9L Gliosarcoma Research Articles

DDEL-03. ASSESSING THE DEPTH OF DRUG PENETRATION INTO BRAIN PARENCHYMA FROM LOCAL DRUG DELIVERY SYSTEMS IN AN ORTHOTOPIC HIGH GRADE GLIOMA ALLOGRAFT USING ORBITRAP-SECONDARY ION MASS SPECTROMETRY

Abstract Local intracranial drug delivery for high-grade glioma has been developed over the last two decades with chemotherapeutic agents administered immediately adjuvant to neurosurgery to overcome poor blood brain barrier permeability. In addition to this, the incorporation of nanoparticles (NPs) into local drug delivery systems (DDS) can result in increased penetration of chemotherapeutics through brain parenchyma, transporting the drug further from the administration site than by drug molecule diffusion alone.Here we adopted two model local DDS containing NPs to compare the depth of penetration through brain parenchyma. Approach one consisted of a thermosensitive in situ assembling polymer matrix loaded with polymer-doxorubicin conjugated NPs which can line the resection cavity walls, while approach two consisted of drug-loaded (etoposide and olaparib) poly(lactic acid)-poly(ethylene glycol) polymeric NPs held within a bioadhesive pectin hydrogel and delivered via a spray device.We assessed the efficacy of both DDS at preventing recurrence of high-grade glioma in an orthotopic rat 9L gliosarcoma model following surgical resection. In addition to monitoring long-term survival, the effect of each DDS was measured histologically by H&E and by caspase-1 (inflammatory response) and ki67 (cell proliferation) immunostaining staining. Depth of drug penetration was evaluated on post-sacrificial sections using Orbitrap-Secondary Ion Mass Spectroscopy (OrbiSIMS) and fluorescent microscopy. In vivo data suggests that the delivery mechanism of the NPs affects the efficacy of the DDS, whereby long-term survival was observed in rats treated with the sprayed olaparib/etoposide NP formulation, relative to rats where olaparib/etoposide was simply pipetted into the resection cavity. OrbiSIMS confirmed the presence of doxorubicin, olaparib and etoposide in brain tissue. Our work encourages consideration of mass spectrometry modalities to complement in vivo efficacy studies, as an analytical tool to assess brain distribution of systemically administered drugs, or localised brain penetration of drugs released biomaterial-based DDS.

Towards differentiation of brain tumor from radiation necrosis using multi-parametric MRI: Preliminary results at 4.7 T using rodent models

BackgroundIt remains a clinical challenge to differentiate brain tumors from radiation-induced necrosis in the brain. Despite significant improvements, no single MRI method has been validated adequately in the clinical setting. MethodsMulti-parametric MRI (mpMRI) was performed to differentiate 9L gliosarcoma from radiation necrosis in animal models. Five types of MRI methods probed complementary information on different scales i.e., T2 (relaxation), CEST based APT (probing mobile proteins/peptides) and rNOE (mobile macromolecules), qMT (macromolecules), diffusion based ADC (cell density) and SSIFT iAUC (cell size), and perfusion based DSC (blood volume and flow). ResultsFor single MRI parameters, iAUC and ADC provide the best discrimination of radiation necrosis and brain tumor. For mpMRI, a combination of iAUC, ADC, and APT shows the best classification performance based on a two-step analysis with the Lasso and Ridge regressions. ConclusionA general mpMRI approach is introduced to choosing candidate multiple MRI methods, identifying the most effective parameters from all the mpMRI parameters, and finding the appropriate combination of chosen parameters to maximize the classification performance to differentiate tumors from radiation necrosis.

Rodent Brain Tumor Models for Studies Focusing on Boron Neutron Capture Therapy.

Rodent brain tumor models have been very useful in advancing the treatment of glioblastomas. This review focuses on the four most widely used rodent brain tumor models: the C6, 9L, and F98 rat gliomas, and the GL261 murine glioma. All of these have been used in studies relating to boron neutron capture therapy. The most important of these studies were those using the 9L gliosarcoma, which led to the clinical use of boronophenylalanine, and the F98 glioma, which has been used for the preclinical evaluation of new boron delivery agents and methods of optimizing their delivery.

Optimization of a flexible fiber-optic probe for epi-mode quantitative phase imaging.

