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Cancer Research 68, 4990, July 1, 2008. doi: 10.1158/0008-5472.CAN-07-5984
© 2008 American Association for Cancer Research
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Priority Reports

Noninvasive Imaging and Quantification of Epidermal Growth Factor Receptor Kinase Activation In vivo

Wenrong Li1,4, Fang Li1, Qian Huang1,5, Barbara Frederick1, Shideng Bao1,2 and Chuan-Yuan Li1,3

Departments of 1 Radiation Oncology, 2 Neurosurgery, and 3 Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado; 4 Xinjiang Academy of Animal Sciences, Urumqi, Xinjiang, China; and 5 No. 1 People's Hospital, Shanghai Jiaotong University, Shanghai, China

Requests for reprints: Chuan-Yuan Li, Department of Radiation Oncology, University of Colorado Health Sciences Center, P.O. Box 6511, MS 8123, Aurora, CO 80010. Phone: 303-724-1542; Fax: 303-724-1554; E-mail: Chuan.Li{at}uchsc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) critical in tumor growth and a major target for anticancer drug development. However, thus far, there is no effective system to monitor its activities in vivo. Here, we report a novel approach to monitor EGFR activation based on the bifragment luciferase reconstitution system. The EGFR receptor and its interacting partner proteins (EGFR, growth factor receptor binding protein 2, and Src homology 2 domain-containing) were fused to NH2 terminal and COOH terminal fragments of the firefly luciferase. After establishing tumor xenograft from cells transduced with the reporter genes, we show that the activation of EGFR and its downstream factors could be quantified through optical imaging of reconstituted luciferase. Changes in EGFR activation could be visualized after radiotherapy or EGFR inhibitor treatment. Rapid and sustained radiation-induced EGFR activation and inhibitor-mediated signal suppression were observed in the same xenograft tumors over a period of weeks. Our data therefore suggest a new methodology where activities of RTKs can be imaged and quantified optically in mice. This approach should be generally applicable to study biological regulation of RTK, as well as to develop and evaluate novel RTK-targeted therapeutics. [Cancer Res 2008;68(13):4990–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The use of bioluminescent firefly luciferase to genetically label cells and proteins has greatly advanced biomedical research. For example, by use of commercially available optical imaging devices, it is now possible to image a few hundred to a few thousand luciferase-expressing tumor cells anywhere in mice (1–3). This capability has made it possible to track the fate of small numbers of tumor cells over the course of days, weeks, or even months, greatly facilitating the studies of tumor development and tumor metastases. As this can be accomplished in the same animals over the course of the experiments, the savings in animal costs and increases in experimental reproducibility are considerable. In addition, biological insights that were otherwise unavailable were often gained from noninvasive imaging studies. For these reasons, there is a surge in interest in developing novel luciferase-based assays that can image ever more sophisticated biological processes, especially in vivo in live animals (4–6).

In this study, we attempt to image the activities of the epidermal growth factor (EGF) receptor (EGFR) by use of the bioluminescent imaging approach. The EGF family of proteins is one of the most important in regulating mammalian cellular growth and proliferation (7). They also play crucial roles in tumor development and tumor response to therapy (8–10). However, thus far, there is no effective system where the in vivo activation of EGFR can be monitored effectively. This is because EGFR activation leads to complicated cascades of molecular events with no specific transcriptional activation of any downstream genes that are amenable for constructing the commonly used promoter-based luciferase reporter systems. However, it is now recognized that ligand binding to EGFR leads the dimerization of the EGFR and activation of the receptor tyrosine kinase (RTK) activities of EGFR (10). This, in turn, leads to the activation and association of downstream factors, such as Src homology 2 domain-containing (Shc) and growth factor receptor binding protein 2 (Grb2).

