Rat Glioma Model Research Articles

Biomineral Nano-Theranostic agent for Magnetic Resonance Image Guided, Augmented Radiofrequency Ablation of Liver Tumor

Theranostic nanoparticles based on biocompatible mineral compositions can significantly improve the translational potential of image guided cancer nano-therapy. Here, we report development of a single-phase calcium phosphate biomineral nanoparticle (nCP) with dual-mode magnetic resonance contrast (T1-T2) together with radiofrequency (RF) mediated thermal response suitable for image-guided RF ablation of cancer. The nanoparticles (NP) are engineered to provide dual MR contrast by an optimized doping concentration (4.1 at%) of paramagnetic ion, Fe3+, which also renders lossy dielectric character for nCP leading to thermal response under RF exposure. In vivo compatibility and dual-mode MR contrast are demonstrated in healthy rat models. MRI and T2 mapping suggest hepatobiliary clearance by ~96 hours. MRI guided intratumoral injection in subcutaneous rat glioma and orthotopic liver tumor models provide clear visualization of NP in MRI which also helps in quantifying NP distribution within tumor. Furthermore, by utilising RF mediated thermal response, NP treated tumor could be ablated using clinically approved RF ablation system (10 W,13.3 GHz). Real-time in vivo thermal imaging exhibits 119 ± 10% increase in temperature change (ΔT) for NP treated orthotopic liver tumor (ΔT = 51.5 ± 2 °C), compared to untreated healthy liver control (ΔT = 21.5 ± 2 °C). In effect, we demonstrate a promising nano-biomineral theranostic agent for dual-mode MRI combined with radiofrequency ablation of solid tumors.

PET Imaging with S-[11C]Methyl-L-Cysteine and L-[Methyl-11C]Methionine in Rat Models of Glioma, Glioma Radiotherapy, and Neuroinflammation

PurposeS-[11C]-methyl-L-cysteine ([11C]MCYS) has been claimed to offer higher tumor selectivity than L-[methyl-11C]methionine ([11C]MET). We examined this claim in animal models.ProceduresRats with implanted untreated (n = 10) or irradiated (n = 7, 1 × 25 Gy, on day 8) orthotopic gliomas were scanned after 6, 9, and 12 days, using positron emission tomography. Rats with striatal injections of saline (n = 9) or bacterial lipopolysaccharide (n = 9) were scanned after 3 days.ResultsUptake of the two tracers in untreated gliomas was similar. [11C]MCYS was not accumulated in salivary glands, nasal epithelium, and healing wounds, in contrast to [11C]MET, but showed 40 % higher accumulation in the healthy brain. Both tracers showed a reduced tumor uptake 4 days after irradiation and minor accumulation in inflamed striatum. [11C]MCYS indicated higher lesion volumes than [11C]MET (untreated tumor + 47 %; irradiated tumor up to + 500 %; LPS-inflamed striatum + 240 %).Conclusions[11C]MCYS was less accumulated in some non-tumor tissues than [11C]MET, but showed lower tumor-to-brain contrast.

Direct saturation-corrected chemical exchange saturation transfer MRI of glioma: Simplified decoupling of amide proton transfer and nuclear overhauser effect contrasts.

Chemical exchange saturation transfer (CEST) MRI has shown promise in tissue characterization in diseases like stroke and tumor. However, in vivo CEST imaging such as amide proton transfer (APT) MRI is challenging because of concomitant factors such as direct water saturation, macromolecular magnetization transfer, and nuclear overhauser effect (NOE), which lead to a complex contrast in the commonly used asymmetry analysis (MTRasym). Here, we propose a direct saturation-corrected CEST (DISC-CEST) analysis for simplified decoupling and quantification of in vivo CEST effects. CEST MRI and relaxation measurements were carried out on a classical 2-pool creatine-gel CEST phantom and normal rat brains (N = 6) and a rat model of glioma (N = 8) at 4.7T. The proposed DISC-CEST quantification was carried out and compared with conventional MTRasym and the original three-offset method. We demonstrated that the DISC-CEST contrast in the phantom had much stronger correlation with MTRasym than the three-offset method, which showed substantial underestimation. In normal rat brains, the DISC-CEST approach revealed significantly stronger APT effect in gray matter and higher NOE effect in white matter. Furthermore, the APT and NOE maps derived from DISC-CEST showed significantly higher APT effect in the tumors than contralateral normal tissue but no apparent difference in NOE. The proposed DISC-CEST method, by correction of nonlinear direct water saturation effect, serves as a promising alternative to both the commonly used MTRasym and the simplistic three-offset analyses. It provides simple yet reliable in vivo CEST quantification such as APT and NOE mapping in brain tumor, which is promising for clinical translation. Magn Reson Med 78:2307-2314, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Neurosurgical microscopic solid laser-based light inhibits photobleaching during fluorescence-guided brain tumor removal with 5-aminolevulinic acid

