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Inactivation of the LKB1 tumor suppressor is a frequent event in non–small cell lung carcinoma (NSCLC) leading to the activation of mTOR complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing comutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas and squamous cell carcinomas (SCC). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1–mutant human cell lines and genetically engineered mouse models of NSCLC that develop both adenocarcinomas and SCCs. Specifically, we found that KRAS/LKB1–mutant lung adenocarcinomas responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors, thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1–mutant adenocarcinomas and squamous cell tumors. Cancer Res; 75(22); 4910–22. ©2015 AACR.

Treatment of LKB1 mutant and LKB1 wildtype NSCLC lines with phenformin, MLN0128 or combination leads to loss of cellular ATP (S1); Metabolite analysis of NSCLC human cell line expressing dox inducible 4E-BP1 4Ala protein and control vector (S2); Biomarker analysis of normal lung tissue in response to drug treatments (S3); Treatment of Kluc mice with phenformin, MLN0128 or combination does not result in therapeutic benefit (S4); Tumor burden does not decrease in response to phenformin, MLN0128 or combination therapy in Kluc mice (S5); Biomarker analysis in tumors treated with phenformin, MLN0128 or combination (S6); Biomarker analysis of mouse tumors lysates, human NSCLC cell lines and mouse embryonic fibroblasts (S7).

A pretargeted oncologic positron emission tomography (PET) imaging that leverages the power of supramolecular nanoparticles with in vivo bioorthogonal chemistry was demonstrated for the clinically relevant problem of tumor imaging. The advantages of this approach are that (i) the pharmacokinetics (PKs) of tumor-targeting and imaging agents can be independently altered via chemical alteration to achieve the desired in vivo performance and (ii) the interplay between the two PKs and other controllable variables confers a second layer of control toward improved PET imaging. In brief, we utilized supramolecular chemistry to synthesize tumor-targeting nanoparticles containing transcyclooctene (TCO, a bioorthogonal reactive motif), called TCO⊂SNPs. After the intravenous injection and subsequent concentration of the TCO⊂SNPs in the tumors of living mice, a small molecule containing both the complementary bioorthogonal motif (tetrazine, Tz) and a positron-emitting radioisotope ((64)Cu) was injected to react selectively and irreversibly to TCO. High-contrast PET imaging of the tumor mass was accomplished after the rapid clearance of the unreacted (64)Cu-Tz probe. Our nanoparticle approach encompasses a wider gamut of tumor types due to the use of EPR effects, which is a universal phenomenon for most solid tumors.

The presence, growth, or decline of transfected cell populations expressing the enzyme Luciferase can be followed in live mice using bioluminescence optical imaging techniques. This protocol describes how to verify the imaging equipment, options for injecting the substrate Luciferin into mice, image acquisition considerations, and commonly used data analysis techniques.

Creation of an animal imaging center is rarely as simple as purchasing a system, placing it in a room somewhere, and turning people loose to use it. There are very few imaging devices that are appropriate to place on a laboratory bench top and use right out of the box without further integration, for example, a microscope. Most systems require training of users and a specialized environment for handling data and animals. Use of animals requires approval of physical spaces, imaging systems, and protocols by the Institutional Animal Care and Use Committee (IACUC), radiation use requires approval from the Radiation Safety Officer (RSO), local institutional veterinary considerations may apply, and usually biosafety approval is required. There may also be other environmental health and safety (EHS) issues to consider, such as magnetic field safety and airflow requirements. The process of comprehensive planning and oversight is now commonplace to house, use, and experiment with animals (Stout et al. 2005; Klaunberg and Davis 2008).

This paper presents a deformable mouse atlas of the laboratory mouse anatomy. This atlas is fully articulated and can be positioned into arbitrary body poses. The atlas can also adapt body weight by changing body length and fat amount.A training set of 103 micro-CT images was used to construct the atlas. A cage-based deformation method was applied to realize the articulated pose change. The weight-related body deformation was learned from the training set using a linear regression method. A conditional Gaussian model and thin-plate spline mapping were used to deform the internal organs following the changes of pose and weight.The atlas was deformed into different body poses and weights, and the deformation results were more realistic compared to the results achieved with other mouse atlases. The organ weights of this atlas matched well with the measurements of real mouse organ weights. This atlas can also be converted into voxelized images with labeled organs, pseudo CT images and tetrahedral mesh for phantom studies.With the unique ability of articulated pose and weight changes, the deformable laboratory mouse atlas can become a valuable tool for preclinical image analysis.

We have developed an efficient, streamlined, cost-effective approach to obtain Investigational New Drug (IND) approvals from the Food and Drug Administration (FDA) for positron emission tomography (PET) imaging probes (while the FDA uses the terminology PET drugs, we are using “PET imaging probes,” “PET probes,” or “probes” as the descriptive terms). The required application and supporting data for the INDs were collected in a collaborative effort involving appropriate scientific disciplines. This path to INDs was successfully used to translate three [18 F]fluoro-arabinofuranosylcytosine (FAC) analog PET probes to phase 1 clinical trials. In doing this, a mechanism has been established to fulfill the FDA regulatory requirements for translating promising PET imaging probes from preclinical research into human clinical trials in an efficient and cost-effective manner. Electronic supplementary material The online version of this article (doi:10.1007/s11307-014-0735-2) contains supplementary material, which is available to authorized users.

Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%-100% of local prostate cancers and 87%-100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using (124)I- and (89)Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer.A11 anti-PSCA minibody was alternatively labeled with (124)I- or (89)Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1×PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient-based partial-volume correction, and the results were compared with ex vivo biodistribution.Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing (124)I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1×PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing (89)Zr-labeled minibody (7.87 ± 0.52 %ID/g 22Rv1×PSCA, 9.33 ± 0.87 %ID/g LAPC-9, P0.0001 for each), but the (124)I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor-to-soft-tissue ratios (22Rv1×PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 (124)I-A11 and 4.87 ± 0.52 (89)Zr-A11, P = 0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides.Both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the (124)I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor-to-soft-tissue contrast. Partial-volume correction was found to be essential for robust quantification of immunoPET imaging with both (124)I- and (89)Zr-labeled A11 minibody.

