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Clinical Cancer Research 14, 2492, April 15, 2008. doi: 10.1158/1078-0432.CCR-07-0782
© 2008 American Association for Cancer Research
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Cancer Therapy: Preclinical

Noninvasive Imaging of Apoptosis and Its Application in Cancer Therapeutics

Julia M. Coppola1, Brian D. Ross1,2 and Alnawaz Rehemtulla2

Authors' Affiliations: Departments of 1 Biological Chemistry and 2 Radiation Oncology, University of Michigan Medical School, Ann Arbor, Michigan

Requests for reprints: Alnawaz Rehemtulla, Department of Radiation Oncology, University of Michigan Medical Center, 109 Zina Pitcher Place, BSRB Level A, Room A528, Ann Arbor, MI 48109. Phone: 734-764-4209; Fax: 734-763-1581; E-mail: alnawaz{at}umich.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Activation of the apoptotic cascade plays an important role in the response of tumors to therapy. Noninvasive imaging of apoptosis facilitates optimization of therapeutic protocols regarding dosing and schedule and enables identification of efficacious combination therapies.

Experimental Design: We describe a hybrid polypeptide that reports on caspase-3 activity in living cells and animals in a noninvasive manner. This reporter, ANLucBCLuc, constitutes a fusion of small interacting peptides, peptide A and peptide B, with the NLuc and CLuc fragments of luciferase with a caspase-3 cleavage site (DEVD) between pepANLuc (ANLuc) and pepBCLuc (BCLuc). During apoptosis, caspase-3 cleaves the reporter, enabling separation of ANLuc from BCLuc. A high-affinity interaction between peptide A and peptide B restores luciferase activity by NLuc and CLuc complementation. Using a D54 glioma model, we show the utility of the reporter in imaging of apoptosis in living subjects in response to various chemotherapy and radiotherapy regimens.

Results: Treatment of live cells and mice carrying D54 tumor xenografts with chemotherapeutic agents such as temozolomide and perifosine resulted in induction of bioluminescence activity, which correlated with activation of caspase-3. Treatment of mice with combination therapy of temozolomide and radiation resulted in increased bioluminescence activity over individual treatments and increased therapeutic response due to enhanced apoptosis.

Conclusion: The data provided show the utility of the ANLucBCLuc reporter in dynamic, noninvasive imaging of apoptosis and provides a rationale for use of this technology to optimize dose and schedule of novel therapies or to develop novel combination therapies using existing drugs.

Programmed cell death or apoptosis is an important physiologic process multicellular organisms use to maintain homeostasis by providing a means for elimination of redundant cells during development (1, 2). Furthermore, cells that have become damaged or are defective undergo apoptosis to prevent disease. Apoptosis may also be initiated in response to cellular stresses, such as growth factor deprivation, DNA-damaging events, hypoxia, heat, cold, or chemical injury (3). Although there are many events that direct a cell to apoptose, the pathways and players that orchestrate the process are a well-characterized few. Apoptosis may proceed by activation of one of two disparate pathways, both requiring sequential activation of zymogen cysteine proteases known as caspases (4, 5). Whether apoptosis proceeds via the intrinsic or extrinsic apoptotic pathway, activation of initiator caspases culminates in activation of executioner caspases, such as caspase-3, whose activity results in the appearance of apoptotic morphology due to cleavage of target proteins containing the caspase-3–specific DEVD cleavage motif (6). Thus, monitoring caspase-3 activity permits comprehensive apoptosis detection.

Dysregulation of apoptotic pathways contributes directly to a wide variety of pathologic conditions, including stroke, dementia, bone marrow disease, and cancer (1, 7, 8). Over the last decade, it has become clear that neoplastic transformation is a result of defects in both apoptosis and proliferation, resulting in aberrant cell growth, as proliferation proceeds unchecked (9, 10). For example, Korsmeyer (11) first showed that constitutive activation of the antiapoptotic gene bcl-2 leads to B-cell lymphoma. Additionally, overexpression of antiapoptotic proteins, such as XIAP and MDM2, occurs in many cancers (4). The transcription factor p53, which controls cell cycle arrest in response to cellular stress or DNA damage, is commonly mutated in many different cancers (12). Whether proteins are constitutively active, overexpressed, or mutated, genetic alterations ultimately attenuate apoptotic responses and facilitate neoplastic transformation through unregulated cellular proliferation. A great majority of modern cancer therapies target these genetically altered proteins or signaling cascades and invariably result in the induction of apoptosis by restoring a means for the apoptotic pathway to ensue (4, 13).

Because many current cancer therapies promote cell death by reinstituting the apoptotic cascade, the ability to detect apoptosis in live cells and animals would aid significantly in development of new cancer therapies and enhance our understanding of various disease processes, such as cancer, wherein dysregulation of apoptosis is involved. Development of a reporter system that monitors apoptosis noninvasively would facilitate drug discovery and preclinical evaluation of therapeutic protocols regarding dosing, schedule, and efficacy of drug combination therapies.

