Fluoro-D-glucose-micro positron emission tomography as a diagnostic tool to confirm brain death in a murine donor lung injury model.

PURPOSE: Because brain death (BD)-related donor lung injury is still poorly understood, a reliable mouse model can help in understanding the immunologic mechanisms behind this lung injury. The purpose of our study was to validate BD in mice using small-animal positron emission tomography. PROCEDURES: BD was induced in male Balb/c mice (27.1 ± 0.9 g) with an intracranial balloon catheter inflated rapidly (<1 min) [BD](R) or gradually (36 ± 5 min) [BD](G), and compared with sham-operated [SH] and control animals [C] (n = 6/group). Ten minutes after balloon insertion 10.4 ± 1.0 MBq 2-deoxy-2-[(18)F]-fluoro-D-glucose ((18)FDG) was administered intravenously and static images were performed and quantified. RESULTS: Coronal, sagittal, and transaxial sections of cerebral (18)FDG activity revealed significant differences when comparing [BD](R) and [BD](G) with [C] and [SH] animals. No significant (18)FDG uptake was visually detectable in [BD](R) and [BD](G). The percentage injected dose showed significant differences between BD groups and [C] and [SH] (P < 0.0001). No significant difference was seen between [C] versus [SH] nor between [BD](R)versus [BD](G) (P > 0.05). CONCLUSIONS: (18)FDG micro positron emission tomography imaging is a valuable tool to demonstrate brain functionality and can therefore be used as a surrogate test to confirm BD in mice.

A potent combination of the novel PI3K Inhibitor, GDC-0941, with imatinib in gastrointestinal stromal tumor xenografts: long-lasting responses after treatment withdrawal.

PURPOSE: Oncogenic signaling in gastrointestinal stromal tumors (GIST) is sustained via PI3K/AKT pathway. We used a panel of six GIST xenograft models to assess efficacy of GDC-0941 as single agent or in combination with imatinib (IMA). EXPERIMENTAL DESIGN: Nude mice (n = 136) were grafted bilaterally with human GIST carrying diverse KIT mutations. Mice were orally dosed over four weeks, grouped as follows: (A) control; (B) GDC-0941; (C) imatinib, and (D) GDC+IMA treatments. Xenografts regrowth after treatment discontinuation was assessed in groups C and D for an additional four weeks. Tumor response was assessed by volume measurements, micro-PET imaging, histopathology, and immunoblotting. Moreover, genomic alterations in PTEN/PI3K/AKT pathway were evaluated. RESULTS: In all models, GDC-0941 caused tumor growth stabilization, inhibiting tumor cell proliferation, but did not induce apoptosis. Under GDC+IMA, profound tumor regression, superior to either treatment alone, was observed. This effect was associated with the best histologic response, a nearly complete proliferation arrest and increased apoptosis. Tumor regrowth assays confirmed superior activity of GDC+IMA over imatinib; in three of six models, tumor volume remained reduced and stable even after treatment discontinuation. A positive correlation between response to GDC+IMA and PTEN loss, both on gene and protein levels, was found. CONCLUSION: GDC+IMA has significant antitumor efficacy in GIST xenografts, inducing more substantial tumor regression, apoptosis, and durable effects than imatinib. Notably, after treatment withdrawal, tumor regression was sustained in tumors exposed to GDC+IMA, which was not observed under imatinib. Assessment of PTEN status may represent a useful predictive biomarker for patient selection.

Molecular imaging of therapy response with (18)F-FLT and (18)F-FDG following cyclophosphamide and mTOR inhibition.

