Metabolic imaging allows early prediction of response to vandetanib.

UNLABELLED: The RET (rearranged-during-transfection protein) protooncogene triggers multiple intracellular signaling cascades regulating cell cycle progression and cellular metabolism. We therefore hypothesized that metabolic imaging could allow noninvasive detection of response to the RET inhibitor vandetanib in vivo. METHODS: The effects of vandetanib treatment on the full-genome expression and the metabolic profile were analyzed in the human medullary thyroid cancer cell line TT. In vitro, transcriptional changes of pathways regulating cell cycle progression and glucose, dopa, and thymidine metabolism were correlated to the results of cell cycle analysis and the uptake of (3)H-deoxyglucose, (3)H-3,4-dihydroxy-L-phenylalanine, and (3)H-thymidine under vandetanib treatment. In vivo, the tumor metabolism under vandetanib was monitored by small-animal PET of tumor-bearing mice. RESULTS: Vandetanib treatment resulted in the transcriptional downregulation of various effector pathways with consecutive downregulation of cyclin expression and a G(0)/G(1) arrest. In vitro, vandetanib treatment resulted in the decreased expression of genes regulating glucose, 3,4-dihydroxy-L-phenylalanine, and thymidine metabolism, with a subsequent reduction in the functional activity of the corresponding pathways. In vivo, metabolic imaging with PET was able to assess changes in the tumoral glucose metabolism profile as early as 3 d after initiation of vandetanib treatment. CONCLUSION: We describe a metabolic imaging approach for the noninvasive detection of successful vandetanib treatment. Our results suggest that PET may be useful for identifying patients who respond to vandetanib early in the course of treatment.

Small-animal PET/CT for monitoring the development and response to chemotherapy of thymic lymphoma in Trp53-/- mice.

UNLABELLED: Transgenic mouse models of human cancers represent one of the most promising approaches to elucidate clinically relevant mechanisms of action and provide insights into the treatment efficacy of new antitumor drugs. The use of Trp53 transgenic mice (Trp53 knockout [Trp53(-/-)] mice) for these kinds of studies is, so far, restricted by limitations in detecting developing tumors and the lack of noninvasive tools for monitoring tumor growth, progression, and treatment response. METHODS: We hypothesized that quantitative small-animal PET with (18)F-FDG was able to detect the onset and location of tumor development, follow tumor progression, and monitor response to chemotherapy. To test these hypotheses, C57BL/6J Trp53(-/-) mice underwent longitudinal small-animal PET during lymphoma development and gemcitabine treatment. Trp53 wild-type (Trp53(+/+)) mice were used as controls, and histology after full necropsy served as the gold standard. RESULTS: In Trp53(+/+) mice, the thymic standardized uptake value (SUV) did not exceed 1.0 g/mL, with decreasing (18)F-FDG uptake over time. Conversely, all Trp53(-/-) mice that developed thymic lymphoma showed increasing thymic glucose metabolism, with a mean SUV doubling time of 9.0 wk (range, 6.0-17.5 wk). Using an SUV of 3.0 g/mL as a criterion provided a sensitivity of 78% and a specificity of 100% for the detection of thymic lymphoma. Treatment monitoring with (18)F-FDG PET correctly identified all histologic responses and relapses to gemcitabine. CONCLUSION: (18)F-FDG small-animal PET can be used to visualize onset and progression of thymic lymphomas in Trp53(-/-) mice and monitor response to chemotherapy. Thus, (18)F-FDG small-animal PET provides an in vivo means to assess intervention studies in the Trp53 transgenic mouse model.

The AMPK agonist AICAR inhibits the growth of EGFRvIII-expressing glioblastomas by inhibiting lipogenesis.

