Rodent Glioma Models Research Articles

Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib’s effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1–10 µM and 25 mg/kg) and in combination with doxil (10–100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell–cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a − 53% versus − 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib’s ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

Implantable theranostic device for in vivo real-time NMR evaluation of drug impact in brain tumors.

The evaluation of the efficacy of a drug is a fundamental step in the development of new treatments or in personalized therapeutic strategies and patient management. Ideally, this evaluation should be rapid, possibly in real time, easy to perform and reliable. In addition, it should be associated with as few adverse effects as possible for the patient. In this study, we present a device designed to meet these goals for assessing therapeutic response. This theranostic device is based on the use of magnetic resonance imaging and spectroscopy for the diagnostic aspect and on the application of the convection-enhanced delivery technique for the therapeutic aspect. The miniaturized device is implantable and can be used in vivo in a target tissue. In this study, the device was applied to rodent glioma models with local administration of choline kinase inhibitor and acquisition of magnetic resonance images and spectra at 7 Tesla. The variations in the concentration of key metabolites measured by the device during the administration of the molecules demonstrate the relevance of the approach and the potential of the device.

INNV-14. INTERIM REPORT OF A PHASE 1/2 STUDY OF SONODYNAMIC THERAPY (SDT) USING AMINOLEVULINIC ACID (ALA) AND LOW-INTENSITY FOCUSED ULTRASOUND IN PEDIATRIC PATIENTS WITH DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract BACKGROUND DIPGs are devastating pediatric brain tumors with limited treatment options, poor prognosis, and survival of 9-11 months. Preclinical studies demonstrate that performing SDT through MR-guided focused ultrasound (MRgFUS) activates ALA metabolite, protoporphyrin IX (PpIX), leading to tumor cell death and prolonging survival in rodent glioma models through generation of reactive oxygen species and lipoperoxidation, without affecting normal tissue. MEDTHODS: Herein we report preliminary data from an ongoing first-in-child dose-escalation trial of ALA-SDT post-radiation therapy in children ≥ 5 years with DIPG. The trial aims to evaluate the safety and tolerability of ALA-SDT, determine the recommended-phase-2-dose and assess preliminary efficacy and pharmacokinetics (PK) of ALA administered intravenously. RESULTS The first study cohort completed enrollment of 3 subjects. Six hours prior to SDT, patients received an infusion of ALA (SONALA-001) at a dose of 5 mg/kg, followed by sonication with 200J. The first subject was treated in 2 sessions: each to half of the pons 30 days apart. Two subsequent subjects had the entire pons treated in a single session. No dose-limiting toxicity (DLT) or related adverse event grade ≥ 3 were observed. For SONALA-001, the Cmax occurred at the end of infusion, followed by rapid clearance (15.7 mL/min/kg), with a plasma half-life of &amp;lt; 1 hour, indicating rapid distribution (5530 ml/kg). For PpIX, the Cmax occurred 6 hours post-dose and declined with a longer mean half-life of 3.8 hours. Both Cmax and AUCall demonstrate circulating PpIX and systemic exposure were significantly lower than for ALA. CONCLUSION SDT signals an innovative and well-tolerated approach for patients with DIPG in our first study cohort. As expected, the PK for SONALA-001 demonstrate rapid infusion and clearance, whereas for PpIX, the PK are consistent with formation of a metabolite. Clinical trial enrollment is ongoing and dose-escalation cohorts will provide additional PK and safety data.