We reasoned that the use of the bifragment luciferase reconstitution system (4, 11–16), which was shown to be able to image interacting protein pairs in tissue culture and live animals (11, 14, 17), will allow us to assess EGFR activities through monitoring the reconstitution of the luciferase activities brought about by the interaction of activated EGFR with its downstream protein partners. Our results show this approach provides a powerful tool to study the biological regulation of EGFR activity in vivo, as well as to develop/evaluate novel therapeutics targeting the EGFR pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Construction of the reporter plasmids. Full-length genes (EGFR, Grb2, and Shc) required to construct the reporters were obtained through different channels. The full-length sequences coding for human EGFR (Genbank accession X00588) and the adaptor protein Grb2 (accession BC000631) were cloned through reverse transcription–PCR from cDNA derived from HCT116 cells, respectively. In using PCR to amplify these genes, the stop codons were removed whereas the native Kozak sequences were preserved. PCR was also used to amplify the human p52Shc (accession BC014158) cDNA from a plasmid generously provided by Dr. Alexander Sorkin (Departments of Pharmacology, University of Colorado Health Science Center). In all three cases, restriction enzyme sites were engineered into the primer to facilitate subsequent fusion with the luciferase moieties. Before cloning into the expression vectors pLNCX/pLPCX, the PCR-amplified genes were verified through sequencing. The NH2 terminal (NLuc, aa2-416) and COOH terminal (CLuc, aa398-550) halves of the luciferase gene were generated by PCR from pGL3 (Promega) by use of 5' primers containing the (Gly4/Ser)3 flexible linker and restriction enzyme sites EcoRI, MluI, and XhoI and 3' primers with stop codon and restriction enzyme sites EcoRI and XhoI (Supplementary Fig. S1). The fusion genes EGFR-NLuc and Grb2-Nluc were then created (through restriction enzyme digestions and ligations; please see supplementary data for more details on the cloning strategy) and subcloned into retrovirus vector pLPCX (Clontech) in proper order, whereas fused EGFR-CLuc and Shc-Cluc genes were ligated into retrovirus vector pLNCX (Clontech) downstream of the cytomegalovirus promoter. pLPCX contains the puromycin resistance gene, and pLNCX confers resistance to G418 for selection of stable integration of the transgenes in target cells. The primers used for cloning the plasmids are provided in the supplementary data.

Transduction of the reporter into tumor cells. Stable transfectants of the non–small cell lung adenocarcinoma cell line H322 (kindly provided by Barbara A. Helfrich of Department of Medicine, University of Colorado Health Sciences Center) were established by serial infections of retroviruses containing the reporter genes. Three transduced cell lines were derived as a result. These include cell lines that contain the binary reporters EGFR/EGFR-luc (EGFR-Nluc + EGFR-CLuc), EGFR/Shc-luc (EGFR-Nluc + Shc-Cluc), and Grb2/Shc-luc (Grb2-Nluc + Shc-Cluc). More details on establishing reporter-transduced cells are provided in the supplementary data.

Imaging of the reporter in vitro. Stably transfected EGFR/Shc-Cluc and Grb2/Shc-Cluc cells (5 x 104 per well in 250 µL of complete growth RPMI 1640 supplemented with 10% fetal bovine serum) were seeded into 48-well plates. When they grew to 80% confluence, growth media were replaced with media containing no serum to starve the cells for 14 to 16 h. Subsequently, cells were treated with EGF (Cell Signal Technology) at 20 ng/mL EGF in RPMI 1640 at 37°C for 15 min. After incubation with EGF, the media were replaced to luciferin-containing media (125 µL of 150 µg/mL lucifercin in DMEM without sodium pyruvate; luciferin was obtained commercially from Xenogen). Photon output for each well was measured 10 min after addition of luciferin with the Xenogen IVIS200 optical imaging system.

For EGFR inhibitor (gefitinib or erbitux) assays, cells were treated with inhibitor after 14 to 16 h of serum starvation and before EGF induction. For gefitinib treatment, cells were treated at 37°C for 8 h. For erbitux treatment, cells exposed at 37°C for 15 min. EGF was then added to the cells at 20 ng/mL for 15 min. Subsequently, lucifercin was added and photon output for each well was measured 10 min later in IVIS200.

Western blot analysis. Standard protocols for Western blot analysis were followed. Detailed protocols, as well as antibody information, are provided in the supplementary data.

Imaging the reporter in vivo. To image EGFR activity in tumors, H322 cells transduced with the reporters were s.c. injected into 4-wk-old to 6-wk-old female athymic nude mice, which were obtained from National Cancer Institute and maintained in accordance with University of Colorado Health Sciences Center Institutional Animal Care and Use Committee guidelines. About 5 x 106 reporter-bearing H322 tumor cells were injected s.c. into mice in 50 µL of Matrigel dissolved in PBS solution in the legs. When tumors reached 5 to 7 mm in diameter, mice were randomly assigned to experimental groups.

In some groups, animals were treated with X-rays (1x or 3x 6 Gy radiation). In addition, the drug gefitinib was given to some groups at 50 mg/kg i.p. daily for 10 d. In cases of combined X-ray and gefitinib group, the drug was given 1 d before irradiation. Luciferase activities in the mice were then imaged at various time points 10 min after i.p. injection of 200 µL of 15 mg/mL luciferin in H2O.

