INTRODUCTION: A new noninvasive oscillating magnetic field (OMF)-generating device called Oncomag... more INTRODUCTION: A new noninvasive oscillating magnetic field (OMF)-generating device called Oncomagnetic (OM) device, developed in our laboratory, disrupts mitochondrial electron transport to elevate intracellular reactive oxygen species (ROS) levels in patient derived glioblastoma (GBM) cells. METHODS: GBM cell death, ROS elevation and caspase 3 activation were studied by bright-field and fluorescence microscopy involving specific fluorescent probes. Device treatment of FOXN1-Nude mice with GBM xenografts was for 4 hours twice a day. Treatment of a 53-year-old male patient with recurrent GBM was done under a compassionate use protocol for 2–6 hours each weekday. Contrast-enhanced MRI scans assessed tumor size. RESULTS: OMF caused selective death of GBM cells, but not normal human cortical neurons and astrocytes. It altered mitochondrial oxygen consumption, increased mitochondrial and cellular ROS, and activated caspase 3. The overall survival of xenografted mice was prolonged by 6 da...
Increased cell proliferation in glioblastoma (GBM) leads to hypoxia in the tumor microenvironment... more Increased cell proliferation in glioblastoma (GBM) leads to hypoxia in the tumor microenvironment. This is a major concern in GBM patients as it promotes tumor invasion. Glutaminolysis is a hallmark of cancer cells and under hypoxic conditions glutamine metabolism proceeds through reductive carboxylation pathway. Recently, we have shown that oscillating magnetic field (OMF) produces oncolytic effects which can influence cellular metabolism. Here, we have explored the effect of OMF on glutamine metabolism in GBM cells. Patient-derived GBM cells were grown in high glucose (25 mM) DMEM supplemented with 20% fetal bovine serum (FBS), 2.0 mM glutamine and 1.0 mM pyruvate at 37 °C under humidified air and 5% CO2. Cells were divided into 2 groups (Test and Sham; n = 4 each group). When reached confluency (~2.0×106 cells/mL), cells in both groups were treated with 4.0 mM of [U-13C]glutamine in DMEM (supplemented with 20% FBS, and 1.0 mM pyruvate). The “Test” group was subjected to OMF for 3...
Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of brain tumor in children, compris... more Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of brain tumor in children, comprising >10% of all pediatric brain tumors. The median survival after diagnosis is < 1 year. Since DIPG tumors infiltrate brainstem and pons, they are inoperable. Currently radiotherapy is the mainstay of treatment, and there is a great need for novel therapies for the treatment of DIPG. Cellular metabolism plays a key role in carcinogenesis, unravelling active metabolic pathways in DIPG would help in developing targeted therapies. Glucose and glutamine are the two major nutrients necessary for the growth and proliferation of cancer cells. In this study, we have investigated the glucose and glutamine metabolism in SF8628 DIPG cells using 1H/13C NMR and GC-MS based metabolic flux analysis. SF8628 cells were grown in DMEM containing 11.0 mM glucose, supplemented with 10% FBS, and 2.0 mM glutamine at 37 °C under humidified air and 5% CO2. When cells reached confluency (replicates = 4), tr...
BACKGROUND We have recently shown that GBM use D-galactose (Gal) as a substrate, in vitro and in ... more BACKGROUND We have recently shown that GBM use D-galactose (Gal) as a substrate, in vitro and in vivo. Gal is imported via Glut3 and/or Glut14 and metabolized through the Leloir pathway. We investigated 4-deoxy-4-fluorogalactose (4DFG) as the lead compound in a family of galactose-based antimetabolites. 4DFG is a potent chemotherapeutic in monotherapy and can bolster existing therapies. METHODS We examined the alteration of glioma metabolism in vitro and in vivo induced by 4DFG. 1H/13C-NMR and optical probes were used to interrogate the effects of 4DFG on glycolysis and mitochondrial respiration in primary glioma cell cultures. Labeled lectins were used to assay for the disruption of glycan synthesis induced by 4DFG. An intracranial model of primary GBM was used to assess efficacy and toxicity in vivo. RESULTS NMR reveals that at physiological concentrations of glucose, low concentrations of 4DFG (5 μM) is able to inhibit glycolytic and mitochondrial flux by approximately 12%, p<...