The EGFR/PI3K/Akt/mTOR signaling pathway is activated in many cancers including glioblastoma, yet mTOR inhibitors have largely failed to show efficacy in the clinic. Rapamycin promotes feedback activation of Akt in some patients, potentially underlying clinical resistance and raising the need for alternative approaches to block mTOR signaling. AMPK is a metabolic checkpoint that integrates growth factor signaling with cellular metabolism, in part by negatively regulating mTOR. We used pharmacological and genetic approaches to determine whether AMPK activation could block glioblastoma growth and cellular metabolism, and we examined the contribution of EGFR signaling in determining response in vitro and in vivo. The AMPK-agonist AICAR, and activated AMPK adenovirus, inhibited mTOR signaling and blocked the growth of glioblastoma cells expressing the activated EGFR mutant, EGFRvIII. Across a spectrum of EGFR-activated cancer cell lines, AICAR was more effective than rapamycin at blocking tumor cell proliferation, despite less efficient inhibition of mTORC1 signaling. Unexpectedly, addition of the metabolic products of cholesterol and fatty acid synthesis rescued the growth inhibitory effect of AICAR, whereas inhibition of these lipogenic enzymes mimicked AMPK activation, thus demonstrating that AMPK blocked tumor cell proliferation primarily through inhibition of cholesterol and fatty acid synthesis. Most importantly, AICAR treatment in mice significantly inhibited the growth and glycolysis (as measured by (18)fluoro-2-deoxyglucose microPET) of glioblastoma xenografts engineered to express EGFRvIII, but not their parental counterparts. These results suggest a mechanism by which AICAR inhibits the proliferation of EGFRvIII expressing glioblastomas and point toward a potential therapeutic strategy for targeting EGFR-activated cancers.

Pharmacokinetics and tumor dynamics of the nanoparticle IT-101 from PET imaging and tumor histological measurements.

IT-101, a cyclodextrin polymer-based nanoparticle containing camptothecin, is in clinical development for the treatment of cancer. Multiorgan pharmacokinetics and accumulation in tumor tissue of IT-101 is investigated by using PET. IT-101 is modified through the attachment of a 1,4,7,10-tetraazacyclododecane-1,4,7-Tris-acetic acid ligand to bind (64)Cu(2+). This modification does not affect the particle size and minimally affects the surface charge of the resulting nanoparticles. PET data from (64)Cu-labeled IT-101 are used to quantify the in vivo biodistribution in mice bearing Neuro2A s.c. tumors. The (64)Cu-labeled IT-101 displays a biphasic plasma elimination. Approximately 8% of the injected dose is rapidly cleared as a low-molecular-weight fraction through the kidneys. The remaining material circulates in plasma with a terminal half-life of 13.3 h. Steadily increasing concentrations, up to 11% injected dose per cm(3), are observed in the tumor over 24 h, higher than any other tissue at that time. A 3-compartment model is used to determine vascular permeability and nanoparticle retention in tumors, and is able to accurately represent the experimental data. The calculated tumor vascular permeability indicates that the majority of nanoparticles stay intact in circulation and do not disassemble into individual polymer strands. A key assumption to modeling the tumor dynamics is that there is a "sink" for the nanoparticles within the tumor. Histological measurements using confocal microscopy show that IT-101 localizes within tumor cells and provides the sink in the tumor for the nanoparticles.

Derivation of a compartmental model for quantifying 64Cu-DOTA-RGD kinetics in tumor-bearing mice.

