Disturbance in cerebral blood microcirculation and hypoxic-ischemic microenvironment are associated with the development of brain metastasis.

Brain metastases (BM) constitute an increasing challenge in oncology due to their impact on neurological function, limited treatment options, and poor prognosis. BM occur through extravasation of circulating tumor cells across the blood-brain barrier. However, the extravasation processes are still poorly understood. We here propose a brain colonization process which mimics infarction-like microenvironmental reactions, that is dependent on Angiopoietin (Ang-2) and vascular endothelial growth factor (VEGF). In this study, intracardiac BM models were used, and cerebral blood microcirculation was monitored by 2-photon microscopy through a cranial window. BM formation was observed using cranial magnetic resonance, bioluminescent imaging, and post-mortem autopsy. Ang-2/VEGF targeting strategies and Ang-2 gain-of-function (GOF) mice were employed to interfere with BM formation. In addition, vascular and stromal factors as well as clinical outcome were analyzed in BM patients. Blood vessel occlusions by cancer cells were detected, accompanied by significant disturbances of cerebral blood microcirculation, and focal stroke-like histological signs. Cerebral endothelial cells showed an elevated Ang-2 expression both in mouse and human BM. Ang-2 GOF resulted in an increased BM burden. Combined anti-Ang-2/anti-VEGF therapy led to a decrease in brain metastasis size and number. Ang-2 expression in tumor vessels of established human brain metastases negatively correlated with survival. Our observations revealed a relationship between disturbance of cerebral blood microcirculation and brain metastasis formation. This suggests that vessel occlusion by tumor cells facilitates brain metastatic extravasation and seeding, while combined inhibition of microenvironmental effects of Ang-2 and VEGF prevent the outgrowth of macrometastases.

DistSNE: Distributed computing and online visualization of DNA methylation-based central nervous system tumor classification.

The current state-of-the-art analysis of central nervous system (CNS) tumors through DNA methylation profiling relies on the tumor classifier developed by Capper and colleagues, which centrally harnesses DNA methylation data provided by users. Here, we present a distributed-computing-based approach for CNS tumor classification that achieves a comparable performance to centralized systems while safeguarding privacy. We utilize the t-distributed neighborhood embedding (t-SNE) model for dimensionality reduction and visualization of tumor classification results in two-dimensional graphs in a distributed approach across multiple sites (DistSNE). DistSNE provides an intuitive web interface (https://gin-tsne.med.uni-giessen.de) for user-friendly local data management and federated methylome-based tumor classification calculations for multiple collaborators in a DataSHIELD environment. The freely accessible web interface supports convenient data upload, result review, and summary report generation. Importantly, increasing sample size as achieved through distributed access to additional datasets allows DistSNE to improve cluster analysis and enhance predictive power. Collectively, DistSNE enables a simple and fast classification of CNS tumors using large-scale methylation data from distributed sources, while maintaining the privacy and allowing easy and flexible network expansion to other institutes. This approach holds great potential for advancing human brain tumor classification and fostering collaborative precision medicine in neuro-oncology.

Wnt/ß-Catenin-Signaling Modulates Megakaryopoiesis at the Megakaryocyte-Erythrocyte Progenitor Stage in the Hematopoietic System.

The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Many steps of the cell-fate decision remain to be elucidated, being important for cancer treatment. To explore the role of Wnt/ß-catenin for MK and RBC differentiation, we activated ß-catenin signaling in platelet-derived growth factor b (Pdgfb)-expressing cells of the HS using a Cre-lox approach (Ctnnb1BM-GOF). FACS analysis revealed that Pdgfb is mainly expressed by megakaryocytic progenitors (MKPs), MKs and platelets. Recombination resulted in a lethal phenotype in mutants (Ctnnb1BM-GOFwt/fl, Ctnnb1BM-GOFfl/fl) 3 weeks after tamoxifen injection, showing an increase in MKs in the BM and spleen, but no pronounced anemia despite reduced erythrocyte counts. BM transplantation (BMT) of Ctnnb1BM-GOF BM into lethally irradiated wildtype recipients (BMT-Ctnnb1BM-GOF) confirmed the megakaryocytic, but not the lethal phenotype. CFU-MK assays in vitro with BM cells of Ctnnb1BM-GOF mice supported MK skewing at the expense of erythroid colonies. Molecularly, the runt-related transcription factor 1 (RUNX1) mRNA, known to suppress erythropoiesis, was upregulated in Ctnnb1BM-GOF BM cells. In conclusion, ß-catenin activation plays a key role in cell-fate decision favoring MK development at the expense of erythroid production.