The intrinsic nature of small-molecule chemotherapeutics, including i) limited aqueous solubility, ii) systemic toxicity due to non-specific whole-body distribution, and iii) potential development of drug resistance after initial administration, compromises their treatment efficacy.[1] Recently, nanoparticle (NP)-based drug delivery systems have been considered as promising alternatives to overcome some of these limitations and begin to resolve obstacles in the disease management in clinical oncology.[2] The intraparticular space of a NP vector can be employed to package drug payloads without constrain associated with their solubility. Further, NP vectors exhibit enhanced permeability and retention (EPR) effects[3] that facilitate the differential uptake, leading to preferential spatio-distribution in tumor.[4] However, conventional NP drug delivery systems tend to passively release drug payloads, limiting the ability to release an effective drug concentration at a desired time window. Therefore, there is a need to develop next-generation NP drug delivery system such as a stimuli-responsive drug release system with a goal of achieving spatio-temporal control, by which an acute level of drug concentration can be delivered at the time point the NP vectors reach maximum tumor accumulation.[5] By doing so, it is expected to dramatically improve therapeutic effects in tumor and effectively reduce systematic toxicity at a minute drug dosage.[6]

Complete arginase I deficiency is the least severe urea cycle disorder, characterized by hyperargininemia and infrequent episodes of hyperammonemia. Patients suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation and seizures, and is associated with intellectual disability. In mice, onset is heralded by weight loss beginning around day 15; gait instability follows progressing to inability to stand and development of tail tremor with seizure-like activity and death. Here we report that hyperargininemic mice treated neonatally with an adeno-associated virus (AAV)-expressing arginase and followed long-term lack any presentation consistent with brain dysfunction. Behavioral and histopathological evaluation demonstrated that treated mice are indistinguishable from littermates, and that putative compounds associated with neurotoxicity are diminished. In addition, treatment results in near complete resolution of metabolic abnormalities early in life; however, there is the development of some derangement later with decline in transgene expression. Ammonium challenging revealed that treated mice are affected by exogenous loading much greater than littermates. These results demonstrate that AAV-based therapy for hyperargininemia is effective and prevents development of neurological abnormalities and cognitive dysfunction in a mouse model of hyperargininemia; however, nitrogen challenging reveals that these mice remain impaired in the handling of waste nitrogen.

Multimodality small-animal molecular imaging has become increasingly important as transgenic and knockout mice are produced to model human diseases. With the ever-increasing number and importance of human disease models, particularly in rodents (mice and rats), the ability of high-resolution multimodality molecular imaging instrumentation to contribute unique information is becoming more common and necessary. Multimodality imaging with high spatial resolution and good sensitivity, which combines modalities and records sequentially or simultaneously complementary information, offers many advantages in certain research experiments. This article discusses the current trends and new horizons in preclinical multimodality imaging in-vivo and its role in biomedical research.

This paper introduces a mouse atlas registration system (MARS), composed of a stationary top-view x-ray projector and a side-view optical camera, coupled to a mouse atlas registration algorithm. This system uses the x-ray and optical images to guide a fully automatic co-registration of a mouse atlas with each subject, in order to provide anatomical reference for small animal molecular imaging systems such as positron emission tomography (PET). To facilitate the registration, a statistical atlas that accounts for inter-subject anatomical variations was constructed based on 83 organ-labeled mouse micro-computed tomography (CT) images. The statistical shape model and conditional Gaussian model techniques were used to register the atlas with the x-ray image and optical photo. The accuracy of the atlas registration was evaluated by comparing the registered atlas with the organ-labeled micro-CT images of the test subjects. The results showed excellent registration accuracy of the whole-body region, and good accuracy for the brain, liver, heart, lungs and kidneys. In its implementation, the MARS was integrated with a preclinical PET scanner to deliver combined PET/MARS imaging, and to facilitate atlas-assisted analysis of the preclinical PET images.

This study investigates methodologies for the estimation of small animal anatomy from non-tomographic modalities, such as planar X-ray projections, optical cameras, and surface scanners. The key goal is to register a digital mouse atlas to a combination of non-tomographic modalities, in order to provide organ-level anatomical references of small animals in 3D.A 2D/3D registration method was developed to register the 3D atlas to the combination of non-tomographic imaging modalities. Eleven combinations of three non-tomographic imaging modalities were simulated, and the registration accuracy of each combination was evaluated.Comparing the 11 combinations, the top-view X-ray projection combined with the side-view optical camera yielded the best overall registration accuracy of all organs. The use of a surface scanner improved the registration accuracy of skin, spleen, and kidneys.The methodologies and evaluation presented in this study should provide helpful information for designing preclinical atlas-based anatomical data acquisition systems.

The effects of the intrinsic activity of lutetium-based scintillators used in PET imaging such as LSO have been well documented and are not usually a concern in most in vivo situations. This activity can, however, become an issue when using a wide energy window or in low count rate scenarios as seen in cell trafficking studies in small animal imaging. To date there has been no systematic validation of Monte Carlo simulations that compare a model of intrinsic 176Lu activity to measured data, making it difficult to include these effects in simulations of proposed scanners. This work seeks to validate a GATE model of intrinsic 176Lu activity against results from a bench-top system of two detector modules from a Siemens Inveon preclinical PET system and also against results from a complete Inveon system. The bench-top measurements and simulations agree well in terms of both count rates and energy spectra, with small differences in the photopeak positions at 202 and 307 keV attributed to LSO non-proportionality, which is not modeled. Measurements and simulations for the full Inveon system exhibited similar discrepancies at low energies attributed to LSO non-proportionality as well as non-uniform detector triggering. For energy thresholds above 200 keV the simulation and measurements exhibited good agreement in count rates and energy spectra. Simulations are performed demonstrating the effect of the intrinsic activity on the minimum detectable activity for the Inveon system, demonstrating agreement with previously published measured results. The results demonstrate that this GATE model of 176Lu activity can be used to accurately simulate the effects of this intrinsic activity in PET systems.

The purpose of this study was to investigate the influence of combined inhibition of receptor activator of nuclear factor kappa-B ligand (RANKL) and bone morphogenetic protein (BMP) activity in a mixed lytic/blastic prostate cancer lesion in bone. Human prostate cancer cells (C4 2b) were injected into immunocompromised mice using an intratibial injection model to create mixed lytic/blastic lesions. RANK-Fc, a recombinant RANKL antagonist, was injected subcutaneously three times a week (10 mg/kg) to inhibit RANKL and subsequent formation, function and survival of osteoclasts. Inhibition of BMP activity was achieved by transducing prostate cancer cells ex vivo with a retroviral vector expressing noggin (retronoggin; RN). There were three treatment groups (RANK-Fc treatment, RN treatment and combined RN and RANK-Fc treatment) and two control groups (untreated control and empty vector control for the RN treatment group). The progression of bone lesion and tumor growth was evaluated using plain radiographs, hindlimb tumor size, 18F-Fluorodeoxyglucose and 18F-fluoride micro PET-CT, histology and histomorphometry. Treatment with RANK-Fc alone inhibited osteolysis and transformed a mixed lytic/blastic lesion into an osteoblastic phenotype. Treatment with RN alone inhibited the osteoblastic component in a mixed lytic/blastic lesion and resulted in formation of smaller osteolytic bone lesion with smaller soft tissue size. The animals treated with both RN and RANK-Fc demonstrated delayed development of bone lesions, inhibition of osteolysis, small soft tissue tumors and preservation of bone architecture with less tumor induced new bone formation. This study suggests that combined inhibition of the RANKL and the BMP pathway may be an effective biologic therapy to inhibit the progression of established mixed lytic/blastic prostate cancer lesions in bone.

