDH-mutant diffuse gliomas, each with matched WHO grade 2, 3, and 4 tumours. Spatial transcriptomics (NanoString GeoMx and 10X Visium), combined with differential expression, spatially variable gene analysis, and gene ontology enrichment, were used to define transcriptional programs underlying progression. Unexpectedly, across both patients, higher-grade tumours demonstrated reduced proliferation at the infiltrative margin as measured by Ki67. Spatial transcriptomic analysis identified an enrichment of invasion-associated genes associated with increasing tumour grade. In an independent bulk RNA-seq cohort, expression of this spatially derived program did not outperform grade-only PFS or OS analysis. These observations underscore the need to develop new metrics that account for spatial evolution that occurs across progression, but escapes detection in bulk analyses and provide the rationale for prioritizing migration-, cytoskeletal- and stress-associated programs for further investigation.

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.

Glioblastoma (GBM) is a highly aggressive brain tumor with an extremely poor 5-year survival rate of 6.9%, largely attributable to the lack of reliable biomarkers. While competing endogenous RNA (ceRNA) and copy number variation (CNV) analyses offer unique biomarker identification potential, current approaches neglect the integration of multiple regulatory mechanisms for biomarker detection. To address this limitation, we applied relational graph convolutional networks (RGCNs) to ceRNA and CNV knowledge graphs through a novel late fusion ensemble architecture. The proposed architecture outperformed baseline models and identified five novel biomarkers, including hsa-miR-196a and hsa-miR-224. Kaplan-Meier survival analysis and Cox regression indicated that the identified genes hold significant prognostic and diagnostic power. The early stratification of the Kaplan-Meier curves indicates the potential these genes hold for patient survival prediction. The results illustrate that a late fusion RGCN ensemble effectively captures complex gene interactions, overcoming limitations of existing models and providing a framework for biomarker discovery. The novel biomarkers serve as prospective targets for future GBM therapeutic development and candidates for non-invasive diagnostic assays.

Glioblastoma (GBM) is the most common primary brain tumour, with a median survival of 12-18 months, highlighting a need for new treatment targets. We observed that pannexin 1 (PANX1), a channel-forming glycoprotein important in purinergic signalling, is upregulated in GBM compared to normal tissue and expressed throughout patient tumours. Western blot analysis of patient-derived GBM cell lines revealed significantly increased PANX1 expression in these primary lines compared to brain tissue and control glial cells. Bulk RNA-sequencing compared the gene expression of GBM cells devoid of PANX1 via CRISPR/Cas9 deletion (PANX1-KO) compared to controls. Gene Ontology and KEGG gene set analyses revealed PANX1-KO in GBM cells affects cell surface and cell junction components, processes, and pathways, including the HIPPO pathway, in addition to critically downregulating {beta}-catenin mRNA and other components of the Wnt pathway. The deletion of PANX1 resulted in a disruption of the {beta}-catenin protein and a dramatic reduction in migration and cell growth. Pharmacological inhibition of PANX1 in GBM cells with Probenecid (PBN) and Spironolactone (SPIR) demonstrated a significant reduction in live cell numbers and migration via scratch assay. Both blockers dramatically decreased F-actin filament formation, and the cellular localization of beta-catenin became more intracellular compared to controls. Xenografted GBM tumours showed a reduction in tumour cell viability by bioluminescent imaging and reduced hemorrhaging incidence when treated with PBN. These new insights support further investigation of PANX1 as a potential GBM therapeutic target and its role in multiple cancer signaling pathways that regulate this devastating disease.

