Multimodality imaging of gene transfer with a receptor-based reporter gene.
Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Noninvasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor-enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging.


METHODS
An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of (111)In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice.


RESULTS
The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of (111)In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2. Although fluorescence was observed, there were discrepancies between in vivo imaging and ex vivo imaging as well as between nuclear imaging and fluorescent imaging.


CONCLUSIONS
These studies showed that the SSTR2-EGFP fusion construct can be used for in vivo nuclear and optical imaging of gene transfer.
Chen
Ron
R
Department of Radiation Oncology, School of Medicine, Washington University, St. Louis, Missouri 63108, USA.
Parry
Jesse J
JJ
Akers
Walter J
WJ
Berezin
Mikhail Y
MY
El Naqa
Issam M
IM
Achilefu
Samuel
S
Edwards
W Barry
WB
Rogers
Buck E
BE
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D045744:Cell Line, Tumor; D005260:Female; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics*; D018773:Genes, erbB-1 / Q000235:genetics; D006801:Humans; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D017481:Receptors, Somatostatin / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D015899:Tomography, Emission-Computed, Single-Photon
D010456:Peptides, Cyclic; D017481:Receptors, Somatostatin; C470036:octreotate, Tyr(3)-; C093733:somatostatin receptor 2