Publications

Osting et al.
Intraoperative magnetic resonance imaging (MRI) has been proposed as a method to optimize intracerebral targeting and for tracking infusate distribution in gene therapy trials for nervous system disorders. We thus investigated possible effects of two MRI contrast agents, gadoteridol (Gd) and galbumin (Gab), on the distribution and levels of transgene expression in the rat striatum and their effect on integrity and stability of recombinant adeno-associated virus (rAAV) particles. MRI studies showed that contrast agent distribution did not predict rAAV distribution. However, green fluorescent protein (GFP) immunoreactivity revealed an
increase in distribution of rAAV5-GFP, but not rAAV2-GFP, in the presence of Gd when compared with viral vector injected alone. In contrast, Gab increased the distribution of rAAV2-GFP not rAAV5-GFP. These observations pointed to a direct effect of infused contrast agent on the rAAV particles. Negative-stain electron microscopy (EM), DNAase treatment, and differential scanning calorimetry (DSC) were used to monitor rAAV2 and rAAV5 particle integrity and stability following contrast agent incubation. EMs of rAAV2-GFP and rAAV5-GFP particles pretreated with Gd appear morphologically similar to the untreated sample; however,
Gab treatment resulted in surface morphology changes and aggregation. A compromise of particle integrity was suggested by sensitivity of the packaged genome to DNAase treatment following Gab incubation but not Gd for both vectors. However, neither agent significantly affected particle stability when analyzed by DSC. An increase in Tm was observed for AAV2 in lactated Ringer’s buffer. These results thus highlight potential interactions between MRI contrast agents and AAV that might affect vector distribution and stability, as well as the stabilizing effect of lactated Ringer’s solution on AAV2.

Leystra et al.
Aberrations in the phosphoinositide 3-kinase (PI3K) signaling pathway play a key role in the pathogenesis of numerous cancers by altering cellular growth, metabolism, proliferation, and apoptosis. Mutations in the catalytic domain of PI3K that generate a dominantly active kinase are commonly found in human colorectal cancers and have been thought to drive tumor progression but not initiation. However, the effects of constitutively activated PI3K upon the intestinal mucosa have not been previously studied in animal models. Here, we show that the expression of a dominantly active form of the PI3K protein in the mouse intestine results in hyperplasia and advanced neoplasia. Mice expressing constitutively active PI3K in the epithelial cells of the distal small bowel and colon rapidly developed invasive adenocarcinomas in the colon that spread into the mesentery and adjacent organs. The histologic characteristics of these tumors were strikingly similar to invasive mucinous colon cancers in humans. Interestingly, these tumors formed without a benign polypoid intermediary, consistent with the lack of aberrant WNT signaling observed. Together, our findings indicate a noncanonical mechanism of colon tumor initiation that is mediated through activation of PI3K. This unique model has the potential to further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification.

Hong et al.
Purpose High tumor microvessel density correlates with a poor prognosis in multiple solid tumor types. The clinical gold standard for assessing microvessel density is CD105 immunohistochemistry on paraffin-embedded tumor specimens. The goal of this study was to develop an 89Zr-based PET tracer for noninvasive imaging of CD105 expression.
Methods TRC105, a chimeric anti-CD105 monoclonal antibody, was conjugated to p-isothiocyanatobenzyldesferrioxamine (Df-Bz-NCS) and labeled with 89Zr. FACS analysis and microscopy studies were performed to compare the CD105 binding affinity of TRC105 and Df-TRC105. PET imaging, biodistribution, blocking, and ex-vivo histology studies were performed on 4T1 murine breast tumor-bearing mice to evaluate the pharmacokinetics and tumor-targeting of 89Zr-Df-TRC105. Another chimeric antibody, cetuximab, was used as an isotype-matched control. Results FACS analysis of HUVECs revealed no difference in CD105 binding affinity between TRC105 and Df-TRC105, which was further validated by fluorescence microscopy. 89Zr labeling was achieved with high yield and specific activity. Serial PET imaging revealed that the
4T1 tumor uptake of 89Zr-Df-TRC105 was 6.1±1.2, 14.3±1.2, 12.4±1.5, 7.1±0.9, and 5.2±0.3 %ID/g at 5, 24, 48, 72, and 96 h after injection, respectively (n=4), higher than all organs starting from 24 h after injection, which provided excellent tumor contrast. Biodistribution data as measured by gamma counting were consistent with the PET findings. Blocking experiments, control studies with 89Zr-Df-cetuximab, and ex-vivo histology all confirmed the in vivo target specificity of 89Zr-Df-TRC105.
Conclusion We report here the first successful PET imaging of CD105 expression with 89Zr as the radiolabel. Rapid, persistent, CD105-specific uptake of 89Zr-Df-TRC105 in the 4T1 tumor was observed.

