Cell-specific crosstalk proteomics reveals cathepsin B signaling as a driver of glioblastoma malignancy near the subventricular zone.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially due to subventricular zone (SVZ) contact. Despite this, crosstalk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. Additionally, GBM brain tumor initiating cells (BTICs) increase expression of CTSB upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Finally, we show LV-proximal CTSB upregulation in patients, showing the relevance of this crosstalk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM. Highlights: Periventricular GBM is more malignant and disrupts neurogenesis in a rodent model.Cell-specific proteomics elucidates tumor-promoting crosstalk between GBM and NPCs.NPCs induce upregulated CTSB expression in GBM, promoting tumor progression.GBM stalls neurogenesis and promotes NPC senescence via CTSB.

Glioblastoma disrupts the ependymal wall and extracellular matrix structures of the subventricular zone.

BACKGROUND: Glioblastoma (GBM) is the most aggressive and common type of primary brain tumor in adults. Tumor location plays a role in patient prognosis, with tumors proximal to the lateral ventricles (LVs) presenting with worse overall survival, increased expression of stem cell genes, and increased incidence of distal tumor recurrence. This may be due in part to interaction of GBM with factors of the subventricular zone (SVZ), including those contained within the cerebrospinal fluid (CSF). However, direct interaction of GBM tumors with CSF has not been proved and would be hindered in the presence of an intact ependymal cell layer. METHODS: Here, we investigate the ependymal cell barrier and its derived extracellular matrix (ECM) fractones in the vicinity of a GBM tumor. Patient-derived GBM cells were orthotopically implanted into immunosuppressed athymic mice in locations distal and proximal to the LV. A PBS vehicle injection in the proximal location was included as a control. At four weeks post-xenograft, brain tissue was examined for alterations in ependymal cell health via immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. RESULTS: We identified local invading GBM cells within the LV wall and increased influx of CSF into the LV-proximal GBM tumor bulk compared to controls. In addition to the physical disruption of the ependymal cell barrier, we also identified increased signs of compromised ependymal cell health in LV-proximal tumor-bearing mice. These signs include increased accumulation of lipid droplets, decreased cilia length and number, and decreased expression of cell channel proteins. We additionally identified elevated numbers of small fractones in the SVZ within this group, suggesting increased indirect CSF-contained molecule signaling to tumor cells. CONCLUSIONS: Our data is the first to show that LV-proximal GBMs physically disrupt the ependymal cell barrier in animal models, resulting in disruptions in ependymal cell biology and increased CSF interaction with the tumor bulk. These findings point to ependymal cell health and CSF-contained molecules as potential axes for therapeutic targeting in the treatment of GBM.

Functional Characterization of Brain Tumor-Initiating Cells and Establishment of GBM Preclinical Models that Incorporate Heterogeneity, Therapy, and Sex Differences.

Glioblastoma (GBM) is the most common primary brain cancer in adults where tumor cell heterogeneity and sex differences influence clinical outcomes. Here, we functionally characterize three male and three female patient-derived GBM cell lines, identify protumorigenic BTICs, and create novel male and female preclinical models of GBM. Cell lines were evaluated on the following features: proliferation, stemness, migration, tumorigenesis, clinical characteristics, and sensitivity to radiation, TMZ, rhTNFSF10 (rhTRAIL), and rhBMP4 All cell lines were classified as GBM according to epigenetic subtyping, were heterogenous and functionally distinct from one another, and re-capitulated features of the original patient tumor. In establishing male and female preclinical models, it was found that two male-derived GBM cell lines (QNS108 and QNS120) and one female-derived GBM cell line (QNS315) grew at a faster rate in female mice brains. One male-derived GBM cell line (QNS108) decreased survival in female mice in comparison with male mice. However, no survival differences were observed for mice injected with a female-derived cell line (QNS315). In summary, a panel of six GBM patient-derived cell lines were functionally characterized, and it was shown that BTIC lines can be used to construct sex-specific models with differential phenotypes for additional studies.

