A method to accurately inject tumor cells into the caudate/putamen nuclei of the mouse brain

OBJECTIVE: To improve currently used techniques to implant tumor cells into the parenchyma of the mouse brain. MATERIALS AND METHODS: The stereotactic injection of 0.5 to 5 microl of indigo carmine over 5 to 40 minutes into the caudate/putamen nuclei of the mouse was done followed by sacrifice and examination of the brain injection site. 1 microl containing 10(5) U87MG glioma cells were stereotactically implanted into the caudate/ putamen nuclei over 20 minutes. The animals were sacrificed from one hour to 63 days after implantation and the brain examined and tumor size measured. RESULTS: An injection of 1 microl of indigo carmine over 20 minutes produced a spherical deposit of dye within the caudate/putamen nuclei. Larger volumes of indigo carmine or shorter injection times resulted in dye spreading along the injection tract or into the ventricles or subarachnoid space. Using the results of the dye studies, the same parameters were used to successfully inject and confine the glioma cells to the caudate/putamen nuclei in 30 of 32 mice. No tumor was found in 2 animals and appears to be explained by obstruction of the injection cannula. The tumor cells appeared viable an hour after injection. However by day three, considerable necrosis of tumor cells were noted, the effects of which resolved by day five. On day six, the injection site was comparable to that at one hour. In the early phase, until the fifth week, tumor volume doubling time was ten days while afterward it was only five days. CONCLUSION: The technique described allows the highly accurate and reproducible introduction of a given number of cells into a specific area of the mouse brain. This should reduce the intragroup variability, be it control or therapeutic, allowing better assessment of outcome with fewer number of mice.     

Analysing the Contact Conduction Influence on the Heat Transfer Intensity in the Rectangular Steel Bars Bundle

Bundles of steel bars, besides metal foams, are an example of cellular solids. Such bundles constitute a charge during the heat treatment of bars. The paper presents a mathematical model of transient heat transfer in a bundle of rectangular steel bars based on the energy balance method. The key element of this model is the procedure of determining the effective thermal conductivity using the electrical analogy. Different mechanisms of heat transfer occurring within the analysed medium (conduction in steel and contact conduction) are assigned corresponding thermal resistances. The discussed procedure involves expressing these resistances with the use of arithmetic relationships describing their changes in the temperature function. Thermal contact resistance has been described with the use of the relationships determined experimentally. As a result of the performed calculations, the influence of contact conduction between the adjacent bars and bundle arrangement on its heating time was established. The results of the calculations show that the heating time of bundles can be lowered by 5–40% as a result of a decrease in the thermal contact resistance. This effect depends on the bar size and bundle arrangement. From the practical point of view, the analysed problem is connected with the optimization of the heat treatment processes of steel bars.     

18F-labeled RGD peptide: initial evaluation for imaging brain tumor angiogenesis

Brain tumors are highly angiogenesis dependent. The cell adhesion receptor integrin alpha(v)beta(3) is overexpressed in glioma and activated endothelial cells and plays an important role in brain tumor growth, spread and angiogenesis. Suitably labeled alpha(v)beta(3)-integrin antagonists may therefore be useful for imaging brain tumor associated angiogenesis. Cyclic RGD peptide c(RGDyK) was labeled with (18)F via N-succinimidyl-4-[(18)F]fluorobenzoate through the side-chain epsilon-amino group of the lysine residue. The radiotracer was evaluated in vivo for its tumor targeting efficacy and pharmacokinetics in subcutaneously implanted U87MG and orthotopically implanted U251T glioblastoma nude mouse models by means of microPET, quantitative autoradiography and direct tissue sampling. The N-4-[(18)F]fluorobenzoyl-RGD ([(18)F]FB-RGD) was produced in less than 2 h with 20-25% decay-corrected yields and specific activity of 230 GBq/micromol at end of synthesis. The tracer showed very rapid blood clearance and both hepatobiliary and renal excretion. Tumor-to-muscle uptake ratio at 30 min was approximately 5 in the subcutaneous U87MG tumor model. MicroPET imaging with the orthotopic U251T brain tumor model revealed very high tumor-to-brain ratio, with virtually no uptake in the normal brain. Successful blocking of tumor uptake of [(18)F]FB-RGD in the presence of excess amount of c(RGDyK) revealed receptor specific activity accumulation. Hence, N-4-[(18)F]fluorobenzoyl labeled cyclic RGD peptide [(18)F]FB-RGD is a potential tracer for imaging alpha(v)beta(3)-integrin positive tumors in brain and other anatomic locations.     