Hilmer et al. Lobeline is a potential smoking cessation drug with affinity for the α4β2 nicotinic acetylcholine receptor and may inhibit the blood–brain barrier (BBB) amine transporter. The goal of this work was to use PET imaging to evaluate the effects of lobeline on the kinetic properties of [18F]nifene in the rat brain. Methods: Direct α4β2* competition of lobeline with [18F]nifene was evaluated using imaging experiments with both displacing and blocking doses of lobeline (1 mg/kg, i.v.) given between two injections of [18F]nifene separated by 50 min. Inhibition of the BBB amine transporter was examined using a separate imaging protocol with three injections of [18F]nifene, first at baseline, then following (−)nicotine blocking, and finally following lobeline blocking. Results: Rapid displacement of [18F]nifene was observed in the α4β2*-rich thalamus following lobeline administration, suggesting direct competition of the drug at α4β2* sites. Slight decreases in BBB transport of [18F]nifene were observed when the α4β2* system was first saturated with (−)nicotine and then given lobeline. This perturbation may be due to inhibition of the BBB amine transporter by lobeline or reductions in blood flow. Significant cerebellar displacement of [18F]nifene was found following the administration of both lobeline and (−)nicotine, indicating detectable specific binding in the rat cerebellum. Conclusion: The competition of lobeline with [18F]nifene is largely dominated at the α4β2* binding site and only small perturbations in BBB transport of [18F]nifene are seen at the 1 mg/kg dose. Similar experiments could be used to study other drugs as therapeutic agents for smoking cessation with PET.

Hafeez et al. We present here first time that Plumbagin (PL), a medicinal plant-derived 1,4-naphthoquinone, inhibits the growth and metastasis of human prostate cancer (PCa) cells in an orthotopic xenograft mouse model. In this study, human PCa PC-3M-luciferase cells (2
106) were injected into the prostate of athymic nude mice. Three days post cell implantation, mice were treated with PL (2 mg/kg body wt. i.p. five days in a week) for 8 weeks. Growth and metastasis of PC-3M-luciferase cells was examined weekly by bioluminescence imaging of live mice. PL-treatment significantly ( p ¼ 0.0008) inhibited the growth of orthotopic xenograft tumors. Results demonstrated a significant inhibition of metastasis into liver ( p ¼ 0.037), but inhibition of metastasis into the lungs ( p ¼ 0.60) and lymph nodes ( p ¼ 0.27) was not observed to be significant. These results were further confirmed by histopathology of these organs. Results of histopathology demonstrated a significant inhibition of metastasis into lymph nodes ( p ¼ 0.034) and lungs ( p ¼ 0.028), and a trend to significance in liver ( p ¼ 0.075). None of the mice in the PL-treatment group showed PCa metastasis into the liver, but these mice had small metastasis foci into the lymph nodes and lungs. However, control mice had large metastatic foci into the lymph nodes, lungs, and liver. PL-caused inhibition of the growth and metastasis of PC-3M cells accompanies inhibition of the expression of: 1) PKCε, pStat3Tyr705, and pStat3Ser727, 2) Stat3 downstream target genes (survivin and BclxL), 3) proliferative markers Ki-67 and PCNA, 4) metastatic marker MMP9, MMP2, and uPA, and 5) angiogenesis markers CD31 and VEGF. Taken together, these results suggest that PL inhibits tumor growth and metastasis of human PCa PC3-M-luciferase cells, which could be used as a therapeutic agent for the prevention and treatment of human PCa.

Haffeez et al. Protein kinase C epsilon (PKCe), a novel PKC isoform, is overexpressed in prostate cancer (PCa) and correlates with disease aggressiveness. However, the functional contribution of PKCe to development or progression of PCa remained to be determined. Here we present the first in vivo genetic evidence that PKCe is essential for both the development and metastasis of PCa in the transgenic mouse model of prostate adenocarcinoma (TRAMP). Heterozygous or homozygous genetic deletions of PKCe in FVB/N TRAMP inhibited PCa development and metastasis as analyzed by positron emission tomography/computed tomography, tumor weight determinations, and histopathology. We also examined biomarkers associated with tumor progression in this model, including markers of survival, proliferation, angiogenesis, inflammation, and metastatic progression. To find clues about the genes regulated by PKCe and linked to the Stat3 signaling pathway, we carried out focused PCR arrays of JAK/STAT signaling in excised PCa tissues from PKCe wild-type and nullizygous TRAMP mice. Notably, PKCe loss was associated with significant downregulation of proliferative and metastatic genes C/EBPb (CCAAT/enhancer binding protein b), CRP (C-reactive protein), CMK, EGFR (epidermal growth factor receptor), CD64, Jun B, and gp130. Taken together, our findings offer the first genetic evidence of the role of PKCe in PCa development and metastasis. PKCe may be potential target for prevention and/or treatment of PCa.

