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. 2010 Aug;16(8):921-6.
doi: 10.1038/nm.2185. Epub 2010 Jul 18.

A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography

Tarik F Massoud  1 , Ramasamy Paulmurugan, Sanjiv S Gambhir
Affiliations

A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography

Tarik F Massoud et al. Nat Med. 2010 Aug.

Abstract

Improved techniques to noninvasively image protein-protein interactions (PPIs) are essential. We molecularly engineered a positron emission tomography (PET)-based split reporter (herpes simplex virus type 1 thymidine kinase), cleaved between Thr265 and Ala266, and used this in a protein-fragment complementation assay (PCA) to quantify PPIs in mammalian cells and to microPET image them in living mice. An introduced point mutation (V119C) markedly enhanced thymidine kinase complementation in PCAs, on the basis of rapamycin modulation of FKBP12-rapamycin-binding domain (FRB) and FKBP12 (FK506 binding protein), the interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor, and in an estrogen receptor intramolecular protein folding assay. Applications of this unique split thymidine kinase are potentially far reaching, including, for example, considerably more accurate monitoring of immune and stem cell therapies, allowing for fully quantitative and tomographic PET localization of PPIs in preclinical small- and large-animal models of disease.

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Figures

Figure 1
Figure 1
Principle of the TK PCA, expression vectors used, and evaluation of plasmid vector constructs containing the V119C mutation. (a) Schematic diagram showing the PCA strategy using split HSV1-TK (here abbreviated as TK) to monitor the hypothetical X–Y heterodimeric protein-protein interaction. This is accomplished by fusing each of the reporter fragments to heterologous X–Y protein domains to generate two chimeric proteins that have the capacity to interact with one another. If the interaction of the two heterologous protein domains (first and foremost) restores the activity of the reporter by bringing the two reporter fragments into close spatial proximity (as a secondary consequence), then this restoration of reporter activity can be used to monitor the interaction of the two heterologous protein domains. Dimerization of the two proteins restores TK activity through protein complementation and produces a PET imaging signal in the presence of radiolabeled TK substrate. If the reporter protein is an enzyme, then an additional strength of this PCA approach is the capacity to amplify the signal associated with each protein-protein interaction event. Note that this is a simplified schematic diagram representing the forward or ‘folding’ mechanism underlying a PCA. The reverse or ‘unfolding’ mechanism is not depicted here for the sake of simplification. A more realistic and detailed graphic depiction of the interplay between these two mechanisms within a PCA has been published previously by Michnick et al.. (b) Schematic representation of the plasmid vector constructs made for transient expression of the seven genes transfected individually or in combinations described in the text and Supplementary Methods, for evaluation of the PCA strategy. Each vector was cloned into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. (c) Graph to show comparison of coexpressed chimeras carrying nTK or cTK with FRB/FKBP12 (with and without rapamycin), and chimeras containing the TK point mutations V119C and R318C on enzyme activity in a PCA, measured by TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein) in transiently transfected 293T cells, with mock (negative) and full length HSV1-sr39TK (positive) controls. The error bar is the standard error of the mean for three samples. Introducing the point mutation V119C to the nTK fragment resulted in an increase (at limit of statistical significance) in measured TK activity upon addition of rapamycin, after co-transfecting nTK(V119C)-FRB and FKBP12-cTK. NS: not significant. Western blot analysis using antibody to TK in the presence or absence of Rapamycin shows adequate expression levels in 293T cells.