TRLS-03. IBRUTINIB DISRUPTS BLOOD-TUMOR BARRIER INTEGRITY TO ENHANCE CNS DRUG DELIVERY AND RODENT GLIOMA MODEL SURVIVAL

Abstract BACKGROUND In the setting of malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-brain-barrier (BBB). Identifying agents that transiently increase BBB permeability would advance glioma treatment. Ibrutinib, an FDA approved lymphoma treatment, has previously impaired aortic endothelial adhesion and in combination with cytotoxic therapy, increased rodent glioma model survival. In this study, we propose ibrutinib inhibits brain endothelial cell junctional expression to disrupt BBB integrity and increase chemotherapy delivery to malignant glioma cells. METHODS Using rat glioma cells, S635, we evaluated treatment effects with doxil (3mg/kg), ibrutinib (25mg/kg) and combination therapy. We measured effects with serial brain MRIs, doxil plasma and brain regional concentrations, along with 3kD dextran permeability. To evaluate the effects of ibrutinib on in vitro brain endothelial cells, we measured changes in cell-cell electrical impedance and rhodamine efflux, as a surrogate of Abcb1 transporter function, over time (0 to 8h) and at varied drug concentrations (1, 5, and 10µM ibrutinib). RESULTS Ibrutinib in combination with doxil, prolonged median survival in rodent glioma models (18 vs. 24days, p&amp;lt;0.05). MRI findings demonstrated a -14 or -53% vs -75% tumor volume change with doxil or ibrutinib alone vs. combination therapy, respectively (p&amp;lt;0.05). Comparing combination therapy vs. doxil alone, ibrutinib increased CNS concentrations of doxil in the tumor injected brain regions with statistical significance (56ng/mL vs. 74.6ng/mL, p&amp;lt;0.05). In in vitro studies, ibrutinib decreased brain endothelial cell-cell impedance maximally at 2 hours, in a dose dependently without affecting cell viability. Additionally, we observed ibrutinib dose dependently inhibit Abcb1 activity in both endothelial and glioma cells. CONCLUSION Our results suggest ibrutinib increases brain endothelial permeability by causing junctional disruption and inhibition of Abcb1, resulting in increased CNS drug entry and prolonged survival in glioma rat models.

CNSC-03. EXPLORING STRUCTURAL DISRUPTION OF TRICELLULAR JUNCTIONS TO ENHANCE BLOOD-BRAIN-BARRIER PERMEABILITY

Abstract The blood-brain barrier (BBB) hinders CNS chemotherapy entry for malignant glioma treatment. It is predominantly composed of brain endothelium linked by bicellular (BJ) and tricellular (TJ) tight junctions. Previous studies demonstrated,angubindin-1 inhibited TJ angulin-1 to increase brain and CSF targeted therapy in rodent lung cancer models. We hypothesize angubindin-1 can transiently decrease BBB junctional integrity to increase chemotherapy permeability and prolong rodent glioma model survival. Rat brain endothelial cells were treated with TJ and BJ inhibitors against angulin-1 (angubindin-1 600µg/mL), claudin-3 (C-CPE 200µg/mL) or claudin-5 (C-CPE-MT 200µg/mL). Endothelial integrity was assessed by immunoblotting and cell-cell electrical impedance. Effects of angubindin-1 on efflux transporter P-glycoprotein (PGP) and migration were studied using rat and human derived glioma cells. Rat glioma models were treated with doxil (3 mg/kg), angubindin-1 (10 mg/kg or 30 mg/kg), or combination therapy for assessments on tumor volume, BBB permeability and survival. We observed decreased angulin-1 expression 5 hours after angubindin-1 treatment, with return to baseline by 24 hours (p&amp;lt;0.05). Angubindin-1, CCPE, and CCPE-mt globally reduced endothelial cell-cell integrity, maximally at 4 hours with a return to baseline by 12 hours; with angubindin-1 demonstrating the largest decreased cell-adhesion (angubindin-1 vs control, p&amp;lt; 0.0001). Angubindin-1 also decreased PGP efflux of rhodamine in both endothelial and glioma cells, along with demonstrating a pro-migratory dose-dependent effect on rat glioma cells. Combined angubindin-1 and doxil increased survival in rat glioma models compared with doxil alone (24 days vs. 18 days, p &amp;lt; 0.0001). Day 14 tumor volume was significantly decreased with angubindin-1 and combination treatment respectively (77.5 % vs 81.6 %, p&amp;lt;0.05). Ongoing studies are exploring, angubindin-1’s effect on large CNS drug permeability, TJ localization post BBB disruption and additional combinational treatments in glioma models. Collectively, these findings pose a unique opportunity to disrupt BBB integrity and improve malignant glioma treatment options.

Magnolol and Temozolomide exhibit a synergistic anti-glioma activity through MGMT inhibition

Temozolomide (TMZ) is the leading chemotherapeutic agent used for glioma therapy due to its good oral absorption and blood-brain barrier permeability. However, its anti-glioma efficacy may be limited due to its adverse effects and resistance development. O6-Methylguanine-DNA-methyltransferase (MGMT), an enzyme associated with TMZ resistance, is activated via the NF-κB pathway, which is found to be upregulated in glioma. TMZ also upregulates NF-κB signaling like many other alkylating agents. Magnolol (MGN), a natural anti-cancer agent, has been reported to inhibit NF-κB signaling in multiple myeloma, cholangiocarcinoma, and hepatocellular carcinoma. MGN has already shown promising results in anti-glioma therapy. However, the synergistic action of TMZ and MGN has not been explored. Therefore, we investigated the effect of TMZ and MGN treatment in glioma and observed their synergistic pro-apoptotic action in both in vitro and in vivo glioma models. To explore the mechanism of this synergistic action, we found that MGN inhibits MGMT enzyme both in vitro and in vivo glioma. Next, we established the link between NF-κB signaling and MGN-induced MGMT inhibition in glioma. MGN inhibits the phosphorylation of p65, a subunit of NF-κB, and its nuclear translocation to block NF-κB pathway activation in glioma. MGN-induced NF-κB inhibition results in the transcriptional inhibition of MGMT in glioma. TMZ and MGN combinatorial treatment also impedes p65 nuclear translocation to inhibit MGMT in glioma. We observed a similar effect of TMZ and MGN treatment in the rodent glioma model. Thus, we concluded that MGN potentiates TMZ-induced apoptosis in glioma by inhibiting NF-κB pathway-mediated MGMT activation.