PETbox is a low-cost benchtop PET scanner dedicated to high throughput preclinical imaging that is currently under development at our institute. This paper presents the design and characterization of the detectors that are used in the PETbox system. In this work, bismuth germanate scintillator was used for the detector, taking advantage of its high stopping power, high photoelectric event fraction, lack of intrinsic background radiation and low cost. The detector block was segmented into a pixelated array consisting of 20 × 44 elements, with a crystal pitch of 2.2 mm and a crystal cross section of 2 mm × 2 mm. The effective area of the array was 44 mm × 96.8 mm. The array was coupled to two Hamamatsu H8500 position sensitive photomultiplier tubes, forming a flat-panel type detector head with a sensitive area large enough to cover the whole body of a typical laboratory mouse. Two such detector heads were constructed and their performance was characterized. For one detector head, the energy resolution ranged from 16.1% to 38.5% full width at half maximum (FWHM), with a mean of 20.1%; for the other detector head, the energy resolution ranged from 15.5% to 42.7% FWHM, with a mean of 19.6 %. The intrinsic spatial resolution was measured to range from 1.55 mm to 2.39 mm FWHM along the detector short axis and from 1.48 mm to 2.33 mm FWHM along the detector long axis, with an average of 1.78 mm. Coincidence timing resolution for the detector pair was measured to be 4.1 ns FWHM. These measurement results show that the detectors are suitable for our specific application.

The biodistribution profiles in mice of two pyrrole-imidazole polyamides were determined by PET. Pyrrole-imidazole polyamides are a class of small molecules that can be programmed to bind a broad repertoire of DNA sequences, disrupt transcription factor-DNA interfaces, and modulate gene expression pathways in cell culture experiments. The 18 F-radiolabeled polyamides were prepared by oxime ligation between 4-[ 18 F]-fluorobenzaldehyde and a hydroxylamine moiety at the polyamide C terminus. Small animal PET imaging of radiolabeled polyamides administered to mice revealed distinct differences in the biodistribution of a 5-ring β-linked polyamide versus an 8-ring hairpin, which exhibited better overall bioavailability. In vivo imaging of pyrrole-imidazole polyamides by PET is a minimum first step toward the translation of polyamide-based gene regulation from cell culture to small animal studies.

The ability to reproducibly and repeatedly image rodents in noninvasive imaging systems, such as small-animal positron emission tomography (PET) and computed tomography (CT), requires a reliable method for anesthetizing, positioning, and heating animals in a simple reproducible manner. In this paper, we demonstrate that mice and rats can be reproducibly and repeatedly imaged using an imaging chamber designed to be rigidly mounted on multiple imaging systems.Mouse and rat imaging chambers were made of acrylic plastic and aluminum. MicroCT scans were used to evaluate the positioning reproducibility of the chambers in multimodality and longitudinal imaging studies. The ability of the chambers to maintain mouse and rat body temperatures while anesthetized with gas anesthesia was also evaluated.Both the mouse and rat imaging chambers were able to reproducibly position the animals in the imaging systems with a small degree of error. Placement of the mouse in the mouse imaging chamber resulted in a mean distance of 0.23 mm per reference point in multimodality studies, whereas for longitudinal studies the mean difference was 1.11 mm. The rat chamber resulted in a mean difference of 0.46 mm in multimodality studies and a mean difference of 4.31 mm in longitudinal studies per reference point. The chambers maintained rodent body temperatures at the set point temperature of 38 degrees C.The rodent imaging chambers were able to reproducibly position rodents in tomographs with a small degree of variability and were compatible with routine use. The embedded anesthetic line and heating system was capable of maintaining the rodent's temperature and anesthetic state, thereby enhancing rodent health and improving data collection reliability.

The accurate assessment of nodal involvement in prostate cancer is crucial to planning treatment, yet there is a shortage of noninvasive imaging techniques capable of visualizing nodal lesions directly. This study demonstrates the feasibility of using recombinant human adenoviral vectors to detect nodal metastases in a human prostate cancer model. This was achieved by the prostate-restricted expression of optical and positron emission tomography (PET) imaging reporter genes by the viral vector coupled with the innate lymphotropic properties of adenovirus. We show that peritumoral administration of these vectors results in the direct detection of reporter gene expression in metastatic lesions within sentinel lymph nodes. Notably, this approach parallels the current lymphoscintigraphy method but enables the direct PET visualization of sentinel lymph node metastases, eliminating the need for invasive lymphadenectomy. These findings may lead to more effective diagnostic and therapeutic strategies for individuals with advanced-stage prostate cancer.

The combination of small-animal PET/CT scans and conventional imaging methods may enhance the evaluation of in vivo biologic interactions of murine models in the study of prostate cancer metastasis to bone.Small-animal PET/CT scans using (18)F-fluoride ion and (18)F-FDG coregistered with high-resolution small-animal CT scans were used to longitudinally assess the formation of osteoblastic, osteolytic, and mixed lesions formed by human prostate cancer cell lines in a severe combined immunodeficient (SCID) mouse tibial injection model. These scans were correlated with plain radiographs, histomorphometry, and soft-tissue measurements.Small-animal PET/CT scans were able to detect biologic activity of cells that induced an osteoblastic lesion 2 wk earlier than on plain radiographs. Furthermore, both the size and the activity of the lesions detected on PET/CT images significantly increased at each successive time point (P0.05). (18)F-FDG lesions strongly correlated with soft-tissue measurements, whereas (18)F-fluoride ion activity correlated with bone volume measured on histomorphometric analysis (P0.005). Osteolytic lesions were successfully quantified using small-animal CT, whereas lesion sizes measured on (18)F-FDG PET scans also strongly correlated with soft-tissue tumor burden (P0.05). In contrast, for mixed lesions, (18)F-fluoride ion and (18)F-FDG PET/CT scans detected only minimal activity.(18)F-FDG and (18)F-fluoride ion PET/CT scans can be useful tools in characterizing pure osteolytic and osteoblastic lesions induced by human prostate cancer cell lines. The value of this technology needs further evaluation to determine whether these studies can be used effectively to detect more subtle responses to different treatment regimens in animal models.

The aim of this study was to examine strain differences in the uptake of Fenestra liver contrast agent (LC) in Nude, C57, and severe-combined immunodeficient (SCID) mice. In addition, we aimed to determine optimum dosing and to determine if there are positron emission tomography (PET)-attenuation effects on 2-deoxy-2[F-18]fluoro-d-glucose (FDG) values due to Fenestra LC. Nude, C57, and SCID mice were injected via tail vein at 5.0, 7.5, 10.0, and 15.0 ml/kg with contrast agent and imaged using micro computed tomography (microCT) 2 h after uptake and then daily up to 7 days. Mice were imaged by microPET/CT with FDG, with or without contrast agent. Significant variations in contrast were observed between mouse strains. SCID mice had significantly more spleen uptake of contrast than the other strains, and C57 mice had significantly more liver uptake of contrast than other strains. Across all strains, the spleen showed significantly higher uptake and duration of contrast than liver. Only the heart showed a significant attenuation of FDG uptake following contrast administration. Strain-specific variations in Fenestra LC uptake and signal duration were observed. At 7.5 ml/kg, this contrast agent is effective for imaging for 1 day, whereas at 15 ml/kg, it can be used up to 1 week. Fenestra LC does not appear to attenuate FDG uptake at 15 ml/kg for most tissues; therefore, it can be used in conjunction with microPET imaging studies.

