[en] Introduction: High-dose ¹⁸F-FDG can provide targeted nuclear therapy of cancer. Endothelial cell injury is a key determinant of tumor response to radiotherapy. Here, we tested the hypothesis that activation of endothelial cell glycolytic metabolism with nitric oxide can enhance the therapeutic effect of high-dose ¹⁸F-FDG. Methods: Calf pulmonary artery endothelial (CPAE) cells were treated with graded doses of ¹⁸F-FDG. Glycolysis was stimulated by 24 h of exposure to the nitric oxide donor, sodium nitroprusside (SNP). Cell viability was assessed by MTT and clonogenic assays. Apoptosis was evaluated by ELISA of cytosolic DNA fragments and Western blots of cleaved caspase-3. Results: SNP stimulation (0.1 and 1 mM) augmented CPAE cell ¹⁸F-FDG uptake to 2.6- and 4.6-fold of controls without adverse effects. Treatment with 333 μCi/ml ¹⁸F-FDG alone reduced viable cell number to 35.4% of controls by Day 3. Combining 0.1 mM SNP stimulation significantly enhanced the killing effect, reducing cell numbers to 19.2% and 39.2% of controls by 333 and 167 μCi/ml of ¹⁸F-FDG, respectively. ¹⁸F-FDG also suppressed clonogenic survival to 80.8% and 43.2% of controls by 83 and 167 μCi/ml, which was again intensified by SNP to 59.7% and 21.1% of controls. The cytotoxic effect of ¹⁸F-FDG was attributed to induction of apoptosis as shown by increased cytosolic fragmented DNA and cleaved caspase-3 levels (26.4% and 30.7% increases by 167 μCi/ml). Combining SNP stimulation significantly increased both of these levels to 1.8-fold of control cells. Conclusion: High-dose ¹⁸F-FDG combined with nitric oxide-stimulated glycolysis is an effective method to inhibit endothelial cell survival and promote apoptosis. These results suggest a potential role of this strategy for targeted radiotherapy of angiogenic vasculature.

[en] Since the ability to monitor in vivo monocyte trafficking would contribute to our understanding of the pathophysiology of various inflammatory disorders, we investigated the feasibility of labeling human monocytes with ¹⁸F-FDG. Human monocytes were separated by Ficoll/Hypaque gradient and purity was assessed by flow cytometry. The influence of insulin and/or glucose on labeling efficiency was evaluated. Cell viability and activation was measured with trypan blue exclusion and hydrogen peroxide assays, respectively. Label stability was measured for up to 18 hr, and the effect of insulin pre-incubation on FDG washout was investigated. PET images were acquired in SD rats at various time points after injection of FDG labeled monocytes. Monocytes were >85% pure, and labeling efficiency was 35% for 1x106 cells after 40 min incubation with 2 mCi ¹⁸F-FDG without insulin. Pre-incubation with 10∼100 nM insulin significantly increased FDG uptake which reached 400% of baseline levels, whereas presence of glucose or serum decreased FDG uptake. Labeled cells were >90% viable for up to 22 hr, and the labeling process did appear to significantly activate cells, Washout studies however, demonstrated gradual washout of the FDG from monocytes after initial uptake PET images of FDG labeled monocytes in SD rats showed consistent findings. Utilizing insulin effects on cellular glucose metabolism may be a feasible way of labeling monocytes with ¹⁸F-FDG for PET imaging. However, gradual washout of FDG after initial uptake poses as a potential problem which needs to be addressed before practical application