To develop this apoptosis reporter, we have adapted the split firefly reporter strategy (14), which effectively abolishes luciferase activity and fused NH2-terminal and COOH-terminal domains of luciferase to two strongly interacting peptides, peptide A (pepA) and peptide B (pepB), respectively (15, 16). Constructing a polypeptide wherein pepA-N-Luciferase (ANLuc) and pepB-C-Luciferase (BCLuc) are positioned with an intervening caspase-3 cleavage site significantly reduces bioluminescence activity as the NH2-terminal and COOH-terminal portions of luciferase are unable to interact. When caspase-3 becomes active, the reporter is cleaved, and pepA and pepB associate by a high-affinity interaction and facilitate complementation of NLuc and CLuc domains, thus reconstituting luciferase.

To test this novel reporter system, we created stable cell lines using D54 cells derived from a patient with glioblastoma multiforme (GBM). GBM is a common type of brain tumor with limited survival beyond 1 year after diagnosis in patients despite aggressive treatment (17–19). New classes of chemotherapeutic drugs, such as perifosine and temozolomide, are being used clinically, independently, and in conjunction with radiation and surgery to combat GBM in patients (20–22). Perifosine is a member of the alkylphospholipid class of chemotherapeutic drugs, which exerts its cytotoxic effects by interfering with Akt activation (23). Temozolomide is an alkylating agent that induces DNA methylation, ultimately resulting in cell death (24). By treating our D54 reporter cell line with these drugs, we show that this apoptosis reporter system is a sensitive, dynamic, and quantitative reporter of caspase-3 activity both in vitro and in vivo. In addition, we show that this reporter system can be used to optimize dosing and scheduling of novel therapies in a dynamic, noninvasive manner.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Enzymes for cloning were purchased from New England Biolabs. Perifosine (Cayman Chemicals), temozolomide (Schering Corp.), staurosporine (Sigma), and luciferin (Promega) were purchased for use. Perifosine was dissolved in saline, and temozolomide was dissolved in DMSO. Drugs, with the exception of staurosporine, were prepared fresh on the day of treatment. Fugene 6, WST-1 cell viability kit, and Rapid DNA Ligation kit were purchased from Roche Diagnostics Corp. All media, fetal bovine serum, and medium components used for cell culture were purchased from Life Technologies.

Cell culture and transfections. COS cells were maintained in DMEM, and D54 cells were maintained in RPMI 1640. The medium of both cell lines contained 10% fetal bovine serum, 1% L-glutamine, 100 µg/mL penicillin, and 100 µg/mL streptomycin. Cells were transfected with plasmids using Fugene 6 according to the manufacturer's protocol and stable D54 clones were selected with 200 µg/mL G418 (Invitrogen). Cell lines were incubated at 37°C with 5% CO2..

Plasmid construction. The intact luciferase plasmid was constructed by ligation of luciferase into the vector pEF. The split luciferase fusion plasmid was constructed by ligation of pepANLuc DEVD to pepBCLuc in the vector pEF. Firefly luciferase fragments (and intact luciferase) were obtained by PCR of pGL2-luciferase plasmid (Promega) as described (14). pepA (with linker) was added to the NH2-terminal fragment of luciferase (1-1,245 bp) using primer 5'-ATGAACGAAGCATATGTACATGACGGTCCTGTACGCTCACTGAACAGCGGCCGCAGAAGTATAGCAACAGAAGAC-3' by PCR. The caspase-3 cleavage site (with triglycine linkers on each side) was added to the COOH-terminal end of pepANLuc fragment using primer 5'-CCTCCTCCATCGACTTCGTCGCCTCCTCCTCCATCCTTGTCAATCAAGGC-3'. pepB (with linker) was added NH2 terminally to the COOH-terminal fragment of luciferase (1,197-1,653 bp) using primer 5'-AAGGCACGAAAGGAAGCAGAACTGGCAGCAGCAACTGCAGAACAGAGCGGCCGCAGACCAGCATGCAAAATACCA-3' by PCR. The complete plasmid was created by ligation of SalI-pepANlucDEVD-Xba to Xba-pepBCLuc-EcoRI as a SalI-EcoRI fragment into the vector pEF.

Drug treatment assays. D54 cells stably expressing ANLucBCLuc were seeded in 12- or 24-well plates. Approximately 48 h later, medium was changed to RPMI 1640 without indicator with 1% fetal bovine serum and drug (or vehicle). Cells were assayed using bioluminescence imaging, cell viability assays, and/or Western blot analysis 3 h (perifosine or staurosporine) or 24 h (temozolomide) after treatment.

