Neuro-Oncology Advances

BackgroundGlioblastoma is an aggressive and incurable brain tumor. Clinical trials of immune checkpoint inhibitors showed no clinical benefit in glioblastoma when given after surgery. However, a clinical trial in which PD1 inhibition was given prior to second surgery did show pharmacodynamic evidence for activity. This suggests the possibility that immune checkpoint inhibitors may be more effective in a setting where large tumors are present. Here we have studied immune responses to large tumors in an autochthonous mouse model of glioblastoma. MethodsGlioblastoma was induced by transfection with oncogenic plasmids injected directly into the lateral ventricle of neonatal mice. Immune responses were assessed using a combination of spectral flow cytometry and immunohistochemistry. ResultsThere was a marked immune response to large tumors, with significant increases in CD4 T cells and dendritic cells. T cell changes occurred primarily at leptomeningeal/perivascular border sites. A large proportion of CD4 T cells expressed PD1 and half of these were regulatory T cells. NK cells were also increased in mice with large tumors, but were predominantly in immature states. The mouse model accurately recapitulates the formation of palisading necroses. These contain apoptotic cells and avidly recruit myeloid cells that are induced to express large amounts of TGF{beta}. ConclusionsLarge glioblastoma tumors generate a border site population of PD1 positive T cells that may explain the pharmacodynamic response in neoadjuvant trials, and a palisading necrosis-driven immunosuppressive mechanism that may explain why responses are insufficient to provide a significant clinical benefit. KEY POINTSThe SB mouse model accurately recapitulates immune features of human glioblastoma Large tumors induce a significant border site immune response Palisading necroses in large tumors counter this with a strong immunosuppressive response IMPORTANCE OF STUDYImmune checkpoint inhibitors have not shown efficacy in glioblastoma when used post-surgery, but do show pharmacodynamic activity when used in patients prior to second surgery (i.e. neoadjuvant). This suggest the possibility that immune checkpoint inhibition is more effective when large tumors are present. Using a clinically-relevant autochthonous mouse model, we show here that large tumors induce an immune response that is evident in leptomeningeal border sites. Large tumors in this mouse model also generate palisading necroses, a well-known diagnostic feature in glioblastoma tumors. These palisading necroses generate large amounts of TGF{beta}, providing a mechanism by which large tumors can suppress border site immune responses. This further supports the concept that palisading necroses are drivers of glioblastoma malignancy and suggests novel strategies to enhance responses to immune checkpoint inhibition in this cancer.

Background: Extent of resection remains central to meningioma management, yet Simpson grading is subjective and may not reflect measurable postoperative residual disease. We compared surgeon-reported Simpson grade, report-derived radiological grading, and residual tumour volumetry across a multicentre cohort. Methods: We performed a retrospective study across two tertiary neurosciences centres comprising four hospitals, including patients undergoing primary cranial meningioma resection from 2006 to 2025. Postoperative magnetic resonance imaging (MRI) reports were harmonised using weakly supervised natural language processing based on term frequency-inverse document frequency (TF-IDF) and a linear support vector machine classifier. Residual tumour volume was segmented from contrast-enhanced postoperative MRI and log-transformed. Concordance between Simpson and radiological gross-total/subtotal resection classification was assessed using absolute agreement and prevalence-adjusted bias-adjusted kappa (PABAK). Cox models assessed recurrence-free survival, with bootstrap validation and anatomical and scan-timing sensitivity analyses. Results: Among 912 patients, recurrence or residual progression occurred in 281. Surgical-radiological agreement was substantial but imperfect (absolute agreement 74%; PABAK 0.61), with lower agreement in skull-base and parafalcine-parasagittal tumours. In adjusted models, recurrence hazard increased with Simpson grade (hazard ratio 1.54, 95% confidence interval 1.37-1.72), radiological grade (1.92, 1.68-2.20), and log-transformed residual volume (1.20, 1.16-1.24; all p<0.0005). Optimism corrected concordance increased from Simpson grade to radiological grade and log-volumetry (0.692, 0.733, and 0.748), with this ranking preserved across sensitivity analyses. Conclusions: Imaging-based postoperative residual disease measures outperformed Simpson grade. TF-IDF-assisted report-derived grading provides a scalable bridge to volumetry, while quantitative residual volume offers the strongest prognostic representation.

Background: Meningiomas are the most common primary intracranial tumors in adults, and volumetric assessment increasingly guides surveillance and treatment decisions. Automated segmentation could enable standardized volumetry but requires robust validation. Purpose: To develop a fully automated three-dimensional deep learning model for meningioma segmentation on multiparametric MRI, and to evaluate segmentation accuracy, external generalizability, failure modes, radiologist-rated clinical plausibility, and workflow feasibility. Methods: From 2024 to 2026, this retrospective study trained a custom 3D nnU-Net residual encoder model. Expert segmentations covered enhancing tumor (ET), tumor core (TC), and whole tumor (WT). Dice similarity coefficient (DSC) was the primary metric. External validation used an independent single-institution dataset (n = 310 intracranial cases) with incomplete MRI protocols. Failure modes, model equity, and inference time were assessed. A blinded multi-rater study (10 radiologists; 510 cases) rated TC segmentations using a 0-10 Likert scale, analyzed with linear mixed-effects models. Results: Model training used the BraTS Meningioma 2023 dataset (n = 1000; mean age 60.2 {+/-} 14.5; 705 female). In cross-validation, mean DSC was 0.939 for ET, 0.937 for TC, and 0.921 for WT. In external validation, mean DSC was 0.872 for TC and 0.842 for WT, despite heterogeneous protocols and incomplete sequences. Predicted TC volumes correlated strongly with reference volumes in cross-validation (r = 0.995) and external validation (r = 0.971). Most common failure modes were skull base and intraosseous tumors with performance equitable across demographic subgroups. Mean inference time was 1.2 seconds. In blinded evaluation (1120 ratings), model segmentations received higher scores than reference annotations (+0.32 BraTS; +1.38 external validation). Conclusion: A fully automated deep-learning model achieved high meningioma segmentation accuracy across multi-institutional training data and external clinical imaging. In a blinded study, model segmentation quality exceeded reference annotations, and 1.2-second inference supported workflow integration. Prospective evaluation is warranted before routine deployment.

