A tale of two tumors: differential, but detrimental, effects of glioblastoma extracellular vesicles (EVs) on normal human brain cells.

Glioblastomas (GBMs) are dreadful brain tumors with abysmal survival outcomes. GBM EVs dramatically affect normal brain cells (largely astrocytes) constituting the tumor microenvironment (TME). EVs from different patient-derived GBM spheroids induced differential transcriptomic, secretomic, and proteomic effects on cultured astrocytes/brain tissue slices as GBM EV recipients. The net outcome of brain cell differential changes nonetheless converges on increased tumorigenicity. GBM spheroids and brain slices were derived from neurosurgical patient tissues following informed consent. Astrocytes were commercially obtained. EVs were isolated from conditioned culture media by ultrafiltration, ultraconcentration, and ultracentrifugation. EVs were characterized by nanoparticle tracking analysis, electron microscopy, biochemical markers, and proteomics. Astrocytes/brain tissues were treated with GBM EVs before downstream analyses. EVs from different GBMs induced brain cells to alter secretomes with pro-inflammatory or TME-modifying (proteolytic) effects. Astrocyte responses ranged from anti-viral gene/protein expression and cytokine release to altered extracellular signal-regulated protein kinase (ERK1/2) signaling pathways, and conditioned media from EV-treated cells increased GBM cell proliferation. Thus, astrocytes/brain slices treated with different GBM EVs underwent non-identical changes in various 'omics readouts and other assays, indicating "personalized" tumor-specific GBM EV effects on the TME. This raises concern regarding reliance on "model" systems as a sole basis for translational direction. Nonetheless, net downstream impacts from differential cellular and TME effects still led to increased tumorigenic capacities for the different GBMs.

Rapid volume pulsations of the extracellular space accompany epileptiform activity in trauma-injured neocortex and depend on the sodium-bicarbonate cotransporter NBCe1.

Post traumatic epilepsy (PTE) is a treatment-resistant consequence of traumatic brain injury (TBI). Recently, it has been revealed that epileptiform activity in acute chemoconvulsant seizure models is accompanied by transient shrinkages of extracellular space (ECS) called rapid volume pulsations (RVPs). Shrinkage of the ECS surrounding neurons and glia may contribute to ictogenic hyperexcitability and hypersynchrony during the chronic phase of TBI. Here, we identify the phenomenon of RVPs occurring spontaneously in rat neocortex at ≥ 3 weeks after injury in the controlled cortical impact (CCI) model for PTE. We further report that blocking the electrogenic action of the astrocytic cotransporter NBCe1 with 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminates both RVPs and epileptiform activity in ex-vivo CCI neocortical brain slices. We conclude that NBCe1-mediated extracellular volume shrinkage may represent a new target for therapeutic intervention in PTE.

High-Throughput Screening of Epigenetic Inhibitors in Meningiomas Identifies HDAC, G9a, and Jumonji-Domain Inhibition as Potential Therapies.

Background Epigenetics may predict treatment sensitivity and clinical course for patients with meningiomas more accurately than histopathology. Nonetheless, targeting epigenetic mechanisms is understudied for pharmacotherapeutic development for these tumors. The bio-molecular insights and potential therapeutic development of meningioma epigenetics led us to investigate epigenetic inhibition in meningiomas. Methods We screened a 43-tumor cohort using a 139-compound epigenetic inhibitor library to assess sensitivity of relevant meningioma subgroups to epigenetic inhibition. The cohort was composed of 5 cell lines and 38 tumors cultured directly from surgery; mean patient age was 56.6 years ± 13.9 standard deviation. Tumor categories: 38 primary tumors, 5 recurrent; 33 from females, 10 from males; 32 = grade 1; 10 = grade 2; 1 = grade 3. Results Consistent with our previous results, histone deacetylase inhibitors (HDACi) were the most efficacious class. Panobinostat significantly reduced cell viability in 36 of 43 tumors; 41 tumors had significant sensitivity to some HDACi. G9a inhibition and Jumonji-domain inhibition also significantly reduced cell viability across the cohort; tumors that lost sensitivity to panobinostat maintained sensitivity to either G9a or Jumonji-domain inhibition. Sensitivity to G9a and HDAC inhibition increased with tumor grade; tumor responses did not separate by gender. Few differences were found between recurrent and primary tumors, or between those with prior radiation versus those without. Conclusions Few efforts have investigated the efficacy of targeting epigenetic mechanisms to treat meningiomas, making the clinical utility of epigenetic inhibition largely unknown. Our results suggest that epigenetic inhibition is a targetable area for meningioma pharmacotherapy.

