Pituitary tumors are rare in the pediatric population, accounting for less than 10% of childhood tumors. Craniopharyngiomas are the most common pediatric sellar lesions, but pituitary adenomas/PitNETs also occur, representing 2-8% of all sellar lesions. This study explores a decade of transsphenoidal microsurgery on pediatric patients, examining perioperative features including complications, clinical outcomes and anatomical peculiarities. This retrospective study included a total of 147 transsphenoidal surgeries performed by a single surgeon on 122 pediatric patients under the age of 18 between 2013 and 2023. Parameters assessed included age, gender, clinical hormone production, histopathological and radiographic parameters, surgical features, postoperative complications and recurrences rate. Among 122 pediatric patients with pituitary lesions, we found that craniopharyngiomas were the most common entity (34.4%), especially in children under 10 years old (58.6%). Pituitary adenomas/PitNETs accounted for 31.1% and were more prevalent in older children, with corticotrophic and lactotrophic adenomas being the most common subtypes. Younger patients needed significantly more time for surgery, with the longest durations observed in the 3-9 years age group and the shortest in the 16-17 years age group. Additionally, sphenoid sinus pneumatization increased with age and was inversely correlated with the duration of surgery. The current study is one of the largest to date on pediatric pituitary region lesions shedding light on demographic, clinical and histopathological features. The age-dependent distribution pattern highlights the prominence of craniopharyngiomas in younger patients, while PPAs became increasingly prevalent in those older than 10 years.

Abstract Corticotroph pituitary neuroendocrine tumours (PitNETs)/adenomas are heterogeneous sellar neoplasms. Currently established histopathological classification approaches are often considered limited in fully capturing the clinical and biological complexity of these tumours. Thus far, a molecular-based classification has not been established in corticotroph PitNETs. We compile molecular data of 270 corticotroph PitNETs (111 internal, 159 external), encompassing epigenome, transcriptome, and proteome profiles. Comprehensive integrative analyses are performed to identify, validate and characterise definitive molecular subgroups. Corticotroph PitNETs separate into four robust and clinicopathologically distinct molecular subgroups, which are broadly distinguishable by microscopy using SSTR1, GATA3 and SSTR5 immunohistochemistry. An integrated stratification model incorporating these molecular subgroups demonstrates significant prognostic utility. Our findings support the establishment of a refined molecular-based corticotroph PitNET classification, the full clinical value of which will require validation in prospective studies. To facilitate future research, we provide an easy-to-use epigenomic classifier for corticotroph PitNETs.

Background: The Comprehensive Complication Index (CCI) is a numerical scale based on the Clavien-Dindo classification (CDC) and both are widely used to assess outcome. However, the CCI had not been described for neurosurgical procedures. Objective: We aimed to investigate the value of the CCI to predict outcome in patients who underwent elective intracranial surgery. Methods: Prospective cohort study including patients who underwent elective intracranial surgery. Complications were graded using CDC, and the CCI was calculated daily. Neurological outcome was assessed using mRS, NANO, NIHSS, and KPS. Results: Of 294 patients who underwent an elective intracranial procedure, 211 (71.8%) patients underwent a craniotomy, while 28 (9.5%) patients had a burr hole procedure and 55 (18.7%) patients a transsphenoidal approach. A higher blood loss was the only significant predictor for a higher CCI (OR 1.09, 95% CI 1.00-1.12, P < 0.01). Patients with a higher CCI had a longer LOS (mean: 5.4 versus 10.9 days, P < 0.01) and ICU stay (mean: 0.8 versus 2.4 days, P < 0.01). Daily CCI revealed a risk for complications after craniotomies until postoperative day (POD) 7. In patients who underwent brain tumor surgery, the resection of meningiomas and metastasis showed a similar developmental course of CCI. A significantly higher CCI was observed in patients who had a neurological deterioration at discharge ( P < 0.01). Conclusion: CCI is a valid scale to measure outcome after intracranial procedures and correlates with neurological outcome. Risk for adverse events after craniotomy is at highest until postoperative day 7.