Phase 1/2, first-in-child study of sonodynamic therapy (SDT) using low intensity focused ultrasound and 5-aminolevulinic acid (ALA) for patients with diffuse intrinsic pontine glioma.

TPS10070 Background: Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain tumor with poor prognosis. Sonodynamic therapy (SDT) with ALA is a non-invasive strategy that sensitizes target tissues with a non-toxic chemical agent followed by exposure to low intensity MR-guided focused ultrasound (MRgFUS). Preclinical studies have shown that ALA is preferentially taken up by glioma cells and metabolized to protoporphyrin IX (PpIX). SDT activates protoporphyrin IX and induces tumor cell death via photodynamic effect (lipoperoxidation, apoptosis). Suheiro et al and Wu et al demonstrated that SDT killed tumors via apoptosis and lipoperoxidation in rodent glioma models with minimal damage to surrounding normal brain.1,2 ALA SDT was well-tolerated in an ongoing first-in-human trial in adults with recurrent high-grade gliomas and demonstrated biomarker evidence of the photodynamic effect3. Methods: A Phase 1/2, multicenter, open label study (NCT05123534) is enrolling patients at Children’s National Hospital. The study employs the Bayesian optimal interval (BOIN) algorithm to evaluate safety and tolerability of treatment with SDT in newly diagnosed DIPG subjects to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of MRgFUS energy in combination with ALA. The study will evaluate 3 ALA doses and 3 acoustic energy doses. ALA is administered IV six to nine hours prior to MRgFUS with the Exablate type-2 device. The first cohort, B1C1, evaluates the lowest drug-device dose combination and if no dose-limiting toxicity (DLT) is encountered after 3 patients, ascending dose-escalation will proceed to the second cohort, B2C1, treated with lowest dose of ALA with the second level of MRgFUS energy. Again, if no DLT is experienced, the next cohort will receive a higher dose combination. The initial patient in each cohort is treated in 2 sessions 21 to 28 days apart, each covering one-half of the pons. Subsequent patients are treated in 1 session to the whole pons. The primary objective is to evaluate the safety and tolerability of SDT in DIPG subjects. Secondary outcomes include preliminary efficacy assessments, pharmacokinetics of ALA and PpIX, mechanical performance of the Exablate Type-2 device, and radiographic evidence of tumor physiological changes associated with SDT. All patients at least 5 years of age with newly diagnosed, radiographically typical DIPG between 4-24 weeks after completion of standard radiotherapy are eligible for the trial. Patients with evidence of disease progression, diagnosis of porphyria, or prior or concurrent therapy with any other anticancer or investigational intervention will be excluded. Cohort 1 has been completed without DLT.

