BackgroundSpontaneous intracerebral abscess formation is a rare condition presenting with a disabling sequela. The origin of infection can either be primary or secondary to an infection at another location. The site of primary infection - due to the proximity, often the oral cavity, the sinuses, and the orbit - determines the causative pathogens. Treatment often combines surgical and antimicrobial therapies. To determine the microbiology and respective changes and treatment outcome, we performed this retrospective monocentric cohort study of patients requiring surgical treatment of brain abscesses.MethodsPatients undergoing surgical treatment of a primary intracranial abscess between January 2000 and January 2021 in the Department of Neurosurgery, Freiburg University Hospital were included. Demographic, clinical and imaging data were extracted from patients' medical records and databases. Treatment approaches were also analyzed, and surgical therapy and antibiotic therapy were reported. Outcome was assessed by the modified Rankin score (mRS) and was dichotomized into good (mRS 0-3) and poor (mRS 4-6) outcome.ResultsWe included 65 patients with spontaneous intracerebral abscess that were treated with neurosurgical intervention at our institution. Analysis of the causative pathogens showed an increasing dominance of rare pathogens such as fungi, parasites, mycobacteria and anaerobes. Outcome measured by the mRS was similar from 2005 to 2021.ConclusionsThe pathogen spectrum of spontaneous intracerebral abscess at our institution is shifting with rarer pathogens being increasingly detected. This retrospective analysis highlights the need for microbiological diagnosis and of combined surgical and antibiological treatment.

Background: Falcotentorial meningiomas are exceptionally uncommon tumors, presenting a challenge for neurosurgeons due to their close proximity to vital structures. Gross total resection represents the standard of treatment for these tumors. However, care must be taken when surgically approaching these lesions, since damaging neurovascular structures may cause unacceptable morbidity. Selecting the optimal surgical approach for each tumor is of paramount importance when treating these patients. Methods: The authors reviewed medical records to identify all patients with falcotentorial meningiomas who underwent resection at the University Hospital of Freiburg between January 2001 and December 2021. Clinical and imaging data, surgical management, and clinical outcomes were analyzed. Results: Falcotentorial meningiomas occurred in 0.7% (15 of 2124 patients) of patients with intracranial meningiomas. Of these 15 patients, 8 were female and 7 male. The occipital interhemispheric approach was used in nine patients, the supracerebellar infratentorial approach in five patients, and the retrosigmoidal approach in one patient. Three patients developed visual field deficits after surgical resection. Incomplete resection was significantly associated with tumor progression (p < 0.05). Conclusions: Individualized surgical strategies, guided by preoperative imaging and classification systems, play a crucial role in optimizing patient care. Among the available approaches, the occipital interhemispheric and supracerebellar infratentorial approaches are frequently employed and considered among the safest options for these tumors.

Single-cell RNA-sequencing (scRNA-seq) is becoming a ubiquitous method in profiling the cellular transcriptomes of both malignant and non-malignant cells from the human brain. Here, we present a protocol to isolate viable tumor cells from human ex vivo glioblastoma cultures for single-cell transcriptomic analysis. We describe steps including surgical tissue collection, sectioning, culturing, primary tumor cells inoculation, growth tracking, fluorescence-based cell sorting, and population-enriched scRNA-seq. This comprehensive methodology empowers in-depth understanding of brain tumor biology at the single-cell level.For complete details on the use and execution of this protocol, please refer to Ravi et al.1

Despite recent advances in cancer immunotherapy, certain tumor types, such as Glioblastomas, are highly resistant due to their tumor microenvironment disabling the anti-tumor immune response. Here we show, by applying an in-silico multidimensional model integrating spatially resolved and single-cell gene expression data of 45,615 immune cells from 12 tumor samples, that a subset of Interleukin-10-releasing HMOX1 + myeloid cells, spatially localizing to mesenchymal-like tumor regions, drive T-cell exhaustion and thus contribute to the immunosuppressive tumor microenvironment. These findings are validated using a human ex-vivo neocortical glioblastoma model inoculated with patient derived peripheral T-cells to simulate the immune compartment. This model recapitulates the dysfunctional transformation of tumor infiltrating T-cells. Inhibition of the JAK/STAT pathway rescues T-cell functionality both in our model and in-vivo, providing further evidence of IL-10 release being an important driving force of tumor immune escape. Our results thus show that integrative modelling of single cell and spatial transcriptomics data is a valuable tool to interrogate the tumor immune microenvironment and might contribute to the development of successful immunotherapies.