Targeting DNA Methyl Transferases with Decitabine in Cultured Meningiomas.

OBJECTIVE: Meningiomas are a common primary central nervous system tumor that lack a U.S. Food and Drug Administration-approved pharmacotherapy. Approximately 20%-35% of meningiomas are classified as higher grade with poor outcome, whereas patients with lower-grade meningiomas are known to have long-term neurologic deficits and reduced overall survival. Recent efforts to understand the epigenetic landscape of meningiomas have highlighted the importance of DNA methylation for predicting tumor outcomes and prognosis; therefore, inhibition of these pathways may present a viable therapy for these tumors. METHODS: In this study, we perform dose-response curves of decitabine, a DNA methyltransferase inhibitor, on patient-cultured tumors and meningioma cell lines. RESULTS: Thirty total samples were evaluated, including 24 patient-cultured tumors and 6 established meningioma cell lines. Meningiomas were found to have a significant reduction in cell viability after decitabine treatment in a dose dependent manner. The effect was primarily driven by 11 of the 30 tumors in our cohort, or 36.7%. Decitabine significantly reduced cell viability across all grades, tumors from different sexes, recurrent and primary tumors, as well as tumors without a history of previous radiation. Surprisingly, our single radiation-induced tumor did demonstrate greater viability after decitabine treatment. CONCLUSIONS: Our work has identified a potential drug candidate in decitabine for the treatment of meningiomas regardless of clinical subgroup. These data require further evaluation in preclinical models, and the conclusions based on clinical subgroups need to be evaluated in a larger cohort to achieve appropriate statistical power.

Higher Levels of IgG3 Antibodies in Serum, But Not in CSF, Distinguish Multiple Sclerosis From Other Neurological Disorders.

Increased intrathecal IgG and oligoclonal bands (OCB) are seminal features of multiple sclerosis (MS). Although no such differences in MS blood total IgG antibodies have been reported, serum OCB are a common and persistent finding in MS and have a systemic source. Recent studies showed that IgG3+ B cells and higher levels of serum IgG3 are linked to the development of MS. Additionally, intrathecal IgG synthesis in MS is associated with IgG3 heavy chain gene single nucleotide polymorphisms, and there is a strong relationship between susceptibility to MS and an IgG3 restriction fragment length polymorphism. These studies support the role of IgG3 in disease pathogenesis. Using multiple immunoassays, we investigated levels of total IgG, IgG1, and IgG3 in sera and CSF of 102 MS patients (19 paired CSF and sera), 76 patients with other neurological disorders (9 paired CSF and sera), and 13 healthy controls. We show that higher levels of total IgG and IgG3 antibodies were detected in MS serum, but not in CSF, which distinguishes MS from other inflammatory and non-inflammatory neurological disorders, with Receiver Operating Characteristic (ROC) Curves 0.79 for both IgG3 & total IgG. Our data support the notion that IgG3 antibodies may be a potential candidate for MS blood biomarker development.

Aberrant Immunoglobulin G Glycosylation in Multiple Sclerosis.

