Search

Background: We studied the regional characteristics within tumor masses using PET tracers and immunohistochemical methods., Methods: The intratumoral distribution of (64)Cu-diacetyl-bis(N4-methylthiosemicarbazone) ([(64)Cu]Cu-ATSM) and [(18)F] 2-fluoro-2-deoxyglucose ((18)F]FDG) in mice with tumors of four different origins (LLC1, Meth-A, B16 and colon26) was compared with the immunohistochemical staining of proliferating cells (Ki67), blood vessels (CD34 or von Willebrand factor), and apoptotic cells (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling method)., Results: With all cell lines, [(64)Cu]Cu-ATSM and [(18)F]FDG were distributed with different gradation in the tumor mass. The immunohistochemical study demonstrated that the high [(64)Cu]Cu-ATSM uptake regions were hypovascular and consisted of tumor cells arrested in the cell cycle, whereas the high [(18)F]FDG uptake regions were hypervascular and consisted of proliferating cells., Conclusion: In our study, it was revealed that one tumor mass contained two regions with different characteristics, which could be distinguished by [(64)Cu]Cu-ATSM and [(18)F]FDG. Because hypoxia and cell cycle arrest are critical factors to reduce tumor sensitivity to radiation and conventional chemotherapy, regions with such characteristics should be treated intensively as one of the primary targets. [(64)Cu]Cu-ATSM, which can delineate hypoxic and cell cycle-arrested regions in tumors, may provide valuable information for cancer treatment as well as possibly for treating such regions directly as an internal radiotherapy reagent.

16 alpha-[(18)F]fluoro-17beta-estradiol ([(18)F]FES) is a radiotracer for imaging estrogen receptors by positron emission tomography. We developed a clinically applicable automatic preparation system for [(18)F]FES by modifying a cassette-type [(18)F]fluorodeoxyglucose synthesizer. Two milligrams of 3-O-methoxymethyl-16,17-O-sulfuryl-16-epiestriol in acetonitrile was heated at 105 degrees C for 10 min with dried [(18)F]fluoride. The resultant solution was evaporated and hydrolyzed with 0.2 N HCl in 90% acetonitrile/water at 95 degrees C for 10 min under pressurized condition. The neutralization was carried out with 2.8% NaHCO(3), and then the high-performance liquid chromatography (HPLC) purification was performed. The desired radioactive fraction was collected and the solvent was replaced by 10 ml of saline, and then passed through a 0.22-microm filter into a pyrogen-free vial as the final product. The HPLC purification data demonstrated that [(18)F]FES was synthesized with a yield of 76.4+/-1.9% (n=5). The yield as the final product for clinical use was 42.4+/-3.2% (n=5, decay corrected). The total preparation time was 88.2+/-6.4 min, including the HPLC purification and the solvent replacement process. The radiochemical purity of the final product was >99%, and the specific activity was more than 111 GBq/micromol. The final product was stable for more than 6 h in saline containing sodium ascorbate. This new preparation system enables us to produce [(18)F]FES safe for clinical use with high and reproducible yield.

A simple, rapid and fully automated preparation of sodium [(18)F]fluoroacetate has been developed by taking advantage of the similarities between the reaction pathways of [(18)F]fluoroacetate and [(18)F]-2-fluoro-deoxyglucose (FDG). The automated synthesis of sodium [(18)F]fluoroacetate was achieved with a commercial [(18)F]FDG synthesizer, the TRACERlab MX(FDG). The method produced the desired compound in a short synthesis time (32 min) and with a high and reproducible radiochemical yield (50.2 +/- 4.8%, decay corrected). The radiochemical purity of sodium [(18)F]fluoroacetate was greater than 99%.