Currently available diagnostic techniques can be unreliable in the diagnosis of delayed fracture healing in certain clinical situations, which can lead to increased complication rates and costs to the health care system. This study sought to determine the utility of positron emission tomography (PET) scanning with (18)F-fluoride ion, which localizes in regions of high osteoblastic activity, and (18)F-fluorodeoxyglucose (FDG), an indicator of cellular glucose metabolism, in assessing bone healing in a rat femur fracture model.Fractures were created in the femurs of immunocompetent rats. Animals in group I had a fracture produced via a manual three-point bending technique. Group II animals underwent a femoral osteotomy with placement of a 2-mm silastic spacer at the fracture site. Fracture healing was assessed with plain radiographs, (18)F-fluoride, and (18)F-FDG PET scans at 1, 2, 3, and 4-week time points after surgery. Femoral specimens were harvested for histologic analysis and manual testing of torsional and bending strength 4 weeks after surgery.All fractures in group I revealed abundant callus formation and bone healing, while none of the nonunion femurs were healed via assessment with manual palpation, radiographic, and histologic evaluation at the 4-week time point. (18)F-fluoride PET images of group I femurs at successive 1-week intervals revealed progressively increased signal uptake at the union site during fracture repair. In contrast, minimal tracer uptake was seen at the fracture sites in group II at all time points after surgery. Data analysis revealed statistically significant differences in mean signal intensity between groups I and II at each weekly interval. No significant differences between the two groups were seen using (18)F-FDG PET imaging at any time point.This study suggests that (18)F-fluoride PET imaging, which is an indicator of osteoblastic activity in vivo, can identify fracture nonunions at an early time point and may have a role in the assessment of longitudinal fracture healing. PET scans using (18)F-FDG were not helpful in differentiating metabolic activity between successful and delayed bone healing.

Inactivation of the LKB1 tumor suppressor is a frequent event in non–small cell lung carcinoma (NSCLC) leading to the activation of mTOR complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing comutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas and squamous cell carcinomas (SCC). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1–mutant human cell lines and genetically engineered mouse models of NSCLC that develop both adenocarcinomas and SCCs. Specifically, we found that KRAS/LKB1–mutant lung adenocarcinomas responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors, thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1–mutant adenocarcinomas and squamous cell tumors. Cancer Res; 75(22); 4910–22. ©2015 AACR.

Many research institutions have a full suite of preclinical tomographic scanners to answer biomedical questions in vivo. Routine multi-modality imaging requires robust registration of images generated by various tomographs. We have implemented a hardware registration method for preclinical imaging that is similar to that used in the combined positron emission tomography (PET)/computed tomography (CT) scanners in the clinic. We designed an imaging chamber which can be rigidly and reproducibly mounted on separate microPET and microCT scanners. We have also designed a three-dimensional grid phantom with 1288 lines that is used to generate the spatial transformation matrix from software registration using a 15-parameter perspective model. The imaging chamber works in combination with the registration phantom synergistically to achieve the image registration goal. We verified that the average registration error between two imaging modalities is 0.335 mm using an in vivo mouse bone scan. This paper also estimates the impact of image misalignment on PET quantitation using attenuation corrections generated from misregistered images. Our technique is expected to produce PET quantitation errors of less than 5%. The methods presented are robust and appropriate for routine use in high throughput animal imaging facilities.

We developed a clinically applicable approach for noninvasive monitoring of reporter–therapeutic linked gene expression in the whole heart of large animals using PET imaging and further validated the efficacy and cardiac adverse effects of reporter–therapeutic linked gene transfer in a rabbit cervical heterotopic functional heart transplant model. Cationic liposome complexed with a vector containing a herpes simplex virus type 1 mutant thymidine kinase (HSV1-sr39tk) as the reporter gene and a recombinant human immunosuppressive cytokine, interleukin-10 (hIL-10), as the therapeutic gene was ex vivo intracoronarily delivered into cardiac allografts before implantation. Long-term HSV1-sr39tk and hIL-10 transgene and protein overexpression associated with myocardial PET reporter probe 9-(4-[ 18 F]fluoro-3-hydroxymethylbutyl)guanine ([ 18 F]FHBG) accumulation was observed in the allografts. The expression of the HSV1-sr39tk gene was significantly correlated with the hIL-10 gene expression and the total myocardial[ 18 F]FHBG accumulation quantified as a percentage of intravenously injected[ 18 F]FHBG dose. A homogeneous distribution of[ 18 F]FHBG accumulation was seen in the whole heart similar to the distribution of[ 18 F]fluorodeoxyglucose, a PET glucose metabolism probe. The immunosuppressive therapeutic efficacy remained the same in allografts treated with reporter–therapeutic linked gene and therapeutic gene only. No cardiac adverse effect was found. Our results demonstrate for the first time that PET reporter–therapeutic linked gene imaging is applicable for noninvasively monitoring ex vivo intracoronarily delivered therapeutic transgene expression in the whole heart.

Background— This study examines the quantitative accuracy, detection sensitivity, and time course of imaging the expression of a mutant herpes simplex type-1 virus thymidine kinase (HSV1-sr39tk) PET reporter gene in rat myocardium by using the PET reporter probe 9-(4-[ 18 F]-Fluoro-3-Hydroxymethylbutyl)-Guanine ([ 18 F]-FHBG) and a small-animal PET (microPET). Methods and Results— In 40 rats, adenovirus expressing HSV1-sr39tk driven by a cytomegalovirus promoter (Ad-CMV-HSV1-sr39tk, 1×10 6 to 1×10 9 pfu) was injected through a thoracotomy directly into the left ventricular myocardium. After 3 days, myocardial perfusion was imaged with [ 13 N]-ammonia for delineating the left ventricular myocardium, followed by imaging the expression of the reporter gene with intravenous [ 18 F]-FHBG. The total myocardial [ 18 F]-FHBG accumulation was quantified in percent of injected dose (%ID). Immunohistochemistry and autoradiography demonstrated HSV1-sr39tk enzyme (HSV1-sr39TK) and accumulation of [ 18 F]-FHBG in the inoculated myocardium in 3 rats each. In 24 rats with various viral titers, the %ID was correlated with ex vivo well counting ( r 2 =0.981, P r 2 =0.790, P 18 F]-FHBG accumulation was identified at viral titers down to 1×10 7 pfu. In 6 rats serially imaged up to day 17, myocardial [ 18 F]-FHBG accumulation on microPET peaked on days 3 to 5 and was no longer identified on days 10 to 17. Conclusions— HSV1-sr39tk reporter gene expression can be monitored with [ 18 F]-FHBG and microPET in rat myocardium quantitatively and serially with high detection sensitivity. Cardiac PET reporter gene imaging offers the potential of monitoring the expression of therapeutic genes in cardiac gene therapy.

Molecular Imaging refers to observations and measurements of molecular processes in living cells or animals [1]. Anything that alters physiological processes can change molecular pharmokinetics (PK) and pharmodynamics (PD), thus these factors are important to understand, control or eliminate wherever possible. Even with in-vitro or ex-vivo measurements, the environment during formation of the molecular target and conditions during exposure to the imaging probe may alter the experimental results. The capacity for biological systems to modify themselves, sometimes very quickly, means that the biological status is a potentially changeable condition.