Quantitative oblique back-illumination microscopy (qOBM) is an emerging label-free optical imaging technology that enables 3D, tomographic quantitative phase imaging (QPI) with epi-illumination in thick scattering samples. In this work, we present a robust optimization of a flexible, fiber-optic-based qOBM system. Our approach enables in silico optimization of the phase signal-to-noise-ratio over a wide parameter space and obviates the need for tedious experimental optimization which could easily miss optimal conditions. Experimental validations of the simulations are also presented and sensitivity limits for the probe are assessed. The optimized probe is light-weight (∼40g) and compact (8mm in diameter) and achieves a 2µm lateral resolution, 6µm axial resolution, and a 300µm field of view, with near video-rate operation (10Hz, limited by the camera). The phase sensitivity is <20nm for a single qOBM acquisition (at 10Hz) and a lower limit of ∼3 nm via multi-frame averaging. Finally, to demonstrate the utility of the optimized probe, we image (1) thick, fixed rat brain samples from a 9L gliosarcoma tumor model and (2) freshly excised human brain tissues from neurosurgery. Acquired qOBM images using the flexible fiber-optic probe are in excellent agreement with those from a free-space qOBM system (both in-situ), as well as with gold-standard histopathology slices (after tissue processing).

Rat and Mouse Brain Tumor Models for Experimental Neuro-Oncology Research.

Rodent brain tumor models have been useful for developing effective therapies for glioblastomas (GBMs). In this review, we first discuss the 3 most commonly used rat brain tumor models, the C6, 9L, and F98 gliomas, which are all induced by repeated injections of nitrosourea to adult rats. The C6 glioma arose in an outbred Wistar rat and its potential to evoke an alloimmune response is a serious limitation. The 9L gliosarcoma arose in a Fischer rat and is strongly immunogenic, which must be taken into consideration when using it for therapy studies. The F98 glioma may be the best of the 3 but it does not fully recapitulate human GBMs because it is weakly immunogenic. Next, we discuss a number of mouse models. The first are human patient-derived xenograft gliomas in immunodeficient mice. These have failed to reproduce the tumor-host interactions and microenvironment of human GBMs. Genetically engineered mouse models recapitulate the molecular alterations of GBMs in an immunocompetent environment and “humanized” mouse models repopulate with human immune cells. While the latter are rarely isogenic, expensive to produce, and challenging to use, they represent an important advance. The advantages and limitations of each of these brain tumor models are discussed. This information will assist investigators in selecting the most appropriate model for the specific focus of their research.

Meloxicam can Potentiate the Therapeutic Effects of Synchrotron Microbeam Radiation Therapy on High-Grade Glioma Bearing Rats.

The microbeam radiation therapy (MRT), a spatially micro-fractionated synchrotron radiotherapy, leads to better control of incurable high-grade glioma than that obtained upon homogeneous radiotherapy. We evaluated the effect of meloxicam, a non-steroidal anti-inflammatory drug (NSAID), to increase the MRT response. Survival of rats bearing intracranial 9L gliosarcoma treated with meloxicam and/or MRT (400 Gy, 50 µm-wide microbeams, 200 µm spacing) was monitored. Tumor growth was assessed on histological tissue sections and COX-2 transcriptomic expression was studied 1 to 25 days after radiotherapy. Meloxicam significantly extended the median survival of microbeam-irradiated rats (from +10.5 to +20 days). Dual treatment led to last survivors until D90 (D39 for the MRT group) and to tumor 9.5 times smaller than MRT alone. No significant modification of COX-2 expression was induced by MRT in normal and tumor tissues. The meloxicam reinforced the anti-tumor effect of MRT for glioma treatment. Although the mechanisms of interaction between meloxicam and MRT remain to be elucidated, the addition of this NSAID, easily implemented as a supplement to water for example, is a very favorable therapeutic regimen since it doubled the survival benefit compared to MRT alone.

