Acquisition of Immune Privilege in GBM Tumors: Role of Prostaglandins and Bile Salts.

Based on the postulate that glioblastoma (GBM) tumors generate anti-inflammatory prostaglandins and bile salts to gain immune privilege, we analyzed 712 tumors in-silico from three GBM transcriptome databases for prostaglandin and bile synthesis/signaling enzyme-transcript markers. A pan-database correlation analysis was performed to identify cell-specific signal generation and downstream effects. The tumors were stratified by their ability to generate prostaglandins, their competency in bile salt synthesis, and the presence of bile acid receptors nuclear receptor subfamily 1, group H, member 4 (NR1H4) and G protein-coupled bile acid receptor 1 (GPBAR1). The survival analysis indicates that tumors capable of prostaglandin and/or bile salt synthesis are linked to poor outcomes. Tumor prostaglandin D2 and F2 syntheses are derived from infiltrating microglia, whereas prostaglandin E2 synthesis is derived from neutrophils. GBMs drive the microglial synthesis of PGD2/F2 by releasing/activating complement system component C3a. GBM expression of sperm-associated heat-shock proteins appears to stimulate neutrophilic PGE2 synthesis. The tumors that generate bile and express high levels of bile receptor NR1H4 have a fetal liver phenotype and a RORC-Treg infiltration signature. The bile-generating tumors that express high levels of GPBAR1 are infiltrated with immunosuppressive microglia/macrophage/myeloid-derived suppressor cells. These findings provide insight into how GBMs generate immune privilege and may explain the failure of checkpoint inhibitor therapy and provide novel targets for treatment.

Method for noninvasive whole-body stimulation with spinning oscillating magnetic fields and its safety in mice.

We recently reported shrinkage of untreatable recurrent glioblastoma (GBM) in an end-stage patient using noninvasive brain stimulation with a spinning oscillating magnetic field (sOMF)-generating device called the Oncomagnetic device. Our in vitro experiments demonstrated selective cancer cell death while sparing normal cells by sOMF-induced increase in intracellular reactive oxygen species (ROS) levels due to magnetic perturbation of mitochondrial electron transport. Here, we describe the results of an in vivo study assessing the toxicity of chronic sOMF stimulation in mice using a newly constructed apparatus comprised of the sOMF-generating active components of the Oncomagnetic device. We chronically stimulated 10 normal 60-day old female C57BL/6 mice in their housing cages for 2 h 3 times a day, as in the patient treatment protocol, over 4 months. We also studied the effects of 2-h acute sOMF stimulation. Our observations and those of blinded independent veterinary staff observers, indicated no significant adverse effects of chronic or acute sOMF stimulation on the health, behavior, electrocardiographic and electroencephalographic activities, hematologic profile, and brain and other tissue and organ morphology of treated mice compared to age-matched untreated control mice. These findings suggest that short- and long-term therapies with the Oncomagnetic device are safe and well tolerated.

Hijacking Sexual Immuno-Privilege in GBM-An Immuno-Evasion Strategy.

Regulatory T-cells (Tregs) are immunosuppressive T-cells, which arrest immune responses to 'Self' tissues. Some immunosuppressive Tregs that recognize seminal epitopes suppress immune responses to the proteins in semen, in both men and women. We postulated that GBMs express reproductive-associated proteins to manipulate reproductive Tregs and to gain immune privilege. We analyzed four GBM transcriptome databases representing ≈900 tumors for hypoxia-responsive Tregs, steroidogenic pathways, and sperm/testicular and placenta-specific genes, stratifying tumors by expression. In silico analysis suggested that the presence of reproductive-associated Tregs in GBM tumors was associated with worse patient outcomes. These tumors have an androgenic signature, express male-specific antigens, and attract reproductive-associated Related Orphan Receptor C (RORC)-Treg immunosuppressive cells. GBM patient sera were interrogated for the presence of anti-sperm/testicular antibodies, along with age-matched controls, utilizing monkey testicle sections. GBM patient serum contained anti-sperm/testicular antibodies at levels > six-fold that of controls. Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are associated with estrogenic tumors which appear to mimic placental tissue. We demonstrate that RORC-Tregs drive poor patient outcome, and Treg infiltration correlates strongly with androgen levels. Androgens support GBM expression of sperm/testicular proteins allowing Tregs from the patient's reproductive system to infiltrate the tumor. In contrast, estrogen appears responsible for MDSC/TAM immunosuppression.