Alternating electric field therapy has been approved for glioblastoma (GBM). We have preclinical ... more 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.
Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of G... more Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of GBM to use galactose (Gal) as a fuel via the Leloir pathway is investigated. Methods: Gene transcript data were accessed to determine the association between expression of genes of the Leloir pathway and patient outcomes. Growth studies were performed on five primary patient-derived GBM cultures using Glc-free media supplemented with Gal. The role of Glut3/Glut14 in sugar import was investigated using antibody inhibition of hexose transport. A specific inhibitor of GALK1 (Cpd36) was used to inhibit Gal catabolism. Gal metabolism was examined using proton, carbon and phosphorous NMR spectroscopy, with 13C-labeled Glc and Gal as tracers. Results: Data analysis from published databases revealed that elevated levels of mRNA transcripts of SLC2A3 (Glut3), SLC2A14 (Glut14) and key Leloir pathway enzymes correlate with poor patient outcomes. GBM cultures proliferated when grown solely on Gal in ...
Regulatory T-cells (Tregs) are immunosuppressive T-cells, which arrest immune responses to ‘Self’... more 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-ma...
Background We postulate that meningiomas undergo distinct metabolic reprogramming in tumorigenesi... more 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...
The FDA has approved tumor treating fields (Optune) to treat glioblastoma (GBM) and unresectable ... more The FDA has approved tumor treating fields (Optune) to treat glioblastoma (GBM) and unresectable mesothelioma. Optune uses scalp electrodes to pass a strong alternating electric field through the head, slowing GBM growth. We have recently developed and tested a novel device that uses an oscillating magnetic field (OMF) generated by rotating magnets to treat GBM. Our first GBM patient had a recurrent GBM with leptomeningeal spread. A custom-made Oncomagnetic device consisting of a multi-sport helmet with three OMF generators called oncoscillators, was worn by the patient at home. Changes in the brain and GBM tumor metabolism were examined using in vivo1H MRS on a 7T Siemens MRI scanner. 1H MRS data of the “tumor” and “non-tumor” regions during OMF treatment was obtained using sLASER pulse sequences. Both non–tumor and tumor regions showed the most common brain metabolites – myo-inositol, creatine, choline, glutamine/glutamate, and N-acetyl aspartate (NAA). 30 days after initiation of...
Oscillating magnetic fields (OMF) produced by a new device with rotating magnets produces rapid a... more Oscillating magnetic fields (OMF) produced by a new device with rotating magnets produces rapid apoptosis of patient derived glioblastoma (GBM) cells in culture. We have proposed that this is due to a sequence of events triggered by the well-established mT-range magnetic influence on the radical electron pair mechanism in the mitochondrial respiratory electron transport chain (ETC). Here we present results of preclinical studies in support of this mitochondrial hypothesis, accounting for the oncolytic effects of OMF. We tested a range of in vitro treatment protocols, involving changes in frequency and timing of OMF, to arrive at an optimally effective set of parameters. After hours of exposure to this optimized intermittent pattern of OMF killed not only patient derived GBM cells, but cells of 4 other malignant tumors in a cell culture dish. This treatment was not lethal to normal human astrocytes, bronchial epithelial cells, and B lymphocytes. Exposure to static magnetic fields and...
Magnetic fields in the mT range influence spin state pairing in redox-active radical pairs genera... more Magnetic fields in the mT range influence spin state pairing in redox-active radical pairs generating spin-forbidden quantum states which are kinetically inert. Studies examining the effects of static magnetic fields on mitochondrial electron transfer kinetics have demonstrated only modest effects. When neodymium super-magnets are securely attached to and precision-balanced on the shafts of electronically-controlled motors it is possible to generate rotating magnetic fields of desirable strengths and frequencies. Unlike a static magnetic field or an alternating field in a static electromagnetic coil, oscillating magnetic field (OMF) produced by rotating lines of force of a spinning permanent magnet can dynamically couple the electron spins of radical pairs within proteins whose orientations are ‘fixed’. The frequencies of rotation of magnets can also be tuned to appropriate electron cycling resonances within the proteins. Using OMF of appropriate field strength, frequency and on/off...