TMIC-23. EXPLORING IBRUTINIB'S EFFECTS ON BLOOD-BRAIN BARRIER INTEGRITY AND GLIOMA PROGRESSION

Abstract BACKGROUND The blood-brain barrier (BBB), among malignant gliomas limits certain agents from impairing tumor growth. Thus, identifying agents that can transiently increase BBB permeability may prove useful in enhancing glioma treatment response. Previous ibrutinib studies showed it independently disrupted gut epithelial integrity and hampered glioma growth through BMX (bone marrow x-linked tyrosine kinase) inhibition. We propose BMX inhibition via ibrutinib disrupts BBB integrity while impairing glioma progression. METHODS To evaluate ibrutinib’s effect on brain endothelium, we evaluated electrical cell-cell impedance, junctional/cytoskeletal expression, downstream protein expression and functional ABC transporter activity; monitoring changes over time (0 to 8h) and at varied drug concentrations (1-10μM ibrutinib). Using rat glioma cells (S635) in vitro and in models, we examined cytotoxicity, apoptosis, model survival, and drug concentrations with ibrutinib alone or combined with poorly permeable Abcb1 substrate doxorubicin. RESULTS Ibrutinib dose-dependently decreased brain endothelial cell-cell impedance by 60% at 2h, without affecting cell viability. We observed decreased ZO-1 junctions, actin cytoskeletal rearrangement, and downstream pErk and pMek decreased expression optimally 2h (10μM ibrutinib). Dose-dependently ibrutinib inhibited Abcb1 efflux activity, further favoring a more permeable endothelium. Synergy with doxorubicin was seen in rat glioma cells treated with ibrutinib via increased apoptosis and cytotoxicity. However, while combined treatment resulted in decreased systemic doxorubicin concentrations, no differences in brain:plasma or tumor:plasma concentrations were evident. Combination therapy also did not increase rodent glioma model survival nor decrease tumor size. CONCLUSION Our results suggest that while ibrutinib induces brain endothelial permeability by junctional/cytoskeletal disruption and inhibition of Abcb1 efflux, no sustained effect can be seen in rodent glioma models in combination with a cytotoxic agent. Additional studies exploring similar agents that can enhance BBB permeability while slowing glioma growth are warranted, so as to improve upon current bleak malignant glioma treatment options.

OKN-007 Alters Protein Expression Profiles in High-Grade Gliomas: Mass Spectral Analysis of Blood Sera

Current therapies for high-grade gliomas, particularly glioblastomas (GBM), do not extend patient survival beyond 16–22 months. OKN-007 (OKlahoma Nitrone 007), which is currently in phase II (multi-institutional) clinical trials for GBM patients, and has demonstrated efficacy in several rodent and human xenograft glioma models, shows some promise as an anti-glioma therapeutic, as it affects most aspects of tumorigenesis (tumor cell proliferation, angiogenesis, migration, and apoptosis). Combined with the chemotherapeutic agent temozolomide (TMZ), OKN-007 is even more effective by affecting chemo-resistant tumor cells. In this study, mass spectrometry (MS) methodology ESI-MS, mass peak analysis (Leave One Out Cross Validation (LOOCV) and tandem MS peptide sequence analyses), and bioinformatics analyses (Ingenuity® Pathway Analysis (IPA®)), were used to identify up- or down-regulated proteins in the blood sera of F98 glioma-bearing rats, that were either untreated or treated with OKN-007. Proteins of interest identified by tandem MS-MS that were decreased in sera from tumor-bearing rats that were either OKN-007-treated or untreated included ABCA2, ATP5B, CNTN2, ITGA3, KMT2D, MYCBP2, NOTCH3, and VCAN. Conversely, proteins of interest in tumor-bearing rats that were elevated following OKN-007 treatment included ABCA6, ADAMTS18, VWA8, MACF1, and LAMA5. These findings, in general, support our previous gene analysis, indicating that OKN-007 may be effective against the ECM. These findings also surmise that OKN-007 may be more effective against oligodendrogliomas, other brain tumors such as medulloblastoma, and possibly other types of cancers.

Lentivirus vector‑mediated genetic manipulation of oncogenic pathways induces tumor formation in rabbit brain.

Translation of promising experimental therapies from rodent models to clinical success has been complicated as the novel therapies often fail in clinical trials. Existing rodent glioma models generally do not allow for preclinical evaluation of the efficiency of novel therapies in combination with surgical resection. Therefore, the aim of the present study was to develop a larger animal model utilizing lentivirus vector-mediated oncogenic transformation in the rabbit brain. Lentiviruses carrying constitutively active AKT and H-Ras oncogenes, and p53 small interfering (si)RNA were introduced into newborn rabbit neural stem cells (NSCs) and intracranially implanted into rabbits' brains to initiate tumor formation. In one of the ten rabbits a tumor was detected 48 days after the implantation of transduced NSCs. Histological features of the tumor mimic was similar to a benign Grade II ganglioglioma. Immunostaining demonstrated that the tissues were positive for AKT and H-Ras. Strong expression of GFAP and Ki-67 was also detected. Additionally, p53 expression was notably lower in the tumor area. The implantation of AKT, H-Ras and p53 siRNA transduced NSCs for tumor induction resulted in ganglioglioma formation. Despite the low frequency of tumor formation, this preliminary data provided a proof of principle that lentivirus vectors carrying oncogenes can be used for the generation of brain tumors in rabbits. Moreover, these results offer noteworthy insights into the pathogenesis of a rare brain tumor, ganglioglioma.

Monitoring Early Changes in Tumor Metabolism in Response to Therapy Using Hyperpolarized 13C MRSI in a Preclinical Model of Glioma.