PURPOSE: Evaluation and comparison of 3'-[(18)F]-fluoro-3'-deoxy-L-thymidine (FLT) and 2-[(18)F]-fluoro-2-deoxyglucose (FDG)-PET to monitor early response following both cyclophosphamide and temsirolimus treatment in a mouse model of Burkitt lymphoma. METHODS: Daudi xenograft mice were treated with either cyclophosphamide or temsirolimus and imaged with FLT-PET and FDG-PET on appropriate days post therapy inititiation. Immunohistochemical (IHC) studies (H&E, TUNEL, CD20, PCNA and ki-67) and DNA flow cytometry studies were performed. RESULTS: FDG tumor uptake decreased immediately after cyclophosphamide treatment while FLT-PET showed only a late and less pronounced decrease. A fast induction of apoptosis was observed together with an early accumulation of cells in the S-phase of the cell cycle, suggesting DNA repair. Temsirolimus treatment reduced both FDG and FLT tumor uptake immediately after therapy and resulted in a fast induction of apoptosis and G(0)-G(1) phase accumulation. CONCLUSION: FLT response was less distinct than FDG response and may be controlled by DNA repair early after cyclophosphamide. Nevertheless, FLT-PET was able to reflect decreased proliferation following temsirolimus.

Preliminary validation of varicella zoster virus thymidine kinase as a novel reporter gene for PET.

INTRODUCTION: Imaging of gene expression with positron emission tomography (PET) has emerged as a powerful tool for biomedical research during the last decade. The prototypical herpes simplex virus type 1 thymidine kinase (HSV1-TK) PET reporter gene (PRG) is widely used and many other PRGs have also been validated. We investigated varicella zoster virus thymidine kinase (VZV-tk) as new PRG with radiolabeled bicyclic nucleoside analogues (BCNAs) as PET tracers. METHODS: The uptake and washout of four different radiolabeled BCNAs was evaluated in cells expressing VZV-tk after lentiviral vector (LV) transduction and in control cells. Metabolism of the tracers was assayed by high pressure liquid chromatography (HPLC). Mice bearing VZV-TK expressing xenografts were imaged with PET. RESULTS: High uptake in VZV-tk expressing cells was seen for 3 of the 4 tracers tested. The uptake of the tracers could be blocked by the presence of excess thymidine in the incubation solution. Cellular retention was variable, with one tracer showing an acceptable half-life of ~1 hour. The amount of intracellular tracer correlated with the titer of LV used to transduce the cells. VZV-TK dependent conversion into metabolites was shown by HPLC. No specific accumulation was observed in cells expressing a fusion protein containing an HSV1-TK moiety. VZV-tk expression in xenografts resulted in a 60% increase in uptake in vivo as measured with PET. CONCLUSIONS: We have validated the combination of VZV-tk and radiolabeled BCNAs as new PRG/PRP system. Further optimization of the PRPs and the PRG are warranted to increase the signal.

The effect of whole-body vibration on peri-implant bone healing in rats.

PURPOSE: The aim of this study was to evaluate the effect of low-magnitude, high-frequency (LMHF) loading, applied by means of whole-body vibration (WBV), on peri-implant bone healing and implant osseointegration in rat tibiae. MATERIALS AND METHODS: A custom-made titanium implant was inserted into the proximal metaphysis of the tibiae of 42 rats and left to heal for 3, 7, 14 or 25 days. Half of the animals received LMHF mechanical vibration for 5 days per week (test), whereas the others served as unloaded controls. The WBV consisted of 15 consecutive frequency steps (12, 20, 30, … to 150 Hz). Each of the 15 frequencies was applied for 2000 cycles, at an acceleration of 0.3 g. In the group with a 25-day healing period, PET images were taken at -1 (day before surgery), 3, 5, 7, 10, 14 and 21 days of loading, after an injection of [(18)F]sodium fluoride, a positron-emitting tracer. The ratio of the metabolic activity around the implants to that of a reference site (uptake ratio) was calculated as a measure of bone metabolism. Bone-to-implant contact (BIC) and peri-implant bone fraction (BF) were analysed for histomorphometrical measurement. RESULTS: The mean BIC and BF were significantly influenced by both the loading and the healing time (ANOVA, P<0.01). The PET images did not reveal any significant difference in uptake ratio between the test and the control implants. CONCLUSION: LMHF loading increased BIC and BF significantly. The results confirm the bone-stimulating potential of LMHF loading, through WBV, on peri-implant bone healing and osseointegration.

18F-FLT and 18F-FDG PET to measure response to radiotherapy combined with celecoxib in two colorectal xenograft models.