Noninvasive treatment with alternating electric fields at 200 kHz (Optune therapy) has been appro... more Noninvasive treatment with alternating electric fields at 200 kHz (Optune therapy) has been approved for recurrent and newly diagnosed glioblastoma (GBM). We have recently tested and confirmed in vitro, and in patient derived xenografted mouse models, the oncolytic potential of powerful newly developed device generating oscillating magnetic fields (OMF) using rotating magnets. This device is completely different and has different mechanisms of action than Optune therapy. We report the successful treatment of an endstage recurrent GBM in a 53-year-old man. The patient had undergone radical surgical excision and standard of care chemoradiotherapy, as well as experimental gene therapy for a left frontal tumor. 18 months after this treatment, there was recurrence with progressive enlargement and leptomeningeal spread, making the patient’s condition not suitable for further treatment. We conducted a 5-week treatment (followed by a pause of one week) consisting of daily exposure (on weekd...
Noninvasive cancer therapy with minimal side effects would be ideal for improving patient outcome... more Noninvasive cancer therapy with minimal side effects would be ideal for improving patient outcome in the clinic. We have developed a novel therapy using strong rotating magnets mounted on a helmet. They generate oscillating magnetic fields (OMF) that penetrate through the skull and cover the entire brain. We have demonstrated that OMF can effectively kill patient derived glioblastoma (GBM) cells in cell culture without having cytotoxic effects on cortical neurons and normal human astrocytes (NHA). Exposure of GBM cells to OMF reduced the cell viability by 33% in comparison to sham-treated cells (p< 0.001), while not affecting NHA cell viability. Time lapse video-microscopy for 16 h after OMF exposure showed a marked elevation of mitochondrial reactive oxygen species (ROS), and rapid apoptosis of GBM cells due to activation of caspase 3. Addition of a potent antioxidant vitamin E analog Trolox effectively blocked OMF-induced GBM cell death. Furthermore, OMF significantly potentiat...
Various pathways can repair DNA alkylation by chemotherapeutic agents such as temozolomide (TMZ).... more Various pathways can repair DNA alkylation by chemotherapeutic agents such as temozolomide (TMZ). The enzyme O6-methylguanine methyltransferase (MGMT) removes O6-methylated DNA adducts, leading to the failure of chemotherapy in resistant glioblastomas. Because of the anti-chemotherapeutic activities of MGMT previously described, estimating the levels of active MGMT in cancer cells can be a significant predictor of response to alkylating agents. Current methods to detect MGMT in cells are indirect, complicated, time-intensive, or utilize molecules that require complex and multistep chemistry synthesis. Our design simulates DNA repair by the transfer of a clickable propargyl group from O6-propargyl guanine to active MGMT and subsequent attachment of fluorescein-linked PEG linker via ”click chemistry.” Visualization of active MGMT levels reveals discrete active and inactive MGMT populations with biphasic kinetics for MGMT inactivation in response to TMZ-induced DNA damage.