Western blotting. Western blotting was done as described (25). Briefly, cells were washed in PBS and lysed with a buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, and 1% NP40 supplemented with Complete protease inhibitor cocktail (Roche Diagnostics). Protein was estimated by detergent-compatible protein assay kit from Bio-Rad. Media and lysates were separated by SDS-PAGE, and protein expression was detected by Western blot analysis using antibodies. ANLucBCLuc was detected using a goat polyclonal antibody to luciferase (Chemicon), with horseradish peroxidase–conjugated secondary antibody followed by detection by chemiluminescent horseradish peroxidase substrate (Pierce). Active caspase-3 was detected using a cleaved caspase-3 (Asp175) rabbit polyclonal antibody (clone 9661, Cell Signaling Technology). Glyceraldehyde-3-phosphate dehydrogenase was detected using a mouse monoclonal antibody (Abcam). Actin was detected using a rabbit polyclonal antibody (Sigma).

Cell viability assay. Cells were seeded in 24-well plates and allowed to incubate for 48 h, after which drug was added. WST-1 reagent was added at indicated time after drug treatment and assayed according to the manufacturer's protocol. Absorbance was monitored at 460 nm and background was monitored at 600 nm using a Fluorostar Optima plate reader (BMG Labtech). Final absorbance units were computed by background subtraction.

Immunostaining. Tumors were excised from mice, immediately placed in OCT compound (Tissue-Tek, Sakura), and frozen at –20°C within 10 min after excision. After freezing, the tumors were stored at –80°C. Tumors were later cryosectioned and stored at –80°C. For assay, slides were fixed in 3.75% paraformaldehyde (Sigma) for 15 min at room temperature, washed once with PBS, and permeabilized with methanol for 10 min at –20°C. Slides were then rinsed once with PBS containing 0.05% Tween 20 (PBS-T). Slides were blocked in 5% donkey serum with 1 mg/mL bovine serum albumin in PBS-T for 1 h at room temperature and then washed once with PBS-T. Cleaved caspase-3 (Asp175) rabbit polyclonal antibody caspase-3 (clone 9661) was added to slides at 1:150 dilution and allowed to incubate overnight at 4°C. Slides were washed thrice in PBS-T and incubated with Cy3-coupled anti-rabbit secondary antibody (Jackson ImmunoResearch) at 1:400 dilution at room temperature for 30 min. The slides were washed thrice in PBS-T and costained with 1 µg/mL 4',6-diamidino-2-phenylindole, mounted, and visualized with a Nikon Eclipse TE2000-U fluorescence microscope. Fluorescent images were acquired with MetaMorph software (Molecular Devices Corp.) using identical exposure times. To determine the percent active caspase-3, eight randomly chosen apoptotic fields from each tumor (>200 cells) were imaged. Total cells (4',6-diamidino-2-phenylindole positive) and active caspase-3–positive cells present in these regions were counted using MetaMorph software and expressed as mean percentage.

Cellular bioluminescence assay. Live cells were plated and treated as described in drug treatment assays. D-luciferin (40 mg/mL in PBS) was added to each well and photon counts were collected by a Xenogen IVIS charge-coupled device camera system 30 to 40 min after luciferin addition. A gray-scale image was collected followed by acquisition and overlay of a pseudocolor image representing the spatial distribution of the detected photons emitted from the active luciferase within the cells. A signal averaging time of 1 min was used for luminescent image acquisition. Signal intensity was quantified as the sum of all detected photon counts (photons per second) within a region of interest prescribed over the tumor site using living image software (Xenogen).

Irradiation. Mice that underwent radiation therapy were given 4 or 2 Gy at a dose rate of ~1.5 Gy/min by a Philips RT-250 Orthovoltage unit (Philips Medical Systems). Dosimetry was carried out using an ionization chamber connected to an electrometer system, which is directly traceable to a National Institute of Standards and Technology calibration. Before radiation, mice were anesthetized by i.p. injection of a mixture of 60 mg/kg ketamine (Fort Dodge Animal Heath) and 3 mg/kg xylazine (Lloyd Laboratories).

Mouse bioluminescence imaging. S.c. D54 tumors were induced in athymic nude mice (Charles River) by implantation of 106 cells suspended in 0.1 mL. Before imaging, animals bearing palpable tumors were anesthetized with a 1% isoflurane/air mixture and given a single i.p. dose of 150 mg/kg luciferin in normal saline. Bioluminescence imaging was accomplished 8 to 14 min after luciferin administration and data were collected at the time of peak luminescence. During image acquisition, isoflurane anesthesia was maintained using a nose cone delivery system, and animal body temperature was regulated by using a temperature-controlled bed. A gray-scale body image was collected followed by acquisition and overlay of a pseudocolor image representing the spatial distribution of the detected photons emitted from the active luciferase within the tumor cells. A signal averaging time of 10 or 30 s was used for acquisition of the luminescent image. Signal intensity was quantified as the sum of all detected photon counts within a region of interest prescribed over the tumor site (photons per second) using living image software. Perifosine was delivered to mice by oral gavage of 30 mg/kg, and temozolomide was delivered i.p. at either 70 or 140 mg/kg. Controls were given either saline (perifosine) or DMSO (temozolomide).