Grudzinski et al. We have previously developed a model that provides relative dosimetry estimates for targeted radionuclide therapy (TRT) agents. The whole-body and tumor pharmacokinetic (PK) parameters of this model can be noninvasively measured with molecular imaging, providing a means of comparing potential TRT agents. Parameter sensitivities and noise will affect the accuracy and precision of the estimated PK values and hence dosimetry estimates. The aim of this work is to apply a PK model for TRT to two agents with different magnitudes of clearance rates, NM404 and FLT, explore parameter sensitivity with respect to time and investigate the effect of noise on parameter precision and accuracy. Twenty-three tumor bearing mice were injected with a ‘slow-clearing’ agent, 124I-NM404 (n = 10), or a ‘fast-clearing’ agent, 18FFLT (3-deoxy-3-fluorothymidine) (n = 13) and imaged via micro-PET/CT pseudo-dynamically or dynamically, respectively. Regions of interest were drawn within the heart and tumor to create time-concentration curves for blood pool and tumor. PK analysis was performed to estimate the mean and standard error of the central compartment efflux-to-influx ratio (k12/k21), central elimination rate constant (kel), and tumor influx-to-efflux ratio (k34/k43), as well as the mean and standard deviation of the dosimetry estimates. NM404 and FLT parameter estimation results were used to analyze model accuracy and parameter sensitivity. The accuracy of the experimental sampling schedule was compared to that of an optimal sampling schedule found using Cramer–Rao lower bounds theory. Accuracy was assessed using correlation coefficient, bias and standard error of the estimate normalized to the mean (SEE/mean). The PK parameter estimation of NM404 yielded a central clearance, kel (0.009 ± 0.003 h−1), normal body retention, k12/k21 (0.69 ± 0.16), tumor retention, k34/k43 (1.44 ± 0.46) and predicted dosimetry, Dtumor (3.47 ± 1.24 Gy). The PK parameter estimation of FLT yielded a central elimination rate constant, kel (0.050 ± 0.025 min−1), normal body retention, k12/k21 (2.21 ± 0.62) and tumor retention, k34/k43 (0.65 ± 0.17), and predicted dosimetry, Dtumor (0.61 ± 0.20 Gy). Compared to experimental sampling, optimal sampling decreases the dosimetry bias and SEE/mean for NM404; however, it increases bias and decreases SEE/mean for FLT. For both NM404 and FLT, central compartment efflux rate constant, k12, and central compartment influx rate constant, k21, possess mirroring sensitivities at relatively early time points. The instantaneous concentration in the blood, C0, was most sensitive at early time points; central elimination, kel, and tumor efflux, k43, are most sensitive at later time points. A PK model for TRT was applied to both a slowclearing, NM404, and a fast-clearing, FLT, agents in a xenograft murine model. NM404 possesses more favorable PK values according to the PK TRT model. The precise and accurate measurement of k12, k21, kel, k34 and k43 will translate into improved and precise dosimetry estimations. This work will guide the future use of this PK model for assessing the relative effectiveness of potential TRT agents.

García-Rodríguez et al. Trabecular bone tissue is a three-dimensional structure that is difficult to duplicate with in vitro cell cultures or animal models. In an attempt to better understand the underlying
mechanisms of tissue response to load, a system to load isolated bone preparations was developed. This ex vivo bone culture and loading system, given the name of ZETOS, compressively loads trabecular bone (10 mm diameter, 5.0 mm height) to evaluate its morphological and physiological responses while keeping cells viable. Compliance of the system may change with time, thus requiring recalibration. The purpose of this research
was to develop and validate a recalibration protocol for the ZETOS system. Ten reference bodies (RBs) were designed and machined out of aluminum 7075-T6, with a structural rigidity range representative of trabecular bone (0.628–28.3 N/
m, or apparent elastic modulus of 40 MPa–1.80 GPa). Finite element analysis (FEA) was used to calculate the rigidity of each RB and was validated with physical testing in a universal testing machine.
Results from FEA were then used to calibrate the system and relate force, piezoelectric actuator expansion, and specimen compressive deformation through a surface generated by spline interpolation, thus creating a calibration table. Calibration of ZETOS was verified by testing the RBs as well as three custom-made, metal springs and comparing measured rigidity to that calculated by FEA. Mean percent difference of FEA results with respect to those from physical testing was 3.28%. The mean percent difference of RB rigidity found with ZETOS with respect to rigidity found with FEA was 1.12% and for the metal springs, the mean percent difference was 1.74%. The calibration procedure for the ZETOS bone loading system has been successfully applied and verified. The use of RBs and FEA allows users to easily and periodically evaluate and recalibrate
the system. Accuracy in studies of human bone mechanotransduction in a controlled environment can therefore be achieved. The recalibration procedure is relevant for other ZETOS users and may serve as the basis for calibration of other testing systems for small specimens of compliant materials.