BackgroundThe Netrin-1 dependence receptor pathway plays critical roles in neural development, but its expression landscape and prognostic significance in glioblastoma (GBM) remain poorly characterized. MethodsSingle-cell RNA-seq data from 148,019 cells across 34 tumors (Neftel et al., 2019) were analyzed to map Netrin-1 pathway gene expression across GBM cellular states. Differential gene expression and pathway enrichment analyses were performed on NEO1-defined subpopulations. Bulk RNA-seq survival analysis was conducted across three independent GBM cohorts TCGA (n=106), CGGA mRNAseq_325 (n=137), and CGGA mRNAseq_693 (n=237), totaling 480 patients. Primary analysis used continuous Cox regression (per-SD hazard ratios); meta-analysis employed fixed-effects inverse-variance weighting. ResultsIn GBM single-cell data, Netrin-1 pathway genes showed state-specific enrichment --NEO1, DCC, NTN1, and RGMB were predominantly expressed in oligodendrocyte-precursor (OPC) and neural-progenitor (NPC) states. Cells positive for NEO1 were enriched for neural differentiation programs (nervous system development, p=9.6x10-; Axon Guidance, p=2.8x10-), whereas NEO1-negative cells were dominated by ribosomal/translational and immune activation programs. In the 3-cohort survival meta-analysis, NTN1 (Netrin-1 ligand) emerged as the sole gene reaching meta-analytic significance as a risk factor (Meta HR=1.163 per SD, 95% CI 1.056-1.281, p=0.0021, I{superscript 2}=0%, 3/3 cohorts concordant), while DCC and RGMB showed directionally consistent protective trends (DCC: Meta HR=0.938, 95% CI 0.858-1.025, p=0.156; RGMB: Meta HR=0.979, 95% CI 0.881-1.087, p=0.686; both 3/3 cohorts concordant). NEO1 itself did not independently predict survival (Meta HR=1.008, 95% CI 0.885-1.147, p=0.910). After Bonferroni correction for 10 genes tested (threshold p<0.005), only NTN1 met strict significance. In exploratory sex-stratified analysis of a single cohort (CGGA 693, n=237), NEO1 and NTN1 exhibited female-specific risk enhancement (NEO1: HR=1.417, p=0.014; NTN1: HR=1.249, p=0.019), with minimal effects in males. UNC5B showed context-dependent risk in MGMT-unmethylated tumors (HR=1.331, p=0.037). These sex-dimorphic findings require independent validation. ConclusionsThe Netrin-1 pathway exhibits divergent prognostic trends in GBM, with NTN1 as a risk factor and DCC trending toward protection--consistent with the dependence receptor model. These findings, which should be interpreted as hypothesis-generating, nominate NTN1 as a candidate therapeutic target and highlight the potential importance of sex-stratified evaluation in future Netrin-1-directed trials. Independent replication in larger cohorts is warranted.

Oligodendroglioma is a primary central nervous system tumor classified by the presence of isocitrate dehydrogenase (IDH) mutations and codeletion of 1p/19q. Here we describe the generation of an IDH-mutant 1p/19q-codeleted oligodendroglioma mouse model using in utero electroporation. We identified IDH1R132H, PIK3CAE545K, CicKO, Fubp1KO and Cdkn2aKO as the optimal combination (termed OligoCdkn2a) to drive fully penetrant tumors that histologically resemble human grade II/III IDH-mutant, 1p/19q-codeleted oligodendroglioma. Replacing Cdkn2a with Trp53 loss in this mouse model shifted tumor histology towards high grade astrocytoma. OligoCdkn2a tumors displayed metabolic and transcriptional changes associated with IDH and CIC mutations, and single cell sequencing identified a bias towards oligodendrocyte differentiation compared to an IDH wild-type glioblastoma mouse model. OligoCdkn2a tumors represent the first mouse model system to recapitulate the genetic, histological and transcriptional features of human IDH-mutant 1p/19q-codeleted oligodendrogliomas, offering a platform to further dissect tumor biology and test new therapeutic strategies.