Striatal 6-[ 18 F]fluoro-L-DOPA (FDOPA) kinetic rate constants were measured by positron emission tomography (PET) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated squirrel monkeys. After scanning, stereological counts of dopaminergic neurons were done in substantia nigra, and dopamine (DA) and metabolite concentrations were determined in the caudate, putamen, and substantia nigra. Graded doses of MPTP produced animals with mild to moderate reductions (10-35%) in dopaminergic neurons, where the percent of cell loss was proportional to the amount of MPTP given. Striatal DA and metabolite concentrations were relatively unchanged in animals given 1.0 and 1.5 mg/kg of MPTP, but were significantly reduced after 2.0 mg/kg of MPTP. All animals injected with a single dose of MPTP showed no overt signs of parkinsonism. In contrast, DA and metabolite concentrations in the substantia nigra were significantly reduced for all MPTP-treated animals. Reduction of dopaminergic indices in the substantia nigra did not parallel reductions in the striatum, indicating differential sensitivity of the nigrostriatal pathway to the neurotoxic effects of MPTP. The percent change in FDOPA uptake (Ki) and decarboyxlation (k 3 ) after MPTP showed significant positive correlations to striatal DA levels, but not to the number of dopaminergic neurons. This suggests that FDOPA is a good index of striatal DA levels.

Individual ventilated cages (IVC) are increasing in popularity. Although mice avoid IVC in preference testing, they show no aversion when provided additional nesting material or the cage is not ventilated. Given the high ventilation rate in IVC, we developed 3 hypotheses: that mice housed in IVC experience more cold stress than do mice housed in static cages; that IVC-induced cold stress affects the results of experiments using mice; and that, when provided shelters, mice behaviorally thermoregulate and thereby rescue the cold-stress effects of IVC. To test these hypotheses, we housed mice in IVC, IVC with shelters, and static cages maintained at 20 to 21 °C. We quantified the cold stress of each housing system on mice by assessing nonshivering thermogenesis and brown adipose vacuolation. To test housing effects in a common, murine model of human disease, we implanted mice with subcutaneous epidermoid carcinoma cells and quantified tumor growth, tumor metabolism, and adrenal weight. Mice housed in IVC had histologic signs of cold stress and significantly higher nonshivering thermogenesis, smaller subcutaneous tumors, lower tumor metabolism, and larger adrenal weights than did mice in static cages. Shelters rescued IVC-induced nonshivering thermogenesis, adrenal enlargement, and phenotype-dependent cold-mediated histologic changes in brown adipose tissue and tumor size. IVC impose chronic cold stress on mice, alter experimental results, and are a source of systemic confounders throughout rodent-dependent research. Allowing mice to exhibit behavioral thermoregulation through seeking shelter markedly rescues the experiment-altering effects of housing-imposed cold stress, improves physiologic uniformity, and increases experimental reproducibility across housing systems.

The distribution volume (DY) of 6-[F-18]fluoro-L-DOPA (FDOPA) in the cerebellum recently has been linked using positron emission tomography (PET) to plasma large neutral amino acid (LNAA) concentrations in monkeys. In this article the authors provide additional experimental support for this relation by directly measuring the DY as the steady-state tissue to plasma radioactivity ratio in rats using a labeled LNAA analog 3-O-methyl-6-[F-18]FDOPA (OMFD), a compound that has no known specific enzyme or receptor interactions in brain tissue. The measured DY for OMFD (tissue OMFD concentration/plasma OMFD concentration) was found to be inversely related to plasma LNAA concentrations. The relation (DY = 1.5−0.00094* [LNAA], R^2 = 0.79) resulted in an 8% DY decrease per 100 nmol/mL plasma LNAA increase within the observed range of 330 to 510 nmol/mL. This was similar to recent noninvasive observations with FDOPA PET in vervet monkeys and with 6-[F-18]Fluoro-m-tyrosine PET in squirrel monkeys. The OMFD striatum to cerebellum (Str/Cb) ratio was greater than 1.0 for all measurements, averaging 1.09 ± 0.04, and was approximately equal to the Str/Cb LNAA ratio of 1.12 ± 0.05. This current study verifies the variation of DV of OMFD or FDOPA as a function of plasma LNAA concentrations and suggests the possibility of using OMFD for measuring cerebral LNAA noninvasively with PET.

The peripherally born metabolite of FDOPA, 3-O-Methyl-FDOPA (3OMFD), crosses the blood-brain barrier, thus complicating positron emission tomography-FDOPA (PET-FDOPA) data analysis. In previous reports the distribution volume (DV) of 3OMFD was constrained to unity. We have recently shown that the forward transport rate-constant of FDOPA ( KS1) and the cerebellum-to-plasma ratio ( Cb/ Cp), a measure for the DV of 3OMFD, are functions of plasma large neutral amino acid (LNAA) concentration. Given large interstudy and intersubject differences in plasma LNAA levels, variations in the DV of 3OMFD are significant. In this report, the authors propose a constraint on the DV of 3OMFD that accounts for these variations. Dynamic PET-FDOPA scans were performed on 12 squirrel monkeys and 12 vervet monkeys. Two sets of constraints were employed on the compartmental model—M1 or M2. In M1, the striatal DV of 3OMFD was constrained to unity; in M2, the striatal DV of 3OMFD was constrained to an estimate derived from the cerebellum. Striatal and cerebellar time-activity curves were fitted using FDOPA and 3OMFD plasma input functions. The estimate of KS1 and that of the compartmental FDOPA uptake-constant ( Ki), both obtained using M2, were adjusted to values corresponding to average LNAA levels. Finally, Ki was compared with the graphical uptake-constant ( PKi). With the use of constraint M2, intersubject variability of squirrel monkey kS3 and Ki was reduced by 45% and 53%, respectively; and for vervet monkeys, by 54% and 44%, respectively. Intersubject variability of Kl and Ki was further reduced after correction for variations in intersubject plasma LNAA levels (for squirrel monkeys, by 67% and 41%; for vervet monkeys, by 40% and 36%, respectively). Ki correlation to PKi was enhanced to identity. Finally, average cerebellar kC2 estimates were more than 2.5-fold higher than striatal kS2 estimates ( P < 0.0001). In modeling of PET-FDOPA data, it cannot be assumed that the DV of 3OMFD is unity. The cerebellar-derived constraint furnishes a reliable estimate for the DV of 3OMFD. Invoking the constraint and correcting for variations in plasma LNAA significantly reduced interstudy and intersubject variations in parameter estimates.

Variations in the cerebellum to plasma ratio at late times in 6-[18F]fluoro-l-DOPA studies are shown to be consistent with competitive binding of large neutral amino acids for a common transporter in the blood—brain barrier and the stability of brain tissue large neutral amino acid level in the presence of plasma level changes. The distribution volume of an inert large neutral amino acid can be estimated from plasma and tissue large neutral amino acid levels and apparent half-saturation concentrations ( Km) of the transporter in the blood—brain barrier. Stability of brain large neutral amino acid levels is supported by literature findings and can be explained by high saturation of the large neutral amino acid transporter at physiologic conditions.

