Differentiation of Glioma and Radiation Injury in Rats Using In Vitro Produce Magnetically Labeled Cytotoxic T-Cells and MRI

A limitation with current imaging strategies of recurrent glioma undergoing radiotherapy is that tumor and radiation injury cannot be differentiated with post contrast CT or MRI, or with PET or other more complex parametric analyses of MRI data. We propose to address the imaging limitation building on emerging evidence indicating that effective therapy for recurrent glioma can be attained by sensitized T-cells following vaccination of primed dendritic cells (DCs). The purpose of this study was to determine whether cord blood T-cells can be sensitized against glioma cells (U-251) and if these sensitized cytotoxic T-cells (CTLs) can be used as cellular magnetic resonance imaging probes to identify and differentiate glioma from radiation necrosis in rodent models.Cord blood T and CD14+ cells were collected. Isolated CD14+ cells were then converted to dendritic cells (DCs), primed with glioma cell lysate and used to sensitize T-cells. Phenotypical expression of the generated DCs were analyzed to determine the expression level of CD14, CD86, CD83 and HLA-DR. Cells positive for CD25, CD4, CD8 were determined in generated CTLs. Specificity of cytotoxicity of the generated CTLs was also determined by lactate dehydrogenase (LDH) release assay. Secondary proliferation capacity of magnetically labeled and unlabeled CTLs was also determined. Generated CTLs were magnetically labeled and intravenously injected into glioma bearing animals that underwent MRI on days 3 and 7 post- injection. CTLs were also administered to animals with focal radiation injury to determine whether these CTLs accumulated non-specifically to the injury sites. Multi-echo T2- and T2*-weighted images were acquired and R2 and R2* maps created. Our method produced functional, sensitized CTLs that specifically induced U251 cell death in vitro. Both labeled and unlabeled CTLs proliferated equally after the secondary stimulation. There were significantly higher CD25 positive cells (p = <0.006) in CTLs. In addition, T2- and T2*-weighted MR images showed increased low signal intensity areas in animals that received labeled CTLs as compared to the images from animals that received control cells. Histological analysis confirmed the presence of iron positive cells in sites corresponding to MRI low signal intensity regions. Significant differences (p = <0.001) in tumor R2 and R2* values were observed among the groups of animals. Animals with radiation injury exhibited neither MRI hypointense areas nor presence of iron positive cells.Our results indicate that T-cells can be effectively sensitized by in vitro methods and used as cellular probes to identify and differentiate glioma from radiation necrosis

Inhibition of Telomerase Activity by Oleanane Triterpenoid CDDO-Me in Pancreatic Cancer Cells is ROS-Dependent

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Changes in vascular permeability and expression of different angiogenic factors following anti-angiogenic treatment in rat glioma.

Anti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies.Seven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (K(trans)), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased K(trans) at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1alpha, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors.These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot

Concentration-independent MRI of pH with a dendrimer-based pH-responsive nanoprobe

The measurement of extracellular pH (pHe ) has significant clinical value for pathological diagnoses and for monitoring the effects of pH-altering therapies. One of the major problems of measuring pHe with a relaxation-based MRI contrast agent is that the longitudinal relaxivity depends on both pH and the concentration of the agent, requiring the use of a second pH-unresponsive agent to measure the concentration. Here we tested the feasibility of measuring pH with a relaxation-based dendritic MRI contrast agent in a concentration-independent manner at clinically relevant field strengths. The transverse and longitudinal relaxation times in solutions of the contrast agent (GdDOTA-4AmP)44 -G5, a G5-PAMAM dendrimer-based MRI contrast agent in water, were measured at 3 T and 7 T magnetic field strengths as a function of pH. At 3 T, longitudinal relaxivity (r1 ) increased from 7.91 to 9.65 mM(-1) s(-1) (on a per Gd(3+) basis) on changing pH from 8.84 to 6.35. At 7 T, r1 relaxivity showed pH response, albeit at lower mean values; transverse relaxivity (r2 ) remained independent of pH and magnetic field strengths. The longitudinal relaxivity of (GdDOTA-4AmP)44 -G5 exhibited a strong and reversible pH dependence. The ratio of relaxation rates R2 /R1 also showed a linear relationship in a pH-responsive manner, and this pH response was independent of the absolute concentration of (GdDOTA-4AmP)44 -G5 agent. Importantly, the nanoprobe (GdDOTA-4AmP)44 -G5 shows pH response in the range commonly found in the microenvironment of solid tumors