Magnetic resonance imaging. For magnetic resonance imaging examination, mice were anesthetized with a 1% isoflurane/air mixture inside a 7T Varian Unity Inova imaging system as previously described (26). A single-slice gradient-echo sequence was used to confirm proper animal positioning and to prescribe subsequent imaging. Anatomic images were acquired using a standard T2-weighted fast spin echo series (repetition time/echo time = 4,000/60 ms, 128 x 128 matrix, 3-cm field of view) to assess tumor volume throughout the study. Fifteen to 17 0.5-mm slices were used to cover the whole tumor. The image acquisition was respiratory gated using a specialized mouse sled (Dazai) connected to a monitoring and gating system (SA Instruments) and the z-gradient first moment was zeroed to reduce the dominant source of motion artifact. Images were acquired before treatment and at 2- to 3-d intervals thereafter. The tumor boundary was manually defined on each slice using a region-of-interest tool (Matlab, MathWorks) and then integrated across slices to provide a volume estimate. Mean volumetric values were computed and error is expressed as SE.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Strategy for noninvasive imaging of caspase-3 and in vitro validation of apoptosis reporter. In an effort to develop an improved reporter of apoptosis, we constructed a hybrid molecule wherein NLuc- and CLuc-containing pepA and pepB at their respective NH2 termini were fused head to tail with an intervening caspase-3 cleavage site (DEVD). We hypothesized that, in this form, interaction between NLuc and CLuc would be minimized due to steric constraints. On induction of apoptosis and caspase-3 activation, cleavage at the DEVD site would free ANLuc and BCLuc and enable reconstitution of luciferase through interaction of pepA and pepB, resulting in bioluminescence activity (Fig. 1A ; ref. 16). Preliminary studies were conducted in the transiently transfected glioblastoma cell line, D54. Induction of apoptosis in transfected cells by treatment with staurosporine resulted in the expected cleavage of ANLucBCLuc (69.3 kDa) to fragments ANLuc (47.8 kDa) and BCLuc (20.4 kDa) in D54 cells as shown in Fig. 1B. The ANLuc and BCLuc polypeptides were only detected 3 h after treatment with staurosporine, wherein a significant fraction of cells were undergoing apoptosis (Fig. 1B). At the 1- and 2-h time points, the reporter molecule was detected as the intact anti-luciferase reactive polypeptide. Inhibition of caspase-3 activation in response to staurosporine treatment in the presence of ZVAD-fmk resulted in an inhibition of ANLucBCLuc cleavage, indicating that ANLuc and BCLuc are derived from cleavage at the DEVD motif (data not shown; ref. 27). To evaluate functional reconstitution of the caspase-3 reporter molecule luciferase domains in an apoptosis-dependent manner, stably transfected D54-ANLucBCLuc cells were treated with increasing doses of temozolomide (Fig. 1C). At 24 h, cells treated with 2 mmol/L temozolomide seemed significantly apoptotic compared with cells treated with vehicle or 1 mmol/L temozolomide. The entire population of cells treated with 4 mmol/L temozolomide had undergone apoptosis. Western blot analysis of these samples revealed detectable activation of caspase-3 in cells treated with 2 and 4 mmol/L of temozolomide but not cells treated with vehicle or 1 mmol/L temozolomide. Western blot analysis for actin was done to ensure equal loading of protein. Treatment of the D54-ANLucBCLuc cells with increasing doses of perifosine resulted in a dose-dependent increase in bioluminescence compared with cells treated with vehicle alone (Fig. 1D). The highest dose of perifosine (24 µmol/L) was cytotoxic, and thus, no significant increase in bioluminescence was detected. Western blot analysis of D54-ANLucBCLuc cells treated in parallel showed a dose-dependent increase in caspase-3 activation as well as corresponding increase in cleavage of ANLucBCLuc to its polypeptides. Glyceraldehyde-3-phosphate dehydrogenase was used as control to ensure equal loading of samples. Similar analysis done in squamous cell carcinoma (SCC-1) cells and prostate carcinoma (DU-145) cells revealed analogous results (data not shown).


