Inhibition of protein phosphatase-2A with LB-100 enhances antitumor immunity against glioblastoma.

PURPOSE: Glioblastoma (GBM) carries a dismal prognosis despite standard multimodal treatment with surgery, chemotherapy and radiation. Immune checkpoint inhibitors, such as PD1 blockade, for treatment of GBM failed to show clinical benefit. Rational combination strategies to overcome resistance of GBM to checkpoint monotherapy are needed to extend the promise of immunotherapy to GBM management. Emerging evidence suggests that protein phosphatase 2A (PP2A) plays a critical role in the signal transduction pathways of both adaptive and innate immune cells and that inhibition of PP2A could enhance cancer immunity. We investigated the use of a PP2A inhibitor, LB-100, to enhance antitumor efficacy of PD1 blockade in a syngeneic glioma model. METHODS: C57BL/6 mice were implanted with murine glioma cell line GL261-luc or GL261-WT and randomized into 4 treatment arms: (i) control, (ii) LB-100, (iii) PD1 blockade and (iv) combination. Survival was assessed and detailed profiling of tumor infiltrating leukocytes was performed. RESULTS: Dual PP2A and PD1 blockade significantly improved survival compared with monotherapy alone. Combination therapy resulted in complete regression of tumors in about 25% of mice. This effect was dependent on CD4 and CD8 T cells and cured mice established antigen-specific secondary protective immunity. Analysis of tumor lymphocytes demonstrated enhanced CD8 infiltration and effector function. CONCLUSION: This is the first preclinical investigation of the effect of combining PP2A inhibition with PD1 blockade for GBM. This novel combination provided effective tumor immunotherapy and long-term survival in our animal GBM model.

Chronic neuronal activation leads to elevated lactate dehydrogenase A through the AMP-activated protein kinase/hypoxia-inducible factor-1α hypoxia pathway.

Recent studies suggest that changes in neuronal metabolism are associated with epilepsy. High rates of ATP depletion, lactate dehydrogenase A and lactate production have all been found in epilepsy patients, animal and tissue culture models. As such, it can be hypothesized that chronic seizures lead to continuing elevations in neuronal energy demand which may lead to an adapted metabolic response and elevations of lactate dehydrogenase A. In this study, we examine elevations in the lactate dehydrogenase A protein as a long-term cellular adaptation to elevated metabolic demand from chronic neuronal activation. We investigate this cellular adaptation in human tissue samples and explore the mechanisms of lactate dehydrogenase A upregulation using cultured neurones treated with low Mg2+, a manipulation that leads to NMDA-mediated neuronal activation. We demonstrate that human epileptic tissue preferentially upregulates neuronal lactate dehydrogenase A, and that in neuronal cultures chronic and repeated elevations in neural activity lead to upregulation of neuronal lactate dehydrogenase A. Similar to states of hypoxia, this metabolic change occurs through the AMP-activated protein kinase/hypoxia-inducible factor-1α pathway. Our data therefore reveal a novel long-term bioenergetic adaptation that occurs in chronically activated neurones and provide a basis for understanding the interplay between metabolism and neural activity during epilepsy.

FKBP38 Regulates Self-Renewal and Survival of GBM Neurospheres.