We observed fine fibrin deposition along the paravascular spaces in naive animals, which increase... more We observed fine fibrin deposition along the paravascular spaces in naive animals, which increased dramatically following subarachnoid hemorrhage (SAH). Following SAH, fibrin deposits in the areas remote from the hemorrhage. Traditionally it is thought that fibrinogen enters subarachnoid space through damaged blood brain barrier. However, deposition of fibrin remotely from hemorrhage suggests that fibrinogen chains Aα, Bβ, and γ can originate in the brain. Here we demonstrate in vivo and in vitro that astroglia and neurons are capable of expression of fibrinogen chains. SAH in mice was induced by the filament perforation of the circle of Willis. Four days after SAH animals were anesthetized, transcardially perfused and fixed. Whole brain was processed for immunofluorescent (IF) analysis of fibrin deposition on the brain surface or in brains slices processed for fibrinogen chains Aα, Bβ, γ immunohistochemical detection. Normal human astrocytes were grown media to confluency and stimulated with NOC-18 (100 µM), TNF-α (100 nM), ATP-γ-S (100 µM) for 24 h. Culture was fixed and washed/permeabilized with 0.1% Triton and processed for IF. Four days following SAH fibrinogen chains Aα IF associated with glia limitans and superficial brain layers increased 3.2 and 2.5 times (p < 0.05 and p < 0.01) on the ventral and dorsal brain surfaces respectively; fibrinogen chains Bβ increased by 3 times (p < 0.01) on the dorsal surface and fibrinogen chain γ increased by 3 times (p < 0.01) on the ventral surface compared to sham animals. Human cultured astrocytes and neurons constitutively expressed all three fibrinogen chains. Their expression changed differentially when exposed for 24 h to biologically significant stimuli: TNFα, NO or ATP. Western blot and RT-qPCR confirmed presence of the products of the appropriate molecular weight and respective mRNA. We demonstrate for the first time that mouse and human astrocytes and neurons express fibrinogen chains suggesting potential presence of endogenous to the brain fibrinogen chains differentially changing to biologically significant stimuli. SAH is followed by increased expression of fibrinogen chains associated with glia limitans remote from the hemorrhage. We conclude that brain astrocytes and neurons are capable of production of fibrinogen chains, which may be involved in various normal and pathological processes.
NEURO-ONCOLOGY • NOVEMBER 2018 with tumor-immune interactions through multiple mechanisms: i) CD2... more NEURO-ONCOLOGY • NOVEMBER 2018 with tumor-immune interactions through multiple mechanisms: i) CD200 is secreted from tumors inducing an immunosuppressive environment, ii) CD200 is upregulated in tumor-associated vascular endothelial cells, creating an immunological barricade around the tumor microenvironment. We are targeting the activation receptor with a peptide ligand (CD200AR-L) activates antigen-presenting cells, enhancing dendritic cell maturation, cytokine production and antigen specific T cell activation. Treatment with CD200AR-L significantly extends survival in two murine glioma models. Directing the immune system to "fight the tumor", requires introduction of an antigen such as autologous or allogeneic tumor lysate for non-immunogenic tumors such as CNS tumors. In an ongoing pilot study treating companion dogs with high-grade glioma, patients receiving a canine specific CD200 peptide inhibitor in combination with the autologous tumor lysate vaccine increased median survival to 330 days, compared to 194 with lysate alone. Currently, 41% of the dogs are alive; the longest living dog is now 810 days post-surgery. 28% of dogs died of non-tumor related deaths. In contrast, 100% of the dogs in the tumor lysate-only group died of tumor recurrence. Furthermore, serum chemistry profiles and physical examinations showed that the peptide did not induce any systemic toxicity. Importantly, we have developed human CD200 peptide ligands that enhance cytokine secretion, dendritic cell maturation, and antigen-specific immune response. This innovative research may provide a significant breakthrough for the field of cancer immunotherapy. We have also demonstrated that the use of a CD200AR-L in a murine breast carcinoma model resulted in a significant survival benefit. In this light, CD200AR-L may be a powerful immunotherapy platform for other solid tumors.
Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of gli... more Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of glioblastoma with respect to patient survival. In addition to elevation of O-methylguanine DNA methyltransferase (MGMT), down-regulation of three DNA repair pathways; canonical mismatch repair (MMR), Non-Homologous End-Joining (NHEJ), and Homologous Recombination (HR) are correlated with poor patient outcome. We have designed and tested both and , a monoamine oxidase B (MAOB) specific prodrug, PAM-OBG, that is converted by glioma MAOB into the MGMT inhibitor O-benzylguanine (OBG) and the DNA crosslinking agent acrolein. In cultured glioma cells, we show that PAM-OBG is converted to OBG, inhibiting MGMT and sensitizing cells to DNA alkylating agents such as BCNU, CCNU, and Temozolomide (TMZ). In addition, we demonstrate that the acrolein generated is highly toxic in glioma treated with an inhibitor of Nucleotide Excision Repair (NER). In mouse intracranial models of primary human glioma, we ...