Rationale:Magnetic resonance imaging (MRI) is essential for visualizing the healthy and diseased brain, yet the cellular basis of MRI signal and how it changes over time remain poorly understood. Methods:Here, we present BRIDGE (Brain Radiological Imaging with Deep-learning based Ground-Truth Exploration), a platform integrating in vivo MRI with in vivo two-photon (2P) and ex vivo super-resolution microscopy using a multi-step, iterative co-registration pipeline. It enables in vivo, longitudinal and voxel-precise mapping of MRI signals to their biological ground truth for the first time. The registered overlay reveals the cellular and anatomical origins of MRI signals and enables training of convolutional neural networks to enhance the effective resolution of MRI. Results:Using BRIDGE, we identified a microenvironmental vessel biomarker for early metastatic colonization in patient-derived xenograft models of breast cancer brain metastasis. In particular, we found that distinct T2*-weighted hypointense lesions correspond to reduced blood flow and erythrostasis in perimetastatic capillaries. In glioma, longitudinal intravital studies further demonstrated direct correlations between non-vasogenic T2-weighted signal changes and patient-dependent tumor growth dynamics. Conclusions:Taken together, BRIDGE advances radiological interpretation by establishing a microscopic ground truth for MRI signatures over time, enabling deep learning-based predictive histology and providing cellular level insights into tumor microenvironment with direct clinical imaging implications.

Abstract Background Analysis of cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) holds great potential as a diagnostic tool and surrogate marker for minimal residual disease in patients with medulloblastoma. Low-coverage Nanopore sequencing is a cost-efficient technique with clinically actionable turn-around time yielding both global DNA methylation patterns and copy number variations (CNV). Methods CSF supernatants were obtained during routine clinical care from patients with medulloblastoma enrolled in HIT trials/registries or treated at our center. DNA methylation patterns and CNV were analyzed in cfDNA from CSF using Nanopore sequencing. Molecular findings were correlated with clinical characteristics and disease course. Results In total, 129 CSF samples from 62 patients were analyzed, including serial samples from 19 patients (2-16 samples/patient). All medulloblastoma groups were represented (WNT, 6/62; SHH 16/62; Group 3, 14/62; Group 4, 26/62). Circulating tumor DNA (ctDNA) was detected in 58/129 samples (45.0%, “molecular M1”) by CNV (n = 55/58) and/or DNA methylation profiling (n = 47/58; both positive in 44/58), of which only nine corresponding CSF cytologies were positive (15.5%, M1). Detection of ctDNA was most frequent pre- or intraoperatively (6/10), at initial staging (17/38), and at radiological progression (16/21). In one patient with suspected medulloblastoma relapse, liquid biopsy revealed a radiation-induced glioma. The only patient with detectable ctDNA at end of therapy developed metastatic relapse three months later despite complete remission on MRI. ctDNA detection (“molecular M1”) at initial staging (n = 11/30 patients) was not associated with inferior survival (2-y-PFS/-OS 59%/69% vs. 80%/93%, p = 0.24/0.41). After initial staging, ctDNA detection (n = 16/33 patients) was not associated with differences in PFS or OS (2-y-PFS/-OS 45%/93% vs. 72%/100%, p = 0.12/0.31). Conclusion Nanopore-based cfDNA analysis from CSF enables sensitive ctDNA detection and early molecular diagnosis. Prospective evaluation at standardized time points in future clinical trials is warranted to validate the clinical relevance of positive liquid biopsy results.

OBJECTIVE:Despite advances in technical approaches, microsurgical resection remains the gold standard for treating drug-resistant mesial temporal lobe epilepsy (MTLE). However, current multicenter data on the risk of new focal neurological deficits following MTLE surgery and on factors predicting the likelihood of seizure freedom postsurgery are limited. This study aimed to evaluate the safety and efficacy of surgery by providing reliable data on the predictors of favorable postoperative outcomes. METHODS:The authors conducted a retrospective multicenter analysis across 20 epilepsy centers on 5 continents. Detailed standardized clinical data were collected, encompassing the preoperative status of patients, presurgical diagnostics, surgical techniques, complications, and neurological outcomes. Predictive factors for postoperative neurological deficits and a satisfactory response to surgery (defined as International League Against Epilepsy [ILAE] classes 1 and 2) were analyzed using a logistic regression model. Additionally, the authors assessed the relationship between neurological deficits, seizure outcomes, and neuropsychological performance. RESULTS:A total of 1167 patients were included in this study. Postoperative new neurological deficits were observed in 22.2% of cases, with new quadrantanopia being the most common (11.2%). No in-hospital mortality or 30-day mortality was recorded. Surgical revision was necessary in 4.3% of cases within the 1st year. A younger age and surgical intervention on the nondominant brain hemisphere were associated with a reduced risk of postoperative neurological deficits. After 1 year, 74.2% of patients achieved seizure outcomes classified as ILAE class 1 or 2. Known positive predictors of seizure outcomes, such as identifiable MRI lesions and a history of febrile seizures, were supported by data. Furthermore, even after adjusting for preoperative MRI findings, hemisphere dominance, occurrence of bilateral tonic-clonic seizures, age, and sex, anterior temporal lobe resection was linked to improved seizure outcomes. CONCLUSIONS:This study offers extensive multicenter data on outcomes following MTLE surgery from a large international patient cohort. The authors' analysis indicates a strong safety profile and high efficacy for epilepsy surgery in this patient group. The comprehensive breakdown of results facilitates the assessment of individual success prospects and improves informed patient counseling.

