Helper-dependent adenovirus-mediated gene transfer of a secreted LDL receptor/transferrin chimeric protein reduces aortic atherosclerosis in LDL receptor-deficient mice.

Familial hypercholesterolemia (FH) is a genetic hyperlipidemia characterized by elevated concentrations of plasma LDL cholesterol. Statins are not always effective for the treatment of FH patients; unresponsive patients have poor prognosis and rely on LDL apheresis. In the past, we developed safe and effective gene therapy strategies for the expression of anti-atherogenic proteins using PEGylated helper-dependent adenoviral (HD-Ad) vectors. We recently developed a HD-Ad vector for the expression of the soluble form of the extracellular portion of the human LDL receptor (LDLR) fused with a rabbit transferrin dimer (LDLR-TF). We evaluated the efficacy of the LDLR-TF chimeric protein  in CHOLDLA7, a cell line lacking LDLR expression, restoring the ability to uptake LDL. Subsequently, we administered intravenously 1 × 10E13 vp/kg of this vector in LDLR-deficient mice and observed amelioration of lipid profile and reduction of aortic atherosclerosis. Finally, we studied LDL distribution after HD-Ad vector-mediated expression of LDLR-TF in LDLR-deficient mice and found LDL accumulation in liver, and in heart and intestine. These results support the possibility of lowering LDL-C levels and reducing aortic atherosclerosis using a secreted therapeutic transgene; the present strategy potentially can be modified and adapted to non-systemic gene transfer with expression of the secreted chimeric protein in muscle or other tissues. Intramuscular or local administration strategies could improve the safety profile of this strategy and facilitate applicability.

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-ß-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

Multimodal imaging for a theranostic approach in a murine model of B-cell lymphoma with engineered nanoparticles.

Nanoparticles (NPs) are a promising tool for in vivo multimodality imaging and theranostic applications. Hyaluronic acid (HA)-based NPs have numerous active groups that make them ideal as tumor-targeted carriers. The B-lymphoma neoplastic cells express on their surfaces a clone-specific immunoglobulin receptor (Ig-BCR). The peptide A20-36 (pA20-36) selectively binds to the Ig-BCR of A20 lymphoma cells. In this work, we demonstrated the ability of core-shell chitosan-HA-NPs decorated with pA20-36 to specifically target A20 cells and reduce the tumor burden in a murine xenograft model. We monitored tumor growth using high-frequency ultrasonography and demonstrated targeting specificity and kinetics of the NPs via in vivo fluorescent reflectance imaging. This result was also confirmed by ex vivo magnetic resonance imaging and confocal microscopy. In conclusion, we demonstrated the ability of NPs loaded with fluorescent and paramagnetic tracers to act as multimodal imaging contrast agents and hence as a non-toxic, highly specific theranostic system.

Advances in multimodal molecular imaging.

Preclinical molecular imaging is an emerging field. Improving the ability of scientists to study the molecular basis of human pathology in animals is of the utmost importance for future advances in all fields of human medicine. Moreover, the possibility of developing new imaging techniques or of implementing old ones adapted to the clinic is a significant area. Cardiology, neurology, immunology and oncology have all been studied with preclinical molecular imaging. The functional techniques of photoacoustic imaging (PAI), fluorescence molecular tomography (FMT), positron emission tomography (PET), and single photon emission computed tomography (SPECT) in association with each other or with the anatomic reference provided by computed tomography (CT) as well as with anatomic and functional information provided by magnetic resonance (MR) have all been proficiently applied to animal models of human disease. All the above-mentioned imaging techniques have shown their ability to explore the molecular mechanisms involved in animal models of disease. The clinical translatability of most of the techniques motivates the ongoing study of their possible fields of application. The ability to combine two or more techniques allows obtaining as much information as possible on the molecular processes involved in pathologies, reducing the number of animals necessary in each experiment. Merging molecular probes compatible with various imaging technique will further expand the capability to achieve the best results.

Molecular imaging of neuroinflammation in preclinical rodent models using positron emission tomography.

