Takanori Sasaki

A Novel PET Imaging Using 64Cu-Labeled Monoclonal Antibody against Mesothelin Commonly Expressed on Cancer Cells

Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

Positron emission tomography to elucidate pharmacokinetic differences of regioisomeric retinoid x receptor agonists.

RXR partial agonist NEt-4IB (2a, 6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: EC50 = 169 nM, E max = 55%) showed a blood concentration higher than its E max after single oral administration at 30 mg/kg to mice, and repeated oral administration at 10 mg/kg/day to KK-A(y) mice afforded antitype 2 diabetes activity without the side effects caused by RXR full agonists. However, RXR full agonist NEt-3IB (1a), in which the isobutoxy and isopropyl groups of 2a are interchanged, gave a much lower blood concentration than 2a. Here we used positron emission tomography (PET) with tracers [(11)C]1a, [(11)C]2a and fluorinated derivatives [(18)F]1b, [(18)F]2b, which have longer half-lives, to examine the reason why 1a and 2a exhibited significantly different blood concentrations. As a result, the reason for the high blood concentration of 2a after oral administration was found to be linked to higher intestinal absorbability together with lower biliary excretion, compared with 1a.

Robotically Driven CT-guided Needle Insertion: Preliminary Results in Phantom and Animal Experiments

Purpose To evaluate the accuracy of the remote-controlled robotic computed tomography (CT)-guided needle insertion in phantom and animal experiments. Materials and Methods In a phantom experiment, 18 robotic and manual insertions each were performed with 19-gauge needles by using CT fluoroscopic guidance for the evaluation of the equivalence of accuracy of insertion between the two groups with a 1.0-mm margin. Needle insertion time, CT fluoroscopy time, and radiation exposure were compared by using the Student t test. The animal experiments were approved by the institutional animal care and use committee. In the animal experiment, five robotic insertions each were attempted toward targets in the liver, kidneys, lungs, and hip muscle of three swine by using 19-gauge or 17-gauge needles and by using conventional CT guidance. The feasibility, safety, and accuracy of robotic insertion were evaluated. Results The mean accuracies of robotic and manual insertion in phantoms were 1.6 and 1.4 mm, respectively. The 95% confidence interval of the mean difference was -0.3 to 0.6 mm. There were no significant differences in needle insertion time, CT fluoroscopy time, or radiation exposure to the phantom between the two methods. Effective dose to the physician during robotic insertion was always 0 μSv, while that during manual insertion was 5.7 μSv on average (P < .001). Robotic insertion was feasible in the animals, with an overall mean accuracy of 3.2 mm and three minor procedure-related complications. Conclusion Robotic insertion exhibited equivalent accuracy as manual insertion in phantoms, without radiation exposure to the physician. It was also found to be accurate in an in vivo procedure in animals.

In vivo distribution of single chain variable fragment (scFv) against atherothrombotic oxidized LDL/β2-glycoprotein I complexes into atherosclerotic plaques of WHHL rabbits: Implication for clinical PET imaging

BACKGROUND Oxidized LDL (oxLDL) can exist as a complex with β2-glycoprotein I (β2GPI) in plasma/serum of patients with non-autoimmune atherosclerotic disease or antiphospholipid syndrome (APS). Nonetheless, direct in vivo evidence supporting the pathophysiological involvement of oxLDL/β2GPI complexes and specific autoantibody against the complexes in developing atherothrombosis has yet been established. In the present study, we demonstrated in vivo distribution of single chain variable fragment of IgG anti-oxLDL/β2GPI complexes (3H3-scFv) in Watanabe heritable hyperlipidemic (WHHL) rabbits by PET/CT imaging. METHODS An antibody-based PET probe, 64Cu-3H3-scFv, was established, and WHHL rabbits were applied for a non-autoimmune atherosclerotic model to demonstrate in vivo distribution of the probe. RESULTS 3H3-scFv has exhibits specificity towards β2GPI complexed with oxLDL but neither a free form of β2GPI nor oxLDL alone. Post-intravenous administration of 64Cu-3H3-scFv into WHHL rabbits has demonstrated a non-invasive approach for in vivo visualization of atherosclerotic lesion. The imaging probe achieved ideal blood clearance and distribution for optimal imaging capacity in 24h, significantly shorter than that of an intact IgG-based imaging probe. 64Cu-3H3-scFv targeted on atherosclerotic plaques in aortas of WHHL rabbits where extensive accumulation of lipid deposits was observed by lipid staining and autoradiography. The accumulation of 64Cu-3H3-scFv in aortic segments of WHHL rabbits was 2.8-folds higher than that of controls (p=0.0045). CONCLUSIONS The present in vivo evidence supports the pathophysiological involvement of oxLDL/β2GPI complexes in atherosclerotic complications of WHHL rabbits. 64Cu-3H3-scFv represents a novel PET imaging probe for non-invasive pathophysiological assessment of oxLDL/β2GPI complexes accumulated in atherosclerotic plaques.