Sex-Specific Differences in Glioblastoma.

Sex differences have been well identified in many brain tumors. Even though glioblastoma (GBM) is the most common primary malignant brain tumor in adults and has the worst outcome, well-established differences between men and women are limited to incidence and outcome. Little is known about sex differences in GBM at the disease phenotype and genetical/molecular level. This review focuses on a deep understanding of the pathophysiology of GBM, including hormones, metabolic pathways, the immune system, and molecular changes, along with differences between men and women and how these dimorphisms affect disease outcome. The information analyzed in this review shows a greater incidence and worse outcome in male patients with GBM compared with female patients. We highlight the protective role of estrogen and the upregulation of androgen receptors and testosterone having detrimental effects on GBM. Moreover, hormones and the immune system work in synergy to directly affect the GBM microenvironment. Genetic and molecular differences have also recently been identified. Specific genes and molecular pathways, either upregulated or downregulated depending on sex, could potentially directly dictate GBM outcome differences. It appears that sexual dimorphism in GBM affects patient outcome and requires an individualized approach to management considering the sex of the patient, especially in relation to differences at the molecular level.

Glioblastoma Proximity to the Lateral Ventricle Alters Neurogenic Cell Populations of the Subventricular Zone.

Despite current strategies combining surgery, radiation, and chemotherapy, glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. Tumor location plays a key role in the prognosis of patients, with GBM tumors located in close proximity to the lateral ventricles (LVs) resulting in worse survival expectancy and higher incidence of distal recurrence. Though the reason for worse prognosis in these patients remains unknown, it may be due to proximity to the subventricular zone (SVZ) neurogenic niche contained within the lateral wall of the LVs. We present a novel rodent model to analyze the bidirectional signaling between GBM tumors and cells contained within the SVZ. Patient-derived GBM cells expressing GFP and luciferase were engrafted at locations proximal, intermediate, and distal to the LVs in immunosuppressed mice. Mice were either sacrificed after 4 weeks for immunohistochemical analysis of the tumor and SVZ or maintained for survival analysis. Analysis of the GFP+ tumor bulk revealed that GBM tumors proximal to the LV show increased levels of proliferation and tumor growth than LV-distal counterparts and is accompanied by decreased median survival. Conversely, numbers of innate proliferative cells, neural stem cells (NSCs), migratory cells and progenitors contained within the SVZ are decreased as a result of GBM proximity to the LV. These results indicate that our rodent model is able to accurately recapitulate several of the clinical aspects of LV-associated GBM, including increased tumor growth and decreased median survival. Additionally, we have found the neurogenic and cell division process of the SVZ in these adult mice is negatively influenced according to the presence and proximity of the tumor mass. This model will be invaluable for further investigation into the bidirectional signaling between GBM and the neurogenic cell populations of the SVZ.

Analysis of intraoperative human brain tissue transcriptome reveals putative risk genes and altered molecular pathways in glioma-related seizures.

BACKGROUND: The pathogenesis of glioma-related seizures (GRS) is poorly understood. Here in, we aim to identify putative molecular pathways that lead to the development of GRS. METHODS: We determined brain transcriptome from intraoperative human brain tissue of patients with either GRS, glioma without seizures (non-GRS), or with idiopathic temporal lobe epilepsy (iTLE). We performed transcriptome-wide comparisons between disease groups tissue from non-epileptic controls (non-EC) to identify differentially-expressed genes (DEG). We compared DEGs to identify those that are specific or common to the groups. Through a gene ontology analysis, we identified molecular pathways enriched for genes with a Log-fold change ≥1.5 or ≤-1.5 and p-value <0.05 compared to non-EC. RESULTS: We identified 110 DEGs that are associated with GRS vs. non-GRS: 80 genes showed high and 30 low expression in GRS. There was significant overexpression of genes involved in cell-to-cell and glutamatergic signaling (CELF4, SLC17A7, and CAMK2A) and down-regulation of genes involved immune-trafficking (CXCL8, H19, and VEGFA). In the iTLE vs GRS analysis, there were 1098 DEGs: 786 genes were overexpressed and 312 genes were underexpressed in the GRS samples. There was significant enrichment for genes considered markers of oncogenesis (GSC, MYBL2, and TOP2A). Further, there was down-regulation of genes involved in the glutamatergic neurotransmission (vesicular glutamate transporter-2) in the GRS vs. iTLE samples. CONCLUSIONS: We identified a number of altered processes such as cell-to-cell signaling and interaction, inflammation-related, and glutamatergic neurotransmission in the pathogenesis of GRS. Our findings offer a new landscape of targets to further study in the fields of brain tumors and seizures.