Neuroinflammation (NI) is an adaptive response to different noxious stimuli, involving microglia, astrocytes and peripheral immune cells. NI is a hallmark of several acute and chronic diseases of central nervous system (CNS) and contributes to both damage and repair of CNS tissue. Interventional or genetically modified rodent models mimicking human neuropathologies may provide valuable insights on basic mechanisms of NI, but also for improving the development of new diagnostic and therapeutic strategies. Preclinical positron emission tomography (PET) allows to investigate noninvasively the inflammatory response in CNS of rodent models at a molecular level, validating innovative probes for early diagnosis, and characterizing the time course of neuroinflammatory changes and their relationship with disease progression, as well as the effects of experimental treatments with high translational potential. In particular, recent efforts of preclinical PET field are intended to develop specific and selective radiotracers that target the activation of innate immune system in CNS. Here, we have reviewed the state of art for PET in relevant rodent models of acute and chronic neuropathologies associated with NI, with particular regard on imaging of activated microglia and astrocytes.

Molecular Imaging of Vulnerable Atherosclerotic Plaques in Animal Models.

Atherosclerosis is characterized by intimal plaques of the arterial vessels that develop slowly and, in some cases, may undergo spontaneous rupture with subsequent heart attack or stroke. Currently, noninvasive diagnostic tools are inadequate to screen atherosclerotic lesions at high risk of acute complications. Therefore, the attention of the scientific community has been focused on the use of molecular imaging for identifying vulnerable plaques. Genetically engineered murine models such as ApoE(-/-) and ApoE(-/-)Fbn1C1039G(+/-) mice have been shown to be useful for testing new probes targeting biomarkers of relevant molecular processes for the characterization of vulnerable plaques, such as vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, intercellular adhesion molecule (ICAM)-1, P-selectin, and integrins, and for the potential development of translational tools to identify high-risk patients who could benefit from early therapeutic interventions. This review summarizes the main animal models of vulnerable plaques, with an emphasis on genetically altered mice, and the state-of-the-art preclinical molecular imaging strategies.

Genetic deletion in uncoupling protein 3 augments 18F-fluorodeoxyglucose cardiac uptake in the ischemic heart.

BACKGROUND: We investigated the effects of uncoupling protein 3 (UCP3) genetic deletion on 18F-fluorodeoxyglucose (FDG) cardiac uptake by positron emission tomography (PET)/computed tomography (CT) dedicated animal system after permanent coronary artery ligation. METHODS: Cardiac 18F-FDG PET/CT was performed in UCP3 knockout (UCP3-/-) and wild-type (WT) mice one week after induction of myocardial infarction or sham procedure. RESULTS: In sham-operated mice no difference in left ventricular (LV) volume was detectable between WT and UCP3-/-. After myocardial infarction, LV volume was higher in both WT and UCP3-/- compared to sham animals, with a significant interaction (p < 0.05) between genotype and myocardial infarction. In sham-operated animals no difference in FDG standardized uptake value (SUV) was detectable between WT (1.8 ± 0.6) and UCP3-/- (1.8 ± 0.6). After myocardial infarction SUV was significantly higher in remote areas than in infarcted territories in both UCP3-/- and WT mice (both p < 0.01). Moreover, in remote areas, SUV was significantly higher (p < 0.001) in UCP3-/- as compared to WT, while in the infarcted territory SUV was comparable (p = 0.29). A significant relationship (r = 0.68, p < 0.001) between LV volume and SUV was found. CONCLUSIONS: In a mice model of permanent coronary occlusion, UCP3 deficiency results in a metabolic shift that favored glycolytic metabolism and increased FDG uptake in remote areas.

High-resolution positron emission tomography/computed tomography imaging of the mouse heart.

Different animal models have been used to reproduce coronary heart disease, but in recent years mice have become the animals of choice, because of their short life cycle and the possibility of genetic manipulation. Various techniques are currently used for cardiovascular imaging in mice, including high-resolution ultrasound, X-ray computed tomography (CT), magnetic resonance imaging and nuclear medicine procedures. In particular, molecular imaging with cardiac positron emission tomography (PET) allows non-invasive evaluation of changes in myocardial perfusion, metabolism, apoptosis, inflammation and gene expression or measurement of changes in left ventricular functional parameters. With technological advances, dedicated small laboratory PET/CT imaging has emerged in cardiovascular research, providing in vivo a non-invasive, serial and quantitative assessment of left ventricular function, myocardial perfusion and metabolism at a molecular level. This non-invasive methodology might be useful in longitudinal studies to monitor cardiac biochemical parameters and might facilitate studies to assess the effect of different interventions after acute myocardial ischaemia.

