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. 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.

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.

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.

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.

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 Chronic subdural hematoma (CSDH) is common and increasing in incidence. Short-term post-surgical outcomes are good, but longer term, patients experience increased morbidity and early mortality than controls and the general population. Deprivation and ethnicity affect many health conditions, but to our knowledge, this is the first UK study investigating their association with CSDH outcomes. Methods This single-centre retrospective cohort study used Secondary Uses Service Admitted Patient Care Spells (SUS+ APCS) data. Adult subdural hematoma (SDH) patients admitted to St Georges Hospital, London treated with burr-hole surgery between April 2022 and April 2025 were included. Primary outcomes were initial length of stay (LoS) and days spent hospitalised in the subsequent year. Deprivation was measured using Index of Multiple Deprivation (IMD). Linear regression models estimated initial LoS and subsequent days spent hospitalised by category, adjusted for factors including age, ethnicity and comorbidities. Results In this single-centre cohort of 449 patients, IMD was not associated with initial LoS, but was significantly associated with subsequent time spent hospitalised. In a log-transformed linear regression model, the most deprived spent 4.5-times longer hospitalised during the year after CSDH than the least deprived (fold change = 4.45, 95% confidence interval (95%CI) 2.20 to 9.01). Conclusions Higher neighbourhood-level deprivation was associated with time spent hospitalised following CSDH. This was robust to adjustment for potential mediating factors. This is the first UK study demonstrating health inequalities in CSDH outcomes, highlighting a need for future research to gain further insights, and to improve equity of outcomes after CSDH.

Purpose: Existing prognostic models for brain metastases from lung cancer rely on clinical variables and do not capture the biological heterogeneity of host or tumor factors underlying survival outcomes. We evaluated whether MRI-derived radiomic biomarkers improve prognostic stratification beyond clinical variables. We also investigated their biological correlates using matched histopathology. Experimental Design: This retrospective study included 103 patients with brain metastases from lung cancer and matched pre-operative MRI and histopathology. Radiomic features from T1-weighted contrast-enhanced and T2-weighted fluid-attenuated inversion recovery sequences were combined with clinical variables to train Cox proportional hazards, random survival forest, and DeepSurv models for overall survival (OS), CNS progression, and leptomeningeal (LM) progression. Risk groups from an imaging-only model were compared for clinical and histopathologic differences. Results: Imaging features improved prognostic performance beyond clinical variables across all modeling frameworks, with the combined clinical-imaging DeepSurv model achieving the highest concordance index for OS (0.728). Risk groups from the best-performing model for each endpoint differed significantly in OS (median 26.7 vs. 9.1 months, p < 0.001) and LM progression (p = 0.003), but not in CNS progression. Imaging-only risk groups did not differ significantly in any clinical variable, but high-risk patients were less likely to have tumor-infiltrating lymphocytes (46% vs. 77%, p = 0.010) and PD-L1 expression [&ge;] 50% (10% vs. 32%, p = 0.049). Conclusions: MRI radiomic biomarkers provide prognostic information beyond established clinical variables in lung cancer brain metastases and correspond to features of the tumor immune microenvironment, supporting their potential to refine risk stratification.

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.

Objectives: Seizure freedom follows surgery in 67% of children with drug-resistant epilepsy, but imaging biomarkers could improve patient selection, epileptogenic zone localisation, and outcome prediction. We assessed whether diffusion MRI abnormalities predicted seizure freedom when resected; aligned with stereo-EEG seizure-onset and interictal zones; distinguished patients with/without an identified onset zone; and localised the epileptogenic zone in MRI-negative cases. Methods: We retrospectively studied children undergoing focal resection (n=110) or stereo-EEG (n=61) between 2015-2023, alongside healthy controls aged 6-18 (n=69), using identical 3T MRI diffusion protocols. Age- and sex-adjusted normative models generated mean diffusivity (MD) and fractional anisotropy (FA) voxelwise z-score maps to identify dominant abnormalities. In resection patients, abnormality-resection overlap was related to 12-month or longer Engel outcome. In stereo-EEG patients, z-scores were compared across seizure-onset, interictal, and uninvolved grey-matter contacts. In lesion-negative patients, abnormalities were compared with PET hypometabolism and stereo-EEG findings. Results: Greater resection of MD abnormalities predicted seizure freedom, especially with overlap >0 (mean differences 17%, 23%; Pperm=0.0086, <0.001), and higher odds of Engel I (OR 1.31/10%; AUC 0.723; Youden threshold 36%). FA showed similar associations. Seizure-onset contacts had higher MD and lower FA z-scores than uninvolved grey matter (+0.65, 0.18; Pperm<0.001, =0.001), with interictal contacts intermediate. Abnormalities were more widespread and contralateral in patients without an identified onset zone. In lesion-negative patients, abnormalities co-localised with PET (20/28 fully, 6/28 partially) and stereo-EEG (17/24 fully, 3/24 partially). Interpretation: Diffusion MRI abnormalities aligned with invasive electrophysiology, predicted seizure freedom when resected, and co-localized with PET/stereo-EEG, supporting an important role in presurgical evaluation.

