From in vitro evaluation to human postmortem pre-validation of a radiopaque and resorbable internal biliary stent for liver transplantation applications.

The implantation of an internal biliary stent (IBS) during liver transplantation has recently been shown to reduce biliary complications. To avoid a potentially morbid ablation procedure, we developed a resorbable and radiopaque internal biliary stent (RIBS). We studied the mechanical and radiological properties of RIBS upon in vivo implantation in rats and we evaluated RIBS implantability in human anatomical specimens. For this purpose, a blend of PLA50-PEG-PLA50 triblock copolymer, used as a polymer matrix, and of X-ray-visible triiodobenzoate-poly(ε-caprolactone) copolymer (PCL-TIB), as a radiopaque additive, was used to design X-ray-visible RIBS. Samples were implanted in the peritoneal cavity of rats. The radiological, chemical, and biomechanical properties were evaluated during degradation. Further histological studies were carried out to evaluate the degradation and compatibility of the RIBS. A human cadaver implantability study was also performed. The in vivo results revealed a decline in the RIBS mechanical properties within 3 months, whereas clear and stable X-ray visualization of the RIBS was possible for up to 6 months. Histological analyses confirmed compatibility and resorption of the RIBS, with a limited inflammatory response. The RIBS could be successfully implanted in human anatomic specimens. The results reported in this study will allow the development of trackable and degradable IBS to reduce biliary complications after liver transplantation. STATEMENT OF SIGNIFICANCE: Biliary reconstruction during liver transplantation is an important source of postoperative morbidity and mortality although it is generally considered as an easy step of a difficult surgery. In this frame, internal biliary stent (IBS) implantation is beneficial to reduce biliary anastomosis complications (leakage, stricture). However, current IBS are made of non-degradable silicone elastomeric materials, which leads to an additional ablation procedure involving potential complications and additional costs. The present study provides in vitro and human postmortem implantation data related to the development and evaluation of a resorbable and radiopaque internal biliary stent (RIBS) that could tackle these drawbacks.

In Vivo Assessment of VCAM-1 Expression by SPECT/CT Imaging in Mice Models of Human Triple Negative Breast Cancer.

Recent progress in breast cancer research has led to the identification of Vascular Cell Adhesion Molecule-1 (VCAM-1) as a key actor of metastatic colonization. VCAM-1 promotes lung-metastases and is associated with clinical early recurrence and poor outcome in triple negative breast cancer (TNBC). Our objective was to perform the in vivo imaging of VCAM-1 in mice models of TNBC. The Cancer Genomic Atlas (TCGA) database was analyzed to evaluate the prognostic role of VCAM-1 in TNBC. MDA-MB-231 (VCAM-1+) and control HCC70 (VCAM-1-) TNBC cells were subcutaneously xenografted in mice and VCAM-1 expression was assessed in vivo by single-photon emission computed tomography (SPECT) imaging using 99mTc-cAbVCAM1-5. Then, MDA-MB-231 cells were intravenously injected in mice and VCAM-1 expression in lung metastasis was assessed by SPECT imaging after 8 weeks. TCGA analysis showed that VCAM-1 is associated with a poor prognosis in TNBC patients. In subcutaneous tumor models, 99mTc-cAbVCAM1-5 uptake was 2-fold higher in MDA-MB-231 than in HCC70 (p < 0.01), and 4-fold higher than that of the irrelevant control (p < 0.01). Moreover, 99mTc-cAbVCAM1-5 uptake in MDA-MB-231 lung metastases was also higher than that of 99mTc-Ctl (p < 0.05). 99mTc-cAbVCAM1-5 is therefore a suitable tool to evaluate the role of VCAM-1 as a marker of tumor aggressiveness of TNBC.

Preclinical Evaluation of Mesothelin-Specific Ligands for SPECT Imaging of Triple-Negative Breast Cancer.