Glioblastoma is the most common malignant primary brain tumor. The outcome is dismal, despite the multimodal therapeutic approach that includes surgical resection, followed by radiation and chemotherapy. The quest for novel therapeutic targets to treat glioblastoma is underway. FKBP38, a member of the immunophilin family of proteins, is a multidomain protein that plays an important role in the regulation of cellular functions, including apoptosis and autophagy. In this study, we tested the role of FKBP38 in glioblastoma tumor biology. Expression of FKBP38 was upregulated in the patient-derived primary glioblastoma neurospheres (GBMNS), compared to normal human astrocytes. Attenuation of FKBP38 expression decreased the viability of GBMNSs and increased the caspase 3/7 activity, indicating that FKBP38 is required for the survival of GBMNSs. Further, the depletion of FKBP38 significantly reduced the number of neurospheres that were formed, implying that FKBP38 regulates the self-renewal of GBMNSs. Additionally, the transient knockdown of FKBP38 increased the LC3-II/I ratio, suggesting the induction of autophagy with the depletion of FKBP38. Further investigation showed that the negative regulation of autophagy by FKBP38 in GBMNSs is mediated through the JNK/C-Jun-PTEN-AKT pathway. In vivo, FKBP38 depletion significantly extended the survival of tumor-bearing mice. Overall, our results suggest that targeting FKBP38 imparts an anti-glioblastoma effect by inducing apoptosis and autophagy and thus can be a potential therapeutic target for glioblastoma therapy.

Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma.

Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated provirus HERV-K (HML-2) can be reactivated in certain cancers. Here, we report pathological expression of HML-2 in malignant gliomas in both cerebrospinal fluid and tumor tissue that was associated with a cancer stem cell phenotype and poor outcomes. Using single-cell RNA-Seq, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells (NPC-like) that drive cellular plasticity. Using CRISPR interference, we demonstrate that HML-2 critically maintained glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models. Additionally, we demonstrate that HML-2 critically regulated embryonic stem cell programs in NPC-derived astroglia and altered their 3D cellular morphology by activating the nuclear transcription factor OCT4, which binds to an HML-2-specific long-terminal repeat (LTR5Hs). Moreover, we discovered that some glioblastoma cells formed immature retroviral virions, and inhibiting HML-2 expression with antiretroviral drugs reduced reverse transcriptase activity in the extracellular compartment, tumor viability, and pluripotency. Our results suggest that HML-2 fundamentally contributes to the glioblastoma stem cell niche. Because persistence of glioblastoma stem cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target.

Targeting neural precursors in the adult brain rescues injured dopamine neurons.

In Parkinson's disease, multiple cell types in many brain regions are afflicted. As a consequence, a therapeutic strategy that activates a general neuroprotective response may be valuable. We have previously shown that Notch ligands support neural precursor cells in vitro and in vivo. Here we show that neural precursors express the angiopoietin receptor Tie2 and that injections of angiopoietin2 activate precursors in the adult brain. Signaling downstream of Tie2 and the Notch receptor regulate blood vessel formation. In the adult brain, angiopoietin2 and the Notch ligand Dll4 activate neural precursors with opposing effects on the density of blood vessels. A model of Parkinson's disease was used to show that angiopoietin2 and Dll4 rescue injured dopamine neurons with motor behavioral improvement. A combination of growth factors with little impact on the vasculature retains the ability to stimulate neural precursors and protect dopamine neurons. The cellular and pharmacological basis of the neuroprotective effects achieved by these single treatments merits further analysis.

IDH-mutated gliomas promote epileptogenesis through d-2-hydroxyglutarate-dependent mTOR hyperactivation.

BACKGROUND: Uncontrolled seizures in patients with gliomas have a significant impact on quality of life and morbidity, yet the mechanisms through which these tumors cause seizures remain unknown. Here, we hypothesize that the active metabolite d-2-hydroxyglutarate (d-2-HG) produced by the IDH-mutant enzyme leads to metabolic disruptions in surrounding cortical neurons that consequently promote seizures. METHODS: We use a complementary study of in vitro neuron-glial cultures and electrographically sorted human cortical tissue from patients with IDH-mutant gliomas to test this hypothesis. We utilize micro-electrode arrays for in vitro electrophysiological studies in combination with pharmacological manipulations and biochemical studies to better elucidate the impact of d-2-HG on cortical metabolism and neuronal spiking activity. RESULTS: We demonstrate that d-2-HG leads to increased neuronal spiking activity and promotes a distinct metabolic profile in surrounding neurons, evidenced by distinct metabolomic shifts and increased LDHA expression, as well as upregulation of mTOR signaling. The increases in neuronal activity are induced by mTOR activation and reversed with mTOR inhibition. CONCLUSION: Together, our data suggest that metabolic disruptions in the surrounding cortex due to d-2-HG may be a driving event for epileptogenesis in patients with IDH-mutant gliomas.