UNLABELLED: Radiolabeled arginine-glycine-aspartate (RGD) peptides are increasingly used in preclinical and clinical studies to assess the expression and function of the alphavbeta3 integrin, a cellular adhesion molecule involved in angiogenesis and tumor metastasis formation. To better understand the PET signal obtained with radiolabeled RGD peptides, we have constructed a compartmental model that can describe the time-activity curves in tumors after an intravenous injection. METHODS: We analyzed 60-min dynamic PET scans obtained with 64Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-RGD in 20 tumor-bearing severe combined immunodeficient (SCID) mice after a bolus dose (18,500 kBq [500 microCi]), using variations of the standard 2-compartment (4k) tissue model augmented with a compartment for irreversible tracer internalization. alphavbeta3 binding sites were blocked in 5 studies with a coinjection of cold peptide. In addition, 20 h after injection, static PET was performed on 9 of 20 mice. We fitted 2k (k3=k4=0), 3k (k4=0), 4k, and 4kc (k4=constant) models to the PET data and used several criteria to determine the best model structure for describing 64Cu-DOTA-RGD kinetics in mice. Akaike information criteria (AIC), calculated from model fits and the ability of each model to predict tumor concentration 20 h after tracer injection, were considered. RESULTS: The 4kc model has the best profile in terms of AIC values and predictive ability, and a constant k4 is further supported by Logan-Patlak analysis and results from iterative Bayesian parameter estimation. The internalization compartment allows quantification of the putative tracer internalization rate for each study, which is estimated here to be approximately an order of magnitude less than k3 and thus does not confound the apparent specific binding of the tracer to the tumor integrin during the first 60 min of the scan. Analysis of specific (S) and nonspecific or nondisplaceable (ND) binding using fitted parameter values showed that the 4kc model provided expected results when comparing alphavbeta3 blocked and nonblocked studies. That is, specific volume of distribution, [VS=(K1k3)/(k2k4)], is much higher than is nondisplaceable volume of distribution, [VND=(K1/k2)], in nonblocking studies (2.2+/-0.6 vs. 0.85+/-0.14); VS and VND are about the same in the blocking studies (0.46+/-1.6 vs. 0.56+/-0.09). Also, the ratio of static tumor and plasma measurements at 60 and 10 min [CT(60)/CP(10)] is highly correlated (RS=0.92) to tumor VS. CONCLUSION: We have developed and tested a compartmental model for use with the 64Cu-DOTA-RGD PET tracer and demonstrated its potential as a tool for analysis and design of preclinical and clinical imaging studies.

Engineered antibody fragments with infinite affinity as reporter genes for PET imaging.

UNLABELLED: Reporter gene imaging has great potential for many clinical applications including the tracking of transplanted cells and monitoring of gene therapy. However, currently available reporter gene-reporter probe combinations have significant limitations with the biodistribution of the reporter probe and the specificity and immunogenicity of the reporter gene. The objective of the present study was to evaluate a new approach for reporter gene imaging based on cell surface expression of antibody fragments that can irreversibly bind to radiometal chelates. METHODS: We developed a new reporter gene, designated 1,4,7,10-tetraazacyclodocecane-N,N',N'',N'''-tetraacetic acid (DOTA) antibody reporter 1 (DAbR1), which consists of the single-chain Fv (scFv) fragment of the anti-Y-DOTA antibody 2D12.5/G54C fused to the human T cell CD4 transmembrane domain. The corresponding reporter probe is yttrium-(S)-2-(4-acrylamidobenzyl)-DOTA (*Y-AABD), a DOTA complex that binds irreversibly to a cysteine residue in the 2D12.5/G54C antibody. U-87 glioma cells were stably transfected with a DAbR1 expression vector. Binding of *Y-AABD to transfected and wild-type cells was studied in vitro and in vivo. RESULTS: Flow cytometry revealed high expression of the DAbR1 protein on the cell surface of tumor cells. Uptake of 90Y-AABD in DAbR1-expressing human U-87 glioma xenografts was 6.2 (+/-1.3) percentage injected dose per gram (%ID/g) at 1 h and 4.9 (+/-0.62) %ID/g at 24 h after injection. The corresponding tumor-to-plasma ratios were 45:1 and 428:1, respectively. Uptake by U-87 tumors without the DAbR1 gene was 0.16 (+/-0.02) %ID/g at 1 h and 0.05 (+/-0.03) %ID/g at 24 h. PET images in mice with 86Y-AABD demonstrated intense uptake in DAbR1-positive tumors and low background activity in the liver. CONCLUSION: These findings indicate that cell surface expression of radiometal chelate binding antibodies such as 2D12.5/G54C is a promising strategy for reporter gene imaging.

Changes in tumor metabolism as readout for Mammalian target of rapamycin kinase inhibition by rapamycin in glioblastoma.

