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1 Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California;2 University of California Los Angeles-Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, California;3 Department of Biomathematics, David Geffen School of Medicine, University of California, Los Angeles, California;4 Howard Hughes Medical Institute and Department of Pharmacology, University of California, San Diego, California; and5 Department of Radiology and the Bio-X Program, Stanford University
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.74–0.85) of signal from each component by imaging tumor xenografts in living mice with all three modalities. Furthermore, metastases of a human melanoma cell line (A375M) stably expressing the triple fusion were imaged by microPET and optical technologies over a 40–50-day time period in living mice. Imaging of reporter gene expression from single cells to living animals with the help of a single tri-fusion reporter gene will have the potential to accelerate translational cancer research. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A specific strategy for combining the different modalities, including a cell-based technique with an animal imaging technique, is to build a unified fusion gene composed of different reporter genes whose expression can be imaged with different imaging modalities in both individual cells and living subjects. This approach is useful, provided that the fusion protein could retain at least moderate levels of activity of each individual reporter protein and be stable and not broken down into its specific constituents. This approach also opens the possibility of merging PET and optical imaging techniques for applications in a single living subject. Previously, we reported construction and validation of a fusion reporter vector bearing HSV1-sr39 thymidine kinase and Renilla luciferase (tk20rl) joined by a 20-amino acid (aa)-long spacer for imaging with microPET and bioluminescence optical charge-coupled device (CCD) modalities in living mice (7) . This strategy was limited by the inability to image individual cells due to the relatively low light yield from bioluminescence. In the current study, we developed and tested several triple fusion vectors bearing a bioluminescence, a fluorescence, and a PET reporter gene joined by a 14-aa-long and an 8-aa-long spacer, respectively. Among all of the vectors tested, hrl-mrfp-ttk triple fusion reporter vector containing a synthetic Renilla luciferase [hrl (8) ], monomeric rfp [mrfp1 (9) ], and a truncated version of sr39tk (ttk; the first 135 bp of sr39tk were deleted) could best preserve the activities of all three component proteins and was therefore pursued for additional studies. We then imaged reporter gene expression in individual living cells by inverted fluorescence microscopy and also in living mice by both microPET and a cooled CCD camera compatible with both bioluminescence and fluorescence imaging. We also developed a lentiviral vector carrying the triple fusion reporter construct to infect dividing and nondividing cells and isolated stable expressers of 293T cells by fluorescence-activated cell sorting (FACS). Finally, we used a cancer metastatic model by introducing human melanoma cells (A375M) stably expressing the triple fusion reporter gene in living mice via tail vein and imaged metastases over a period of 50 days by all three of the imaging modalities. The unique tri-fusion vector should facilitate rapid translation of approaches developed in cells to preclinical models and, eventually, clinical applications.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of hrl-mrfp-ttk and Other Fusion Genes.
PCR amplification and standard cloning techniques were used to insert the hrl and mrfp genes from plasmid pCDNA 3.1-CMV-hrl (Promega, Madison, WI) and pCDNA3.1-CMV-mrfp1 in frame with the ttk gene into the pCDNA3.1-sr39-truncated tk (a kind gift of Dr. D. Kaufman; University of California, Los Angeles, CA). The CMV-wtk vector was obtained from Dr. M. Black and modified to truncated wtk (wttk) by deleting first 135 bp through PCR and cloned in pCDNA3.1 backbone to generate CMV-wttk plasmid. CMV-fl and CMV-egfp were purchased from Promega and BD Sciences-Clontech (Palo Alto, CA) respectively. For PCR amplifications, different 5' and 3' end primers were used to generate the fusion vectors. Standard cloning techniques were used to generate the lentiviral (CS-hrl-mrfp-ttk) vector as performed previously in our laboratory (11)
.
Cell Lines and Transient Transfection Procedures.
Neuro 2a (N2a) neuronal cell lines (a gift from Dr. Vincent Mauro; Scripps Research Institute, La Jolla, CA), 293T human embryonic kidney cells (American Type Culture Collection, Manassas, VA), and A375M human melanoma cells (a gift from Dr. M. Kolodny; University of California, Los Angeles, CA) were used. The N2a and A375M cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin (100 µg/ml) and streptomycin (292 µg/ml), and 293T cells were grown in MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution. All transient transfections were carried out using the Superfect transfection reagent (Qiagen, Valencia, CA) following the protocol recommended by the manufacturer.
tTK, hRL, and ß-Galactosidase (ß-Gal) Activity.
