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Glioblastoma remains one of the deadliest brain malignancies. First-line therapy consists of maximal surgical tumor resection, accompanied by chemotherapy and radiotherapy. Malignant cells escape surgical resection by migrating into the surrounding healthy brain tissue, where they give rise to the recurrent tumor. Based on gene expression, tumor cores can be subtyped into mesenchymal, proneural, and classical tumors, each being associated with differences in genetic alterations and cellular composition. In contrast, the adjacent brain parenchyma where infiltrating malignant cells escape surgical resection is less characterized in patients. Using spatial transcriptomics (n = 11), we show that malignant cells within proneural or mesenchymal tumor cores display spatially organized differences in gene expression, although such differences decrease within the infiltrated brain tissue. Malignant cells residing in infiltrated brain tissue have increased expression of genes related to neurodevelopmental pathways and glial cell differentiation. Our findings provide an updated view of the spatial landscape of glioblastomas and further our understanding of the malignant cells that infiltrate the healthy brain, providing new avenues for the targeted therapy of these cells after surgical resection., (© 2024. The Author(s).)

Purpose: Mesenchymal stromal cells (MSCs) within the glioblastoma microenvironment have been shown to promote tumor progression. Tumor Treating Fields (TTFields) are alternating electric fields with low intensity and intermediate frequency that exhibit anti-tumorigenic effects. While the effects of TTFields on glioblastoma cells have been studied previously, nothing is known about the influence of TTFields on MSCs., Methods: Single-cell RNA sequencing and immunofluorescence staining were employed to identify glioblastoma-associated MSCs in patient samples. Proliferation and clonogenic survival of human bone marrow-derived MSCs were assessed after TTFields in vitro. MSC' characteristic surface marker expression was determined using flow cytometry, while multi-lineage differentiation potential was examined with immunohistochemistry. Apoptosis was quantified based on caspase-3 and annexin-V/7-AAD levels in flow cytometry, and senescence was assessed with ß-galactosidase staining. MSCs' migratory potential was evaluated with Boyden chamber assays., Results: Single-cell RNA sequencing and immunofluorescence showed the presence of glioblastoma-associated MSCs in patient samples. TTFields significantly reduced proliferation and clonogenic survival of human bone marrow-derived MSCs by up to 60% and 90%, respectively. While the characteristic surface marker expression and differentiation capacity were intact after TTFields, treatment resulted in increased apoptosis and senescence. Furthermore, TTFields significantly reduced MSCs' migratory capacity., Conclusion: We could demonstrate the presence of tumor-associated MSCs in glioblastoma patients, providing a rationale to study the impact of TTFields on MSCs. TTFields considerably increase apoptosis and senescence in MSCs, resulting in impaired survival and migration. The results provide a basis for further analyses on the role of MSCs in glioblastoma patients receiving TTFields., (© 2024. The Author(s).)

In this issue of Cancer Cell, Zhong et al. explore the dual role of TREM2 in glioblastoma-associated myeloid cells, demonstrating its function in promoting inflammation at the tumor-neural interface and suppression within the tumor core, influenced by the local microenvironment. These findings open up promising prospects for advancements in neuro-oncological immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Glioblastoma, the most aggressive and prevalent form of primary brain tumor, is characterized by rapid growth, diffuse infiltration, and resistance to therapies. Intrinsic heterogeneity and cellular plasticity contribute to its rapid progression under therapy; therefore, there is a need to fully understand these tumors at a single-cell level. Over the past decade, single-cell transcriptomics has enabled the molecular characterization of individual cells within glioblastomas, providing previously unattainable insights into the genetic and molecular features that drive tumorigenesis, disease progression, and therapy resistance. However, despite advances in single-cell technologies, challenges such as high costs, complex data analysis and interpretation, and difficulties in translating findings into clinical practice persist. As single-cell technologies are developed further, more insights into the cellular and molecular heterogeneity of glioblastomas are expected, which will help guide the development of personalized and effective therapies, thereby improving prognosis and quality of life for patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yabo, Heiland. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking., Methods: Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging., Results: High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging., Conclusions: We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background: Primary brain tumors and metastases in the central nervous system (CNS) are characterized by their unique microenvironment, which interacts with neuronal structures and influences structural and adaptive immunity., Objective: How significant are various tumor-host interactions from a prognostic and therapeutic perspective?, Material and Method: A literature search was carried out for relevant articles on the topic: microenvironment glioblastoma or metastasis through PubMed and Medline., Results: Modern high-throughput methods, such as spatial and single-cell resolution molecular characterization of tumors and their microenvironment enable a detailed mapping of changes and adaptation of individual cells within the microenvironment of tumors; however, treatment approaches based on altered tumor-host cell interactions, such as immune modeling, cell-based treatment methods or checkpoint inhibition have so far not shown any significant advantages for survival., Conclusion: A deeper understanding of the complex immune landscape and the microenvironment of metastases of the CNS and intracerebral tumors is essential to optimize future treatment strategies., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