Figure 1
Figure 1
Principle of the TK PCA, expression vectors used, and evaluation of plasmid vector constructs containing the V119C mutation. (a) Schematic diagram showing the PCA strategy using split HSV1-TK (here abbreviated as TK) to monitor the hypothetical X–Y heterodimeric protein-protein interaction. This is accomplished by fusing each of the reporter fragments to heterologous X–Y protein domains to generate two chimeric proteins that have the capacity to interact with one another. If the interaction of the two heterologous protein domains (first and foremost) restores the activity of the reporter by bringing the two reporter fragments into close spatial proximity (as a secondary consequence), then this restoration of reporter activity can be used to monitor the interaction of the two heterologous protein domains. Dimerization of the two proteins restores TK activity through protein complementation and produces a PET imaging signal in the presence of radiolabeled TK substrate. If the reporter protein is an enzyme, then an additional strength of this PCA approach is the capacity to amplify the signal associated with each protein-protein interaction event. Note that this is a simplified schematic diagram representing the forward or ‘folding’ mechanism underlying a PCA. The reverse or ‘unfolding’ mechanism is not depicted here for the sake of simplification. A more realistic and detailed graphic depiction of the interplay between these two mechanisms within a PCA has been published previously by Michnick et al.. (b) Schematic representation of the plasmid vector constructs made for transient expression of the seven genes transfected individually or in combinations described in the text and Supplementary Methods, for evaluation of the PCA strategy. Each vector was cloned into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. (c) Graph to show comparison of coexpressed chimeras carrying nTK or cTK with FRB/FKBP12 (with and without rapamycin), and chimeras containing the TK point mutations V119C and R318C on enzyme activity in a PCA, measured by TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein) in transiently transfected 293T cells, with mock (negative) and full length HSV1-sr39TK (positive) controls. The error bar is the standard error of the mean for three samples. Introducing the point mutation V119C to the nTK fragment resulted in an increase (at limit of statistical significance) in measured TK activity upon addition of rapamycin, after co-transfecting nTK(V119C)-FRB and FKBP12-cTK. NS: not significant. Western blot analysis using antibody to TK in the presence or absence of Rapamycin shows adequate expression levels in 293T cells.
Figure 1
Figure 1
Principle of the TK PCA, expression vectors used, and evaluation of plasmid vector constructs containing the V119C mutation. (a) Schematic diagram showing the PCA strategy using split HSV1-TK (here abbreviated as TK) to monitor the hypothetical X–Y heterodimeric protein-protein interaction. This is accomplished by fusing each of the reporter fragments to heterologous X–Y protein domains to generate two chimeric proteins that have the capacity to interact with one another. If the interaction of the two heterologous protein domains (first and foremost) restores the activity of the reporter by bringing the two reporter fragments into close spatial proximity (as a secondary consequence), then this restoration of reporter activity can be used to monitor the interaction of the two heterologous protein domains. Dimerization of the two proteins restores TK activity through protein complementation and produces a PET imaging signal in the presence of radiolabeled TK substrate. If the reporter protein is an enzyme, then an additional strength of this PCA approach is the capacity to amplify the signal associated with each protein-protein interaction event. Note that this is a simplified schematic diagram representing the forward or ‘folding’ mechanism underlying a PCA. The reverse or ‘unfolding’ mechanism is not depicted here for the sake of simplification. A more realistic and detailed graphic depiction of the interplay between these two mechanisms within a PCA has been published previously by Michnick et al.. (b) Schematic representation of the plasmid vector constructs made for transient expression of the seven genes transfected individually or in combinations described in the text and Supplementary Methods, for evaluation of the PCA strategy. Each vector was cloned into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. (c) Graph to show comparison of coexpressed chimeras carrying nTK or cTK with FRB/FKBP12 (with and without rapamycin), and chimeras containing the TK point mutations V119C and R318C on enzyme activity in a PCA, measured by TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein) in transiently transfected 293T cells, with mock (negative) and full length HSV1-sr39TK (positive) controls. The error bar is the standard error of the mean for three samples. Introducing the point mutation V119C to the nTK fragment resulted in an increase (at limit of statistical significance) in measured TK activity upon addition of rapamycin, after co-transfecting nTK(V119C)-FRB and FKBP12-cTK. NS: not significant. Western blot analysis using antibody to TK in the presence or absence of Rapamycin shows adequate expression levels in 293T cells.