PurposeGlioblastoma (GBM) is characterized by poor clinical outcomes and marked resistance to radiotherapy. Because effective radiosensitizing strategies for GBM remain limited, we investigated whether inhibition of KRAS/RAS signaling could enhance radiation response in GBM. In particular, we evaluated the radiosensitizing potential of RMC-6236, an RAS(ON) multiselective inhibitor that suppresses active RAS signaling across multiple RAS-dependent states. Experimental DesignHuman GBM cell lines (U251, LN-18, ACPK1, and OSU61) were treated with radiation, with or without genetic or pharmacological KRAS inhibition. KRAS signaling was suppressed by siRNA-mediated knockdown or RMC-6236 treatment. Radiation-induced KRAS activation and downstream MAPK signaling were assessed by Raf-RBD pull-down assays and immunoblotting. Radiosensitivity was evaluated using clonogenic survival assay. DNA damage persistence, cell cycle distribution, and mitotic catastrophe were analyzed by {gamma}H2AX immunofluorescence, flow cytometry, and nuclear morphology assessment, respectively. In vivo therapeutic efficacy was examined in an orthotopic U251 xenograft model. ResultsRadiation-induced transient activation and increased KRAS protein expression of KRAS, accompanied by activation of ERK, JNK, and p38 signaling in GBM cells. siKRAS suppressed radiation-induced KRAS and MAPK activation, and significantly enhanced radiosensitivity in all four GBM cell lines. Similarly, RMC-6236 inhibited radiation-induced KRAS activation and attenuated downstream MAPK signaling without reducing the total KRAS protein expression. RMC-6236 significantly increased the radiosensitivity across all GBM cell lines, with dose enhancement factors ranging from 1.33 1.46. Mechanistically, combined treatment with RMC-6236 and radiation increased persistent {gamma}H2AX foci and enhanced mitotic catastrophe without producing consistent redistribution of cells into radiosensitive cell cycle phases. In an orthotopic GBM model, the combination of RMC-6236 and radiation significantly prolonged survival compared to that of the control and radiation alone. ConclusionsThese findings indicate that radiation-induced KRAS signaling is a functionally important mediator of radioresistance in GBM and demonstrate that inhibition of KRAS/RAS signaling enhances the radiation response in vitro and in vivo. RMC-6236 may represent a promising radiosensitizing strategy for GBM by suppressing adaptive RAS/MAPK signaling and promoting persistent DNA damage and mitotic catastrophe following irradiation. However, clinical trials of this combination are warranted.

Background: Reirradiation (reRT) is increasingly offered following progression in diffuse intrinsic pontine glioma (DIPG) and diffuse midline glioma (DMG), though optimal patient selection remains a challenge. This study evaluated clinical outcomes after reRT in a contemporary cohort of patients with DIPG/DMG. Methods: Patients <26 years old with DMG/DIPG treated with radiation therapy between 2011-2025 were retrospectively reviewed. Primary endpoints included overall survival (OS2) and progression-free survival (PFS2), measured from first progression, and change in neurologic symptoms after reRT. Survival was estimated using Kaplan Meier methods, with Cox proportional hazards modeling for prognostic factors. Results: Fifty eight patients were included; 37 (63.8%) underwent reRT. Tumors were predominantly pontine (74.1%). ReRT was associated with improvement in motor function (51.4% vs. 9.5%, p=0.002), cranial nerve function (29.7% vs. 4.8%, p=0.044), and gait ataxia (35.1% vs. 9.5%, p=0.059). Median OS2 and PFS2 were improved with reRT (OS2: 9.67 vs. 2.57 months, p<0.001; PFS2: 5.63 vs. 1.57 months, p<0.001). OS2 was independently associated with reRT (HR 0.27, p<0.0001), pontine location (HR 2.94, p=0.004), and steroid use at progression (HR 4.12, p=0.001). PFS2 was independently associated with reRT (HR 0.23, p < .0001) and distant pattern of failure (HR 2.83, p=.037). Among reRT patients, non-pontine location was associated with improved OS2 (p=0.02), and local failure was associated with improved PFS2 (p=0.003). Conclusion: ReRT was associated with neurologic improvement and prolonged survival. Patients with non-pontine tumors or local-only failure might derive the greatest benefit. Prospective studies are warranted to define optimal dose/fractionation and refine patient selection.

Glioblastoma progression is spatially heterogeneous, but conventional imaging provides limited information about where subsequent tumor progression is likely to occur. We developed a directional magnetic resonance elastography framework to test whether local post-treatment tumor-brain interface mechanics are associated with later spatial tumor progression. In a secondary analysis of a prospectively acquired glioblastoma cohort, wedge-level viscoelastic instability features were extracted from the first post-treatment MRE scan and related to novel tumor burden on the second post-treatment scan after excluding tumor already present on pretreatment or first post-treatment imaging. Nine patients had longitudinal imaging suitable for spatial comparison; six lesions showed net interval growth and were included in the primary wedge-level directional analysis, while three non-growing lesions were retained for descriptive comparison. In growing lesions, several directional mechanical features were descriptively associated with later novel tumor burden. In cluster-aware models accounting for within-patient dependence among wedges, mean {Delta}tan{delta} ; showed the most consistent association with later wedge-level novel tumor fraction across mixed-effects and generalized estimating equation analyses. Associations were directionally stable across wedge-width sensitivity analyses. These findings provide proof of principle that post-treatment glioblastoma interface mechanics contain spatially resolved information related to where later tumor emergence occurs, supporting further validation of directional MRE as a framework for longitudinal mapping of progression geometry.