Targeting protein arginine methyltransferase 5 sensitizes glioblastoma to trametinib.

Background: The prognosis of glioblastoma (GBM) remains dismal because therapeutic approaches have limited effectiveness. A new targeted treatment using MEK inhibitors, including trametinib, has been proposed to improve GBM therapy. Trametinib had a promising preclinical effect against several cancers, but its adaptive treatment resistance precluded its clinical translation in GBM. Previously, we have demonstrated that protein arginine methyltransferase 5 (PRMT5) is upregulated in GBM and its inhibition promotes apoptosis and senescence in differentiated and stem-like tumor cells, respectively. We tested whether inhibition of PRMT5 can enhance the efficacy of trametinib against GBM. Methods: Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, ELISA, and western blot were analyzed. In vivo, NSG mice were intracranially implanted with PRMT5-intact and -depleted GBMNS, treated with trametinib by daily oral gavage, and observed for tumor progression and mice survival rate. Results: PRMT5 depletion enhanced trametinib-induced cytotoxicity in GBMNS. PRMT5 knockdown significantly decreased trametinib-induced AKT and ERBB3 escape pathways. However, ERBB3 inhibition alone failed to block trametinib-induced AKT activity suggesting that the enhanced antitumor effect imparted by PRMT5 knockdown in trametinib-treated GBMNS resulted from AKT inhibition and not ERBB3 inhibition. In orthotopic murine xenograft models, PRMT5-depletion extended the survival of tumor-bearing mice, and combination with trametinib further increased survival. Conclusion: Combined PRMT5/MEK inhibition synergistically inhibited GBM in animal models and is a promising strategy for GBM therapy.

Adenosine A2A Receptor Activation Enhances Blood-Tumor Barrier Permeability in a Rodent Glioma Model.

The blood-tumor barrier (BTB) limits the entry of effective chemotherapeutic agents into the brain for treatment of malignant tumors like glioblastoma. Poor drug entry across the BTB allows infiltrative glioma stem cells to evade therapy and develop treatment resistance. Regadenoson, an FDA-approved adenosine A2A receptor (A2AR) agonist, has been shown to increase drug delivery across the blood-brain barrier in non-tumor-bearing rodents without a defined mechanism of enhancing BTB permeability. Here, we characterize the time-dependent impact of regadenoson on brain endothelial cell interactions and paracellular transport, using mouse and rat brain endothelial cells and tumor models. In vitro, A2AR activation leads to disorganization of cytoskeletal actin filaments by 30 minutes, downregulation of junctional protein expression by 4 hours, and reestablishment of endothelial cell integrity by 8 hours. In rats bearing intracranial gliomas, regadenoson treatment results in increase of intratumoral temozolomide concentrations, yet no increased survival noted with combined temozolomide therapy. These findings demonstrate regadenoson's ability to induce brain endothelial structural changes among glioma to increase BTB permeability. The use of vasoactive mediators, like regadenoson, which transiently influences paracellular transport, should further be explored to evaluate their potential to enhance central nervous system treatment delivery to aggressive brain tumors. IMPLICATIONS: This study provides insight on the use of a vasoactive agent to increase exposure of the BTB to chemotherapy with intention to improve glioma treatment efficacy.

GL261 luciferase-expressing cells elicit an anti-tumor immune response: an evaluation of murine glioma models.

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

Convection-enhanced delivery of botulinum toxin serotype A into the nonhuman primate cisterna magna and hippocampus.