Defects of the cranial vault often require cosmetic reconstruction with patient-specific implants, particularly in cases of craniofacial involvement. However, fabrication takes time and is expensive; therefore, efforts must be made to develop more rapidly available and more cost-effective alternatives. The current study investigated the feasibility of an augmented reality (AR)-assisted single-step procedure for repairing bony defects involving the facial skeleton and the skull base. In an experimental setting, nine neurosurgeons fabricated AR-assisted and conventionally shaped ("freehand") implants from polymethylmethacrylate (PMMA) on a skull model with a craniofacial bony defect. Deviations of the surface profile in comparison with the original model were quantified by means of volumetry, and the cosmetic results were evaluated using a multicomponent scoring system, each by two blinded neurosurgeons. Handling the AR equipment proved to be quite comfortable. The median volume deviating from the surface profile of the original model was low in the AR-assisted implants (6.40 cm3) and significantly reduced in comparison with the conventionally shaped implants (13.48 cm3). The cosmetic appearance of the AR-assisted implants was rated as very good (median 25.00 out of 30 points) and significantly improved in comparison with the conventionally shaped implants (median 14.75 out of 30 points). Our experiments showed outstanding results regarding the possibilities of AR-assisted procedures for single-step reconstruction of craniofacial defects. Although patient-specific implants still represent the gold standard in esthetic aspects, AR-assisted procedures hold high potential for an immediately and widely available, cost-effective alternative providing excellent cosmetic outcomes.

Glioblastomas are malignant tumors of the central nervous system hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies. The source of dynamic reorganization within the spatial context of these tumors remains elusive. Here, we characterized glioblastomas by spatially resolved transcriptomics, metabolomics, and proteomics. By deciphering regionally shared transcriptional programs across patients, we infer that glioblastoma is organized by spatial segregation of lineage states and adapts to inflammatory and/or metabolic stimuli, reminiscent of the reactive transformation in mature astrocytes. Integration of metabolic imaging and imaging mass cytometry uncovered locoregional tumor-host interdependence, resulting in spatially exclusive adaptive transcriptional programs. Inferring copy-number alterations emphasizes a spatially cohesive organization of subclones associated with reactive transcriptional programs, confirming that environmental stress gives rise to selection pressure. A model of glioblastoma stem cells implanted into human and rodent neocortical tissue mimicking various environments confirmed that transcriptional states originate from dynamic adaptation to various environments.

Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.

Despite recent advances in cancer immunotherapy, its efficacy in Glioblastoma (GBM) is limited due to poor understanding of molecular states and cellular plasticity of immune cells within the tumor microenvironment. Here, we combined spatial and single-cell transcriptomics of 47.284 immune cells, to map the potential cellular interactions leading to the immunosuppressive microenvironment and dysfunction of T cells. Computational approach identified a subset of IL10 releasing HMOX1+ myeloid cells which activates transcriptional programs towards a dysfunctional state in T cells, and was found to be localized within mesenchymal dominated subregions of the tumor. These findings were further validated by a human ex-vivo neocortical GBM model (n=6) coupled with patient derived peripheral T-cells. Finally, the dysfunctional transformation of T cells was shown to be rescued by JAK/STAT inhibition in both our model and in-vivo. We strongly believe that our findings would be the stepping stone towards successful development of immunotherapeutic approaches in GBM.