Incorporating Clinical Imaging into the Delivery of Microbeam Radiation Therapy

Synchrotron microbeam radiation therapy is a promising pre-clinical radiation treatment modality; however, it comes with many technical challenges. This study describes the image guidance protocol used for Australia’s first long-term pre-clinical MRT treatment of rats bearing 9L gliosarcoma tumours. The protocol utilises existing infrastructure available at the Australian Synchrotron and the adjoining Monash Biomedical Imaging facility. The protocol is designed and optimised to treat small animals utilising high-resolution clinical CT for patient specific tumour identification, coupled with conventional radiography, using the recently developed SyncMRT program for image guidance. Dosimetry performed in small animal phantoms shows patient dose is comparable to standard clinical doses, with a CT associated dose of less than 1.39cGy and a planar radiograh dose of less than 0.03cGy. Experimental validation of alignment accuracy with radiographic film demonstrates end to end accuracy of less than ±0.34mm in anatomical phantoms. Histological analysis of tumour-bearing rats treated with microbeam radiation therapy verifies that tumours are targeted well within applied treatment margins. To date, this technique has been used to treat 35 tumour-bearing rats.

Label-free detection of brain tumors in a 9L gliosarcoma rat model using stimulated Raman scattering-spectroscopic optical coherence tomography.

.Significance: In neurosurgery, it is essential to differentiate between tumor and healthy brain regions to maximize tumor resection while minimizing damage to vital healthy brain tissue. However, conventional intraoperative imaging tools used to guide neurosurgery are often unable to distinguish tumor margins, particularly in infiltrative tumor regions and low-grade gliomas.Aim: The aim of this work is to assess the feasibility of a label-free molecular imaging tool called stimulated Raman scattering-spectroscopic optical coherence tomography (SRS-SOCT) to differentiate between healthy brain tissue and tumor based on (1) structural biomarkers derived from the decay rate of signals as a function of depth and (2) molecular biomarkers based on relative differences in lipid and protein composition extracted from the SRS signals.Approach: SRS-SOCT combines the molecular sensitivity of SRS (based on vibrational spectroscopy) with the spatial and spectral multiplexing capabilities of SOCT to enable fast, spatially and spectrally resolved molecular imaging. SRS-SOCT is applied to image a 9L gliosarcoma rat tumor model, a well-characterized model that recapitulates human high-grade gliomas, including high proliferative capability, high vascularization, and infiltration at the margin. Structural and biochemical signatures acquired from SRS-SOCT are extracted to identify healthy and tumor tissues.Results: Data show that SRS-SOCT provides light-scattering-based signatures that correlate with the presence of tumors, similar to conventional OCT. Further, nonlinear phase changes from the SRS interaction, as measured with SRS-SOCT, provide an additional measure to clearly separate tumor tissue from healthy brain regions. We also show that the nonlinear phase signals in SRS-SOCT provide a signal-to-noise advantage over the nonlinear amplitude signals for identifying tumors.Conclusions: SRS-SOCT can distinguish both spatial and spectral features that identify tumor regions in the 9L gliosarcoma rat model. This tool provides fast, label-free, nondestructive, and spatially resolved molecular information that, with future development, can potentially assist in identifying tumor margins in neurosurgery.

Study of the Structural and Magnetic Properties of COFE2O4 Nanoparticles for Therapeutic Agent for Cancer Therapy

The cobalt ferrite (CoFe2O4) nanoparticles were synthesized by chemical co-precipitation technique and characterized by X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), and Mössbauer spectroscopy. The CoFe2O4 nanoparticles coated with biocompatible chitosan at different concentrations to produce homogeneous suspensions were also characterized by dynamic light scattering (DLS) and hyperthermia set-up. XRD data of the sample explored the particle size near about 7 nm in as-dried condition. TEM micrograph of bare CoFe2O4 nanoparticles provided the particle size nearly too about 8 nm which is in good agreement with the XRD data. This particle size after coating with chitosan became 14 nm. EDX results of this sample confirmed its nano dimension with spinel structure. VSM results of the sample in as-dried condition showed the ferromagnetic character which has been beyond proved by Mössbauer spectroscopy. The hydrodynamic diameter (Hd) and polydispersity index (PDI) of the chitosan-coated samples also provided promising results. The samples were tested for their induction heating properties with an RF magnetic field of 20 mT and a frequency of 342 kHz. We also studied therapeutic efficiency in-vitro on 9L gliosarcoma cancer cells, which revealed &gt; 98% of mortality through hyperthermia protocol with the same RF field and frequency using chitosan-coated CoFe2O4nanoparticles.&#x0D; GUB JOURNAL OF SCIENCE AND ENGINEERING, Vol 7, Dec 2020 P 69-75

Synthesis, Radiolabeling, and Biological Evaluation of the trans-Stereoisomers of 1-Amino-3-(fluoro-18F)-4-fluorocyclopentane-1-carboxylic Acid as PET Imaging Agents.