A hallmark of the inflammatory response in multiple sclerosis (MS) is the presence of intrathecal Immunoglobulin G (IgG) antibodies and oligoclonal bands (OCBs). The biological activity of IgGs is modulated by changes in glycosylation. Using multiple immunoassays with common lectins for sialylation and galactosylation, we investigated levels of IgG glycosylation in 28 MS and 37 control sera as well as paired CSF and serum. We demonstrated the presence of significantly lower levels of IgG sialylation in MS CSF compared to paired serum. Further, we showed that in MS there was no correlation between sialylated IgG and total IgG antibodies, or between sialylated IgG in CSF and serum. ELISA with native IgG antibodies showed significantly higher levels of sialylated and galactosylated IgG in MS compared to other neurological disorders and normal healthy controls. We conclude that lower levels of sialylated intrathecal IgG and higher levels of serum IgG galactosylation in MS may play significant role in disease pathogenesis. The unique IgG glycosylation profiles suggest a complexed nature of the IgG antibodies which may influence its effector functions in MS.

Cytokine-Laden Extracellular Vesicles Predict Patient Prognosis after Cerebrovascular Accident.

BACKGROUND: A major contributor to disability after hemorrhagic stroke is secondary brain damage induced by the inflammatory response. Following stroke, global increases in numerous cytokines-many associated with worse outcomes-occur within the brain, cerebrospinal fluid, and peripheral blood. Extracellular vesicles (EVs) may traffic inflammatory cytokines from damaged tissue within the brain, as well as peripheral sources, across the blood-brain barrier, and they may be a critical component of post-stroke neuroinflammatory signaling. METHODS: We performed a comprehensive analysis of cytokine concentrations bound to plasma EV surfaces and/or sequestered within the vesicles themselves. These concentrations were correlated to patient acute neurological condition by the Glasgow Coma Scale (GCS) and to chronic, long-term outcome via the Glasgow Outcome Scale-Extended (GOS-E). RESULTS: Pro-inflammatory cytokines detected from plasma EVs were correlated to worse outcomes in hemorrhagic stroke patients. Anti-inflammatory cytokines detected within EVs were still correlated to poor outcomes despite their putative neuroprotective properties. Inflammatory cytokines macrophage-derived chemokine (MDC/CCL2), colony stimulating factor 1 (CSF1), interleukin 7 (IL7), and monokine induced by gamma interferon (MIG/CXCL9) were significantly correlated to both negative GCS and GOS-E when bound to plasma EV membranes. CONCLUSIONS: These findings correlate plasma-derived EV cytokine content with detrimental outcomes after stroke, highlighting the potential for EVs to provide cytokines with a means of long-range delivery of inflammatory signals that perpetuate neuroinflammation after stroke, thus hindering recovery.

High-Throughput Mechanistic Screening of Epigenetic Compounds for the Potential Treatment of Meningiomas.

BACKGROUND: Meningiomas are the most common primary central nervous system tumors. 20-30% of these tumors are considered high-grade and associated with poor prognosis and high recurrence rates. Despite the high occurrence of meningiomas, there are no FDA-approved compounds for the treatment of these tumors. METHODS: In this study, we screened patient-cultured meningiomas with an epigenetic compound library to identify targetable mechanisms for the potential treatment of these tumors. Meningioma cell cultures were generated directly from surgically resected patient tumors and were cultured on a neural matrix. Cells were treated with a library of compounds meant to target epigenetic functions. RESULTS: Although each tumor displayed a unique compound sensitivity profile, Panobinostat, LAQ824, and HC toxin were broadly effective across most tumors. These three compounds are broad-spectrum Histone Deacetylase (HDAC) inhibitors which target class I, IIa, and IIb HDACs. Panobinostat was identified as the most broadly effective compound, capable of significantly decreasing the average cell viability of the sample cohort, regardless of tumor grade, recurrence, radiation, and patient gender. CONCLUSIONS: These findings strongly suggest an important role of HDACs in meningioma biology and as a targetable mechanism. Additional validation studies are necessary to confirm these promising findings, as well to identify an ideal HDAC inhibitor candidate to develop for clinical use.

Recombinant antibodies derived from laser captured single plasma cells of multiple sclerosis brain identified phage peptides which may be used as tools for characterizing intrathecal IgG response.