6-[F-18]Fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) has been used to measure the central dopaminergic function in many species, including humans and monkeys. For transport across the blood brain barrier (BBB), FDOPA competes with plasma large neutral amino acids (LNAA). In this article we evaluate the effects of normal physiological LNAA concentration variation on BBB transport (K1) and the FDOPA uptake measurement, Ki. We also investigate a method for reducing the dependency of FDOPA quantitation on LNAA. Adult vervet monkeys ( Cercopithecus aethiops sabaeus, n = 19) were fasted overnight before FDOPA positron emission tomography scans. Blood samples were drawn for LNAA determination, metabolite analysis, and compartmental modeling. The estimated K1 and Ki were both negatively correlated with LNAA concentrations (r2= 0.51 and 0.62, respectively). Using an adjustment to K1 and Ki based on these correlations, the LNAA dependency was reduced (SD of the data for K1 was reduced by 33%, for Ki by 40%). Experiments with amino acid loading on an additional six animals indicate that BBB transport can be described using Michaelis-Menten kinetics. Results show a clear dependence of FDOPA uptake on plasma LNAA concentrations, which can be removed to increase the precision of FDOPA quantitation.

In six vervet monkeys, presynaptic striatal dopamine function was assessed longitudinally by [18F]fluoro-L-DOPA (FDOPA)-positron emission tomography (PET) after administration (2 x 2 mg/kg, i.m., 4 h apart) of either amphetamine (Amp), n = 3, or methamphetamine (MeAmp), n = 3. At 1-2 weeks postdrug, both Amp and MeAmp exposure effected similar decreases (60-70%) in the FDOPA influx rate constant (FDOPA Ki), an index of striatal dopamine synthesis capacity. Subsequent studies in these subjects showed that FDOPA Ki values were decreased by 45-67% at 3-6 weeks, by 25% at 10-12 weeks and by 16% in one Amp-treated subject at 32 weeks. Biochemical analysis showed that striatal dopamine concentrations were decreased by 75% at 3-4 weeks and by 55% at 10-12 weeks. These results indicate that in vervet monkey striatum, an acute Amp or MeAmp drug dosage produces extensive striatal dopamine system neurotoxicity. However, these effects were reversible; observed time-dependent recovery in both FDOPA Ki and dopamine concentrations indicates that neurochemical plasticity remains active in the adult primate striatum. At 3-4 and 10-12 weeks postdrug, the concurrent characterization of the striatal FDOPA Ki and dopamine concentrations for individual subjects showed that Ki decreases between 24 and 67% corresponded to dopamine depletions of 55-85%. These relatively larger postdrug decrements in steady-state striatal dopamine concentrations suggest that compensatory increases in dopamine synthesis capacity develop in the partially lesioned striatum. In contrast to the dopamine depletion in striatum, substantia nigra concentrations remained unchanged from referent values at both 3-4 and 10-12 weeks postdrug. Thus, the integrity of the substantia nigra could not be inferred from decreases in the striatal FDOPA Ki parameter. This disparity between striatum and substantia nigra reactivity to systemic administration of amphetamines suggests that each has unique dopamine system regulatory mechanisms.

A chronic 10-day amphetamine (Amp) protocol was used to induce significant long-term decrements of the striatal [18F]fluoro-L-DOPA influx rate constant (FDOPA Ki) in the vervet monkey. Longitudinal FDOPA-positron emission tomography (PET) assessment in Amp-treated subjects subsequently revealed a gradual recovery of striatal dopamine function: FDOPA Ki values were decreased by approximately 70% at 1 month, approximately 45% at 6 months, approximately 20% at 12 months and were similar to pre-Amp values at 24 months. Motoric and social behavioral measures were obtained on all subjects within a species-typical group setting. Behavioral observations were conducted during both basal and stressor-challenge conditions, the latter being created by placing a potential intruder-animal in an individual cage adjacent to the subject's group enclosure. During basal conditions, post-Amp stereotypies were present at 2 weeks and locomotor behaviors were increased throughout 1 month; both alterations occurred while FDOPA Ki values were significantly decreased. Social behaviors were also significantly affected; affiliative behavior was decreased up to 6 months while aggressive behavior was increased for 12 months. However, a different pattern of behavioral changes emerged under stressor-challenge conditions. Motoric and social changes were of greater magnitude and persisted longer than in basal settings while aggressive behavior remained elevated at 24 months. These results indicate that chronic Amp-induced decreases in FDOPA Ki values and behavioral alterations are reversible. Changes in striatal dopamine function as indexed with FDOPA-PET are not correlated with post-Amp alterations in behaviors and moreover, expression of those behaviors is context-dependent.

Laboratory mice routinely are housed at 20 to 22 °C-well below the murine thermoneutral zone of 29 to 34 °C. Chronic cold stress requires greater energy expenditure to maintain core body temperature and can lead to the failure of mouse models to emulate human physiology. We hypothesized that mice housed at ambient temperatures of 20 to 22 °C are chronically cold-stressed, have greater energy expenditure, and have high glucose utilization in brown adipose tissue. To test our hypotheses, we used indirect calorimetry to measure energy expenditure and substrate utilization in C57BL/6J and Crl:NU-Foxn1(nu) nude mice at routine vivarium (21 °C), intermediate (26 °C), and heated (31 °C) housing temperatures. We also examined the activation of interscapular brown adipose tissue, the primary site of nonshivering thermogenesis, via thermography and glucose uptake in this region by using positron emission tomography. Energy expenditure of mice was significantly higher at routine vivarium temperatures compared with intermediate and heated temperatures and was associated with a shift in metabolism toward glucose utilization. Brown adipose tissue showed significant activation at routine vivarium and intermediate temperatures in both hirsuite and nude mice. Crl:NU-Foxn1(nu) mice experienced greater cold stress than did C57BL/6J mice. Our data indicate mice housed under routine vivarium conditions are chronically cold stress. This novel use of thermography can measure cold stress in laboratory mice housed in vivaria, a key advantage over classic metabolic measurement tools. Therefore, thermography is an ideal tool to evaluate novel husbandry practices designed to alleviate murine cold stress.

In this chapter we delve into the primary components and operational issues that need to be considered in the setup and operation of a dedicated animal imaging facility with the use of PET, SPECT, CT, MR, and other platforms. The special conditions required for successful animal models, needs of the animals during imaging, and the avoidance of environmental and physiologic artifacts to the images are discussed.

Murine models are used extensively in biological and translational research. For many of these studies it is necessary to access the vasculature for the injection of biologically active agents. Among the possible methods for accessing the mouse vasculature, tail vein injections are a routine but critical step for many experimental protocols. To perform successful tail vein injections, a high skill set and experience is required, leaving most scientists ill-suited to perform this task. This can lead to a high variability between injections, which can impact experimental results. To allow more scientists to perform their own tail vein injections and to decrease the variability between injections a vascular access system (VAS) that semi-automatically inserts a needle into the tail vein of a mouse was developed. The VAS uses near infrared (NIR) light, image processing techniques, computer controlled motors, and a pressure feedback system to insert the needle and to validate its proper placement within the vein. The VAS was tested by injecting a commonly used radiolabeled probe (FDG) into the tail veins of five mice. These mice were then imaged using micro-positron emission tomography (PET) to measure the percentage of the injected probe remaining in the tail. These studies showed that, on average, the VAS leaves 3.4% of the injected probe in the tail. With these preliminary results, the VAS system demonstrates the potential for improving the accuracy of tail vein injections in mice.