OBJECTIVE: Botulinum toxin serotype A (BoNT/A) was reported to raise the seizure threshold when injected into the seizure focus of a kindled rodent model. Delivering BoNT/A to the nonhuman primate (NHP) central nervous system via convection-enhanced delivery (CED) has not been performed. The objective of this study was to determine the toxicity and distribution characteristics of CED of BoNT/A into the NHP hippocampus and cisterna magna. METHODS: Escalating BoNT/A doses were delivered by CED into the NHP hippocampus (n = 4) and cisterna magna (n = 5) for behavioral and histological assessment and to determine the highest nonlethal dose (LD0) and median lethal dose (LD50). Hippocampal BoNT/A was coinfused with Gd-albumin, a surrogate MRI tracer. Gd-albumin and radioiodinated BoNT/A (125I-BoNT/A) were coinfused into the hippocampus of 3 additional NHPs to determine BoNT/A distribution by in vivo MRI and postmortem quantitative autoradiography. Scintillation counting of CSF assessed the flow of 125I-BoNT/A from the hippocampus to CSF postinfusion. RESULTS: LD0 and LD50 were 4.2 and 18 ng/kg, and 5 and > 5 ng/kg for the NHP hippocampus and cisterna magna, respectively. Gd-albumin and 125I-BoNT/A completely perfused the hippocampus (155-234 mm3) in 4 of 7 NHPs. Fifteen percent of BoNT/A entered CSF after hippocampal infusion. The MRI distribution volume of coinfused Gd-albumin (VdMRI) was similar to the quantitative autoradiography distribution of 125I-BoNT/A (VdQAR) (mean VdMRI = 139.5 mm3 [n = 7]; VdQAR = 134.8 mm3 [n = 3]; r = 1.00, p < 0.0001). No infusion-related toxicity was identified histologically except that directly attributable to needle placement. CONCLUSIONS: Gd-albumin accurately tracked BoNT/A distribution on MRI. BoNT/A did not produce CNS toxicity. BoNT/A LD0 exceeded 10-fold the dose administered safely to humans for cosmesis and dystonia.

Convection-Enhanced Delivery of Muscimol Into the Bilateral Subthalamic Nuclei of Nonhuman Primates.

BACKGROUND: Muscimol is a gamma-aminobutyric acid receptor agonist that selectively and temporarily inhibits neurons. Local bolus injection of muscimol has been used experimentally to inhibit neuronal populations within discrete anatomical structures and discern their physiological function. OBJECTIVE: To determine the safety and behavioral effects of convection-enhanced delivery of muscimol into the bilateral subthalamic nuclei (STN) of nonhuman primate rhesus macaques (NHPs). METHODS: Six awake NHPs underwent co-infusion of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA), a surrogate magnetic resonance imaging (MRI) tracer, with increasing concentrations of muscimol for behavioral and histological assessment. Three other NHPs were co-infused with Gd-DTPA and 3H-muscimol into the STN to determine muscimol distribution by MRI and autoradiography. Two NHPs underwent microcatheter implantation without muscimol infusion for control comparison. RESULTS: MRI revealed selective and complete perfusion of the bilateral STN in animals infused with Gd-DTPA and muscimol. No abnormal movements occurred at 0.125 mM. Muscimol doses between 0.25 and 4.4 mM resulted in transient, dose-dependent hyperkinesia. Muscimol (8.8 mM) resulted in severe bilateral dyskinesias, ballistic movements, and sedation. An 88.8 mM dose produced unresponsiveness in 1 animal. Infusion-related pathological abnormities or toxicity was not present on histological examination. MRI distribution of co-infused Gd-DTPA was similar to autoradiographic distribution of 3H-muscimol (Vd; R = 0.94). Mean Vd of infused animals was 37.9 mm3 ± 11.7 mm3 and mean Vd: Vi 7.6 ± 2.3. CONCLUSION: Bilateral convection-enhanced delivery of muscimol into the primate STN resulted in dose-related hyperkinetic movements that resolved after stopping the infusion. Muscimol was not toxic to brain tissue. Gd-DTPA accurately tracked muscimol distribution.