The enantiomeric non-natural cyclic amino acids (3R,4R)-1-amino-3-fluoro-4-(fluoro-18F)cyclopentane-1-carboxylic acid and (3S,4S)-1-amino-3-fluoro-4-(fluoro-18F)cyclopentane-1-carboxylic acid ([ 18 F]5) have been prepared as a racemic mixture in 1.3% decay corrected radiochemical yield and in greater than 99% radiochemical purity. [ 18 F]5 is transported primarily via system L with some transport occurring via system ASC, as assessed in rat 9L gliosarcoma, human U87 ΔEGFR glioblastoma, and human DU145 androgen-independent prostate carcinoma tumor cells. In rats bearing intracranial 9L gliosarcoma, [ 18 F]5 gave tumor to contralateral brain tissue ratios of up to 2.8. Biodistribution studies in healthy rats demonstrated that bladder accumulation is delayed until 10 min postinjection.

Synthesis, Radiolabeling, and Biological Evaluation of the cis Stereoisomers of 1-Amino-3-Fluoro-4-(fluoro-18F)Cyclopentane-1-Carboxylic Acid as PET Imaging Agents.

The non-natural cyclic amino acids (1S,3R,4S)-1-amino-3-fluoro-4-(fluoro-18F)cyclopentane-1-carboxylic acid ([18F]9) and (1S,3S,4R)-1-amino-3-fluoro-4-(fluoro-18F)cyclopentane-1-carboxylic acid ([18F]28) have been prepared in 10 and 1.7% decay corrected radiochemical yield, respectively, and in greater than 99% radiochemical purity. Cell assays in rat 9L gliosarcoma, human U87 ΔEGFR glioblastoma, and human DU145 androgen-independent prostate carcinoma tumor cells indicated that both compounds are substrates for amino acid transport primarily by system L, with some transport taking place via system ASC. In rats with 9L gliosarcoma, [18F]9 and [18F]28 provided high tumor to normal brain tissue ratios, with maximal ratios of 3.5 and 4.1, respectively. Biodistribution studies in healthy rats confirmed that both compounds are BBB permeable and that bladder accumulation is low until at least 5 min post injection.

An in vivo multimodal feasibility study in a rat brain tumour model using flexible multinuclear MR and PET systems

BackgroundIn addition to the structural information afforded by 1H MRI, the use of X-nuclei, such as sodium-23 (23Na) or phosphorus-31 (31P), offers important complementary information concerning physiological and biochemical parameters. By then combining this technique with PET, which provides valuable insight into a wide range of metabolic and molecular processes by using of a variety of radioactive tracers, the scope of medical imaging and diagnostics can be significantly increased. While the use of multimodal imaging is undoubtedly advantageous, identifying the optimal combination of these parameters to diagnose a specific dysfunction is very important and is advanced by the use of sophisticated imaging techniques in specific animal models.MethodsIn this pilot study, rats with intracerebral 9L gliosarcomas were used to explore a combination of sequential multinuclear MRI using a sophisticated switchable coil set in a small animal 9.4 T MRI scanner and, subsequently, a small animal PET with the tumour tracer O-(2-[18F]-fluoroethyl)-L-tyrosine ([18F]FET). This made it possible for in vivo multinuclear MR-PET experiments to be conducted without compromising the performance of either multinuclear MR or PET.ResultsHigh-quality in vivo images and spectra including high-resolution 1H imaging, 23Na-weighted imaging, detection of 31P metabolites and [18F]FET uptake were obtained, allowing the characterisation of tumour tissues in comparison to a healthy brain. It has been reported in the literature that these parameters are useful in the identification of the genetic profile of gliomas, particularly concerning the mutation of the isocitrate hydrogenase gene, which is highly relevant for treatment strategy.ConclusionsThe combination of multinuclear MR and PET in, for example, brain tumour models with specific genetic mutations will enable the physiological background of signal alterations to be explored and the identification of the optimal combination of imaging parameters for the non-invasive characterisation of the molecular profile of tumours.