PETbox4 is a new, fully tomographic bench top PET scanner dedicated to high sensitivity and high resolution imaging of mice. This manuscript characterizes the performance of the prototype system using the National Electrical Manufacturers Association (NEMA) NU 4–2008 standards, including studies of sensitivity, spatial resolution, energy resolution, scatter fraction, count-rate performance and image quality. The PETbox4 performance is also compared with the performance of PETbox, a previous generation limited angle tomography system. PETbox4 consists of four opposing flat-panel type detectors arranged in a box like geometry. Each panel is made by a 24 × 50 pixelated array of 1.82 × 1.82 × 7mm bismuth germanate (BGO) scintillation crystals with a crystal pitch of 1.90mm. Each of these scintillation arrays is coupled to two Hamamatsu H8500 photomultiplier tubes via a glass light guide. Volumetric images for a 45 × 45 × 95 mm field of view (FOV) are reconstructed with a maximum likelihood expectation maximization (ML-EM) algorithm incorporating a system model based on a parameterized detector response. With an energy window of 150–650 keV, the peak absolute sensitivity is approximately 18% at the center of FOV. The measured crystal energy resolution ranges from 13.5% to 48.3% full width at half maximum (FWHM), with a mean of 18.0%. The intrinsic detector spatial resolution is 1.5mm FWHM in both transverse and axial directions. The reconstructed image spatial resolution for different locations in the FOV ranges from 1.32mm to 1.93 mm, with an average of 1.46 mm. The peak noise equivalent count rate for the mouse-sized phantom is 35 kcps for a total activity of 1.5 MBq (40 µCi) and the scatter fraction is 28%. The standard deviation in the uniform region of the image quality phantom is 5.7%. The recovery coefficients range from 0.10 to 0.93. In comparison to the first generation two panel PETbox system, PETbox4 achieves substantial improvements on sensitivity and spatial resolution. The overall performance demonstrates that the PETbox4 scanner is suitable for producing high quality images for molecular imaging based biomedical research.

The acute and long-term effects of chronic amphetamine administration on the striatal dopamine system in monkeys were assessed with 6-[18F]fluoro-L-DOPA (FDOPA) and positron emission tomography (PET). Vervet monkeys (Cerecopithecus aethiops) were administered amphetamine doses, i.m., that increased from 4 mg/kg/d to 18 mg/kg/d over a 10 day period. Post-amphetamine FDOPA-PET scans at 1-2, 3-4, and 6 week time points in individual subjects showed persistent decrements in dopamine synthesis capacity as reflected by FDOPA influx rate constant (Ki) values being approximately 30% that of pre-drug assessment. In other animals that were administered the same drug regimen, biochemical analysis of striatal regions at 1-2 weeks post-drug indicated that dopamine concentrations were decreased by approximately 95% throughout caudate and putamen regions, while the homovanillic acid/dopamine level ratio was increased 3-10-fold. Post-drug FDOPA-PET Ki values remained consistently low up to 6 weeks; however, at the 5-6 month time point, relative increases in FDOPA-Ki values (approximately 53% of pre-drug values) were observed for all subjects, indicative of partial recovery of striatal dopamine synthesis capacity. These results demonstrate that FDOPA-PET can reveal temporal activity changes within the striatal dopamine system of individual subjects. The apparent, partial reversibility of amphetamine's neurotoxic effects suggests a plasticity of dopaminergic function that may include regeneration of dopaminergic terminals and compensatory increases in residual dopamine synthesis rates. The persistence of the partial decrement in dopamine synthesis capacity, however, may indicate a long term component of amphetamine's toxic effects.

To investigate in vivo transcription of the facilitative glucose transporter isoform-GLUT3 gene, we created GLUT3-firefly luciferase transgenic mouse lines that demonstrate tissue-specific [adult: brain > testis ≥ skeletal muscle > placenta; postnatal (PN): skeletal muscle > brain = skin], temporal, and spatial distribution of the reporter gene/enzyme activity that is unique from endogenous GLUT3 mRNA/protein. In this mouse model, luciferase expression/activity serving as a readout of in vivo transcription peaked at 12 days gestation along with proliferating cell nuclear antigen (cell replication) in placenta and embryonic brain preceding peak GLUT3 protein expression at 18–19 days gestation. In contrast, a postnatal increase in brain luciferase mRNA peaked with endogenous GLUT3 mRNA, but after that of NeuroD6 protein (neurogenesis) at PN7. Luciferase activity paralleled GLUT3 protein expression with Na+-K+-ATPase (membrane expansion) and synaptophysin (synaptogenesis) proteins, peaking at PN14 and lasting until 60 days in the adult. Thus GLUT3 transcription in placenta and embryonic brain coincided with cell proliferation and in postnatal brain with synaptogenesis. Longitudinal noninvasive bioluminescence (BLI) monitoring of in vivo brain GLUT3 transcription reflected cross-sectional ex vivo brain luciferase activity only between PN7 and PN21. Hypoxia/reoxygenation at PN7 revealed transcriptional increase in brain GLUT3 expression reflected by in vivo BLI and ex vivo luciferase activity. These observations collectively support a temporal contribution by transcription toward ensuring adequate tissue-specific, developmental (placenta and embryonic brain), and postnatal hypoxic brain GLUT3 expression.

Arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia. Human patients suffer from neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation. In a murine model, onset of the phenotypic abnormality is heralded by weight loss beginning around day 15 with death occurring typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. The goal of this study was to address the development of a gene therapy approach for arginase deficiency beginning in the neonatal period. Lifespan extension, body weight, circulating amino acids and ammonia levels were examined as outcome parameters after gene therapy with an adeno-associated viral vector expressing arginase was administered to mice on the second day of life (DOL). One-hundred percent of untreated arginase-deficient mice died by DOL 24, whereas 89% of the adeno-associated virus (AAV)-treated arginase deficient mice have survived for >8 months. While animals at 8 months demonstrate elevated glutamine levels, ammonia is less than three times that of controls and arginine levels are normal. These studies are the first to demonstrate that AAV-based therapy for arginase deficiency is effective and supports the development of gene therapy for this and the other urea cycle disorders.

Postnatal ad libitum caloric intake superimposed on intrauterine growth restriction (IUGR) is associated with adult-onset obesity, insulin resistance, and type 2 diabetes mellitus (T2DM). We hypothesized that this paradigm of prenatal nutrient deprivation–induced programming can be reversed with the introduction of early postnatal calorie restriction. Ten-month-old male rats exposed to either prenatal nutrient restriction with ad libitum postnatal intake (IUGR), pre- and postnatal nutrient restriction (IPGR), or postnatal nutrient restriction limited to the suckling phase (50% from postnatal [PN]1 to PN21) (PNGR) were compared with age-matched controls (CON). Visceral adiposity, metabolic profile, and insulin sensitivity by hyperinsulinemic-euglycemic clamps were examined. The 10-month-old male IUGR group had a 1.5- to 2.0-fold increase in subcutaneous and visceral fat (P < 0.0002) while remaining euglycemic, insulin sensitive, inactive, and exhibiting metabolic inflexibility (Vo2) versus CON. The IPGR group remained lean, euglycemic, insulin sensitive, and active while maintaining metabolic flexibility. The PNGR group was insulin sensitive, similar to IPGR, but less active while maintaining metabolic flexibility. We conclude that IUGR resulted in obesity without insulin resistance and energy metabolic perturbations prior to development of glucose intolerance and T2DM. Postnatal nutrient restriction superimposed on IUGR was protective, restoring metabolic normalcy to a lean and active phenotype.