BackgroundFluorescence image guided surgery (FIGS) with 5-aminolevulinic acid for malignant gliomas improves surgical outcome. One of the problems during FIGS is photobleaching under surgical microscopic white light. A solid laser-based white light source for neurosurgery that we developed does not include light with a wavelength of around 405nm, which is strongly absorbed by protoporphyrin IX. In the present study, we examined the efficacy of this light source to prevent the photobleaching of protoporphyrin IX-induced fluorescence. MethodsFilter papers transfused with protoporphyrin IX solution and a coronally sectioned F98 glioma rat model pretreated with 50mg/kg 5-aminolevulinic acid were continuously exposed to white light. One group was exposed to conventional xenon-based white light and another group was exposed to laser-based white light. Fluorescence at a wavelength of 635nm was measured with a radiospectrometer (in vitro study) and the relative fluorescence brightness was also measured in digital images (in vivo study) under excitation from violet blue light emitted from diodes every 5min. Results and conclusionEstimated time for 50% photobleaching was prolonged about two times in the laser-based white light exposure group compared with that in the xenon-based white light exposure group (9.1/18.7min). In the brain tumor rat model, it was also prolonged about 2.7 times (15.1/40.7min). A laser-based white light source may inhibit photobleaching during FIGS for malignant gliomas. This light source for neurosurgical microscopy has the potential to prolong the prognosis of malignant glioma patients.

Effect of Rat C6 Glioma Cells in Varying Concentrations of Cultured in Isorhamnetin

Objective: To investigate the effects of different concentrations of isorhamnetin on C6 rat glioma cells in vitro from January 2015 to June 2015. Methods: The blank control group, blank solvent control group and four concentration groups were used to observe the cell growth status under a microscope. MTT colorimetric assay was used to detect the effect of isorhamnetin on C6 glioma cells in vitro and the cell inhibition rate And survival rate were measured. The apoptotic and apoptotic rates were measured by flow cytometry in the treatment group and the control group. The relationship between the different concentrations of isorhamnetin and C6 glioma cell apoptosis was analyzed the total protein was extracted and the total AKT protein and Ser473 AKT protein content were detected by Western blotting. The rat model of glioma was constructed by SD rats.Five days of isorhamnetin was continuously fed and the plasma was detected by high-performance liquid chromatography,liver, brain tissue isorhamnetin content.Â

T11TS immunotherapy repairs PI3K-AKT signaling in T-cells: Clues toward enhanced T-cell survival in rat glioma model.

Malignant glioma is the most fatal of astrocytic lineage tumors despite therapeutic advances. Onset and progression of gliomas is accompanied by severe debilitation of T-cell defense and T-cell survival. One of the chief contributors to T-cell survival downstream of activation is the PI3K-AKT pathway. Our prior studies showed that the novel immunotherapeutic molecule T11-target structure (T11TS) blocks T-cell apoptosis in glioma. We also showed activation of immunological synapse components and calcineurin-NFAT pathway following T11TS immunotherapy of glioma-bearing rats. This lead to investigations whether such T-cell activation upon T11TS therapy translates into activation of downstream PI3K/AKT signals which may be related to observed blockade of T-cell apoptosis. For the purpose, we assessed by flowcytometry and immunoblotting, expressions of PI3K, PDK1, AKT, p-AKT, and PTEN in splenic T-cells of normal, experimentally-induced glioma-bearing rats and glioma-bearing rats receiving first, second and third doses of T11TS. We also determined comparative nuclear translocation of NF-κB across groups. We found significant increases in T-cell expressions of PDK1, PI3K, and p-AKT in T11TS-treated animal groups compared to sharp downregulations in glioma. AKT levels remained unchanged across groups. PTEN levels declined sharply after T11TS immunotherapy. T11TS also caused enhanced NF-κB translocation to the T-cell nucleus compared to glioma group. Results showed heightened activation of the PI3K-AKT pathway in glioma-bearing rats following T11TS immunotherapy. These results illustrate the novel role of T11TS immunotherapy in ameliorating the PI3K pathway in T-cells in glioma-bearing animals to enhance T-cell survival, according greater defense against glioma. The study thus has far-reaching clinical outcomes.