Integrative multi-omics reveals two biologically distinct groups of pilocytic astrocytoma.

Pilocytic astrocytoma (PA), the most common pediatric brain tumor, is driven by aberrant mitogen-activated protein kinase signaling most commonly caused by BRAF gene fusions or activating mutations. While 5-year overall survival rates exceed 95%, tumor recurrence or progression constitutes a major clinical challenge in incompletely resected tumors. Here, we used similarity network fusion (SNF) analysis in an integrative multi-omics approach employing RNA transcriptomic and mass spectrometry-based proteomic profiling to molecularly characterize PA tissue samples from 62 patients. Thereby, we uncovered that PAs segregated into two molecularly distinct groups, namely, Group 1 and Group 2, which were validated in three non-overlapping cohorts. Patients with Group 1 tumors were significantly younger and showed worse progression-free survival compared to patients with group 2 tumors. Ingenuity pathways analysis (IPA) and gene set enrichment analysis (GSEA) revealed that Group 1 tumors were enriched for immune response pathways, such as interferon signaling, while Group 2 tumors showed enrichment for action potential and neurotransmitter signaling pathways. Analysis of immune cell-related gene signatures showed an enrichment of infiltrating T Cells in Group 1 versus Group 2 tumors. Taken together, integrative multi-omics of PA identified biologically distinct and prognostically relevant tumor groups that may improve risk stratification of this single pathway driven tumor type.

Immuno-PET Imaging of Tumour PD-L1 Expression in Glioblastoma.

There is no established method to assess the PD-L1 expression in brain tumours. Therefore, we investigated the suitability of affibody molecule (ZPD-L1) radiolabelled with F-18 (Al18F) and Ga-68 to measure the expression of PD-L1 in xenograft mouse models of GBM. Mice bearing subcutaneous and orthotopic tumours were imaged 1 h post-radioconjugate administration. Ex vivo biodistribution studies and immunohistochemistry (IHC) staining were performed. Tumoural PD-L1 expression and CD4+/CD8+ tumour-infiltrating lymphocytes were evaluated in human GBM specimens. ZPD-L1 was radiolabelled with radiochemical yields of 32.2 ± 4.4% (F-18) and 73.3 ± 1.8% (Ga-68). The cell-associated radioactivity in vitro was consistent with PD-L1 expression levels assessed with flow cytometry. In vivo imaging demonstrated that 18F-AlF-NOTA-ZPD-L1 can distinguish between PD-L1 high-expressing tumours (U87-MGvIII) and PD-L1-negative ones (H292PD-L1Ko). The radioconjugate was quickly cleared from the blood and normal tissues, allowing for high-contrast images of brain tumours as early as 1 h post-injection. 68Ga-NOTA-ZPD-L1 showed heterogeneous and diffuse accumulation that corresponded to the extensively infiltrating GCGR-E55 tumours involving contiguous lobes of the brain. Lastly, 39% of analysed GBM patient samples showed PD-L1+ staining of tumour cells that was associated with elevated levels of CD4+ and CD8+ lymphocytes. Our results suggest that the investigated radioconjugates are very promising agents with the potential to facilitate the future design of treatment regimens for GBM patients.