The innate immune compartment of the human central nervous system (CNS) is highly diverse and includes several immune-cell populations such as macrophages that are frequent in the brain parenchyma (microglia) and less numerous at the brain interfaces as CNS-associated macrophages (CAMs). Due to their scantiness and particular location, little is known about the presence of temporally and spatially restricted CAM subclasses during development, health and perturbation. Here we combined single-cell RNA sequencing, time-of-flight mass cytometry and single-cell spatial transcriptomics with fate mapping and advanced immunohistochemistry to comprehensively characterize the immune system at human CNS interfaces with over 356,000 analyzed transcriptomes from 102 individuals. We also provide a comprehensive analysis of resident and engrafted myeloid cells in the brains of 15 individuals with peripheral blood stem cell transplantation, revealing compartment-specific engraftment rates across different CNS interfaces. Integrated multiomic and high-resolution spatial transcriptome analysis of anatomically dissected glioblastoma samples shows regionally distinct myeloid cell-type distributions driven by hypoxia. Notably, the glioblastoma-associated hypoxia response was distinct from the physiological hypoxia response in fetal microglia and CAMs. Our results highlight myeloid diversity at the interfaces of the human CNS with the periphery and provide insights into the complexities of the human brain's immune system., (© 2023. The Author(s).)

Growth factor receptors rank among the most important oncogenic pathways, but pharmacologic inhibitors often demonstrate limited benefit as monotherapy. Here, we show that epidermal growth factor receptor (EGFR) signaling repressed N 6 -methyladenosine (m 6 A) levels in glioblastoma stem cells (GSCs), whereas genetic or pharmacologic EGFR targeting elevated m 6 A levels. Activated EGFR induced non-receptor tyrosine kinase SRC to phosphorylate the m 6 A demethylase, AlkB homolog 5 (ALKBH5), thereby inhibiting chromosomal maintenance 1 (CRM1)-mediated nuclear export of ALKBH5 to permit sustained mRNA m 6 A demethylation in the nucleus. ALKBH5 critically regulated ferroptosis through m 6 A modulation and YTH N6-methyladenosine RNA binding protein (YTHDF2)-mediated decay of the glutamate-cysteine ligase modifier subunit (GCLM). Pharmacologic targeting of ALKBH5 augmented the anti-tumor efficacy of EGFR and GCLM inhibitors, supporting an EGFR-ALKBH5-GCLM oncogenic axis. Collectively, EGFR reprograms the epitranscriptomic landscape through nuclear retention of the ALKBH5 demethylase to protect against ferroptosis, offering therapeutic paradigms for the treatment of lethal cancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: In glioblastoma (GBM), the effects of altered glycocalyx are largely unexplored. The terminal moiety of cell coating glycans, sialic acid, is of paramount importance for cell-cell contacts. However, sialic acid turnover in gliomas and its impact on tumor networks remain unknown., Methods: We streamlined an experimental setup using organotypic human brain slice cultures as a framework for exploring brain glycobiology, including metabolic labeling of sialic acid moieties and quantification of glycocalyx changes. By live, 2-photon and high-resolution microscopy we have examined morphological and functional effects of altered sialic acid metabolism in GBM. By calcium imaging we investigated the effects of the altered glycocalyx on a functional level of GBM networks., Results: The visualization and quantitative analysis of newly synthesized sialic acids revealed a high rate of de novo sialylation in GBM cells. Sialyltrasferases and sialidases were highly expressed in GBM, indicating that significant turnover of sialic acids is involved in GBM pathology. Inhibition of either sialic acid biosynthesis or desialylation affected the pattern of tumor growth and lead to the alterations in the connectivity of glioblastoma cells network., Conclusions: Our results indicate that sialic acid is essential for the establishment of GBM tumor and its cellular network. They highlight the importance of sialic acid for glioblastoma pathology and suggest that dynamics of sialylation have the potential to be targeted