Abstract 4067: BPM31510, a clinical stage metabolic modulator demonstrates therapeutic efficacy in an in vivo C6 rat glioma model and synergizes with temozolomide

Abstract BPM31510, a parenteral nanodispersion of ubidecarenone that is in clinical testing for solid tumors, effectuates an anti-cancer effect in highly metabolic cancers by eliciting an anti-Warburg effect. Intraperitoneal (i.p.) administration of BPM31510 reverses paraplegia in a rat CNS leukemia model demonstrating the bioavailability of the drug to the central nervous system. The study describes data demonstrating the therapeutic utility of BPM31510 in glioblastoma multiforme (GBM). BPM31510 decreased cell proliferation rates in patient derived GBM cell lines compared to temozolamide (TMZ), pretreatment with BPM31510 followed by TMZ challenge was associated with significant synergy in reducing cell proliferation compared to monotherapy. In a C6 glioma allograft rat model treatment with BPM31510 alone (n=30/group) (i.p., 50mg/kg, b.i.d) increased overall survival compared to untreated control group. BPM31510 in combination with procarbazine (oral, 60mg/kg; d 8, 21) and vincristine (IV, 1.4mg/kg; d 8, 29) significantly improved overall survival compared to BPM31510 alone or chemotherapy. Furthermore, long term survival was achieved in four of twenty rats with C6 gliomas receiving BPM31510 doses between 10 and 50 mg/kg compared with none treated with either vehicle or irradiation (P < 0.02, Fisher exact test). Interestingly, the long term survival persisted for over two months after all treatment was discontinued; MR imaging confirmed the presence of static masses in these long term survivors. Collectively, the data demonstrate that BPM31510 has a robust effect in a preclinical model of GBM. Intravenous BPM31510 is currently in Phase 1/2 clinical trial for TMZ+BEV refractory GBM patients. Citation Format: Stephane Gesta, Semma Nagpal, Tulin Dadali, Milton Merchant, Taichang Jan, Anne R. Diers, Michael Kiebish, Joaquin Jiminez, Vivek K. Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan, Lawrence Recht. BPM31510, a clinical stage metabolic modulator demonstrates therapeutic efficacy in an in vivo C6 rat glioma model and synergizes with temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4067. doi:10.1158/1538-7445.AM2017-4067

Abstract 1869: Improved image-guided surgical resection of glioblastoma with [18F]-fluoroethyltyrosine Cerenkov luminescence imaging

Abstract The ability of a surgeon to completely resect a tumor is directly related to outcome in high- and low-grade gliomas. However current intraoperative navigational tools are useful only in a subset of glioma patients and do not detect the full extent of disease. We modelled orthotopic brain tumors by stereotactically implanting human U87, rat F98 and C6 glioblastoma cells into the forebrains of rats. We show here that a new hybrid PET-Cerenkov luminescence imaging approach using O-(2-[18F]fluoroethyl)-L-tyrosine (FET) can accurately delineate tumor margins pre- and intra-operatively. We demonstrate consistency in the successful localisation and quantification of tumor burden using PET and Cerenkov luminescence imaging. The Cerenkov signal in individual tumors was directly proportional to the signal detected in corresponding FET PET scans (y = 1.06x - 0.01; R2 = 0.98; p < 0.0001) and subsequent autoradiography indicated equivalence between modalities. Cerenkov luminescence was better able to discriminate tumor from healthy brain tissue than the current ‘gold standard’ for intraoperative mapping, 5-ALA (5-aminolevulinic acid), indicated by a greater area under the ROC curve in human xenograft (0.968 ± 0.003 vs. 0.893 ± 0.019; p = 0.003) and syngeneic rat glioma models (0.970 ± 0.010 vs. 0.774 ± 0.046; p = 0.006). The quantitative accuracy of Cerenkov luminescence enabled us to determine a threshold of 2.1 x 102 p-1 sec-1 cm-2 sr-1 MBq-1 that separated U87 human glioblastoma and normal brain tissue, more precisely guiding tumor excision than 5-ALA. At the optimal threshold the specificity for detecting syngeneic rat and human xenograft gliomas was better with FET Cerenkov than 5-ALA (91.1 ± 2.7 % vs. 67.2 ± 5.2 %; p = 0.007 and 91.2 ± 1.0 % vs. 82.9 ± 1.8 %; p = 0.005 respectively). We confirmed and extended these findings at higher spatial resolution on cryosections using FET autoradiography and 5-ALA confocal microscopy, with FET demonstrating higher specificity and sensitivity for tumor detection than 5-ALA. FET PET-Cerenkov luminescence imaging has the potential for guiding resections in a much broader range of glioma patients than current approaches. Previous clinical experience with FET offers a facile route for clinical translation of this technology. Citation Format: David Y. Lewis, Richard Mair, Alan J. Wright, Kieren Allinson, Scott K. Lyons, Tom Booth, Robert Bielik, Dmitry Soloviev, Kevin M. Brindle. Improved image-guided surgical resection of glioblastoma with [18F]-fluoroethyltyrosine Cerenkov luminescence imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1869. doi:10.1158/1538-7445.AM2017-1869