Effect of ependymal and pial surfaces on convection-enhanced delivery.

OBJECT: Convection-enhanced delivery (CED) is increasingly used to investigate new treatments for central nervous system disorders. Although the properties of CED are well established in normal gray and white matter central nervous system structures, the effects on drug distribution imposed by ependymal and pial surfaces are not precisely defined. To determine the effect of these anatomical boundaries on CED, the authors infused low MW and high MW tracers for MR imaging near ependymal (periventricular) and pial (pericisternal) surfaces. METHODS: Five primates underwent CED of Gd-diethylenetriamine pentaacetic acid (Gd-DTPA; MW 590 D) or Gd-bound albumin (Gd-albumin; MW 72,000 D) during serial real-time MR imaging (FLAIR and T1-weighted sequences). Periventricular (caudate) infusions were performed unilaterally in 1 animal (volume of infusion [Vi] 57 microl) and bilaterally in 1 animal with Gd-DTPA (Vi = 40 mul on each side), and bilaterally in 1 animal with Gd-albumin (Vi = 80 microl on each side). Pericisternal infusions were performed in 2 animals with Gd-DTPA (Vi = 190 microl) or with Gd-albumin (Vi = 185 microl) (1 animal each). Clinical effects, MR imaging, and histology were analyzed. RESULTS: Large regions of the brain and brainstem were perfused with both tracers. Intraparenchymal distribution was successfully tracked in real time by using T1-weighted MR imaging. During infusion, the volume of distribution (Vd) increased linearly (R2 = 0.98) with periventricular (mean Vd/Vi ratio +/- standard deviation; 4.5 +/- 0.5) and pericisternal (5.2 +/- 0.3) Vi, but did so only until the leading edge of distribution reached the ependymal or pial surfaces, respectively. After the infusate reached either surface, the Vd/Vi decreased significantly (ependyma 2.9 +/- 0.8, pia mater 3.6 +/- 1.0; p < 0.05) and infusate entry into the ventricular or cisternal cerebrospinal fluid (CSF) was identified on FLAIR but not on T1-weighted MR images. CONCLUSIONS: Ependymal and pial boundaries are permeable to small and large molecules delivered interstitially by convection. Once infusate reaches these surfaces, a portion enters the adjacent ventricular or cisternal CSF and the tissue Vd/Vi ratio decreases. Although T1-weighted MR imaging is best for tracking intraparenchymal infusate distribution, FLAIR MR imaging is the most sensitive and accurate for detecting entry of Gd-labeled imaging compounds into CSF during CED.

Image-guided convection-enhanced delivery of gemcitabine to the brainstem.

OBJECT: To determine if the potent antiglioma chemotherapeutic agent gemcitabine could be delivered to the brainstem safely at therapeutic doses while monitoring its distribution using a surrogate magnetic resonance (MR) imaging tracer, the authors used convection-enhanced delivery to perfuse the primate brainstem with gemcitabine and Gd-diethylenetriamine pentaacetic acid (DTPA). METHODS: Six primates underwent convective brainstem perfusion with gemcitabine (0.4 mg/ml; two animals), Gd-DTPA (5 mM; two animals), or a coinfusion of gemcitabine (0.4 mg/ml) and Gd-DTPA (5 mM; two animals), and were killed 28 days afterward. These primates were observed over time clinically (six animals), and with MR imaging (five animals), quantitative autoradiography (one animal), and histological analysis (all animals). In an additional primate, 3H-gemcitabine and Gd-DTPA were coinfused and the animal was killed immediately afterward. In the primates there was no histological evidence of infusate-related tissue toxicity. Magnetic resonance images obtained during infusate delivery demonstrated that the anatomical region infused with Gd-DTPA was clearly distinguishable from surrounding noninfused tissue. Quantitative autoradiography confirmed that Gd-DTPA tracked the distribution of 3H-gemcitabine and closely approximated its volume of distribution (mean volume of distribution difference 13.5%). Conclusions. Gemcitabine can be delivered safely and effectively to the primate brainstem at therapeutic concentrations and at volumes that are higher than those considered clinically relevant. Moreover, MR imaging can be used to track the distribution of gemcitabine by adding Gd-DTPA to the infusate. This delivery paradigm should allow for direct therapeutic application of gemcitabine to brainstem gliomas while monitoring its distribution to ensure effective tumor coverage and to maximize safety.