Figure 2
Figure 2
Subcellular localization of components of the PCA. (a) Fluorescence micrographs (×40 magnification) after immunohistochemical staining of negative control (293T-Mock) cells, positive control (Full length sr39TK [V119C] transfected 293T cells) cells, and 293T cells transiently transfected with component vectors of the PCA. These show considerable levels of TK protein expression in both nucleus and cytoplasm regardless of whether the TK reporter was split or intact. Fusion with interacting proteins FRB and FKBP12 did not perceptively hinder normal translocation of functional TK fragments within cells. Scale bar, 50 μm. (b) Western blot analysis reveals expression levels of components of the TK PCA determined in both nuclear and cytoplasmic fractions from lysates of 293T cells transfected accordingly, after immunoblotting with antibody to TK. Antibody to β-actin was used as an internal control for loading. Again, considerable levels of TK protein expression in both nucleus and cytoplasm showed that that there was no perceptible hindrance to normal translocation of functional TK fragments within cells.
Figure 2
Figure 2
Subcellular localization of components of the PCA. (a) Fluorescence micrographs (×40 magnification) after immunohistochemical staining of negative control (293T-Mock) cells, positive control (Full length sr39TK [V119C] transfected 293T cells) cells, and 293T cells transiently transfected with component vectors of the PCA. These show considerable levels of TK protein expression in both nucleus and cytoplasm regardless of whether the TK reporter was split or intact. Fusion with interacting proteins FRB and FKBP12 did not perceptively hinder normal translocation of functional TK fragments within cells. Scale bar, 50 μm. (b) Western blot analysis reveals expression levels of components of the TK PCA determined in both nuclear and cytoplasmic fractions from lysates of 293T cells transfected accordingly, after immunoblotting with antibody to TK. Antibody to β-actin was used as an internal control for loading. Again, considerable levels of TK protein expression in both nucleus and cytoplasm showed that that there was no perceptible hindrance to normal translocation of functional TK fragments within cells.
Figure 3
Figure 3
Testing the TK PCA in separate cell lines and PPI systems. (a) Graph to show comparison of coexpressed chimeras carrying nTK or cTK with FRB/FKBP12 (with and without rapamycin) in different cell lines, as measured by TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein) in transiently transfected 293T cells, SKBr3 cells, and SKOV3 cells. The error bar is the standard error of the mean for three samples. There was a statistically significant increase in measured TK activity upon addition of rapamycin to all 3 cell lines. Despite lack of normalization of transfection efficiency, there was a similar approximate 3-fold rise in split-TK complementation upon PPI regardless of which cell line was used. Western blot analysis of all 3 cells using antibody to TK before and after addition of rapamycin shows adequate expression levels. (b) A vector was constructed that transiently expresses a fusion protein with split TK fragments (containing a V119C point mutation) and substituting HIF1α for FRB, and VHL for FKBP12. Each construct was cloned into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently co-transfected (200 ng DNA each vector per well) to express nTK(V119C)-HIF1α and VHL-cTK, an empty vector, and full length HSV1-sr39TK as a positive control for 48 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with increasing doses of desferrioxamine (DFO) resulted in a proportional reduction in interaction of HIF1α and VHL. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to TK after treatment with the different doses of DFO shows adequate expression levels in 293T cells. (c) Schematic diagram of the intramolecular folding sensor construct with the split TK fragments on either side of the estrogen receptor ligand binding domain (ER–LBD), and each construct was made by cloning into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently transfected (500 ng DNA per well) to express the intramolecular folding sensor and treated with the indicated ER ligands or carrier control (dymethyl sulfoxide, DMSO) for 24 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with the ER ligands 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), raloxifene (RAL), methyl piperidinylethoxy pyrazole (MPP), genistein (GEN), diethylstilbestrol (DES), and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) led to levels of intramolecular-folding-assisted complementation that were significantly higher than that of carrier control-treated cells (P < .05) except for genistein. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to ERα after treatment with the different ligands shows adequate expression levels in 293T cells.