Reactive transformation of astrocytes in IDH wild-type glioma contribute to anti-tumor immunity and support pro-oncogenic signaling. The role of intra- and peritumoral astrocytes in IDH1/2 mutated glioma, a prognostically beneficial subgroup with oncogene-driven enrichment of R-2-hydroxygluterate (2-HG) remains sparsely explored. Here, we characterized the transcriptomic signature of IDH1/2-mutant glioma associated astrocytes and determined a unique inflammatory transformation, profoundly different to astrocytes in IDH wildtype glioma patients. IDH-mutated glioma inoculation into human neocortical sections or treatment with R-2-HG resulted in an oncometabolite-mediated transcriptional shift towards inflammation in astrocytes. This transcriptional shift was found to be mediated by myeloid cell polarization through R-2-HG in the tumor microenvironment, which was further confirmed by selective depletion of myeloid cells. Integrative analysis of bulk RNA-sequencing of purified microglia, single-cell transcriptomics and spatially resolved transcriptomics confirmed this microglia-mediated inflammatory activation of astrocytes. This inflammatory activation is accompanied by loss of neurotransmitter homeostasis. Further, treatment of cortical sections with RH2G results in increased neuronal activity and LFP activity, pointing to the excitotoxic nature of RH2G in the neural microenvironment, with a significant loss of neurons with chronic treatment. The presented findings provide insights into the role that R-2HG plays in the reactive transformation of astrocytes within the IDH-mutated tumor environment and is fundamentally different that seen in IDH wildtype glioma.

Elderly patients constitute an expanding part of our society. Due to a continuously increasing life expectancy, an optimal quality of life is expected even into advanced age. Glioblastoma (GBM) is more common in older patients, but they are still often withheld from efficient treatment due to worry of worse tolerance and have a significantly worse prognosis compared to younger patients. Our retrospective observational study aimed to investigate the therapeutic benefit from a second resection in recurrent glioblastoma of elderly patients. We included a cohort of 39 elderly patients (> 65 years) with a second resection as treatment option in the case of a tumor recurrence. A causal inference model was built by multiple non- and semiparametric models, which was used to identify matched patients from our elderly GBM database which comprises 538 patients. The matched cohorts were analyzed by a Cox-regression model adjusted by time-dependent covariates. The Cox-regression analysis showed a significant survival benefit (Hazard Ratio: 0.6, 95% CI 0.36–0.9, p-value = 0.0427) for the re-resected group (18.0 months, 95% CI 13.97–23.2 months) compared to the group without re-resection (10.1 months, 95% CI 8.09–20.9 months). No differences in the co-morbidities or hemato-oncological side effects during chemotherapy could be detected. Anesthetic- and surgical complications were rare and comparable to the complication rate of patients undergoing the first-line resection. Taken together, in elderly patients, re-resection is an acceptable treatment option in the recurrent state of a glioblastoma. The individual evaluation of the patients′ medical status as well as the chances of withstanding general anesthesia needs to be done in close interdisciplinary consultation. If these requirements are met, elderly patients benefit from a re-resection.

The role of tumor-associated astrocytes in the microenvironment of glioma has long been underestimated but is moving into the focus of current research. We explored the role of reactive astrocytes in IDH-mutated glioma using RNA-sequencing of purified astrocytes and microglia and single-nucleus RNA-sequencing of infiltrating tumor regions. Mapping of the transcriptional phenotype of astrocytes along developmental and reactive trajectories revealed an inflammatory transformation of IDH-mutated associated astrocytes. The major proportion of astrocytes is marked by complement-activation similar to findings in neuroinflammatory diseases. A human neocortical slices model with injected IDH-mutated patient-derived cells or D-2HG treatment (+/- microglia depletion) was used to map shared and unique transcriptional adaptation in astrocytes promoted by either tumor cells or metabolic alteration. High-dimensional electrophysiological profiling was used to investigate alterations in neural response to tumor-induced microenvironmental transformation. We showed that 2HG alone promote the inflammatory pattern of astrocytes, which causes neurotoxicity and seizures in our neocortical slice model. Depletion of microglia rescued the neurotoxicity suggesting that microglia predominantly drive inflammatory astrogliosis as a response to metabolic alteration the tumor environment. We showed that neurotoxic astrogliosis induced by the oncometabolite D-2HG via distinct microglia activation promote the evolution of frequently observed seizures in IDH-mutated glioma patients.