therapeutically., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intra-tumoral heterogeneity and cell-state plasticity are key drivers for the therapeutic resistance of glioblastoma. Here, we investigate the association between spatial cellular organization and glioblastoma prognosis. Leveraging single-cell RNA-seq and spatial transcriptomics data, we develop a deep learning model to predict transcriptional subtypes of glioblastoma cells from histology images. Employing this model, we phenotypically analyze 40 million tissue spots from 410 patients and identify consistent associations between tumor architecture and prognosis across two independent cohorts. Patients with poor prognosis exhibit higher proportions of tumor cells expressing a hypoxia-induced transcriptional program. Furthermore, a clustering pattern of astrocyte-like tumor cells is associated with worse prognosis, while dispersion and connection of the astrocytes with other transcriptional subtypes correlate with decreased risk. To validate these results, we develop a separate deep learning model that utilizes histology images to predict prognosis. Applying this model to spatial transcriptomics data reveal survival-associated regional gene expression programs. Overall, our study presents a scalable approach to unravel the transcriptional heterogeneity of glioblastoma and establishes a critical connection between spatial cellular architecture and clinical outcomes., (© 2023. The Author(s).)

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

Background: Recent efforts have described the evolution of glioblastoma from initial diagnosis to post-treatment recurrence on a genomic and transcriptomic level. However, the evolution of the proteomic landscape is largely unknown., Methods: Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used to characterize the quantitative proteomes of two independent cohorts of paired newly diagnosed and recurrent glioblastomas. Recurrence-associated proteins were validated using immunohistochemistry and further studied in human glioma cell lines, orthotopic xenograft models, and human organotypic brain slice cultures. External spatial transcriptomic, single-cell, and bulk RNA sequencing data were analyzed to gain mechanistic insights., Results: Although overall proteomic changes were heterogeneous across patients, we identified BCAS1, INF2, and FBXO2 as consistently upregulated proteins at recurrence and validated these using immunohistochemistry. Knockout of FBXO2 in human glioma cells conferred a strong survival benefit in orthotopic xenograft mouse models and reduced invasive growth in organotypic brain slice cultures. In glioblastoma patient samples, FBXO2 expression was enriched in the tumor infiltration zone and FBXO2-positive cancer cells were associated with synaptic signaling processes., Conclusions: These findings demonstrate a potential role of FBXO2-dependent glioma-microenvironment interactions to promote tumor growth. Furthermore, the published datasets provide a valuable resource for further studies., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Enhanced fatty acid synthesis is a hallmark of tumors, including glioblastoma. SREBF1/2 regulate the expression of enzymes involved in fatty acid and cholesterol synthesis. Yet, little is known about the precise mechanism regulating SREBP gene expression in glioblastoma. Here, we show that a novel interaction between the co-activator/co-repressor CTBP and the tumor suppressor ZBTB18 regulates the expression of SREBP genes. In line with our findings, metabolic assays and glucose tracing analysis confirm the reduction in several phospholipid species upon ZBTB18 expression. Our study identifies CTBP1/2 and LSD1 as co-activators of SREBP genes and indicates that the functional activity of the CTBP-LSD1 complex is altered by ZBTB18. ZBTB18 binding to the SREBP gene promoters is associated with reduced LSD1 demethylase activity of H3K4me2 and H3K9me2 marks. Concomitantly, the interaction between LSD1, CTBP, and ZNF217 is increased, suggesting that ZBTB18 promotes LSD1 scaffolding function. Our results outline a new epigenetic mechanism enrolled by ZBTB18 and its co-factors to regulate fatty acid synthesis that could be targeted to treat glioblastoma patients., (© 2022 Ferrarese et al.)