Intracranial injection of natural killer cells engineered with a HER2-targeted chimeric antigen receptor in patients with recurrent glioblastoma.

BACKGROUND: Glioblastoma (GB) is incurable at present without established treatment options for recurrent disease. In this phase I first-in-human clinical trial we investigated safety and feasibility of adoptive transfer of clonal chimeric antigen receptor (CAR)-NK cells (NK-92/5.28.z) targeting HER2, which is expressed at elevated levels by a subset of glioblastomas. METHODS: Nine patients with recurrent HER2-positive GB were treated with single doses of 1 × 107, 3 × 107, or 1 × 108 irradiated CAR-NK cells injected into the margins of the surgical cavity during relapse surgery. Imaging at baseline and follow-up, peripheral blood lymphocyte phenotyping and analyses of the immune architecture by multiplex immunohistochemistry and spatial digital profiling were performed. RESULTS: There were no dose-limiting toxicities, and none of the patients developed a cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. Five patients showed stable disease after relapse surgery and CAR-NK injection that lasted 7 to 37 weeks. Four patients had progressive disease. Pseudoprogression was found at injection sites in 2 patients, suggestive of a treatment-induced immune response. For all patients, median progression-free survival was 7 weeks, and median overall survival was 31 weeks. Furthermore, the level of CD8+ T-cell infiltration in recurrent tumor tissue prior to CAR-NK cell injection positively correlated with time to progression. CONCLUSIONS: Intracranial injection of HER2-targeted CAR-NK cells is feasible and safe in patients with recurrent GB. 1 × 108 NK-92/5.28.z cells was determined as the maximum feasible dose for a subsequent expansion cohort with repetitive local injections of CAR-NK cells.

A flow cytometry-based protocol for syngenic isolation of neurovascular unit cells from mouse and human tissues.

The neurovascular unit (NVU), composed of endothelial cells, pericytes, juxtaposed astrocytes and microglia together with neurons, is essential for proper central nervous system functioning. The NVU critically regulates blood-brain barrier (BBB) function, which is impaired in several neurological diseases and is therefore a key therapeutic target. To understand the extent and cellular source of BBB dysfunction, simultaneous isolation and analysis of NVU cells is needed. Here, we describe a protocol for the EPAM-ia method, which is based on flow cytometry for simultaneous isolation and analysis of endothelial cells, pericytes, astrocytes and microglia. This method is based on differential processing of NVU cell types using enzymes, mechanical homogenization and filtration specific for each cell type followed by combining them for immunostaining and fluorescence-activated cell sorting. The gating strategy encompasses cell-type-specific and exclusion markers for contaminating cells to isolate the major NVU cell types. This protocol takes ~6 h for two sets of one or two animals. The isolation part requires experience in animal handling, fresh tissue processing and immunolabeling for flow cytometry. Sorted NVU cells can be used for downstream applications including transcriptomics, proteomics and cell culture. Multiple cell-type analyses using UpSet can then be applied to obtain robust targets from single or multiple NVU cell types in neurological diseases associated with BBB dysfunction. The EPAM-ia method is also amenable to isolation of several other cell types, including cancer cells and immune cells. This protocol is applicable to healthy and pathological tissue from mouse and human sources and to several cell types compared with similar protocols.

The neurovasculature as a target in temporal lobe epilepsy.

The blood-brain barrier (BBB) is a physiological barrier maintaining a specialized brain micromilieu that is necessary for proper neuronal function. Endothelial tight junctions and specific transcellular/efflux transport systems provide a protective barrier against toxins, pathogens, and immune cells. The barrier function is critically supported by other cell types of the neurovascular unit, including pericytes, astrocytes, microglia, and interneurons. The dysfunctionality of the BBB is a hallmark of neurological diseases, such as ischemia, brain tumors, neurodegenerative diseases, infections, and autoimmune neuroinflammatory disorders. Moreover, BBB dysfunction is critically involved in epilepsy, a brain disorder characterized by spontaneously occurring seizures because of abnormally synchronized neuronal activity. While resistance to antiseizure drugs that aim to reduce neuronal hyperexcitability remains a clinical challenge, drugs targeting the neurovasculature in epilepsy patients have not been explored. The use of novel imaging techniques permits early detection of BBB leakage in epilepsy; however, the detailed mechanistic understanding of causes and consequences of BBB compromise remains unknown. Here, we discuss the current knowledge of BBB involvement in temporal lobe epilepsy with the emphasis on the neurovasculature as a therapeutic target.