Deming et al. The treatment of localized colorectal cancer (CRC) depends on resection of the primary tumor with adequate margins and sufficient lymph node sampling. A novel imaging agent that accumulates in CRCs and the associated lymph nodes is needed. Cellectar Biosciences has developed a phospholipid ether analog platform that is both diagnostic and therapeutic. CLR1502 is a near-infrared fluorescent molecule, whereas 124/131I-CLR1404 is under clinical investigation as a PET tracer/therapeutic agent imaged by SPECT. We investigated the use of CLR1502 for the detection of intestinal cancers in a murine model and 131I-CLR1404 in a patient with metastatic CRC. Mice that develop multiple intestinal tumors ranging from adenomas to locally advanced adenocarcinomas were utilized. After 96 hours post CLR1502 injection, the intestinal tumors
were analyzed using a Spectrum IVIS (Perkin Elmer) and a Fluobeam (Fluoptics). The intensity of the fluorescent signal was correlated with the histological characteristics for each tumor. Colon adenocarcinomas demonstrated increased accumulation of CLR1502 compared to non-invasive lesions (total radiant efficiency: 1.7661010 vs 3.276109 respectively, p = 0.006). Metastatic mesenteric tumors and uninvolved lymph nodes were detected with CLR1502. In addition, SPECT imaging with 131I-CLR1404 was performed as part of a clinical trial in patients with advanced solid tumors. 131I-CLR1404 was shown to accumulate in metastatic tumors in a patient with colorectal adenocarcinoma. Together, these compounds might enhance our ability to properly resect CRCs through better localization of the primary tumor and improved lymph node identification as well as detect distant disease.

As part of a larger experiment investigating serotonergic regulation of female marmoset sexual behavior, this study was designed to (1) advance methods for PET imaging of common marmoset monkey brain, (2) measure normalized FDG uptake as an index of local cerebral metabolic rates for glucose, and (3) study changes induced in this index of cerebral glucose metabolism by chronic treatment of female marmosets with a serotonin 1A receptor (5-HT1A) agonist. We hypothesized that chronic treatment with the 5-HT1A agonist 8-OH-DPAT would alter the glucose metabolism index in dorsal raphe (DR), medial prefrontal cortex (mPFC),
medial preoptic area of hypothalamus (mPOA), ventromedial nucleus of hypothalamus (VMH), and field CA1 of hippocampus.
Eight adult ovariectomized female common marmosets (Callithrix jacchus) were studied with and without estradiol replacement. In a crossover design, each subject was treated daily with 8-OH-DPAT (0.1 mg/kg SC daily) or saline. After 42–49 days of treatment, the glucose metabolism radiotracer FDG was administered to each female immediately prior to 30 min of interaction with her male pairmate, after which the subject was anesthetized and imaged by PET. Whole brain normalized PET images were analyzed with anatomically defined regions of interest (ROI). Whole brain voxelwisemappingwas also used to explore treatment effects and correlations between alterations in the glucose metabolism index and pairmate interactions.
The rank order of normalized FDG uptake was VMH/mPOA>DR>mPFC/CA1 in both conditions. 8-OH-DPAT did not induce alterations in the glucose metabolism index in ROIs. Voxelwise mapping showed a significant reduction in normalized FDG uptake in response to 8-OH-DPAT in a cluster in medial occipital cortex as well as a significant correlation between increased rejection of mount attempts and reduced normalized FDG uptake in an overlapping cluster.
In conclusion, PET imaging has been used to measure FDG uptake relative to whole brain in marmoset monkeys. Voxelwise mapping shows that 8-OH-DPAT reduces this index of glucose metabolism in medial occipital cortex, consistent with alterations in female sexual behavior.