Research Article
Comparison of semiquantitative fluorescence imaging and PET tracer uptake in mesothelioma models as a monitoring system for growth and therapeutic effects

https://doi.org/10.1016/j.nucmedbio.2008.08.002Get rights and content

Abstract

Introduction

Various techniques are available for in vivo imaging, and precise understanding of their characteristics is essential for effective use of the imaging results. We established human mesothelioma cell lines expressing red fluorescent protein (RFP) and examined their fluorescence intensity and uptake of positron emission tomography (PET) tracer analogs to compare their characteristics and assess their usefulness in the evaluation of therapeutics.

Method

A human mesothelioma cell line was stably transfected to express RFP. Fluorescence, cell number and protein amount were measured during cell growth and treatment with cytotoxic reagents. In in vivo experiments, RFP-expressing cells were injected subcutaneously or into the pleural cavity of nude mice, and fluorescence images were taken with or without pemetrexed treatment. The uptake of [3H]3′-deoxy-3′-fluorothymidine ([3H]FLT) and [14C]2-fluoro-2-deoxy-d-glucose ([14C]FDG) under treatment with the above reagents in vitro and in vivo were examined.

Results

Strong correlation was observed between fluorescence intensity and total cell number with or without cytotoxic treatment. The uptake of [3H]FLT and [14C]FDG decreased rapidly after the initiation of treatment with actinomycin D or cycloheximide. When treated with pemetrexed, the uptake of [3H]FLT temporarily increased. The cells formed subcutaneous and orthotopic tumors, with fluorescence intensity correlating with tumor volume. The correlation was sustained under pemetrexed treatment. The uptake of [3H]FLT in vivo increased significantly early after pemetrexed treatment.

Conclusion

Fluorescence imaging could be used to semiquantitatively monitor tumor size, whereas PET could be used to monitor tumor response to therapeutic treatments, and especially, FLT might be a good marker of the response to anti-folate chemotherapeutics.

Introduction

Mesothelioma is a very aggressive cancer that arises from mesothelial cells lining pleural, peritoneal, and pericardial cavities [1]. Asbestos exposure is the main cause of mesothelial carcinogenesis [2], [3] and is an issue of major concern in Japan. Since mesothelioma develops 30–50 years after the first asbestos exposure, its incidence is expected to peak 10–20 years from now. Prognosis of mesothelioma is very poor, the median survival being less than 1 year from diagnosis [4]. Because there are still few optimal diagnostic and therapeutic protocols, the development of new diagnostic, preventive and therapeutic options for mesothelioma are urgently needed.

In the early stage of developing new therapeutics and diagnostics, animal models play critical roles, where sensitive and reliable detection of tumor growth and response to treatments is essential. Mouse tumor models have undergone profound improvements in the fidelity of emulating human disease and have allowed the possibility of in vivo molecular studies. For monitoring in vivo event, several modalities are used, such as optical imaging (fluorescence and bioluminescence), positron emission tomography (PET), single photon emission tomography (SPECT), computed tomography, magnetic resonance imaging (MRI), and ultrasonography. Fluorescence imaging is one of the non-invasive techniques for capturing fluorescent molecules both in vitro and in vivo. Although it suffers from low spatial resolution (100 μm–1 mm in vivo), low permeability of the fluorescence emitted from inside the body and autofluorescence that sometimes influences the detection of the signal in vivo, there are many advantages in the use of fluorescence, such as its high sensitivity and high time-resolution (0.05 s to a few min). It is also less expensive than other imaging tools, as visualization of fluorescence images requires no preparative procedures or contrast agents [5], [6], and the procedure is less stressful to mice. In addition, fluorescent protein is stable enough to allow monitoring of drug effects in a single animal over a long period in a semiquantitative manner by measuring fluorescence intensity. The above advantages make the cells and animal models expressing fluorescent protein very attractive for the evaluation of therapeutics, and especially for high through-put screening, although the imaging of fluorescent protein is hardly applicable for humans. PET (and SPECT) is unique among the imaging modalities in that it can capture the functions of cells and tissues through the accumulation of radiolabeled compounds. PET, in contrast to fluorescence imaging, can be applied to humans, enabling the monitoring of the therapy effect and response in individual patients. The cost and time needed for each imaging, however, makes PET less attractive than fluorescence imaging in the screening of therapeutics.

In this study, we established human mesothelioma cells expressing red fluorescent protein (RFP) and their mouse xenograft model and compared their fluorescence and the uptake of PET tracer analogs in vitro and in vivo to distinguish their characteristics and usefulness in monitoring tumor growth and therapy response. We examined the effects of pemetrexed, an antifolate antineoplastic agent approved for mesothelioma treatment combined with cisplatin, along with the typical cytotoxic reagents actinomycin D, a transcription inhibitor, and cycloheximide, a protein synthesis inhibitor, on the fluorescence intensity and uptake of two radiolabeled metabolic tracers, [3H]3′-deoxy-3′-fluorothymidine ([3H]FLT) and [14C]2-fluoro-2-deoxy-d-glucose ([14C]FDG), that simulate the widely used PET tracers [18F]3′-deoxy-3′-fluorothymidine ([18F]FLT) and [18F]2-fluoro-2-deoxy-d-glucose, in cultured cells. In the xenograft model, the response to pemetrexed was examined.

Section snippets

Cell culture and vectors

The human mesothelioma cell line MSTO211H was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI medium 1640 (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS, SAFC Biosciences, Lenexa, KS, USA), 50 U/ml of penicillin and 50 μg/ml of streptomycin (GIBCO Invitrogen, Carlsband, CA, USA) in a humidified atmosphere of 5% CO2 in air at 37°C. The pDsRed2-C1 and pTurboRFP vectors were purchased from Clontech (Mountain View, CA, USA) and

Cell growth and treatment with cytotoxic reagents

We selected clones emitting the brightest fluorescent signal, Ds#4 and Tu#6, respectively, from DsRed- and TurboRFP-transfected colonies. Although fluorescent proteins are generally thought to be toxic for cells [7], there was no significant difference in the in vitro growth rate and cell doubling time of RFP-expressing cells compared with parental MSTO211H cells (doubling time of Ds#4 was 21.9 h, Tu#6: 22.9 h, and MSTO211H: 23.0 h). When the cells were seeded at various concentrations into

Discussion

Noninvasive imaging can be a powerful tool in the development and evaluation of therapeutic and diagnostic protocols, where the key issue is an accurate interpretation of imaging results based on a thorough understanding of the characteristics of each imaging method. Also important in the development and evaluation of therapeutics and diagnostics are practical disease models. Various types of mesothelioma models have been reported [8], [9], [10], [11], and tumor growth and drug effect have been

Acknowledgments

We would like to thank the members of the Diagnostic Imaging Group, Molecular Imaging Center, NIRS, for helpful discussions and valuable suggestions. We would also like to thank Ms. Sogawa for her help with the animal experiments and Mr. Gerz for English correction. Pemetrexed (Alimta) was kindly provided by Eli Lilly Japan K.K. This research was supported by grants from the National Institute of Radiological Sciences.

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