TK enzyme activity assays were performed as described previously (12)
, and ß-Gal and Renilla or firefly luciferase assays were done using the ß-Gal enzyme assay system and Dual-Luciferase Reporter Assay System from Promega, respectively. Each of the luciferase reactions was measured in a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA) for a period of 10 s.
Western Blot Analysis.
The levels of tTK and hRL were evaluated by Western blotting with a rabbit polyclonal anti-TK antiserum and a mouse monoclonal anti-Renilla antibody using cell lysates prepared from 293T cells transfected with CMV-hrl-mrfp-ttk, CMV-ttk, and CMV-hrl plasmids (12)
.
Lentiviral Production.
Lentivirus was developed and used to infect 293T and A375M cells as described previously (11)
.
Fluorescence Microscopy, CCD Imaging, and FACS.
Expression of mRFP1 was observed under a Zeiss Axiovert 200M fluorescence microscope (Carl Zeiss Microimaging Inc., Thornwood, NY) with DsRed filter setting (
ex, 546 nm; em, 605 nm) and analyzed with MetaMorph software (University Imaging Corp., Downingtown, PA). For quantification of the expression level of mRFP1 present in the CMV-hrl-mrfp-ttk and CMV-mrfp1, 1 x 104 and 1 x 105 of 293T, A375M, or N2a cells expressing the vectors were seeded in black-bottomem clear 96-well plates and imaged in the Xenogen IVIS optical imaging system (Xenogen Corp., Almeda, CA) with an excitation filter at 500–550 nm and an emission filter at 575–650 nm. Regions of interest (ROIs) were drawn over the cell area and quantified by using Living Image Software version 2.20. For FACS, 1 x 106 of CS-hrl-mrfp-ttk infected 293T and A375M cells were sorted by using a Becton Dickinson FACSvantage SE cell sorter.
MicroPET Imaging of Mice.
Animal care and euthanasia were performed with the approval of the University of California Animal Research Committee. Male 12–14-week-old nude mice (nu/nu) received s.c. injection with 10 x 106 293T cells transiently expressing the CMV-hrl-mrfp-ttk fusion, CMV-ttk, CMV-hrl, and CMV-mrfp1 on the ventral side, and mice (n = 4) were scanned the next day using a microPET as described previously (12)
. Additionally, 10 x 106 of each of four differentially expressing clones of 293T cells stably expressing hrl-mrfp-ttk gene were implanted in three mice and scanned in the microPET 24 h later. The microPET images were reconstructed by using three-dimensional filtered back projection and an iterative maximum a posteriori algorithm (13)
. ROIs were drawn over the tumor area. The ROI counts were converted to percentage of injected dose/g (ID/g) using filtered back projection as described previously (12)
, and images shown were reconstructed with maximum a posteriori algorithm.
Bioluminescence and Fluorescence Imaging of mRFP1 and RL Expression in Living Mice.
For in vivo fluorescence imaging, mice implanted with the cells described above were anesthetized, and each mouse was placed in a light tight chamber equipped with a halogen light source, and whole body image was acquired for 1 s using the Xenogen IVIS optical imaging system with an excitation filter at 500–550 nm and an emission filter at 575–650 nm. ROIs were drawn over implanted cell area and quantified by using Living Image Software version 2.20. For bioluminescence imaging, each mouse next received injection with 10 µl (2 µg/µl dissolved in methanol) of coelenterazine diluted in 90 µl of PBS (pH 7) via tail vein. Each animal was then placed supine in the same light tight chamber, and whole body images were obtained and quantified as described previously (8)
. Both bioluminescence and fluorescence signals were recorded as maximum [photons/second/centimeter2/steradian (photons/s/cm2/sr)].
Multimodality Imaging of Cancer Metastasis in Living Mice Using a Human Melanoma Cell Line (A375M) Stably Expressing the Triple Fusion Vector.