Statistical method. Student's t test was used where necessary. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Design of a bifragment luciferase reconstitution system for noninvasive observation of EGFR activation. We set out to develop a novel system to examine EGFR activation in vivo noninvasively. To achieve this goal, a bifragment luciferase reconstitution scheme was used. We took advantage of the fact that EGFR activation necessitates (a) dimerization of EGFR on the surface of cells and (b) activation of the kinase activities of EGFR and the subsequent phosphorylation of its downstream partners, such as Shc. This leads to the physical association of EGFR, Shc, and the adaptor protein Grb2.

We designed a system where EGFR and its interaction partners Shc and Grb2 were fused to either NH2 terminal or COOH terminal fragments of the firefly luciferase (Fig. 1A ). We reasoned that in cells stably transduced with these reporters, activation of the EGFR would lead to the physical interaction of EGFR with its partner proteins (EGFR itself, Shc, or Grb2), which could potentially reconstitute the active luciferase enzyme that could be imaged optically (Fig. 1B). A set of chimeric genes with EGFR, Grb2, and Shc fused with either NH2 terminal or COOH terminal luciferase fragments were (Fig. 1A) transduced into a human non–small cell lung adenocarcinoma line H322. The expressions of the recombinant proteins were then examined through Western blotting, and clear evidence for the presence of both endogenous and recombinant proteins were observed (Fig. 1C).


Figure 1
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  		Figure 1. Structure of a bifragment luciferase reconstitution system for detecting EGFR pathway activation. A, structures of various fusion reporter proteins between EGFR pathway proteins (EGFR, Shc, Grb2) and the fragments of firefly luciferase (Nluc, Cluc represent the NH2 terminal and COOH terminal halves of the firefly luciferase gene). The retroviral vector pLPCX and pLNCX were used to carry the reporter genes (with resistance genes for puromycin and neomycin, respectively). B, graphic illustration of the principles of EGFR activity reporters used in this study. Three different versions of the reporters were shown. C, Western blot analyses of the expression of endogenous and recombinant EGFR pathway proteins after transduction of one or both components of the reporter system into the human lung cancer line H322.

 
Validation of EGF-induced reporter gene activation in tissue culture. We next measured luciferase activities in H322 cells stably transduced with pairs of recombinant reporter genes in the presence or absence of EGF. After initial screening, we found that EGF addition induced no increase in luciferase signals in cells transduced with EGFR/EGFR-luc fragments (data not shown), indicating no reconstitution under these circumstances, which perhaps reflected the unfavorable spatial conditions for the luciferase moieties to reconstitute when the EGFR receptors dimerize. On the other hand, cells transduced with EGFR/Shc-luc fragments or Grb2/Shc-luc fragments showed robust EGF-mediated induction of luciferase signals, indicating reconstitution of the enzyme accompanied by EGFR activation (Fig. 2A and B ). The functional relevance of the reporter system was then examined through optical imaging and Western blotting. It is clear that EGF-induced activation of both EGFR/Shc-luc and Grb2/Shc-luc reporters were correlated with phosphorylation/activation of the endogenous or recombinant EGFR and Shc in cells (Fig. 2C).


Figure 2
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  		Figure 2. In vitro characterization of the kinetics of EGF-induced activation of the bifragment luciferase reporter. A, the dose-response curve for the EGFR/Shc-luc reporter and the Grb2/Shc-luc reporter. Top, representative images of reporter-transduced cells (in 48-well plates) treated with different concentrations of EGF at 37°C for 15 min and then imaged in the IVIS200 instrument; bottom, quantitative dose response of the reporter activation after EGF addition. Error bars, SDs derived from three to five data points. B, the time course of reporter activation for the same EGFR reporters. Top, representative images of reporter-transduced cells treated with 20 ng/mL of EGF for various lengths of time and imaged in the IVIS200 instrument for reconstituted luciferase gene activities; bottom, time course of reporter activation. Error bars, SDs derived from three to five data points. C, Western blot analyses of EGF-mediated activation of endogenous and recombinant EGFR and downstream factors. Cells transduced with various recombinant reporter genes were incubated with EGF (20 ng/mL for 15 min) and then lysed. Antibodies against total and phosphorylated EGFR and Shc proteins were used to analyze total and activated forms of these proteins in Western blots. EN + SC, EGFR-Nluc + Shc-Cluc; GN + SC, Grb2-Nluc+Shc-Cluc. The phosphorylation of the proteins correlated well with optical imaging of the reporter cells (A and B).