Human Cerebrospinal Fluid Modulates Pathways Promoting Glioblastoma Malignancy.

Glioblastoma (GBM) is the most common and devastating primary cancer of the central nervous system in adults. High grade gliomas are able to modify and respond to the brain microenvironment. When GBM tumors infiltrate the Subventricular zone (SVZ) they have a more aggressive clinical presentation than SVZ-distal tumors. We suggest that cerebrospinal fluid (CSF) contact contributes to enhance GBM malignant characteristics in these tumors. We evaluated the impact of human CSF on GBM, performing a transcriptome analysis on human primary GBM cells exposed to CSF to measure changes in gene expression profile and their clinical relevance on disease outcome. In addition we evaluated the proliferation and migration changes of CSF-exposed GBM cells in vitro and in vivo. CSF induced transcriptomic changes in pathways promoting cell malignancy, such as apoptosis, survival, cell motility, angiogenesis, inflammation, and glucose metabolism. A genetic signature extracted from the identified transcriptional changes in response to CSF proved to be predictive of GBM patient survival using the TCGA database. Furthermore, CSF induced an increase in viability, proliferation rate, and self-renewing capacity, as well as the migratory capabilities of GBM cells in vitro. In vivo, GBM cells co-injected with human CSF generated larger and more proliferative tumors compared to controls. Taken together, these results provide direct evidence that CSF is a key player in determining tumor growth and invasion through the activation of complex gene expression patterns characteristic of a malignant phenotype. These findings have diagnostic and therapeutic implications for GBM patients. The changes induced by CSF contact might play a role in the increased malignancy of SVZ-proximal GBM.

Alpha 1-antichymotrypsin contributes to stem cell characteristics and enhances tumorigenicity of glioblastoma.

BACKGROUND: Glioblastomas (GBMs) are the main primary brain tumors in adults with almost 100% recurrence rate. Patients with lateral ventricle proximal GBMs (LV-GBMs) exhibit worse survival compared to distal locations for unknown reasons. One hypothesis is the proximity of these tumors to the cerebrospinal fluid (CSF) and its chemical cues that can regulate cellular phenotype. We therefore investigated the role of CSF on GBM gene expression and the role of a CSF-induced gene, SERPINA3, in GBM malignancy in vitro and in vivo. METHODS: We utilized human CSF and GBM brain tumor-initiating cells (BTICs). We determined the impact of SERPINA3 expression in glioma patients using The Cancer Genome Atlas (TCGA) database. SERPINA3 expression changes were evaluated at mRNA and protein levels. The effects of knockdown (KD) and overexpression (OE) of SERPINA3 on cell migration, viability and cell proliferation were evaluated. Stem cell characteristics on KD cells were evaluated by differentiation and colony formation experiments. Tumor growth was studied by intracranial and flank injections. RESULTS: GBM-CSF increased BTIC migration accompanied by upregulation of the SERPINA3 gene. In patient samples and TCGA data, we observed SERPINA3 to correlate directly with brain tumor grade and indirectly with GBM patient survival. SERPINA3 KD induced a decrease in cell proliferation, migration, invasion, and stem cell characteristics, while SERPINA3 OE increased cell migration. In vivo, SERPINA3 KD BTICs showed increased survival in a murine model. CONCLUSIONS: SERPINA3 plays a key role in GBM malignancy and its inhibition results in a better outcome using GBM preclinical models.