Multi-layered core-sheath fiber membranes for controlled drug release in the local treatment of brain tumor

Interstitial chemotherapy plays a pivotal role in the treatment of glioblastoma multiforme (GBM), an aggressive form of primary brain cancer, by enhancing drug biodistribution to the tumor and avoiding systemic toxicities. The use of new polymer structures that extend the release of cytotoxic agents may therefore increase survival and prevent recurrence. A novel core-sheath fiber loaded with the drug carmustine (BCNU) was evaluated in an in vivo brain tumor model. Three-dimensional discs were formed from coaxially electrospun fiber membranes and in vitro BCNU release kinetics were measured. In vivo survival was assessed following implantation of discs made of compressed core-sheath fibers (NanoMesh) either concurrently with or five days after intracranial implantation of 9L gliosarcoma. Co-implantation of NanoMesh and 9L gliosarcoma resulted in statistically significant long-term survival (>150 days). Empty control NanoMesh confirmed the safety of these novel implants. Similarly, Day 5 studies showed significant median, overall, and long-term survival rates, suggesting optimal control of tumor growth, confirmed with histological and immunohistochemical analyses. Local chemotherapy by means of biodegradable NanoMesh implants is a new treatment paradigm for the treatment for brain tumors. Drug delivery with coaxial core-sheath structures benefits from high drug loading, controlled long-term release kinetics, and slow polymer degradation. This represents a promising evolution for the current treatment of GBM.

Polo-like kinase 1 inhibitor BI6727 sensitizes 9L gliosarcoma cells to ionizing irradiation

Surgery, chemotherapy and radiotherapy remain as the major treatment strategies for cancers. Some agents such as anti-cancer drugs have capacity to enhance the radiation sensitivity of cancer cells at G2/M phase, leading to an improved radiotherapeutic efficacy. BI6727 is an ATP-competitive polo-like kinase 1 (Plk 1) inhibitor and an anti-cancer drug. Using the radio-resistant 9L rat gliosarcoma cells as model, we examined the effect of BI6727 on cell growth and assessed the chemo-radiotherapeutic efficiency between 150 kVp conventional irradiation (dose rate of 0.76 Gy min−1) and 66 keV synchrotron x-ray broad beam irradiation (dose rate of 46 Gy s−1). Our studies showed that BI6727 significantly caused cell growth arrest at G2/M phase and inhibited 9L cell proliferation with EC50 of 58.1 nM. In combinatory treatment, irradiation of BI6727-treated 9L cells with synchrotron x-rays at a dose rate of 46 Gy s−1 resulted in significant reduction of the cell survival compared to the conventional x-rays at a dose rate of 0.76 Gy min−1. These results indicated that Plk1 inhibitor BI6727 enhanced radio-sensitization of 9L cells in a dose rate dependent manner. For clinical application, irradiation with high dose rate is a promising strategy to improve chemo-radiotherapeutic efficacy for gliosarcoma cancer.

Overall Survival in Malignant Glioma Is Significantly Prolonged by Neurosurgical Delivery of Etoposide and Temozolomide from a Thermo-Responsive Biodegradable Paste.

High-grade glioma (HGG) treatment is limited by the inability of otherwise potentially efficacious drugs to penetrate the blood-brain barrier. We evaluate the unique intracavity delivery mode and translational potential of a blend of poly(DL-lactic acid-co-glycolic acid; PLGA) and poly(ethylene glycol; PEG) paste combining temozolomide and etoposide to treat surgically resected HGG. To prolong stability of temozolomide prodrug, combined in vitro drug release was quantitatively assessed from low pH-based PLGA/PEG using advanced analytic methods. In vitro cytotoxicity was measured against a panel of HGG cell lines and patient-derived cultures using metabolic assays. In vivo safety and efficacy was evaluated using orthotopic 9L gliosarcoma allografts, previously utilized preclinically to develop Gliadel. Combined etoposide and temozolomide in vitro release (22 and 7 days, respectively) was achieved from a lactic acid-based PLGA/PEG paste, used to enhance stability of temozolomide prodrug. HGG cells from central-enhanced regions were more sensitive to each compound relative to primary lines derived from the HGG-invasive margin. Both drugs retained cytotoxic capability upon release from PLGA/PEG. In vivo studies revealed a significant overall survival benefit in postsurgery 9L orthotopic gliosarcomas, treated with intracavity delivered PLGA/PEG/temozolomide/etoposide and enhanced with adjuvant radiotherapy. Long-term survivorship was observed in over half the animals with histologic confirmation of disease-free brain. The significant survival benefit of intracavity chemotherapy demonstrates clinical applicability of PLGA/PEG paste-mediated delivery of temozolomide and etoposide adjuvant to radiotherapy. PLGA/PEG paste offers a future platform for combination delivery of molecular targeted compounds.