Background: Long non-coding RNAs (lncRNAs) are emerging regulators of tumor initiation and progression through their effects on stemness, lineage commitment, and transcriptional plasticity. The contribution of lncRNA reprogramming during early p53-deficient gliomagenesis remains poorly understood. Methods: Human induced pluripotent stem cells (iPSCs) carrying a complete TP53 knockout were differentiated into neural progenitor cells (NPCs) and cerebral organoids. Bulk RNA sequencing was performed at multiple differentiation stages and compared with established glioblastoma cell lines (U87 and U118) to identify lncRNA expression changes associated with p53 loss and tumor-like transformation. Results: p53 deficiency induced a progressive and stage-specific remodeling of the lncRNA landscape, with the most pronounced transcriptional shift observed in 40-day NPCs. This late-stage profile partially overlapped with glioblastoma-like cell lines and was characterized by the upregulation of five lncRNAs enriched in both p53-deficient neural models and glioma cells. Principal component analysis showed that these lncRNAs clustered with tumor-associated samples and were strongly co-expressed with mitotic cell-cycle genes. In particular, LINC00973 and LINC01583 were highly expressed in primary glioblastoma datasets, while RP5-875H18.9 and RP11-1094H24.4 emerged as previously unrecognized glioma-associated transcripts. Conclusion: Loss of p53 drives stage-dependent lncRNA reprogramming during neural differentiation and promotes the emergence of glioma-associated transcriptional features. These findings identify candidate lncRNAs that may contribute to early gliomagenesis and represent potential biomarkers or therapeutic targets in glioblastoma.

Glioblastoma is among the most aggressive primary brain tumors, with the invasion of surrounding tissue driven by coordinated remodeling of the extracellular matrix (ECM) by proteolytic enzymes, including the matrix metalloproteinase (MMP) family. The gap junction protein connexin43 (Cx43) has emerged as an unexpected promoter of this invasive phenotype. However, the downstream molecular mechanisms linking Cx43 to ECM proteolysis remain poorly understood. We previously demonstrated selective upregulation of MMP3 within the secretome of Cx43-overexpressing glioma cells, implicating Cx43 in the regulation of ECM protease activity. To identify the molecular intermediaries responsible, we carried out a proteomic survey of gap junction (GJ)-associated signaling networks. Amongst these, we identified a Cx43 protein-protein interaction involving annexin A2 (ANXA2), a known regulator of ECM proteases. SDS-PAGE/Western blot and confocal imaging demonstrated that elevated Cx43 expression drives selective recruitment of ANXA2 to gap junction membranes, forming a junctional signaling platform rather than increasing total ANXA2 abundance. This recruitment coincided with co-localization of S100A10, suggesting assembly of an intact ANXA2/S100A10 complex at gap junctions. Using ANXA2-mimetic peptides targeting the S100A10-binding domain, we demonstrate that disruption of the ANXA2/S100A10 complex leads to a significant reduction in MMP3 activity. Furthermore, in 3D spheroids grown in ECM-replicating gels, ANXA2 peptide treatment significantly reduced the invasive potential of C6-13 (Cx43-overexpressing) cells. Together, these data define a novel Cx43[-&gt;]ANXA2[-&gt;]S100A10[-&gt;]MMP3 signaling axis, advancing our understanding of ECM remodeling in glioblastoma and establishing the ANXA2/S100A10 complex as a promising therapeutic target for disrupting Cx43-driven glioblastoma progression.