Three 8-week-old Beige severe combined immunodeficient mice received injection with 7 x 105 A375M cells stably expressing the hrl-mrfp-ttk gene via tail vein and were imaged repeatedly with fluorescence, bioluminescence, and microPET. At day 40, the mice were first imaged with microPET and bioluminescence (as described above) and then sacrificed and imaged; the chest was cut open with Illuminatool Tunable lighting system using the 540 nm excitation filter and RFP viewing glass (Lightools Research). Fluorescence imaging and light photograph of mice were digitally captured with a Nikon camera for 2 s.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid DNA prepared from four to five clones of CMV-hrl-mrfp-ttk triple fusion were transiently transfected in 293T cells, and the cells were first observed in a fluorescence microscope for mRFP1 activity and further assayed for hRL and tTK activity. The plasmid clone exhibiting the highest mRFP1, hRL, and tTK activities was selected for additional studies. To extend our study to different variants of bioluminescence/fluorescence/PET reporter genes, we also generated several functionally active multimodality reporter fusion vectors, i.e., fl-mrfp-ttk [by replacing hrl with firefly luciferase (fl)], fl/hrl-egfp/rfp-ttk (mrfp1 is replaced with egfp or tetrameric rfp known as DsRed2), and fl/hrl-rfp-wttk [by replacing the truncated HSV1-sr39tk (ttk) with wild-type HSV1-truncated thymidine kinase (wttk)]. The nature and the order of the spacers for all these constructs were equivalent to CMV-hrl-mrfp-ttk vector described earlier. The ttk, wttk, fl, hrl, rfp, gfp, and mrfp1 genes were also cloned in pCDNA3.1+ backbone to generate positive control plasmids to directly compare the results of each fusion. All these fusion vectors were functionally active with respect to each individual protein, however the level of activity varied for each construct (Table 1)
. Overall, the hrl-mrfp-ttk fusion construct showed the highest activity for all three of the component proteins in comparison with other vectors and thus was further studied for multimodality imaging.
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were transiently transfected with the triple fusion plasmid along with proper positive controls (pCDNA3.1-ttk, pCDNA3.1-mrfp1, pCDNA3.1-hrl) and negative controls (mock-transfected control cells). Each cell line was also cotransfected with the CMV-ß-gal reporter gene to normalize for transfection efficiency. After 24 h, the expression of mrfp1 was observed in the inverted fluorescence microscope, and then activity of the other three reporter genes was assayed from the same cell lysates, and tTK and hRL activities were normalized to ß-Gal activity. In all three of the different cell lines, CMV-hrl-mrfp-ttk showed equal or slightly higher tTK activity (not statistically significant) compared with the positive control (pCDNA3.1-ttk) but had a lower hRL activity [33% (A375M), 27.4% (N2a), and 54% (293T)] compared with that of the positive control pCDNA3.1-hrl plasmid (P < 0.05). The expression level of mrfp1 of this triple fusion vector in all of the cell lines was 60–70% of the positive control pCDNA3.1-mrfp1 vector, as determined by the fluorescence signal using the CCD camera.
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. The predominant band in the triple fusion sample recognized by anti-TK (Fig. 2A.1)
and anti-RL (Fig. 2
.A.2) antibodies is about 100 kDa, the expected size of the hRL-mRFP-tTK fusion protein. The tTK and the hRL proteins were recognized at about 36 kDa band by their specific antibodies. Similar results were also obtained from N2a and A375M cell extracts (data not shown).
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. The sorted and unsorted cells were then assayed for tTK and hRL expression (Fig. 2B.2)
and clearly show a significant (P < 0.05) gain in expression in the sorted cell population. Similarly, the A375M cells were transduced with the lentivirus carrying the triple fusion reporter gene, and positive expressers were selected by two rounds of FACS sorting and verifed to have significant hRL and tTK activities (data not shown).
293T Cells Transiently Expressing the hrl-mrfp-ttk Fusion Reporter Gene Can Be Imaged in Living Mice with the MicroPET and Optical Cooled CCD Imaging Systems.