Mesothelin is a cell-surface glycoprotein restricted to mesothelial cells overexpressed in several types of cancer, including triple-negative breast cancer not responding to trastuzumab or hormone-based therapies. Mesothelin-targeting therapies are currently being developed. However, the identification of patients potentially eligible for such a therapeutic strategy remains challenging. The objective of this study was to perform the radiolabeling and preclinical evaluation of 99mTc-A1 and 99mTc-C6, two antimesothelin single-domain antibody (sdAb)-derived imaging agents. Methods: A1 and C6 were radiolabeled with 99mTc and evaluated in vitro on recombinant protein and cells, as well as in vivo in xenograft mouse models of the triple-negative breast cancer cell lines HCC70 (mesothelin-positive) and MDA-MB-231 (mesothelin-negative). Results: Both 99mTc-A1 and 99mTc-C6 bound mesothelin with high affinity in vitro, with 99mTc-A1 affinity being 2.4-fold higher than that of 99mTc-C6 (dissociation constant, 43.9 ± 4.0 vs. 107 ± 16 nM, P < 0.05). 99mTc-A1 and 99mTc-C6 remained stable in vivo in murine blood (>80% at 2 h) and ex vivo in human blood (>90% at 6 h). In vivo 99mTc-A1 uptake (percentage injected dose) in HCC70 tumors was 5-fold higher than in MDA-MB-231 tumors and 1.5-fold higher than that of 99mTc-C6 (2.34% ± 0.36% vs. 0.48% ± 0.18% and 1.56% ± 0.43%, respectively, P < 0.01) and resulted in elevated tumor-to-background ratios. In vivo competition experiments demonstrated the specificity of 99mTc-A1 uptake in HCC70 tumors. Conclusion: Mesothelin-positive tumors were successfully identified by SPECT using 99mTc-A1 and 99mTc-C6. Considering its superior characteristics, 99mTc-A1 was selected as the most suitable tool for further clinical translation.

Sympathetic cardiac function in early sepsis: Noninvasive evaluation with [123I]-meta-iodobenzylguanidine (123I-MIBG) in vivo SPECT imaging.

BACKGROUND: Sympathetic system abnormalities have been reported in sepsis-related cardiac dysfunction. The present study aimed at evaluating the potential of the norepinephrine radiolabeled analogue [123I]-meta-iodobenzylguanidine (123I-MIBG) for the noninvasive assessment of modifications in cardiac sympathetic activity occurring in lipopolysaccharide (LPS)-induced experimental acute sepsis by single-photon emission computed tomographic imaging (SPECT). METHODS AND RESULTS: Sepsis was induced in male Wistar rats by intraperitoneal injection of 10 mg·kg-1 lipopolysaccharide (n = 16), whereas control animals (n = 7) were injected with vehicle (NaCl 0.9%). Echocardiography in LPS-injected animals (n = 8) demonstrated systolic and diastolic cardiac dysfunction. 123I-MIBG was injected 1 hour after LPS or vehicle administration (n = 8 and 7, respectively), and in vivo SPECT imaging was performed early and late (20 and 180 minutes) after tracer injection prior to animal euthanasia and ex vivo assessment of 123I-MIBG biodistribution. Global and 17-segment SPECT image analysis indicated that early 123I-MIBG activity was not affected by LPS treatment, whereas late cardiac tracer activity was significantly decreased in LPS-treated animals. Consequently, the cardiac washout of 123I-MIBG was significantly higher in LPS-treated (63.3% ± 4.0%) than that in control animals (56.7% ± 5.8%) (P < .05). CONCLUSION: Sepsis-induced modifications in cardiac sympathetic nervous system activity were evidenced by noninvasive in vivo 123I-MIBG SPECT imaging.

Evaluation of Antiatherogenic Properties of Ezetimibe Using 3H-Labeled Low-Density-Lipoprotein Cholesterol and 99mTc-cAbVCAM1-5 SPECT in ApoE-/- Mice Fed the Paigen Diet.