Effect of BPM31510 on radiosensitivity of temozolomide-resistant glioblastoma cell model and survival in in vivo C6 glioma rat model supporting phase I clinical investigation in GBM.

e13509 Background: Glioblastoma (GB) is characterized by dysregulated metabolism, utilizing glycolysis for energy production to support unrestricted growth. BPM 31510, an ubidecarenone containing lipid nanodispersion effectuates a switch in cancer energy sourcing from glycolysis towards mitochondrial OXPHOS, i.e. reverses Warburg effect, providing rationale for its potential utility in treatment of GB. The current study investigated utility of BPM31510 alone and in combination with temozolomide. Methods: In vitro (U251-MG human GB cell line) and in vivo (C6 glioma rat model) preclinical models of GB were used to assess the anti-cancer activity of BPM 31510 alone (100 mg/kg/d) and combination with TMZ/bevacizumab (BEV). In addition, an in vitro model of acquired TMZ chemo-resistance was established by progressive adaptation of parental U251-MG cells to increasing doses of TMZ. Parental and resistant subclones (TMZ-R) were used to define activity of BPM31510 in the TMZ-refractory setting. Results: In vitro results demonstrated that BPM 31510 has anti-cancer activity in both parental and TMZ-R U251-MG cells with EC50 values of ~400 µM and 800 µM, respectively. Importantly, BPM 31510 treatment also resensitized TMZ-R cell lines to TMZ. In vivo, BPM 31510 treatment was associated with increasing duration of survival; one fifth of the rats treated achieved survival greater than 15 days post implantation, a response not observed in the control or irradiation arms of the study. Assessment of the combination of BPM 31510 with TMZ or BEV in the in vivoC6 glioma rat model is ongoing. A phase I open-label, non-randomized clinical trial to evaluate the safety and tolerability of BPM31510 in patients with recurrent BEV-refractory GB, as well as the changes in GB metabolism by SUV-PET imaging in response to treatment is under investigation. Conclusions: Preclinical data demonstrate that BPM 31510 has potential anti-cancer activity alone and in combination with standard therapy regimens and alleviates TMZ chemo-resistance in preclinical models of GB. These results provide support of a Phase 1 clinical study of BPM31510 in GB; this study is actively enrolling.

Farnesylthiosalicylic acid-loaded lipid-polyethylene glycol-polymer hybrid nanoparticles for treatment of glioblastoma.

We aimed to develop lipid-polyethylene glycol (PEG)-polymer hybrid nanoparticles, which have high affinity to tumour tissue with active ingredient, a new generation antineoplastic drug, farnesylthiosalicylic acid (FTA) for treatment of glioblastoma. Farnesylthiosalicylic acid-loaded poly(lactic-co-glycolic acid)-1,2 distearoyl-glycerol-3-phospho-ethanolamine-N [methoxy (PEG)-2000] ammonium salt (PLGA-DSPE-PEG) with or without 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) hybrid nanoparticles has been prepared and evaluated for in-vitro characterization. Cytotoxicity of FTA-loaded nanoparticles along with its efficacy on rat glioma-2 (RG2) cells was also evaluated both invitro (in comparison with non-malignant cell line, L929) and invivo. Scanning electron microscopy studies showed that all formulations prepared had smooth surface and spherical in shape. FTA and FTA-loaded nanoparticles have cytotoxic activity against RG2 glioma cell lines in cell culture studies, which further increases with addition of DOTAP. Magnetic resonance imaging and histopathologic evaluation on RG2 tumour cells in rat glioma model (49 female Wistar rats, 250-300g) comparing intravenous and intratumoral injections of the drug have been performed and FTA-loaded nanoparticles reduced tumour size significantly in in-vivo studies, with higher efficiency of intratumoral administration than intravenous route. Farnesylthiosalicylic acid-loaded PLGA-DSPE-PEG-DOTAP hybrid nanoparticles are proven to be effective against glioblastoma in both in-vitro and in-vivo experiments.