Our aim of building a fusion reporter vector was to test its efficacy for simultaneous imaging of reporter gene expression quantitatively and repeatedly in living subjects using different modalities. We therefore injected 10 x 106 293T cells transiently transfected with either CMV-hrl-mrfp-ttk, CMV-ttk, CMV-hrl, or CMV-mrfp1 vectors s.c. at four different sites on the ventral sides of four 12–14-week-old nu/nu nude mice. The mice were first scanned using the cooled CCD camera for fluorescence followed by a bioluminescence scan after injection of 20 µg of coelenterazine via tail vein. Fluorescence imaging of these mice reveals that the cells expressing the hrl-mrfp-ttk fusion (Fig. 3A.1, a)
show lower fluorescence [11.03 ± 6.03 x 108 maximum (p/s/cm2/sr)] in comparison with the cells expressing mrfp1 vector (Fig. 3A.1, c)
alone [65.8 ± 32.29 x 108 maximum (p/s/cm2/sr); Fig. 3A.1
]. No significant signal is observed from the other two sites of implantation carrying the CMV-ttk (Fig. 3A.1, b)
- and CMV-hrl (Fig. 3A.1, d)
-expressing cells. A bioluminescence scan of the mice shows a signal of 5.8 ± 3.7 x 106 maximum (p/s/cm2/sr) from the cells expressing the fusion reporter gene (Fig. 3A.2, a)
and about 7.37 ± 4 x 106 maximum (p/s/cm2/sr) from the CMV-hrl-expressing cells (Fig. 3A.2, d)
. Similar to fluorescence imaging, the other two sites carrying CMV-mrfp1 (Fig. 3A.2, c)
- and CMV-ttk (Fig. 3A.2, b)
-expressing cells did not show any significant bioluminescence signal. Because the FHBG mass used for PET imaging is 1000-fold lower (due to the presence of radioactive isotope) than the coelenterazine mass used for bioluminescence imaging, PET imaging is not as sensitive as bioluminescence at superficial depths (1)
. Furthermore, PET imaging benefits from well-vascularized tissues with relatively high levels of reporter gene expression. We therefore implanted the cells expressing the fusion gene and ttk gene in the right and left axillary region of the mice. We quantified the signal from each of the sites expressing the CMV-hrl-mrfp-ttk and CMV-ttk directly from the microPET images to determine the percentage of ID/g tumor for FHBG. This percentage of ID/g is a measure of the amount of tracer accumulated in a given tissue site normalized to the injected amount and to the mass of the tissue examined. The mean percentage of ID/g for FHBG accumulation in the CMV-hrl-mrfp-ttk-expressing cells (0.303 ± 0.09; Fig. 3A.3, a
and Fig. 3A.4, a
) did not differ significantly from that of the CMV-ttk-expressing cells (0.313 ± 0.09; Fig 3A.3, b
and Fig. 3A.4, b
) for the four mice (Fig. 3A.3 and 3A.4)
. Preservation of a high level of tTK activity and moderate levels of hRL and mRFP1 activities by this tri-fusion vector thus allows simultaneous imaging of transient expression of all three of the reporter genes in living mice with all three of the imaging techniques. Repetitive imaging of the same mouse over a 10-day period produced signals for all three of the reporter genes that increased with time as the tumor burden increased (data not shown).
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); R2 = 0.74 (hRL:mRFP1; Fig. 3
.B.2); and R2 = 0.74 (tTK:mRFP1; Fig. 3B.3
).
Metastasis of A375M Human Melanoma Cells Expressing the hrl-mrfp-ttk Reporter Gene Can Be Imaged by MicroPET and an Optical CCD Camera in Living Mice.
To apply the tri-fusion strategy to a relevant preclinical cancer study, we used a melanoma metastatic model. A375M human melanoma cells are known to metastasize to other organs once injected in the animal i.v. and form pulmonary and brain metastases with some rare occurrence of bone metastases (14
, 15)
. A375M cells (7 x 105) stably expressing the hrl-mrfp-ttk reporter gene were injected in three 8-week-old Beige severe combined immunodeficient mice via tail vein. On the first day of cell injection, bioluminescence signal was detectable from the lungs (the primary route of cell migration; Fig. 4A
), but not from microPET images (data not shown). The mice were then subsequently imaged over time every 6–7 days for a period of 40–50 days. At day 40, moderate microPET signal (0.35% ID/g) from the lungs and strong signal (0.78% ID/g) from the chest region are detected from one of the three mice (Fig. 4C)
. A corresponding bioluminescence signal (2 x 105 p/s/cm2/sr) is detected from the lungs of the same mouse on the same day (Fig. 4B)
. The relatively high PET signal from the chest region (Fig. 4C)
is not evident with bioluminescence imaging (Fig. 4C)