BackgroundRadiation therapy is integral to the curative treatment of childhood brain tumors but contributes to late neurocognitive impairment in survivors. FLASH (ultra-high dose rate, >40Gy/s) reduces normal-tissue toxicity in preclinical models, and proton-FLASH is currently the only modality capable of delivering ultra-high dose rates to the deep targets, such as pediatric brain tumors. However, two questions remain unresolved before clinical translation: (1) whether the FLASH effect can be achieved on synchrotron-based proton systems, which deliver protons in discrete spills that may be insufficient to cover a clinical target within a single delivery, and (2) which dose-rate metric, among the multiple definitions currently used in the field, best predicts the biological FLASH effect. MethodsC57BL/6 mice (7-8 weeks) received 10 Gy whole-brain RT via a clinical Hitachi ProBEAT synchrotron with CBCT-guided delivery, using three transmission-beam techniques: single-spill pencil beam scanning (SS PBS), multi-spill PBS with [~]2-second inter-spot delay (MS PBS), and passive scatter (PS), compared to conventional (CONV) delivery and unirradiated controls (n=24-28/group, equal sex distribution). Dose rate was quantified using three frameworks: field dose rate (FDR), PBS dose rate (PBSDR), and dose-averaged dose rate (DADR). Recognition memory was assessed by novel object recognition (NOR) at 6 weeks post-RT, and cognitive flexibility was assessed via touchscreen visual discrimination and reversal learning at 14 weeks. Hippocampal neuroinflammation was evaluated by immunofluorescence and immunohistochemistry for Iba1, NeuN, and GFAP. ResultsFLASH conditions were met by SS PBS and PS under all three dose-rate definitions, but MS PBS qualified as FLASH only by DADR. Despite this, neuroprotection was preserved across all three FLASH techniques: discrimination index was significantly higher for SS PBS (P=0.021), MS PBS (P=0.008), and PS (P<0.001) versus CONV, with no significant difference between FLASH techniques. On touchscreen testing, FLASH-treated females demonstrated preserved cognitive flexibility (P=0.047 vs. CONV on reversal learning correct trials). Iba1+ microglia were reduced in FLASH compared to CONV mice, with morphology suggestive of preserved homeostatic state. ConclusionsSynchrotron-based proton FLASH preserves neurocognitive function across all delivery techniques, including under multi-spill delivery essential for treating clinical-scale pediatric brain tumors. Critically, this neuroprotection was observed even for deliveries that qualified as FLASH only by DADR, identifying DADR as the dose-rate metric most relevant to the biological FLASH effect with direct implications for clinical trial design and dose-rate reporting standards.

AbstractO_ST_ABSBackgroundC_ST_ABSGlioblastoma (GB) is the most aggressive primary brain tumor, characterized by therapy resistance, attributed to a multitude of epi-genetic changes resulting in phenotypic plasticity with altered cell states. To uncover druggable epigenetic vulnerabilities, we disturbed GB-derived spheres and observed coordinated repression of the aberrantly activated hemopoietic stem-like cell signature, dominated by HOXA genes. This signature has been associated with poor prognosis and resistance to therapy in GB. Here we investigate biological vulnerabilities associated with the deregulated epigenetic landscape in high-HOX GB. MethodsGB-derived spheres (GS) were treated with an inhibitor of Bromodomain and extra-terminal motif proteins (BETi) (JQ1) or transduced with inducible constructs to genetically modulate HOXA10 expression (shRNA for knockdown, ectopic HOXA10). Functional effects were evaluated through proliferation, neurosphere formation, and senescence assays. Epigenomic profiling incorporated RNA-seq, ChIP-seq, ATAC-seq, promoter capture MicroC, and DNA methylation. ResultsBETi-mediated rapid, coordinated downregulation of the HOX-signature, suggested direct transcriptional regulation. Knockdown of HOXA10 alone yielded similar effects, decreasing expression of HOXA genes, reducing proliferation, self-renewal capacity, and triggering senescence. Conversely, ectopic HOXA10 expression was ineffective in reactivating the HOXA cluster, or reverse BETi-mediated biological effects. Integrative epigenomic analysis of high-HOX-GS revealed concerted activation of the HOXA region, with broad domains of H3K27ac/H3K4me3 associated with super-enhancer activity, open chromatin (ATAC) and focal DNA hypomethylation. Architectural changes included altered CTCF interactions and increased promoter-anchored looping. ConclusionThese results position the HOX-signature as a potential therapeutic target and offer a mechanistic rationale for disrupting BET-dependent transcriptional regulation in high-HOX GB. Key pointsO_LIEpigenetic activation of stem cell-related high-HOX signature in GB is associated with a super-enhancer encompassing the HOXA locus. C_LIO_LITargeting this vulnerability by BETi or HOXA10 knockdown results in concerted repression and loss of stemness features. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/727851v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@5bf4b9org.highwire.dtl.DTLVardef@11fa405org.highwire.dtl.DTLVardef@497a2eorg.highwire.dtl.DTLVardef@1f47084_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Chiesi, D. (2026) https://BioRender.com/eknk0ez Importance of studyGlioblastoma (GB) are the most aggressive brain tumors in adults that are difficult to treat, due to their high plasticity resulting invariably to resistance to therapies. Here we report on the identification of epigenetic vulnerabilities that may be leveraged in combination therapies. Disturbing GB-derived stem-like cells with epigenetic drugs, we uncovered that a HOXA gene dominated hematopoietic stem cell-related signature, previously associated with aggressiveness and treatment resistance, can be repressed in a coordinated manner, resulting in loss of stem cell features. Analysis of the underlying epigenetic landscape revealed that the HOXA region was activated in high-HOX glioblastoma through the formation of a super-enhancer. This feature presents a particular vulnerability that may be leveraged by BETi as strategy of a combination therapy.