SPECT analysis of <i>in vivo</i> accumulation of Tc-99m-HYNIC-VEGF-c.

<p>VEGF-c (which targets both VEGFR2 and VEGFR3) was tagged with HYNIC chelators and then labeled with Tc-99m-pertechnetate (Tc-99m) and injected intravenously in PTK787 and vehicle treated rats. One hour after injection, SPECT images were obtained using dedicated animal scanner. All PTK787 treated rats showed increased accumulation of Tc-99m-HYNIC-VEGF-c in the tumors (lower panel, arrows) compared to that of vehicle treated tumors (upper panel, arrows).</p

Western blot images and densitometry analysis of western blots.

<p>(<b>A</b>) Expression of different angiogenic factors (left panel) in the vehicle- and PTK787 treated tumors from representative cases at the peripheral (P), central part of the tumors (C) and contralateral brains (B). Note the increased expression of VEGF, SDF-1 and HIF-1α at the peripheral part of PTK787 treated tumors. Right panel shows the densitometry analysis of the blot (normalized to β-actin and contralateral brain). The analysis also confirmed the finding of the blot. Note the patterns of VEGF, SDF-1 and HIF-1α in PTK787 treated tumors which are different from that of vehicle treated tumors. (<b>B</b>) Densitometry analysis of VEGFR2, VEGFR3 and EGFR blot (normalized to β-actin and contralateral brain). Expression of VEGFR2, VEGFR3 and EGFR showed higher normalized values at the peripheral part of the PTK787 treated tumors compared to that of central part and the expression patterns are different in vehicle treated tumors. Please note that PTK787 treated tumors showed lower normalized values of VEGFR2 and EGFR both at the periphery and central parts of the tumors compared to that of corresponding contralateral brain; whereas vehicle treated tumors did not show any changes in the normalized values of VEGFR2 and EGFR compared to that of corresponding contralateral brain. Data are expressed as mean ± SEM, n = 3.</p

Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGF, HIF-1α, SDF-1 and vessel morphology.

<p>Expression of vascular endothelial growth factor (VEGF) (dark brown colored) at different parts of the PTK787 treated and vehicle treated tumors. There were no differences observed in the expression of VEGF on immunohistochemistry at different parts of the tumors treated with either PTK787 or vehicle. However, delineation of vessels using FITC tagged tomato lectin indicated higher number of dilated vessels at the tumor periphery in rats that received PTK787 treatment. These dilated vessels may be indicative of increased permeability, fPV and signal intensity changes in PTK787 treated tumors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008727#pone-0008727-g001" target="_blank"><b>Figures 1</b></a><b> and </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008727#pone-0008727-g002" target="_blank"><b>2</b></a>). Increased permeability may also be due to increased VEGF expression. HIF-1α expression was mostly seen in the central part of the vehicle treated tumors (arrows), however, SDF-1 expressions were observed both in PTK787 and vehicle treated tumors (arrows).</p

Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGFR2, VEGFR3 and EGFR.

<p>Immunohistochemistry confirmed the findings of SPECT studies, where PTK787 treated tumors showed increased expression of VEGFR2 and VEFGR3 at the peripheral parts of the tumors, especially around the vessels (arrows) compared to that of vehicle treated tumors (right column). Both PTK787 (lower panel, left column) and vehicle treated (lower panel, right column) showed expression of EGFR in the tumors. Lower panel, middle column show no brown cells in negative control slide.</p