Pediatric high-grade gliomas (HGGs) are the leading cause of brain cancer-related death in children. HGGs include distinct subtypes defined by anatomical location as well as molecular characteristics that stratify into H3K27M-altered diffuse midline gliomas (DMG) and hemispheric HGGs such as isocitrate dehydrogenase-wild type (IDH-WT) glioblastoma. Neuronal activity drives HGG progression both through paracrine signaling and direct neuron-to-glioma synapses. Using whole-cell patch clamp electrophysiology, in vivo optogenetics, and patient-derived glioma xenograft mouse models, we identify functional, tumor-promoting GABAergic neuron-to-glioma synapses mediated by GABAA receptors in DMGs. GABAergic input has a depolarizing effect on DMG cells due to chloride uptake by the Na-K-2Cl cotransporter NKCC1 and consequent elevated intracellular chloride concentration. In contrast, depolarizing GABAergic currents were not detected in IDH-WT HGG. Membrane depolarization is known to increase glioma proliferation and accordingly, we find that the activity of GABAergic interneurons promotes DMG proliferation in vivo. Increasing GABA signaling with the benzodiazepine lorazepam increases glioma proliferation and reduces survival in xenograft models of DMG but not IDH-WT HGG. Conversely, we find that the anti-seizure medication levetiracetam attenuates low-frequency depolarizing GABAergic synaptic currents in a glioma-specific manner, reducing those in DMG but not in healthy neurons. The effect in DMG is independent of action on synaptic vesicle glycoprotein 2A (SV2A), the chief mechanism by which levetiracetam suppresses seizures. Levetiracetam reduces glioma proliferation and extends survival of mice bearing DMG xenografts, but not IDH-WT HGG xenografts. Retrospective real-world clinical data demonstrate longer overall survival for children with DMG who were taking levetiracetam, which was not evident in pediatric hemispheric HGG. These findings uncover growth-promoting GABAergic synaptic communication between GABAergic neurons and DMG cells, underscoring a tumor subtype-specific mechanism of brain cancer neurophysiology with potentially important implications for commonly used drugs in this disease context, which should be further studied in future prospective clinical studies.

Background: Ependymoma (EPN) is not a uniform disease but represents different disease types with biological and clinical heterogeneity. However, the pattern of when and where different types of EPN relapse is not yet comprehensively described. Methods: We assembled 269 relapsed intracranial EPN from pediatric (n = 233) and adult (n = 36) patients from European and Northern American cohorts and correlated DNA methylation patterns and copy-number alterations with clinical information. Results: The cohort comprised the following molecular EPN types: PF-EPN-A (n = 177), ST-EPN-ZFTA (n = 45), PF-EPN-B (n = 31), PF-EPN-SE (n = 12), and ST-EPN-YAP (n = 4). First relapses of PF-EPN-B (PF: posterior-fossa) and PF-EPN-SE (SE: subependymoma) occurred later than of PF-EPN-A, ST-EPN-YAP (ST: supratentorial), or ST-EPN-ZFTA (median time to relapse: 4.3 and 6.0 years vs. 1.9/1.0/2.4 years; P < .01). Metastatic or combined recurrences in PF-EPN-B and -A more often involved the spinal cord than in ST-EPN-ZFTA (72.7% and 40.0 vs. 12.5%; P < .01). No distant relapses were observed in ST-EPN-YAP (n = 4) or PF-EPN-SE (n = 12). Post-relapse survival (PRS) was poor for PF-EPN-A and ST-EPN-ZFTA (5-year PRS: 44.5% +/- 4.4%/47.8% +/- 9.1%), whereas PF-EPN-B and PF-EPN-SE displayed a 5-year PRS of 89.5% +/- 7.1%/90.0% +/- 9.5% (P = .03). However, 10-year PRS for PF-EPN-B dropped to 45.8% +/- 17.3%. Neither between the radiation field and relapse pattern nor between the radiation field and spinal involvement at relapse an impact was identified. Notably, all patients with relapsed ST-EPN-YAP did not receive upfront radiotherapy but were successfully salvaged using irradiation at relapse. Conclusions: Relapse patterns of specific EPN types are different. Future clinical trials, treatment adaptions, duration of surveillance, and diagnostics should be planned to incorporate entity-specific relapse information.