Background: Prostate-specific membrane antigen (PSMA) PET/CT is central to prostate cancer staging and theranostic workflows. To our knowledge, no direct within-patient comparison of [18F]FC303 ([18F]Florastamin) and [68Ga]Ga-PSMA-11 has been reported. We performed a preliminary paired method-comparison study under non-harmonized acquisition protocols. Patients and Methods: Twenty patients with histologically confirmed prostate cancer underwent [68Ga]Ga-PSMA-11 PET/CT (185 +/- 37 MBq, 60 +/- 10 min) followed by [18F]FC303 PET/CT (370 +/- 37 MBq, 105 +/- 15 min) on the same PET/CT system within each patient (median interval, 29.5 days). Index targets were anatomically matched to the biopsied or surgically sampled lesion or target region. The primary malignant set included 18 histologically malignant targets; two histology-negative or indeterminate targets were included only in sensitivity analysis. Fixed [68Ga]Ga-PSMA-11-first scan order and the 45-min uptake-time difference were central interpretive constraints. Results: Across five predefined reference organs, [18F]FC303 showed lower SUVmean than [68Ga]Ga-PSMA-11 (all Benjamini-Hochberg-adjusted p < 0.001; [68Ga]/[18F]FC303 geometric mean ratio [GMR], 1.29-3.89). In the primary malignant set, [18F]FC303 lesion SUVmax was lower than [68Ga]Ga-PSMA-11 (median, 11.3 vs 18.1; paired median difference, -5.50; 95% CI, -6.85 to -2.90; Wilcoxon p = 8.4 x 10-4), with strong rank correlation (Spearman {rho} = 0.90). Passing-Bablok regression yielded {beta} = 1.13 (95% CI, 1.04-1.45), and log-Bland-Altman GMR (FC303/[68Ga]) was 0.75, consistent with proportional non-interchangeability. Tumor-to-liver and tumor-to-mediastinum ratios did not differ significantly (GMR, 1.17 [95% CI, 0.94-1.45] and 0.96 [0.80-1.15], respectively); the study was not powered for equivalence. The n = 20 sensitivity analysis showed consistent directionality. Conclusions: Under non-harmonized acquisition conditions, [18F]FC303 showed lower physiologic reference-organ SUVmean and malignant target-region SUVmax than [68Ga]Ga-PSMA-11, whereas tumor-to-liver and tumor-to-mediastinum ratios were not significantly different. Absolute SUVs were not interchangeable; [68Ga]Ga-PSMA-11-derived SUV thresholds should not be directly transferred to [18F]FC303 without tracer-specific calibration.

BackgroundGlioblastoma (GBM) is a sex-biased disease characterized by higher incidence and poorer survival in males. These sex differences are primarily driven by metabolic and immune signatures, with iron metabolism playing a major role. While iron is essential for tumor cell proliferation, it is also critical for T cell recruitment and function within the tumor microenvironment (TME). Clinical data indicates that iron deficiency impacts GBM survival in a sex-biased manner; however, the underlying mechanisms remain unexplored. MethodsWe employed a FTH1 heterozygous knockdown mouse model to induce tumor iron deficiency in GBM-bearing mice. TME dynamics were interrogated using flow cytometry and spatial transcriptomics (10X Xenium) to analyze immune infiltration, localization, and ligand-receptor signaling between GBM and immune cells. ResultsFTH1 knockdown resulted in iron deficiency in the GBM TME. Iron deficiency altered the TME dynamics in a sex biased manner. FTH1 knockdown in females caused an anti-inflammatory, cytokine deficient TME which failed to recruit CD4 and CD8 T cells. In males, FTH1 knockdown caused a proinflammatory environment by activating the innate immune response. Additionally, FTH1 knockdown increased the density of TAMs in the immediate surrounding of GBM cells in a sex biased manner. ConclusionThese results demonstrate that tumor iron plays a sex-biased role in immune infiltration and anti-tumor immunity.