Figure 1
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  		Fig. 1. Strategy for noninvasive imaging of caspase-3 and in vitro validation of apoptosis reporter. A, the ANLucBCLuc apoptosis imaging reporter constitutes the split luciferase (NLuc and CLuc) domains fused to interacting peptides, pepA and pepB, with an intervening caspase-3 cleavage motif. On induction of apoptosis, the reporter molecule is proteolytically cleaved by caspase-3 at the DEVD motif. This cleavage enables interaction between pepANLuc and pepBCLuc, thus reconstituting luciferase activity. B, a representative Western blot of lysates of D54 cells transfected with ANLucBCLuc and treated with 0.5 µmol/L staurosporine (STS) or vehicle for the indicated time periods and probed with luciferase antibody. C, a representative Western blot of D54-ANLucBCLuc cell lysates treated with increasing concentrations of temozolomide for 24 h and probed with luciferase and active caspase-3 antibodies. Lysates were also probed with actin antibody to control for loading. D, bioluminescence imaging was done on whole D54-ANLucBCLuc cells treated with increasing concentrations of perifosine or vehicle after 3 h (n = 4) and expressed as percent change in bioluminescence activity compared with vehicle control after normalizing to viable cells. Columns, mean; bars, SD. A representative Western blot of D54-ANLucBCLuc cell lysates treated as above and probed with luciferase and active caspase-3–specific antibodies. Lysates were also probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody to control for loading.

 
Validation of the ANLucBCLuc apoptosis reporter in vivo. To evaluate the utility of the reporter in a live subject, D54-ANLucBCLuc cells were implanted s.c. in flanks of mice. When tumors reached ~100 mm3, mice were randomly sorted into two groups. Bioluminescence imaging was done before treatment and 12 h after treatment with 30 mg/kg perifosine or vehicle control. An ~6-fold increase in bioluminescence activity was observed in animals treated with perifosine, whereas those in vehicle control group had no significant increase in bioluminescence activity (Fig. 2A and B ). To investigate the quantitative nature of the reporter, mice bearing D54-ANLucBCLuc tumors were treated with 70 mg/kg temozolomide, 140 mg/kg temozolomide, or vehicle alone (Fig. 2C and D, top). As above, vehicle-treated animals had no significant increase in bioluminescence signal after drug administration. Animals treated with 70 mg/kg temozolomide showed a peak bioluminescent activity at the 3-h time point, after which a gradual decline was observed. Similarly, animals treated with 140 mg/kg temozolomide had a peak in bioluminescence activity at the 3-h time point, which declined subsequently over time. The average increase in bioluminescence activity at the 3-h time point was ~5-fold in animals treated with 70 mg/kg temozolomide and ~12-fold in animals treated with 140 mg/kg temozolomide. To confirm that bioluminescence observed with temozolomide treatment was due to caspase-3 activity and not to substrate treatment/delivery or promoter-related effects on the reporter system, we treated mice bearing D54-ANLucBCLuc tumors or intact, constitutively active luciferase (D54-Luc) tumors with 140 mg/kg temozolomide and monitored bioluminescence over time. Here, D54-ANLucBCLuc tumor-bearing animals had an ~8-fold increase in bioluminescent activity 3 h after temozolomide administration, whereas animals bearing D54-Luc tumors had no significant increase in bioluminescence activity over time (Fig. 2D, bottom).


Figure 2
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  		Fig. 2. Validation of the ANLucBCLuc apoptosis reporter in vivo. A, bioluminescence images of representative animals harboring D54-ANLucBCLuc xenografts from control-treated (n = 6) and perifosine-treated (n = 6) groups both before and 12 h after treatment. B, the mean induction of bioluminescence activity in response to perifosine (30 mg/kg; n = 6) or vehicle-treated (n = 6) animals was calculated and plotted as the mean fold increase from pretreatment values. C, bioluminescence images of representative animals with D54-ANLucBCLuc xenografts from control-treated, 70 mg/kg temozolomide (TMZ)–treated, and 140 mg/kg temozolomide–treated groups before and 3 h after treatment. D, the mean induction of bioluminescence activity from control-treated (n = 4), 70 mg/kg temozolomide–treated (n = 4), and 140 mg/kg temozolomide–treated (n = 4) groups (top) and from animals bearing D54-Luc (n = 4) or D54-ANLucBCLuc (n = 4) tumors treated with 140 mg/kg temozolomide (bottom) was calculated and plotted over time. Data represent the mean fold increase from pretreatment values. Points, mean; bars, SD.

 
Bioluminescence activity parallels levels of active caspase-3 in tumor xenografts. To validate that increases in bioluminescence activity were coincident with activated caspase-3, tumors from animals treated with vehicle, 70 mg/kg temozolomide, and 140 mg/kg temozolomide were excised, cryosectioned, and immunostained for active caspase-3 (Fig. 3A ). Eight randomly chosen apoptotic fields from treated tumors were imaged and used to determine the percentage of cells with activated caspase-3. Tumors from animals treated with 70 mg/kg temozolomide had ~5-fold increase in cells staining positive for active caspase-3 compared with control. Active caspase-3 levels were ~2-fold higher in tumors from animals treated with 140 mg/kg temozolomide than in tumors from animals treated with 70 mg/kg temozolomide and ~12-fold higher than tumors from animals treated with vehicle (Fig. 3B).