Figure 3
Figure 3
Testing the TK PCA in separate cell lines and PPI systems. (a) Graph to show comparison of coexpressed chimeras carrying nTK or cTK with FRB/FKBP12 (with and without rapamycin) in different cell lines, as measured by TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein) in transiently transfected 293T cells, SKBr3 cells, and SKOV3 cells. The error bar is the standard error of the mean for three samples. There was a statistically significant increase in measured TK activity upon addition of rapamycin to all 3 cell lines. Despite lack of normalization of transfection efficiency, there was a similar approximate 3-fold rise in split-TK complementation upon PPI regardless of which cell line was used. Western blot analysis of all 3 cells using antibody to TK before and after addition of rapamycin shows adequate expression levels. (b) A vector was constructed that transiently expresses a fusion protein with split TK fragments (containing a V119C point mutation) and substituting HIF1α for FRB, and VHL for FKBP12. Each construct was cloned into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently co-transfected (200 ng DNA each vector per well) to express nTK(V119C)-HIF1α and VHL-cTK, an empty vector, and full length HSV1-sr39TK as a positive control for 48 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with increasing doses of desferrioxamine (DFO) resulted in a proportional reduction in interaction of HIF1α and VHL. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to TK after treatment with the different doses of DFO shows adequate expression levels in 293T cells. (c) Schematic diagram of the intramolecular folding sensor construct with the split TK fragments on either side of the estrogen receptor ligand binding domain (ER–LBD), and each construct was made by cloning into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently transfected (500 ng DNA per well) to express the intramolecular folding sensor and treated with the indicated ER ligands or carrier control (dymethyl sulfoxide, DMSO) for 24 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with the ER ligands 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), raloxifene (RAL), methyl piperidinylethoxy pyrazole (MPP), genistein (GEN), diethylstilbestrol (DES), and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) led to levels of intramolecular-folding-assisted complementation that were significantly higher than that of carrier control-treated cells (P < .05) except for genistein. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to ERα after treatment with the different ligands shows adequate expression levels in 293T cells.
Figure 3
Figure 3
Testing the TK PCA in separate cell lines and PPI systems. (a) Graph to show comparison of coexpressed chimeras carrying nTK or cTK with FRB/FKBP12 (with and without rapamycin) in different cell lines, as measured by TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein) in transiently transfected 293T cells, SKBr3 cells, and SKOV3 cells. The error bar is the standard error of the mean for three samples. There was a statistically significant increase in measured TK activity upon addition of rapamycin to all 3 cell lines. Despite lack of normalization of transfection efficiency, there was a similar approximate 3-fold rise in split-TK complementation upon PPI regardless of which cell line was used. Western blot analysis of all 3 cells using antibody to TK before and after addition of rapamycin shows adequate expression levels. (b) A vector was constructed that transiently expresses a fusion protein with split TK fragments (containing a V119C point mutation) and substituting HIF1α for FRB, and VHL for FKBP12. Each construct was cloned into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently co-transfected (200 ng DNA each vector per well) to express nTK(V119C)-HIF1α and VHL-cTK, an empty vector, and full length HSV1-sr39TK as a positive control for 48 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with increasing doses of desferrioxamine (DFO) resulted in a proportional reduction in interaction of HIF1α and VHL. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to TK after treatment with the different doses of DFO shows adequate expression levels in 293T cells. (c) Schematic diagram of the intramolecular folding sensor construct with the split TK fragments on either side of the estrogen receptor ligand binding domain (ER–LBD), and each construct was made by cloning into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently transfected (500 ng DNA per well) to express the intramolecular folding sensor and treated with the indicated ER ligands or carrier control (dymethyl sulfoxide, DMSO) for 24 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with the ER ligands 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), raloxifene (RAL), methyl piperidinylethoxy pyrazole (MPP), genistein (GEN), diethylstilbestrol (DES), and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) led to levels of intramolecular-folding-assisted complementation that were significantly higher than that of carrier control-treated cells (P < .05) except for genistein. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to ERα after treatment with the different ligands shows adequate expression levels in 293T cells.