Effects of some anesthetic agents on skin microcirculation evaluated by laser Doppler perfusion imaging in mice.

BACKGROUND: Anesthetic agents alter microcirculation, influencing tissue oxygenation and delivery of vital substrates. Laser Doppler perfusion imaging is a widespread technique in the field of microvascular research that can evaluate noninvasively and in real time the effects of environmental conditions, physical manipulations, diseases and treatments on peripheral perfusion. This study aims to evaluate laser Doppler perfusion imaging as a means to detect changes in skin microcirculation induced by some popular anesthetic agents in a murine model. Twenty-four age- and gender-matched healthy CD1 mice were examined by laser Doppler perfusion imaging. The skin microcirculatory response was measured at the level of plantar surfaces during isoflurane anesthesia with or without subsequent dexmedetomidine or acepromazine. At the end of the procedure, dexmedetomidine was reversed by atipamezole administration. RESULTS: In all mice, skin blood flow under isoflurane anesthesia did not show significant differences over time (P = 0.1). The serial perfusion pattern and values following acepromazine or dexmedetomidine administration differed significantly (P < 0.05). CONCLUSIONS: We standardized a reliable laser Doppler perfusion imaging protocol to non-invasively assess changes in skin microcirculation induced by anesthesia in mice, considering the advantages and drawbacks of this technique and its translational value.

PET/CT imaging in mouse models of myocardial ischemia.

Different species have been used to reproduce myocardial infarction models but in the last years mice became the animals of choice for the analysis of several diseases, due to their short life cycle and the possibility of genetic manipulation. Many techniques are currently used for cardiovascular imaging in mice, including X-ray computed tomography (CT), high-resolution ultrasound, magnetic resonance imaging, and nuclear medicine procedures. Cardiac positron emission tomography (PET) allows to examine noninvasively, on a molecular level and with high sensitivity, regional changes in myocardial perfusion, metabolism, apoptosis, inflammation, and gene expression or to measure changes in anatomical and functional parameters in heart diseases. Currently hybrid PET/CT scanners for small laboratory animals are available, where CT adds high-resolution anatomical information. This paper reviews mouse models of myocardial infarction and discusses the applications of dedicated PET/CT systems technology, including animal preparation, anesthesia, radiotracers, and images postprocessing.

Repeatability, reproducibility and standardisation of a laser Doppler imaging technique for the evaluation of normal mouse hindlimb perfusion.

BACKGROUND: Preclinical perfusion studies are useful for the improvement of diagnosis and therapy in dermatologic, cardiovascular and rheumatic human diseases. The Laser Doppler Perfusion Imaging (LDPI) technique has been used to evaluate superficial alterations of the skin microcirculation in surgically induced murine hindlimb ischemia. We assessed the reproducibility and the accuracy of LDPI acquisitions and identified several critical factors that could affect LDPI measurements in mice. METHODS: Twenty mice were analysed. Statistical standardisation and a repeatability and reproducibility analysis were performed on mouse perfusion signals with respect to differences in body temperature, the presence or absence of hair, the type of anaesthesia used for LDPI measurements and the position of the mouse body. RESULTS: We found excellent correlations among measurements made by the same operator (i.e., repeatability) under the same experimental conditions and by two different operators (i.e., reproducibility). A Bland-Altman analysis showed the absence of bias in repeatability (p = 0.29) or reproducibility (p = 0.89). The limits of agreement for repeatability were -0.357 and -0.033, and for reproducibility, they were -0.270 and 0.238. Significant differences in perfusion values were observed in different experimental groups. CONCLUSIONS: Different experimental conditions must be considered as a starting point for the evaluation of new drugs and strategic therapies.

Therapeutic Potential of a Novel αvß3 Antagonist to Hamper the Aggressiveness of Mesenchymal Triple Negative Breast Cancer Sub-Type.