The endothelial-enriched lncRNA LINC00607 mediates angiogenic function.

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.

MR imaging profile and histopathological characteristics of tumour vasculature, cell density and proliferation rate define two distinct growth patterns of human brain metastases from lung cancer.

PURPOSE: Non-invasive prediction of the tumour of origin giving rise to brain metastases (BMs) using MRI measurements obtained in radiological routine and elucidating the biological basis by matched histopathological analysis. METHODS: Preoperative MRI and histological parameters of 95 BM patients (female, 50; mean age 59.6 ± 11.5 years) suffering from different primary tumours were retrospectively analysed. MR features were assessed by region of interest (ROI) measurements of signal intensities on unenhanced T1-, T2-, diffusion-weighted imaging and apparent diffusion coefficient (ADC) normalised to an internal reference ROI. Furthermore, we assessed BM size and oedema as well as cell density, proliferation rate, microvessel density and vessel area as histopathological parameters. RESULTS: Applying recursive partitioning conditional inference trees, only histopathological parameters could stratify the primary tumour entities. We identified two distinct BM growth patterns depending on their proliferative status: Ki67high BMs were larger (p = 0.02), showed less peritumoural oedema (p = 0.02) and showed a trend towards higher cell density (p = 0.05). Furthermore, Ki67high BMs were associated with higher DWI signals (p = 0.03) and reduced ADC values (p = 0.004). Vessel density was strongly reduced in Ki67high BM (p < 0.001). These features differentiated between lung cancer BM entities (p ≤ 0.03 for all features) with SCLCs representing predominantly the Ki67high group, while NSCLCs rather matching with Ki67low features. CONCLUSION: Interpretable and easy to obtain MRI features may not be sufficient to predict directly the primary tumour entity of BM but seem to have the potential to aid differentiating high- and low-proliferative BMs, such as SCLC and NSCLC.

Anti-osteopontin therapy leads to improved edema and infarct size in a murine model of ischemic stroke.

Ischemic stroke is a serious neurological disorder that is associated with dysregulation of the neurovascular unit (NVU) and impairment of the blood-brain barrier (BBB). Paradoxically, reperfusion therapies can aggravate NVU and BBB dysfunction, leading to deleterious consequences in addition to the obvious benefits. Using the recently established EPAM-ia method, we identified osteopontin as a target dysregulated in multiple NVU cell types and demonstrated that osteopontin targeting in the early acute phase post-transient middle cerebral artery occlusion (tMCAO) evolves protective effects. Here, we assessed the time course of osteopontin and CD44 receptor expression in NVU cells and examined cerebroprotective effects of osteopontin targeting in early and late acute phases of ischemic stroke. Expression analysis of osteopontin and CD44 receptor post-tMCAO indicated increased levels of both, from early to late acute phases, which was supported by their co-localization in NVU cells. Combined osteopontin targeting in early and late acute phases with anti-osteopontin antibody resulted in further improvement in BBB recovery and edema reduction compared to targeting only in the early acute phase comprising the reperfusion window. Combined targeting led to reduced infarct volumes, which was not observed for the single early acute phase targeting. The effects of the therapeutic antibody were confirmed both in vitro and in vivo in reducing osteopontin and CD44 expression. Osteopontin targeting at the NVU in early and late acute phases of ischemic stroke improves edema and infarct size in mice, suggesting anti-osteopontin therapy as promising adjunctive treatment to reperfusion therapy.

Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood-brain barrier in acute ischemic stroke.

Blood-brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database ( https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke ). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

New Fully Automated Preparation of High Apparent Molar Activity 68Ga-FAPI-46 on a Trasis AiO Platform.

A large number of applications for fibroblast activation protein inhibitors (FAPI)-based PET agents have been evaluated in conditions ranging from cancer to non-malignant diseases such as myocardial infarction. In particular, 68Ga-FAPI-46 was reported to have a high specificity and affinity for FAP-expressing cells, a fast and high accumulation in tumor lesions/injuries together with a fast body clearance when investigated in vivo. Due to the increasing interest in the use of the agent both preclinically and clinically, we developed an automated synthesis for the production of 68Ga-FAPI-46 on a Trasis AiO platform. The new synthetic procedure, which included the processing of the generator eluate using a strong cation exchange resin and a final purification step through an HLB followed by a QMA cartridge, yielded 68Ga-FAPI-46 with high radiochemical purity (>98%) and apparent molar activity (271.1 ± 105.6 MBq/nmol). Additionally, the in vitro and in vivo properties of the product were assessed on glioblastoma cells and mouse model. Although developed for the preparation of 68Ga-FAPI-46 for preclinical use, our method can be adapted for clinical production as a reliable alternative to the manual (i.e., cold kit) or modular systems preparations already described in the literature.

DNA methylation-based prediction of response to immune checkpoint inhibition in metastatic melanoma.

BACKGROUND: Therapies based on targeting immune checkpoints have revolutionized the treatment of metastatic melanoma in recent years. Still, biomarkers predicting long-term therapy responses are lacking. METHODS: A novel approach of reference-free deconvolution of large-scale DNA methylation data enabled us to develop a machine learning classifier based on CpG sites, specific for latent methylation components (LMC), that allowed for patient allocation to prognostic clusters. DNA methylation data were processed using reference-free analyses (MeDeCom) and reference-based computational tumor deconvolution (MethylCIBERSORT, LUMP). RESULTS: We provide evidence that DNA methylation signatures of tumor tissue from cutaneous metastases are predictive for therapy response to immune checkpoint inhibition in patients with stage IV metastatic melanoma. CONCLUSIONS: These results demonstrate that LMC-based segregation of large-scale DNA methylation data is a promising tool for classifier development and treatment response estimation in cancer patients under targeted immunotherapy.

Role of angiopoietin-2 in venous thrombus resolution and chronic thromboembolic disease.

BACKGROUND: Defective angiogenesis, incomplete thrombus revascularisation and fibrosis are considered critical pathomechanisms of chronic thromboembolic pulmonary hypertension (CTEPH) after pulmonary embolism. Angiopoietin-2 (ANGPT2) has been shown to regulate angiogenesis, but its importance for thrombus resolution and remodelling is unknown. METHODS: ANGPT2 plasma concentrations were measured in patients with CTEPH (n=68) and acute pulmonary embolism (n=84). Tissue removed during pulmonary endarterectomy (PEA) for CTEPH was analysed (immuno)histologically. A mouse model of inferior vena cava ligation was used to study the kinetics of venous thrombus resolution in wild-type mice receiving recombinant ANGPT2 via osmotic pumps, and in transgenic mice overexpressing ANGPT2 in endothelial cells. RESULTS: Circulating ANGPT2 levels were higher in CTEPH patients compared to patients with idiopathic pulmonary arterial hypertension and healthy controls, and decreased after PEA. Plasma ANGPT2 levels were elevated in patients with pulmonary embolism and diagnosis of CTEPH during follow-up. Histological analysis of PEA specimens confirmed increased ANGPT2 expression, and low levels of phosphorylated TIE2 were observed in regions with early-organised pulmonary thrombi, myofibroblasts and fibrosis. Microarray and high-resolution microscopy analysis could localise ANGPT2 overexpression to endothelial cells, and hypoxia and transforming growth factor-ß1 were identified as potential stimuli. Gain-of-function experiments in mice demonstrated that exogenous ANGPT2 administration and transgenic endothelial ANGPT2 overexpression resulted in delayed venous thrombus resolution, and thrombi were characterised by lower TIE2 phosphorylation and fewer microvessels. CONCLUSION: Our findings suggest that ANGPT2 delays venous thrombus resolution and that overexpression of ANGPT2 contributes to thrombofibrosis and may thus support the transition from pulmonary embolism to CTEPH.

