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Review
. 2000 Jan-Apr;2(1-2):118-38.
doi: 10.1038/sj.neo.7900083.

Imaging transgene expression with radionuclide imaging technologies

S S Gambhir  1 , H R Herschman, S R Cherry, J R Barrio, N Satyamurthy, T Toyokuni, M E Phelps, S M Larson, J Balatoni, R Finn, M Sadelain, J Tjuvajev, R Blasberg
Affiliations
Review

Imaging transgene expression with radionuclide imaging technologies

S S Gambhir et al. Neoplasia. 2000 Jan-Apr.

Abstract

A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review "marker/reporter gene" imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications.

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Figures

Figure 1
Figure 1
(A) Schematic for imaging HSV1-tk marker/reporter gene expression with marker/reporter probes 5-iodo-2′-fluoro-2′deoxy-1-β-D-arabino-furanosyl-uracil (FIAU) or 8-[18F]fluoropenciclovir (FPCV). The HSV1-tk gene complex is transfected into target cells by a vector, which could be a retrovirus, an adenovirus, a liposome or any other transfer vector. Inside the transfected cell, the HSV1-tk gene is transcribed to HSV1-tk mRNA and then translated on the ribosomes to the protein enzyme, HSV1-TK. After FIAU or FPCV phosphorylation by the HSV1-TK enzyme, the radiolabeled marker/reporter probes do not readily cross the cell membrane and are “trapped” within the cell. Thus, the magnitude of marker/reporter probe accumulation in the cell reflects the level of HSV1-TK enzyme activity and level of HSV1-tk gene expression. FIAU-PO4 is incorporated into the DNA of dividing cells, whereas FPCV-PO4 and other phosphorylated acycloguanosine compounds act as DNA chain terminators and DNA polymerase inhibitors. (B) Schematic for imaging D2R marker/reporter gene expression with the marker/reporter probe 3-(2′-[18F]fluoroethyl)-spiperone (FESP). The dopamine type 2 receptor (D2R) marker/reporter gene once delivered to a cell by a vector of choice is transcribed to D2R mRNA and then translated to D2R. FESP can bind to extracellular and intracellular D2R receptors leading to probe accumulation in cells expressing D2R. Levels of accumulation of FESP reflect the level of D2R gene expression.
Figure 2
Figure 2
MicroPET scanner developed at UCLA for high-resolution PET imaging of small animals. Shown is the first-generation microPET scanner. The scanner has a transverse field of view of 11 cm and an axial field of view of 1.8 cm. The spatial resolution of the scanner is ∼1.83 mm3. An entire mouse can be scanned using multiple bed positions in 30 to 60 minutes. Newer versions of this scanner will allow for ∼13 mm3 spatial resolution and scanning times for an entire mouse in under 10 minutes.
Figure 3
Figure 3
Examples of microPET images. Left column (mice images): (top) [11C]-carfentanil binding to striata in mouse brain; (bottom) [18F]-fluoride mouse bone scan. Right column (rat images): (top) [18F]fluorodeoxyglucose (FDG) coronal images of rat brain; (bottom) FDG transverse sections at level of rat myocardium.
Figure 4
Figure 4
Chemical structures for marker/reporter probes for HSV1-TK and D2R. Derivatives of thymidine as substrates for HSV1-TK (top panel), derivatives of acycloguanosine as substrates for HSV1-TK (middle panel), and FESP as a ligand for D2R (bottom panel). Abbreviations are as stated in the main body of the paper.
Figure 5
Figure 5
Comparative assays of HSV1-tk expression. The radiotracer accumulation ratio (FIAU/TdR) that normalizes FIAU uptake for cell proliferation is compared to a functional assay of HSV1-tk expression; namely, sensitivity (IC50) to the antiviral drug, ganciclovir. Four different sets of stably transduced cell lines (clones) expressing HSV1-tk were studied. A highly reproducible relationship between these two assays is demonstrated that is independent of cell line and retroviral transduction vector.
Figure 6
Figure 6
[14C]FIAU imaging in tumors with autoradiography. The histology and autoradiographic images were generated from the same tissue section (panels A and B). The HSV1-tk-transduced RG2TK+ and wild-type RG2 tumors in each hemisphere of the rat brain are clearly seen in the toluidine blue-stained histologic section (panel A). Twenty four hours after administration of [14C]FIAU, the RG2TK+ tumor is clearly visualized in the autoradiographic image (panel B), whereas the RG2 tumor is barely detectable and the surrounding brain is at background levels. Rinsing an adjacent tissue section for 4 hours in 10% TCA had little effect on the distribution and amount of radioactivity measured in the autoradiogram (compare panels B and C).