Combination of Aß Suppression and Innate Immune Activation in the Brain Significantly Attenuates Amyloid Plaque Deposition.

Anti-Aß clinical trials are currently under way to determine whether preventing amyloid deposition will be beneficial in arresting progression of Alzheimer disease. Both clinical and preclinical studies suggest that antiamyloid strategies are only effective if started at early stages of the disease process in a primary prevention strategy. Because this approach will be difficult to deploy, strategies for secondary prevention aimed at later stages of disease are also needed. In this study, we asked whether combining innate immune activation in the brain with concurrent Aß suppression could enhance plaque clearance and could improve pathologic outcomes in mice with moderate amyloid pathologic disorder. Starting at 5 months of age, tet-off amyloid precursor protein transgenic mice were treated with doxycycline (dox) to suppress further amyloid precursor protein/Aß production, and at the same time mice were intracranially injected with adeno-associated virus 1 expressing murine IL-6 (AAV1-mIL-6). Three months later, mice treated with the combination of Aß suppression and AAV1-mIL-6 showed significantly less plaque pathologic disorder than dox or AAV1-mIL-6 only groups. The combination of AAV1-mIL-6 + dox treatment lowered total plaque burden by >60% versus untreated controls. Treatment with either dox or AAV1-mIL-6 alone was less effective than the combination. Our results suggest a synergistic mechanism by which the up-regulation of mIL-6 was able to improve plaque clearance in the setting of Aß suppression.

Altered Innate Immune and Glial Cell Responses to Inflammatory Stimuli in Amyloid Precursor Protein Knockout Mice.

Amyloid precursor protein (APP) and its cleaved products have been reported to have important functions in CNS health, including in memory and synapse formation, cell survival and neuroprotection. Furthermore APP and its cleaved products have been shown to be transiently increased in response to various CNS stressors, suggesting a role in response to acute cellular injury. In an attempt to further understand the function of APP in response to CNS injury, we have used intracranial LPS injection as an inflammatory injury model in APP knock out mice (APPKO). Our data show that innate immune responses to LPS injection is significantly blunted in APPKO mice compared to APP sufficient wild type (BL6) mice. Morphologically, glial cells in APPKO mice appear less reactive, with shorter ramified processes and smaller cell bodies in response to LPS. Additionally, quantitative RT-PCR analysis for several glia markers and innate immune cytokine levels (e.g. TNFα, IL-6, IL-1ß and IL-10) showed significantly reduced expression levels in LPS injected APPKO mice. In vitro cell culture assays confirmed this attenuated response to LPS stimulation by primary microglial cells isolated from APPKO mice. Our data suggests that APP full length protein and/or its cleaved products are necessary to mount a complete and effective innate immune cell response to inflammatory injury.

ATP-binding cassette transporters P-glycoprotein and breast cancer related protein are reduced in capillary cerebral amyloid angiopathy.

Alzheimer's disease (AD) is the most common form of dementia and marked by deposition of amyloid-ß (Aß) within the brain. Alterations of Aß transporters at the neurovasculature may play a role in the disease process. We investigated the expression of ABC transporters P-glycoprotein (P-gp) and breast cancer related protein (BCRP) in non-neurologic controls, AD, and severe capillary cerebral amyloid angiopathy (capCAA) cases, which are characterized by deposition of Aß within cerebral capillaries. Our data show that microvascular expression of P-gp and BCRP is strikingly decreased in capCAA-affected vessels but not in AD and control samples. Messenger RNA levels of P-gp, but not of BCRP, were downregulated in brain endothelial cells on exposure to oligomeric Aß42, but not fibrillar Aß42 or Aß40. Coincubating Aß42 together with clusterin, an amyloid-associated protein highly expressed in capCAA-affected vessels, strongly reduced levels of P-gp. In conclusion, accumulation of Aß, in combination with clusterin, within and around cerebral capillaries, may further aggravate the disease process in AD by affecting P-gp expression. Loss of P-gp expression or activity may serve as a selective biomarker for ongoing capCAA.

