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Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. GL261 murine glioma cells are widely used as a syngeneic animal model of glioma, however, it has become common practice to transfect these cells with luciferase for fluorescent tumor tracking. The aim of this study was to compare the survival of mice injected with fluorescent or non-fluorescent GL261 cells and characterize the differences in their tumor microenvironment. Mice were intracranially implanted with GL261, GL261 Red-FLuc or GL261-Luc2 cells at varying doses. Cytokine profiles were evaluated by proteome microarray and Kaplan–Meier survival analysis was used to determine survival differences. Median survival for mice implanted with 5 × 104 GL261 cells was 18 to 21 days. The GL261 Red-FLuc implanted mice cells did not reach median survival at any tumor dose. Mice injected with 3 × 105 GL261-Luc2 cells reached median survival at 23 days. However, median survival was significantly prolonged to 37 days in mice implanted with 5 × 104 GL261-Luc2 cells. Additionally, proteomic analyses revealed significantly elevated inflammatory cytokines in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Our data suggest that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators.

Mutations in EPAS1, encoding hypoxia-inducible factor-2α (HIF-2α), were previously identified in a syndrome of multiple paragangliomas, somatostatinoma, and polycythemia. HIF-2α, when dimerized with HIF-1β, acts as an angiogenic transcription factor. Patients referred to the NIH for new, recurrent, and/or metastatic paraganglioma or pheochromocytoma were confirmed for EPAS1 gain-of-function mutation; imaging was evaluated for vascular malformations. We evaluated the Epas1A529V transgenic syndrome mouse model, corresponding to the mutation initially detected in the patients (EPAS1A530V), for vascular malformations via intravital 2-photon microscopy of meningeal vessels, terminal vascular perfusion with Microfil silicate polymer and subsequent intact ex vivo 14T MRI and micro-CT, and histologic sectioning and staining of the brain and identified pathologies. Further, we evaluated retinas from corresponding developmental time points (P7, P14, and P21) and the adult dura via immunofluorescent labeling of vessels and confocal imaging. We identified a spectrum of vascular malformations in all 9 syndromic patients and in all our tested mutant mice. Patient vessels had higher variant allele frequency than adjacent normal tissue. Veins of the murine retina and intracranial dura failed to regress normally at the expected developmental time points. These findings add vascular malformation as a new clinical feature of EPAS1 gain-of-function syndrome., We discovered vascular malformations due to failure of developmental vascular regression in patients with EPAS1 gain-of-function mutation syndrome and the corresponding transgenic mouse model.

Objective Patients with intracerebral hemorrhage (ICH) may elaborate varying degrees of perihematomal edema (PHE), requiring closer monitoring and a higher intensity of treatment. Here, we explore whether the soluble form of CD163, a scavenger receptor responsible for hemoglobin sequestration, can serve as a prognostic biomarker of PHE development and poor outcome after ICH. Methods Our study cohort was comprised of 51 primary age- and sex-matched ICH patients with moderate-sized, hypertensive deep hemorrhages. Patients were part of a prospective ICH registry cataloguing admission data along with functional outcomes. We measured sCD163 levels in serial serum and cerebrospinal fluid (CSF) samples obtained at prespecified timepoints. Descriptive statistics, including a generalized estimating equation for longitudinal data, were used to analyze sCD163 in relation to ICH outcomes. Results Acute serum sCD163 (

Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. Intracranially injected GL261 cells are widely used as an immunocompetent animal model of glioma, but it is common practice to transfect these with luciferase to facilitate tumor monitoring during treatment. Our group has previously shown that the luciferase-expressing GL261 Red-FLuc cells create an inflammatory response when implanted intracranially. Now, we additionally explore the inflammatory response of GL261-Luc2 cells and demonstrate a similar host immune response occurs with this model as well. In our in vivo evaluation, C57BL/6 mice underwent stereotaxic, intracranial implantation with GL261, GL261 Red-FLuc or GL261-Luc2 cells at doses of 5x104cells/5mL or 3x105cells/5uL.MRIs were performed to monitor relative tumor growth. To assess intrinsic differences between cell lines, in vitro cytokine profiles were evaluated by proteome microarray. Kaplan-Meier survival analyses demonstrated median survival for mice implanted with GL261 cells at 5x104cells was 18 to 21 days. The GL261-Red FLuc implanted mice cells did not reach median survival at either tumor dose with greater than 60% of mice termed long-term survivors. Finally, mice injected with GL261-Luc2 cells at 3x105cells reached median survival at 23 days, but median survival was significantly prolonged for mice implanted with GL261-Luc2 at a dose of 5x104cells (37 days, with 40% becoming long-term survivors) compared to GL261 implanted mice. MRIs reveal differences in tumor growth that correspond with the differences in median survival between groups. In addition, proteomic analyses revealed significantly elevated inflammatory cytokines such as IFN-gamma, IL-7 and TNF-alpha in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Further immune characterization is ongoing. Our data suggests that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators which stimulate the tumoricidal function of immune cells in the tumor microenvironment.