The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis.

The tumor microenvironment in brain metastases is characterized by high myeloid cell content associated with immune suppressive and cancer-permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T-cell-directed therapies fail to elicit effective anti-tumor immune responses. Here, we seek to evaluate the applicability of radio-immunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti-cancer activity. Radiotherapy-induced immune modulation resulted in an increase in cytotoxic T-cell numbers and prevented the induction of lymphocyte-mediated immune suppression. Radio-immunotherapy led to significantly improved tumor control with prolonged median survival in experimental breast-to-brain metastasis. However, long-term efficacy was not observed. Recurrent brain metastases showed accumulation of blood-borne PD-L1+ myeloid cells after radio-immunotherapy indicating the establishment of an immune suppressive environment to counteract re-activated T-cell responses. This finding was further supported by transcriptional analyses indicating a crucial role for monocyte-derived macrophages in mediating immune suppression and regulating T-cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio-immunotherapy in brain metastases.

Influence of VEGF-A, VEGFR-1-3, and neuropilin 1-2 on progression-free: and overall survival in WHO grade II and III meningioma patients.

Higher grade meningiomas tend to recur. We aimed to evaluate protein levels of vascular endothelial growth factor (VEGF)-A with the VEGF-receptors 1-3 and the co-receptors Neuropilin (NRP)-1 and -2 in WHO grade II and III meningiomas to elucidate the rationale for targeted treatments. We investigated 232 specimens of 147 patients suffering from cranial meningioma, including recurrent tumors. Immunohistochemistry for VEGF-A, VEGFR-1-3, and NRP-1/-2 was performed on tissue micro arrays. We applied a semiquantitative score (staining intensity x frequency). VEGF-A, VEGFR-1-3, and NRP-1 were heterogeneously expressed. NRP-2 was mainly absent. We demonstrated a significant increase of VEGF-A levels on tumor cells in WHO grade III meningiomas (p = 0.0098). We found a positive correlation between expression levels of VEGF-A and VEGFR-1 on tumor cells and vessels (p < 0.0001). In addition, there was a positive correlation of VEGF-A and VEGFR-3 expression on tumor vessels (p = 0.0034). VEGFR-2 expression was positively associated with progression-free survival (p = 0.0340). VEGF-A on tumor cells was negatively correlated with overall survival (p = 0.0084). The VEGF-A-driven system of tumor angiogenesis might still present a suitable target for adjuvant therapy in malignant meningioma disease. However, its role in malignant tumor progression may not be as crucial as expected. The value of comprehensive testing of the ligand and all receptors prior to administration of anti-angiogenic therapy needs to be evaluated in clinical trials.

Compensatory CSF2-driven macrophage activation promotes adaptive resistance to CSF1R inhibition in breast-to-brain metastasis.

Tumor microenvironment-targeted therapies are emerging as promising treatment options for different cancer types. Tumor-associated macrophages and microglia (TAMs) represent an abundant nonmalignant cell type in brain metastases and have been proposed to modulate metastatic colonization and outgrowth. Here we demonstrate that targeting TAMs at distinct stages of the metastatic cascade using an inhibitor of colony-stimulating factor 1 receptor (CSF1R), BLZ945, in murine breast-to-brain metastasis models leads to antitumor responses in prevention and intervention preclinical trials. However, in established brain metastases, compensatory CSF2Rb-STAT5-mediated pro-inflammatory TAM activation blunted the ultimate efficacy of CSF1R inhibition by inducing neuroinflammation gene signatures in association with wound repair responses that fostered tumor recurrence. Consequently, blockade of CSF1R combined with inhibition of STAT5 signaling via AC4-130 led to sustained tumor control, a normalization of microglial activation states and amelioration of neuronal damage.