Metabolism of fructose in B-cells: A 13C NMR spectroscopy based stable isotope tracer study.

Earlier studies on glucose metabolism in B-cells suggested an active TCA cycle in both naïve B cells and differentiated IgA plasma cells. Glycolysis was shown to be more active in IgA plasma cells than naïve B-cells. There have been no reports on the metabolism of fructose in B-cells. Fructose is a major sugar present in the western diet. Thus, we have investigated the metabolism of fructose in B-cells including the effect of glucose on the metabolism of fructose. In this study, using 13C NMR spectroscopy and [U-13C]fructose and [U-13C]glucose as stable 13C isotope tracers, we investigated the metabolic fate of fructose and glucose in B-cells. B-cells showed mitochondrial oxidation of fructose when administered alone, but showed diminished oxidation of fructose in the presence of glucose. On the other hand, fructose did not significantly affect the mitochondrial metabolism of glucose.

A Motion Simulator Ride Associated With Headache and Subdural Hematoma: First Case Report.

We report the first case report of symptomatic bilateral subdural hematomas (SDH) associated with riding a centrifugal motion simulator ride. A previously healthy 55-year-old male developed new onset daily headaches 1 week after going on the ride that were due to symptomatic bilateral SDH requiring operative intervention with a full recovery. There was no history of other trauma or other systemic or intracranial abnormality to account for the development of the SDH. We review the headaches and other clinical features associated with chronic SDH. Twelve cases of roller coaster headaches due to SDH associated with riding roller coasters have been reported. The pathophysiology is reviewed, which we believe is the same mechanism that may be responsible in this case. Although it is possible that this neurovascular injury is truly rare, it is also possible that this injury is underreported as patients and physicians may not make the association or physicians have not reported additional cases. The risk of this injury likely increases with age, as the size of the subdural space increases, and may support the maxim that "roller coasters and simulators are for kids."

Epithelial and organ-related marker expression in pituitary adenomas.

The histologic expression of epithelial and organ-related immunohistochemical markers in primary sellar region tumors has received little attention to date. This lack of empirical data may lead to mistaken assumptions in the evaluation of sellar region neoplasms. To address this issue, the frequency and specificity of epithelial (cytokeratin 7(CK7), CK20) and organ-related differentiation markers (gross cystic disease fluid protein-15 (GCDFP-15), thyroid transcription factor-1 (TTF-1), Napsin A, paired box 8 (PAX-8), hepatocyte paraffin 1 (HepPar1) and estrogen receptor (ER)) were studied in 40 patients with adenomas comprising five hormonal sub-types. Non-parametric statistical procedures were used to examine associations between marker expression and tumor sub-type. CK7 and CK20 immunoreactivity were seen in 48% and 8% of tumors, respectively, although never in a diffuse pattern. CK20 expression was nearly exclusive to corticotrophs, whereas CK7 frequently highlighted cells with dendritic-type morphology. The specificity of organ-related differentiation markers was 100% (monoclonal Napsin A, GCDFP-15 and TTF-1), 97% (HepPar1 and PAX-8), 90% (polyclonal Napsin A) and 72% (ER); no tumors demonstrated significant co-expression of these organ-related markers with either CK7 or CK20. The first major conclusion of this study is that CK7 staining in adenoma is more frequent than has been previously than has been previously described. CK7 immunoreactive cells often displayed a dendritic-type morphology, including within large macroadenomas, which raises the question as to whether these represent tumor cells with folliculo-stellate cell-type differentiation, as these also have dendritic cell-type morphology and express CK7 in non-neoplastic glands. The second major conclusion, which confirms earlier findings, is that CK20 staining is a very infrequent immunohistochemical finding in adenomas that is virtually limited to corticotrophs and thus is helpful in diagnostic confirmation of that sub-type. The final conclusion is in regard to those features that separate adenomas from sellar region metastases: adenomas do not demonstrate significant expression of TTF-1, monoclonal Napsin A, PAX-8, HepPar1 or GCDFP-15, nor do they exhibit diffuse expression of CK7 and CK20.

Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred T cells in glioblastoma.

Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.

Failure of porous tantalum cervical interbody fusion devices: two-year results from a prospective, randomized, multicenter clinical study.

OBJECTIVE: The objective of this study was to assess the safety and efficacy of 2 novel cervical interbody fusion devices in the treatment of single-level degenerative cervical disk disease. Both devices were fabricated from a porous tantalum material. The high overall porosity of the devices was intended to facilitate anterior cervical interbody fusion. METHODS: A prospective, randomized, 3-armed, clinical study was initiated with the following treatment groups: porous tantalum ring device packed with autograft, porous tantalum block device, and iliac crest autograft control. All the patients had single-level symptomatic cervical disk disease that had failed to respond to nonoperative therapy. Clinical and radiographic data were collected preoperatively, during surgery, before hospital discharge, and at 6 weeks, 3 months, 6 months, 12 months, and 24 months postoperatively. RESULTS: Six investigators participated in the clinical study at 6 investigational centers in the United States. Enrollment into the study was terminated after 39 patients had been accrued because of concerns over delayed fusion in the porous tantalum treatment groups. Of the 39 patients enrolled into the clinical study, 11 patients received the control treatment of iliac autograft fusion, 13 patients received the porous tantalum ring device with the center cavity packed with cancellous iliac crest autograft, and 15 patients received the porous tantalum block device. These patients were evaluated for 24 months as per the study protocol. There were no significant differences in any of the patient demographic variables collected. The mean operative times for both the ring and block device groups were slightly lower than the control treatment. Two patients in the block treatment group were determined to be nonunion between the 6- and 12-month time points and underwent additional surgery. Five patients with porous tantalum devices showed radiographic evidence of device fragmentation, and one patient in addition had radiographic evidence of erosion of the involved vertebral bodies by the device. One patient in the ring treatment group died from a myocardial infarction and kidney failure subsequent to the 12-month follow-up visit, which was unrelated to the device or the spinal fusion procedure. Fusion rate at 2 years for the tantalum device was very low as compared with the control arm (44% vs. 100%). Patient Neck Disability Index scores, Short Form-36 scores, and neurological status scores were similar between the 3 treatment groups. CONCLUSIONS: This study demonstrates that stand-alone porous tantalum material is not ideal for a cervical spine interbody fusion because of the low rate of arthrodesis and the risk of device fragmentation in patients who fail to fuse.

Spinning magnetic field patterns that cause oncolysis by oxidative stress in glioma cells.

Raising reactive oxygen species (ROS) levels in cancer cells to cause macromolecular damage and cell death is a promising anticancer treatment strategy. Observations that electromagnetic fields (EMF) elevate intracellular ROS and cause cancer cell death, have led us to develop a new portable wearable EMF device that generates spinning oscillating magnetic fields (sOMF) to selectively kill cancer cells while sparing normal cells in vitro and to shrink GBM tumors in vivo through a novel mechanism. Here, we characterized the precise configurations and timings of sOMF stimulation that produce cytotoxicity due to a critical rise in superoxide in two types of human glioma cells. We also found that the antioxidant Trolox reverses the cytotoxic effect of sOMF on glioma cells indicating that ROS play a causal role in producing the effect. Our findings clarify the link between the physics of magnetic stimulation and its mechanism of anticancer action, facilitating the development of a potential new safe noninvasive device-based treatment for GBM and other gliomas.

Recurrent petit mal seizures in Erdheim-Chester disease mimicking an intra-axial brain tumor: illustrative case.