Real-time imaging of convection-enhanced delivery of viruses and virus-sized particles.

OBJECT: Despite recent evidence showing that convection-enhanced delivery (CED) of viruses and virus-sized particles to the central nervous system (CNS) is possible, little is known about the factors influencing distribution of these vectors with convection. To better define the delivery of viruses and virus-sized particles in the CNS, and to determine optimal parameters for infusion, the authors coinfused adeno-associated virus ([AAV], 24-nm diameter) and/or ferumoxtran-10 (24 nm) by using CED during real-time magnetic resonance (MR) imaging. METHODS: Sixteen rats underwent intrastriatal convective coinfusion with 4 microl of 35S-AAV capsids (0.5-1.0 x 10(14) viral particles/ml) and increasing concentrations (0.1, 0.5, 1, and 5 mg/ml) of a similar sized iron oxide MR imaging agent (ferumoxtran-10). Five nonhuman primates underwent either convective coinfusion of 35S-AAV capsids and 1 mg/ml ferumoxtran-10 (striatum, one animal) or infusion of 1 mg/ml ferumoxtran-10 alone (striatum in two animals; frontal white matter in two). Clinical effects, MR imaging studies, quantitative autoradiography, and histological data were analyzed. RESULTS: Real-time, T2-weighted MR imaging of ferumoxtran-10 during infusion revealed a clearly defined hypointense region of perfusion. Quantitative autoradiography confirmed that MR imaging of ferumoxtran-10 at a concentration of 1 mg/ml accurately tracked viral capsid distribution in the rat and primate brain (the mean difference in volume of distribution [Vd] was 7 and 15% in rats and primates, respectively). The Vd increased linearly with increasing volume of infusion (Vi) (R2 = 0.98). The mean Vd/Vi ratio was 4.1 +/- 0.2 (mean +/- standard error of the mean) in gray and 2.3 +/- 0.1 in white matter (p < 0.01). The distribution of infusate was homogeneous. Postinfusion MR imaging revealed leakback along the cannula track at infusion rates greater than 1.5 microl/minute in primate gray and white matter. No animal had clinical or histological evidence of toxicity. CONCLUSIONS: The CED method can be used to deliver AAV capsids and similar sized particles to the CNS safely and effectively over clinically relevant volumes. Moreover, real-time MR imaging of ferumoxtran-10 during infusion reveals that AAV capsids and similar sized particles have different convective delivery properties than smaller proteins and other compounds.

Real-time image-guided direct convective perfusion of intrinsic brainstem lesions. Technical note.

Recent preclinical studies have demonstrated that convection-enhanced delivery (CED) can be used to perfuse the brain and brainstem with therapeutic agents while simultaneously tracking their distribution using coinfusion of a surrogate magnetic resonance (MR) imaging tracer. The authors describe a technique for the successful clinical application of this drug delivery and monitoring paradigm to the brainstem. Two patients with progressive intrinsic brainstem lesions (one with Type 2 Gaucher disease and one with a diffuse pontine glioma) were treated with CED of putative therapeutic agents mixed with Gd-diethylenetriamene pentaacetic acid (DTPA). Both patients underwent frameless stereotactic placement of MR imaging-compatible outer guide-inner infusion cannulae. Using intraoperative MR imaging, accurate cannula placement was confirmed and real-time imaging during infusion clearly demonstrated progressive filling of the targeted region with the drug and Gd-DTPA infusate. Neither patient had clinical or imaging evidence of short- or long-term infusate-related toxicity. Using this technique, CED can be used to safely perfuse targeted regions of diseased brainstem with therapeutic agents. Coinfused imaging surrogate tracers can be used to monitor and control the distribution of therapeutic agents in vivo. Patients with a variety of intrinsic brainstem and other central nervous system disorders may benefit from a similar treatment paradigm.

