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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. Klemm et al. show that resistance to CSF1R inhibition in breast cancer brain metastasis is driven by compensatory activation of the CSF2Rb–STAT5 axis in macrophages, which can be alleviated by combined targeting of CSF1R and STAT5.

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.

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.

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.

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

While the central nervous system is considered an immunoprivileged site and brain tumors display immunosuppressive features, both innate and adaptive immune responses affect glioblastoma (GBM) growth and treatment resistance. However, the impact of the major immune cell population in gliomas, represented by glioma-associated microglia/macrophages (GAMs), on patients' clinical course is still unclear. Thus, we aimed at assessing the immunohistochemical expression of selected microglia and macrophage markers in 344 gliomas (including gliomas from WHO grade I-IV). Furthermore, we analyzed a cohort of 241 IDH1R132H-non-mutant GBM patients for association of GAM subtypes and patient overall survival. Phenotypical properties of GAMs, isolated from high-grade astrocytomas by CD11b-based magnetic cell sorting, were analyzed by immunocytochemistry, mRNA microarray, qRT-PCR and bioinformatic analyses. A higher amount of CD68-, CD163- and CD206-positive GAMs in the vital tumor core was associated with beneficial patient survival. The mRNA expression profile of GAMs displayed an upregulation of factors that are considered as pro-inflammatory M1 (eg, CCL2, CCL3L3, CCL4, PTGS2) and anti-inflammatory M2 polarization markers (eg, MRC1, LGMN, CD163, IL10, MSR1), the latter rather being associated with phagocytic functions in the GBM microenvironment. In summary, we present evidence that human GBMs contain mixed M1/M2-like polarized GAMs and that the levels of different GAM subpopulations in the tumor core are positively associated with overall survival of patients with IDH1R132H-non-mutant GBMs.

Aims The paired box gene 8 (PAX8) plays crucial roles in organ patterning and cellular differentiation during development and tumorigenesis. Although its function is partly understood in vertebrate development, there is poor data concerning human central nervous system (CNS) development and brain tumours. Methods We investigated developing human (n = 19) and mouse (n = 3) brains as well as medulloblastomas (MBs) (n = 113) for PAX8 expression by immunohistochemistry. Human MB cell lines were assessed for PAX8 expression using polymerase chain reaction and immunoblotting and analysed for growth and migration following PAX8 knock-down by small interfering ribonucleic acid (siRNA). Results PAX8 protein expression was associated with germinal layers in human and murine forebrain and hindbrain development. PAX8 expression significantly decreased over time in the external granule cell layer but increased in the internal granule cell layer. In MB subtypes, we observed an association of PAX8 expression with sonic hedgehog (SHH) and wingless int subtypes but not with group 3 and 4 MBs. Beyond that, we detected high PAX8 levels in desmoplastic MB subtypes. Univariate analyses revealed high PAX8 levels as a prognostic factor associated with a significantly better patient prognosis in human MB (overall survival: Log-Rank P = 0.0404, Wilcoxon P = 0.0280; progression-free survival: Log-Rank P = 0.0225; Wilcoxon P = 0.0136). In vitro assays revealed increased proliferation and migration of MB cell lines after PAX8 siRNA knock-down. Conclusion In summary, high PAX8 expression is linked to better prognosis in MBs potentially by suppressing both proliferative and migratory properties of MB cells. The distinct spatio-temporal expression pattern of PAX8 during brain development might contribute to the understanding of distinct MB subtype histogenesis.