BACKGROUND: Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis characterized histologically by foamy histiocytes and Touton giant cells in a background of fibrosis. Bone pain with long bone osteosclerosis is highly specific for ECD. Central nervous system involvement is rare, although dural, hypothalamic, cerebellar, brainstem, and sellar region involvement has been described. OBSERVATIONS: A 59-year-old man with a history of ureteral obstruction, medically managed petit mal seizures, and a left temporal lesion followed with serial magnetic resonance imaging (MRI) presented with worsening seizure control. Repeat MRI identified bilateral amygdala region lesions. Gradual growth of the left temporal lesion over 1 year with increasing seizure frequency prompted resection. A non-Langerhans cell histiocytosis with a BRAF V600E mutation was identified on pathology. Imaging findings demonstrated retroperitoneal fibrosis and long bone osteosclerosis with increased fluorodeoxyglucose uptake that, together with the neuropathologic findings, were diagnostic of ECD. LESSONS: This case of biopsy-proven ECD is unique in that the singular symptom was seizures well controlled with medical management in the presence of similarly located bilateral anterior mesial temporal lobe lesions. Although ECD is rare intracranially, its variable imaging presentation, including the potential to mimic seizure-associated medial temporal lobe tumors, emphasizes the need for a wide differential diagnosis.

mRNA stability alterations mediated by HuR are necessary to sustain the fast growth of glioma cells.

Regulation of mRNA decay is an important mechanism controlling gene expression. Steady state levels of mRNAs can be markedly altered by changes in the decay rate. The control of mRNA stability depends on sequences in the transcript itself and on RNA-binding proteins that dynamically bind to these sequences. A well characterized sequence motif, which has been shown to be present in many short-lived mRNAs, is the de-stabilizing adenylate/uridylate-rich element (ARE) located at the 3' untranslated region (3'UTR) of mRNAs. HuR is an RNA-binding protein, which binds to AREs and in doing so, increases the half-life and steady state levels of the corresponding mRNA. Using tissue microarray technology, we found that HuR is over-expressed in human gliomas. We also found that there is a change in HuR localization from being solely in the nucleus to being expressed at high levels in the cytosol. Moreover, a positive correlation was found between total HuR levels, cytosolic localization and tumor grade. We also studied the decay rate of several HuR target mRNAs and found that these mRNAs have a slower rate of decay in glioma cell lines than in astrocytes. Finally, we have been able to decrease both the stability and steady state level of these transcripts in glioma cells using an RNA decoy. More importantly, the decoy transfected cells and cells exposed to a HuR inhibitor have reduced cell growth. In addition, pharmacological inhibition of HuR also resulted in glioma cell growth inhibition. In conclusion, our data suggest that post-transcriptional control abnormalities mediated by HuR are necessary to sustain the rapid growth of this devastating type of cancer.

Indolent multicentric chordoma - A previously undescribed entity: A Case report and literature review.

Background: Chordomas are rare neuraxial tumors arising from remnants of primitive notochord. They are generally slow-growing malignant neoplasms. Only four adult cases of multicentric chordomas have been reported, all with aggressive and rapid growth. Here, we present an unusual case of indolent multicentric chordomas involving cervical and thoracic spine, sacrum, and calvarium. Case Description: A 60-year-old male was found to have multiple lesions throughout his neuroaxis incidentally on workup for colitis. A needle biopsy documented the diagnosis of chordoma. This has been followed for more than 4 years with no progression. Conclusion: We present the first reported case of indolent multicentric chordomas. Due to the extreme rarity of indolent multicentric chordomas, close follow-up is needed and recommended.

Glutamine anaplerosis is required for amino acid biosynthesis in human meningiomas.