[en] While ¹⁸F-FDG PET has an important role for detecting and monitoring breast carcinoma, the metabolic effects of estrogen stimulation on breast cancer cells may potentially affect their glucose handling. We therefore investigated the effect of estrogen stimulation on FDG uptake in a breast cancer cell line. Estrogen responsive human breast cancer MCF-7 cells were cultured in estrogen free media and were stimulated with 10^-6∼10^-11 M 17β-estradial (E₂) for 2 to 72 hours. Levels of ¹⁸F-FDG uptake was measured after 40 min incubation. Cell number was assessed with MTT assays. The effect of P13-kinase inhibition was evaluated with 200 nM wortmannin added immediately before E₂ stimulation. FDG uptake was significantly augmentated after 48 ∼ 72 hr stimulation by E₂, with peak effects at concentrations of 10^-8∼10^-10M. After 48 hr stimulation with 10^-9 M E₂. FDG uptake averaged 176 ±6% of control levels (p<0.000). MTT assays did not show significant differences in cell number between groups at this stage. FDG uptake was also increased to 140±16% of controls after 24 hr stimulation (p<0.02), but the effect was significantly inhibited by wortmannin. Estradiol stimulation significantly enhances FDG uptake in estrogen responsive breast cancer cells, which appears to be at least partially mediated by the P13 kinase pathway. This phenomena may have important implication in interpretation of FDG uptake values for monitoring treatment response, since the patient may have different estrogen levels or may undergo antiestrogen treatment

[en] Since angiostatin is a promising anticancer agent that target tumor endothelial cells, it may have advantages over many current tumor imaging agents by overcoming problems such as poor delivery or multi-drug resistance. We therefore synthesized radiolabeled agniostatin and tested it in vivo. ¹²³-angiostatin was synthesized using the Bolton Hunter method. ¹²³I labeled plasminogen lysin-binding-site (LBS) was also synthesized. Blood clearance of he radiotracer was measured in SD rats, while tissue distribution was assessed in ICR mice at 1,4, and 18 hr. Pinhole scintigraphy was performed in SD rats and in nude mice bearing RR 1022 tumors at various time points. Radiochemical yield of ¹²³I-angiostatin approximated 20%. In vivo distribution demonstrated stability of the label for at least 20 hr. ¹²³I-angiostatin was cleared from the circulation in a biexponential manner with rapid early clearance followed by a slower rate of elimination Tissue distribution in mice showed the highest uptake in the kidneys which was the major route of excretion. This was followed by the lung, liver, and myocardium whose uptake of 1.5∼2% ID/gm at 1 hrs gradually decreased over time (all p<0.05). Skeletal muscle uptake was relatively low (<0.3 %ID/gm). ¹²³I-angiostatin and ¹²³I-LBS images in SD rates showed a similar distribution. Blood pool activity gradually cleared while tumor uptake increased over time, resulting in a high tumor to non tumor ratio at 20 hr. ¹²³I-angiostatin has promising potential as a new tumor imaging agent. Further study is warranted to assess its mechanism of uptake and precise role in cancer imaging

[en] Nicotinic acetylcholine receptors (nAChRs), which mediate excitatory neurotransmission, are known to participate in various neurophysiological functions. Severe losses of nAChRs have been noted in Alzheimer's and Parkinson's diseases. Therefore, noninvasive and quantitative imaging of nAChRs would offer a better understanding on the function of these receptors. In this study, 2 -(¹⁸F)fluoro-A85380 ([¹⁸F]1), an α₄β₂ nAChRs radioligand, was prepared using one HPLC purification and evaluated in mouse brain, and the results were compared with those in the literature. (¹⁸F)1 was prepared by (¹⁸F)fluorination of the iodo precursor followed by acidic deprotection and then purified by HPLC. Tissue distribution studies were performed in mouse brain at the indicated time points and the result was expressed as %ID/g. Inhibition studies were also carried out with pretreatment of various ligands. One HPLC purification method gave the desired product in 15-20% radiochemical yield and with high specific activity (38-55 GBq/μmol). Tissue distribution studies showed that (¹⁸F)1 specifically labeled nAChRs in mouse brain with a high thalamus to cerebellum uptake ratio (13.8 at 90 min). Inhibition studies demonstrated selective binding of (¹⁸F)1 to nAChRs, blocking the uptake of the (¹⁸F) 1 in nAChR-rich regions by selective ligands such as cytisine and nicotine which are well-known nAChRs agonists. This study demonstrated that the (¹⁸F)1 produced by the method using one HPLC purification gave the results similar to those reported in the lieterature. Therefore, this synthetic method can be readily applied to the routine preparation of (¹⁸F)1, a PET radioligand for α₄β₂ nAChRs imaging