SummaryThe diversity of molecular states and cellular plasticity of immune cells in the glioblastoma environment is still poorly understood. Here, we performed scRNA sequencing of the immune compartment and mapped potential cellular interactions leading to an immunosuppressive microenvironment and dysfunction of T cells. Through inferring the dynamic adaptation during T cell activation, we identified three different terminal states with unique transcriptional programs. Modeling of driver genes for terminal T cell fate identified IL-10 signaling alterations in a subpopulation of HAVCR2(+) T cells. To explore in depth cellular interactions, we established anin-silicomodel by the integration of spatial transcriptomic and scRNA-sequencing, and identified a subset of HMOX1+myeloid cells defined by IL10 release leading to T cell exhaustion. We found a spatial overlap between HMOX(+) myeloid and HAVCR2(+) T cells, suggesting that myeloid-lymphoid interaction causes immunosuppression present in tumor regions with enriched mesenchymal gene expression. Using human neocortical GBM model, coupled with patient-derived T cells, we confirmed that the functional interaction between myeloid and lymphoid cells, leads to a dysfunctional state of T cells. This IL-10 driven T cell exhaustion was found to be rescued by JAK/STAT inhibition. A comprehensive understanding of the cellular states and plasticity of lymphoid cells in GBM will aid towards successful immunotherapeutic approaches.

Reactive astrocytes evolve after brain injury, inflammatory and degenerative diseases, whereby they undergo transcriptomic re-programming. In malignant brain tumors, their function and crosstalk to other components of the environment is poorly understood. Here we report a distinct transcriptional phenotype of reactive astrocytes from glioblastoma linked to JAK/STAT pathway activation. Subsequently, we investigate the origin of astrocytic transformation by a microglia loss-of-function model in a human organotypic slice model with injected tumor cells. RNA-seq based gene expression analysis of astrocytes reveals a distinct astrocytic phenotype caused by the coexistence of microglia and astrocytes in the tumor environment, which leads to a large release of anti-inflammatory cytokines such as TGFβ, IL10 and G-CSF. Inhibition of the JAK/STAT pathway shifts the balance of pro- and anti-inflammatory cytokines towards a pro-inflammatory environment. The complex interaction of astrocytes and microglia cells promotes an immunosuppressive environment, suggesting that tumor-associated astrocytes contribute to anti-inflammatory responses.

When it comes to the human brain, models that closely mimic in vivo conditions are lacking. Living neuronal tissue is the closest representation of the in vivo human brain outside of a living person. Here, we present a method that can be used to maintain therapeutically resected healthy neuronal tissue for prolonged periods without any discernible changes in tissue vitality, evidenced by immunohistochemistry, genetic expression, and electrophysiology. This method was then used to assess glioblastoma (GBM) progression in its natural environment by microinjection of patient-derived tumor cells into cultured sections. The result closely resembles the pattern of de novo tumor growth and invasion, drug therapy response, and cytokine environment. Reactive transformation of astrocytes, as an example of the cellular nonmalignant tumor environment, can be accurately simulated with transcriptional differences similar to those of astrocytes isolated from acute GBM specimens. In a nutshell, we present a simple method to study GBM in its physiological environment, from which valuable insights can be gained. This technique can lead to further advancements in neuroscience, neuro-oncology, and pharmacotherapy.

Although reactive astrocytes constitute a major component of the cellular environment in glioblastoma, their function and crosstalk to other components of the environment is still poorly understood. Gene expression analysis of purified astrocytes from both the tumor core and non-infiltrated cortex reveals a tumor-related up-regulation of Chitinase 3-like 1 (CHI3L1), a cytokine which is related to inflammation, extracellular tissue remodeling, and fibrosis. Further, we established and validated a co-culture model to investigate the impact of reactive astrocytes within the tumor microenvironment. Here we show that reactive astrocytes promote a subtype-shift of glioblastoma towards the mesenchymal phenotype, driving mitogen-activated protein kinases (MAPK) signaling as well as increased proliferation and migration. In addition, we demonstrate that MAPK signaling is directly caused by a CHI3L1-IL13RA2 co-binding, which leads to increased downstream MAPK and AKT signaling. This novel microenvironmental crosstalk highlights the crucial role of non-neoplastic cells in malignant brain tumors and opens up new perspectives for targeted therapies in glioblastoma.