BackgroundEpidermal growth factor receptor (EGFR) amplification occurs in [~]50% of IDH-wildtype glioblastoma (GBM) cases, frequently accompanied by expression of the oncogenic EGFRvIII variant. Although EGFR represents an attractive therapeutic target, EGFR-directed therapies have shown limited clinical efficacy in GBM. Resistance to kinase inhibitors is frequently attributed to activation of compensatory signaling pathways ("kinome rewiring"). We therefore investigated whether EGFR inhibition in GBM induces broad adaptive kinase responses that could be co-targeted to overcome resistance. MethodsWe molecularly profiled 29 patient-derived GBM cell lines for EGFR status and selected five representative models spanning EGFR amplification states for functional analyses. Cells were treated with EGFR inhibitors and responses were assessed using viability assays, time-resolved immunoblotting, and phosphoproteomics (LC-MS/MS) with kinase activity inference. ResultsEGFR inhibitors preferentially impaired viability in EGFR-driven models and transiently reduced EGFR phosphorylation during the initial response. However, partial restoration of EGFR phosphorylation and downstream signaling occurred after 24 hours of inhibitor exposure. Phosphoproteomics revealed no evidence of broad kinome rewiring within this timeframe but instead identified increased EGFR abundance, associated with partial restoration of EGFR pathway activity. The phosphorylated-to-total EGFR ratio remained stable, indicating that increased EGFR abundance may enable persistent residual kinase activity despite continued, but incomplete, target inhibition. ConclusionsEarly responses to EGFR inhibition in GBM were not characterized by broad kinome rewiring but by restoration of EGFR signaling associated with increased EGFR abundance. These findings suggest that adaptive signaling remains largely EGFR-dependent despite inhibitor exposure, identifying regulation of EGFR abundance as a potential contributor to therapeutic resistance. Key points- Early responses to EGFR inhibition occur without evidence of broad kinome rewiring. - EGFR signaling is restored during sustained inhibitor exposure. - Increased EGFR abundance is associated with restoration of pathway activity. Importance of the studyAdaptive resistance to EGFR-targeted therapies in GBM is commonly attributed to activation of alternative signaling pathways. Using patient-derived GBM models and phosphoproteomic profiling, we show that early adaptive responses to EGFR inhibition are not characterized by broad kinome signaling rewiring but instead remain centered on reactivation of EGFR signaling. Our findings suggest that increased EGFR abundance in response to inhibitor exposure may enhance residual EGFR signaling sufficiently to partially restore downstream pathway activity. These results indicate that early adaptive responses to EGFR inhibition may remain largely EGFR-dependent, potentially limiting the effectiveness of strategies primarily aimed at co-targeting alternative signaling pathways. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/744581v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@8d4ea3org.highwire.dtl.DTLVardef@125e3eeorg.highwire.dtl.DTLVardef@9742c0org.highwire.dtl.DTLVardef@9f4fa8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: IDH1 mutations in diffuse gliomas undergo epigenetic transformation and metabolic stress, through accumulation of 2-hydroxyglutarate. While these tumours generally exhibit a less aggressive phenotype, significant prognostic heterogeneity persists. The extent to which the One Carbon Metabolism (OCM) network is rewired in IDH1-MT gliomas and its contribution to this heterogeneity remain poorly understood. Methods: We analysed the expression of 64 OCM genes using TCGA and CGGA transcriptomic datasets (n = 709). Composite OCM score, Cohen's d, PCA and driver gene analysis were used to quantify effect sizes between IDH1-WT and MT cohorts. Prognostic relevance was assessed using Kaplan-Meier and Cox regression analyses. OCM metabolic rewiring and dependency were validated using in vitro U87 IDH1-WT and IDH1-R132H mutant glioblastoma cell line models. Furthermore, immune microenvironment interactions were evaluated using CIBERSORTx to dissect OCM-associated correlations. Results: Using transcriptional profiling and targeted in-vitro validation, we reveal a distinct two-state metabolic phenotype governed by the OCM network. We demonstrate that while IDH1-MT tumors primarily rely on a suppressed metabolic phenotype, they are supported by compensatory mitochondrial pathways and an aggressive subset aberrantly hyperactivates specific proliferative OCM genes. This high aggregate OCM score was significantly associated with poor overall survival in the IDH1-MT cohort (CGGA HR = 1.61, p < 0.001), identifying an aggressive subset that predominates the favourable IDH mutation status. Functional validation showed aggressive IDH1-MT cells lose their baseline metabolic flexibility with changes in critical OCM genes and exhibit a strict auxotrophic dependence. Further, we also define that OCM rewiring was also associated with immune cell abundance, suggesting high OCM activity correlating with monocyte infiltration and low M2 macrophage polarisation. Conclusion: Our findings offer a framework to identify high-risk patients and propose translational strategies for IDH1-MT tumors that may be refractory to standard IDH inhibitors. Therefore, the study presents a conceptual model for metabolic precision therapy defining OCM metabolic subsets that distinguish indolent, mitochondrial-dependent tumours from aggressive, proliferation-dependent phenotypes in IDH1-MT gliomas.