BackgroundConvection-enhanced delivery of topotecan enables sustained local chemotherapy for recurrent glioblastoma and was associated with reduced tumor proliferation in our previous phase 1B clinical trial. That trial incorporated a paired pre- and post-treatment biopsy design - rare in glioblastoma clinical research - enabling tissue-anchored assessment of drug effect without reliance on radiographic or survival endpoints, which are notoriously difficult to interpret in this disease. However, the cellular and molecular consequences of local chemotherapy within the treated tumor microenvironment remain incompletely defined. MethodsWe integrated paired, MRI-localized pre- and post-treatment biopsies from a first-in-human CED-topotecan trial (n=5), leveraging the paired biopsy architecture, in which each patient serves as their own control and post-treatment specimens are spatially annotated relative to the MRI-defined infusion zone, to generate tissue-based evidence of drug effect without requiring large patient numbers. These biopsies were integrated with complementary experimental models, including a time-resolved syngeneic murine glioma CED model, acute patient-derived glioblastoma slice cultures, and in vitro human microglial and glioma systems. Clinical biopsies were analyzed by bulk RNA-seq, cell-type deconvolution, and multiplex immunofluorescence. Murine tumors were analyzed by survival, immunofluorescence, and single-cell RNA-seq; patient-derived slice cultures were profiled by single-cell RNA-seq. ResultsIn paired human biopsies, CED-topotecan induced spatially restricted transcriptional remodeling within the infusion zone, characterized by suppression of proliferative tumor programs and enrichment of inflammatory, interferon, hypoxia, and mesenchymal signatures. Cell-type deconvolution and immunofluorescence linked this response to myeloid remodeling, including enrichment of monocyte-derived tumor-associated macrophage states, increased MARCO-positive myeloid populations, and pH2AX-positive genotoxic stress within Iba1-positive myeloid cells. In the murine CED model, topotecan prolonged survival and reduced tumor cellularity, while also inducing inflammatory and DNA-damage programs in tumor-associated macrophages that evolved by 7-days toward hypoxia, angiogenesis, TGF-{beta} signaling, and mesenchymal/tissue-remodeling programs. Human slice culture and in vitro microglial systems confirmed stress-coupled inflammatory and DNA-damage responses in human myeloid cells. ConclusionsLocal topotecan delivery produces spatially structured tumor cytotoxicity together with a genotoxic, stress-coupled inflammatory myeloid response that evolves toward mesenchymal macrophage remodeling. By integrating paired clinical biopsies with time-resolved and mechanistic experimental models, this study provides a framework for understanding how local chemotherapy reshapes the glioblastoma microenvironment and for future studies evaluating dose, schedule, treatment duration, and combination strategies. These findings demonstrate that paired, spatially annotated tissue sampling from small, precisely characterized clinical cohorts can yield mechanistic insight that conventional radiographic and survival endpoints cannot provide, and support tissue-based response assessment as the appropriate paradigm for evaluating novel locoregional therapies in glioblastoma.

Glioblastoma (GBM) is a highly aggressive type of glioma that is resistant to immunotherapy and is associated with poor prognosis, largely due to its immunosuppressive tumor microenvironment. Brutons tyrosine kinase (BTK) is a non-receptor kinase that not only plays an important role in oncogenic signaling, particularly in tumor growth, but also regulates the activity of tumor-infiltrating myeloid cells, including dendritic cells, macrophages, and microglia in brain tumors. High BTK expression is associated with poor survival in patients with glioma. Oncolytic herpes simplex virus type 1 (oHSV)-derived virotherapy, a novel treatment strategy, has demonstrated effectiveness against GBM; however, its efficacy is limited by the tumor microenvironment. In this study, we found that BTK is predominantly expressed in GBM-infiltrating myeloid cells. Intratumoral injection of oHSV not only promotes infiltration of myeloid cells and T cells but also activates BTK in these myeloid cells, thereby limiting oHSV infection and replication in tumor cells. Combination treatment with BTK inhibitor ibrutinib improves anti-tumor efficacy of oHSV in both human GBM12 xenograft and syngeneic murine GSC005 models. Mechanistically, BTK inhibition increases oHSV-mediated tumor cell death (cleaved caspase-3) and cytotoxic CD8 T cell infiltration, while decreasing tumor cell proliferation (Ki-67). BTK inhibition not only suppresses oHSV clearance by tumor-infiltrating microglia and macrophages but also reduces their pro-invasive effects on tumor cells. Addition of IDO inhibitor, an immune modulator, further prolongs survival in tumor-bearing mice in a syngeneic GBM model. Single-cell mRNA sequencing (scRNA-seq) analysis indicates that combination treatment modifies key signaling pathways in both tumor-infiltrating myeloid cells (macrophages and microglia) and CD8 T cells. Further analysis shows that BTK inhibition, with or without IDO inhibition, promotes the formation of tumor-infiltrating tertiary lymphoid structures (TLS) during intratumoral oHSV treatment, subsequently remodeling T cell, NKT cell, and monocyte-macrophage populations. These results indicate that BTK inhibition exerts multifaceted effects in enhancing the anti-tumor efficacy of oHSV therapy.