Immunocompetent murine models of glioma, such as GL261, are essential to assess immune therapy efficacy. The GL261 cell line expressing firefly luciferase (GL261RFluc), has been used to enable non-invasive tumor monitoring in preclinical studies. However, it is unclear if the GL261 and GL261RFluc cells are equivalent, particularly when applied to tumor immunology. C57BL/6 mice (n=20 per group, repeated once) underwent stereotaxic, intracranial implantation with GL261 or GL261RFLuc cells at 5x10^4cells/5uL and were assessed for survival. GL261 and GL261RFluc cell lines were assessed by CFSE proliferation assay. TGF-Beta2 ELISA and proteome profiler immunoassay were performed on equivalent cell culture lysates for cytokine analysis. Mice were implanted with GL261 or GL261RFluc cell lines, sacrificed at day 10, and brains were either disassociated, FACS stained, and sorted for immune cell surface antigens (n=4 each group) or formalin-fixed and paraffin embedded for immunohistochemistry (IHC, n=6 each group). Median survival for GL261 implanted mice was 20.9 ± 1.3 days with all animals progressing to a moribund state, while median survival was not reached for animals implanted with GL261RFluc cells (P=0.001). Quantitative IHC analyses of brains implanted with GL261RFluc cells showed significant increases in CD4 and CD8 cells but decrease in FoxP3 positive cells. Proliferation assays were equivalent. TGF-Beta2 ELISA showed significantly elevated levels in the GL261RFLuc cells. Proteomic profiler results demonstrated differential cytokine expression greater than 2-fold for over 25% of cytokines evaluated. The detected increases in chemoattractants CCL2 and CCL5 were particularly pronounced in GL261RFLuc cells. FACS analysis showed a trend of increased PD-L1 positive cells in brains implanted with GL261 cells, but this did not reach significance. GL261RFluc cells create an inflammatory immune microenvironment when implanted intracranially in C57BL/6 mice compared to GL261 cells. These findings suggest that investigators should avoid GL261RFluc cells when evaluating immune therapeutics in a C57BL/6 background.

Von Hippel-Lindau (VHL)-associated hemangioblastomas (VHL-HB) arise in the central nervous system (CNS), and are a leading cause of morbidity and mortality in VHL disease. Currently, surgical resection is the most effective way to manage symptomatic VHL-HBs. Surgically unresectable VHL-HBs or those in frail patients are challenging problems. Therapies targeting oncologic and vascular endothelial growth factor (VEGF) pathways have failed to demonstrate tumor control. Our experience and previous reports on VHL-HB avidity to somatostatin analogues suggested somatostatin receptor (SSTR) expression in VHL-HBs, offering an alternative therapeutic strategy. We explored this possibility by demonstrating consistent histologic expression of SSTR1, 2a, 4, and 5 in VHL-HBs. We found that somatostatin analogue octreotide induces apoptosis in VHL-HB stromal cells in a dose-dependent fashion by BAX – caspase-3 pathway unrelated to canonical VHL pathway. When administered to a patient with unresectable symptomatic suprasellar hemangioblastoma, octreotide resulted in tumor volume reduction, symptom stabilization, and tumor cytopenia on repeat 68Ga-DOTA-TATE positron emission tomography (PET) within 6 months, suggesting tumor infarction. We conclude that VHL-HBs harbor multiple SSTR subtypes that offer actionable chemo-therapeutic strategy for management of symptomatic, unresectable tumors by somatostatin analogue therapy.

von Hippel-Lindau (VHL) patients develop multiple central nervous system hemangioblastomas (HB). Some HBs become symptomatic with exponential growth or cyst formation following long periods of quiescence. Understanding the factors underlying growth in hemangioblastoma may lead to better strategies to arrest or prevent tumor growth. In 5 VHL patients, we resected quiescent hemangioblastomas (Q-HB) that were en-route during surgical access to symptomatic hemangioblastomas (S-HB), for matched tumor analysis. Quantitative reverse transcriptase analysis demonstrated a 2-fold increase in EPO expression in all S-HB, while 4/5 showed either Hypoxia Inducible Factor-1α or 2α upregulation. Additionally, all S-HB had increased phosphorylated erythropoietin (EPO) receptor and phosphorylated STAT-5 relative to matched Q-HB, with increased phosphorylated JAK-2 largely confined to the stromal cells in clusters within the tumors. These findings suggest that Q-HB to S-HB conversion may be associated with an erythropoietin-signaling loop. Furthermore, we found that EPO is detectable in cyst fluid from S-HB (n = 14), while absent in CSF (n = 1). Additionally, S-HB presentation or S-HB resection does not result in discernible change in serum EPO or hemoglobin (n = 60). These observations suggest that the altered erythropoietin signaling is focal and suggests that studying modulation of erythropoietin receptor pathway may lead to strategies in preventing HB growth.