Endogenous Leu332Gln mutation in p53 disrupts the tetramerization ability in a canine mammary gland tumor cell line

Mutations in the p53 gene are associated with more than half of all human cancers. These mutations often cause a disruption of the tumor-suppressor function of p53 and induce genomic instabilities. Wild‑type p53 requires tetramerization to function as an initiator of cell cycle arrest and apoptosis. Although alterations in p53 tetramerization caused by mutation have been well studied, there are few cell lines containing an endogenous mutation in the tetramerization domain of p53. Here, we report the discovery of a canine mammary gland tumor cell line CTB‑m2, which contains the Leu332Gln (L332Q) mutation corresponding to Leu344 in the tetramerization domain of human p53. Although CTB‑m2 cells are genetically heterozygous for the Leu332Gln mutation, the mutant mRNA was almost exclusively expressed. CTB‑m2 cells showed enhanced cell proliferation compared to wild‑type p53-expressing CTB‑m cells of the same lineage. A p53 tetramerization reporter assay showed that the ability of the p53 mutant to form tetramers was significantly lower than that of wild‑type p53. An immunoblot analysis of cross-linked p53 oligomerized forms demonstrated that the L332Q mutant lacked the ability to form tetramers but retained the ability to form dimers. These data suggest that the p53 mutant cell line CTB‑m2 could be a useful tool for analyzing the precise tetramerization mechanisms of p53 and verifying the effects of therapeutic agents against tumors expressing p53 mutants that lack the ability to tetramerize.

Robotic Insertion of Various Ablation Needles Under Computed Tomography Guidance: Accuracy in Animal Experiments

OBJECTIVE To evaluate the accuracy of robotic insertion of various ablation needles at various locations under computed tomography (CT) guidance in swine. MATERIALS AND METHODS The robot was used for CT-guided insertion of four ablation needles, namely a single internally cooled radiofrequency ablation (RFA) needle (Cool-tip), a multi-tined expandable RFA needle (LeVeen), a cryoablation needle (IceRod), and an internally cooled microwave ablation needle (Emprint). One author remotely operated the robot with the operation interface in order to orient and insert the needles under CT guidance. Five insertions of each type of ablation needle towards 1.0-mm targets in the liver, kidney, lung, and hip muscle were attempted on the plane of an axial CT image in six swine. Accuracy of needle insertion was evaluated as the three-dimensional length between the target centre and needle tip. The accuracy of needle insertion was compared according to the type of needle used and the location using one-way analysis of variance. RESULTS The overall mean accuracy of all four needles in all four locations was 2.8 mm. The mean accuracy of insertion of the Cool-tip needle, LeVeen needle, IceRod needle, and Emprint needle was 2.8 mm, 3.1 mm, 2.5 mm, and 2.7 mm, respectively. The mean accuracy of insertion into the liver, kidney, lung, and hip muscle was 2.7 mm, 2.9 mm, 2.9 mm, and 2.5 mm, respectively. There was no significant difference in insertion accuracy among the needles (P = .38) or the locations (P = .53). CONCLUSION Robotic insertion of various ablation needles under CT guidance was accurate regardless of type of needle or location in swine.