Early detection of tumor response to chemotherapy is of great importance for appropriate treatment of tumors. In this study, characteristics of two positron emission tomography (PET) tracers, [(18)F]2-fluoro-2-deoxy-D-glucose (FDG) and[(18)F]3'-fluoro-3'-deoxy-thymidine (FLT), in the early detection of tumor cell response as well as tolerance development to chemotherapy was compared using rat C6 glioma cells and 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosoureahydrochloride (ACNU). ACNU is an alkylating agent known to induce drug resistance through expression of O(6)-methylguanine-deoxyribonucleic acid methyl transferase (O(6)-MGMT). We established an ACNU-resistant C6 glioma cell line (C6/ACNU) and investigated the effect of ACNU on the uptake of FLT and FDG. In C6 cells, DNA synthesis presented as [(3)H]thymidine ([(3)H]Thd) incorporation into DNA was quickly suppressed by ACNU. In C6/ACNU cells, the suppression was recovered promptly, indicating that DNA alkylation occurs initially but highly expressed O(6)-MGMT repairs DNA, leading to the recovery of DNA synthesis. The patterns of FLT uptake in C6 and C6/ACNU were difficult to distinguish in the very early stage of the treatment, though it was reported that FLT uptake well correlated with proliferation in certain conditions. FDG uptake showed different patterns between the resistant and control cells, with significantly decreased uptake in C6 cells and unchanged uptake in C6/ACNU cells at 18-24 h after the treatment. Though difficult to be directly translated into clinical situation, the present study will provide a base to develop an appropriate protocol to assess tumor response to treatment by PET and to design effective treatment plans.

The transplantation of functional cells such as dopaminergic cells into damaged tissue is now clinically ongoing, but at present the population of surviving cells at the transplantation site mostly cannot be noninvasively examined. To visualize surviving transplanted functional cells using a noninvasive method, we chose the estrogen receptor ligand binding domain (ERL) as a reporter molecule and 16alpha-[18F]-fluoro-17beta-estradiol (FES) for its ligand. We used a mouse embryonic stem (ES) cell line for recipient cells as a model. To obtain ES cells that constitutively or inducibly express ERL, we transfected two types of expression vectors into EB5 parental ES cell line using the lipofection method and obtained about 30 clones for each of the two types of transfectants. Then, to examine the expression level of ERL, we performed Western blotting analysis. Ligand uptake experiments were carried out using [3H]-estradiol with or without excessive unlabeled estradiol for control cells and ERL transfectants. Each selected clone was also used for in vivo positron emission tomography (PET) imaging studies involving FES in nude mice transplanted with control cells and ERL transfectants. In some of the clones transfected with the inducible-type ERL gene, protein was expressed much higher than in the controls. However, constitutive-type ERL gene-transfected ES cells showed no protein production in spite of their gene expression activity being considerably high. All clones also expressed equal levels of the Oct-3/4 gene, a marker of pluripotency, in comparison with the parental cells. Also, the specific uptake of [3H]-estradiol was over 30 times higher in inducer-treated ERL-expressing ES cells compared to untreated control cells. Finally, by performing dynamic PET imaging, we successfully visualized ERL-expressing teratomas using FES.

64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) is a promising radiotherapy agent for the treatment of hypoxic tumors. In an attempt to elucidate the radiobiological basis of 64Cu-ATSM radiotherapy, we have investigated the cellular response patterns in vitro cell line models. Cells were incubated with 64Cu-ATSM, and the dose-response curves were obtained by performing a clonogenic survival assay. Radiation-induced damage in DNA was evaluated using the alkali comet assay and apoptotic cells were detected using Annexin V-FITC and propidium iodide staining methods. Washout rate and subcellular distribution of 64Cu in cells were investigated to further assess the effectiveness of 64Cu-ATSM therapy on a molecular basis. A direct comparison of subcellular localization of Cu-ATSM was made with the flow tracer analog Cu-pyruvladehyde-bis(N4-methylthiosemicarbazone). In this study, 64Cu-ATSM was shown to reduce the clonogenic survival rate of tumor cells in a dose-dependent manner. Under hypoxic conditions, cells took up 64Cu-ATSM and radioactive 64Cu was highly accumulated in the cells. In the 64Cu-ATSM-treated cells, DNA damage by the radiation emitted from 64Cu was detected, and inhibition of cell proliferation and induction of apoptosis was observed at 24 and 36 h after the treatment. The typical features of postmitotic apoptosis induced by radiation were observed following 64Cu-ATSM treatment. The majority of the 64Cu taken up into the cells remained in the postmitochondrial supernatant (the cellular residue after removal of the nuclei and mitochondria), which indicates that the beta- particle emitted from 64Cu may be as effective as the Auger electrons in 64Cu-ATSM therapy. These data allow us to postulate that 64Cu-ATSM will be able to attack the hypoxic tumor cells directly, as well as potentially affecting the peripheral nonhypoxic regions indirectly by the beta- particle decay of 64Cu.