Purpose: Most chimeric antigen receptor (CAR) T-cell strategies against glioblastoma have demonstrated only modest therapeutic activity and are based on persistent gene modification strategies that have limited transgene capacity, long manufacturing processes, and the risk for uncontrollable off-tumor toxicities. mRNA-based T-cell modifications are an emerging safe, rapid, and cost-effective alternative to overcome these challenges, but are underexplored against glioblastoma., Experimental Design: We generated mouse and human mRNA-based multifunctional T cells coexpressing a multitargeting CAR based on the natural killer group 2D (NKG2D) receptor and the proinflammatory cytokines IL12 and IFNα2 and assessed their antiglioma activity in vitro and in vivo., Results: Compared with T cells that either expressed the CAR or cytokines alone, multifunctional CAR T cells demonstrated increased antiglioma activity in vitro and in vivo in three orthotopic immunocompetent mouse glioma models without signs of toxicity. Mechanistically, the coexpression of IL12 and IFNα2 in addition to the CAR promoted a proinflammatory tumor microenvironment and reduced T-cell exhaustion as demonstrated by ex vivo immune phenotyping, cytokine profiling, and RNA sequencing. The translational potential was demonstrated by image-based single-cell analyses of mRNA-modified T cells in patient glioblastoma samples with a complex cellular microenvironment. This revealed strong antiglioma activity of human mRNA-based multifunctional NKG2D CAR T cells coexpressing IL12 and IFNα2 whereas T cells that expressed either the CAR or cytokines alone did not demonstrate comparable antiglioma activity., Conclusions: These data provide a robust rationale for future clinical studies with mRNA-based multifunctional CAR T cells to treat malignant brain tumors., (©2022 American Association for Cancer Research.)

Glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. We investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti-apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non-malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti-apoptotic BCL-2 family members. High anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein-targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3-mimetics., (© 2022. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

Introduction: 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., Materials and Methods: 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., Results: 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., Conclusion: 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.

Fusion proteins involving the BRAF serine/threonine kinase occur in many cancers. The oncogenic potential of BRAF fusions has been attributed to the loss of critical N-terminal domains that mediate BRAF autoinhibition. We used whole-exome and RNA sequencing in a patient with glioblastoma multiforme to identify a rearrangement between TTYH3, encoding a membrane-resident, calcium-activated chloride channel, and BRAF intron 1, resulting in a TTYH3-BRAF fusion protein that retained all features essential for BRAF autoinhibition. Accordingly, the BRAF moiety of the fusion protein alone, which represents full-length BRAF without the amino acids encoded by exon 1 (BRAF ΔE1 ), did not induce MEK/ERK phosphorylation or transformation. Likewise, neither the TTYH3 moiety of the fusion protein nor full-length TTYH3 provoked ERK pathway activity or transformation. In contrast, TTYH3-BRAF displayed increased MEK phosphorylation potential and transforming activity, which were caused by TTYH3-mediated tethering of near-full-length BRAF to the (endo)membrane system. Consistent with this mechanism, a synthetic approach, in which BRAF ΔE1 was tethered to the membrane by fusing it to the cytoplasmic tail of CD8 also induced transformation. Furthermore, we demonstrate that TTYH3-BRAF signals largely independent of a functional RAS binding domain, but requires an intact BRAF dimer interface and activation loop phosphorylation sites. Cells expressing TTYH3-BRAF exhibited increased MEK/ERK signaling, which was blocked by clinically achievable concentrations of sorafenib, trametinib, and the paradox breaker PLX8394. These data provide the first example of a fully autoinhibited BRAF protein whose oncogenic potential is dictated by a distinct fusion partner and not by a structural change in BRAF itself.