Figure 3
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  		Fig. 3. Bioluminescence activity parallels levels of active caspase-3 in tumor xenografts. A, representative images of x20 magnified tumor samples from D54 tumor-bearing animals treated with vehicle (control), 70 mg/kg temozolomide, and 140 mg/kg temozolomide. Tumor samples were immunostained with 4',6-diamidino-2-phenylindole (DAPI) nuclear stain (blue) and active caspase-3–specific antibody (Cell Signaling Technology; red). B, eight randomly chosen apoptotic fields from each tumor were used to determine the percentage of active caspase-3–positive tumor cells from animals treated with vehicle, 70 mg/kg temozolomide, and 140 mg/kg temozolomide as indicated. Columns, mean; bars, SD.

 
Temozolomide in combination with radiation therapy enhances apoptosis in GBM tumor xenografts. To investigate the potential use of the reporter in evaluation of combination therapies, animals bearing D54-ANLucBCLuc tumors were treated with radiation therapy (4 Gy), temozolomide (70 mg/kg), and a combination of temozolomide and radiation therapy (Fig. 4A-C ). As observed previously, animals treated with vehicle only had no significant increase in bioluminescence activity, whereas animals treated with temozolomide had ~4-fold increase in bioluminescence. Animals treated with radiation therapy alone had no significant increase in bioluminescence activity. In contrast, pretreatment of animals with temozolomide 18 h before treatment with radiation therapy resulted in an increase in bioluminescence of ~8-fold above controls, which peaked 6 h after radiation therapy administration and diminished over time.


Figure 4
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  		Fig. 4. Temozolomide in combination with radiation therapy (RT) enhances apoptosis in GBM tumor xenografts. A, bioluminescence images of representative animals harboring D54-ANLucBCLuc xenografts from radiation therapy–treated, temozolomide-treated, and temozolomide/radiation therapy combination therapy–treated groups both before and 6 h after treatment with radiation therapy. B, the mean induction of bioluminescence activity from radiation therapy–treated (n = 4), temozolomide-treated (n = 4), and temozolomide/radiation therapy combination therapy–treated (n = 4) animals was calculated and plotted as the mean fold increase from pretreatment values over time. C, the mean induction of bioluminescence activity from vehicle (n = 4), radiation therapy (n = 4), temozolomide (n = 4), and temozolomide/radiation therapy combination therapy (n = 4) animals was calculated 6 h after radiation therapy and plotted as the mean fold increase from pretreatment values. Columns, mean; bars, SD.

 
Temozolomide in combination with radiation therapy enhances apoptosis in GBM tumor xenografts in a therapeutic setting. To evaluate the efficacy of temozolomide and radiation therapy in combination in a context that approximates clinical treatment of patients with GBM, D54-ANLucBCLuc tumor-bearing animals were separated into four groups. One group of animals was treated with vehicle and served as control, one group was treated with only radiation therapy, one group was treated with only temozolomide, and the final group was treated with a combination of radiation therapy and temozolomide (Fig. 5A-C ). Each animal was treated five times a week for 2 weeks. Radiation therapy given at a fractionated schedule did not result in significant increase in bioluminescence activity over time. In contrast, repeat treatment of tumor-bearing animals with temozolomide resulted in a cumulative increase in bioluminescence activity until day 12, after which a decline in bioluminescence activity was observed. Animals treated with a fractionated schedule of both therapies had significant enhancement in bioluminescence activity compared with animals treated with single therapies. Determination of bioluminescence activity for each animal revealed that control-treated and radiation therapy–treated animals had a minimal increase in bioluminescence activity, whereas temozolomide-treated animals had an ~50-fold increase. Animals treated with both temozolomide and radiation therapy showed >150-fold increase in bioluminescence activity.


Figure 5
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  		Fig. 5. Temozolomide in combination with radiation therapy enhances apoptosis in GBM tumor xenografts in a therapeutic setting. A, bioluminescence images of representative animals harboring D54-ANLucBCLuc xenografts treated with vehicle, radiation therapy, temozolomide, or temozolomide/radiation therapy combination therapy before treatment and at peak maximal induction of bioluminescence activity after treatment. B, change in bioluminescence activity in vehicle-treated, radiation therapy–treated, temozolomide-treated, and temozolomide/radiation therapy–treated group was calculated, normalized to tumor volume, and plotted as the mean fold increase from pretreatment values over time. Mean tumor volumes were assessed on alternate days and are expressed in relation to the initial volume. Horizontal lines, a period wherein daily doses of 70 mg/kg temozolomide were given. Arrows, 2 Gy daily dose of radiation therapy. C, the mean induction of bioluminescence activity from vehicle (n = 6), radiation therapy (n = 4), temozolomide (n = 8), and temozolomide/radiation therapy combination therapy (n = 10) animals was calculated and plotted as the mean fold increase from pretreatment values. Columns, mean; bars, SE.