, likely due to relatively poor penetration of light produced by Renilla luciferase from greater depths. A faint bioluminescence signal (5 x 103 p/s/cm2/sr) was also seen from the pelvic region of the mouse that was undetectable in microPET, likely due to hindrance by the nonspecific signal due to FHBG tracer clearance from the kidneys and gastrointestinal tract. In vivo fluorescence imaging of metastases did not produce good images due to significant autofluorescence caused by the presence of hair. However, when the mouse was sacrificed, and internal organs were exposed, several small metastatic tumors were found with fluorescence (Fig. 4E)
. Among the other two mice, one showed bioluminescence signal in the abdominal region at day 48; however we could not detect specific microPET signal, likely due to the presence of moderate levels of nonspecific signal resulting from the clearence of FHBG through kidneys and the gastrointestinal tract.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although our previous bi-fusion reporter construct (tk20rl) showed well-correlated expression of PET and bioluminescence imaging modalities (7) , it was somewhat limited by decreased TK activity and was susceptible to enzymatic cleavage into its component proteins. By changing the orientation of the fusion partner in the current vector, we could gain a significant amount of TK activity, indicating that the COOH-terminal end of thymidine kinase protein may be crucial for ensuring TK enzyme activity. In contrast, the hRL activity of the current construct showed a decrease in enzyme activity as opposed to our previous vector, which showed a gain in RL activity. However, this new synthetic version of Renilla luciferase (hRL) is 40–50-fold more active than original Renilla luciferase (18) , and therefore a drop in RL activity did not affect the efficacy of this vector significantly. Moreover, the tk gene in this triple fusion vector has a deletion of the first 135 bp that contains a nuclear localization signal and a cryptic testis-specific transcriptional start point (19 , 20) . Thus, this deletion leads to more cytoplasmic localization of TK enzyme, likely resulting in more TK activity (21) due to the availability of greater amount of substrate (FHBG). This deletion mutant will also likely overcome the problem of male sterility in transgenic mice carrying the thymidine kinase gene due to production of a shorter transcript in testis from a cryptic transcriptional initiation site (20) present in the first 135 bp of the gene. Another added advantage of this vector over our previous one and other vectors reported in the literature is that it can retain its integrity as a fusion protein when expressed, so that signal from each component of the tri-fusion protein will not be susceptible to problems related to cleavage. The absence of cleavage of the triple fusion vector is likely due to change of certain amino acids (Cys-Gly to Ser-Thr) present in the spacer in contrast to the previously reported 20-aa spacer of the tk20rl vector.
In the process of building a better multimodality vector, we constructed several other fusion vectors (see Table 1
). Most of these vectors had lower tTK, luciferase, and RFP activity, probably due to the inherent nature of RFP (DsRed2) of forming obligate tetramer for proper maturation of the flurophores (22)
present in the fusion genes. The tetrameric nature of RFP present in hrl/fl-rfp-ttk fusions might impose structural and functional constrains on the other partner proteins resulting in decreased TK and luciferase activity. Our hrl/fl-egfp-ttk vectors did show a better TK and luciferase activity than hrl/fl-rfp-ttk vectors, due to the monomeric nature of eGFP proteins, but did not show better activity than the hrl/fl-mrfp-ttk fusion vectors. Moreover, the excitation and emission spectra of GFP (ex, 489 nm; em, 508 nm) is not as favorable for fluorescence imaging in living subjects as compared with RFP and mRFP because of the better penetration of red and near-infrared light in tissues (1)
. We also consistently observed a drop in tTK and RFP activity of the triple fusions with firefly luciferase in comparison with the fusions bearing Renilla luciferase. Fusion reporter vectors bearing truncated wild-type thymidine kinase also preserved a better wild-type thymidine kinase and luciferase activity (with both firefly and Renilla), and these vectors should be useful in the future when using other substrates (e.g., 2'-fluoro- 5-fluoro-1-ß-D-arbinofuranosyluracil/2'-deoxy-2'-fluoro-5-fluoro- 1-ß-D-arbinofuranosyluracil) that are more sensitive when used with wild-type thymidine kinase (23)
. It is likely that one tri-fusion will not serve the needs for all applications, and investigators will need to choose from a library of tri-fusions for a given application.