Longitudinal evaluation of tumor microenvironment in rat focal brainstem glioma using diffusion and perfusion MRI.

Brainstem gliomas are aggressive and difficult to treat. Growth of these tumors may be characterized with MRI methods. To visualize longitudinal changes in tumor volume, vascular leakiness, and tissue microstructure in an animal model of brainstem glioma. Prospective animal model. Male Sprague-Dawley rats (n = 9) were imaged with 9L gliosarcoma cells infused into the pontine reticular formation of the brainstem. The MRI tumor microenvironment was studied at 3 and 10 days postimplantation of tumor cells. Diffusion tensor imaging (DTI) and dynamic contrast-enhanced (DCE)-MRI were performed at 4.7T using spin-echo multislice echo planar imaging and gradient echo multislice imaging, respectively. Tumor leakiness was assessed by the forward volumetric transfer constant, Ktrans , estimated from DCE-MRI data. Tumor structure was evaluated with fractional anisotropy (FA) obtained from DTI. Tumor volumes, delineated by a T1 map, T2 -weighted image, FA, and DCE signal enhancement were compared. Changes in the assessed parameters within and across the groups (ie, rats 3 and 10 days post tumor cell implantation) were evaluated with Wilcoxon rank-sum tests. Day 3 tumors were visible mainly on contrast-enhanced images, while day 10 tumors were visible in both contrast-enhanced and diffusion-weighted images. Mean Ktrans at day 10 was 41% lower than at day 3 (P = 0.23). In day 10 tumors, FA was regionally lower in the tumor compared to normal tissue (P = 0.0004), and tumor volume, segmented based on FA map, was significantly smaller (P ≤ 0.05) than that obtained from other contrasts. Contrast-enhanced MRI was found to be more sensitive in detecting early-stage tumor boundaries than other contrasts. Areas of the tumor outlined by DCE-MRI and DTI were significantly different. Over the observed period of tumor growth, average vessel leakiness decreased with tumor progression. 2 Technical Efficacy: Stage 3 J. Magn. Reson. Imaging 2019;49:1322-1332.

Radiosensitisation enhancement effect of BrUdR and Ta2O5 NSPs in combination with 5-Fluorouracil antimetabolite in kilovoltage and megavoltage radiation

This article demonstrates in vitro a synergistic effect on 9L gliosarcoma cells when exposed to bromodeoxyuridine (BrUdR) and a low concentration (100 times lower than the IC50) of 5-Fluorouracil (5-FU), in combination with x-ray irradiation. The synergy is brought about by several important factors including the x-ray beam energy, atomic number of the BrUdR (Z = 35), effectiveness of 5-FU in reducing the available repair processes and distribution of the BrUdR in and around the 9L cells (32% of the total substitution of BrUdR for thymidine into nucleus DNA). Our results show that the synergistic effect, evident in an optimised 125 kVp x-ray field, leads to a radiosensitisation enhancement ratio at the 10% survival level (SER10%) of 2.11. We highlight the importance of the aforementioned factors by similarly performed experiments for higher Z (Z = 73) tantalum pentoxide nano-structured particles (Ta2O5 NSPs) that are substituted for the BrUdR in larger concentration (∼10 times). In the Ta2O5 NSPs experiments, no synergistic effect is observed in the kVp irradiation with optimal energy spectrum even though the effective Z and NSP concentration is much higher than for BrUdR. All experiments were repeated using a MV x-ray irradiation field and no synergistic effect is observed for either the BrUdR or Ta2O5 case. We therefore hypothesise that the synergistic outcome is due to more drastic and complex damages induced by BrUdR under the exposure of kVp radiation. Such damages are achieved by the localisation of the BrUdR in the DNA and the high LET (very short range) secondary electrons in combination with the 5-FU. In order to achieve similar synergistic effects in the more clinically relevant x-ray energy field the concentration of the high Z material needs to be greater in order to create a higher LET electron environment.