The addition of ezetimibe, an intestinal cholesterol absorption inhibitor, to statin therapy has recently shown clinical benefits in the Improved Reduction of Outcomes: Vytorin Efficacy International Trial by reducing low-density-lipoprotein (LDL) cholesterol levels more than statin therapy alone. Here, we investigated the mechanisms by which inhibition of intestinal cholesterol absorption might contribute to the clinically observed reduction in cardiovascular events by evaluating its effect on inflammatory plaque development in apolipoprotein E-/- mice. Methods: Apolipoprotein E-/- mice were fed the Paigen diet (1.25% cholesterol, 0.5% cholic acid, and 15% fat) without or with ezetimibe (7 mg/kg/d) for 6 wk. In a first set of mice (n = 15), we intravenously injected 3H-cholesteryl oleate-labeled human LDL to test whether ezetimibe promotes LDL-derived cholesterol fecal excretion. In a second set (n = 20), we used the imaging agent 99mTc-cAbVCAM1-5 to evaluate expression of an inflammatory marker, vascular cell adhesion molecule 1 (VCAM-1), in atherosclerotic plaques. In a third set (n = 21), we compared VCAM-1 expression with 99mTc-cAbVCAM1-5 uptake in various tissues. Results: Mice treated with ezetimibe showed a 173% higher LDL-cholesteryl ester plasma disappearance rate (P < 0.001 vs. control) after 3H-cholesteryl oleate-labeled LDL injection. At 96 h after injection, the hepatic fraction of 3H-tracer was 61% lower in mice treated with ezetimibe (P < 0.001). Meanwhile, LDL-derived 3H-cholesterol excretion in the feces was 107% higher (P < 0.001). The antiatherogenic effect of ezetimibe monitored by 99mTc-cAbVCAM1-5 SPECT showed a 49% reduction in aortic tracer uptake (percentage injected dose per cubic centimeter, 0.95 ± 0.04 vs. 1.87 ± 0.11; P < 0.01). In addition to hypercholesterolemia, the proinflammatory Paigen diet significantly increased VCAM-1 expression with respect to the control group in various tissues, including the aorta, and this expression correlated strongly with 99mTc-cAbVCAM1-5 uptake (r = 0.75; P < 0.05). Conclusion: Inhibition of intestinal cholesterol absorption with ezetimibe promotes antiatherosclerotic effects through increased LDL cholesterol catabolism and LDL-derived cholesterol fecal excretion and reduces inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding ezetimibe to a statin therapy.

99mTc-cAbVCAM1-5 imaging is a sensitive and reproducible tool for the detection of inflamed atherosclerotic lesions in mice.

UNLABELLED: (99m)Tc-cAbVCAM1-5, a single-domain antibody fragment directed against mouse or human vascular cell adhesion molecule 1 (VCAM-1), recently has been proposed as a new imaging agent for the detection of inflamed atherosclerotic lesions. Indeed, in a mouse model of atherosclerosis, (99m)Tc-cAbVCAM1-5 specifically bound to VCAM-1-positive lesions, thereby allowing their identification on SPECT images. The purpose of the present study was to investigate (99m)Tc-cAbVCAM1-5 imaging sensitivity using a reference statin therapy. METHODS: Thirty apolipoprotein E-deficient mice were fed a western-type diet. First, the relationship between the level of VCAM-1 expression and (99m)Tc-cAbVCAM1-5 uptake was evaluated in 18 mice using immunohistochemistry and autoradiography. Second, longitudinal SPECT/CT imaging was performed on control (n = 9) or atorvastatin-treated mice (0.01% w/w, n = 9). RESULTS: (99m)Tc-cAbVCAM1-5 uptake in atherosclerotic lesions correlated with the level of VCAM-1 expression (P < 0.05). Atorvastatin exerted significant antiatherogenic effects, and (99m)Tc-cAbVCAM1-5 lesion uptake was significantly reduced in 35-wk-old atorvastatin-treated mice, as indicated by ex vivo γ-well counting and autoradiography (P < 0.05). SPECT imaging quantification based on contrast-enhanced CT was reproducible (interexperimenter intraclass correlation coefficient, 0.97; intraexperimenter intraclass correlation coefficient, 0.90), and yielded results that were highly correlated with tracer biodistribution (r = 0.83; P < 0.0001). Therefore, reduced (99m)Tc-cAbVCAM1-5 uptake in atorvastatin-treated mice was successfully monitored noninvasively by SPECT/CT imaging (0.87 ± 0.06 vs. 1.11 ± 0.09 percentage injected dose per cubic centimeter in control group, P < 0.05). CONCLUSION: (99m)Tc-cAbVCAM1-5 imaging allowed the specific, sensitive, and reproducible quantification of VCAM-1 expression in mouse atherosclerotic lesions. (99m)Tc-cAbVCAM1-5 therefore exhibits suitable characteristics for the evaluation of novel antiatherogenic agents.