Figure 4
Figure 4
Imaging of tumors containing the split TK constructs. (a) Transaxial tomographic microPET images through a representative prone-positioned mouse implanted subcutaneously over the left shoulder with mock transfected 293T cells, and over the right shoulder with 293T cells stably expressing both nTK(V119C)-FRB and FKBP12-cTK. The mouse was injected with 200 μCi of [18F]-FHBG prior to imaging on days 1, 2, and 5 into the imaging protocol (i.e., after 7 days of initial xenograft growth). Elliptical dotted white line outlines the surface of the mouse’s upper thorax. Color intensity is a reflection on probe accumulation after its phosphorylation by the complemented TK enzyme. Quantitative analysis of this probe accumulation shows a mean %ID g−1 (obtained from 5 tomographic slices through each tumor for all animals) as displayed in accompanying graph (b). The difference between accumulation in tumors exhibiting split TK complementation and control tumors was statistically significant (P = 0.02) on Day 5. Also shown is the optical CCD imaging on day 5 of the imaging protocol of the same mouse immediately before its subsequent microPET imaging of bilateral shoulder region subcutaneous xenografts, and is shown as a visible light image superimposed on the CCD bioluminescence image with a scale in photons/sec/cm2/steradian. Mice were imaged in the prone position after tail-vein injection of 4 mg D-Luciferin per animal. Each mouse was implanted subcutaneously over the left shoulder with 5 × 106 mock transfected 293T cells admixed with 50,000 293T stable cells expressing Firefly luciferase, and subcutaneously over the right shoulder with 5 × 106 293T stable cells expressing both nTK(V119C)-FRB and FKBP12-cTK admixed with 50,000 293T stable cells expressing Firefly luciferase. Bioluminescence imaging shows equivalent viable tumor load in both xenografts. Angeled black dotted line shows the transaxial plane, bisecting each tumor, through which the microPET images were obtained. (c) Coronal tomographic microPET images through two representative prone mice implanted subcutaneously over the left shoulder (circle 1) with control tumors of 293T cells stably expressing nTK plus cTK only (in a single vector), and over the right shoulder (circle 2) with 293T cells expressing nTK(V119C)-FRB plus FKBP12-cTK in a single vector. Unlike tumors containing the complemented TK enzyme, there was minimal FHBG accumulation (%ID g−1) in the control tumors on the fifth day of the imaging protocol upon systemic administration of rapamycin (see text). Intense accumulation in centre of image is due to non-specific probe excretion in the gut.
Figure 4
Figure 4
Imaging of tumors containing the split TK constructs. (a) Transaxial tomographic microPET images through a representative prone-positioned mouse implanted subcutaneously over the left shoulder with mock transfected 293T cells, and over the right shoulder with 293T cells stably expressing both nTK(V119C)-FRB and FKBP12-cTK. The mouse was injected with 200 μCi of [18F]-FHBG prior to imaging on days 1, 2, and 5 into the imaging protocol (i.e., after 7 days of initial xenograft growth). Elliptical dotted white line outlines the surface of the mouse’s upper thorax. Color intensity is a reflection on probe accumulation after its phosphorylation by the complemented TK enzyme. Quantitative analysis of this probe accumulation shows a mean %ID g−1 (obtained from 5 tomographic slices through each tumor for all animals) as displayed in accompanying graph (b). The difference between accumulation in tumors exhibiting split TK complementation and control tumors was statistically significant (P = 0.02) on Day 5. Also shown is the optical CCD imaging on day 5 of the imaging protocol of the same mouse immediately before its subsequent microPET imaging of bilateral shoulder region subcutaneous xenografts, and is shown as a visible light image superimposed on the CCD bioluminescence image with a scale in photons/sec/cm2/steradian. Mice were imaged in the prone position after tail-vein injection of 4 mg D-Luciferin per animal. Each mouse was implanted subcutaneously over the left shoulder with 5 × 106 mock transfected 293T cells admixed with 50,000 293T stable cells expressing Firefly luciferase, and subcutaneously over the right shoulder with 5 × 106 293T stable cells expressing both nTK(V119C)-FRB and FKBP12-cTK admixed with 50,000 293T stable cells expressing Firefly luciferase. Bioluminescence imaging shows equivalent viable tumor load in both xenografts. Angeled black dotted line shows the transaxial plane, bisecting each tumor, through which the microPET images were obtained. (c) Coronal tomographic microPET images through two representative prone mice implanted subcutaneously over the left shoulder (circle 1) with control tumors of 293T cells stably expressing nTK plus cTK only (in a single vector), and over the right shoulder (circle 2) with 293T cells expressing nTK(V119C)-FRB plus FKBP12-cTK in a single vector. Unlike tumors containing the complemented TK enzyme, there was minimal FHBG accumulation (%ID g−1) in the control tumors on the fifth day of the imaging protocol upon systemic administration of rapamycin (see text). Intense accumulation in centre of image is due to non-specific probe excretion in the gut.