Local relapse along the resection cavity is the most frequent mode of glioblastoma progression. Surgically induced molecular alterations and transformation of residual tumor ecosystems driving local tumor relapse remain elusive. Here, we applied MERFISH and Visium spatial transcriptomics to 203 specimens from 179 patients obtained at first surgery, early re-resection (2–40d) and late recurrence, and trained a spatial graph-attention network (sGAT) that predicted time-after-surgery from microenvironmental neighborhoods (AUC=0.83). Explainable-AI approaches (Integrated-gradient/attention) revealed a stereotyped cascade dominated by a hypoxia/HIF-1α-program with glycolytic switch and lactate-fueled-coupling between residual tumor cells, neurons and infiltrating myeloid cells within 2–10d at peri-cavity-rim. Between days 10–30 the niche transitions to TREM2⁺/P2RY12⁺ microglia, CXCL13⁺ T-cell influx and endothelial proliferation. Multiparametric MRI confirmed a stroke-like-hypoperfusion and blood-brain-barrier breach peaking at days 1–5 and resolving by days 60–90. To validate time-resolved tissue adaptation and causality, we performed spatial metabolomic and transcriptomic profiling on our murine-cortical-resection model. The sGAT classifier trained on human data assigned the exact post-operative status to murine samples (R² = 0.91). MALDI-imaging mass spectrometry confirmed the enhanced lactate consumption in proximity to the resection cavity enriched for neurons and altered microglia. Knockout of the lactate transporter MCT2 in neurons (rAAV:::hSyn:::shRNA-MCT2) demonstrated reduced tumor proliferation and lower-invasive growth (padj<0.05) with reduced tumor-neuron synaptic contacts in a rabies-ΔG-GFP retrograde tracing model using human neocortical slices (padj < 0.01). Similar reduction of tumor-neuron synaptic contacts was observed after depletion of microglia with PLX5622, supporting a co-operative metabolic-inflammatory-axis. Post-resection metabolic adaptation and tumor regrowth link was confirmed in vivo leveraging a LDH inhibitor or ROS scavenger demonstrating a significant decrease in tumor relapse. Our data delineate a narrow, druggable window immediately after surgery in which hypoxic-inflammatory-reprogramming metabolically and synoptically primes glioblastoma for recurrence, nominating peri-cavity lactate-shuttling or microglial activation as actionable adjuvant targets.

Glioblastomas are invasive brain tumors with high therapeutic resistance. Neuron-to-glioma synapses have been shown to promote glioblastoma progression. However, a characterization of tumor-connected neurons has been hampered by a lack of technologies. Here, we adapted retrograde tracing using rabies viruses to investigate and manipulate neuron-tumor networks. Glioblastoma rapidly integrated into neural circuits across the brain, engaging in widespread functional communication, with cholinergic neurons driving glioblastoma invasion. We uncovered patient-specific and tumor-cell-state-dependent differences in synaptogenic gene expression associated with neuron-tumor connectivity and subsequent invasiveness. Importantly, radiotherapy enhanced neuron-tumor connectivity by increased neuronal activity. In turn, simultaneous neuronal activity inhibition and radiotherapy showed increased therapeutic effects, indicative of a role for neuron-to-glioma synapses in contributing to therapeutic resistance. Lastly, rabies-mediated genetic ablation of tumor-connected neurons halted glioblastoma progression, offering a viral strategy to tackle glioblastoma. Together, this study provides a framework to comprehensively characterize neuron-tumor networks and target glioblastoma.