Figure 7
Figure 7
[131I]FIAU imaging of tumors with a gamma camera. Gamma camera imaging was performed at 4, 24 and 36 hours after [131I]FIAU injection in animals bearing bilateral RG2 flank tumors; the images have been normalized to a reference standard (not shown in the field of view). The arrow indicates the inoculation site of HSV1-tk retroviral vector producer cells (gp-STK-A2) in the left flank tumor. The images demonstrate washout of radioactivity from the body with specific retention of activity in the area of gp-STK-A2 cell inoculation (see the 24- and 36-hour images). The non-transduced contralateral tumor (right flank) and other tissues did not show any retention of radioactivity.
Figure 8
Figure 8
[124I]FIAU PET images of HSV1-tk gene expression. Imaging multiple tumors (produced from stably transduced cell lines expressing different levels of HSV1-tk) in the same animal provides direct image and quantitative comparisons, since all tumors are exposed to the same levels of [124I]FIAU in the blood (same input function). PET imaging (over 40 min in 3D, septa-out mode) was performed 32 hours after [124I]FIAU injection with a General Electric Advance PET tomograph (Milwaukee, WI). Note the different levels of radioactivity (%dose/g) in the tumors. The relationship between radioactivity concentration (%dose/g) in the tumors and two independent assays of HSV1-tk expression in the cell lines used to produce the tumors is shown in Figure 9.
Figure 9
Figure 9
Quantitative relationships between the FIAU radioactivity (%dose/g) and two independent assays of HSV1-tk expression. The FIAU radioactivity (%dose/g) data were obtained from imaging experiments shown in Figure 8. Highly significant relationships were observed between the level of [124I]FIAU accumulation (%dose/g) and HSV1-tk mRNA levels (y = 0.005xe(0.071x), r = 0.993; left panel), and between the level of [124I]FIAU accumulation (%dose/g) and sensitivity (IC50) to the antiviral drug, GCV (y = 0.050xx(-0.422), r = 0.974; right panel).
Figure 10
Figure 10
MicroPET and DWBA images of mice after administration of control virus and Ad-CMV-HSV1-tk. Swiss-webster mice were injected through the tail vein with (a) 1.5x109 pfu of control virus or (b) 1.5x109 pfu of Ad-CMV-HSV1-tk virus. For each mouse, a whole body mean coronal projection image of the Fluorine-18 activity distribution is displayed on the left. The liver outline, in white, was determined from both the FGCV signal and cryostat slices. The second images from the left are coronal sections, approximately 2 mm thick, from the microPET. After their PET scans, the mice were killed, frozen and sectioned. The next images are photographs of the tissue sections (45 µm thick) corresponding to approximately the mid-thickness of the microPET coronal section. The images on the right are DWBA (autorad) of these tissue sections. The color scale represents the FGCV %ID/g tissue. Images are displayed on the same quantitative color scale, to allow signal intensity comparisons among the panels.
Figure 11
Figure 11
FGCV retention in liver as a function of HSV1-tk gene expression. Fourteen adult Swiss-webster mice were injected through the tail vein with 0 to 2.0x109 pfu of Ad-CMV-HSV1-tk virus and additional control virus to maintain the total viral burden to be fixed to 2.0x109 pfu. Forty-eight (±1) hours later, animals received a tail vein injection of FGCV. Animals were sacrificed 180 minutes later. Livers were removed and liver samples were analyzed for (i) Fluorine-18 retained in tissue; (ii) HSV1-tk mRNA levels normalized to GAPDH; and (iii) HSV1-TK enzyme levels. (Panel A) HSV1-TK enzyme levels versus HSV1-tk mRNA levels (y = 0.76x + 0.30, r2 = 0.81). (Panel B) FGCV %ID/g liver versus HSV1-tk mRNA levels (y = 0.71x + 0.44, r2 = 0.81). (Panel C) FGCV %ID/g liver versus HSV1-TK enzyme activity (y = 0.79x + 0.31, r2 = 0.71). Each point represents data from a different mouse.
Figure 12
Figure 12