PURPOSE: To determine the dependence of celecoxib on the tumour micro-environment in vitro and in vivo and to compare the use of (18)F-Fluorodeoxyglucose ((18)F-FDG) and (18)F- 3'-deoxy-3-fluorothymidine ((18)F-FLT) to measure tumour response. MATERIALS AND METHODS: In vitro, colony assays were performed on a cyclo-oxygenase 2 (COX-2) negative (HCT116) and a COX-2 positive cell line (HCA7). Xenograft models of these cell lines were treated with celecoxib and/or radiotherapy. Micro Positron Emission Tomography (microPET) scans with (18)F-FDG and (18)F-FLT were performed at different time-points. RESULTS: In vitro, no radiosensitising effect was seen in either of the cell lines. In vivo results showed a significant effect of celecoxib in the COX-2 negative tumours (HCT116) (enhancement ratio 1.5, p = 0.02) while no significant effect was observed in the COX-2 positive model (HCA7). A good correlation between (18)F-FDG and (18)F-FLT uptake was seen in both tumour models (r = 0.48, p = 0.002; r = 0.41, p = 0.005). After irradiation, a decrease in the uptake of both tracers was observed in both tumour models, which was more pronounced in the combination group, confirming the growth delay data. CONCLUSIONS: The contradicting in vitro and in vivo results suggest a major role of the tumour micro-environment. (18)F-FLT seems a good alternative for (18)F-FDG to follow tumour growth after radiation treatment.

Disparity between in vivo EGFR expression and 89Zr-labeled cetuximab uptake assessed with PET.

UNLABELLED: The epidermal growth factor receptor (EGFR) is highly expressed in a significant number of human malignancies, and its expression is associated with tumor aggressiveness and overall treatment resistance. The monoclonal antibody cetuximab is increasingly used in clinical settings as a treatment modality in combination with more conventional therapies, such as radio- and chemotherapy. Currently, little is known about tumor-specific uptake and overall pharmacokinetics. Noninvasive quantification of cetuximab uptake could provide important diagnostic information for patient selection and therapy evaluation. To this end, we have developed and validated a novel probe using cetuximab labeled with the long-lived positron emitter 89Zr for PET imaging. METHODS: Tumor cell lines with varying EGFR expression levels were used for in vivo tumor imaging experiments. PET with 89Zr-labeled cetuximab (3.75+/-0.14 MBq) was performed on tumor-bearing NMRI-nu mice at multiple time points after injection (ranging from 1 to 120 h) and quantified by drawing regions of interest on selected tissues. Uptake was compared by biodistribution gamma-counting, and ex vivo EGFR expression levels were quantified using Western blot analysis. RESULTS: Uptake of 89Zr-labeled cetuximab was demonstrated in the EGFR-positive tumors. However, the EGFR levels measured in vivo did not correlate with the relative signal obtained by PET. Tumor-to-blood ratios were significantly higher in the cell lines with intermediate (compared with the high) EGFR expression starting from 24 h after injection. Normal tissue uptake was unaffected by the different tumor types. Ex vivo gamma-counting experiments confirmed the observed in vivo PET results. A similar disparity was found between 89Zr-labeled cetuximab tumor uptake and in vivo EGFR expression levels as demonstrated by Western blotting. CONCLUSION: The 89Zr-labeled cetuximab imaging probe is a promising tool for noninvasive evaluation of cetuximab uptake. Our results demonstrate a disparity between in vivo EGFR expression levels and cetuximab uptake. In a general sense, the results indicate a disparity between antibody uptake and expression levels of a biologic target in a tumor, suggesting that additional pharmacokinetic or pharmacodynamic mechanisms influence tumor delivery of this therapy. These additional mechanisms may explain why receptor expression levels alone are not sufficient to predict patient response.

Synthesis and evaluation of a (99m)Tc-MAMA-propyl-thymidine complex as a potential probe for in vivo visualization of tumor cell proliferation with SPECT.