This study shows the use of hyperpolarized 13C magnetic resonance spectroscopic imaging (MRSI) to assess therapeutic efficacy in a preclinical tumor model. 13C-labeled pyruvate was used to monitor early changes in tumor metabolism based on the Warburg effect. High-grade malignant tumors exhibit increased glycolytic activity and lactate production to promote proliferation. A rodent glioma model was used to explore altered lactate production after therapy as an early imaging biomarker for therapeutic response. Rodents were surgically implanted with C6 glioma cells and separated into 4 groups, namely, no therapy, radiotherapy, chemotherapy and combined therapy. Animals were imaged serially at 6 different time points with magnetic resonance imaging at 3 T using hyperpolarized [1-13C]pyruvate MRSI and conventional 1H imaging. Using hyperpolarized [1-13C]pyruvate MRSI, alterations in tumor metabolism were detected as changes in the conversion of lactate to pyruvate (measured as Lac/Pyr ratio) and compared with the conventional method of detecting therapeutic response using the Response Evaluation Criteria in Solid Tumors. Moreover, each therapy group expressed different characteristic changes in tumor metabolism. The group that received no therapy showed a gradual increase of Lac/Pyr ratio within the tumor. The radiotherapy group showed large variations in tumor Lac/Pyr ratio. The chemo- and combined-therapy groups showed a statistically significant reduction in tumor Lac/Pyr ratio; however, only combined therapy was capable of suppressing tumor growth, which resulted in low endpoint mortality rate. Hyperpolarized 13C MRSI detected a prompt reduction in Lac/Pyr ratio as early as 2 days post combined chemo- and radiotherapies.

Therapeutic Targeting of TGFβ Ligands in Glioblastoma Using Novel Antisense Oligonucleotides Reduces the Growth of Experimental Gliomas.

Transforming growth factor (TGF)-β is expressed at high levels by glioma cells and contributes to the malignant phenotype of glioblastoma. However, its therapeutic targeting remains challenging. Here, we examined an alternative therapeutic approach of TGFβ inhibition using two novel phosphorothioate-locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, ISTH1047 and ISTH0047, which specifically target TGFβ1 and TGFβ2. We characterized the effects of ISTH1047 and ISTH0047 on TGFβ1/2 expression, downstream signaling and growth of human LN-308, LN-229, and ZH-161 cells as well as murine SMA-560 glioma cells in vitro. Furthermore, we assessed their target inhibition and effects on survival in orthotopic xenogeneic and syngeneic rodent glioma models in vivo. Both antisense oligonucleotides specifically silenced their corresponding target and abrogated SMAD2 phosphorylation in several glioma cell lines. Moreover, inhibition of TGFβ1 or TGFβ2 expression by ISTH1047 or ISTH0047 reduced the migration and invasiveness of LN-308 and SMA-560 glioma cells. Systemic antisense oligonucleotide administration to glioma-bearing mice suppressed TGFβ1 or TGFβ2 mRNA expression as well as the expression of the downstream target PAI-1 in orthotopic gliomas. Glioma-bearing mice had significantly prolonged survival upon systemic treatment with ISTH1047 or ISTH0047, which was associated with a reduction of intratumoral SMAD2 phosphorylation and, in a fully immunocompetent model, with increased immune cell infiltration. Targeting TGFβ expression with the novel LNA antisense oligonucleotides ISTH1047 or ISTH0047 results in strong antiglioma activity in vitro and in vivo, which may represent a promising approach to be examined in human patients with glioma.

SCIDOT-21. IMPROVING DRUG DELIVERY TO GLIOBLASTOMA BY TARGETING CANONICAL WNT/β-CATENIN SIGNALING IN THE BLOOD-BRAIN BARRIER