 
Radiation therapy/temozolomide combination therapy enhances tumor regression in GBM tumor xenografts. Determination of tumor volumes over time in each of the treatment groups described above revealed that animals treated with radiation therapy alone exhibited a tumor growth delay compared with control group, and animals treated with temozolomide or combination therapy exhibited an enhanced tumor growth delay compared with radiation therapy–treated and control-treated animals (Fig. 6A ). After completion of the treatment regimen, tumors of animals treated with vehicle alone had a mean volume increase of 150% over the volume recorded before the start of treatment, whereas animals treated with radiation therapy had a mean tumor volume increase of only 50%. Animals receiving temozolomide alone or a combination of the two therapies had tumors shrink to –30% and –75% of their original volume, respectively (Fig. 6B).


Figure 6
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  		Fig. 6. Radiation therapy/temozolomide combination therapy enhances tumor regression in GBM tumor xenografts. A, mice harboring D54-ANLucBCLuc xenografts were treated with vehicle (n = 6), radiation therapy (n = 4), temozolomide (n = 8), or temozolomide/radiation therapy (n = 10). Mean tumor volumes were assessed on alternate days and are expressed in relation to the initial volume. Horizontal lines, a period wherein daily doses of 70 mg/kg temozolomide were given. Arrows, 2 Gy daily dose of radiation therapy. B, similarly, changes in tumor volume were calculated 16 d after the start of treatment and are expressed as a percent change in tumor volume. Columns, mean; bars, SE.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Molecular imaging provides the ability to dynamically and noninvasively monitor specific biological pathways in a single living subject. Recent advances in the field have enabled imaging of protein-protein interactions using split reporter constructs (14, 28–31), and others (32–34) have used molecular imaging to monitor processes such as cell proliferation or protein degradation. Here, we describe a strategy for imaging apoptosis using a luciferase complementation assay with enhanced signal to noise over our previous efforts (35). Increased signal to noise was achieved by reducing interaction between NLuc and CLuc fragments by steric hindrance (to decrease background) and by increasing NLuc and CLuc fragment complementation via the high-affinity interaction of pepA and pepB (to increase signal). Although some self-complementation is observed by NLuc and CLuc proteins when coexpressed in culture (14), addition of pepA and pepB to the split luciferase fragments increased bioluminescence activity observed by ~20-fold (data not shown). Additionally, expression of ANLuc and BCLuc components in parallel as a hybrid protein (ANLucBCLuc) hindered self-complementation between the NLuc and CLuc fragments, reducing bioluminescence activity observed to ~38% of coexpressed NLuc and CLuc fragments (data not shown). Bioluminescence observed by expression of ANLucBCLuc was reduced to ~2% of that observed by coexpression of pepANLuc and pepBCLuc fragments (data not shown). This approach both lowered background and increased luciferase signal, greatly enhancing the signal to noise of the ANLucBCLuc reporter construct. The highest bioluminescence activity exhibited in early studies was approximately 6- to 8-fold over pretreatment values (35, 36). In contrast, activation of the ANLucBCLuc reporter has consistently resulted in an induction of bioluminescence activity of 6- to 75-fold over pretreatment values under various conditions.

Time- and dose-dependent studies revealed that bioluminescence activity and cleavage of ANLucBCLuc occurs in conditions wherein caspase-3 is active. In dose-dependent studies, increased bioluminescence activity was observed at concentrations lower than detectable active caspase-3 by Western blot analysis (Fig. 1D). This suggests that the reporter may be a more sensitive measure of caspase-3 activation than traditional Western blot analysis. Although not investigated here, this reporter may also be activated by active caspase-7, which cleaves proteins containing the DEVD cleavage motif (37).

The quantitative nature of this reporter technology was shown by comparing bioluminescence activity and active caspase-3 staining of tumors from animals treated with vehicle, 70 mg/kg temozolomide, or 140 mg/kg temozolomide as shown in Figs. 2C and D and 3A and B. Bioluminescence activity in tumors of animals receiving 140 mg/kg temozolomide increased ~12-fold over that observed in tumors of animals treated with vehicle, which correlated well with the ~12-fold increase in tumor cells staining positive for active caspase-3. Additionally, tumors from animals treated with 70 mg/kg temozolomide had an ~5-fold increase in bioluminescence activity compared with control, which also paralleled the ~5-fold increase in cells staining positive for active caspase-3 from these tumors. This suggests that ANLucBCLuc can be used in a quantitative manner to determine whether increasing doses of drug are adding therapeutic benefit by enhancing apoptosis.