Background: To minimize blood sampling necessary for the autoradiographic (ARG) method using alpha-[(14)C]methyl-tryptophan (alpha-MTrp), a simplified method using a standard exposure time (theta(S)) was proposed and the accuracy of the method was evaluated., Methods: A total of 168 rats from three sets of experiments with different serotonergic drugs to evaluate changes in cerebral serotonin (5-hydroxytryptamine; 5-HT) synthesis rate were used. In the acute treatment study, rats received a drug injection 30 min before the tracer experiment. In the repeated treatment study, the same dose of drug was injected for 7 days. The two-time point method provided a parameter of brain trapping constant (K*) from the slope of linear regression of the Patlak plot, from which 5-HT synthesis rate is calculated. The measured exposure time (theta(M)) for 60 and 150 min after the tracer injection obtained from individual blood sampling in the original method and the theta(S) calculated from theta(M) were used for evaluation of differences in K* values., Results: No significant difference in theta(M) was noted among different experiments, although plasma radioactivity at the end of experiment was significantly different between the acute and the repeated treatments for one of the three drugs. No difference in K* for each treatment was observed between the original method and the simplified single blood sampling method because there was no difference in theta(M) among three experiments and between theta(M) and theta(S)., Conclusion: The simplified alpha-MTrp ARG method, which uses theta(S) and a single arterial blood sample at the end of each experiment, can be used for the measurement of 5-HT synthesis rate in the rat brain.

[11C]Choline is a potential tracer to detect tumors, especially brain and prostate cancers. The metabolism of [11C]choline defines the accumulation pattern of [11C]choline in tumors depicted by positron emission tomography. Choline is a precursor of phosphatidylcholine that is a major constituent of membrane lipids. Membrane lipid synthesis as well as DNA synthesis is activated during cell proliferation. We investigated the relation between [14C]choline metabolism and proliferative activity using 10 tumor cell lines and fibroblasts. [14C]Choline uptake was higher in tumor cells than in fibroblasts and was correlated with the proliferative activity, though the sensitivity of [14C]choline uptake to proliferative activity was less than that of [1-14C]acetate. [14C]Phosphocholine produced from [14C]choline by phosphorylation mainly contributed to this accumulation. [11C]Choline can be used for the evaluation of tumor proliferation through estimating choline kinase activity.

The aim of this study was to determine the most suitable iodonucleoside analogs for use in tissue proliferation imaging by means of single photon emission tomography (SPECT). In this study, 5-[(125)I]iodo-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)uracil ([(125)I]FITAU, 1E) and 5-[(125)I]iodo-1-methyl-(2-deoxy-2-bromo-beta-D-arabinofuranosyl)uracil ([(125)I]IMBAU, 1F) were synthesized and their biological data were compared with previously published results regarding 4'-thio nucleoside analogs and the reference compound 5-[(125)I]iodo-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil ([(125)I]FIAU, 1D). 5-Iodo-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (FIAU, 2D), 5-iodo-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)uracil (FITAU, 2E), and 5-iodo-1-methyl-(2-deoxy-2-bromo-beta-D-arabinofuranosyl)uracil (IMBAU, 2F) were successfully labeled with (125)I and their in vitro cytosolic thymidine kinase (TK(1)) phosphorylation, recombinant thymidine phosphorylase enzymatic catabolism, TK(1)-dependent cell uptake, and in vivo biodistribution in normal mice were evaluated. Five compounds (1B, 1C, 1D, 1E, and 1F) were stable against C-N glycoside degradation induced by recombinant thymidine phosphorylase. However, 5-[(125)I]iodo-2'-deoxyuridine ([(125)I]IUdR, 1A) was not shown to be stable against such degradation. The TK(1) assay showed that [(125)I]FIAU (1D) expressed 16% of the phosphorylation potential of [(125)I]IUdR (1A). Furthermore, [(125)I]FITAU (1E) was shown to have reduced phosphorylation potential, in comparison with that of [(125)I]IUdR (1A) (<0.01). [(125)I]IMBAU (1F) did not show any phosphorylation. In vitro cell uptake and in vivo proliferation-selective uptake of each nucleoside was largely dependent on its potential as a TK(1) substrate. Neither [(125)I]FITAU (1E) nor [(125)I]IMBAU (1F) were shown to have distinct TK(1)-dependent cell uptake and retention in the proliferating tissues. From these results, we concluded that [(125)I]FITAU (1E) and [(125)I]IMBAU (1F) are not effective as imaging agents of cell proliferation. The biological data obtained with these nucleosides were compared, and requirements for the design of pharmaceutically useful radioiodinated nucleoside analogs were also considered.