Abstract BACKGROUND Glioblastoma (GBM) patient survival and therapy response is greatly hindered by the presence of invasive glioma stem cells (GSC) and the blood-brain barrier (BBB) which limits effective drug delivery. WNT/beta-catenin signaling is important in the development and maintenance of the BBB by mediating transcription of growth factors, multidrug resistance proteins, and junctional proteins. In WNT-subtype medulloblastoma, activating mutations of beta-catenin lead to reciprocal secretion of WNT antagonists such as WIF1 and DKK1 into the tumor microenvironment. These WNT antagonists can act upon the surrounding endothelium and induce a leaky BBB. Therefore, we hypothesize that pharmacological inhibition of WNT/beta-catenin signaling in brain endothelial cells will decrease BBB integrity, enabling enhanced paracellular drug delivery to infiltrative GSCs. METHODS We recapitulated the WNT-medulloblastoma phenotype in GBM by activating WNT/beta-catenin signaling in primary human GSCs, inducing secretion of downstream WNT antagonists. Conditioned-media (CM) from GSCs was then applied to human brain microvascular endothelial cells (HBMEC) to indirectly inhibit WNT signaling. Additionally, we directly inhibited WNT/beta-catenin signaling in HBMECs with the small molecule inhibitor ICG-001. Endothelial cell-cell interaction was measured by electrical impedance using the ACEA xCELLigence system. Fenestration and junctional expression were evaluated by immunoblotting and immunofluorescence. RESULTS ICG-001 or WNT-GSC-CM, but not control GSC-CM, upregulated fenestration related protein, PLVAP, and downregulated junctional proteins claudin-5, ZO-1, and VE-Cadherin in HBMECs. Endothelial cell-cell interaction was transiently decreased by ICG-001 or WNT-GSC-CM. Pre-clinical studies are underway to evaluate the functional impact of WNT/beta-catenin inhibition on BBB integrity and permeability in rodent glioma models. Altogether, these results support targeting WNT/beta-catenin signaling in brain endothelial cells to enhance drug delivery to CNS tumors. CONCLUSION Modulation of intratumoral Wnt/beta-catenin signaling, particularly in highly resistant GSCs, may enhance chemotherapy drug delivery, potentially expanding the drug portfolio and improving the prognosis of GBM.

Progress in Confocal Laser Endomicroscopy for Neurosurgery and Technical Nuances for Brain Tumor Imaging With Fluorescein.

Background: Previous studies showed that confocal laser endomicroscopy (CLE) images of brain tumors acquired by a first-generation (Gen1) CLE system using fluorescein sodium (FNa) contrast yielded a diagnostic accuracy similar to frozen surgical sections and histologic analysis. We investigated performance improvements of a second-generation (Gen2) CLE system designed specifically for neurosurgical use.Methods: Rodent glioma models were used for in vivo and rapid ex vivo CLE imaging. FNa and 5-aminolevulinic acid were used as contrast agents. Gen1 and Gen2 CLE images were compared to distinguish cytoarchitectural features of tumor mass and margin and surrounding and normal brain regions. We assessed imaging parameters (gain, laser power, brightness, scanning speed, imaging depth, and Z-stack [3D image acquisition]) and evaluated optimal values for better neurosurgical imaging performance with Gen2.Results: Efficacy of Gen1 and Gen2 was similar in identifying normal brain tissue, vasculature, and tumor cells in masses or at margins. Gen2 had smaller field of view, but higher image resolution, and sharper, clearer images. Other advantages of the Gen2 were auto-brightness correction, user interface, image metadata handling, and image transfer. CLE imaging with FNa allowed identification of nuclear and cytoplasmic contours in tumor cells. Injection of higher dosages of FNa (20 and 40 mg/kg vs. 0.1–8 mg/kg) resulted in better image clarity and structural identification. When used with 5-aminolevulinic acid, CLE was not able to detect individual glioma cells labeled with protoporphyrin IX, but overall fluorescence intensity was higher (p < 0.01) than in the normal hemisphere. Gen2 Z-stack imaging allowed a unique 3D image volume presentation through the focal depth.Conclusion: Compared with Gen1, advantages of Gen2 CLE included a more responsive and intuitive user interface, collection of metadata with each image, automatic Z-stack imaging, sharper images, and a sterile sheath. Shortcomings of Gen2 were a slightly slower maximal imaging speed and smaller field of view. Optimal Gen2 imaging parameters to visualize brain tumor cytoarchitecture with FNa as a fluorescent contrast were defined to aid further neurosurgical clinical in vivo and rapid ex vivo use. Further validation of the Gen2 CLE for microscopic visualization and diagnosis of brain tumors is ongoing.

Tumor growth under rhGM-CSF application in an orthotopic rodent glioma model.