Nicotine induces weight loss in both humans and rodents consuming a regular diet; however, the effect of nicotine on body weight and fat composition in rodents consuming a high-fat diet (HFD) has not been well studied. Thus, this study examined the effect of nicotine vs saline on body weight and fat composition in mice fed with either an HFD (62% of kcal from fat) or a standard normal chow diet (NCD) for 7 weeks. Nicotine dose dependently reduced body weight gain in mice that consumed both diets, but this effect was significantly greater in mice on the HFD. Caloric intake was decreased in nicotine-treated mice. Estimates of energy intake suggested that decreased caloric intake accounted for all the reduced weight gain in mice on an NCD and 66% of the reduced weight gain on an HFD. Computed tomography analysis for fat distribution demonstrated that nicotine was effective in reducing abdominal fat in mice that consumed the HFD, with nicotine treatment leading to lower visceral fat. The effect of nicotine on weight loss in mice on an HFD was completely blocked by mecamylamine, a nonselective nicotinic acetylcholine receptor (nAChR) antagonist, but only partially blocked by the α4β2 nAChR partial agonist/antagonist, varenicline. We conclude that nicotine is effective in preventing HFD-induced weight gain and abdominal fat accumulation.

PETBox4, currently under development at the Crump Institute is a new tomograph dedicated to preclinical imaging of mice. This system presents a significant improvement on sensitivity and spatial resolution compared to the first generation PETBox. We report here on its design and initial performance characteristics.

Micro-CT is widely used in preclinical studies of small animals. Due to the low soft-tissue contrast in typical studies, segmentation of soft tissue organs from noncontrast enhanced micro-CT images is a challenging problem. Here, we propose an atlas-based approach for estimating the major organs in mouse micro-CT images. A statistical atlas of major trunk organs was constructed based on 45 training subjects. The statistical shape model technique was used to include inter-subject anatomical variations. The shape correlations between different organs were described using a conditional Gaussian model. For registration, first the high-contrast organs in micro-CT images were registered by fitting the statistical shape model, while the low-contrast organs were subsequently estimated from the high-contrast organs using the conditional Gaussian model. The registration accuracy was validated based on 23 noncontrast-enhanced and 45 contrast-enhanced micro-CT images. Three different accuracy metrics (Dice coefficient, organ volume recovery coefficient, and surface distance) were used for evaluation. The Dice coefficients vary from 0.45 ± 0.18 for the spleen to 0.90 ± 0.02 for the lungs, the volume recovery coefficients vary from 0.96 ± 0.10 for the liver to 1.30 ± 0.75 for the spleen, the surface distances vary from 0.18 ± 0.01 mm for the lungs to 0.72 ± 0.42 mm for the spleen. The registration accuracy of the statistical atlas was compared with two publicly available single-subject mouse atlases, i.e., the MOBY phantom and the DIGIMOUSE atlas, and the results proved that the statistical atlas is more accurate than the single atlases. To evaluate the influence of the training subject size, different numbers of training subjects were used for atlas construction and registration. The results showed an improvement of the registration accuracy when more training subjects were used for the atlas construction. The statistical atlas-based registration was also compared with the thin-plate spline based deformable registration, commonly used in mouse atlas registration. The results revealed that the statistical atlas has the advantage of improving the estimation of low-contrast organs.

OBJECTIVE β-Cells that express an imaging reporter have provided powerful tools for studying β-cell development, islet transplantation, and β-cell autoimmunity. To further expedite diabetes research, we generated transgenic C57BL/6 “MIP-TF” mice that have a mouse insulin promoter (MIP) driving the expression of a trifusion (TF) protein of three imaging reporters (luciferase/enhanced green fluorescent protein/HSV1-sr39 thymidine kinase) in their β-cells. This should enable the noninvasive imaging of β-cells by charge-coupled device (CCD) and micro-positron emission tomography (PET), as well as the identification of β-cells at the cellular level by fluorescent microscopy. RESEARCH DESIGN AND METHODS MIP-TF mouse β-cells were multimodality imaged in models of type 1 and type 2 diabetes. RESULTS MIP-TF mouse β-cells were readily identified in pancreatic tissue sections using fluorescent microscopy. We show that MIP-TF β-cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed a progressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing proinsulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse β-cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, providing a new experimental model of type 1 diabetes. Mice fed a high-fat diet to model early type 2 diabetes displayed a progressive increase in their pancreatic bioluminescent signals, which were positively correlated with area under the curve–intraperitoneal glucose tolerance test (AUC-IPGTT). CONCLUSIONS MIP-TF mice provide a new tool for monitoring β-cells from the single cell level to noninvasive assessments of β-cells in models of type 1 diabetes and type 2 diabetes.

Better intraprostatic cancer imaging techniques are needed to guide clinicians in prostate cancer treatment decisions. Because many genes are specifically overexpressed in cancer cells, one strategy to improve prostate cancer detection is to image intraprostatic cancer–specific transcriptional activity. Because of the obstacles of weak cancer- or tissue-specific promoter activity and bladder clearance of many PET tracers, intraprostatic PET of gene transcriptional activity has not been previously reported. Methods: The two-step transcriptional amplification (TSTA) system that amplifies the prostate-specific antigen promoter activity was used for PET imaging of the reporter gene herpes simplex virus type-1 sr39 thymidine kinase (HSV1-sr39tk). The TSTA-sr39tk system was injected directly into prostates or prostatic tumors as a replication-incompetent adenovirus (AdTSTA-sr39tk) and imaged using PET. Results: AdTSTA-sr39tk was able to image prostate-specific antigen promoter transcriptional activity by 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine PET, in both mouse and canine prostates in vivo. Ex vivo small-animal PET images, scintigraphic counts, and sr39tk expression analysis confirmed the specificity of the observed signal. Conclusion: Here, by combining the TSTA-amplified signal with a protocol for tracer administration, we show that in vivo PET detection of transcriptional activity is possible in both mouse and immunocompetent canine prostates. These results suggest that imaging applications using transcription-based tumor-specific promoters should be pursued to better visualize cancer foci that escape detection by conventional biopsies.

By measuring the efficiencies of each radioactivity sensing device, the resulting data can be converted into a common unit of reference, usually either Becquerel or Curies. This conversion into the same unit makes it possible to compare values across different types of measurement systems, making use of the value of each system, whether it be high spatial resolution or in vivo measurements over hours or days. To be accurate, the various corrections and calibration factors must be carefully measured and applied. Fortunately, regular assessments of the calibration factors are an excellent quality control measure and are relatively simple to obtain. Combining autoradiography with in vivo imaging is challenging but certainly possible, enabling more exact knowledge of the precise location of the imaging probe used in vivo.