The mesenchymal sub-type of triple negative breast cancer (MES-TNBC) has a highly aggressive behavior and worse prognosis, due to its invasive and stem-like features, that correlate with metastatic dissemination and resistance to therapies. Furthermore, MES-TNBC is characterized by the expression of molecular markers related to the epithelial-to-mesenchymal transition (EMT) program and cancer stem cells (CSCs). The altered expression of αvß3 integrin has been well established as a driver of cancer progression, stemness, and metastasis. Here, we showed that the high levels of αvß3 are associated with MES-TNBC and therefore exploited the possibility to target this integrin to reduce the aggressiveness of this carcinoma. To this aim, MES-TNBC cells were treated with a novel peptide, named ψRGDechi, that we recently developed and characterized for its ability to selectively bind and inhibit αvß3 integrin. Notably, ψRGDechi was able to hamper adhesion, migration, and invasion of MES-TNBC cells, as well as the capability of these cells to form vascular-like structures and mammospheres. In addition, this peptide reversed EMT program inhibits mesenchymal markers. These findings show that targeting αvß3 integrin by ψRGDechi, it is possible to inhibit some of the malignant properties of MES-TNBC phenotype.

Aptamer-mediated impairment of EGFR-integrin αvß3 complex inhibits vasculogenic mimicry and growth of triple-negative breast cancers.

Current treatment options for triple-negative breast cancers (TNBCs) is limited by the absence of well-defined biomarkers, excluding a targeted therapy. Notably, epidermal growth factor receptor (EGFR) is overexpressed in a great proportion of TNBCs and is a negative prognostic factor. In clinical trials, however, existing EGFR inhibitors showed disappointing outcome. Oligonucleotide aptamers are a valid alternative to antibodies for diagnostic and therapeutic uses. Here, we prove that, when applied to aggressive TNBC cell lines with unique stem-like plasticity, the anti-EGFR CL4 aptamer, but not erlotinib or cetuximab, prevents the vasculogenic mimicry (VM) capability of the cells and destroys previously formed channels in three-dimensional culture. Notably, we found that CL4 impairs the matrix-induced integrin αvß3 interaction with EGFR and integrin αvß3-dependent cell adhesion. Consistently, the aptamer strongly inhibits VM and tumor growth in a xenograft TNBC model. These data suggest that in TNBC cells, EGFR may cooperate with integrin αvß3 to regulate integrin binding to extracellular ligands required for VM, and EGFR-targeting by CL4 aptamer may counteract this event. Overall, we demonstrate a novel mechanism of action for CL4 related with integrin αvß3-EGFR interaction, that may help to develop new oligonucleotide-based strategy addressing unmet need for TNBCs therapy.

Inhibition of Bone Marrow-Derived Mesenchymal Stem Cells Homing Towards Triple-Negative Breast Cancer Microenvironment Using an Anti-PDGFRß Aptamer.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are shown to participate in tumor progression by establishing a favorable tumor microenvironment (TME) that promote metastasis through a cytokine networks. However, the mechanism of homing and recruitment of BM-MSCs into tumors and their potential role in malignant tissue progression is poorly understood and controversial. Here we show that BM-MSCs increase aggressiveness of triple-negative breast cancer (TNBC) cell lines evaluated as capability to migrate, invade and acquire stemness markers. Importantly, we demonstrate that the treatment of BM-MSCs with a nuclease-resistant RNA aptamer against platelet-derived growth factor receptor ß (PDGFRß) causes the inhibition of receptor-dependent signaling pathways thus drastically hampering BM-MSC recruitment towards TNBC cell lines and BM-MSCs trans-differentiation into carcinoma-associated fibroblast (CAF)-like cells. Moreover, in vivo molecular imaging analysis demonstrated the aptamer ability to prevent BM-MSCs homing to TNBC xenografts. Collectively, our results indicate the anti-PDGFRß aptamer as a novel therapeutic tool to interfere with BM-MSCs attraction to TNBC providing the rationale to further explore the aptamer in more complex pre-clinical settings.

A novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) specifically detects CXCR4 expressing tumors.

C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.

Combined microcomputed tomography, biomechanical and histomorphometric analysis of the peri-implant bone: a pilot study in minipig model.