Regulation of the host immune response serves a pivotal role in the persistence and progression of malignant glioma. To date, cytotoxic cluster of differentiation (CD)-8+ T and natural killer cells are considered the main cellular components of host tumor control. The influence of macrophages in an orthotropic C6 tumor implantation model was investigated and the aim of the present study was to characterize the effects of systemic macrophage-activation on glioma growth by using the granulocyte macrophage colony stimulating factor (rhGM-CSF). A total of 20 male Sprague-Dawley rats were orthotopically implanted with C6 glioma spheroids and treated subcutaneously with 10 µg/kg rhGM-CSF every other day; 9 animals served as controls. Serial magnetic resonance imaging was performed on days 7, 14, 21, 28, 32 and 42 post-implantation to monitor tumor volume. Histological work-up included hematoxylin and eosin, CD68/ED-1 macrophage, CD8 T-cell and Ki-67 MIB1 proliferation staining in gliomas and spleen. Experimental C6-gliomas developed in 15/20 (75%) animals. In rhGM-CSF treated rats, tumors developed significantly later and reached a smaller size (median, 134 mm3) compared with the controls (median, 262 mm3). On day 14, solid tumors presented in 11/17 (65%) rhGM-CSF-treated animals; in control animals tumor growth was detected in 3/9 animals on day 7 and in all animals on day 14. The mean survival time was 35 days in the rhGM-CSF group and significantly longer when compared with the control group (24 days). Immunohistochemistry exhibited significantly more macrophages in tumors, particularly in the perivascular zone of the rhGM-CSF group when compared with untreated animals; intratumoral CD8+ counts were equal in both groups. A systemic stimulation of macrophages by rhGM-CSF resulted in significantly reduced and delayed tumor growth in the rodent C6 glioma model. The present data suggested a significant role of macrophages in host control of experimental gliomas on the innate immune response. Until now, the role of macrophages may have been underestimated in host glioma control.

Phase I study of procaspase activating compound-1 (PAC-1) for treatment of advanced malignancies.

TPS2621 Background: Dysregulation in apoptotic pathways is a hallmark feature of cancer, offering opportunities for pharmacologic intervention in its treatment. Caspases, a family of proteases, play several roles in apoptosis. Caspase-3 catalyzes the intra-cellular proteolysis that characterizes part of the apoptotic process. Caspase-3 levels are low in many tumors including: glioblastoma; breast, colon, lung, and liver cancers: lymphoma: neuroblastoma; and melanoma. In contrast, procaspase-3, the precursor of caspase-3 is elevated in these tumors. We describe a phase I study of PAC-1, a drug that catalyzes the conversion of procaspase -3 to caspase-3. It induces apoptosis in tumor cell lines in vitro. In vivo activity has also been shown, when combined with alkylating chemotherapy, in rodent glioma models and in canines diagnosed with malignant glioma. Methods: This Phase I dose escalation study has two components: the first (C1) to determine the maximum tolerated dose (MTD) of PAC-1 in advanced malignancies; the second (C2) to determine the MTD of PAC-1 when combined with temozolomide (TMZ) in patients with recurrent anaplastic astrocytoma (AA) or glioblastoma (GBM). A modified Fibonacci 3 + 3 design is used, expanding to nine subjects at the MTD level in each component. PAC-1 pharmacokinetics is assessed in all subjects during the first cycle. Secondary objectives include pharmacodynamics and correlations of PAC-1 activity with procaspase-3 expression in tumor tissue. Neurologic toxicity is closely monitored throughout the study. Inclusion criteria: diagnoses of advanced malignancies (C1) and recurrent AA or GBM (C2), ECOG PS 0-2, adequate organ function. Exclusion criteria: prior cytotoxic therapy in the last 3-6 weeks (varying with drug class) or uncontrolled chronic illness. Administration and design: For C1, PAC-1 (orally administered) is dosed at 75-1,000 mg daily on days 1-21 of each 28 day cycle. For C 2, the first PAC-1 dose is 375 mg daily with potential for escalation to 1,000 mg daily. TMZ, PO, is dosed at 150 mg/m2 daily, days 8-12, of each cycle. Enrollment to date for C1 is 27, (dose level 6); and for C2 is 4 (dose level 1). The study is open to accrual. Clinical trial information: NCT02355535.

Synergistic and targeted therapy with a procaspase-3 activator and temozolomide extends survival in glioma rodent models and is feasible for the treatment of canine malignant glioma patients.

PurposeGlioblastoma is a deadly brain cancer with a median survival time of ∼15 months. Ionizing radiation plus the DNA alkylator temozolomide (TMZ) is the current standard therapy. PAC-1, a procaspase-3 activating small molecule, is blood-brain barrier penetrant and has previously demonstrated ability to synergize with diverse pro-apoptotic chemotherapeutics. We studied if PAC-1 could enhance the activity of TMZ, and whether addition of PAC-1 to standard treatment would be feasible in spontaneous canine malignant gliomas.Experimental DesignUsing cell lines and online gene expression data, we identified procaspase-3 as a potential molecular target for most glioblastomas. We investigated PAC-1 as a single agent and in combination with TMZ against glioma cells in culture and in orthotopic rodent models of glioma. Three dogs with spontaneous gliomas were treated with an analogous human glioblastoma treatment protocol, with concurrent PAC-1.ResultsProcaspase-3 is expressed in gliomas, with higher gene expression correlating with increased tumor grade and decreased prognosis. PAC-1 is cytotoxic to glioma cells in culture and active in orthotopic rodent glioma models. PAC-1 added to TMZ treatments in cell culture increases apoptotic death, and the combination significantly increases survival in orthotopic glioma models. Addition of PAC-1 to TMZ and radiation was well-tolerated in 3 out of 3 pet dogs with spontaneous glioma, and partial to complete tumor reductions were observed.ConclusionsProcaspase-3 is a clinically relevant target for treatment of glioblastoma. Synergistic activity of PAC-1/TMZ in rodent models and the demonstration of feasibility of the combined regime in canine patients suggest potential for PAC-1 in the treatment of glioblastoma.