Real-time, image-guided, convection-enhanced delivery of interleukin 13 bound to pseudomonas exotoxin.

PURPOSE: To determine if the tumor-targeted cytotoxin interleukin 13 bound to Pseudomonas exotoxin (IL13-PE) could be delivered to the brainstem safely at therapeutic doses while monitoring its distribution in real-time using a surrogate magnetic resonance imaging tracer, we used convection-enhanced delivery to perfuse rat and primate brainstems with IL13-PE and gadolinium-bound albumin (Gd-albumin). EXPERIMENTAL DESIGN: Thirty rats underwent convective brainstem perfusion of IL13-PE (0.25, 0.5, or 10 microg/mL) or vehicle. Twelve primates underwent convective brainstem perfusion of either IL13-PE (0.25, 0.5, or 10 microg/mL; n = 8), co-infusion of 125I-IL13-PE and Gd-albumin (n = 2), or co-infusion of IL13-PE (0.5 microg/mL) and Gd-albumin (n = 2). The animals were permitted to survive for up to 28 days before sacrifice and histologic assessment. RESULTS: Rats showed no evidence of toxicity at all doses. Primates showed no toxicity at 0.25 or 0.5 microg/mL but showed clinical and histologic toxicity at 10 microg/mL. Quantitative autoradiography confirmed that Gd-albumin precisely tracked IL13-PE anatomic distribution and accurately showed the volume of distribution. CONCLUSIONS: IL13-PE can be delivered safely and effectively to the primate brainstem at therapeutic concentrations and over clinically relevant volumes using convection-enhanced delivery. Moreover, the distribution of IL13-PE can be accurately tracked by co-infusion of Gd-albumin using real-time magnetic resonance imaging.

Histone Deacetylase Inhibitor SAHA Is a Promising Treatment of Cushing Disease.

Context: Remission failure following transsphenoidal surgery in Cushing disease (CD) from pituitary corticotroph tumors (CtTs) remains clinically challenging. Histone deacetylase inhibitors (HDACis) are antitumor drugs approved for clinical use, with the potential to affect adrenocorticotropin hormone (ACTH) hypersecretion by inhibiting pro-opiomelanocortin (POMC) transcription. Objective: Testing the efficacy of suberoylanilide hydroxamic acid (SAHA) on human and murine ACTH-secreting tumor (AtT-20) cells. Design: Cell viability, ACTH secretion (enzyme-linked immunosorbent assay), apoptosis, and gene expression profile were investigated on AtT-20 cells. In vivo efficacy was examined in an athymic nude mouse AtT-20 xenograft model. SAHA efficacy against human-derived corticotroph tumor (hCtT) (n = 8) was tested in vitro. Setting: National Institutes of Health. Intervention: SAHA (0.5 to 8 µM). Main Outcome Measures: AtT-20 and hCtT cell survival, in vitro/invivo ACTH measurements. Results: SAHA (1 µM) reduced AtT-20 viability to 75% at 24 hours, 43% at 48 hours (analysis of variance; P = 0.002). Apoptosis was confirmed with elevated BAX/Bcl2 ratio and FACS. Intriguingly, early (3-hour) significant decline (70%; P < 0.0001) of secreted ACTH and diminished POMC transcription was observed with SAHA (1 µM). Microarray analysis revealed a direct association between liver X receptor alpha (LXRα) and POMC expression. Accordingly, SAHA reduced LXRα in AtT-20 cells but not in normal murine corticotrophs. Xenografted nude-mice tumor involution (126 ± 33/160 ± 35 vs 337 ± 49 mm3; P = 0.0005) was observed with 5-day intraperitoneal SAHA, with reversal of elevated ACTH (P < 0.0001). SAHA did not affect serum ACTH in nontumor mice. Lastly, we confirmed that SAHA (1 µM/24 h) decreased hCtT survival (78.92%; P = 0.0007) and ACTH secretion (83.64%; P = 0.03). Conclusion: Our findings demonstrate SAHA's efficacy in reducing survival and ACTH secretion in AtT-20 and hCtT cells, providing a potential intervention for recurrent/unremitting CD.