INTRODUCTION: Cytosolic thymidine kinase (TK1) catalyzes phosphorylation of thymidine to its monophosphate. TK1 activity is closely related with DNA synthesis, and thymidine analogs derivatized with bulky carboranylalkyl groups at the N-3 position were reported to be good substrates for TK1. Accordingly, we have synthesized (99m)Tc-MAMA-propyl-thymidine and evaluated it as a potential tumor tracer. METHODS: The bis(S-trityl)-protected MAMA-propyl-thymidine precursor (3-N-[S-trityl-2-mercaptoethyl]-N-[N'-(S-trityl-2-mercaptoethyl)amidoacetyl]-aminopropyl-thymidine) was prepared in three steps, and its structure was confirmed with (1)H NMR and mass spectrometry. Deprotection of the thiols and labeling with (99m)Tc were done in a two-step, one-pot procedure, yielding (99m)Tc-MAMA-propyl-thymidine, which was analyzed with high-performance liquid chromatography, radio-LC-MS analysis (ESI+) and electrophoresis, and its log P was determined. The biodistribution in normal mice was evaluated, and its biodistribution in a radiation-induced fibrosarcoma (RIF) tumor mouse was compared with that of 3'-deoxy-3'-[(18)F] fluorothymidine [(18)F]FLT. RESULTS: (99m)Tc-MAMA-propyl-thymidine was obtained with a radiochemical yield of 70%. Electrophoresis indicated that the complex is uncharged, and its log P was 1.0. The molecular ion mass of the Tc complex was 589 Da, which is compatible with the hypothesized N(2)S(2)-oxotechnetium structure. Tissue distribution showed fast clearance from plasma primarily by the hepatobiliary pathway. Whole-body planar imaging after injection of (99m)Tc-MAMA-propyl-thymidine in an RIF tumor-bearing mouse showed high uptake in the liver and the intestines. No uptake was observed in the tumor, in contrast to the clear uptake observed for [(18)F] FLT visualized with muPET. CONCLUSIONS: Although it has been reported that TK1 accepts large substituents at the N-3 position of the thymine ring, the results of this study show that (99m)Tc-MAMA-propyl-thymidine cannot be used as a single photon emission computed tomography tumor tracer, probably because the (99m)Tc-MAMA ligand is too bulky to be tolerated by TK1.

Motor- and food-related metabolic cerebral changes in the activity-based rat model for anorexia nervosa: a voxel-based microPET study.

Anorexia nervosa (AN) is a disorder that is difficult to treat with psycho- or pharmacotherapy. In order to identify involved neurocircuitry, we investigated the cerebral metabolic alterations in the activity-based anorexia (ABA) rat model, where restriction of the food intake period induces hyperactivity and decreased body weight. Cerebral (18)F-fluorodeoxyglucose uptake was investigated in rats in the activity-based anorexia model (n=9) and compared to controls (n=10), using a CTI Focus microPET 220. Regional metabolic changes were investigated using statistical parametric mapping (SPM2) and correlated to weight and hyperactivity measures on a voxel-by-voxel basis. Higher regional metabolism was found in ABA rats in the mediodorsal thalamus, ventral pontine nuclei and cerebellum, while hypometabolism was seen in the left rhinal and bilateral insular cortex, and bilateral ventral striatum (p<0.001). A positive correlation was observed between body weight loss and brain metabolism in the cingulate cortex and surrounding motor and somatosensory cortex (p<0.001). Thus, in the ABA model metabolic changes are present in brain areas related to disease status and weight loss, which share several characteristics with the human disease.

Establishment of a mouse gastrointestinal stromal tumour model and evaluation of response to imatinib by small animal positron emission tomography.

BACKGROUND: Gastrointestinal stromal tumours (GIST) predominantly express activating mutations of the KIT tyrosine kinase receptor and are successfully treated with imatinib mesylate, a KIT inhibitor. As resistance to imatinib causes therapy failure, our aim was to develop an in vivo GIST model to evaluate KIT inhibitors and monitor therapy with small animal positron emission tomography (PET). MATERIALS AND METHODS: The first mouse model of GIST xenografts was successfully established by injecting GIST882 cells subcutaneously into nude mice. RESULTS: Using the small animal PET, FDG up-take in xenografts was significantly decreased after 24 h of treatment with imatinib, which correlated with a response to treatment, e.g., with a decrease in tumour volume, the inhibition of KIT and downstream intermediate phosphorylation and arrest of tumour cell proliferation as evaluated after 7 days of treatment. CONCLUSION: This model is useful to study imatinib resistance and to evaluate novel targeted therapies.