BACKGROUND: We postulate that meningiomas undergo distinct metabolic reprogramming in tumorigenesis and unraveling their metabolic phenotypes provide new therapeutic insights. Glutamine catabolism is key to the growth and proliferation of tumors. Here, we investigated the metabolomics of freshly resected meningiomas and glutamine metabolism in patient-derived meningioma cells. METHODS: 1H NMR spectroscopy of tumor tissues from meningioma patients was used to differentiate the metabolite profiles of grade-I and grade-II meningiomas. Glutamine metabolism was examined using 13C/15N glutamine tracer, in 5 patient-derived meningioma cells. RESULTS: Alanine, lactate, glutamate, glutamine, and glycine were predominantly elevated only in grade-II meningiomas by 74%, 76%, 35%, 75%, and 33%, respectively, with alanine and glutamine levels being statistically significant (P ≤ .02). 13C/15N glutamine tracer experiments revealed that both grade-I and -II meningiomas actively metabolize glutamine to generate various key carbon intermediates including alanine and proline that are necessary for the tumor growth. Also, it is shown that glutaminase (GLS1) inhibitor, CB-839 is highly effective in downregulating glutamine metabolism and decreasing proliferation in meningioma cells. CONCLUSION: Alanine and glutamine/glutamate are mainly elevated in grade-II meningiomas. Grade-I meningiomas possess relatively higher glutamine metabolism providing carbon/nitrogen for the biosynthesis of key nonessential amino acids. GLS1 inhibitor (CB-839) is very effective in downregulating glutamine metabolic pathways in grade-I meningiomas leading to decreased cellular proliferation.

Cancer stem cells, not bulk tumor cells, determine mechanisms of resistance to SMO inhibitors.

The emergence of treatment resistance significantly reduces the clinical utility of many effective targeted therapies. Although both genetic and epigenetic mechanisms of drug resistance have been reported, whether these mechanisms are stochastically selected in individual tumors or governed by a predictable underlying principle is unknown. Here, we report that the dependence of cancer stem cells (CSCs), not bulk tumor cells, on the targeted pathway determines the molecular mechanism of resistance in individual tumors. Using both spontaneous and transplantable mouse models of sonic hedgehog (SHH) medulloblastoma (MB) treated with an SHH/Smoothened inhibitor, sonidegib/LDE225, we show that genetic-based resistance occurs only in tumors that contain SHH-dependent CSCs (SD-CSCs). In contrast, SHH MBs containing SHH-dependent bulk tumor cells but SHH-independent CSCs (SI-CSCs) acquire resistance through epigenetic reprogramming. Mechanistically, elevated proteasome activity in SMOi-resistant SI-CSC MBs alters the tumor cell maturation trajectory through enhanced degradation of specific epigenetic regulators, including histone acetylation machinery components, resulting in global reductions in H3K9Ac, H3K14Ac, H3K56Ac, H4K5Ac, and H4K8Ac marks and gene expression changes. These results provide new insights into how selective pressure on distinct tumor cell populations contributes to different mechanisms of resistance to targeted therapies. This insight provides a new conceptual framework to understand responses and resistance to SMOis and other targeted therapies.

Single-cell analysis of human glioma and immune cells identifies S100A4 as an immunotherapy target.

A major rate-limiting step in developing more effective immunotherapies for GBM is our inadequate understanding of the cellular complexity and the molecular heterogeneity of immune infiltrates in gliomas. Here, we report an integrated analysis of 201,986 human glioma, immune, and other stromal cells at the single cell level. In doing so, we discover extensive spatial and molecular heterogeneity in immune infiltrates. We identify molecular signatures for nine distinct myeloid cell subtypes, of which five are independent prognostic indicators of glioma patient survival. Furthermore, we identify S100A4 as a regulator of immune suppressive T and myeloid cells in GBM and demonstrate that deleting S100a4 in non-cancer cells is sufficient to reprogram the immune landscape and significantly improve survival. This study provides insights into spatial, molecular, and functional heterogeneity of glioma and glioma-associated immune cells and demonstrates the utility of this dataset for discovering therapeutic targets for this poorly immunogenic cancer.

Case Report: End-Stage Recurrent Glioblastoma Treated With a New Noninvasive Non-Contact Oncomagnetic Device.