INTRODUCTION: A new noninvasive oscillating magnetic field (OMF)-generating device called Oncomag... more INTRODUCTION: A new noninvasive oscillating magnetic field (OMF)-generating device called Oncomagnetic (OM) device, developed in our laboratory, disrupts mitochondrial electron transport to elevate intracellular reactive oxygen species (ROS) levels in patient derived glioblastoma (GBM) cells. METHODS: GBM cell death, ROS elevation and caspase 3 activation were studied by bright-field and fluorescence microscopy involving specific fluorescent probes. Device treatment of FOXN1-Nude mice with GBM xenografts was for 4 hours twice a day. Treatment of a 53-year-old male patient with recurrent GBM was done under a compassionate use protocol for 2–6 hours each weekday. Contrast-enhanced MRI scans assessed tumor size. RESULTS: OMF caused selective death of GBM cells, but not normal human cortical neurons and astrocytes. It altered mitochondrial oxygen consumption, increased mitochondrial and cellular ROS, and activated caspase 3. The overall survival of xenografted mice was prolonged by 6 da...
Increased cell proliferation in glioblastoma (GBM) leads to hypoxia in the tumor microenvironment... more Increased cell proliferation in glioblastoma (GBM) leads to hypoxia in the tumor microenvironment. This is a major concern in GBM patients as it promotes tumor invasion. Glutaminolysis is a hallmark of cancer cells and under hypoxic conditions glutamine metabolism proceeds through reductive carboxylation pathway. Recently, we have shown that oscillating magnetic field (OMF) produces oncolytic effects which can influence cellular metabolism. Here, we have explored the effect of OMF on glutamine metabolism in GBM cells. Patient-derived GBM cells were grown in high glucose (25 mM) DMEM supplemented with 20% fetal bovine serum (FBS), 2.0 mM glutamine and 1.0 mM pyruvate at 37 °C under humidified air and 5% CO2. Cells were divided into 2 groups (Test and Sham; n = 4 each group). When reached confluency (~2.0×106 cells/mL), cells in both groups were treated with 4.0 mM of [U-13C]glutamine in DMEM (supplemented with 20% FBS, and 1.0 mM pyruvate). The “Test” group was subjected to OMF for 3...
Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of brain tumor in children, compris... more Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of brain tumor in children, comprising >10% of all pediatric brain tumors. The median survival after diagnosis is < 1 year. Since DIPG tumors infiltrate brainstem and pons, they are inoperable. Currently radiotherapy is the mainstay of treatment, and there is a great need for novel therapies for the treatment of DIPG. Cellular metabolism plays a key role in carcinogenesis, unravelling active metabolic pathways in DIPG would help in developing targeted therapies. Glucose and glutamine are the two major nutrients necessary for the growth and proliferation of cancer cells. In this study, we have investigated the glucose and glutamine metabolism in SF8628 DIPG cells using 1H/13C NMR and GC-MS based metabolic flux analysis. SF8628 cells were grown in DMEM containing 11.0 mM glucose, supplemented with 10% FBS, and 2.0 mM glutamine at 37 °C under humidified air and 5% CO2. When cells reached confluency (replicates = 4), tr...
BACKGROUND We have recently shown that GBM use D-galactose (Gal) as a substrate, in vitro and in ... more BACKGROUND We have recently shown that GBM use D-galactose (Gal) as a substrate, in vitro and in vivo. Gal is imported via Glut3 and/or Glut14 and metabolized through the Leloir pathway. We investigated 4-deoxy-4-fluorogalactose (4DFG) as the lead compound in a family of galactose-based antimetabolites. 4DFG is a potent chemotherapeutic in monotherapy and can bolster existing therapies. METHODS We examined the alteration of glioma metabolism in vitro and in vivo induced by 4DFG. 1H/13C-NMR and optical probes were used to interrogate the effects of 4DFG on glycolysis and mitochondrial respiration in primary glioma cell cultures. Labeled lectins were used to assay for the disruption of glycan synthesis induced by 4DFG. An intracranial model of primary GBM was used to assess efficacy and toxicity in vivo. RESULTS NMR reveals that at physiological concentrations of glucose, low concentrations of 4DFG (5 μM) is able to inhibit glycolytic and mitochondrial flux by approximately 12%, p<...