Transcription factors that induce epithelial-mesenchymal transition (EMT) promote invasion, chemoresistance and a stem-cell phenotype in epithelial tumors, but their roles in central nervous system tumors are not well-understood. We hypothesized these transcription factors have a functional impact in grades II-III gliomas. Using the National Cancer Institute (NCI) Repository for Molecular Brain Neoplasia Data (REMBRANDT) and the Cancer Genome Atlas (TCGA) Lower-Grade Glioma (LGG) data, we determined the impact of EMT-promoting transcription factors (EMT-TFs) on overall survival in grades II-III gliomas, compared their expression across common genetic subtypes and subsequently validated these findings in a set of 31 tumors using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. Increased expression of the gene coding for the transcriptional repressor Zinc Finger E box-binding Homeobox 1 (ZEB1) was associated with a significant increase in overall survival (OS) on Kaplan-Meier analysis. Genetic subtype analysis revealed that ZEB1 expression was relatively increased in IDH1/2-mutant gliomas, and IDH1/2-mutant gliomas expressed significantly lower levels of many ZEB1 transcriptional targets. Similarly, IDH1/2-mutant tumors expressed significantly higher levels of targets of microRNA 200C (MIR200C), a key regulator of ZEB1. In a validation study, ZEB1 mRNA was significantly increased in IDH1-mutant grades II-III gliomas, and ZEB1 protein expression was more pronounced in these tumors. Our findings demonstrate a novel relationship between IDH1/2 mutations and expression of ZEB1 and its transcriptional targets. Therapy targeting ZEB1-associated pathways may represent a novel therapeutic avenue for this class of tumors.

After ischemic stroke, various damage-associated molecules are released from the ischemic core and diffuse to the ischemic penumbra, activating microglia and promoting proinflammatory responses that may cause damage to the local tissue. Here we demonstrate using in vivo and in vitro models that, during sublethal ischemia, local neurons rapidly produce interleukin-4 (IL-4), a cytokine with potent anti-inflammatory properties. One such anti-inflammatory property includes its ability to polarize macrophages away from a proinflammatory M1 phenotype to a “healing” M2 phenotype. Using an IL-4 reporter mouse, we demonstrated that IL-4 expression was induced preferentially in neurons in the ischemic penumbra but not in the ischemic core or in brain regions that were spared from ischemia. When added to cultured microglia, IL-4 was able to induce expression of genes typifying the M2 phenotype and peroxisome proliferator activated receptor γ (PPARγ) activation. IL-4 also enhanced expression of the IL-4 receptor on microglia, facilitating a “feedforward” increase in (1) their expression of trophic factors and (2) PPARγ-dependent phagocytosis of apoptotic neurons. Parenteral administration of IL-4 resulted in augmented brain expression of M2- and PPARγ-related genes. Furthermore, IL-4 and PPARγ agonist administration improved functional recovery in a clinically relevant mouse stroke model, even if administered 24 h after the onset of ischemia. We propose that IL-4 is secreted by ischemic neurons as an endogenous defense mechanism, playing a vital role in the regulation of brain cleanup and repair after stroke. Modulation of IL-4 and its associated pathways could represent a potential target for ischemic stroke treatment. SIGNIFICANCE STATEMENT Depending on the activation signal, microglia/macrophages (MΦ) can behave as “healing” (M2) or “harmful” (M1). In response to ischemia, damaged/necrotic brain cells discharge factors that polarize MΦ to a M1-like phenotype. This polarization emerges early after stroke and persists for days to weeks, driving secondary brain injury via proinflammatory mediators and oxidative damage. Our study demonstrates that, to offset this M1-like polarization process, sublethally ischemic neurons may instead secrete a potent M2 polarizing cytokine, interleukin-4 (IL-4). In the presence of IL-4 (including when IL-4 is administered exogenously), MΦ become more effective in the cleanup of ischemic debris and produce trophic factors that may promote brain repair. We propose that IL-4 could represent a potential target for ischemic stroke treatment/recovery.