PURPOSE: Inhibition of the protein kinase mammalian target of rapamycin (mTOR) is being evaluated for treatment of a variety of malignancies. However, the effects of mTOR inhibitors are cytostatic and standard size criteria do not reliably identify responding tumors. The aim of this study was to evaluate whether response to mTOR inhibition could be assessed by positron emission tomography (PET) imaging of tumor metabolism. EXPERIMENT DESIGN: Glucose, thymidine, and amino acid utilization of human glioma cell lines with varying degrees of sensitivity to mTOR inhibition were assessed by measuring in vitro uptake of [18F]fluorodeoxyglucose ([18F]FDG), [18F]fluorothymidine ([18F]FLT), and [3H]l-tyrosine before and after treatment with the mTOR inhibitor rapamycin. The tumor metabolic activity in vivo was monitored by small-animal PET of tumor-bearing mice. The mechanisms underlying changes in metabolic activity were analyzed by measuring expression and functional activity of enzymes and transporters involved in the uptake of the studied imaging probes. RESULTS: In sensitive cell lines, rapamycin decreased [18F]FDG and [18F]FLT uptake by up to 65% within 24 hours after the start of therapy. This was associated with inhibition of hexokinase and thymidine kinase 1. In contrast, [3H]l-tyrosine uptake was unaffected by rapamycin. The effects of rapamycin on glucose and thymidine metabolism could be imaged noninvasively by PET. In sensitive tumors, [18F]FDG and [18F]FLT uptake decreased within 48 hours by 56 +/- 6% and 52 +/- 8%, respectively, whereas there was no change in rapamycin-resistant tumors. CONCLUSIONS: These encouraging preclinical data warrant clinical trials evaluating [18F]FDG and [18F]FLT-PET for monitoring treatment with mTOR inhibitors in patients.

Anesthesia and other considerations for in vivo imaging of small animals.

The use of small animal imaging is increasing in biomedical research thanks to its ability to localize altered biochemical and physiological processes in the living animal and to follow these processes longitudinally and noninvasively. In contrast to human studies, however, imaging of small animals generally requires anesthesia, and anesthetic agents can have unintended effects on animal physiology that may confound the results of the imaging studies. In addition, repeated anesthesia, animal preparation for imaging, exposure to ionizing radiation, and the administration of contrast agents may affect the processes under study. We discuss this interplay of factors for small animal imaging in the context of four common imaging modalities for small animals: positron emission tomography (PET) and single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and optical imaging. We discuss animal preparation for imaging, including choice of animal strain and gender, the role of fasting and diet, and the circadian cycle. We review common anesthesias used in small animal imaging, such as pentobarbital, ketamine/xylazine, and isoflurane, and describe techniques for monitoring the respiration and circulation of anesthetized animals that are being imaged as well as developments for imaging conscious animals. We present current imaging literature exemplifying how anesthesia and animal handling can influence the biodistribution of PET tracers. Finally, we discuss how longitudinal imaging studies may affect animals due to repeated injections of radioactivity or other substrates and the general effect of stress on the animals. In conclusion, there are many animal handling issues to consider when designing an imaging experiment. Reproducible experimental conditions require clear, consistent reporting, in the study design and throughout the experiment, of the animal strain and gender, fasting, anesthesia, and how often individual animals were imaged.

Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging.

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5' end of the siRNA molecules allows labeling with (64)Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by approximately 50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics.

Characterization of chronic nicotine exposure on the survival of the spastic Han-Wistar rat.

Excitotoxicity has been implicated as a mechanism of cell death in many neurodegenerative disorders. Cell culture studies have shown that neuroprotection can be induced by preincubation with the acetylcholine agonist nicotine. We investigated the possible neuroprotective effects of nicotine in the spastic Han-Wistar rat, which suffers from glutamate excitotoxicity affecting two central nervous system regions: The hippocampus and the cerebellum. To investigate nicotine's possible neuroprotection, we treated 25-day-old mutant and normal siblings with 50-75 mg/l nicotine in their drinking solutions. The 75-ml/l dose significantly improved motor activity and increased longevity of the mutants (p<.05). To assess whether nicotine protected individual neurons, we performed hematoxylin and eosin (H&E) staining of brain sections. The histological data indicated that nicotine increased the survival of Purkinje cells in the mutants by as much as 50% but did not prevent cell death. To investigate whether the neuroprotection by nicotine was due to changes in nicotinic receptor expression, we performed immunohistochemical studies by staining for the alpha 3, alpha 4, and alpha 7 receptor subunits in mutant and normal rats. The alpha 4 subunit was upregulated by nicotine treatment in the cerebellum and was noted to have lower levels throughout the hippocampus of mutant animals. The alpha 3 and alpha 7 subunits showed no change in expression among all groups.