Microglia are tissue-resident macrophages of the CNS that orchestrate local immune responses and contribute to several neurological and psychiatric diseases. Little is known about human microglia and how they orchestrate their highly plastic, context-specific adaptive responses during pathology. Here we combined two high-dimensional technologies, single-cell RNA-sequencing and time-of-flight mass cytometry, to identify microglia states in the human brain during homeostasis and disease. This approach enabled us to identify and characterize a previously unappreciated spectrum of transcriptional states in human microglia. These transcriptional states are determined by their spatial distribution, and they further change with aging and brain tumor pathology. This description of multiple microglia phenotypes in the human CNS may open promising new avenues for subset-specific therapeutic interventions.

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., (© 2019 Ravi et al.)

Background: In recent years, PD-1/PD-L1 immune checkpoint inhibitors have improved cancer therapy in many tumor types, but no benefit of immune checkpoint therapy has been found in glioblastoma multiforme (GBM). Based on the results of our earlier work, which showed a reduction of PD-L1 expression in patients treated with temozolomide (TMZ), we aimed to investigate the link between TMZ therapy and the immune control point target PD-L1., Methods: RNA-sequencing data from de-novo and recurrent glioblastoma were analyzed by AutoPipe algorithm. Results were confirmed either in a cell model by two primary and one established GBM cell line and specimens of de-novo and recurrent GBM. PD-L1 and pathway activation of the JAK/STAT pathway was analyzed by quantitative real-time PCR and western blot., Results: We found a significant downregulation of the JAK/STAT pathway and immune response in recurrent tumors. The cell model showed an upregulation of PD-L1 after IFNγ treatment, while additional TMZ treatment lead to a reduction of PD-L1 expression and JAK/STAT pathway activation. These findings were confirmed in specimens of de-novo and recurrent glioblastoma., Conclusions: Our results suggest that TMZ therapy leads to a down-regulation of PD-L1 in primary GBM cells. These results support the clinical findings where PD-L1 is significantly reduced in recurrent GBMs. If the target is diminished, it may also lead to impaired efficacy of PD-1/PD-L1 inhibitors such as nivolumab.

Introduction: Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults with peak incidence in patients older than 65 years. These patients are mostly underrepresented in clinical trials and often undertreated due to concomitant diseases. Recently, different therapeutic approaches for elderly patients with GBM were discussed. To date, there is no defined standard treatment. The aim of the present study is to evaluate the functional and oncological outcome in surgical treatment of elderly patients., Materials and Methods: A total of 342 elderly patients aged ≥ 65 years were retrospectively analyzed in our neurosurgical center. Surgical therapy, adjuvant treatment, overall survival (OS) and functional outcome using Karnofsky performance scale (KPS) and Neurological assessment of neuro-oncology-score were analyzed., Results: The median age at GBM diagnosis was 73.4 (IQR 9.28) years. Median overall survival was 7.5 (CI 95% 6.0-9.1) months and median preoperative or postoperative KPS was 80 (IQR 20). Surgical resection was performed in 216 (63.2%) patients, in 125 patients (36.5%) patients a stereotactic biopsy was performed. The median OS was significantly higher in patients with gross total resection (GTR) compared to partial resection and biopsy (10.8 months; CI 95% 9.5-12.3). Patients with combined radio- and chemo-therapy (RCT) showed significant longer OS, particularly MGMT-negative GBM. Higher preoperative KPS was found to be associated with improved overall survival., Conclusion: GTR and adjuvant combined RCT provides benefits for overall survival in elderly patients. Therapy decision should be made in regard to preoperative functional status instead of biological age.