One of the potential uses of the multimodality reporter vectors in gene therapy is to target any type of cell line or tissues and then follow gene expression using a multimodality approach. Viral vectors, especially the lentiviral ones, are among the most standardized and widely used vectors to deliver any gene of interest to target tissues or an organism and to isolate cells, particularly nondividing cells stably expressing the gene. Recently, a bicistronic lentiviral vector carrying tk and fl reporter genes has been successfully used for PET and bioluminescence imaging in our laboratory (11) . The new lentiviral construct carrying the triple fusion gene reported in this work has been used successfully with FACS analysis to isolate lentiviral infected 293T and A375M cells stably expressing the triple fusion reporter. This lentiviral construct should have tremendous potential in wide variety of research applications. Our preliminary data with the A375M metastatic melanoma model further confirm the usefulness of this lentiviral vector carrying triple fusion reporter gene to follow progression of cancer metastases by molecular imaging. Extensions of this study with drug treatment are currently in progress (24) .
Use of light is probably the oldest method of analyzing tissues in biomedical science (25) . The various optical imaging approaches including fluorescence microscopy (at the cellular level), diffuse optical tomography, and intravital microscopy (for deeper structures at the organism level) are commonly used (26 , 27) . However, intrinsic absorption and scattering of light through the tissues and autofluorescence properties of biological molecules (e.g., tryptophan, collagen, elastin, nicotineamide adenine dinucleotide, hemoglobin, oxyhemoglobin, and so forth) impose certain restrictions for using fluorescence as an imaging tool in small living subjects. However, both light attenuation and autofluorescence decline as wavelength increases, especially in the red to near-infrared region (>600 nm). A fluorescence protein/fluorochrome with excitation and emission toward red (560 nm onward) has better penetrability through the tissues than that with excitation and emission in the blue or green region. Moreover, hemoglobin and water, which are responsible for the highest absorption of light among all other biological molecules, have their lowest coefficient of absorption in the red and near-infrared region. Therefore, in vivo fluorescence imaging is more suitable in the red and near-infrared region than in the green or yellow region. Optical imaging in living subjects at the near-infrared region (650–900 nm) has therefore been used extensively by applying different fluorochromes that emit light at near-infrared region spectra (28) and in combination with other imaging modalities (29) . However, synthesis and attachment of these fluorochromes to proteins/copolymers require complex chemical procedures and are more difficult to generalize. Also, these strategies are not directly applicable to genetically encoded reporters. The mRFP1 protein used in this study has an excitation and emission range in the far-red region (584–607 nm) and thus is one of the better reporter gene choices for fluorescence imaging in living subjects (9) . The monomeric nature of this protein also confers a better functional preservation as fusion partner as compared with RFP or DsRed2 (tetramer) and HcRed (dimer). However, we still observed a significant amount of autofluorescence from the mouse relative to bioluminescence, which is not limited by autofluorescence, and therefore bioluminescence produced a better signal:background ratio in living animals. However, bioluminescence imaging of gene expression in a single cell is not easily possible due to generation of relatively low amounts of light. Therefore, the current results would support using the fluorescence component for cell imaging/sorting with limited in vivo imaging, bioluminescence for small animal imaging even with a very few number of cells, and PET for tomographically imaging living subjects including larger animals and humans.
Our cancer metastatic model shows that metastases can be imaged by microPET and bioluminescence in living mice using this triple fusion reporter gene, with certain limitations for each technique. The bioluminescence signal from Renilla luciferase is not detectable from metastases present at greater depths but is easily detectable from superficial metastases from any region of the body. On the other hand, microPET reveals metastases from deep inside the body, but signals from metastases in the abdomen/pelvis are somewhat obscured by the nonspecific signal in the gastrointestinal tract and urinary collecting system due to tracer clearance. Finally, autofluorescing properties of biological molecules limit detection of metastases by in vivo fluorescence imaging in living animals. However, metastases can be easily visualized in sacrificed animals with exposed tissues in situ using whole body fluorescence imaging. It is likely that the fluorescence signal to background can be improved by using an excitation source under the animal and imaging with a camera above the animal to help minimize autofluorescence. The bioluminescence signal can also be markedly improved by injecting higher doses of substrate (coelenterazine), as we have demonstrated recently (18) . As red-shifted bioluminescent reporters with high substrate utilization capacity are developed, this will also likely aid in helping to use bioluminescence-based reporter fusions. In addition, the microPET signal can be improved by using tracers with longer half-lives (e.g., 124I-labeled 2'-fluoro-5-fluoro-1-ß-D-arbinofuranosyluracil) to allow the background signal from the gastrointestinal tract and renal collecting system to be reduced by waiting longer after tracer injection before imaging animals. With continued refinement in reporter genes, substrates for reporter proteins, and physical instrumentation, it is likely that higher spatial resolution imaging with greater sensitivity for detecting smaller numbers of cells will eventually be possible.