The utility of this technology in an experimental therapeutic setting was shown in various contexts. First, the ability of this reporter to noninvasively report on the efficacy of combination therapies is shown in Figs. 4 and 5. Treatment of orthotopic GBM tumors to single modality therapy (temozolomide or radiation therapy) as well as combination therapy (temozolomide + radiation therapy) in both short-term (36 h) and long-term (2 week) studies revealed enhanced apoptotic cell death when animals were treated with combination therapy. Second, the utility of the reporter in scheduling optimization was shown. For example, data presented in Fig. 4 show that application of a single dose of radiation therapy to animals pretreated with temozolomide 18 h before was sufficient to induce a significant enhancement in apoptosis, whereas radiation therapy alone did not result in a significant induction of apoptosis.

An apoptosis reporter such as ANLucBCLuc that can be used to monitor apoptosis dynamically and noninvasively provides the opportunity to rationally design fractionated therapeutic protocols. For example, data presented in Fig. 2D show that a single dose of temozolomide results in peak apoptosis 3 h after administration and subsides significantly after 12 h. Retreatment of animals at this time would be rational in that systemic toxicity would be minimized while still providing prolonged periods of increased apoptosis. Indeed, experiments described in Fig. 5 were designed based on this concept, and thus, animals were treated daily with lower doses of temozolomide.

Results obtained from the combination therapy experiments conducted in this study are supported by various published studies using temozolomide and radiation therapy treatment. Treatment of tumors with radiation therapy alone did not induce apoptosis as detected by our reporter (Figs. 4C and 5C); however, these tumors had decreased growth compared with tumors from control mice (Fig. 6B). These data support studies showing that tumor response to radiation therapy results in only modest levels of apoptosis in most tumor cell lines (38, 39). Instead, tumors treated with DNA-damaging therapy such as irradiation undergo cell death via necrosis or "mitotic catastrophe" (40). Administration of the DNA-alkylating agent temozolomide in conjunction with radiation increases dsDNA damage to levels that activate apoptotic machinery and forms the basis of radiosensitizing activity of temozolomide (24). Thus, tumors of animals receiving both temozolomide + radiation therapy had dsDNA damage to an extent that enabled activation of the apoptotic cascade, resulting in increased bioluminescence activity compared with animals treated with individual therapies. Additionally, the results of our temozolomide and radiation combination therapy experiments are consistent with data from a preclinical setting where temozolomide + radiation therapy treatment was found to be synergistic in tumors with low O6-methylguanine-DNA methyltransferase levels. D54 cells express low levels of O6-methylguanine-DNA methyltransferase, which may provide an additional mechanism for the radiosensitization effect of temozolomide (24, 41).

Although bioluminescence imaging of apoptosis using the technology described here is limited to testing of potential chemotherapeutic drugs using animals in a research setting, we believe that information garnered using this type of technology with regard to schedule and efficacy of combination therapies has potential to affect the clinical setting. In fact, temozolomide + radiation therapy combination treatment of brain tumors is showing significant promise in patients.

Although not investigated here, we believe that the ANLucBCLuc reporter would be of significant benefit in studies of other pathologies wherein dysregulation of apoptosis plays an important role and have begun development of transgenic animals that initiate expression of the reporter in a tissue-specific manner. Additionally, we are developing reporters for other cytosolic proteases that play a role in various disease processes. Further, replacing the reporter platform with other reporter molecules (e.g., B-galactosidase) that, unlike luciferase, do not require ATP as a cofactor and would remain active in nonviable cells could be developed. Recently, firefly luciferase fragments with novel split sites enabling more effective silencing of luciferase have been reported (42). We are constructing caspase reporters using these improved split sites to further reduce the background signal of our reporter system. In summary, we have shown that this reporter system can be used to sensitively, dynamically, and quantitatively image apoptosis. This will enable development or enhancement of therapeutic protocols by providing a platform to investigate dosing, scheduling, or the efficacy of combination therapies in living subjects.


   Acknowledgments
 
We thank Bradford Moffat and Mukilan Muthuswami (Molecular Imaging Core, University of Michigan) for assistance with magnetic resonance imaging, Mahaveer Swaroop Bhojani for assistance with figure generation, Mike Dugre for careful reading of the manuscript, and Steven Kronenberg for generation of Fig. 1A.


   Footnotes
 
Grant support: NIH/National Cancer Institute grants P01CA85878, R24CA83099, and P50CA093990.

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.

Received 4/ 4/07; revised 10/10/07; accepted 10/25/07.


   References
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 Abstract
 Materials and Methods
 Results
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 References
 

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