Species-specific urokinase receptor ligands reduce glioma growth and increase survival primarily by an antiangiogenesis mechanism

Species-specific urokinase receptor (uPAR) ligands with improved pharmacokinetics were generated by site-specific mutagenesis and amino-terminal pegylation. These molecules were used to probe the role of uPAR in brain tumor progression and angiogenesis. The ligands blocked endothelial cell tube formation in Matrigel in a species-specific manner and reduced both baseline and uPA amino-terminal fragment-stimulated cell migration on vitronectin gradients. Treatment of U87MG gliomas implanted orthotopically in mice with single species-specific or combination uPAR ligands resulted in significant decreases in tumor size, which translated to increases in survival time, and which were most significant when the murine-specific ligand was included. Further analysis of tumors showed that the reduced sizes were correlated with a decrease in tumor cell proliferation and mean vessel density and an increase in tumor cell apoptosis. In addition, a large increase in collagen deposition was observed in the treated groups. Statistical analysis showed that the combination therapy demonstrated a clear synergy as compared to the individual agent treatments. These results suggest that the major role of the uPAR system in brain tumor progression is in the stromal compartment and particularly in neovascularization, a hallmark of invasive brain tumors.     

Longitudinal MicroPET Imaging of Brain Tumor Growth with F-18-labeled RGD Peptide

Purpose EMD 121974, a potent cyclic RGD peptide inhibitor of α _v-integrins, demonstrated effectiveness in suppressing brain tumor growth in both preclinical models and phases I/II clinical trials. The ability to non-invasively evaluate α _v-integrin expression provides a novel and unique way to better understand brain tumor angiogenesis in relationship to α _v-integrin expression, and allow for direct assessment of anti-integrin treatment efficacy. Procedures We developed a F-18-labeled RGD peptide [F-18]FB-RGD and performed serial microPET imaging scans to follow brain tumor growth and angiogenesis as a function of time in an orthotopic U87MG glioblastoma xenograft model in athymic nude mice. Results The tumor was barely visible on microPET at the size of ≤1.5 mm diameter at which time no angiogenesis was evident on histological examination. When tumor started to grow exponentially by day 35 the activity accumulation in the brain tumor also increased accordingly, with best tumor-to-brain contrast seven weeks after inoculation of 10⁵ U87MG cells into the mice forebrain. Conclusions Longitudinal microPET imaging and [F-18]FB-RGD provides the sensitivity and resolution to visualize and quantify anatomical variations during brain tumor growth and angiogenesis, most likely through interaction with α _v-integrins expressed on tumor cells and angiogenic tumor vessels.     

Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor alphavbeta3-integrin expression.

The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study, we introduced a polyethylene glycol (PEG; molecular weight, 3,400) moiety between DOTA and RGD and evaluated the 64Cu-DOTA-PEG-RGD tracer for microPET imaging in brain tumor models. METHODS: DOTA was activated in situ and conjugated with RGD-PEG-NH2 under slightly basic conditions. alphavbeta3-Integrin-binding affinity was evaluated with a solid-phase receptor-binding assay in the presence of 125I-echistatin. Female nude mice bearing subcutaneous U87MG glioblastoma xenografts were administered 64Cu-DOTA-PEG-RGD, and the biodistributions of the radiotracer were evaluated from 30 min to 4 h after injection. microPET (20 min of static imaging at 1 h after injection) and then quantitative autoradiography were used for tumor visualization and quantification. The same tracer was also applied to an orthotopic U87MG model for tumor detection. RESULTS: The radiotracer was synthesized with a high specific activity (14,800-29,600 GBq/mmol [400-800 Ci/mmol]). The c(RGDyK)-PEG-DOTA ligand showed intermediate binding affinity for alphavbeta3-integrin (50% inhibitory concentration, 67.5 +/- 7.8 nmol/L [mean +/- SD]). The pegylated RGD peptide demonstrated rapid blood clearance (0.57 +/- 0.15 percentage injected dose [%ID]/g [mean +/- SD] at 30 min after injection and 0.03 +/- 0.02 %ID/g at 4 h after injection). Activity accumulation in the tumor was rapid and high at early time points (2.74 +/- 0.45 %ID/g at 30 min after injection), and some activity washout was seen over time (1.62 +/- 0.18 %ID/g at 4 h after injection). Compared with (64)Cu-DOTA-RGD, this tracer showed improved in vivo kinetics, with significantly reduced liver uptake (0.99 +/- 0.08 %ID/g vs. 1.73 +/- 0.39 %ID/g at 30 min after injection and 0.58 +/- 0.07 %ID/g vs. 2.57 +/- 0.49 %ID/g at 4 h after injection). The pegylated RGD peptide showed higher renal accumulation at early time points (3.51 +/- 0.24 %ID/g vs. 2.18 +/- 0.23 %ID/g at 30 min after infection) but more rapid clearance (1.82 +/- 0.29 %ID/g vs. 2.01 +/- 0.25 %ID/g at 1 h after injection) than 64Cu-DOTA-RGD. The integrin receptor specificity of this radiotracer was demonstrated by blocking of tumor uptake by coinjection with nonradiolabeled c(RGDyK). The high tumor-to-organ ratios for the pegylated RGD peptide tracer (at 1 h after injection: tumor-to-blood ratio, 20; tumor-to-muscle ratio, 12; tumor-to-liver ratio, 2.7; and tumor-to-kidney ratio, 1.2) were confirmed by microPET and autoradiographic imaging in a subcutaneous U87MG tumor model. This tracer was also able to detect an orthotopic brain tumor in a model in which U87MG cells were implanted into the mouse forebrain. Although the magnitude of tumor uptake in the orthotopic xenograft was lower than that in the subcutaneous xenograft, the orthotopic tumor was still visualized with clear contrast from normal brain tissue. CONCLUSION: This study demonstrated the suitability of a PEG moiety for improving the in vivo kinetics of a 64Cu-RGD peptide tracer without compromising the tumor-targeting ability and specificity of the peptide. Systematic investigations of the effects of the size and geometry of PEG on tumor targeting and in vivo kinetics will lead to the development of radiotracers suitable for clinical applications such as visualizing and quantifying alphav-integrin expression by PET. In addition, the same ligand labeled with therapeutic radionuclides may be applicable for integrin-targeted internal radiotherapy.     