Additional studies quantitatively comparing fluorescence, bioluminescence, and PET in small living animals should also help to better define the potential roles of each modality in specific applications. Cancer research, including imaging of preclinical models of tumors and metastases, immune cell trafficking, transgenic models, gene therapy, and monitoring therapy in general should all benefit from the strategies developed in the current work.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Sanjiv S. Gambhir, Stanford University, The James H. Clark Center, 318 Campus Drive, East Wing, First Floor, Stanford, California 94305-5427. E-mail: sgambhir{at}stanford.edu
Received 6/19/03. Revised 11/ 6/03. Accepted 12/ 5/03.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 T.F. Massoud, A. Singh, and S.S. Gambhir Noninvasive Molecular Neuroimaging Using Reporter Genes: Part II, Experimental, Current, and Future Applications AJNR Am. J. Neuroradiol., March 1, 2008; 29(3): 409 - 418. [Abstract] [Full Text] [PDF]
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Z. Love, F. Wang, J. Dennis, A. Awadallah, N. Salem, Y. Lin, A. Weisenberger, S. Majewski, S. Gerson, and Z. Lee Imaging of Mesenchymal Stem Cell Transplant by Bioluminescence and PET J. Nucl. Med., December 1, 2007; 48(12): 2011 - 2020. [Abstract] [Full Text] [PDF]
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 Y. Zhang, J. P. Bressler, J. Neal, B. Lal, H.-E. C. Bhang, J. Laterra, and M. G. Pomper ABCG2/BCRP Expression Modulates D-Luciferin Based Bioluminescence Imaging Cancer Res., October 1, 2007; 67(19): 9389 - 9397. [Abstract] [Full Text] [PDF]
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 C. E. Gomez, J. L. Najera, E. Domingo-Gil, L. Ochoa-Callejero, G. Gonzalez-Aseguinolaza, and M. Esteban Virus distribution of the attenuated MVA and NYVAC poxvirus strains in mice J. Gen. Virol., September 1, 2007; 88(9): 2473 - 2478. [Abstract] [Full Text] [PDF]
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P. Ray, R. Tsien, and S. S. Gambhir Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects Cancer Res., April 1, 2007; 67(7): 3085 - 3093. [Abstract] [Full Text] [PDF]
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C. M. Deroose, A. De, A. M. Loening, P. L. Chow, P. Ray, A. F. Chatziioannou, and S. S. Gambhir Multimodality Imaging of Tumor Xenografts and Metastases in Mice with Combined Small-Animal PET, Small-Animal CT, and Bioluminescence Imaging J. Nucl. Med., February 1, 2007; 48(2): 295 - 303. [Abstract] [Full Text] [PDF]
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 J. De Rijck, L. Vandekerckhove, R. Gijsbers, A. Hombrouck, J. Hendrix, J. Vercammen, Y. Engelborghs, F. Christ, and Z. Debyser Overexpression of the Lens Epithelium-Derived Growth Factor/p75 Integrase Binding Domain Inhibits Human Immunodeficiency Virus Replication J. Virol., December 1, 2006; 80(23): 11498 - 11509. [Abstract] [Full Text] [PDF]
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 R. Zhou, P. D. Acton, and V. A. Ferrari Imaging Stem Cells Implanted in Infarcted Myocardium J. Am. Coll. Cardiol., November 21, 2006; 48(10): 2094 - 2106. [Abstract] [Full Text] [PDF]
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 N. Belousova, R. Harris, K. Zinn, M. A. Rhodes-Selser, A. Kotov, O. Kotova, M. Wang, R. Aurigemma, Z. B. Zhu, D. T. Curiel, et al. Circumventing Recombination Events Encountered with Production of a Clinical-Grade Adenoviral Vector with a Double-Expression Cassette Mol. Pharmacol., November 1, 2006; 70(5): 1488 - 1493. [Abstract] [Full Text] [PDF]
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K. Yanagihara, M. Takigahira, F. Takeshita, T. Komatsu, K. Nishio, F. Hasegawa, and T. Ochiya A photon counting technique for quantitatively evaluating progression of peritoneal tumor dissemination. Cancer Res., August 1, 2006; 66(15): 7532 - 7539. [Abstract] [Full Text] [PDF]