 
A careful examination showed that the two reporter combinations showed different kinetics after EGF treatment. In dose response studies, after 15 minutes of incubation, the EGFR/Shc-luc reporter showed a slower dose response at lower EGF concentrations but also reached a higher plateau. In contrast, the Grb2/Shc-luc reporter showed relatively faster EGF dose response but reached a lower plateau (Fig. 2A). In time course studies, EGFR/Shc-luc showed earlier induction but reached a lower plateau (at 2.5-fold over control) 15 minutes after EGF addition (Fig. 2B), whereas Grb2/Shc-luc had slower induction but continued to increase (above 5-fold induction over control) 30 minutes after EGF addition. In addition, Western blot analyses indicate that the activation of reporter genes correlated well with phosphorylation of endogenous and recombinant EGFR and Shc proteins (Fig. 2C).

An important point to note is the impressive ability of the new reporter system to detect the different kinetics of EGF-mediated induction between EGFR/Shc and Grb2/Shc. This has not been possible in the past with Western blot–based approaches. It clearly shows the power of our molecular imaging approach for EGFR activity.

Evaluating the efficacy of known EGFR inhibitors in vitro. The utility of the reporter system was further evaluated in testing the efficacy of known EGFR inhibitors in tissue culture cells. Gefitinib has been shown to be a potent and specific small molecule inhibitor of the tyrosine kinase activities of EGFR (18). As shown in Fig. 3A , significant (P < 0.05, Student's t test) inhibition of EGF-induced activation of both EGFR/Shc-luc and Grb2/EGFR-luc reporters was observed in H322 cells beginning at gefitinib concentration 0.25 µmol/L. Another EGFR inhibitor, the monoclonal antibody erbitux (19), also exhibited potency in inhibiting the activation of the reporters, significantly down-regulating the reporter activities at concentrations of 1 nmol/L or more. Western blot analysis of cell lysates indicated that the activities of the reporters correlated well with the phosphorylation status of the EGFR and Shc proteins, as well as the presence of the inhibitors (Fig. 3C).


Figure 3
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  		Figure 3. Quantitative imaging of the effect of EGFR inhibitors on the EGFR reporter activities. A, effective suppression of both EGFR/Shc-luc and Grb2/Shc-luc reporters by the small molecule EGFR kinase inhibitor gefitinib. Cells transduced with the reporters were incubated with EGF (at 20 ng/mL) and various concentrations of the inhibitors. After 10 min of incubation, the cells were imaged for luciferase activities. Error bars, SDs of three to five triplicate data points. B, effective suppression of the both EGFR/Shc-luc and Grb2/Shc-luc reporters by the monoclonal antibody EGFR inhibitor erbitux (cetuximab). Error bars, SDs of three to five triplicate data points. C, Western blot analyses of the effect of EGFR inhibitors (gefitinib or erbitux) on EGF-induced activation of EGFR and its downstream protein Shc. The phosphorylation status of the proteins, which indicated the activation status, was closely correlated with the addition of the inhibitors and the reconstitution of luciferase activities (A and B).

 
Validation of the EGFR activity reporters in xenogfraft tumors undergoing radiotherapy. Our data in vitro suggest that the bifragment reconstitution system is capable of quantifying EGFR activation. The real test of the utility of the bifragment reconstitution system, however, is whether it is able to report the in vivo activities of EGFR.

To assess the properties of the EGFR reporter systems in vivo, we established xenograft tumors of H322 lung cancer line with the EGFR/Shc-luc reporter and the Grb2/Shc-luc reporter, respectively. When these tumors reached 5 to 7 mm in diameters, they were irradiated and the EGFR activities were followed in the irradiated tumors. Our results showed that ionizing radiation induced a very rapid activation of EGFR activities (~2-fold above background) within 1 hour of irradiation in the tumors (Fig. 4A ). This was true of both reporter systems. The reporter activities rapidly dropped to background levels and then quickly climbed even higher levels (~3-fold above background) at 3 hours after irradiation. For EGFR/Shc-luc, after the initial surge in the first day (P < 0.02 at 30 minutes and 3, 6, 9, and 12 hours), the level dropped to lower but still significantly above control levels at day 2 (P < 0.05). Interestingly, another peak was observed at day 6 (P < 0.04), after which persistently higher EGFR activity levels were seen until day 10. This pattern of twin-peak activation was also observed for the Grb2/Shc-luc reporter, which showed even higher levels of induction (P < 0.05 for all time points in day 1, except at 3 hours and days 2, 3, 5, 6, 8, and 10). These results were generally consistent with published reports indicating that radiation induced activation of the EGFR pathway (20, 21). However, the kinetics of activation is different from the earlier studies due to the fact that different irradiation and observation schedules were used in the earlier studies.