On Determination of the Effective Thermal Conductivity of a Bundle of Steel Bars Using the Krischer Model and Considering Thermal Radiation

Cellular solid materials are commonly found in industrial applications. By definition, cellular solids are porous materials that are built of distinct cells. One of the groups of such materials contains metal foams. Another group of cellular metals contains bundles of steel bars, which create charges during the heat treatment of the bars. A granular structure connected by the lack of continuity of the solid phase is the main feature that distinguishes bundles from metal foams. The boundaries of the bundle cells are made of adjacent bars, with the internal region taking the form of an air cavity. In this paper, we discuss the possibility of using the Krischer model to determine the effective thermal conductivity of heat-treated bundles of steel bars based on the results of experimental tests and calculations. The model allows the k_ef coefficient to be precisely determined, although it requires the weighting parameter f to be carefully matched. It is shown that the value of f depends on the bar diameter, while its changes within the examined temperature range (25–800 °C) can be described using a third-degree polynomial. Determining the coefficients of such a polynomial is possible only when the effective thermal conductivity of the considered charge is known. Moreover, we analyze a simplified solution, whereby a constant value of the f coefficient is used for a given bar diameter; however, the k_ef values obtained thanks to this approach are encumbered with inaccuracy amounting to several dozen percentage points. The obtained results lead to the conclusion that the Krischer model cannot be used for the discussed case.     

MicroPET imaging of brain tumor angiogenesis with <Superscript>18</Superscript>F-labeled PEGylated RGD peptide

We have previously labeled cyclic RGD peptide c(RGDyK) with fluorine-18 through conjugation labeling via a prosthetic 4-[¹⁸F]fluorobenzoyl moiety and applied this [¹⁸F]FB-RGD radiotracer for _v-integrin expression imaging in different preclinical tumor models with good tumor-to-background contrast. However, the unfavorable hepatobiliary excretion and rapid tumor washout rate of this tracer limit its potential clinical applications. The aims of this study were to modify the [¹⁸F]FB-RGD tracer by inserting a heterobifunctional poly(ethylene glycol) (PEG, M.W. =3,400) between the ¹⁸F radiolabel and the RGD moiety and to test this [¹⁸F]FB-PEG-RGD tracer for brain tumor targeting and in vivo kinetics. [¹⁸F]FB-PEG-RGD was prepared by coupling the RGD-PEG conjugate with N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) under slightly basic conditions (pH=8.5). The radiochemical yield was about 20–30% based on the active ester [¹⁸F]SFB, and specific activity was over 100 GBq/mol. This tracer had fast blood clearance, rapid and high tumor uptake in the subcutaneous U87MG glioblastoma model (5.2±0.5%ID/g at 30 min p.i.). Moderately rapid tumor washout was observed, with the activity accumulation decreased to 2.2±0.4%ID/g at 4 h p.i. MicroPET and autoradiography imaging showed a very high tumor-to-background ratio and limited activity accumulation in the liver, kidneys and intestinal tracts. U87MG tumor implanted into the mouse forebrain was well visualized with [¹⁸F]FB-PEG-RGD. Although uptake in the orthotopic tumor was significantly lower (P<0.01) than in the subcutaneous tumor, the maximum tumor-to-brain ratio still reached 5.0±0.6 due to low normal brain background. The results of H&E staining post mortem agreed with the anatomical information obtained from non-invasive microPET imaging. In conclusion, PEGylation suitably modifies the physiological behavior of the RGD peptide. [¹⁸F]FB-PEG-RGD gave improved tumor retention and in vivo kinetics compared with [¹⁸F]FB-RGD.