Reproducibility and relative stability in magnetic resonance imaging indices of tumor vascular physiology over a period of 24 h in a rat 9L gliosarcoma model

PurposeThe objective was to study temporal changes in tumor vascular physiological indices in a period of 24h in a 9L gliosarcoma rat model. MethodsFischer-344 rats (N=14) were orthotopically implanted with 9L cells. At 2weeks post-implantation, they were imaged twice in a 24h interval using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Data-driven model-selection-based analysis was used to segment tumor regions with varying vascular permeability characteristics. The region with the maximum number of estimable parameters of vascular kinetics was chosen for comparison across the two time points. It provided estimates of three parameters for an MR contrast agent (MRCA): i) plasma volume (vp), ii) forward volumetric transfer constant (Ktrans) and interstitial volume fraction (ve, ratio of Ktrans to reverse transfer constant, kep). In addition, MRCA extracellular distribution volume (VD) was estimated in the tumor and its borders, along with tumor blood flow (TBF) and peritumoral MRCA flux. Descriptors of parametric distributions were compared between the two times. Tumor extent was examined by hematoxylin and eosin (H&E) staining. Picrosirus red staining of secreted collagen was performed as an additional index for 9L cells. ResultsTest-retest differences between population summaries for any parameter were not significant (paired t and Wilcoxon signed rank tests). Bland-Altman plots showed no apparent trends between the differences and averages of the test-retest measures for all indices. The intraclass correlation coefficients showed moderate to almost perfect reproducibility for all of the parameters, except vp. H&E staining showed tumor infiltration in parenchyma, perivascular space and white matter tracts. Collagen staining was observed along the outer edges of main tumor mass. ConclusionThe data suggest the relative stability of these MR indices of tumor microenvironment over a 24h duration in this gliosarcoma model.

Co-localization of vimentin with a tumor associated antigen identified by the monoclonal antibody H5 in 9L gliosarcoma cells and normal glial cells from Fischer 344 rats

Abstract Previously, we used a monoclonal antibody, designated H5, to isolate a tumor-associated antigen (TAA) expressed by 9L, a gliosarcoma line isolated from Fischer 344 (F344) rats. This TAA was tentatively identified as vimentin via mass spectrometry. In this project, we identified the location of this TAA and vimentin on 9L gliosarcoma and normal rat glial cells using confocal fluorescent microscopy. Vimentin and the TAA always co-localized which suggests that the TAA bound by H5 is a moiety on the vimentin protein. In addition, 9L expressed an increased level of vimentin compared to normal rat glial cells. Glial cells were examined from neonatal, 6 week, and adult rats. The glial cells expressed less vimentin than 9L gliosarcoma tumor cells at all ages. These results suggest that vimentin is upregulated as part of the tumor phenotype giving a possible mechanism for detecting transformed cells of this tumor type.

Level of expression of vimentin mRNA in 9L gliosarcoma cells compared to microglial cells, astrocytes, and oligodendrocytes isolated from Fischer 344 rats

Abstract Gliosarcomas are an aggressive brain cancer that can become metastatic. Previously, we used a monoclonal antibody, designated H5, to isolate a tumor-associated antigen (TAA) expressed by 9L, a gliosarcoma line isolated from Fischer 344 (F344) rats. This TAA was tentatively identified as vimentin via mass spectrometry. In this project, we compared the level of vimentin mRNA in 9L cells to that of three types of glial cells: microglial cells, astrocytes, and oligodendrocytes isolated from F344 rats via qPCR. The level of vimentin mRNA in 9L cells was significantly upregulated as compared to the expression levels in all three types of glial cells. Similar research on carcinomas has identified vimentin as being involved in metastatic tumors and is now used as a genetic marker of this specific type of cancer. This research suggests that vimentin mRNA in 9L undergoes similar upregulation to that of carcinomas.