Background The Wnt/beta-catenin and the Hedgehog (Hh) pathway interact in various cell types while eliciting opposing or synergistic cellular effects. Both pathways are known as exclusive drivers of two distinct molecular subtypes of medulloblastoma (MB). In sonic hedgehog (Shh)-driven MB, activation of Wnt signaling has been shown to suppress tumor growth by either beta-catenin-dependent or -independent inhibition of Shh signaling. However, mechanistic insight in how beta-catenin inhibits the Hh pathway is not known. Findings Here we show that beta-catenin stabilization by the glycogen synthase kinase 3 inhibitor lithium chloride (LiCl) reduced growth of primary hedgehog-driven MB tumor spheres from patched heterozygous mice (Ptch+/-) in vitro. LiCl treatment of MB spheres down-regulated the Hh target Gli1, whereas the repressive Gli3 protein (Gli3R) was increased. Mechanistically, we show by co-immunoprecipitation and proximity ligation assay that stabilized beta-catenin physically interacts with Gli1, leading to Gli1 sequestration and inhibition of its transcriptional activity. Reduction of Hh signaling upon LiCl stimulation resulted in reduced proliferation, sphere self renewal, a G2/M arrest and induction of a senescent-like state, indicated by p21 upregulation and by increased staining of senescence-associated beta-galactosidase (SA-betaGal). Moreover, LiCl treatment of subcutaneously transplanted MB cells significantly reduced tumor initiation defined as “tumor take”. Although tumor progression was similar, LiCl-treated tumors showed decreased mitotic figures and phospho-histone H3 staining. Conclusion We propose that beta-catenin stabilization increases its physical interaction with Gli1, leading to Gli1 degradation and inhibition of Hh signaling, thereby promoting tumor cell senescence and suppression of “tumor take” in mice. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0294-4) contains supplementary material, which is available to authorized users.

Simple cells in primary visual cortex were famously found to respond to low-level image components such as edges. Sparse coding and independent component analysis (ICA) emerged as the standard computational models for simple cell coding because they linked their receptive fields to the statistics of visual stimuli. However, a salient feature of image statistics, occlusions of image components, is not considered by these models. Here we ask if occlusions have an effect on the predicted shapes of simple cell receptive fields. We use a comparative approach to answer this question and investigate two models for simple cells: a standard linear model and an occlusive model. For both models we simultaneously estimate optimal receptive fields, sparsity and stimulus noise. The two models are identical except for their component superposition assumption. We find the image encoding and receptive fields predicted by the models to differ significantly. While both models predict many Gabor-like fields, the occlusive model predicts a much sparser encoding and high percentages of ‘globular’ receptive fields. This relatively new center-surround type of simple cell response is observed since reverse correlation is used in experimental studies. While high percentages of ‘globular’ fields can be obtained using specific choices of sparsity and overcompleteness in linear sparse coding, no or only low proportions are reported in the vast majority of studies on linear models (including all ICA models). Likewise, for the here investigated linear model and optimal sparsity, only low proportions of ‘globular’ fields are observed. In comparison, the occlusive model robustly infers high proportions and can match the experimentally observed high proportions of ‘globular’ fields well. Our computational study, therefore, suggests that ‘globular’ fields may be evidence for an optimal encoding of visual occlusions in primary visual cortex.

The Sonic Hedgehog (SHH) pathway plays a central role in the developing mammalian CNS. In our study, we aimed to investigate the spatiotemporalSHHpathway expression pattern in human fetal brains. We analyzed 22 normal fetal brains for Shh, Patched, Smoothened, and Gli1-3 expression by immunohistochemistry. In the telencephalon, strongest expression of Shh, Smoothened, and Gli2 was found in the cortical plate (CP) and ventricular zone. Patched was strongly upregulated in the ventricular zone and Gli1 in the CP. In the cerebellum,SHHpathway members were strongly expressed in the external granular layer (EGL).SHHpathway members significantly decreased over time in the ventricular and subventricular zone and in the cerebellar EGL, while increasing levels were found in more superficial telencephalic layers. Our findings show thatSHHpathway members are strongly expressed in areas important for proliferation and differentiation and indicate a temporal expression gradient in telencephalic and cerebellar layers probably due to decreased proliferation of progenitor cells and increased differentiation. Our data about the spatiotemporal expression ofSHHpathway members in the developing human brain serves as a base for the understanding of both normal and pathological CNS development.