OBJECTIVE:Quality monitoring and improvement are crucial in pediatric neurosurgery for effective risk assessment and surgical preparation. This study evaluates the reliability of the ClassIntra classification for intraoperative complications and its potential for predicting postoperative outcomes in pediatric patients. METHODS:In this prospective cohort study at a tertiary care center, we analyzed 47 pediatric patients undergoing various neurosurgical procedures. Data were systematically collected throughout the perioperative period, focusing on preoperative characteristics, intraoperative variables, and postoperative recovery metrics. RESULTS:The cohort was categorized using the ClassIntra grading system into 3 groups: CI = 0 (N = 24), CI = 1 (N = 20), and CI ≥ 2 (N = 3). The mean age was 7.0 years, with no significant demographic differences across groups. Operative times increased with higher ClassIntra grades (126.6 minutes for CI = 0, 227.6 minutes for CI = 1, and 260.7 minutes for CI ≥ 2; P = 0.01). Patients with major intraoperative adverse events showed higher neurological deterioration (P = 0.03) and increased 90-day readmission rates in the CI ≥ 2 group (P < 0.01). CONCLUSIONS:Intraoperative complications significantly affect postoperative recovery in pediatric neurosurgery. Longer operative times and worse neurological outcomes correlate with higher ClassIntra ratings, emphasizing the need for improved monitoring and risk-reduction strategies.

In recent years, it has been increasingly recognized that tumor growth relies not only on support from the surrounding microenvironment but also on the tumors capacity to adapt to – and actively manipulate – its niche. While targeting angiogenesis and modulating the local immune environment have been explored as therapeutic approaches, these strategies have yet to yield effective treatments for brain tumors and remain under refinement. More recently, the nervous system itself has been explored as a critical environmental support for cancer, with extensive neuro-tumoral interactions observed both intracranially and in extracranial sites containing neural components. In the brain, interactions between glioma cells as well as metastatic lesions with neural components have clinical implications for diagnostics, risk assessments, neurological sequelae, and the development of innovative therapeutics. Here, we review these neuro-tumoral dynamics, emphasizing aspects relevant to neurosurgical practice.

Extracellular vesicles (EVs) act as carriers of biological information from tumors to the bloodstream, enabling the detection of circulating tumor material and tracking of disease progression. This is particularly crucial in glioblastoma, a highly aggressive and heterogeneous tumor that is challenging to monitor. Using imaging flow cytometry (IFCM), we conducted an immunophenotyping analysis of eight glioma-associated antigens and tetraspanins in plasma EVs from 37 newly diagnosed glioblastoma patients (pre- and post-surgery), 11 matched individuals with recurrent glioblastoma, and 22 healthy donors (HD). Tenascin-C (TNC) positive EVs displayed the strongest differences in newly diagnosed and recurrent glioblastoma patients, when compared to non-tumor subjects. Among dual-positive subpopulations, TNC+/CD9+ EVs were the most elevated in newly diagnosed (FC = 7.6, p <0.0001, AUC = 81%) and recurrent patients (FC = 16.5, p <0.0001; AUC = 90%) than HD. In comparison with other CNS tumors (n = 25), this subpopulation was also 34.5-fold higher in glioblastoma than in meningioma cases (p <0.01). Additionally, TNC+/CD9+ EV levels were 3.3-fold elevated in cerebrospinal fluid from glioblastoma patients (n = 6) than controls (p <0.05). Aberrant TNC levels were further observed in glioblastoma EVs from different sources and purified via different methods. Immunohistochemical analysis revealed high levels of TNC in tumor tissues. Spatial transcriptomic analysis indicated a TNC overexpression in malignant cell populations of glioblastoma resections, particularly in cells with mesenchymal-like signatures and chromosomal aberrations. Lastly, we purified TNC+ EVs from plasma of 21 glioblastoma patients by magnetic sorting and detected the oncogenic mutation TERT*C228T by droplet digital PCR. The mutant allele frequency was higher in TNC+ EVs vs TNC-negative EVs (FC = 32, p <0.001), total EVs (FC = 5.3, p <0.001) or cell-free DNA (FC = 5.3, p <0.01). In conclusion, circulating TNC+ EVs may have potential as clinical biomarkers in glioblastoma, and their purification could improve the identification of tumor-specific mutations in liquid biopsies.

