Non-hybridization saturable mechanisms play a role in the uptake of (68)Ga-Labeled LNA-DNA mixmer antisense oligonucleotides in rats.

Oligonucleotides (ODN) are key molecules for the aim of preventing translation of a gene product or monitoring gene expression in tissues. However, multiple methodological and biological hurdles need to be solved before in vivo application in humans will be possible. For positron emission tomography (PET) investigations, a 20-mer DNA-locked nucleic acid (LNA) mixmer ODN specific for rat chromogranin-A mRNA was labeled with (68)Ga and its uptake was examined in vivo in rats with and without blocking of scavenger receptors by polyribonucleotides. In addition, uptake studies of (68)Ga-LNA were performed with respect to time and concentration in human and rat cell lines. The human cell lines did not express the target mRNA. Both polyinosinic acid (poly-I) and polyadenylic acid (poly-A) reduced the uptake in rat tissues and in human cell lines. Poly-I was found to be more effective in the liver whereas poly-A was more effective in the kidney. In addition, the blockade by poly-I was statistically significant in the pancreas, adrenal gland, bone marrow, intestine, testis, urinary bladder, muscle, parotid gland, and heart, whereas poly-A also caused significant reduction in pancreas, adrenal gland, and bone marrow but not as much as in kidney. Cell culture study showed a 2-phase dose-dependent uptake characteristic with a saturable and a passive diffusion-like phase; however, these 2 phases were not so well expressed in the rat cell line. The results suggest that scavenger receptors or other saturable processes unrelated to hybridization may be involved in the tissue uptake of (68)Ga-LNA and in the clearance of antisense ODN through the liver, kidney, spleen, and bone marrow. The fact that these processes may be sequence-dependent suggests that proof of in vivo hybridization through imaging may not be obtained by only comparing sense and antisense sequences and proving dose-dependency.

Evaluation of the Hsp90 inhibitor NVP-AUY922 in multicellular tumour spheroids with respect to effects on growth and PET tracer uptake.

BACKGROUND: Molecular targeting has become a prominent concept in cancer treatment and heat shock protein 90 (Hsp90) inhibitors are suggested as promising anticancer drugs. The Hsp90 complex is one of the chaperones that facilitate the refolding of unfolded or misfolded proteins and plays a role for key oncogenic proteins such as Her2, Raf-1, Akt/PKB, and mutant p53. NVP-AUY922 is a novel low-molecular Hsp90 inhibitor, currently under clinical development as an anticancer drug. Disruption of the Hsp90-client protein complexes leads to proteasome-mediated degradation of client proteins and cell death. The aim of the current study was to use a combination of the multicellular tumour spheroid (MTS) model and positron emission tomography (PET) to investigate the effects of NVP-AUY922 on tumour growth and its relation to PET tracer uptake for the selection of appropriate PET tracer. A further aim was to evaluate the concentration and time dependence in the relation between growth inhibition and PET tracer uptake as part of translational imaging activities. METHODS: MTS of two breast cancer cell lines (MCF-7 and BT474), one glioblastoma cell line (U87MG) and one colon carcinoma cell line (HCT116) were prepared. Initially, we investigated MTS growth pattern and (3)H-thymidine incorporation in MTS after continuous exposure to NVP-AUY922 in order to determine dose response. Then the short-term effect of the drug on the four PET tracers 2-[(18)F] fluoro-2-deoxyglucose (FDG), 3'-deoxy-3'-fluorothymidine (FLT), methionine and choline was correlated to the long-term effect (changes in growth pattern) to determine the adequate PET tracer with high predictability. Next, the growth inhibitory effect of different dose schedules was evaluated to determine the optimal dose and time. Finally, the effect of a 2-h exposure to the drug on growth pattern and FDG/FLT uptake was evaluated. RESULTS: A dose-dependent inhibition of growth and decrease of (3)H-thymidine uptake was observed with 100% growth cessation in the dose range 7-52 nM and 50% (3)H-thymidine reduction in the range of 10-23 nM, with the most pronounced effect on BT474 cells. The effect of the drug was best detected by FLT. The results suggested that a complete cessation of growth of the viable cell volume was achieved with about 50% inhibition of FLT uptake 3 days after continuous treatment. Significant growth inhibition was observed at all doses and all exposure time spans. Two-hour exposure to NVP-AUY922 generated a growth inhibition which persisted dose dependently up to 10 days. The uptake of FDG per viable tumour volume was reduced by just 25% with 300 nM treatment of the drug, whereas the FLT uptake decreased up to 75% in correlation with the growth inhibition and recovery. CONCLUSIONS: Our results indicate a prolonged action of NVP-AUY922 in this cell culture, FLT is a suitable tracer for the monitoring of the effect and a FLT PET study within 3 days after treatment can predict the treatment outcome in this model. If relevant in vivo, this information can be used for efficient planning of animal PET studies and later human PET trial.