Neuroinflammation and common mechanism in Alzheimer's disease and prion amyloidosis: amyloid-associated proteins, neuroinflammation and neurofibrillary degeneration.

BACKGROUND: In cases with a long (>1 year) clinical duration of prion disease, the prion protein can form amyloid deposits. These cases do not show accumulation of 4-kDa ß-amyloid, which is observed in amyloid deposits in Alzheimer's disease (AD). In AD, amyloid is associated with inflammation and neurofibrillary degeneration, and it is elusive whether prion amyloid is associated with these changes as well. OBJECTIVES: The presence of inflammation and neurofibrillary degeneration was evaluated in prion amyloidosis. MATERIAL AND METHODS: Cortical areas of variant Creutzfeldt-Jakob disease (CJD; n = 3), young sporadic CJD (n = 4), different Gerstmann-Sträussler-Scheinker's disease patients (n = 5) and AD cases (n = 5) were examined using immunohistochemistry and specific stainings for amyloid. RESULTS: In both AD and prion disease cases, which were negative for 4-kDa ß-amyloid, parenchymal and vascular amyloid deposits were positive for amyloid-associated proteins such as complement protein and were associated with microglia clusters. Tau and ubiquitin were found near prion plaques in some of the Gerstmann-Sträussler-Scheinker's disease and sporadic CJD cases and also near vascular prion amyloid deposits. In variant CJD cases, occasionally, microglia clustering was found in plaques but no ubiquitin or complement proteins and hardly tau protein. CONCLUSIONS: In both AD and prion disease amyloid formation, irrespective of the protein involved, there seems to be a neuroinflammatory response with secondary neurofibrillary degeneration.

Neuroinflammation and blood-brain barrier changes in capillary amyloid angiopathy.

INTRODUCTION: ß-Amyloid (Aß) accumulation in cortical capillaries is a variant of cerebral amyloid angiopathy (CAA) referred to as capillary CAA (capCAA). capCAA is associated with a neuroinflammatory response. In vitro studies indicate that Aß induces reactive oxygen species (ROS) production, mainly generated through NADPH oxidase (NOX), by activated microglia. ROS in turn can induce altered expression of tight junctions (TJ), which are essential for blood-brain barrier (BBB) function. Whether the function of the BBB is affected in the brains of Alzheimer's disease (AD) patients with comorbid capCAA remains elusive. Cases with capCAA and no other AD-related changes allow studying capCAA-associated BBB alterations independent of AD pathology. AIM: In this study, we have investigated BBB alterations in capCAA and addressed the role of the neuroinflammatory response. METHODS: Human postmortem brain tissue with capCAA was analyzed by immunohistochemical staining. RESULTS: In this study, we show for the first time a dramatic loss of TJ proteins claudin-5, occludin and ZO-1 in Aß-laden capillaries. In addition, affected capillaries are associated with clusters of NOX-2-positive activated microglia. Disrupted BBB function was observed by increased presence of fibrinogen around the affected capillaries. CONCLUSIONS: Our data provide support for the early observation that neuroinflammatory response is involved in the altered expression of TJs in endothelial cells and loss of BBB integrity in capCAA.

Small heat shock proteins induce a cerebral inflammatory reaction.