(64)Cu-labeled diacetyl-bis(N(4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a promising agent for internal radiation therapy and imaging of hypoxic tissues. In the present study, the intra-tumoral distribution of (64)Cu-ATSM was investigated by comparing it to that of [(18)F]FDG and histological findings. VX2 tumors were implanted into Japanese white rabbits subcutaneously. (64)Cu-ATSM and [(18)F]FDG were co-injected intravenously and the tumor was dissected and cut into 1 mm thick slices 1 h after the injection. The uptake of (64)Cu-ATSM and [(18)F]FDG was measured using a dual-tracer autoradiographic technique. Histological cell biology was estimated from the optical microscopy of tumor sections. The major accumulation of (64)Cu-ATSM was observed around the outer rim of the tumor masses which consisted mainly of active cells and expected to be hypoxic. [(18)F]FDG was distributed more widely with highest levels in the inner regions where pre-necrotic cells were mainly observed. (64)Cu-ATSM appears to be useful for the detection of hypoxic but active tumor cell regions in vivo.

(64)Cu is a useful radiotracer for positron emission tomography (PET) and a promising radiotherapy agent for the treatment of cancer. Recently, (64)Cu-labeled radiopharmaceuticals were reported to be useful for internal radiation therapy as well as PET monitoring of tumors.(64)Cu was produced at the Fukui Medical University's cyclotron using twelve MeV proton irradiation and the (64)Ni(p,n) (64)Cu nuclear reaction. A (64)Ni target was electroplated on a gold disk at a thickness of 50 to > 100 microm. Electroplating was performed at 2.5 V, at currents between 5-15 mA, and was completed in 12-24 hr. The (64)Ni target was bombarded with a 50 +/- 3 microA proton current. After bombardment, (64)Cu was separated from the (64)Ni target and other contaminants using an anion exchange column. Target (64)Ni was recovered and re-used. The yield of (64)Cu was 0.6 to > 3.0 mCi/microA*h, and averaged 1.983 mCi/microA*h. The radionuclidic purity of (64)Cu was over 99%. In this study, we obtained sufficient qualities and quantities of (64)Cu for therapeutic application and dose monitoring using PET using an ultra-small cyclotron.

The usefulness of radiolabeled 3'-fluoro-3'-deoxythymidine (FLT), a thymidine derivative with affinity to cytoplasmic thymidine kinase 1 (TK1), as a tumor proliferation marker was evaluated using [3H]FLT and 22 cultured tumor cell lines. Asynchronously growing tumor cells were used for studies to mimic in vivo status of tumors. FLT uptake in each cell line was compared with [3H]thymidine ([3H]Thd) uptake and %S-phase fraction, both known as acceptable markers of proliferation. Uptake of the mitochondrial TK2 specific substrate [3H]arabinothymidine ([3H]AraT) was studied as a reference. Metabolic fate of FLT in tumor cells was also analyzed to elucidate the retention mechanism of FLT. [3H]FLT uptake was mildly correlated with the %S-phase fraction (r=0.76, p<0.0001) and correlated better with [3H]Thd uptake (r=0.88, p<0.0001). In contrast, the TK(2) specific substrate, [3H]AraT, was not significantly correlated with the %S-phase fraction (r=0.19, p=0.39), although it showed some correlation with the [3H]Thd uptake (r=0.47, p<0.05). Over 90% of radioactivity of [3H]Thd was found in the DNA fraction after 60 minutes incubation. In contrast, most of the radioactivity of [3H]FLT was found in the acid-soluble fraction (95%). [3H]FLT incorporation into the DNA fraction was negligible (0.2%). The [3H]AraT was mainly distributed in the acid-soluble fraction (70%) and the DNA fraction (20%). From our results, we concluded that FLT uptake in tumor cells reflects tumor cell proliferation. However, much more convincing validation is needed to clarify the difference between FLT and true substrates for DNA synthesis, like thymidine.