Development and optimization of an injectable formulation of copper diethyldithiocarbamate, an active anticancer agent.

Copper diethyldithiocarbamate (Cu(DDC)2) is the active anticancer agent generated when disulfiram (DSF) is provided in the presence of copper. To date, research directed toward repurposing DSF as an anticancer drug has focused on administration of DSF and copper in combination, efforts that have proven unsuccessful in clinical trials. This is likely due to the inability to form Cu(DDC)2 at relevant concentrations in regions of tumor growth. Little effort has been directed toward the development of Cu(DDC)2 because of the inherent aqueous insolubility of the complex. Here, we describe an injectable Cu(DDC)2 formulation prepared through a method that involves synthesis of Cu(DDC)2 inside the aqueous core of liposomes. Convection-enhanced delivery of a Cu(DDC)2 formulation prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol liposomes into a rat model of F98 glioma engendered a 25% increase in median survival time relative to vehicle-treated animals. In a murine subcutaneous MV-4–11 model, treatment resulted in a 45% reduction in tumor burden when compared to controls. Pharmacokinetic studies indicated that the Cu(DDC)2 was rapidly eliminated after intravenous administration while the liposomes remained in circulation. To test whether liposomal lipid composition could increase Cu(DDC)2 circulation lifetime, a number of different formulations were evaluated. Studies demonstrated that liposomes composed of DSPC and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-(carboxy[polyethylene glycol]-2000) (95:5) enhanced Cu(DDC)2 concentrations in the circulation as reflected by a 4.2-fold increase in plasma AUC(0−∞) relative to the DSPC/cholesterol formulation. The anticancer activity of this Cu(DDC)2 formulation was subsequently evaluated in the MV-4–11 model. At its maximum tolerated dose, this formulation exhibited comparable activity to the DSPC/cholesterol formulation. This is the first report demonstrating the therapeutic effects of an injectable Cu(DDC)2 formulation in vivo.

Effects of curcumin-loaded PLGA nanoparticles on the RG2 rat glioma model

BackgroundCurcumin, the active ingredient of turmeric, has a remarkable antitumor activity against various cancers, including glioblastoma. However, it has poor absorption and low bioavailability; thus, to cross the blood–brain barrier and reach tumor tissue, it needs to be transferred to tumor site by special drug delivery systems, such as nanoparticles. ObjectiveWe aimed to evaluate the antitumor activity of curcumin on glioblastoma tissue in the rat glioma-2 (RG2) tumor model when it is loaded on poly(lactic-co-glycolic acid)-1,2-distearoyl-glycerol-3-phospho-ethanolamine-N-[methoxy (polyethylene glycol)-2000] ammonium salt (PLGA-DSPE-PEG) hybrid nanoparticles. MethodsGlioblastoma was induced in 42 adult female Wistar rats (250–300g) by RG2 tumor model. The curcumin-loaded nanoparticles were injected by intravenous (n=6) or intratumoral route (n=6). There were five control groups, each containing six rats. First control group was not applied any treatment. The remaining four control groups were given empty nanoparticles or curcumin alone by intravenous or intratumoral route, respectively. The change in tumor volume was assessed by magnetic resonance imaging and histopathology before and 5days after drug injections. ResultsTumor size decreased significantly after 5days of intratumoral injection of curcumin-loaded nanoparticle (from 66.6±44.6 to 34.9±21.7mm3, p=0.028), whereas it significantly increased in nontreated control group (from 33.9±21.3 to 123.7±41.1mm3, p=0.036) and did not significantly change in other groups (p>0.05 for all). ConclusionIn this in vivo experimental model, intratumoral administration of curcumin-loaded PLGA-DSPE-PEG hybrid nanoparticles was effective against glioblastoma. Curcumine-loaded nanoparticles may have potential application in chemotherapy of glioblastoma.