The evolving and highly heterogeneous nature of malignant brain tumors underlies their limited response to therapy and poor prognosis. In addition to genetic alterations, highly dynamic processes, such as transcriptional and metabolic reprogramming, play an important role in the development of tumor heterogeneity. The current study reports an adaptive mechanism in which the metabolic environment of malignant glioma drives transcriptional reprogramming. Multiregional analysis of a glioblastoma patient biopsy revealed a metabolic landscape marked by varying stages of hypoxia and creatine enrichment. Creatine treatment and metabolism was further shown to promote a synergistic effect through upregulation of the glycine cleavage system and chemical regulation of prolyl-hydroxylase domain. Consequently, creatine maintained a reduction of reactive oxygen species and change of the α-ketoglutarate/succinate ratio, leading to an inhibition of HIF signaling in primary tumor cell lines. These effects shifted the transcriptional pattern toward a proneural subtype and reduced the rate of cell migration and invasion in vitro Transcriptional subclasses of glioblastoma multiforme are heterogeneously distributed within the same tumor. This study uncovered a regulatory function of the tumor microenvironment by metabolism-driven transcriptional reprogramming in infiltrating glioma cells. Implications: Transcriptional subclasses of glioblastoma multiforme are heterogeneously distributed within the same tumor. This study uncovered a regulatory function of the tumor microenvironment by metabolism-driven transcriptional reprogramming in infiltrating glioma cells. Mol Cancer Res; 16(4); 655-68. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The purpose of this study was to map the landscape of metabolic-transcriptional alterations in glioblastoma multiforme. Omic-datasets were acquired by metabolic profiling (1D-NMR spectroscopy n=33 Patient) and transcriptomic profiling (n=48 Patients). Both datasets were analyzed by integrative network modeling. The computed model concluded in four different metabolic-transcriptomic signatures containing: oligodendrocytic differentiation, cell-cycle functions, immune response and hypoxia. These clusters were found being distinguished by individual metabolism and distinct transcriptional programs. The study highlighted the association between metabolism and hallmarks of oncogenic signaling such as cell-cycle alterations, immune escape mechanism and other cancer pathway alterations. In conclusion, this study showed the strong influence of metabolic alterations in the wide scope of oncogenic transcriptional alterations.

Glioblastoma multiforme are highly malignant brain tumours with frequent genetic and epigenetic alterations. The poor clinical outcome of these tumours necessitates the development of new treatment options. Immunotherapies for glioblastoma multiforme including PD1/PD-L1 inhibition are currently tested in ongoing clinical trials. The purpose of this study was to investigate the molecular background of PD-L1 expression in glioblastoma multiforme and to find associated pathway activation and genetic alterations. We show that PD-L1 is up-regulated in IDH1/2 wildtype glioblastoma multiforme compared to lower-grade gliomas. In addition, a strong association of PD-L1 with the mesenchymal expression subgroup was observed. Consistent with that, NF1 mutation and corresponding activation of the MAPK pathway was strongly connected to PD-L1 expression. Our findings may explain different response to PD-L1 inhibition of patients in ongoing trials and may help to select patients that may profit of immunotherapy in the future.

In the past, changes of the Apparent Diffusion Coefficient in glioblastoma multiforme have been shown to be related to specific genes and described as being associated with survival. The purpose of this study was to investigate diffusion imaging parameters in combination with genome-wide expression data in order to obtain a comprehensive characterisation of the transcriptomic changes indicated by diffusion imaging parameters. Diffusion-weighted imaging, molecular and clinical data were collected prospectively in 21 patients. Before surgery, MRI diffusion metrics such as axial (AD), radial (RD), mean diffusivity (MD) and fractional anisotropy (FA) were assessed from the contrast enhancing tumour regions. Intraoperatively, tissue was sampled from the same areas using neuronavigation. Transcriptional data of the tissue samples was analysed by Weighted Gene Co-Expression Network Analysis (WGCNA) thus classifying genes into modules based on their network-based affiliations. Subsequent Gene Set Enrichment Analysis (GSEA) identified biological functions or pathways of the expression modules. Network analysis showed a strong association between FA and epithelial-to-mesenchymal-transition (EMT) pathway activation. Also, patients with high FA had a worse clinical outcome. MD correlated with neural function related genes and patients with high MD values had longer overall survival. In conclusion, FA and MD are associated with distinct molecular patterns and opposed clinical outcomes.