More than 80% of Alzheimer's disease (AD) patients have some degree of cerebral amyloid angiopathy (CAA). In addition to arteries and veins, capillaries can also be affected. Capillary CAA (capCAA), rather than CAA in larger vessels, is associated with flame-like amyloid-beta (Aß) deposits that may extend beyond the vessel wall and radiate into the neuropil, a phenomenon also known as "dyshoric angiopathy." Aß deposits in AD, parenchymal as well as (cap)CAA and dyshoric angiopathy, are associated with a local inflammatory reaction, including activation of microglial cells and astrocytes that, among others, produce cytokines and reactive oxygen species. This neuroinflammatory reaction may account for at least part of the cognitive decline. In previous studies we observed that small heat shock proteins (sHsps) are associated with Aß deposits in AD. In this study the molecular chaperones Hsp20, HspB8 and HspB2B3 were found to colocalize with CAA and capCAA in AD brains. In addition, Hsp20, HspB8 and HspB2B3 colocalized with intercellular adhesion molecule 1 (ICAM-1) in capCAA-associated dyshoric angiopathy. Furthermore, we demonstrated that Hsp20, HspB8 and HspB2B3 induced production of interleukin 8, soluble ICAM-1 and monocyte chemoattractant protein 1 by human leptomeningeal smooth muscle cells and human brain astrocytes in vitro and that Hsp27 inhibited production of transforming growth factor beta 1 and CD40 ligand. Our results suggest a central role for sHsps in the neuroinflammatory reaction in AD and CAA and thus in contributing to cognitive decline.

Amyloid Beta induces oxidative stress-mediated blood-brain barrier changes in capillary amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is frequently observed in Alzheimer's disease (AD) and is characterized by deposition of amyloid beta (Aß) in leptomeningeal and cortical brain vasculature. In 40% of AD cases, Aß mainly accumulates in cortical capillaries, a phenomenon referred to as capillary CAA (capCAA). The aim of this study was to investigate blood-brain barrier (BBB) alterations in CAA-affected capillaries with the emphasis on tight junction (TJ) changes. First, capCAA brain tissue was analyzed for the distribution of TJs. Here, we show for the first time a dramatic loss of occludin, claudin-5, and ZO-1 in Aß-laden capillaries surrounded by NADPH oxidase-2 (NOX-2)-positive activated microglia. Importantly, we observed abundant vascular expression of the Aß transporter receptor for advanced glycation endproducts (RAGE). To unravel the underlying mechanism, a human brain endothelial cell line was stimulated with Aß1-42 to analyze the effects of Aß. We observed a dose-dependent cytotoxicity and increased ROS generation, which interestingly was reversed by administration of exogenous antioxidants, NOX-2 inhibitors, and by blocking RAGE. Taken together, our data evidently show that Aß is toxic to brain endothelial cells via binding to RAGE and induction of ROS production, which ultimately leads to disruption of TJs and loss of BBB integrity.

Characteristics of dyshoric capillary cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) affects brain parenchymal and leptomeningeal arteries and arterioles but sometimes involves capillaries (capCAA) with spread of the amyloid into the surrounding neuropil, that is, dyshoric changes. We determined the relationship between capCAA and larger vessel CAA, ß amyloid (Aß) plaques, neurofibrillary changes, inflammation, and apolipoprotein E (APOE) in 22 cases of dyshoric capCAA using immunohistochemistry. The dyshoric changes contained predominantly Aß1-40, whereas dense bulblike deposits adjacent to the capillary wall contained mostly Aß1-42. There was an inverse local correlation between Aß plaque load and capCAA severity (p = 0.01), suggesting that Aß transport between the neuropil and the circulation may be mechanistically involved. Deposits of hyperphosphorylated tau and ubiquitin and clusters of activated microglia, resembling the changes around Aß plaques, were found around capCAA but were absent around larger vessel CAA. In 14 cases for which APOE genotype was available, there was a high APOE-ε4 allele frequency (54%; 43% homozygous). The severity of CapCAA increased with the number of ε4-alleles; and APOE4 seemed to colocalize with capCAA by immunohistochemistry. These results suggest that capCAA is pathologically and possibly pathogenetically distinct from larger vessel CAA, and that it is associated with a high APOE-ε4 allele frequency.