Nicotinic acid inhibits glioma invasion by facilitating Snail1 degradation

Malignant glioma is a formidable disease that commonly leads to death, mainly due to the invasion of tumor cells into neighboring tissues. Therefore, inhibition of tumor cell invasion may provide an effective therapy for malignant glioma. Here we report that nicotinic acid (NA), an essential vitamin, inhibits glioma cell invasion in vitro and in vivo. Treatment of the U251 glioma cells with NA in vitro results in reduced invasion, which is accompanied by a loss of mesenchymal phenotype and an increase in cell-cell adhesion. At the molecular level, transcription of the adherens junction protein E-cadherin is upregulated, leading to accumulation of E-cadherin protein at the cell-cell boundary. This can be attributed to NA’s ability to facilitate the ubiquitination and degradation of Snail1, a transcription factor that represses E-cadherin expression. Similarly, NA transiently inhibits neural crest migration in Xenopus embryos in a Snail1-dependent manner, indicating that the mechanism of action for NA in cell migration is evolutionarily conserved. We further show that NA injection blocks the infiltration of tumor cells into the adjacent brain tissues and improves animal survival in a rat model of glioma. These results suggest that NA treatment may be developed into a potential therapy for malignant glioma.

Glioblastoma spheroids produce infiltrative gliomas in the rat brainstem.

Diffuse intrinsic pontine glioma (DIPG) is universally fatal without proven therapy other than radiation therapy for palliation. Representative animal models will play an essential role in the preclinical stage of future therapy development. To address the shortage of representative models, we created a novel infiltrative brainstem glioma model in rats based on glioblastoma spheroids. Cells dissociated from glioblastoma spheroids grown from surgical specimens were implanted into the brainstem of NIH nude rats. Animals were serially assessed clinically and radiographically with magnetic resonance imaging (MRI). Tumors were further characterized using histology, immunohistochemistry, and cytogenetics. Tumor generation was successful in all animals receiving glioblastoma spheroid cells. The rats survived 17-25weeks before severe symptoms developed. The tumors showed as diffuse hyperintense lesions on T2-weighted images. Histologically, they demonstrated cellular heterogeneity, and infiltrative and invasive features, with cells engorging vascular structures. The tumors were shown to comprise immature human origin glial tumor cells, with human epidermal growth factor receptor (EGFR) gene amplification and gain. This study showed that cells from glioblastoma spheroids produced infiltrative gliomas in rat brainstem. The rat brainstem gliomas are radiographically and histologically accurate compared to DIPG. These tumors develop over several months that would allow sequential clinical and radiographic assessments of therapeutic interventions. This study demonstrated in principle the feasibility of developing patient-specific animal models based on putative cancer stem cells from biopsy or resection samples.

Convection enhanced delivery of panobinostat (LBH589)-loaded pluronic nano-micelles prolongs survival in the F98 rat glioma model.

BackgroundThe pan-histone deacetylase inhibitor panobinostat is a potential therapy for malignant glioma, but it is water insoluble and does not cross the blood–brain barrier when administered systemically. In this article, we describe the in vitro and in vivo efficacy of a novel water-soluble nano-micellar formulation of panobinostat designed for administration by convection enhanced delivery (CED).Materials and methodsThe in vitro efficacy of panobinostat-loaded nano-micelles against rat F98, human U87-MG and M059K glioma cells and against patient-derived glioma stem cells was measured using a cell viability assay. Nano-micelle distribution in rat brain was analyzed following acute CED using rhodamine-labeled nano-micelles, and toxicity was assayed using immunofluorescent microscopy and synaptophysin enzyme-linked immunosorbent assay. We compared the survival of the bioluminescent syngenic F98/Fischer344 rat glioblastoma model treated by acute CED of panobinostat-loaded nano-micelles with that of untreated and vehicle-only-treated controls.ResultsNano-micellar panobinostat is cytotoxic to rat and human glioma cells in vitro in a dose-dependent manner following short-time exposure to drug. Fluorescent rhodamine-labelled nano-micelles distribute with a volume of infusion/volume of distribution (Vi/Vd) ratio of four and five respectively after administration by CED. Administration was not associated with any toxicity when compared to controls. CED of panobinostat-loaded nano-micelles was associated with significantly improved survival when compared to controls (n=8 per group; log-rank test, P<0.001). One hundred percent of treated animals survived the 60-day experimental period and had tumour response on post-mortem histological examination.ConclusionCED of nano-micellar panobinostat represents a potential novel therapeutic option for malignant glioma and warrants translation into the clinic.