Alternating electric field therapy has been approved for glioblastoma (GBM). We have preclinical ... more 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.
Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of G... more Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of GBM to use galactose (Gal) as a fuel via the Leloir pathway is investigated. Methods: Gene transcript data were accessed to determine the association between expression of genes of the Leloir pathway and patient outcomes. Growth studies were performed on five primary patient-derived GBM cultures using Glc-free media supplemented with Gal. The role of Glut3/Glut14 in sugar import was investigated using antibody inhibition of hexose transport. A specific inhibitor of GALK1 (Cpd36) was used to inhibit Gal catabolism. Gal metabolism was examined using proton, carbon and phosphorous NMR spectroscopy, with 13C-labeled Glc and Gal as tracers. Results: Data analysis from published databases revealed that elevated levels of mRNA transcripts of SLC2A3 (Glut3), SLC2A14 (Glut14) and key Leloir pathway enzymes correlate with poor patient outcomes. GBM cultures proliferated when grown solely on Gal in ...
Regulatory T-cells (Tregs) are immunosuppressive T-cells, which arrest immune responses to ‘Self’... more 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-ma...
Background We postulate that meningiomas undergo distinct metabolic reprogramming in tumorigenesi... more 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...
The FDA has approved tumor treating fields (Optune) to treat glioblastoma (GBM) and unresectable ... more The FDA has approved tumor treating fields (Optune) to treat glioblastoma (GBM) and unresectable mesothelioma. Optune uses scalp electrodes to pass a strong alternating electric field through the head, slowing GBM growth. We have recently developed and tested a novel device that uses an oscillating magnetic field (OMF) generated by rotating magnets to treat GBM. Our first GBM patient had a recurrent GBM with leptomeningeal spread. A custom-made Oncomagnetic device consisting of a multi-sport helmet with three OMF generators called oncoscillators, was worn by the patient at home. Changes in the brain and GBM tumor metabolism were examined using in vivo1H MRS on a 7T Siemens MRI scanner. 1H MRS data of the “tumor” and “non-tumor” regions during OMF treatment was obtained using sLASER pulse sequences. Both non–tumor and tumor regions showed the most common brain metabolites – myo-inositol, creatine, choline, glutamine/glutamate, and N-acetyl aspartate (NAA). 30 days after initiation of...
Oscillating magnetic fields (OMF) produced by a new device with rotating magnets produces rapid a... more Oscillating magnetic fields (OMF) produced by a new device with rotating magnets produces rapid apoptosis of patient derived glioblastoma (GBM) cells in culture. We have proposed that this is due to a sequence of events triggered by the well-established mT-range magnetic influence on the radical electron pair mechanism in the mitochondrial respiratory electron transport chain (ETC). Here we present results of preclinical studies in support of this mitochondrial hypothesis, accounting for the oncolytic effects of OMF. We tested a range of in vitro treatment protocols, involving changes in frequency and timing of OMF, to arrive at an optimally effective set of parameters. After hours of exposure to this optimized intermittent pattern of OMF killed not only patient derived GBM cells, but cells of 4 other malignant tumors in a cell culture dish. This treatment was not lethal to normal human astrocytes, bronchial epithelial cells, and B lymphocytes. Exposure to static magnetic fields and...
Magnetic fields in the mT range influence spin state pairing in redox-active radical pairs genera... more Magnetic fields in the mT range influence spin state pairing in redox-active radical pairs generating spin-forbidden quantum states which are kinetically inert. Studies examining the effects of static magnetic fields on mitochondrial electron transfer kinetics have demonstrated only modest effects. When neodymium super-magnets are securely attached to and precision-balanced on the shafts of electronically-controlled motors it is possible to generate rotating magnetic fields of desirable strengths and frequencies. Unlike a static magnetic field or an alternating field in a static electromagnetic coil, oscillating magnetic field (OMF) produced by rotating lines of force of a spinning permanent magnet can dynamically couple the electron spins of radical pairs within proteins whose orientations are ‘fixed’. The frequencies of rotation of magnets can also be tuned to appropriate electron cycling resonances within the proteins. Using OMF of appropriate field strength, frequency and on/off...