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.

Gliomas, particularly glioblastoma (GBM), are aggressive primary brain tumors associated with dysregulated NLR signaling, a pathway central to innate immunity and inflammation. NLRP1 triggers proinflammatory cytokine release and pyroptotic cell death via autoproteolytic cleavage. The FIIND missense variant rs11651270 (M1184V) may modulate this cleavage process. While NLRP1 polymorphisms are associated with various diseases and cancers, their specific impact on glioma remains to be investigated. In our study, five FIIND-domain single-nucleotide polymorphisms (SNPs) of NLRP1 rs371579423, rs58604457, rs57636751, rs11651270, and rs2301583, were investigated by Sanger sequencing in a clinical cohort of glioma patients and compared against population-matched controls from the GenomeIndia dataset (Rajasthan cohort) using genetic association models. Molecular dynamics simulations were performed to evaluate the structural effects of the missense variant rs11651270 (M1184V) in NLRP1 during pre- and post-cleavage states. The linked variants rs58604457 (G>A) and rs57636751 (C>T) exhibited complete linkage disequilibrium (r2=1.00) and were significantly associated with lower odds of glioma (odds ratio ~0.5). The missense variant rs11651270 (T>C) showed no association with glioma risk. Notably, the SNP rs2301583 had a higher allelic frequency in glioma cases despite being absent in the GenomeIndia population-based control catalogue. Molecular dynamics simulations revealed that the M1184V substitution stabilizes local FIIND architecture by preserving {beta}-strand organization through persistent interactions with neighboring residues. This study provides the first combined genetic and structural analysis of NLRP1 FIIND-domain variants in an Indian glioma cohort. These findings illustrate the potential value of integrating population-based genetic association with structural modelling to generate hypotheses and uncover potential functional mechanisms of inflammasome-gene variants in neuro-oncology.

Objective: To evaluate whether entrepreneurial methodologies applied to system-wide healthcare redesign were associated with improved survival, care timeliness, and rural-urban equity among patients with glioblastoma. Design: Non-randomized pre-post cohort study Setting: Tertiary neuro-oncology centre in Ontario, Canada Participants: Adults aged >18 years with histologically confirmed glioblastoma who underwent surgical resection between January 1, 2018, and February 28, 2025 Interventions: Implementation of the Integrative Brain Tumor Program (IBTP), a system-wide care intervention grounded in user-defined priorities and developed using a design thinking approach (empathize, define, ideate, prototype, test) integrated with operational frameworks adapted from early-stage innovation. System change was treated as a deliberate, deployable intervention that could be designed, launched, iteratively refined, and evaluated. Coordinated changes were embedded across healthcare services within existing infrastructure and resource constraints through a single centralized nurse navigator who standardized referrals, patient education, and real-time care coordination. Main Outcomes and Measures: Primary outcomes were one-year overall survival and time to postoperative MRI completion and radiotherapy initiation. Secondary outcomes assessed rural-urban equity in these measures. Associations were evaluated using Cox proportional hazards and Fine-Gray competing-risk models adjusted for age, sex, rurality, MGMT promoter methylation status, and calendar time. Results: Among 297 patients (244 pre-implementation, 53 post-implementation), baseline demographic and tumor characteristics were similar across cohorts. One-year overall survival was higher in the post-implementation cohort (60.4% vs 42.2%), corresponding to an adjusted hazard ratio of 0.61 (95% CI, 0.38-0.99). Postoperative MRI completion within 48 hours increased from 45.9% to 66.0% (adjusted cause-specific hazard ratio, 1.50; 95% CI, 1.05-2.14) with similar improvements observed at 7 days. Time to radiotherapy initiation did not differ between cohorts. Survival and MRI timeliness did not differ by rural or urban residence in either period, though rural radiotherapy delays were attenuated postimplementation. Conclusions: Systematic application of entrepreneurial methods to health system redesign was associated with clinically meaningful improvements in glioblastoma survival and care timeliness using minimal resources (single nurse navigator). These findings suggest that treating system change as an intervention grounded in user-defined priorities and oriented toward integrated systems rather than sequential process optimization can support sustainable transformation of complex, coordination-dependent care pathways and warrants evaluation in other disease settings.