Anti-high Mobility Group Box 1 Antibody Ameliorates Albuminuria in MRL/lpr Lupus-Prone Mice

We evaluated the efficacy of a neutralizing anti-high mobility group box 1 (HMGB1) monoclonal antibody in MRL/lpr lupus-prone mice. The anti-HMGB1 monoclonal antibody (5 mg/kg weight) or class-matched control immunoglobulin G2a (IgG2a) was administered intravenously twice a week for 4–15 weeks. Urine albumin was monitored, and histological evaluation of the kidneys was conducted at 16 weeks. Lymphadenopathies were evaluated by 1-(2′-deoxy-2′-[18F]fluoro-β-D-arabinofuranosyl)cytosine ([18F]FAC) positron emission tomography/computed tomography (PET/CT) at 12 weeks. Following 4-week treatment, [18F]FAC-PET/CT showed similar accumulation in cervical and axillary lymph nodes at 12 weeks of age. However, anti-HMGB1 monoclonal antibody sufficiently inhibited the increase in albuminuria compared to an isotype control following 15-week treatment. Complement deposition was also improved; however, there were no significant differences in IgG deposition and renal pathological scores between the two groups. Anti-double-stranded DNA (dsDNA) antibody titers and cytokine and chemokine levels were also unaltered. Although there were no significant differences in glomerular macrophage infiltration, neutrophil infiltration was significantly decreased by the anti-HMGB1 monoclonal antibody. Antagonizing HMGB1 treatment suppressed HMGB1 translocation from nuclei in the kidney and suppressed neutrophil extracellular traps. The anti-HMGB1 monoclonal antibody demonstrated therapeutic potential against albuminuria in lupus nephritis by inhibiting neutrophil recruitment and neutrophil extracellular traps.

Synthesis of 11C-Labeled RXR Partial Agonist 1-[(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino]benzotriazole-5-carboxylic Acid (CBt-PMN) by Direct [11C]Carbon Dioxide Fixation via Organolithiation of Trialkyltin Precursor and PET Imaging Thereof

The retinoid X receptor (RXR) partial agonist 1-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino]benzotriazole-5-carboxylic acid (1; CBt-PMN, Emax = 75%, EC50 = 143 nM) is a candidate for treatment of central nervous system (CNS) diseases such as Alzheimers and Parkinsons diseases based on reports that RXR-full agonist 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene) shows therapeutic effects on these disease in rodent models. Here, we synthesized carbon-11-labeled ([11C]1) as a tracer for positron emission tomography (PET) and used it in a PET imaging study to examine the brain uptake and biodistribution of 1. We found that 11CO2 fixation after tin-lithium exchange at -20 °C afforded [11C]1. This methodology may also be useful for synthesizing 11CO2H-PET tracer derivatives of other compounds bearing π-rich heterocyclic rings. A PET/CT imaging study of [11C]1 in mice indicated 1 is distributed to the brain and is thus a candidate for treatment of CNS diseases.

The Function of β2-glycoprotein I in Angiogenesis and Its in Vivo Distribution in Tumor Xenografts

Intact β2-glycoprotein I (iβ2GPI) is a glycoprotein that regulates coagulation and fibrinolysis. Nicked β2GPI (nβ2GPI) possesses an angiogenic property at a relatively low concentration, and an antiangiogenic property at a high concentration. Here we investigated the functions of βi 2GPI and nβ2GPI in vascular endothelial growth factor (VEGF)-A-induced endothelial cell proliferation and tube formation. We used noninvasive PET imaging to analyze the ｉｎ　ｖｉｖｏ distribution of intravenously injected β2GPI variants in tumor lesions in mice. iβ2GPI was incubated with plasmin to obtain nβ2GPI, and its N-terminal sequence was analyzed. nβ2GPI had at least one other cleavage site upstream of the β2GPIs domain V, whereas the former plasmin-cleavage site locates between K317 and T318. Both of intact and nicked β2GPI significantly inhibited the VEGF-A-induced cell proliferation and the tube formation of human umbilical vein endothelial cells (HUVECs). PET imaging visualized considerably distributed intensities of all tested β2GPI variants in tumor lesions of pancreatic tumor cell-xenografts. These results indicate that β2GPI may be physiologically and pathophysiologically important in the regulation of not only coagulation and fibrinolysis, but also angiogenesis.