Background: Despite epidemiological interest in aspirin's chemopreventive potential against glioma, the underlying multi-layered molecular mechanisms -- spanning COX-2/PGE2 signaling, iron metabolism, ferroptosis, epigenetic regulation, and the NEO1/hepcidin regulatory axis -- have not been systematically characterized at the multi-omics level. Methods: We conducted an integrative multi-omics analysis leveraging TCGA-GBM (n=172) and TCGA-LGG (n=534) transcriptomes, CPTAC GBM proteomics (n=99), TCGA HM450K DNA methylation data (GBM n=140, LGG n=516), GEO aspirin perturbation datasets, IEU OpenGWAS summary statistics, and independent single-cell RNA-seq data (GSE131928, 28 GBM patients). Eight analytical tracks were executed: (1) COX-2/PGE2 pathway profiling, (2) BBB tight junction characterization, (3) GEO-derived aspirin response signature projection, (4) gut-brain axis evaluation, (5) Mendelian randomization (MR) using PTGS2 cis-SNPs, (6) iron metabolism and ferroptosis pathway analysis, (7) NEO1/HFE2/BMP6/HAMP regulatory axis characterization with multi-omics validation, and (8) single-cell transcriptomic validation across GBM malignant cell states. Results: Transcriptomic analysis revealed profound reprogramming of the NEO1/hepcidin iron regulatory axis in GBM: HAMP (hepcidin) was massively upregulated (log2FC=+2.92, P=5.0e-37), accompanied by TFRC upregulation (log2FC=+1.38, HR=2.30, P=3.6e-42) and NEO1 downregulation (log2FC=-0.57, HR=0.59, P=4.6e-6). De novo HM450K methylation analysis revealed HAMP as the dominant epigenetic target in the iron network, exhibiting the strongest hypomethylation signal (DeltaBeta=-0.265, P=1.4e-48), while NEO1 and TFRC showed constitutively low baseline methylation (Beta<0.05). Gene set enrichment analysis identified ferroptosis driver genes (NES=+1.861, P=0.030) and the iron deficiency response pathway (NES=+1.698, P=0.010) as the most significantly enriched pathways in GBM. Molecular subtype analysis revealed that the mesenchymal GBM subtype exhibits the highest iron metabolism gene expression. Mendelian randomization established a causal relationship between PTGS2 expression and glioma risk (IVW OR=1.31, P=1.1e-4). Single-cell RNA-seq analysis validated that iron metabolism gene expression is heterogeneously distributed across malignant cell states, with the mesenchymal state exhibiting the highest HAMP expression and elevated ferroptosis vulnerability. GPX4 was universally highly expressed across all cell states, indicating pan-GBM dependence on GPX4-mediated ferroptosis suppression. Conclusions: This multi-omics investigation reveals that the NEO1/hepcidin iron regulatory axis is epigenetically reprogrammed in glioma, driving iron-dependent vulnerability that bridges COX-2 signaling with ferroptosis susceptibility. The convergent evidence from transcriptomics, proteomics, epigenomics, and causal inference provides a comprehensive mechanistic framework for aspirin's protective effects against glioma and identifies the NEO1/HAMP/TFRC axis as a promising therapeutic target.

Background. Meningiomas exhibit well-established hormonal biology, yet no study has examined whether myeloid immune infiltration interacts with estrogen-responsive transcription in this tumor type. Methods. We applied three-method consensus immune deconvolution (EPIC, MCPcounter, CIBERSORTx) to 968 harmonized meningioma RNA-seq transcriptomes from five public datasets, stratified by Thirimanne et al. (2024) transcriptomic subtypes. Competitive gene set enrichment compared macrophage-high versus macrophage-low tertiles with sex-adjusted, purity-adjusted, and method-independent sensitivity analyses. Survival modeling tested both total macrophage burden and a decomposed microglia-to-macrophage ratio validated against single-cell ground truth (pseudo-bulk r = 0.77). Results. Macrophage-high tumors showed significant suppression of estrogen response gene sets (FDR = 4.9 x 10-5) despite paradoxical ESR1 upregulation (log2FC = +0.40, FDR = 2.5 x 10-26) and PGR downregulation (log2FC = -0.34, FDR = 2.7 x 10-3), indicating post-receptor transcriptional disruption. This signal strengthened after sex adjustment (FDR = 1.9 x 10-6) and was confirmed across a multi-layer sensitivity battery (eleven analyses including reference-matrix-independent, purity-adjusted, rotation-based self-contained, and empirical-null tests; all FDR < 3 x 10-4 in the relevant convergent tests). Myeloid infiltration was strongly subtype-dependent (Kruskal-Wallis p = 7.4 x 10-16) but grade-independent (p = 0.399), with CSF1R enriched in the macrophage-dominant Cluster B. Neither total macrophage score (HR = 0.90, p = 0.53; N = 102) nor a decomposed microglia/macrophage ratio (HR = 0.92, p = 0.46; N = 101) predicted recurrence-free survival. Conclusions. The pre-registered primary endpoint - macrophage infiltration score predicting recurrence-free survival - was not supported; the estrogen-immune dissociation emerged from secondary exploratory gene-set analysis and requires independent validation. Macrophage-infiltrated meningiomas exhibit a previously unreported dissociation between maintained ESR1 expression and suppressed estrogen-responsive transcription, with implications for hormonal therapy stratification.

Colony stimulating factor 1 receptor (CSF1R) is a tyrosine kinase receptor that is expressed exclusively in microglia within the CNS. Its endogenous ligands, colony stimulating factor-1 (CSF1) and interleukin-34 (IL-34), are released from neurons, positioning CSF1R as a key mediator receptor of neuron-glia communication. CSF1R is considered not only a potential drug target, but also a biomarker of neuroinflammation. From that perspective, selective radioligands for neuroimaging are of great interest for imaging neuroinflammation and determining drug occupancy. In this study, we have validated the binding characteristics of a CSF1R inhibitor, 4-((5-MethOxy-6-((5-methoxypyridin-2-yl)methoxy)pyridin-3-yl)methyl)-2-(1-methyl-1H-pyrazol-4-yl)pyrimidine (5-MOP) as a novel CSF1R radioligand, by performing in vitro saturation binding experiments in human and murine tissues. 5-MOP was found to be selective for CSF1R among a broad range of kinases. Autoradiography revealed that [3H]5-MOP binds with high affinity (KD = 9.8 nM) to a single saturable binding site in human meningioma tissues, and this binding was displaced with known CSF1R inhibitors, including CPPC, sCSF1inh and GW-2580. In contrast, CPPC, which has been extensively used as a CSF1R radioligand showed substantial cross-reactivity to other brain kinases, including Trk A/B/C, and [3H]CPPC could only be displaced with CPPC itself, not by other ligands, including 5-MOP. These results identify [3H]5-MOP as the most selective radioligand currently available, enabling accurate detection of drug occupancy and activated microglia. Significance of the studyThis study identifies and validates a novel selective radioligand that binds CSF1R with high selectivity and low nanomolar affinity. Because CSF1R is selectively expressed in activated microglia, this radioligand could be useful for detecting neuroinflammatory activity.