[en] There has recently been increasing interest in the development of radioprobes that specifically target proteins transcribed from expression of reporter genes of interest. The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ gene. We synthesised and purified radioiodine-labelled phenylethyl-β-d-thiogalactopyranoside (PETG), a competitive inhibitor specific against Escherichia coli β-galactosidase. We showed that [¹²⁵I]iodo-PETG specifically binds to β-galactosidase as verified by column chromatography and polyacrylamide gel electrophoresis after incubation of radiotracer with the protein. We also showed through enzyme kinetic studies that iodo-PETG retains inhibitory action against β-galactosidase activity. COS-7 cells infected with a recombinant adenovirus expressing the lacZ gene had viral titre-dependent enhancements in [¹²⁵I]iodo-PETG uptake (r²=0.897; P=0.001), which reached up to 642.5%±16.7% of control levels (P<0.00001). Moreover, the level of uptake was highly correlated to luminescent measurements of β-galactosidase activity (r²=0.878; P<0.0001). These results confirm that radioiodine-labelled PETG specifically targets β-galactosidase and that its uptake rates faithfully reflect levels of expression of the lacZ reporter gene. Further investigations were performed in nude mice bearing human neuroblastoma tumours transferred with the lacZ gene. Compared with control tumours, lacZ-expressing tumours were slightly better visualised on [¹²³I]iodo-PETG images and had a modest increase in tumour to muscle count ratio (2.6±0.2 vs 1.9±0.1, P<0.05). The present results provide proof-of-principle for the potential of radiolabelled inhibitors as promising radiotracers to monitor lacZ gene expression levels. Future modifications to improve cell permeability should enhance in vivo contrast levels and may allow the use of radiolabelled β-galactosidase inhibitors for non-invasive monitoring of lacZ gene expression. (orig.)

[en] In this study, we investigated the effects of hydrazinonicotinamide (HYNIC)—a bifunctional crosslinker widely used to ^99mTc radiolabel protein and nanoparticles for imaging studies—on quantum dot opsonization, macrophage engulfment and in vivo kinetics. In streptavidin-coated quantum dots (SA-QDots), conjugation with HYNIC increased the net negative charge without affecting the zeta potential. Confocal microscopy and fluorescence-activated cell sorting showed HYNIC attachment to suppress SA-QDot engulfment by macrophages. Furthermore, HYNIC conjugation suppressed surface opsonization by serum protein including IgG. When intravenously injected into mice, HYNIC conjugation significantly prolonged the circulation of SA-QDots and reduced their hepatosplenic uptake. Diminished reticuloendothelial system clearance of SA-QDots and aminoPEG-QDots by HYNIC conjugation was also demonstrated by in vivo and ex vivo optical imaging. The effects of HYNIC on the opsonization, phagocytosis and in vivo kinetics of quantum dots were reversed by removal of the hydrazine component from HYNIC. Thus, surface functionalization with HYNIC can improve the in vivo kinetics of quantum dots by reducing phagocytosis via suppression of surface opsonization. (paper)