To reveal the metabolic fate of acetate in neoplasms that may characterize the accumulation patterns of [1-(11)C]acetate in tumors depicted by positron emission tomography. Four tumor cell lines (LS174T, RPMI2650, A2780, and A375) and fibroblasts in growing and resting states were used. In uptake experiments, cells were incubated with[1-(14)C]acetate for 40 min. [(14)C]CO(2) was measured in the tight-air chamber, and the metabolites in cells were identified by thin layer chromatography and paper chromatography. The glucose metabolic rate of each cell line was measured with [2,6-(3)H]2-deoxy-glucose (DG), and the growth activity of each cell line was estimated by measuring the incorporation of [(3)H]methyl thymidine into DNA. Compared with resting fibroblasts, all four tumor cell lines showed higher accumulation of (14)C activity from [1-(14)C]acetate. These tumor-to-normal ratios of [1-(14)C]acetate were larger than those of DG. Tumor cells incorporated (14)C activity into the lipid-soluble fraction, mostly of phosphatidylcholine and neutral lipids, more prominently than did fibroblasts. The lipid-soluble fraction of (14)C accumulation in cells showed a positive correlation with growth activity, whereas the water-soluble and CO(2) fractions did not. These findings suggest that the high tumor-to-normal ratio of [1-(14)C]acetate is mainly due to the enhanced lipid synthesis, which reflects the high growth activity of neoplasms. This in vitro study suggests that [1-(11)C]acetate is appropriate for estimating the growth activity of tumor cells.

Copper labeled diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) is a promising agent for the imaging of hypoxic tissues. In the present study 64Cu(t1/2 = 12.8 h) labeled Cu-ATSM was used in combination with 11C (t1/2 = 20.3 min) labeled acetate as a regional perfusion marker to visualize hypoxic rat heart tissue in an acute left anterior descending (LAD) coronary artery occluded rat model using an ex vivo tissue slice imaging technique. 64Cu-ATSM was injected intravenously c.a. 10 min after occlusion and rats were sacrificed by cervical dislocation 10 min after injection. Carbon-11-acetate was injected 1 min before sacrifice to obtain a measure of blood flow. The heart was dissected, frozen, and cut into 1-mm thick slices with a gauged slicer, and 11C images were obtained with an electronic autoradiography instrument. After decay of 11C, 64Cu images were obtained in the same manner. In ischemic regions, where there was low 11C accumulation, 64Cu showed high accumulation when compared with normal regions. In rats with a large occlusion, the center of the ischemia did not show any accumulation of either 11C or 64Cu, indicating no blood supply. Cu-ATSM appears to be useful for the detection of hypoxia with contrast being observed at short times (10 min) postinjection.

A multi-aminolinked oligodeoxynucleotide (ODN) was synthesized by substitution of dT with aminolinked dU in the sequence, following conjugation with isothiocyanobenzyl-EDTA (IBE) for 111In labeling. As a model target gene, the c-erbB-2 protooncogene was used. The probability of the number of aminolinked dU in the 20mer ODN was 5, but there were actually 3 and 4 in the selected antisense and sense ODNs, respectively. The IBE/ODN conjugation levels of probes with multi-chelating sites (MCS-probe) were 1.6 (antisense) and 2.4 (sense), more than 50 times higher than those of our previous studies using 5'-end aminolinked ODNs (IBE/ODN = 0.03). Labeling studies using the MCS-probe and 111In indicated that specific radioactivity as high as 48 MBq/nmol could be obtained with a labeling efficiency of over 90%. The 111In-antisense-MCS-probe could bound to sense ODN under physiological conditions, but the 111In-sense-MCA-probe could not. Thus, side-chain modification of ODN for metal labeling is considered to be useful for antisense techniques.