PET and DWBA images of mice following Ad-CMV-βGal and Ad-CMV-D2R virus administration. Nude mice were injected through the tail vein with (A) 9x109 pfu of Ad-CMV-βGal virus or (B) 9x109 pfu of Ad-CMV-D2R virus. Two days after virus administration, both mice were injected through the tail vein with FESP (200 µCi, 200 µl). Three hours after the FESP injection, the animals were anesthetized, positioned supine with tail on left, and imaged with microPET For each mouse, a whole body coronal projection image of the [18F] activity distribution is displayed on the left. The liver outline, in white, was determined from both the FESP signal and cryostat slices. The second images from the left are coronal sections, approximately 2 mm thick, from the microPET. After their PET scans, the mice were killed, frozen and sectioned. The next images are photographs of the tissue sections (0.2 mm thick) corresponding to approximately the mid-thickness of the microPET coronal section. The images on the right are the DWBA (autorad) of these tissue sections. The color scale represents the %ID/g tissue. Images are displayed on the same quantitative color scale, to allow signal intensity comparisons among the panels.
Figure 13
Figure 13
PET ROI analysis of images from living animals reflects hepatic FESP retention, D2R levels and D2R mRNA levels in Ad-CMV-D2R infected mice. Nude mice were injected through the tail vein with 5x106 to 9x109 pfu of Ad-CMV-D2R virus, to produce varying levels of D2R reporter gene expression. FESP was injected through the tail vein from 2 to 60 days after viral injection. Three hours after FESP injection, the mice were imaged in the ACAT PET scanner. The mice were then sacrificed, and their livers were removed and homogenized. Samples from each liver were analyzed for FESP and labeled FESP metabolites, [3H]spiperone binding activity, and D2R mRNA and GAPDH mRNA levels. FESP metabolites were 1.0 ± 0.6 %ID/g liver. (A) in vivo hepatic [18F] retention (measured by image analysis) as a function of in vitro hepatic FESP retention (measured by well counting). Note that the contribution of hepatic FESP metabolites has been removed from the values determined for FESP retention by well counting, but remain in the image data and contribute to a non-zero intercept. (B) in vivo analysis of hepatic [18F] retention as a function of hepatic D2R levels, measured by [3H]spiperone binding. (C) in vivo analysis of hepatic [18F] retention as a function of GAPDH-normalized levels of hepatic D2R mRNA, measured by Northern blot analysis.
Figure 14
Figure 14
D2R and HSV1-tk tumor-bearing mouse imaged on a microPET with FESP and FPCV. A nude mouse carrying a tumor that expresses D2R on the left shoulder and a tumor that expresses HSV1-tk on the right shoulder was imaged on a microPET with FESP (day 0) and FPCV (day 1). Images are displayed using the same common global maximum. FESP accumulates primarily in the D2R positive tumor and FPCV accumulates primarily in the HSV1-tk positive tumor. Background activity in the gastrointestinal tract is seen due to clearance of both FESP and FPCV from these routes. Renal clearance is not visible in the coronal sections shown.
Figure 15
Figure 15
FPCV and FHBG images of a transgenic mouse imaged with microPET. A transgenic mouse was studied in which the albumin promoter drives the HSV1-tk marker/reporter gene. The mouse was imaged on day 0 with a microPET 1 hour after administration of FPCV (left panel) and on day 1 with FHBG (right panel). Both images are displayed using the same common global maximum and illustrate the higher %ID/g liver when utilizing FHBG compared with FPCV. There is significantly greater hepatic accumulation of FHBG compared to FPCV.
Figure 16
Figure 16
Schematic of transgene co-expression using fusion and internal ribosomal entry site (IRES) -based strategies. In a fusion gene strategy, the mRNA is translated into a single fusion protein containing the therapeutic and reporter proteins. In the IRES-linked gene strategy, the mRNA is translated into two distinct protein products through a cap-dependent and cap-independent process facilitated through the use of an IRES.
Figure 17
Figure 17
IRES-mediated pseudo-bicistronic co-expression of HSV1-tk and Lac Z genes. The MoMVL-based retroviral vector STLEO was used to stably transduce RG2 and W256 tumor cells. The level of Lac Z expression in different RG2-STLEO and W256-STLEO clones (and in tumors produced from the clones) were measured and directly compared with the FIAU radiotracer assay for HSV1-tk gene co-expression in the same clones (tumors). A good correlation between the levels of Lac Z and HSV1-tk gene expression was observed over a wide range of values in two separate cell lines with different transduction efficiencies, and this relationship was observed both in vitro (y = -0.161 + 0.010x, r = 0.977; left panel) and in vivo (y = 0.010xe(0.046x), r = 0.995; right panel).
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