[en] Small animal imaging with meta-iodobenzylguanidine (MIBG) allows characterization of animal models, optimization of tumor treatment strategies, and monitoring of gene expression. Anesthetic agents, however, can affect norepinephrine (NE) transport and systemic sympathetic activity. We thus elucidated the effects of anesthetic agents on MIBG transport and biodistribution. SK-N-SH neuroblastoma and PC-12 pheochromocytoma cells were measured for ¹²³I-MIBG uptake after treatment with ketamine (Ke), xylazine (Xy), Ke/Xy, or pentobarbital (Pb). NE transporters were assessed by Western blots. Normal ICR mice and PC-12 tumor-bearing mice were injected with ¹²³I-MIBG 10 min after anesthesia with Ke/Xy, Ke, Xy, or Pb. Plasma NE levels and MIBG biodistribution were assessed. Cellular ¹²³I-MIBG uptake was dose-dependently inhibited by Ke and Xy but not by Pb. Treatment for 2 h with 300 μM Ke, Xy, and Ke/Xy decreased uptake to 46.0 ± 1.6, 24.8 ± 1.5, and 18.3 ± 1.6% of controls. This effect was completely reversed by fresh media, and there was no change in NE transporter levels. In contrast, mice anesthetized with Ke/Xy showed no decrease of MIBG uptake in target organs. Instead, uptakes and organ-to-blood ratios were increased in the heart, lung, liver, and adrenals. Plasma NE was notably reduced in the animals with corresponding decreases in blood MIBG, which partly contributed to the increase in target organ uptake. In spite of their inhibitory effect at the transporter level, Ke/Xy anesthesia is a satisfactory method for MIBG imaging that allows favorable target tissue uptake and contrast by reducing circulating NE and MIBG. (orig.)

[en] Angiostatin (AS) is a potent antiangiogenic agent which inhibits tumor growth through specific action on proliferating endothelial cells. Imaging of radiolabeled AS would enhance our knowledge on the pharmacokinetics of AS and might provide useful information relating to tumor neovasculature. We therefore investigated the potential of radiolabeled AS as a novel tumor imaging agent. Human angiostatin was radioiodine labeled using the lactoperoxidase method. Competition binding studies showed a dose-dependent inhibition of ¹²⁵I-AS binding to endothelial cells by excess unlabeled AS, and a displacement curve demonstrated that specific binding was dose dependent and saturable, with a K_d value of 169 nM. Gel analysis showed that ¹²⁵I-AS remained stable in serum for up to 24 h without significant degradation. Intravenously injected ¹²⁵I-AS in rats was cleared from the blood in an exponential fashion. Biodistribution data from human colon cancer-bearing Balb/C nude mice showed high uptake in the kidneys, stomach, liver, and lungs. Tumor uptake was 3.2±0.7, 2.6±0.2, and 1.7±0.2%ID/g at 2, 4, and 9 h after injection, respectively. Tumor to muscle count ratio increased from 3.1±0.5 at 2 h to 4.4±0.5 at 9 h. Serial scintigraphy from 1 to 5 h after ¹²³I-AS injection demonstrated high uptake in the kidneys and bladder, consistent with renal excretion. There was clear demarcation of tumor by 1 h, with gradual increase in contrast over time (4-h tumor to contralateral thigh ratio =4.7±1.1). Thus, radioiodine-labeled angiostatin binds specifically to endothelial cells and has potential as a novel tumor imaging agent. (orig.)

[en] Endothelial cells and their metabolic state regulate glucose transport into underlying tissues. Here, we show that low oxygen tension stimulates human umbilical vein endothelial cell ¹⁸F–fluorodeoxyglucose (¹⁸F–FDG) uptake and lactate production. This was accompanied by augmented hexokinase activity and membrane Glut-1, and increased accumulation of hypoxia-inducible factor-1α (HIF1α). Restoration of oxygen reversed the metabolic effect, but this was blocked by HIF1α stabilization. Hypoxia-stimulated ¹⁸F–FDG uptake was completely abrogated by silencing of HIF1α expression or by a specific inhibitor. There was a rapid and marked increase of reactive oxygen species (ROS) by hypoxia, and ROS scavenging or NADPH oxidase inhibition completely abolished hypoxia-stimulated HIF1α and ¹⁸F–FDG accumulation, placing ROS production upstream of HIF1α signaling. Hypoxia-stimulated HIF1α and ¹⁸F–FDG accumulation was blocked by the protein kinase C (PKC) inhibitor, staurosporine. The phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, blocked hypoxia-stimulated ¹⁸F–FDG uptake and attenuated hypoxia-responsive element binding of HIF1α without influencing its accumulation. Thus, ROS-driven HIF1α accumulation, along with PKC and PI3K signaling, play a key role in triggering accelerated glycolysis in endothelial cells under hypoxia, thereby contributing to ¹⁸F–FDG transport.