The purpose of this study was to investigate the molecular background of cerebral blood volume (CBV) and vessel size (VS) of capillaries in glioblastoma multiforme (GBM). Both parameters are derived from extended perfusion MR imaging.A prospective case study including 21 patients (median age 66 years, 10 females) was performed. Before operation, CBV and VS of contrast enhancing tumor were assessed. Tissue was sampled from the assessed areas under neuronavigation control. After RNA extraction, transcriptional data was analyzed by Weighted Gene Co-Expression Network Analysis (WGCNA) and split into modules based on its network affiliations. Gene Set Enrichment Analysis (GSEA) identified biological functions or pathways of the genetic modules. These were applied on 484 GBM samples of the TCGA database.Ten modules were highly correlated to CBV and VS. One module was exclusively associated to VS and highly correlated to hypoxia, another one exclusively to CBV showing strong enrichments in the Epithelial Growth Factor (EGF) pathway and Epithelial-to-Mesenchymal-Transition (EMT). Moreover, patients with increased CBV and VS predominantly showed a mesenchymal gene-expression, a finding that could be corroborated by TCGA data.In conclusion, CBV and VS mirror different genetic pathways and reflect certain molecular subclasses of GBM.

The purpose of this study was to identify markers from perfusion, diffusion, and chemical shift imaging in glioblastomas (GBMs) and to correlate them with genetically determined and previously published patterns of structural magnetic resonance (MR) imaging. Twenty-six patients (mean age 60 years, 13 female) with GBM were investigated. Imaging consisted of native and contrast-enhanced 3D data, perfusion, diffusion, and spectroscopic imaging. In the presence of minor necrosis, cerebral blood volume (CBV) was higher (median ± SD, 2.23% ± 0.93) than in pronounced necrosis (1.02% ± 0.71), p corr  = 0.0003. CBV adjacent to peritumoral fluid-attenuated inversion recovery (FLAIR) hyperintensity was lower in edema (1.72% ± 0.31) than in infiltration (1.91% ± 0.35), p corr  = 0.039. Axial diffusivity adjacent to peritumoral FLAIR hyperintensity was lower in severe mass effect (1.08*10 -3 mm 2 /s ± 0.08) than in mild mass effect (1.14*10 -3 mm 2 /s ± 0.06), p corr  = 0.048. Myo-inositol was positively correlated with a marker for mitosis (Ki-67) in contrast-enhancing tumor, r = 0.5, p corr  = 0.0002. Changed CBV and axial diffusivity, even outside FLAIR hyperintensity, in adjacent normal-appearing matter can be discussed as to be related to angiogenesis pathways and to activated proliferation genes. The correlation between myo-inositol and Ki-67 might be attributed to its binding to cell surface receptors regulating tumorous proliferation of astrocytic cells.

Glioblastomas are characterized by transcriptionally distinct subtypes, but despite possible clinical relevance, their regulation remains poorly understood. The commonly used molecular classification systems for GBM all identify a subtype with high expression of mesenchymal marker transcripts, strongly associated with invasive growth. We used a comprehensive data-driven network modeling technique (augmented sparse inverse covariance selection, aSICS) to define separate genomic, epigenetic, and transcriptional regulators of glioblastoma subtypes. Our model identified Annexin A2 (ANXA2) as a novel methylation-controlled positive regulator of the mesenchymal subtype. Subsequent evaluation in two independent cohorts established ANXA2 expression as a prognostic factor that is dependent on ANXA2 promoter methylation. ANXA2 knockdown in primary glioblastoma stem cell-like cultures suppressed known mesenchymal master regulators, and abrogated cell proliferation and invasion. Our results place ANXA2 at the apex of a regulatory cascade that determines glioblastoma mesenchymal transformation and validate aSICS as a general methodology to uncover regulators of cancer subtypes., (Copyright © 2016. Published by Elsevier B.V.)