Dexamethasone is widely used to control cerebral edema and inflammation in glioblastoma, but its benefits are limited by systemic toxicities and adverse prognostic associations. We evaluated local administration of dexamethasone via convection-enhanced delivery (CED) to maximize intratumoral anti-inflammatory effects by increasing local corticosteroid exposure while minimizing systemic exposure. In two glioma mouse models, continuous intraparenchymal infusion of dexamethasone was well tolerated with no adverse effects. Pharmacokinetic analyses supported preferential intratumoral distribution and reduced systemic exposure with CED compared with systemic dosing. Single-nucleus RNA sequencing (snRNA-seq) and immunohistochemistry showed attenuation of glioma-associated inflammation with downregulation of reactive microglial/macrophage programs and reduced tumor-infiltrating myeloid cells with a morphology consistent with a less activated state. Experiments in human induced pluripotent stem cell (iPSC)-derived microglia confirmed that dexamethasone directly suppresses inflammatory gene expression, indicating a conserved mechanism across species. This inflammatory suppression was recapitulated in both immortalized microglial (HMC3) and macrophage (THP1) cell lines. These findings suggest that localized dexamethasone delivered by CED reprograms the glioma immune microenvironment and achieves control of inflammation without the systemic adverse effects associated with standard systemic dexamethasone therapy. This clinically translatable strategy may improve symptom management and provide a platform for integrating local immunomodulation with future glioblastoma therapies.

Background: Non-invasive diagnosis, reliable recurrence surveillance remain critical unmet needs in gliomas. Glioma induces profound systemic immune alterations despite its anatomical confinement to the central nervous system. Circulating immune cells, particularly monocytes, are key mediators of tumor-host crosstalk and may retain tumor-induced transcriptional imprints. However, their potential clinical utility as blood-based biomarkers for detection and monitoring, remain largely unexplored. Methods and findings: In this study, we performed integrated single-cell RNA sequencing of blood immune cells and demonstrated that circulating CD14+ monocytes are significantly expanded in glioma patients, exhibiting features of differentiation arrest and increased transcriptional plasticity. These cells harbor glioma-specific molecular signatures distinct from those observed in healthy controls and patients with other tumors. Leveraging these findings, we developed an ensemble machine learning diagnostic model based on transcriptomic profiles of circulating CD14+ monocytes (training cohort, n=107), which achieved a mean area under the receiver operating characteristic curve (AUC) of 0.971 during cross-validation. In an independent cohort of 567 participants, the model maintained high diagnostic accuracy, yielding an AUC of 0.877 for distinguishing glioma from controls and other tumors. And it achieved a recurrence detection AUC of 0.969 in 51 postoperative samples. Moreover, in a prospective follow-up study involving 30 glioma patients, lower model-derived scores of postoperation were significantly associated with prolonged progression-free survival (log-rank test, P=0.043), supporting its prognostic utility. Conclusion: We demonstrate circulating CD14+ monocytes undergo glioma-specific transcriptional reprogramming, generating systemic tumor-associated signal captured via transcriptomic profiling. This blood-based diagnostic model provides non-invasive, scalable approach for glioma detection, recurrence surveillance, outcome prediction.

Objectives: AI-based reconstructions can reduce MRI acquisition times and/or improve image quality. Guidelines recommend clinical evaluations and post-deployment monitoring of these novel methods, however, there has been little investigation of the clinical resources required for such assessments. The aim of this study was to evaluate the healthcare resource utilisation and potential savings associated with AI-based reconstructions in rectal MRI. Methods: A retrospective economic costing analysis was conducted from the NHS healthcare perspective. Resource utilisation data were extracted from the Electronic Patient Records for 9 healthy volunteer scans and 104 rectal MRI examinations evaluating an AI-based reconstruction. The resource profile included the MRI scan and the staff time required for data acquisition and analysis. Results: The clinical evaluation of the AI-based reconstruction cost {pound}15,023. Deployment of the AI-based reconstruction reduced the length of an MRI rectum scan by 22 minutes, theoretically saving approximately {pound}3,437 per month. Addition of post-deployment quality control scans reduced this monthly saving to {pound}2,636. If the quality control scans were evaluated using radiologists rather than image quality metrics, monthly savings would be approximately {pound}2,541. With ongoing quality control, the clinical evaluation cost would be recouped between 5.8 and 6 months, compared with 4.4 months without ongoing quality control. Conclusions: Deploying AI-based reconstructions can yield cost savings through reduced scanning times. Quality control tests using image quality metrics would save radiological burden and reduce costs compared with conducting repeated image scoring by radiologists.