Von Hippel-Lindau (VHL) disease is caused by germline mutation of the VHL tumour suppressor gene. Patients frequently develop multiple nervous system tumours, denominated haemangioblastomas. Analysis of affected autopsy tissues suggests that tumourigenesis propagates from developmentally arrested, embryonic cells and progresses with consistent architectural, cytological, and molecular sequences similar to haemangioblastic formation and differentiation in the embryo. In this study, we analysed 156 nervous system tumours, 139 of which had been surgically resected from 83 VHL patients. We demonstrate that large tumours consistently contain epithelioid components characteristic of haemangioblastic differentiation in comparison to small tumours that solely display a poorly differentiated, mesenchymal structure. We further show exclusive activation of HIF2alpha in both small mesenchymal tumours and the mesenchymal component of large tumours, whereas activation of HIF1alpha is associated with epithelioid structure. We also show that the MIB1 proliferative index is variably increased in the epithelioid component of large tumours, with extramedullary haematopoiesis foci within the epithelioid component at 100%. These data provide compelling evidence that nervous system tumourigenesis in VHL disease represents a protracted process of haemangioblastic proliferation and differentiation that parallels haemangioblastic formation and differentiation in the embryo.

Intravenous enzyme replacement therapy (ERT) with purified glucocerebrosidase (GLA) leads to significant improvement of the clinical manifestations in patients with Type 1 Gaucher disease. However, the high doses required, slow response and inability to recover most of the infused enzyme in the target tissues may be attributed to losses occurring during transit en route to the lysosome. Pre-incubation of GLA with isofagomine (IFG), a slow-binding inhibitor, significantly increased stability of the enzyme to heat, neutral pH and denaturing agents in vitro. Preincubation of GLA with isofagomine prior to uptake by cultured cells results in increased intracellular enzyme activity accompanied by an increase in enzyme protein suggesting that reduced denaturation of GLA in the presence of isofagomine leads to a decrease in the degradation of the enzyme after internalization. Pre-incubation of GLA with slow-binding inhibitors before infusion may improve the effectiveness of ERT for Gaucher disease.

Glioblastoma multiformes (GBMs) express increased aquaporin (AQP) 1 compared to normal brain. AQPs may contribute to edema, cell motility, and shuttling of H(2)O and H(+) from intracellular to extracellular space. We sought to gain insight into AQP1 function in GBM. In cultured 9L gliosarcoma cells, AQP1 expression was induced by dexamethasone, platelet-derived growth factor, NaCl, hypoxia, D-glucose (but not L-glucose), and fructose. Induction of AQP1 expression correlated with the level of glycolysis, maximized by increasing medium D-glucose or fructose and decreasing O(2), and was quantified by measuring lactate dehydrogenase (LDH) activity and medium lactate concentration. Upregulation of the protease cathepsin B was also observed in 9L cells cultured under glycolytic conditions. Immunohistochemical staining of human GBM specimens revealed increased coincident expression of AQP1, LDH, and cathepsin B in glioma cells associated with blood vessels at the tumor periphery. GBMs are known to exhibit aerobic glycolysis. Increased glucose metabolism at the tumor periphery may provide a scenario by which upregulation of AQP1, LDH, and cathepsin B contributes to acidification of the extracellular milieu and to invasive potential of glioma cells in perivascular space. The specific upregulation and metabolic consequences of increased AQP1 in gliomas may provide a therapeutic target, both as a cell surface marker and as a functional intervention.

Background IDH mutations have been demonstrated to confer prolonged survival in patients suffering from gliomas, but the mechanisms underlying the improved prognosis are unclear. While some studies have attributed these observations to an enhanced sensitivity to genotoxic therapies, others have postulated that IDH-mutated gliomas exhibit less aggressive intrinsic biological behavior, including the propensity to invade distant sites. Although most gliomas recur local to the site of initial presentation, some tumors demonstrate distant recurrence, the vast majority of which involve the contralateral hemisphere. Trans-tentorial spread has been described once before, in which a supratentorial glioblastoma was reported to recur infratentorially in the cerebellum. Case presentation We describe a patient who underwent surgical resection, followed by adjuvant radiation and temozolomide of a World Health Organization (WHO) III anaplastic astrocytoma in the right temporal lobe, exhibiting an IDH1 (R132H) mutation. Twenty-two months after surgery, he developed a second lesion, located in the right cerebellum, suspicious for recurrent tumor versus radiation necrosis. A second surgery was performed, and pathology demonstrated recurrent tumor, consistent with IDH1-mutated anaplastic astrocytoma. Conclusions This is the first example of trans-tentorial spread in an IDH-mutated glioma, suggesting that despite improved survival, IDH mutations may not preclude gliomas from exhibiting the ability to invade distant sites of the brain.