OBJECTIVE:Tiny intracranial aneurysms (tiAs) pose challenges in detection and management. Recent advances in neuroimaging have improved the detection rates of these diminutive lesions, yet the decision-making process regarding their treatment remains controversial. While larger aneurysm size is a relevant risk factor for a possible rupture in common risk scores, the rupture of tiAs leading to subarachnoid hemorrhage (SAH) is also reported frequently. However, a heterogeneity in practice exists, and clear guidelines for the treatment of incidental tiAs are lacking. The aim of this study was to investigate clinical decision-making for aneurysm repair by investigating the risk factors for rupture in tiny aneurysms. METHODS:This retrospective analysis utilized data from patients admitted to the University Medical Center Hamburg-Eppendorf for aneurysmal SAH (n = 427) between 2010 and 2020 and patients with unruptured intracranial aneurysm (UIAs) discussed by the medical center's aneurysm board (n = 743) between 2012 and 2022. Patients with single tiAs, defined as a maximum diameter of 3 mm, were selected for further analysis. Patient demographics, aneurysm characteristics, and risk factors were compared between the SAH and UIA groups. A modified UIA treatment score (UIATS*) and the PHASES (population, hypertension, age, size of aneurysm, earlier SAH from another aneurysm, site of aneurysm) score were collected in UIA patients and applied retrospectively in SAH patients for the time of admission. RESULTS:The authors identified 79 tiAs in the SAH cohort and 109 tiAs in the UIA cohort. Irregular morphology (OR 4.53, 95% CI 1.84-11.16; p < 0.001) and a high size ratio/aspect ratio (> 3/1.6; OR 14.73, 95% CI 3.55-61.17; p < 0.001) were significantly associated with rupture when comparing tiAs of the SAH and UIA groups. When applying the UIATS* to tiny aneurysms retrospectively, intervention would have been recommended in 20.3% of SAH cases compared with 0.9% of UIA cases (p < 0.001). Comparatively, for nontiny aneurysms, intervention would have been recommended in 33.9% of SAH cases compared with 15.6% of UIA cases (p < 0.001). CONCLUSIONS:In the present collective, several ruptured tiAs could be identified. Nevertheless, only a few would have been recommended for treatment. Especially tiAs with irregular morphology and abnormal configurations should be critically evaluated for rupture risk. Current scoring systems provide valuable guidance but should be used in conjunction with individualized, multidisciplinary treatment approaches.

Immune evasion is a hallmark of gliomas, yet the genetic mechanisms by which tumors escape immune surveillance remain incompletely understood. In this study, we systematically examined the presence of somatic mutations in HLA genes and genes encoding proteins involved in antigen presentation across isocitrate dehydrogenase wild-type and mutant gliomas using targeted next-generation sequencing. To address the challenges associated with detecting somatic mutations in these highly polymorphic and complex regions of the genome, we applied a combination of short-read and long-read sequencing techniques, extended the genetic region of interest (exons and introns), and applied a tailored bioinformatics analysis pipeline, which enabled an accurate evaluation of comprehensive sequencing data. Our analysis identified mutations in HLA class II and nonclassic HLA genes as well as genes associated with antigen presentation, such as TAP1/2 and B2M. Three-dimensional modeling of individual mutations simulated the potential impact of somatic mutations in TAP1 and B2M on the encoded protein configuration. The presence of somatic mutations supports the role of antigen-presenting genes in the pathophysiology and potential immune escape of gliomas. Our data demonstrated an increased frequency of such mutations in recurrent glioblastoma, potentially resulting from a positive selection or mutagenic enrichment of tumor cells during tumor progression. Taken together, this research generates new insights and hypotheses for the functional analysis and optimization of immunotherapy strategies for gliomas, which may guide personalized treatment paradigms.