Alternating electric field therapy has been approved for glioblastoma (GBM). We have preclinical evidence for anticancer effects in GBM cell cultures and mouse xenografts with an oscillating magnetic field (OMF) generating device. Here we report OMF treatment of end-stage recurrent glioblastoma in a 53-year-old man who had undergone radical surgical excision and chemoradiotherapy, and experimental gene therapy for a left frontal tumor. He experienced tumor recurrence and progressive enlargement with leptomeningeal involvement. OMF for 5 weeks was well tolerated, with 31% reduction of contrast-enhanced tumor volume and reduction in abnormal T2-weighted Fluid-Attenuated Inversion Recovery volume. Tumor shrinkage appeared to correlate with treatment dose. These findings suggest a powerful new noninvasive therapy for glioblastoma.

Rotating Magnetic Fields Inhibit Mitochondrial Respiration, Promote Oxidative Stress and Produce Loss of Mitochondrial Integrity in Cancer Cells.

Electromagnetic fields (EMF) raise intracellular levels of reactive oxygen species (ROS) that can be toxic to cancer cells. Because weak magnetic fields influence spin state pairing in redox-active radical electron pairs, we hypothesize that they disrupt electron flow in the mitochondrial electron transport chain (ETC). We tested this hypothesis by studying the effects of oscillating magnetic fields (sOMF) produced by a new noninvasive device involving permanent magnets spinning with specific frequency and timing patterns. We studied the effects of sOMF on ETC by measuring the consumption of oxygen (O2) by isolated rat liver mitochondria, normal human astrocytes, and several patient derived brain tumor cells, and O2 generation/consumption by plant cells with an O2 electrode. We also investigated glucose metabolism in tumor cells using 1H and 13C nuclear magnetic resonance and assessed mitochondrial alterations leading to cell death by using fluorescence microscopy with MitoTracker™ and a fluorescent probe for Caspase 3 activation. We show that sOMF of appropriate field strength, frequency, and on/off profiles completely arrest electron transport in isolated, respiring, rat liver mitochondria and patient derived glioblastoma (GBM), meningioma and diffuse intrinsic pontine glioma (DIPG) cells and can induce loss of mitochondrial integrity. These changes correlate with a decrease in mitochondrial carbon flux in cancer cells and with cancer cell death even in the non-dividing phase of the cell cycle. Our findings suggest that rotating magnetic fields could be therapeutically efficacious in brain cancers such as GBM and DIPG through selective disruption of the electron flow in immobile ETC complexes.

Selective induction of rapid cytotoxic effect in glioblastoma cells by oscillating magnetic fields.

PURPOSE: The mechanisms underlying anticancer effects of electromagnetic fields are poorly understood. An alternating electric field-generating therapeutic device called Optune™ device has been approved for the treatment of glioblastoma (GBM). We have developed a new device that generates oscillating magnetic fields (OMF) by rapid rotation of strong permanent magnets in specially designed patterns of frequency and timing and have used it to treat an end-stage recurrent GBM patient under an expanded access/compassionate use treatment protocol. Here, we ask whether OMF causes selective cytotoxic effects in GBM and whether it is through generation of reactive oxygen species (ROS). METHODS: We stimulated patient derived GBM cells, lung cancer cells, normal human cortical neurons, astrocytes, and bronchial epithelial cells using OMF generators (oncoscillators) of our Oncomagnetic Device and compared the results to those obtained under unstimulated or sham-stimulated control conditions. Quantitative fluorescence microscopy was used to assess cell morphology, viability, and ROS production mechanisms. RESULTS: We find that OMF induces highly selective cell death of patient derived GBM cells associated with activation of caspase 3, while leaving normal tissue cells undamaged. The cytotoxic effect of OMF is also seen in pulmonary cancer cells. The underlying mechanism is a marked increase in ROS in the mitochondria, possibly in part through perturbation of the electron flow in the respiratory chain. CONCLUSION: Rotating magnetic fields produced by a new noninvasive device selectively kill cultured human glioblastoma and non-small cell lung cancer cells by raising intracellular reactive oxygen species, but not normal human tissue cells.