Abstract Oligodendroglioma are defined by a distinct molecular phenotype marked by 1p19q co-deletion and simultaneous presence of an IDH1/2 mutation. These tumors showed a favorable clinical course and long-term survival of around 15 years. Due to the long course of the disease, prospective studies to determine the effectiveness of different therapeutic strategies are difficult, since the percentage of patients with multiple therapies is high. Here we report a computational approach to map the longitudinal growth pattern, to quantify the effect of therapies on tumor growth and to identify similarities and spatial heterogeneity of oligodendroglioma growth. In our study, we included a cohort of 44 histopathologically and molecularly stratified oligodendrogliomas WHO°II (n=23) and WHO°III (n=21). We started our investigation with the longitudinal tumor segmentation. All volumetric data were pinpointed to the times of tumor therapy within all patients. Next, we extracted first-order features of tumor growth and response to chemo- or radiotherapy as well as resection, resulting in a total number of 98 features. An unsupervised cluster was used to identify similarities between patients, which revealed 3 subgroups. The first subgroup contained patients with predominantly frontal oligodendrogliomas marked by increased response to radiotherapy. The second subgroup included temporal oligodendrogliomas with high response rate to PC/PCV chemotherapy and flagged by epilepsy. The third group was heterogeneous with varying growth behaviors. A survival analysis showed a better separation between low- and high-risk patients based on the growth pattern model, in contrast to the WHO grading system. Taken together, our analysis revealed a novel classification of oligodendroglioma based on the longitudinal growth pattern and therapeutical response. We also detected a spatial difference between frontally or temporally localized oligodendrogliomas. We plan to further investigate molecular data that explain these spatial differences, which also may uncover novel therapeutic strategies.

Abstract Reactive astrocytes are caused by multiple pathologies of the central nervous system, whereby they undergo distinct transcriptomic re-programming. Although the role of reactive astrocytes in some inflammatory diseases has been investigated, many central questions regarding the immunoregulatory functions of tumor-associated astrocytes and their crosstalk to microglia remain poorly understood. In our presented study, we purified astrocytes from various pathologies and different brain tumors to map the transcriptional landscape of reactive astrocytes. We identified the marker genes CHI3L1 and CD274 highly enriched in reactive astrocytes of the marginal astrogliosis scar at the tumor boarder. Human neocortical slices along with a microglia loss-of-function model were used to explore the crosstalk of microglia and reactive astrocytes within the tumor environment. Our results revealed that the reactive phenotype mutually arises from both, microglia and tumor cells. This interaction caused JAK/STAT signalling in reactive astrocytes along with a large release of anti-inflammatory cytokines such as TGFß and IL10. Additionally, inhibition of the JAK/STAT pathway recovered the release of anti-inflammatory cytokines and resulted in a pro-inflammatory environment. Besides the immunosuppressive properties, we found evidence that reactive astrocytes drove AKT and MAPK signaling in the tumor through astrocytic released CHI3L1 and consequential binding to IL13RA2. Our findings revealed increased malignant properties arising from astrocytic-tumor interaction, which were rescued by IL13RA2 inhibition. In a nutshell, reactive astrocytes have decisive regulatory tasks in the microenvironment of CNS tumors. Along with microglia, reactive astrocytes cause the evolution of an immunosuppressive environment and support malignant properties of the tumor.

Gap junctions have recently been shown to interconnect glioblastoma cells to a multicellular syncytial network, thereby allowing intercellular communication over long distances as well as enabling glioblastoma cells to form routes for brain microinvasion. Against this backdrop gap junction-targeted therapies might provide for an essential contribution to isolate cancer cells within the brain, thus increasing the tumor cells' vulnerability to the standard chemotherapeutic agent temozolomide. By utilizing INI-0602-a novel gap junction inhibitor optimized for crossing the blood brain barrier-in an oncological setting, the present study was aimed at evaluating the potential of gap junction-targeted therapy on primary human glioblastoma cell populations. Pharmacological inhibition of gap junctions profoundly sensitized primary glioblastoma cells to temozolomide-mediated cell death. On the molecular level, gap junction inhibition was associated with elevated activity of the JNK signaling pathway. With the use of a novel gap junction inhibitor capable of crossing the blood-brain barrier-thus constituting an auspicious drug for clinical applicability-these results may constitute a promising new therapeutic strategy in the field of current translational glioblastoma research.