Comparative Analysis of Human Nucleoside Kinase-Based Reporter Systems for PET Imaging
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Radionuclide-based reporter gene imaging has the sensitivity to monitor gene- and cell-based therapies in human subjects. Potential immunogenicity of current viral transgenes warrants development of human-based reporter systems. We compared human nucleoside kinase reporters to a panel of nucleoside analogs of FEAU, FMAU, and FIAU, including the first in vivo assessment of l-[18F]FEAU.
Human isogenic U87 cell lines were transduced to express different human reporter genes including dCK-R104M/D133A (dCKDM), dCK-R104Q/D133N (dCKep16A), dCK-A100V/R104M/D133A (dCK3M), and TK2-N93D/L109F (TK2DM), and wild-type dCK (dCK) and herpes simplex virus type-1 (HSVTK) reporter gene as references. In vitro cell uptake assays were performed with [18F]FEAU, l-[18F]FEAU, [14C]FMAU, l-[18F]FMAU, and [124I]FIAU. Micro-positron emission tomography/X-ray computed tomography imaging of xenograft-bearing nu/nu mice was conducted with [18F]FEAU, l-[18F]FEAU, l-[18F]FMAU, and [124I]FIAU on consecutive days. A cell viability assay was also performed to assess sensitivities to gemcitabine and bromovinyldeoxyuridine (BVdU).
In vitro, dCKep16A and dCKDM with [18F]FEAU exhibited the highest sensitivity and selectivity of the human reporters, second only to HSVTK/[18F]FEAU. l-[18F]FEAU biodistribution in mice was on par with [18F]FEAU and l-[18F]FMAU. l-[18F]FMAU uptake in isogenic xenografts was highest for all human reporter genes. However, [18F]FEAU was the most selective of the short half-life reporter probes due to its minimal recognition by human dCK and relative sensitivity, whereas [124I]FIAU permitted imaging at a later time point, improving signal-to-background ratio. Of the human reporter genes, dCKep16A consistently outperformed the other tested reporters. Reporter genes of interest increased potency to the nucleoside analog prodrugs gemcitabine and BVdU.
We demonstrate that human nucleoside kinase reporter systems vary significantly in their sensitivity and selectivity for in vivo imaging. The sufficiently high signal-to-background ratios and enhanced suicide gene potential support clinical translation.
Key wordsHuman reporter gene imaging Deoxycytidine kinase Thymidine kinase [18F]FEAU l-[18F]FEAU l-[18F]FMAU [14C]FMAU [124I]FIAU Gene and cell therapy Immunotherapy
We thank Dr. Caius G. Radu at the Ahmanson Translational Imaging Division at UCLA for donating the pMSCV-hTK2 plasmid. We thank Dr. Pat Zanzonico and Valerie Longo at Memorial Sloan Kettering Cancer Center (MSKCC) and Waldemar Ladno and Dr. Olga Sergeeva at the UCLA Crump Institute for Molecular Imaging for their imaging technical assistance and expertise. We thank Eva M. Burnazi at MSKCC for her radiochemistry technical expertise. We thank Dr. Ronald Blasberg for his help in preparing this manuscript. Technical services were provided by the MSKCC Small Animal Imaging Core Facility and the UCLA Crump Institute’s Preclinical Imaging Technology Center.
Compliance with Ethical Standards
This work was supported by the Molecular Imaging: Training for Oncology (MITO) of the National Institutes of Health, Cancer Education and Career Development Program 5R25CA096945-09 grant, NIH P50 CA86438-11, R01 CA161138, and R01 CA163980 grants. Technical services provided by the MSKCC Small-Animal Imaging Core Facility are supported in part by NIH Small-Animal Imaging Research Program (SAIRP), NIH Shared Instrumentation Grant No 1 S10 RR020892-01, NIH Shared Instrumentation Grant No 1 S10 RR028889-01, and NIH Center Grant P30 CA08748. Technical services provided by the UCLA Crump Institute’s Preclinical Imaging Technology Center are supported in part by NIH In Vivo Cellular and Molecular Imaging Centers Grant P50 CA086306, NIH Cancer Center Support Grant P30 CA016042, and NIH SPORE Grant P50 CA092131.
Conflict of Interest
The authors declare that they have no conflict of interest.
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