Modeling spheroid growth, PET tracer uptake, and treatment effects of the Hsp90 inhibitor NVP-AUY922.

UNLABELLED: For a PET agent to be successful as a biomarker in early clinical trials of new anticancer agents, some conditions need to be fulfilled: the selected tracer should show a response that is related to the antitumoral effects, the quantitative value of this response should be interpretable to the antitumoral action, and the timing of the PET scan should be optimized to action of the drug. These conditions are not necessarily known at the start of a drug-development program and need to be explored. We proposed a translational imaging activity in which experiments in spheroids and later in xenografts are coupled to modeling of growth inhibition and to the related changes in the kinetics of PET tracers and other biomarkers. In addition, we demonstrated how this information can be used for planning clinical trials. METHODS: The first part of this concept is illustrated in a spheroid model with BT474 breast cancer cells treated with the heat shock protein 90 (Hsp90) inhibitor NVP-AUY922. The growth-inhibitory effect after a pulse treatment with the drug was measured with digital image analysis to determine effects on volume with high accuracy. The growth-inhibitory effect was described mathematically by a combined E(max) and time course model fitted to the data. The model was then used to simulate a once-per-week treatment; in these experiments the uptake of the PET tracers (18)F-FDG and 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) was determined at different doses and different time points. RESULTS: A drug exposure of 2 h followed by washout of the drug from the culture medium generated growth inhibition that was maximal at the earliest time point of 1 d and decreased exponentially with time during 10-12 d. The uptake of (18)F-FDG per viable tumor volume was minimally affected by the treatment, whereas the (18)F-FLT uptake decreased in correlation with the growth inhibition. CONCLUSION: The study suggests a prolonged action of the Hsp90 inhibitor that supports a once-per-week schedule. (18)F-FLT is a suitable tracer for the monitoring of effect, and the (18)F-FLT PET study might be performed within 3 d after dosing.

A computerized infusion pump for control of tissue tracer concentration during positron emission tomography in vivo pharmacokinetic/pharmacodynamic measurements.

BACKGROUND: A computer controlled infusion pump (UIPump) for regulation of target tissue concentration of radioactive compounds was developed for use in biological research and tracer development for PET. METHODS: Based on observed tissue or plasma kinetics after a bolus injection of the tracer an algorithm calculates the infusion needed to obtain a specified target kinetic curve. A computer feeds this infusion scheme into an infusion pump connected to an animal via a venous catheter. The concept was validated using [11C]Flumazenil administrated to Sprague-Dawley rats where the whole brain distribution and kinetic of the tracer was measured over time using a microPET-scanner. The accuracy and precision of the system was assessed by producing steady-state levels of the tracer and by mimicking kinetics after oral administration. RESULTS: Various kinetic profiles could be generated, including rapid achievement of constant levels, or step-wise increased levels. The resulting tissue curves had low deviation from the target curves according to the specified criteria: AUC (%): 4.2 +/- 2.8, Maximal deviation (%): 13.6 +/- 5.0 and R2: 0.95 +/- 0.02. CONCLUSION: The UIPump-system is suitable for use in PET-research for assessment of PK/PD properties by simulation of different tracer tissue kinetics in vivo.

Application of the multicellular tumour spheroid model to screen PET tracers for analysis of early response of chemotherapy in breast cancer.