Glucose transporter (GLUT) expression and hexokinase activity are thought to be related to high [18F]-fluorodeoxyglucose (FDG) uptake in tumor cells, but their relative importance is still unknown. To determine which is the predominant factor in FDG uptake in tumor cells, cultured tumor cell lines and a normal cell line were studied in vitro with respect to 2-deoxyglucose (DG) uptake, hexokinase activity, and the initial uptake rate of 3-O-methylglucose (3-O-MG) transport, which is generally accepted as indicating the amount of GLUT expressed on the plasma membrane. In 16 types of tumor cells and one fibroblast cell line, DG uptake was assessed for 60 min, the initial uptake rate of 3-O-MG transport was measured for 1 min, and total hexokinase activity, including that in the mitochondrial fraction, was determined. Across all 16 tumor cell lines, there was a significant correlation between DG uptake and 3-O-MG transport (p = 0.0012, F test), but not between DG uptake and hexokinase activity. Hexokinase activity of the tumor cells was comparable to that of the human fibroblast cells in the exponential growth phase. Most tumor cells showed higher DG uptake and 3-O-MG transport than the human fibroblast cells. The results suggest that DG uptake of cultured tumor cells is governed by GLUT expression, which may be a distinct characteristic of the neoplastic process.

The mechanism of technetium-99m-labeled d,l-hexamethylpropylene amine oxime (99mTc-HMPAO) hyperfixation in damaged brain was elucidated using in vitro metabolic studies. Among the subcellular fractions of mouse brain homogenate, the mitochondrial fraction showed dominant metabolic activity with respect to 99mTc-HMPAO, followed by the cytosolic fraction. The metabolic activity of the mitochondrial fraction was enhanced by heat and detergent treatment, being proportional to the leakage of thiol (SH) compound(s) from the granules. The leaked SH compound(s) had a higher metabolic activity than glutathione, a well-known reductant in cells. 99mTc-HMPAO might be metabolized by mitochondrial SH compound(s) exhibiting strong reductant activity, and hyperfixation might be an indication of mitochondrial damage of the brain.

To determine appropriate use of 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) in the diagnosis of malignant tumors, the mechanism of enhanced FDG uptake in tumor cells was reassessed using in vitro cultured cell lines and 3H-deoxyglucose (DG), in combination with possible parameters of aerobic and anaerobic energy production. The high DG uptake in the tumor cells reflected the dependency of energy production on anaerobic glycolysis, and paradoxically on low levels of aerobic oxidative phosphorylation in mitochondria. We discuss here factors underlying anaerobic glycolysis in tumor cells.

Central benzodiazepine receptor binding was studied in spontaneously epileptic rats (SER) and in their parent strain. Kyoto-Wistar (KW) rats, by ex vivo quantitative autoradiography with [125I]iomazenil. Thirteen-week-old SER developed tonic and absence-like seizures following mild stimulation by tapping at 5 min before injection of the radioligand. When compared with age-matched KW rats, a significant 76% elevation of radioactivity was observed in the SER hippocampus, while there was no difference in hippocampal blood flow obtained using [125I]N-isopropyl-p-iodoamphetamine. Since benzodiazepine receptors act to enhance inhibitory GABA transmission, this finding suggests a possible mechanism for seizure-induced enhancement of inhibition.

This study investigates the possible relationship between myocardial [125I]BMIPP accumulation and ATP concentration, in the salt-induced hypertensive Dahl-strain rats. [125I]BMIPP accumulation in the myocardium was inversely related to the degree of hypertension. On the other hand, ATP levels increased in the myocardium of rats with higher blood pressure showing an inverse relationship with BMIPP accumulation. Further studies are required for elucidating these possible inter-related phenomena.