Figure 4
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  		Figure 4. In vivo imaging of EGFR activation during radiotherapy. A, radiotherapy induced activation of EGFR/Shc-luc (broken lines) and Grb2/Shc-luc (solid lines) reporters in H322 xenograft tumors. After reporter-transduced H322 lung tumor cell (5 x 106) implantation (s.c.) and tumor formation (with diameters around 5–7 mm), the tumors were irradiated with X-rays (6 Gy). The activities of EGFR were then imaged. Data obtained 2 wk after radiotherapy. Data of the first 24 h after radiotherapy were plotted in a separate graph (top) to show more details of activation during this period. Error bars, SE (n = 4). B, the effect of the small molecule EGFR inhibitor gefitinib on radiotherapy-induced activation of the EGFR receptors. When reporter-transduced H322 tumors were 5 to 7 mm in diameter, the EGFR inhibitor gefitinib were given on a daily basis for 10 d. Radiation (3 x 6 Gy) was then given every other day starting 1 d after drug administration (days 0, 2, and 4). Quantitative imaging of EGFR activation was carried out, and the data were plotted (left). Top left, data from the first 24 h in more detail. Error bars, SE (n = 3–5). Top right, representative images of mice with the reporter-transduced tumors after various treatments. Bottom right, Western blot autoradiograph of total EGFR, activated (phosphorylated) EGFR, and β-actin levels in tumors obtained (from sacrificed mice) from different times points after they were irradiated in vivo.

 
To simulate multifraction radiotherapy in the clinic, additional experiments were carried out when the tumors were given three doses of radiation (Fig. 4B, left). Similar to the single-dose radiation, the multifraction radiation induced significant increases in EGFR activities. For EGFR/Shc-Luc, the induction is significantly different form controls at all time points observed (P < 0.03 compared with control levels). For Grb2/Shc-luc, multifraction radiation caused even more pronounced increases (e.g., up to 14-fold increase on day 5, P < 0.03 compared with control levels). The increase in EGFR activation induced by additional exposures to radiation were also confirmed by Western blot analysis of EGFR phosphorylation in irradiated tumors (Fig. 4B, lower right) and cells (Supplementary Fig. S3). One interesting aspect of the in vivo Western blot data is the change in total EGFR levels, which are not completely concordant with phosphorylated EGFR levels. Total EGFR levels showed an increase at 1 and 3 hours after the third fraction of radiation but a decrease 24 hours after the third fraction despite a very significant increase in phosphorylated EGFR levels (Fig. 4B, bottom right). This, in contrast to the tissue cultured cells, did not show any total EGFR fluctuations after multiple radiation exposures despite an increase in phosphorylated EGFR (Supplementary Fig. S3).

In addition, the differences between the EGFR/Shc-luc and Grb2/Shc-luc reporters were also amplified with the multifraction radiations, particularly at later time points. The exact mechanisms underlying the differences between the two reporters are not known. However, they probably reflected the fact that the activity of EGFR/Shc-Luc reporter is linear with interaction between EGF and the EGFR-luc containing EGFR dimers whereas the activity of the Grb2/Shc-luc reporter is the catalytic results of activated endogenous and exogenous EGFR. They clearly indicated the more sensitive nature of our reporter system when compared with traditional Western blot or ELISA-based approach for detecting EGFR activities.

Our results also indicated that gefitinib could inhibit Grb2/Shc-luc reporter activation in vivo very effectively both at earlier times (right after the first radiation dose) and at later times during therapy (e.g., P < 0.04 on day 5, the day the induced Grb2/Shc-luc peaked; Fig. 4B). In contrast, the EGFR/Shc-luc reporter was not inhibited effectively right after the first radiation therapy dose. However, its second peak of activation (day 7) was inhibited significantly (P < 0.02). Currently, we do not have a plausible explanation for the lack of EGFR/Shc-luc activities in the first 24 hours.