OBJECTIVES: To present a practical approach that combines biomechanical tests, microcomputed tomography (µCT) and histomorphometry, providing quantitative results on bone structure and mechanical properties in a minipig model, in order to investigate the specific response to an innovative dental biomaterial. METHODS: Titanium implants with innovative three-dimensional scaffolds were inserted in the tibias of 4 minipigs. Primary stability and osseointegration were investigated by means of insertion torque (IT) values, resonance frequency analysis (RFA), bone-to-implant contact (BIC), bone mineral density (BMD) and stereological measures of trabecular bone. RESULTS: A significant positive correlation was found between IT and RFA (r=0.980, p=0.0001). BMD at the implant sites was 18% less than the reference values (p=0.0156). Peri-implant Tb.Th was 50% higher, while Tb.N was 50% lower than the reference zone (p<0.003) and they were negatively correlated (r=-0.897, p=0.006). SIGNIFICANCE: µCT increases evaluation throughput and offers the possibility for qualitative three-dimensional recording of the bone-implant system as well as for non-destructive evaluation of bone architecture and mineral density, in combination with conventional analysis methods. The proposed multimodal approach allows to improve accuracy and reproducibility for peri-implant bone measurements and could support future investigations.

In vivo imaging and characterization of [(18)F]DPA-714, a potential new TSPO ligand, in mouse brain and peripheral tissues using small-animal PET.

INTRODUCTION: The translocator protein 18 kDa (TSPO), a biochemical marker of neuroinflammation, is highly expressed in the brain activated microglia and it is also expressed by peripheral inflammatory cells and normal peripheral tissues. Thus, development of radioligands for the TSPO may contribute to further understanding the in vivo TSPO function in central and peripheral inflammatory processes and other pathologies. Here, we report the biodistribution, the specific binding and the radiometabolites of [(18)F]DPA-714, a promising fluorinated PET radiotracer, in normal mice using a microPET/CT scanner. METHODS: The in vivo biodistribution and kinetics of [(18)F]DPA-714 were measured in mice brain and peripheral tissues. Specific binding to TSPO sites was assessed using pharmacological competitive studies by means of saturation experiments performed by i.v. injection of 1mg/kg of unlabeled DPA-714 or 3mg/kg of unlabeled PK11195. A region of interest analysis was performed to generate time-activity curves in the brain, heart, lung, kidney, spleen and liver. Metabolites assay was performed in the plasma and peripheral organs by radio-HPLC. RESULTS: [(18)F]DPA-714 reached high concentration in lung, heart, kidney and spleen, tissues well known to be rich in TSPO sites. [(18)F]DPA-714 kinetics were faster in the lung and slower in the kidney. Pre-injection of unlabeled DPA-714 or PK11195 inhibited about 80% of [(18)F]DPA-714 uptake in the lung and heart (p<0.0005). The percentage of inhibition in the kidney was lower and achieved at later times only with DPA-714 (p<0.05) but not with PK11195. Sixty minutes after radiotracer injection only unmetabolized radioligand was found in the brain, lung, heart and spleen. CONCLUSION: These results suggest that [(18)F]DPA-714 is a suitable PET ligand for imaging in mice brain and peripheral tissues since it binds with high specificity TSPO binding sites and it is almost unchanged at 60 minutes after radiotracer injection in the brain and TSPO-rich regions.

Evaluation of growth patterns and body composition in C57Bl/6J mice using dual energy X-ray absorptiometry.

The normal growth pattern of female C57BL/6J mice, from 5 to 30 weeks of age, has been investigated in a longitudinal study. Weight, body surface area (BS), and body mass index (BMI) were evaluated in forty mice. Lean mass and fat mass, bone mineral content (BMC), and bone mineral density (BMD) were monitored by dual energy X-ray absorptiometry (DEXA). Weight and BS increased linearly (16.15 ± 0.64-27.64 ± 1.42 g; 51.13 ± 0.74-79.57 ± 2.15 cm(2), P < 0.01), more markedly from 5 to 9 weeks of age (P < 0.001). BMD showed a peak at 17 weeks (0.0548 ± 0.0011 g/cm(2) ∗ m, P < 0.01). Lean mass showed an evident gain at 9 (15.8 ± 0.8 g, P < 0.001) and 25 weeks (20.5 ± 0.3 g, P < 0.01), like fat mass from 13 to 17 weeks (2.0 ± 0.4-3.6 ± 0.7 g, P < 0.01). BMI and lean mass index (LMI) reached the highest value at 21 weeks (3.57 ± 0.02-0.284 ± 0.010 g/cm(2), resp.), like fat mass index (FMI) at 17 weeks (0.057 ± 0.009 g/cm(2)) (P < 0.01). BMI, weight, and BS showed a moderate positive correlation (0.45-0.85) with lean mass from 5 to 21 weeks. Mixed linear models provided a good prediction for lean mass, fat mass, and BMD. This study may represent a baseline reference for a future comparison of wild-type C57BL/6J mice with models of altered growth.