Simulating vasogenic brain edema using chronic VEGF infusion.

OBJECTIVE To study peritumoral brain edema (PTBE), it is necessary to create a model that accurately simulates vasogenic brain edema (VBE) without introducing a complicated tumor environment. PTBE associated with brain tumors is predominantly a result of vascular endothelial growth factor (VEGF) secreted by brain tumors, and VEGF infusion alone can lead to histological blood-brain barrier (BBB) breakdown in the absence of tumor. VBE is intimately linked to BBB breakdown. The authors sought to establish a model for VBE with chronic infusion of VEGF that can be validated by serial in-vivo MRI and histological findings. METHODS Male Fischer rats (n = 182) underwent stereotactic striatal implantation of MRI-safe brain cannulas for chronic infusion of VEGF (2-20 µg/ml). Following a preinfusion phase (4-6 days), the rats were exposed to VEGF or control rat serum albumin (1.5 µl/hr) for as long as 144 hours. Serial MRI was performed during infusion on a high-field (9.4-T) machine at 12-24, 24-36, 48-72, and 120-144 hours. Rat brains were then collected and histological analysis was performed. RESULTS Control animals and animals infused with 2 µg/ml of VEGF experienced no neurological deficits, seizure activity, or abnormal behavior. Animals treated with VEGF demonstrated a significantly larger volume (42.90 ± 3.842 mm3) of T2 hyper-attenuation at 144 hours when compared with the volume (8.585 ± 1.664 mm3) in control animals (mean difference 34.31 ± 4.187 mm3, p < 0.0001, 95% CI 25.74-42.89 mm3). Postcontrast T1 enhancement in the juxtacanalicular region indicating BBB breakdown was observed in rats undergoing infusion with VEGF. At the later time periods (120-144 hrs) the volume of T1 enhancement (34.97 ± 8.99 mm3) was significantly less compared with the region of edema (p < 0.0001). Histologically, no evidence of necrosis or inflammation was observed with VEGF or control infusion. Immunohistochemical analysis demonstrated astrocyte activation, vascular remodeling, and increased claudin-5 expression in juxtacanalicular regions. Aquaporin-4 expression was increased in both control and VEGF animals in the juxtacanalicular regions. CONCLUSIONS The results of this study show that chronic brain infusion of VEGF creates a reliable model of VBE. This model lacks necrosis and inflammation that are characteristic of previous models of VBE. The model allows for a precise investigation into the mechanism of VBE formation. The authors also anticipate that this model will allow for investigation into the mechanism of glucocorticoid action in abrogating VBE, and to test novel therapeutic strategies targeting PTBE.

Technique for enhanced accuracy and reliability in non-human primate stereotaxy.

Despite the importance of enhancing the accuracy and reliability of non-human primate stereotaxy, a number of limitations exist using currently described techniques. To overcome these problems, we present a simple universally available approach that combines pre-operative magnetic resonance imaging and the non-surgical creation of reference points (teeth marking). We have found that this approach improves stereotaxic targeting reliability and permits accurate reproducible stereotaxic localization at time points distant from the pre-operative imaging.