Noninvasive treatment with alternating electric fields at 200 kHz (Optune therapy) has been appro... more Noninvasive treatment with alternating electric fields at 200 kHz (Optune therapy) has been approved for recurrent and newly diagnosed glioblastoma (GBM). We have recently tested and confirmed in vitro, and in patient derived xenografted mouse models, the oncolytic potential of powerful newly developed device generating oscillating magnetic fields (OMF) using rotating magnets. This device is completely different and has different mechanisms of action than Optune therapy. We report the successful treatment of an endstage recurrent GBM in a 53-year-old man. The patient had undergone radical surgical excision and standard of care chemoradiotherapy, as well as experimental gene therapy for a left frontal tumor. 18 months after this treatment, there was recurrence with progressive enlargement and leptomeningeal spread, making the patient’s condition not suitable for further treatment. We conducted a 5-week treatment (followed by a pause of one week) consisting of daily exposure (on weekd...
Noninvasive cancer therapy with minimal side effects would be ideal for improving patient outcome... more Noninvasive cancer therapy with minimal side effects would be ideal for improving patient outcome in the clinic. We have developed a novel therapy using strong rotating magnets mounted on a helmet. They generate oscillating magnetic fields (OMF) that penetrate through the skull and cover the entire brain. We have demonstrated that OMF can effectively kill patient derived glioblastoma (GBM) cells in cell culture without having cytotoxic effects on cortical neurons and normal human astrocytes (NHA). Exposure of GBM cells to OMF reduced the cell viability by 33% in comparison to sham-treated cells (p< 0.001), while not affecting NHA cell viability. Time lapse video-microscopy for 16 h after OMF exposure showed a marked elevation of mitochondrial reactive oxygen species (ROS), and rapid apoptosis of GBM cells due to activation of caspase 3. Addition of a potent antioxidant vitamin E analog Trolox effectively blocked OMF-induced GBM cell death. Furthermore, OMF significantly potentiat...
Various pathways can repair DNA alkylation by chemotherapeutic agents such as temozolomide (TMZ).... more Various pathways can repair DNA alkylation by chemotherapeutic agents such as temozolomide (TMZ). The enzyme O6-methylguanine methyltransferase (MGMT) removes O6-methylated DNA adducts, leading to the failure of chemotherapy in resistant glioblastomas. Because of the anti-chemotherapeutic activities of MGMT previously described, estimating the levels of active MGMT in cancer cells can be a significant predictor of response to alkylating agents. Current methods to detect MGMT in cells are indirect, complicated, time-intensive, or utilize molecules that require complex and multistep chemistry synthesis. Our design simulates DNA repair by the transfer of a clickable propargyl group from O6-propargyl guanine to active MGMT and subsequent attachment of fluorescein-linked PEG linker via ”click chemistry.” Visualization of active MGMT levels reveals discrete active and inactive MGMT populations with biphasic kinetics for MGMT inactivation in response to TMZ-induced DNA damage.