Figure 4
Figure 4
Imaging of tumors containing the split TK constructs. (a) Transaxial tomographic microPET images through a representative prone-positioned mouse implanted subcutaneously over the left shoulder with mock transfected 293T cells, and over the right shoulder with 293T cells stably expressing both nTK(V119C)-FRB and FKBP12-cTK. The mouse was injected with 200 μCi of [18F]-FHBG prior to imaging on days 1, 2, and 5 into the imaging protocol (i.e., after 7 days of initial xenograft growth). Elliptical dotted white line outlines the surface of the mouse’s upper thorax. Color intensity is a reflection on probe accumulation after its phosphorylation by the complemented TK enzyme. Quantitative analysis of this probe accumulation shows a mean %ID g−1 (obtained from 5 tomographic slices through each tumor for all animals) as displayed in accompanying graph (b). The difference between accumulation in tumors exhibiting split TK complementation and control tumors was statistically significant (P = 0.02) on Day 5. Also shown is the optical CCD imaging on day 5 of the imaging protocol of the same mouse immediately before its subsequent microPET imaging of bilateral shoulder region subcutaneous xenografts, and is shown as a visible light image superimposed on the CCD bioluminescence image with a scale in photons/sec/cm2/steradian. Mice were imaged in the prone position after tail-vein injection of 4 mg D-Luciferin per animal. Each mouse was implanted subcutaneously over the left shoulder with 5 × 106 mock transfected 293T cells admixed with 50,000 293T stable cells expressing Firefly luciferase, and subcutaneously over the right shoulder with 5 × 106 293T stable cells expressing both nTK(V119C)-FRB and FKBP12-cTK admixed with 50,000 293T stable cells expressing Firefly luciferase. Bioluminescence imaging shows equivalent viable tumor load in both xenografts. Angeled black dotted line shows the transaxial plane, bisecting each tumor, through which the microPET images were obtained. (c) Coronal tomographic microPET images through two representative prone mice implanted subcutaneously over the left shoulder (circle 1) with control tumors of 293T cells stably expressing nTK plus cTK only (in a single vector), and over the right shoulder (circle 2) with 293T cells expressing nTK(V119C)-FRB plus FKBP12-cTK in a single vector. Unlike tumors containing the complemented TK enzyme, there was minimal FHBG accumulation (%ID g−1) in the control tumors on the fifth day of the imaging protocol upon systemic administration of rapamycin (see text). Intense accumulation in centre of image is due to non-specific probe excretion in the gut.
Figure 5
Figure 5
The relative advantage of PET over optical imaging in imaging sources of signal at depths below 1 cm from the exterior is exemplified in this separate experiment. Five mice were imaged 6 days post implantation with 5 × 106 293T cells stably expressing TK or Firefly luciferase (FLUC). The microPET signal return (FHBG accumulation in %ID g−1) from shoulder tumors (circled) expressing TK did not differ in significance whether the animals were imaged in the supine or prone positions (probe %ID g−1 of 0.89 and 1.02 respectively), as demonstrated in the transaxial and coronal tomographic images of a representative animal (intense accumulation in centre and lower aspect of coronal images is due to non-specific probe excretion in the gut). When imaged in the supine position, the light emanating from subcutaneous xenografts on the shoulder or back showed no penetration through the full thickness of the mouse.
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