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 W. S. El-Deiry, C. C. Sigman, and G. J. Kelloff Imaging and Oncologic Drug Development J. Clin. Oncol., July 10, 2006; 24(20): 3261 - 3273. [Abstract] [Full Text] [PDF]
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 W. S. El-Deiry The Promise of Optical Technologies for Measuring Tumor Response in Cancer Therapy Trials Am. Assoc. Cancer Res. Educ. Book, April 1, 2006; 2006(1): 143 - 146. [Full Text] [PDF]
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J. C. Wu, J. M. Spin, F. Cao, S. Lin, X. Xie, O. Gheysens, I. Y. Chen, A. Y. Sheikh, R. C. Robbins, A. Tsalenko, et al. Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation Physiol Genomics, March 13, 2006; 25(1): 29 - 38. [Abstract] [Full Text] [PDF]
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 F. Cao, S. Lin, X. Xie, P. Ray, M. Patel, X. Zhang, M. Drukker, S. J. Dylla, A. J. Connolly, X. Chen, et al. In Vivo Visualization of Embryonic Stem Cell Survival, Proliferation, and Migration After Cardiac Delivery Circulation, February 21, 2006; 113(7): 1005 - 1014. [Abstract] [Full Text] [PDF]
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P. Padmanabhan, J. Otero, P. Ray, R. Paulmurugan, A. R. Hoffman, S. S. Gambhir, S. Biswal, and G. A. Ulaner Visualization of Telomerase Reverse Transcriptase (hTERT) Promoter Activity Using a Trimodality Fusion Reporter Construct J. Nucl. Med., February 1, 2006; 47(2): 270 - 277. [Abstract] [Full Text] [PDF]
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K. P. Hoeflich, D. C. Gray, M. T. Eby, J. Y. Tien, L. Wong, J. Bower, A. Gogineni, J. Zha, M. J. Cole, H. M. Stern, et al. Oncogenic BRAF Is Required for Tumor Growth and Maintenance in Melanoma Models Cancer Res., January 15, 2006; 66(2): 999 - 1006. [Abstract] [Full Text] [PDF]
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Y. Wang, M. Iyer, A. Annala, L. Wu, M. Carey, and S. S. Gambhir Noninvasive indirect imaging of vascular endothelial growth factor gene expression using bioluminescence imaging in living transgenic mice Physiol Genomics, January 12, 2006; 24(2): 173 - 180. [Abstract] [Full Text] [PDF]
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 R. T. Sadikot and T. S. Blackwell Bioluminescence Imaging Proceedings of the ATS, December 1, 2005; 2(6): 537 - 540. [Abstract] [Full Text] [PDF]
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C. J. Shu, S. Guo, Y. J. Kim, S. M. Shelly, A. Nijagal, P. Ray, S. S. Gambhir, C. G. Radu, and O. N. Witte Visualization of a primary anti-tumor immune response by positron emission tomography PNAS, November 29, 2005; 102(48): 17412 - 17417. [Abstract] [Full Text] [PDF]
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S.-C. Hung, W.-P. Deng, W. K. Yang, R.-S. Liu, C.-C. Lee, T.-C. Su, R.-J. Lin, D.-M. Yang, C.-W. Chang, W.-H. Chen, et al. Mesenchymal Stem Cell Targeting of Microscopic Tumors and Tumor Stroma Development Monitored by Noninvasive In vivo Positron Emission Tomography Imaging Clin. Cancer Res., November 1, 2005; 11(21): 7749 - 7756. [Abstract] [Full Text] [PDF]
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J. H. Shin, J.-K. Chung, J. H. Kang, Y. J. Lee, K. I. Kim, Y. So, J. M. Jeong, D. S. Lee, and M. C. Lee Noninvasive Imaging for Monitoring of Viable Cancer Cells Using a Dual-Imaging Reporter Gene J. Nucl. Med., December 1, 2004; 45(12): 2109 - 2115. [Abstract] [Full Text] [PDF]
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L. Wang, W. C. Jackson, P. A. Steinbach, and R. Y. Tsien Evolution of new nonantibody proteins via iterative somatic hypermutation PNAS, November 30, 2004; 101(48): 16745 - 16749. [Abstract] [Full Text] [PDF]
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 S. X. Lin and F. R. Maxfield Fluorescence imaging in living animals. Focus on "Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy" Am J Physiol Cell Physiol, August 1, 2004; 287(2): C257 - C259. [Full Text] [PDF]
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