INTRODUCTION: Positron emission tomography (PET) is suggested for early monitoring of treatment response, assuming that effective anticancer treatment induces metabolic changes that precede morphology alterations and changes in growth. The aim of this study was to introduce multicellular tumour spheroids (MTS) to study the effect of anticancer drugs and suggest an appropriate PET tracer for further studies. METHODS: MTS of the breast cancer cell line MCF7 were exposed to doxorubicin, paclitaxel, docetaxel, tamoxifen or imatinib for 7 days for growth pattern studies and for 3 or 5 days for PET tracer studies. The effect on growth was computed using the semi-automated size determination method (SASDM). The effect on the uptake of PET tracers [18F]3'-deoxy-3'-fluorothymidine (FLT), [1-11C]acetate (ACE), [11C]choline (CHO), [11C]methionine (MET), and 2-[18F]fluoro-2-deoxyglucose (FDG) was calculated in form of uptake/viable volume of the MTS at the end of the drug exposures, and finally the uptake was related to effects on growth rate. RESULTS: The drugs paclitaxel, docetaxel and doxorubicin gave severe growth inhibition, which correlated well with inhibition of the FLT uptake. FLT had, compared with ACE, CHO, MET and FDG, higher sensitivity in monitoring the therapy effects. CONCLUSION: SASDM provides an effective, user-friendly, time-saving and accurate method to record the growth pattern of the MTS, and also to calculate the effect of the drug on PET tracer uptake. This study demonstrate the use of MTS and SASDM in combination with PET tracers as a promising approach to probe and select PET tracer for treatment monitoring of anticancer drugs and that can hopefully be applied for optimisation in breast cancer treatment.

Multicellular tumour spheroid as a model for evaluation of [18F]FDG as biomarker for breast cancer treatment monitoring.

BACKGROUND: In order to explore a pre-clinical method to evaluate if [18F]FDG is valid for monitoring early response, we investigated the uptake of FDG in Multicellular tumour spheroids (MTS) without and with treatment with five routinely used chemotherapy agents in breast cancer. METHODS: The response to each anticancer treatment was evaluated by measurement of the [18F]FDG uptake and viable volume of the MTSs after 2 and 3 days of treatment. RESULTS: The effect of Paclitaxel and Docetaxel on [18F]FDG uptake per viable volume was more evident in BT474 (up to 55% decrease) than in MCF-7 (up to 25% decrease). Doxorubicin reduced the [18F]FDG uptake per viable volume more noticeable in MCF-7 (25%) than in BT474 MTSs. Tamoxifen reduced the [18F]FDG uptake per viable volume only in MCF-7 at the highest dose of 1 microM. No effect of Imatinib was observed. CONCLUSION: MTS was shown to be appropriate to investigate the potential of FDG-PET for early breast cancer treatment monitoring; the treatment effect can be observed before any tumour size changes occur.The combination of PET radiotracers and image analysis in MTS provides a good model to evaluate the relationship between tumour volume and the uptake of metabolic tracer before and after chemotherapy. This feature could be used for screening and selecting PET-tracers for early assessment of treatment response. In addition, this new method gives a possibility to assess quickly, and in vitro, a good preclinical profile of existing and newly developed anti-cancer drugs.

A new, fast and semi-automated size determination method (SASDM) for studying multicellular tumor spheroids.

BACKGROUND: Considering the width and importance of using Multicellular Tumor Spheroids (MTS) in oncology research, size determination of MTSs by an accurate and fast method is essential. In the present study an effective, fast and semi-automated method, SASDM, was developed to determinate the size of MTSs. The method was applied and tested in MTSs of three different cell-lines. Frozen section autoradiography and Hemotoxylin Eosin (H&E) staining was used for further confirmation. RESULTS: SASDM was shown to be effective, user-friendly, and time efficient, and to be more precise than the traditional methods and it was applicable for MTSs of different cell-lines. Furthermore, the results of image analysis showed high correspondence to the results of autoradiography and staining. CONCLUSION: The combination of assessment of metabolic condition and image analysis in MTSs provides a good model to evaluate the effect of various anti-cancer treatments.