DNA methylation-based classification is increasingly a cornerstone of CNS tumor diagnostics. Recently,nanopore sequencing enables very rapid methylation profiling to support surgical decision making. Time-constrained sequencing yields sparse methylation profiles, necessitating specialized classifiers for accurate predictions. All publicly available sparse-profile classifiers rely on a single reference set of only 2,801 samples published in 2018 and its accompanying classification system. Here we present Sturgeon V2, trained on nearly 10,000 methylation profiles assembled from a range of public repositories and collaborations. To harmonize differences in tumor class labels, we developed an iterative semi-supervised relabeling strategy with a two-tiered concordance filter. We demonstrate that this filtering is essential for optimal performance. Compared to Sturgeon V1, we further redesigned the classifier architecture to provide more stable, and consistent confidence estimates while accumulating sequencing data and we introduce an updated class system including new classes, such as meningioma subtypes, PATZ- fusion neuroepithelial tumors and PLAG1/2-amplified embryonal tumors. In a retrospective intraoperative cohort of 94 pediatric cases, Sturgeon V2 was confidently correct in 85 cases (Sturgeon V1: 78) with one confident misclassification, and in simulated sequencing runs (n=1880) reduced the error rate among confident calls from 7.1% to 2.8%, outperforming publicly available alternatives. Sturgeon V2 is the first openly available, locally executable classifier for sparse methylation profiles trained beyond the 2018 reference era.

Objective To estimate the mortality proportion among patients with severe traumatic brain injury (sTBI) and to assess variation in mortality according to geographical region, study characteristics, patient populations, and mortality assessment time points. Design Systematic review and meta-analysis. Data sources PubMed/MEDLINE, Embase, Scopus, Web of Science, Google Scholar, and Cochrane Library were searched from database inception to January 2026. Eligibility criteria Studies reporting mortality among patients with severe TBI were eligible. Severe TBI was defined as a Glasgow Coma Scale (GCS) score of [&le;]8, equivalent to GCS <9 or GCS 3-8. Studies involving mixed TBI severity were included only when data for patients with severe TBI could be separately extracted. Data extraction and synthesis Two reviewers independently screened studies, extracted data, and assessed methodological quality. Mortality proportions were pooled using a random-effects model. Statistical heterogeneity was assessed using Cochran's Q and the I2 statistic. Subgroup and sensitivity analyses were undertaken where sufficient data were available. Results Twenty-five studies comprising 12,829 patients with severe TBI were included in the systematic review. Across studies, 5,550 deaths were reported. Mortality proportions ranged from 17.5% to 71.1%. The pooled mortality proportion was 40.6% (95% CI 35.7% to 45.7%), with substantial heterogeneity (I2=96.8%, p<0.001). Mortality varied considerably according to geographical region and timing of outcome assessment, with higher estimates generally observed in African settings and at longer follow-up periods. Conclusion Mortality among patients with severe TBI remains substantial, with approximately two in five patients dying across the included studies. However, the very high heterogeneity indicates that the pooled estimate should not be interpreted as a universal mortality risk. Differences in healthcare resources, patient characteristics, injury severity, management practices, and mortality assessment time points may contribute to the observed variation.