Myeloid cells expressing high level of CD45 are associated with a distinct activated phenotype in glioma.

Glioblastoma multiforme is characterized by high accumulation of microglia/macrophages. The function of these tumor-infiltrating myeloid cells is not sufficiently elucidated. Therefore, a better understanding of the precise immune cell composition and function in brain tumors is required. In rodent glioma models, two different myeloid cell populations exist, determined by the expression level of CD45, namely CD11b+CD45low and CD11b+CD45high. Previous analyses of cytokine and marker expression profiles were almost exclusively performed on the entire myeloid cell fraction. Consequently, described pro- and anti-tumoral characteristics were not assigned to the evident subpopulations. In the present study, we used a syngeneic glioblastoma mouse model and subsequent flow cytometric analyses to demonstrate the distinct properties of CD11b+CD45high and the CD11b+CD45low cells. First, the majority of CD11b+CD45high cells expressed high level of GR1 and around 6% of IL10 representing in part features of myeloid-derived suppressor cells, while the CD11b+CD45low fraction displayed no upregulation of these molecules. Second, we detected that specifically the CD11b+CD45high population showed antigen-presenting, co-stimulatory, and inflammatory features. Here, we identified up to 80% of MHCII and approximately 50% of CD86 and TNFα-expressing cells. Investigation of MHCI and CD80 revealed a moderate upregulation. By contrast, in the CD11b+CD45low cell fraction, merely MHCII and TNFα were marginally overexpressed. In summary, these data emphasize the specific phenotype of CD11b+CD45high cells in glioma with suppressive as well as pro-inflammatory characteristics whereas the CD11b+CD45low cells were almost unaffected. Hence, primarily, the subpopulation consisting of CD45high-expressing cells is activated by the tumor and should be considered as therapeutic target.

A reproducible model of intramedullary spinal cord tumor in rats bearing RG2 cells.

Intramedullary spinal cord tumors (IMSCTs) are lethal diseases to many patients. The lack of adequate animal model has hampered the development of novel treatments. In the current study, a rodent intramedullary glioma model is established to study IMSCT progression. Fischer 344 rats received a intramedullary implantation of RG2 glioma cells. The neurological state of each rat was evaluated on daily basis using the Basso, Beattie and Bresnahan (BBB) scale. Rats implanted with RG2 cells developed significant hind limb paraplegia 20 days after implantation. Magnetic resonance imaging (MRI) scans after three weeks revealed significant intramedullary RG2 tumors in the rats. Forty days post implantation, rats were sacrificed for histopathological examination. Neuro-imaging and HE staining cross sections confirmed intramedullary RG2 glioma cells invading to the spinal cord. Thus, our model displayed many of the same invasive characteristics as human IMSCTs. This model should be a reliable and reproducible methodology to correlate well with the features of human IMSCT.

Hypoxia Imaging With PET Correlates With Antitumor Activity of the Hypoxia-Activated Prodrug Evofosfamide (TH-302) in Rodent Glioma Models.

High-grade gliomas are often characterized by hypoxia, which is associated with both poor long-term prognosis and therapy resistance. The adverse role hypoxia plays in treatment resistance and disease progression has led to the development of hypoxia imaging methods and hypoxia-targeted treatments. Here, we determined the tumor hypoxia and vascular perfusion characteristics of 2 rat orthotopic glioma models using 18-fluoromisonidozole positron emission tomography. In addition, we determined tumor response to the hypoxia-activated prodrug evofosfamide (TH-302) in these rat glioma models. C6 tumors exhibited more hypoxia and were less perfused than 9L tumors. On the basis of these differences in their tumor hypoxic burden, treatment with evofosfamide resulted in 4- and 2-fold decreases in tumor growth rates of C6 and 9L tumors, respectively. This work shows that imaging methods sensitive to tumor hypoxia and perfusion are able to predict response to hypoxia-targeted agents. This has implications for improved patient selection, particularly in clinical trials, for treatment with hypoxia-activated cytotoxic prodrugs, such as evofosfamide.