Acute Temporal Changes of MRI-Tracked Tumor Vascular Parameters after Combined Anti-angiogenic and Radiation Treatments in a Rat Glioma Model: Identifying Signatures of Synergism.

In this study we used magnetic resonance imaging (MRI) biomarkers to monitor the acute temporal changes in tumor vascular physiology with the aim of identifying the vascular signatures that predict response to combined anti-angiogenic and radiation treatments. Forty-three athymic rats implanted with orthotopic U-251 glioma cells were studied for approximately 21 days after implantation. Two MRI studies were performed on each animal, pre- and post-treatment, to measure tumor vascular parameters. Two animal groups received treatment comprised of Cilengitide, an anti-angiogenic agent and radiation. The first group received a subcurative regimen of Cilengitide 1 h before irradiation, while the second group received a curative regimen of Cilengitide 8 h before irradiation. Cilengitide was given as a single dose (4 mg/kg; intraperitoneal) after the pretreatment MRI study and before receiving a 20 Gy radiation dose. After irradiation, the post-treatment MRI study was performed at selected time points: 2, 4, 8 and 12 h (n = ≥5 per time point). Significant changes in vascular parameters were observed at early time points after combined treatments in both treatment groups (1 and 8 h). The temporal changes in vascular parameters in the first group (treated 1 h before exposure) resembled a previously reported pattern associated with radiation exposure alone. Conversely, in the second group (treated 8 h before exposure), all vascular parameters showed an initial response at 2-4 h postirradiation, followed by an apparent lack of response at later time points. The signature time point to define the "synergy" of Cilengitide and radiation was 4 h postirradiation. For example, 4 h after combined treatments using a 1 h separation (which followed the subcurative regimen), tumor blood flow was significantly decreased, nearly 50% below baseline (P = 0.007), whereas 4 h after combined treatments using an 8 h separation (which followed the curative regimen), tumor blood flow was only 10% less than baseline. Comparison between the first and second groups further revealed that most other vascular parameters were maximally different 4 h after combined treatments. In conclusion, the data are consistent with the assertion that the delivery of radiation at the vascular normalization time window of Cilengitide improves radiation treatment outcome. The different vascular responses after the different delivery times of combined treatments in light of the known tumor responses under similar conditions would indicate that timing has a crucial influence on treatment outcome and long-term survival. Tracking acute changes in tumor physiology after monotherapy or combined treatments appears to aid in identifying the beneficial timing for administration, and perhaps has predictive value. Therefore, judicial timing of treatments may result in optimal treatment response.

Evaluation of neovascularization patterns in an orthotopic rat glioma model with dynamic contrast-enhanced MRI

Background Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been proved useful in evaluating glioma angiogenesis, but the utility in evaluating neovascularization patterns has not been reported. Purpose To evaluate invivo real-time glioma neovascularization patterns by measuring glioma perfusion quantitatively using DCE-MRI. Material and Methods Thirty Sprague-Dawley rats were used to establish C6 orthotopic glioma model and underwent MRI and pathology detections. As MRI and pathology were performed at six time points (i.e. 4, 8, 12, 16, 20, and 24 days) post transplantation, neovascularization patterns were evaluated via DCE-MRI. Results Four neovascularization patterns were observed in glioma tissues. Sprout angiogenesis and intussusceptive microvascular growth located inside tumor, while vascular co-option and vascular mimicry were found in the tumor margin and necrotic area, respectively. Sprout angiogenesis and intussusceptive microvascular growth increased with Ktrans, Kep, and Vp inside tumor tissue. In addition, Kep and Vp were positively correlated with sprout angiogenesis and intussusceptive microvascular growth. Vascular co-option was decreased at 12 and 16 days post transplantation and correlated negatively with Ktrans and Kep detected in the glioma margin, respectively. Changes of vascular mimicry showed no significant statistical difference at the six time points. Conclusion Our results indicate that DCE-MRI can evaluate neovascularization patterns in a glioma model. Furthermore, DCE-MRI could be an imaging biomarker for guidance of antiangiogenic treatments in humans in the future.

Simultaneous Administration of Statins and Pioglitazone Limits Tumor Growth in a Rat Model of Malignant Glioma.