We observed fine fibrin deposition along the paravascular spaces in naive animals, which increase... more We observed fine fibrin deposition along the paravascular spaces in naive animals, which increased dramatically following subarachnoid hemorrhage (SAH). Following SAH, fibrin deposits in the areas remote from the hemorrhage. Traditionally it is thought that fibrinogen enters subarachnoid space through damaged blood brain barrier. However, deposition of fibrin remotely from hemorrhage suggests that fibrinogen chains Aα, Bβ, and γ can originate in the brain. Here we demonstrate in vivo and in vitro that astroglia and neurons are capable of expression of fibrinogen chains. SAH in mice was induced by the filament perforation of the circle of Willis. Four days after SAH animals were anesthetized, transcardially perfused and fixed. Whole brain was processed for immunofluorescent (IF) analysis of fibrin deposition on the brain surface or in brains slices processed for fibrinogen chains Aα, Bβ, γ immunohistochemical detection. Normal human astrocytes were grown media to confluency and stimulated with NOC-18 (100 µM), TNF-α (100 nM), ATP-γ-S (100 µM) for 24 h. Culture was fixed and washed/permeabilized with 0.1% Triton and processed for IF. Four days following SAH fibrinogen chains Aα IF associated with glia limitans and superficial brain layers increased 3.2 and 2.5 times (p < 0.05 and p < 0.01) on the ventral and dorsal brain surfaces respectively; fibrinogen chains Bβ increased by 3 times (p < 0.01) on the dorsal surface and fibrinogen chain γ increased by 3 times (p < 0.01) on the ventral surface compared to sham animals. Human cultured astrocytes and neurons constitutively expressed all three fibrinogen chains. Their expression changed differentially when exposed for 24 h to biologically significant stimuli: TNFα, NO or ATP. Western blot and RT-qPCR confirmed presence of the products of the appropriate molecular weight and respective mRNA. We demonstrate for the first time that mouse and human astrocytes and neurons express fibrinogen chains suggesting potential presence of endogenous to the brain fibrinogen chains differentially changing to biologically significant stimuli. SAH is followed by increased expression of fibrinogen chains associated with glia limitans remote from the hemorrhage. We conclude that brain astrocytes and neurons are capable of production of fibrinogen chains, which may be involved in various normal and pathological processes.
NEURO-ONCOLOGY • NOVEMBER 2018 with tumor-immune interactions through multiple mechanisms: i) CD2... more NEURO-ONCOLOGY • NOVEMBER 2018 with tumor-immune interactions through multiple mechanisms: i) CD200 is secreted from tumors inducing an immunosuppressive environment, ii) CD200 is upregulated in tumor-associated vascular endothelial cells, creating an immunological barricade around the tumor microenvironment. We are targeting the activation receptor with a peptide ligand (CD200AR-L) activates antigen-presenting cells, enhancing dendritic cell maturation, cytokine production and antigen specific T cell activation. Treatment with CD200AR-L significantly extends survival in two murine glioma models. Directing the immune system to "fight the tumor", requires introduction of an antigen such as autologous or allogeneic tumor lysate for non-immunogenic tumors such as CNS tumors. In an ongoing pilot study treating companion dogs with high-grade glioma, patients receiving a canine specific CD200 peptide inhibitor in combination with the autologous tumor lysate vaccine increased median survival to 330 days, compared to 194 with lysate alone. Currently, 41% of the dogs are alive; the longest living dog is now 810 days post-surgery. 28% of dogs died of non-tumor related deaths. In contrast, 100% of the dogs in the tumor lysate-only group died of tumor recurrence. Furthermore, serum chemistry profiles and physical examinations showed that the peptide did not induce any systemic toxicity. Importantly, we have developed human CD200 peptide ligands that enhance cytokine secretion, dendritic cell maturation, and antigen-specific immune response. This innovative research may provide a significant breakthrough for the field of cancer immunotherapy. We have also demonstrated that the use of a CD200AR-L in a murine breast carcinoma model resulted in a significant survival benefit. In this light, CD200AR-L may be a powerful immunotherapy platform for other solid tumors.
Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of gli... more Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of glioblastoma with respect to patient survival. In addition to elevation of O-methylguanine DNA methyltransferase (MGMT), down-regulation of three DNA repair pathways; canonical mismatch repair (MMR), Non-Homologous End-Joining (NHEJ), and Homologous Recombination (HR) are correlated with poor patient outcome. We have designed and tested both and , a monoamine oxidase B (MAOB) specific prodrug, PAM-OBG, that is converted by glioma MAOB into the MGMT inhibitor O-benzylguanine (OBG) and the DNA crosslinking agent acrolein. In cultured glioma cells, we show that PAM-OBG is converted to OBG, inhibiting MGMT and sensitizing cells to DNA alkylating agents such as BCNU, CCNU, and Temozolomide (TMZ). In addition, we demonstrate that the acrolein generated is highly toxic in glioma treated with an inhibitor of Nucleotide Excision Repair (NER). In mouse intracranial models of primary human glioma, we ...
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Papers by Martyn Sharpe