In summary, both in vitro and in vivo results support the feasibility of our new luciferase-based reporter system for noninvasive imaging of EGFR activities.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
The ability to "see" a biological process in action is always sought after in biomedical research. The advent of sensitive optical imaging system makes it possible to visualize and quantify luminescence and fluorescence in vivo in rodents. This capability, in combination with the availability of natural or artificially "improved" fluorescent proteins (i.e., EGFP) or luminescent proteins (e.g., firefly luciferase), greatly expanded the horizons of molecular imaging in small animals by making it possible to genetically engineer various reporter systems. Our results with artificially engineered EGFR activity reporters provide an important example of the power of the luciferase-based optical imaging reporter in studying sophisticated signal transduction pathways and drug action.

Labeling of individual cell types/proteins to study their trafficking/distribution in vivo or the evaluation of in vivo promoter activities have been the most common usage for luciferase molecular imaging. Whereas many of these studies have greatly advanced our understanding of in vivo biology of the cells/proteins of interest with regard to their regulation at the transcriptional, translational, or posttranslational levels, the approach is limited to cases where the study subjects are whole cells, gene promoters, or proteins with physiologic variations in posttranslational stabilities. For many biological processes, especially ones involving enzymatic activities, the imaging approaches are not well developed. One example where luciferase imaging was used to study in vivo enzymatic activity was the successful engineering of a luciferase-based caspase activity reporter (22, 23). In another example a modified luciferin substrate was used to detect caspase activities (24).

Our study deals with the activity of the EGFR activities. EGFR is an archetypical member of the RTK family. Numerous members of the RTK family play crucial roles in various biological processes. The interest in EGFR and related receptors (i.e., Her2/neu) is especially strong because of the roles they play in tumor biology and in anticancer drug development (10, 25). To date, there are some reports of attempts to image EGFR. Some deal only with the distribution of the receptor protein rather than its biological activities (26). This type of studies usually involve the use of labeled antibodies or ligands that are specific for EGFR (27, 28). EGFR extracellular domain dimerization has been imaged in at the cellular level through reconstitution of the lacZ enzyme (29, 30). However, this strategy does not address the kinase activity of the EGFR protein and is not suitable for imaging in live animals. There are also reports that used the FRET strategies and fluorescent protein pairs to detect EGFR activation (31–34). Again this strategy is not suitable for small animal imaging.

Our study uses the bi-fragment luciferase reconstitution assay to image kinase activity of EGFR through quantification of the interaction between activated EGFR and its downstream partners. It is a novel system that has not previously been reported. The success of this strategy shows a powerful approach for studying the in vivo regulation of the EGFR receptors. In addition, it is generally applicable for studying other member of the RTK family because many of these use similar kinase cascades. Examples of these include the vascular endothelial growth factor receptor, the insulin-like growth factor receptor, and transforming growth factor-β receptor, etc. The availability of a noninvasive and quantitative imaging system should greatly facilitate the study of the biology of these RTKs under normal (i.e., physiologic, developmental) and pathologic (i.e., cancer) conditions. It should also facilitate the development and evaluation of novel therapeutics based on these RTKs.

Finally, our data derived from monitoring EGFR reporter activities during radiotherapy illustrated how the optical imaging reporters could be used to monitor in vivo signal transduction pathways in the course of therapy. For example, the observation of radiation-induced twin-peak pattern of activation over the course of a week in the same tumors could only be achieved with a noninvasive imaging system. Such radiation-induced EGFR activation may provide an explanation as to why combined administration of EGFR inhibitors with radiotherapy has potent antitumor efficacy in a number of malignancies in human patients (9, 35).

In summary, we have developed a novel bifragment luciferase reconstitution-based molecular imaging system to study EGFR. Our method should set a precedent for studying other RTKs by use of the molecular imaging approach.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: U.S. National Institute of Bioimaging and Bioengineering grant EB001882, U.S. National Cancer Institute grant CA81512, Komen Foundation for Breast Cancer Research grant (C-Y. Li), National Basic Research Project of China grant 2004CB518804 (Q. Huang), and National Science Foundation of China for Outstanding Young Investigators grants 30325043 (Q. Huang) and 30428015 (C-Y. Li).

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.

We thank Barbara Helfrich, Department of Medicine, University of Colorado Health Sciences Center, for providing the H322 cell line and Dr. Alexander Sorkin, Department of Pharmacology, University of Colorado Health Sciences Center, for providing the Shc52-encoding plasmid.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 10/24/07. Revised 3/24/08. Accepted 5/ 5/08.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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