Linking epigenetic signature and metabolic phenotype in IDH mutant and IDH wildtype diffuse glioma.

AIMS: Changes in metabolism are known to contribute to tumour phenotypes. If and how metabolic alterations in brain tumours contribute to patient outcome is still poorly understood. Epigenetics impact metabolism and mitochondrial function. The aim of this study is a characterisation of metabolic features in molecular subgroups of isocitrate dehydrogenase mutant (IDHmut) and isocitrate dehydrogenase wildtype (IDHwt) gliomas. METHODS: We employed DNA methylation pattern analyses with a special focus on metabolic genes, large-scale metabolism panel immunohistochemistry (IHC), qPCR-based determination of mitochondrial DNA copy number and immune cell content using IHC and deconvolution of DNA methylation data. We analysed molecularly characterised gliomas (n = 57) for in depth DNA methylation, a cohort of primary and recurrent gliomas (n = 22) for mitochondrial copy number and validated these results in a large glioma cohort (n = 293). Finally, we investigated the potential of metabolic markers in Bevacizumab (Bev)-treated gliomas (n = 29). RESULTS: DNA methylation patterns of metabolic genes successfully distinguished the molecular subtypes of IDHmut and IDHwt gliomas. Promoter methylation of lactate dehydrogenase A negatively correlated with protein expression and was associated with IDHmut gliomas. Mitochondrial DNA copy number was increased in IDHmut tumours and did not change in recurrent tumours. Hierarchical clustering based on metabolism panel IHC revealed distinct subclasses of IDHmut and IDHwt gliomas with an impact on patient outcome. Further quantification of these markers allowed for the prediction of survival under anti-angiogenic therapy. CONCLUSION: A mitochondrial signature was associated with increased survival in all analyses, which could indicate tumour subgroups with specific metabolic vulnerabilities.

HIF-1α is involved in blood-brain barrier dysfunction and paracellular migration of bacteria in pneumococcal meningitis.

Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae, leading to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from blood to the brain across the blood-cerebrospinal fluid barrier or the blood-brain barrier (BBB). The underlying mechanisms are still poorly understood. Current treatment strategies include adjuvant dexamethasone for inflammation and cerebral edema, followed by antibiotics. The success of dexamethasone is however inconclusive, necessitating new therapies for controlling edema, the primary reason for neurological complications. Since we have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is involved in transmigration of pathogens across the BBB. In human, murine meningitis brain samples, HIF-1α activation was observed by immunohistochemistry. S. pneumoniae infection in brain endothelial cells (EC) resulted in in vitro upregulation of HIF-1α/VEGF (Western blotting/qRT-PCR) associated with increased paracellular permeability (fluorometry, impedance measurements). This was supported by bacterial localization at cell-cell junctions in vitro and in vivo in brain ECs from mouse and humans (confocal, super-resolution, electron microscopy, live-cell imaging). Hematogenously infected mice showed increased permeability, S. pneumoniae deposition in the brain, along with upregulation of genes in the HIF-1α/VEGF pathway (RNA sequencing of brain microvessels). Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells or using primary brain ECs from HIF-1α knock-out mice revealed reduced endothelial permeability and transmigration of S. pneumoniae. Therapeutic rescue using the HIF-1α inhibitor echinomycin resulted in increased survival and improvement of BBB function in S. pneumoniae-infected mice. We thus demonstrate paracellular migration of bacteria across BBB and a critical role for HIF-1α/VEGF therein and hence propose targeting this pathway to prevent BBB dysfunction and ensuing brain damage in infections.