The goal of this study was to identify correlations between metabolites from proton MR spectroscopy and genetic pathway activity in glioblastoma multiforme (GBM). Twenty patients with primary GBM were analysed by short echo-time chemical shift imaging and genome-wide expression analyses. Weighed Gene Co-Expression Analysis was used for an integrative analysis of imaging and genetic data. N-acetylaspartate, normalised to the contralateral healthy side (nNAA), was significantly correlated to oligodendrocytic and neural development. For normalised creatine (nCr), a group with low nCr was linked to the mesenchymal subtype, while high nCr could be assigned to the proneural subtype. Moreover, clustering of normalised glutamine and glutamate (nGlx) revealed two groups, one with high nGlx being attributed to the neural subtype, and one with low nGlx associated with the classical subtype. Hence, the metabolites nNAA, nCr, and nGlx correlate with a specific gene expression pattern reflecting the previously described subtypes of GBM. Moreover high nNAA was associated with better clinical prognosis, whereas patients with lower nNAA revealed a shorter progression-free survival (PFS).

Dysembryoplastic neuroepithelial tumors (DNET) are considered to be rare, benign, and associated with chronic epilepsy. We present the case of a 28-year-old man with a history of epilepsy since age 12. Surgery of an occipital cortical lesion in 2009 revealed a DNET. Five years later, a recurrent tumor at the edge of the resection cavity was removed, and the tissue underwent an intensive diagnostic workup. The first tumor was unequivocally characterized as a DNET, but neuropathological diagnostics of the recurrent tumor revealed a glioblastoma. After 6 months, another recurrent tumor was detected next to the location of the original tumor, and this was also resected. An Illumina 450 K beadchip methylation array was performed to characterize all of the tumors. The methylation profile of these tumors significantly differed from other glioblastoma and epilepsy-associated tumor profiles and revealed a DNET-like methylation profile. Thus, molecular characterization of these recurrent tumors suggests malignant transformation of a previously benign DNET. We found increased copy number changes in the recurrent DNET tumors after malignant transformation. Modern high-throughput analysis adds essential molecular information in addition to standard histopathology for proper identification of rare brain tumors that present with an unusual clinical course., (© 2016 American Association of Neuropathologists, Inc. All rights reserved.)

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Simple Summary: Natural killer (NK) cells play a pivotal role in anti-cancer immunity. We conducted an integrated analysis of single-cell RNA-sequencing datasets of malignant brain tumor-associated immune cells from a mouse glioma model and human newly diagnosed and recurrent glioblastomas. We found a robust increase in NK cells during brain tumor production. The cells showed a gene expression phenotype associated with NK cell dysfunction. Notably, the cytokine Lymphotoxin-β (LTB) was highly expressed in these cells. LTB receptors are found on myeloid and structural cells. In summary, we characterize a dysfunctional NK cell phenotype in advanced glioma. Glioblastomas are the most common primary brain tumors. Despite extensive clinical and molecular insights into these tumors, the prognosis remains dismal. While targeted immunotherapies have shown remarkable success across different non-brain tumor entities, they failed to show efficacy in glioblastomas. These failures prompted the field to reassess the idiosyncrasies of the glioblastoma microenvironment. Several high-dimensional single-cell RNA sequencing studies generated remarkable findings about glioblastoma-associated immune cells. To build on the collective strength of these studies, we integrated several murine and human datasets that profiled glioblastoma-associated immune cells at different time points. We integrated these datasets and utilized state-of-the-art algorithms to investigate them in a hypothesis-free, purely exploratory approach. We identified a robust accumulation of a natural killer cell subset that was characterized by a downregulation of activation-associated genes with a concomitant upregulation of apoptosis genes. In both species, we found a robust upregulation of the Lymphotoxin-β gene, a cytokine from the TNF superfamily and a key factor for the development of adaptive immunity. Further validation analyses uncovered a correlation of lymphotoxin signaling with mesenchymal-like glioblastoma regions in situ and in TCGA and CGGA glioblastoma cohorts. In summary, we identify lymphotoxin signaling as a potential therapeutic target in glioblastoma-associated natural killer cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]