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.

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.

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: Schizophrenia is increasingly viewed as a disorder of multi-regional brain networks, but molecular and pathophysiological hypotheses are often studied at the level of single brain regions or neurotransmitter systems. A multi-regional, multi-neurotransmitter analysis is needed to clarify how molecular abnormalities relate to circuit-level hypotheses of the disorder. Methods: Transcriptomic data from 2,209 post-mortem brain samples spanning five brain regions in 1,037 individuals were integrated. Directed connectivity strengths between regions were estimated using co-expression between presynaptic and postsynaptic neurotransmission-related genes. These estimates constitute an "interregional molecular connectome". Macaque transcriptomic data were used to benchmark the estimation pipeline against reference macaque connectomes. Connectivity differences between healthy controls and patients with schizophrenia were calculated, and a directional consistency filter was applied to identify candidate dysregulations. An LLM-augmented literature screening framework was used to assess the level of literature support for our candidates. Results: Our estimation pipeline showed concordance with reference connectomes, outperformed whole-transcriptome and random-gene co-expression measures, and remained stable when applied to macaque data. A total of 10 connections were marked as candidate dysregulations in schizophrenia at a nominal permutation threshold. Three of the candidates align with established anatomical connectivity, and another three with anatomical connections through relay regions. These candidates were characterized by bidirectional abnormalities in the cortico-striatal circuitry. The remaining four candidates had no well-established monosynaptic connection between two regions. These candidates were interpreted as co-regulated co-expression coordination changes. Literature screening quantified the number of articles discussing each candidate and how many of the articles directly support or refute each candidate. Our candidates collectively highlight the role of the cortico-striato-thalamo-cortical circuit in the pathophysiology of schizophrenia. Conclusions: Interregional co-expression of neurotransmission-related genes carries more anatomical information than whole-transcriptome co-expression. Applied to schizophrenia, our analysis recapitulates known hypotheses and also proposes hypothesis-generating data-driven findings for experimental follow-up. This approach complements conventional region-wise analyses. Our approach could support a unified, multi-regional, and multi-neurotransmitter perspective of schizophrenia and point to candidate molecular mechanisms for further studies.

Non-functioning gonadotroph (NFG) tumors are the most common type of non-functioning pituitary adenomas and can cause significant symptoms due to mass effect. However, the molecular programs underlying NFG tumor growth and their relationship to the normal anterior pituitary gland (APG) are poorly understood. To gain a deeper understanding of NFG tumor biology in the context of the normal APG, we performed single-cell/nucleus RNA-sequencing on 21 NFG tumors and 8 APG samples in human, generating the largest transcriptomic dataset of its kind. Single-cell/nucleus sequencing yielded 77,342, cells from APG samples and 152,649 cells from NFG tumor samples. Differential expression analysis within the APG identified novel marker genes of each neuroendocrine cell type and defined distinct transcriptional signatures of anterior and posterior pituitary stem cell populations. Comparison with tumor transcriptomes revealed that NFG tumor cells most closely resemble gonadotrophs, while also showing significant enrichment of thyrotroph and somatotroph markers. Notably, there was no significant overlap with stem cell markers, suggesting that NFG tumors most likely originate from differentiated gonadotrophs but can take on transcriptional characteristics of other neuroendocrine cell types. Pseudobulk profiling of NFG tumor cells demonstrated that tumor volume is significantly positively correlated with 83 genes including known oncogenes CAD, BRF2, and SOX12, along with 16 zinc finger transcription factors. Lastly, analysis of the tumor microenvironment revealed proportional increases in myeloid, endothelial, and mural cell populations in tumor samples compared to APG samples and highlighted cross talk between tumor and endothelial, mesenchymal, and immune populations via VEGF, PDGF, and MIF signaling pathways respectively. This study identifies novel transcriptomic signatures determining cell type identity in both APG neuroendocrine and NFG tumor cells. Our findings elucidate molecular programs driving NFG lineage infidelity and tumor growth, highlighting candidate predictors of patient outcomes and potential targets for therapeutic intervention.

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.

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: 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.