Extracellular vesicles (EVs) transport biomolecules that could serve as biomarkers for disease diagnosis and monitoring. The clinical utility of EVs derived from cerebrospinal fluid (CSF) in patients with intradural spinal tumors (IST) has not yet been investigated. Here, we obtained EVs from CSF of adult patients with intraspinal ependymoma (n = 9), meningioma (n = 9), hemangioma (n = 4) and schwannian tumors (n = 7), as well as comparison group (‘CG’, normal pressure hydrocephalus, n = 7), by ultrafiltration. CSF-EVs were characterized by electron microscopy and nanoparticle tracking analysis. EV populations according to the presence of tetraspanins (CD9, CD63, CD81) were measured by imaging flow cytometry (IFCM). CD81+ EVs were more prevalent in the comparison group, meningioma, ependymoma WHO grade 2, and hemangioma, whereas CD9+ EVs were predominant in ependymoma grade 1 and Schwannian tumors. CD63+ EVs per milliliter/CSF differed between ependymoma WHO grades 1 and 2 (FC = 24.6, AUC = 90%, p < 0.05). Based on results from a bead-based multiplex profiling, we selected ITGB1, CD44, CD133 and HLA-DR/DQ/DP for further phenotyping in CSF-EVs using IFCM, in combination with each tetraspanin as double-positive subpopulations. Compared to CG, CD44+ EVs were the most relevant population in CSF from IST patients, followed by ITGB1. Notable differences in absolute (EVs/mL CSF) and relative (percentages of CSF-EVs) levels were: CD44+/CD81+ for ependymoma grade 1 (FC = 196.5 and 34.5; p < 0.01) and grade 2 (%FC = 6.1, p < 0.05); CD44+/CD63+ for meningioma (abs. and %FC > 1000, p < 0.05); ITGB1+/CD81+ for hemangioma (%FC = 4.8, p < 0.05); and ITGB1+/CD9+ for schwannian tumors (abs.FC = 19.8, p < 0.01). In conclusion, we identified distinct EV subpopulations in the CSF of IST patients, potentially facilitating tumor classification.

Glioblastomas are known for their immunosuppressive tumor microenvironment, which may explain the failure of most clinical trials in the past decade. Recent studies have emphasized the significance of stratifying glioblastoma patients to predict better therapeutic responses and survival outcomes. This study aims to investigate the prognostic relevance of peripheral immune cell counts sampled prior to surgery, with a special focus on methylation-based subclassification. Peripheral blood was sampled in patients with newly diagnosed (n = 176) and recurrent (n = 41) glioblastoma at the time of surgery and analyzed for neutrophils, monocytes, leukocytes, platelets, neutrophil-lymphocyte ratio, lymphocyte-monocyte ratio, and platelet-lymphocyte ratio. Peripheral immune cell counts were correlated with patients' survival after combined radiochemotherapy. In addition, 850 k genome-wide DNA methylation was assessed on tissue for defining tumor subclasses and performing cell-type deconvolution. In newly diagnosed glioblastoma, patients with higher peripheral neutrophil counts had an unfavorable overall survival (OS) (p = 0.01, median overall-survival (mOS) 17.0 vs. 10.0 months). At the time of first recurrence, a significant decrease of peripheral immune cell counts was observed, and elevated monocyte (p = 0.03), neutrophil (p = 0.04), and platelet (p = 0.01) counts were associated with poorer survival outcomes. DNA methylation subclass-stratified analysis revealed a significant survival influence of neutrophils (p = 0.007) and lymphocytes (p = 0.04) in the mesenchymal (MES) subclass. Integrating deconvolution of matched tumor tissue showed that platelets and monocytes were correlated with a more differentiated, tumor-progressive cell state, and peripheral immune cell counts were most accurately reflected in tissue of the MES subclass. This study illustrates a restricted prognostic significance of peripheral immune cell counts in newly diagnosed glioblastoma and a constrained representation in matched tumor tissue, but it demonstrates a more pertinent situation at the time of recurrence and after DNA methylation-based stratification.

Magnetic resonance imaging (MRI) plays a crucial role in visualizing brain structures and pathologies, but the cellular basis of MRI signals remains incompletely understood. Using the BRIDGE co-registration workflow, a platform that integrates in vivo MRI with two-photon (2P) microscopy, in patient-derived glioblastoma models and human glioblastoma tissue, T2w signal intensities were correlated with tumor growth rates and density over time. By leveraging nnU-Net, a convolutional neural network for image segmentation, in combination with BRIDGE the visibility and segmentation of vessels in MRI were improved. In the future, this microscopy-based segmentation approach could be used for predicting tumor growth and analyzing vascular alterations in gliomas and brain metastases. Overall, BRIDGE provides a framework that deciphers microscopic signatures from clinical imaging, offering new opportunities for deep learning-enhanced histology in clinical imaging.