Monitoring reversal of MET-mediated resistance to EGFR tyrosine kinase inhibitors in non-small cell lung cancer using 3'-deoxy-3'-[18F]-fluorothymidine positron emission tomography.

PURPOSE: MET amplification is one of the mechanisms underlying acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in non-small cell lung cancer (NSCLC). Here, we tested whether 3'-deoxy-3'-[(18)F]-fluorothymidine ([(18)F]FLT) positron emission tomography/computerized tomography (PET/CT) can detect MET-mediated resistance to EGFR TKIs and monitor the effects of MET inhibitors in NSCLC. EXPERIMENTAL DESIGN: H1993 and H820 NSCLC cells with high and low levels of MET amplification, respectively, and HCC827-expressing MET, but without gene amplification, were tested for the effects of MET inhibitors on the EGFR pathway and proliferation both in vitro and in vivo. Nude mice bearing NSCLCs with and without MET amplification were subjected to [(18)F]FLT PET/CT before and after treatment with crizotinib or erlotinib (50 mg/kg and 100 mg/kg p.o. for 3 days). RESULTS: H1993 cells showed high responsiveness to MET inhibitors and were resistant to erlotinib. Conversely, HCC827 cells showed high sensitivity to erlotinib and were resistant to MET inhibitors. Accordingly, H1993 tumors bearing MET amplification showed a mean reduction in [(18)F]FLT uptake of 28% and 41% after low- and high-dose treatment with crizotinib for 3 days, whereas no posttherapy changes of [(18)F]FLT uptake were observed in HCC827 tumors lacking MET amplification. Furthermore, a persistently high [(18)F]FLT uptake was observed in H1993 tumors after treatment with erlotinib, whereas HCC827 tumors showed up to 39% reduction of [(18)F]FLT uptake following erlotinib treatment. Imaging findings were confirmed by Ki67 immunostaining of tumor sections. CONCLUSIONS: [(18)F]FLT PET/CT can detect MET-mediated resistance to EGFR TKIs and its reversal by MET inhibitors in NSCLC.

Assessment of dual-energy x-ray absorptiometry for use in evaluating the effects of dietary and environmental management on Hermann's tortoises (Testudo hermanni).

OBJECTIVE: To assess dual-energy x-ray absorptiometry (DXA) for evaluating effects of diet and environment on bone mineral density in Hermann's tortoises (Testudo hermanni). ANIMALS: 26 Hermann's tortoises within 1 month after hatching. PROCEDURES: Group 1 was housed in an artificial setting and fed naturally growing vegetation. Group 2 was housed in an artificial setting and fed vegetables grown for human consumption. Group 3 was maintained in an outside enclosure and fed naturally growing vegetation. After 10 months, pyramidal growth, body weight, and adverse conditions were assessed. Bone mineral density (BMD) of the axial and appendicular skeleton, shell, vertebral column, and pelvis was measured via DXA. RESULTS: Group 2 had the highest mean ± SD body weight (65.42 ± 30.85 g), followed by group 1 (51.08 ± 22.92 g) and group 3 (35.74 ± 7.13 g). Mean BMD of the shell varied significantly among groups (group 1, 0.05 ± 0.03 g/cm(2)•m; group 2, 0.09 ± 0.15 g/cm(2)•m; and group 3, undetectable). The BMD of the axial and appendicular skeleton, vertebral column, and pelvis did not differ significantly among groups. Pyramidal growth was highest in group 1 and not evident in group 3. CONCLUSIONS AND CLINICAL RELEVANCE: Tortoises raised in artificial conditions did not have deficits in BMD, compared with results for outdoor-housed hibernating tortoises. Supplemental calcium was apparently not necessary when an adequate photothermal habitat and plant-based diet were provided. Higher BMD of captive-raised tortoises was morphologically associated with a higher incidence of pyramidal growth in captive-raised groups.