Statins are cholesterol reducers with considerable dose-dependent effect against glioma cells. The apoptotic effect could be increased by combining statins or by adding pioglitazone (PGZ). The last one is an anti-diabetic drug, an agonist of the peroxisome proliferator-activated receptor-gamma (PPARG). We used an animal model to test the effect of such combination in vivo and we investigated the changes on immunological processes. Thirty-three rats (F344/DuCrl) were anesthetized and glioblastoma (F98) cells were implanted stereotactically. Animals were randomized into four groups: i) control (N=9); ii) intraperitoneal injection of PGZ 10 mg/kg/day (N=8); iii) oral administration of atorvastatin (ATVS) 40 mg/kg and lovastatin (LVS) 50 mg/kg (N=8); iv) oral administration of ATVS 40 mg/kg, LVS 50 mg/kg and PGZ 5 mg/kg (N=8). Treatment was started at 3rd postoperative day and continued for 14 days. The animals were followed-up for 30 days after start of therapy. Survival time, tumor volume, proliferation rate, counts of peripheral and tumor infiltrating leukocytes were compared. No difference of survival time or incidence of neurological deficits was observed. The combination of statins with PGZ led to a significant reduction in tumor volume by approximately 40% (p<0.05), statins combination was less effective and PGZ alone did not affect tumor volume. The groups treated with statins displayed significantly lower counts of peripheral CD3+, CD4+ and CD8+ T-cells and lower tumor associated CD68-positive cells (p<0.01, in respect to controls or PGZ alone). The proliferation rate was not statistically different. No relevant toxic effects were observed. Statins and PGZ are well-tolerated in rats and produced a significant tumor reduction, while an impact on neurological deficits or survival improvement could not be demonstrated. The reduction of infiltrating macrophages by using statins and PGZ should be further studied.

Metal Ion Ornamented Ultrafast Light-Sensitive Nanogel for Potential in Vivo Cancer Therapy

A smart, ultrafast, light-responsive nanogel is a potential carrier for on-demand and immediate delivery of therapeutic agents. Here, a novel branched pentaerythritol poly(caprolactone)-b-poly(acrylic acid)-based smart, light-responsive nanogel has been fabricated by using ferric ion (Fe3+) as a cross-linker. The mentioned block copolymer has been synthesized by combining both the ring opening and atom transfer radical polymerization techniques. Branched structure of the polymer offers a minute amount (1.5 mol %) of Fe3+ sufficient for nanogel formation. The nanogel looks like a spherically shaped human brain holding the water molecule as like cerebrospinal fluid in the brain. The particle size of the nanogel has been tailored (between 30 and 450 nm) by separately varying both the molar concentration of Fe3+ and polymer chain length. The highly negative zeta potential (−46 mV) of the nanogel promotes its impressive colloidal stability and prolongs the circulation time in vivo. Nanogels securely hold the DOX molecules (maximum drug loading capacity: 26.2%). Exposure of light onto the nanogel (in the presence of lactic acid) produces immediate initiation of de-cross-linking followed by the release of DOX molecules (85.2% at 120 min). The nanogel shows significantly high uptake and acute toxicity against a cancerous cell line (C6 glioma) in vitro. Administration of the DOX-loaded nanogel on the C6 glioma rat model (in vivo) offered tremendous inhibition (∼91%) of tumor growth without any toxic side effects (confirmed by histopathology).

Inhibitory effects of lapachol on rat C6 glioma in vitro and in vivo by targeting DNA topoisomerase I and topoisomerase II.

BackgroundLapachol is a natural naphthoquinone compound that possesses extensive biological activities. The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo, as well as the potential mechanisms.MethodsThe antitumor effect of lapachol was firstly evaluated in the C6 glioma model in Wistar rats. The effects of lapachol on C6 cell proliferation, apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/ phenazinemethosulfate (PMS) assay, hoechst 33358 staining, annexin V-FITC/PI staining, and comet assay. Effects of lapachol on topoisomerase I (TOP I) and topoisomerase II (TOP II) activities were detected by TOP I and TOP II mediated supercoiled pBR322 DNA relaxation assays and molecular docking. TOP I and TOP II expression levels in C6 cells were also determined.ResultsHigh dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats (P < 0.05). It was showed that lapachol could inhibit proliferation, induce apoptosis and DNA damage of C6 cells in dose dependent manners. Lapachol could inhibit the activities of both TOP I and II. Lapachol-TOP I showed relatively stronger interaction than that of lapachol-TOP II in molecular docking study. Also, lapachol could inhibit TOP II expression levels, but not TOP I expression levels.ConclusionThese results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro, which might be related with inhibiting TOP I and TOP II activities, as well as TOP II expression.