Oligoclonal bands and increased IgG antibody levels can be detected in the cerebrospinal fluid in vast majority of patients with Multiple Sclerosis (MS). However, the antigenic specificity of oligoclonal IgG has yet to be determined. Using laser capture microdissection, we isolated single CD38+ plasma cells from lesion areas in two autopsy MS brains, and generated three recombinant antibodies (rAbs) from clonally expanded plasma cells. Panning phage-displayed random peptide libraries was carried out to determine peptide antigen specificities of these MS brain rAbs. We identified 25 high affinity phage peptides from which 5 peptides are unique. Database searches revealed that they shared sequence homologies with Epstein-Barr nuclear antigens 4 and 6, as well as with other viral proteins. Significantly, these peptides were recognized by intrathecal IgG and oligoclonal IgG bands in other MS patients. Our results demonstrate that functional recombinant antibodies can be generated from clonally expanded plasma cells in MS brain lesions by laser capture microdissection, and that these MS brain rAbs have the potential for determining the targets of intrathecal IgG and oligoclonal bands.

Oligoclonal IgG antibodies in multiple sclerosis target patient-specific peptides.

IgG oligoclonal bands (OCBs) are present in the cerebrospinal fluid (CSF) of more than 95% of patients with multiple sclerosis (MS), and are considered to be the immunological hallmark of disease. However, the target specificities of the IgG in MS OCBs have remained undiscovered. Nevertheless, evidence that OCBs are associated with increased levels of disease activity and disability support their probable pathological role in MS. We investigated the antigen specificity of individual MS CSF IgG from 20 OCB-positive patients and identified 40 unique peptides by panning phage-displayed random peptide libraries. Utilizing our unique techniques of phage-mediated real-time Immuno-PCR and phage-probed isoelectric focusing immunoblots, we demonstrated that these peptides were targeted by intrathecal oligoclonal IgG antibodies of IgG1 and IgG3 subclasses. In addition, we showed that these peptides represent epitopes sharing sequence homologies with proteins of viral origin, and proteins involved in cell stress, apoptosis, and inflammatory processes. Although homologous peptides were found within individual patients, no shared peptide sequences were found among any of the 42 MS and 13 inflammatory CSF control specimens. The distinct sets of oligoclonal IgG-reactive peptides identified by individual MS CSF suggest that the elevated intrathecal antibodies may target patient-specific antigens.

Frailty as a Predictor of Neurosurgical Outcomes in Brain Tumor Patients.

BACKGROUND: Preoperative risk assessment is important, but inexact because physiologic reserves are difficult to measure. When assessing quality of life for patients with brain tumors, having a better predictor of postsurgical outcome would be beneficial in counseling these patients. Frailty is thought to estimate physiologic reserves, and it has been found to predict postoperative complications, length of stay, and discharge to a skilled nursing facility or assisted living facility in patients undergoing various types of surgery. Frailty as an adjunct to preoperative assessment of neurosurgical patients has never been evaluated. This study aimed to determine whether frailty predicts neurosurgical complications in patients with brain tumors and enhances current perioperative risk models. METHODS: Frailty was preoperatively assessed in 260 patients undergoing surgery for brain tumor resection using a validated scale that assessed weakness, weight loss, exhaustion, low physical activity, and slowed walking speed. Patients were classified as nonfrail (score of 0-1), moderately frail (score of 2-3), or frail (score of 4-5). Moderately frail and frail patients were combined for analysis. RESULTS: Preoperative frailty was associated with an increased risk for discharge to a location other than home (10.36; 95% confidence interval, 3.6-30.1), postoperative complications (2.09; 95% confidence interval, 1.09-3.98), and a longer length of stay (1.66; 95% confidence interval, 1.24-2.21). CONCLUSIONS: Frailty independently predicts discharge disposition, postoperative complications, and length of stay in patients undergoing surgery for brain tumor resection. Preoperative assessment of frailty can help neurosurgeons and patients make more informed decisions about pursing surgical treatment.