The paired box gene 8 (PAX8) plays crucial roles in organ patterning and cellular differentiation during development and tumorigenesis. Although its function is partly understood in vertebrate development, there is poor data concerning human central nervous system (CNS) development and brain tumours.We investigated developing human (n = 19) and mouse (n = 3) brains as well as medulloblastomas (MBs) (n = 113) for PAX8 expression by immunohistochemistry. Human MB cell lines were assessed for PAX8 expression using polymerase chain reaction and immunoblotting and analysed for growth and migration following PAX8 knock-down by small interfering ribonucleic acid (siRNA).PAX8 protein expression was associated with germinal layers in human and murine forebrain and hindbrain development. PAX8 expression significantly decreased over time in the external granule cell layer but increased in the internal granule cell layer. In MB subtypes, we observed an association of PAX8 expression with sonic hedgehog (SHH) and wingless int subtypes but not with group 3 and 4 MBs. Beyond that, we detected high PAX8 levels in desmoplastic MB subtypes. Univariate analyses revealed high PAX8 levels as a prognostic factor associated with a significantly better patient prognosis in human MB (overall survival: Log-Rank P = 0.0404, Wilcoxon P = 0.0280; progression-free survival: Log-Rank P = 0.0225; Wilcoxon P = 0.0136). In vitro assays revealed increased proliferation and migration of MB cell lines after PAX8 siRNA knock-down.In summary, high PAX8 expression is linked to better prognosis in MBs potentially by suppressing both proliferative and migratory properties of MB cells. The distinct spatio-temporal expression pattern of PAX8 during brain development might contribute to the understanding of distinct MB subtype histogenesis.

WHO grade III meningiomas are malignant neoplasms for which new and more targeted treatment strategies are urgently needed. Although clinical trials investigating anti-angiogenic vascular endothelial growth factor (VEGF) targeted therapies are currently recruiting, knowledge about the expression of VEGF and VEGF receptors remains to be determined.We investigated the expression of VEGF and its receptors VEGFR1 and VEGFR2 in 32 WHO grade III meningioma samples by immunohistochemistry. Furthermore, we performed in-situ hybridisation for VEGF.We found low VEGF expression in tumor and endothelial cells. Highest VEGF expression levels were seen in peri-necrotic tumor cells potentially suffering from hypoxia. VEGFR1 and 2 were virtually absent on tumor cells, although endothelial cells displayed significantly higher levels reaching stronger expression for VEGFR2 than VEGFR1.Our findings showing constant expression levels of VEGFR2 in endothelial cells serve as a first indication that the use of small tyrosine kinase inhibitors such as Sunitinib directly targeting the VEGF-receptors might be worth testing, also in the clinical context in cases of therapy-refractory meningiomas. Further investigations are needed to study the response to drugs targeting the VEGF pathway in relation to the expression profile of VEGF and its receptors in high grade meningiomas.

The scaffold protein A-kinase anchor protein 12 (AKAP12) exerts tumor suppressor activity and is downregulated in several tumor entities. We characterized AKAP12 expression and regulation in astrocytomas, including pilocytic and diffusely infiltrating astrocytomas. We examined 194 human gliomas and 23 normal brain white matter samples by immunohistochemistry or immunoblotting for AKAP12 expression. We further performed quantitative methylation analysis of the AKAP12 promoter by MassARRAY® of normal brain, World Health Organization (WHO) grade I to IV astrocytomas, and glioma cell lines. Our results show that AKAP12 is expressed in a perivascular distribution in normal CNS, strongly upregulated in tumor cells in pilocytic astrocytomas, and weakly expressed in diffuse astrocytomas of WHO grade II to IV. Methylation analyses revealed specific hypermethylation of AKAP12α promoter in WHO grade II to IV astrocytomas. Restoration experiments using 5-aza-2'-deoxycytidine in primary glioblastoma cells decreased AKAP12α promoter methylation and markedly increased AKAP12α mRNA levels. In summary, we demonstrate that AKAP12 is differentially expressed in human astrocytomas showing high expression in pilocytic but low expression in diffuse astrocytomas of all WHO-grades. Our results further indicate that epigenetic mechanisms are involved in silencing AKAP12 in diffuse astrocytomas; however, a tumor suppressive role of AKAP12 in distinct astrocytoma subtypes remains to be determined.

Wnt modulates glioma vascularization by regulating PDGF-B expression., Endothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood–brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin–Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.