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Bioluminescence imaging has previously been used to monitorthe formation of grafted tumors in vivo and measure cellnumber during tumor progression and response to therapy. Thedevelopment and optimization of successful cancer therapystrategies may well require detailed and specific assessment ofbiological processes in response to mechanistic intervention.Here, we use bioluminescence imaging to monitor the cellcycle in a genetically engineered, histologically accurate modelof glioma in vivo. In these platelet-derived growth factor(PDGF)-driven oligodendrogliomas, G1 cell-cycle arrest isgenerated by blockade of either the PDGF receptor or mTORusing small-molecule inhibitors.

One factor limiting the high-throughput use of mouse models forpreclinical trials is difficulty in performing longitudinal studies ana-lyzing tumor-cell response. One of the most common alterationsfound in human cancer is inactivation of the RB pathway1. The muta-tions leading to this inactivation result in loss of RB-E2F transcrip-tional repressor complexes and activation of E2F-regulatedtranscription2. Here we present a new transgenic mouse for biolumi-nescence imaging (BLI) of the proliferative activity of glioma cells. Inthis mouse line, the gene encoding luciferase is controlled by thehuman E2F1 promoter, which exhibits tumor-specific activity in vivo3. We show that the activity of the RB pathway in endogenouslyinduced brain tumor cells can be detected, followed over time andused to evaluate the efficacy of drug treatment in vivo. We use thistechnology to show that the proliferation of PDGF-induced gliomasis dependent on both PDGF receptor activation and mTOR signaling.

RESULTSThe Ef-luc transgenic constructRecently, use of in vivo BLI has become an established method4–6 thatis more sensitive than other in vivo imaging methods presently avail-able7,8. To image the loss of RB pathway function in tumor cells in vivo we used the human E2F1 promoter controlling the expressionof the firefly luciferase gene, the Ef-luc transgenic construct (seeSupplementary Fig. 1a online). The E2F1 promoter is autoregulatory,

containing four E2F binding sites. In vitro, the E2F1 promoter hasbeen shown to have a strict cell cycle–regulated expression with ele-vated activity during late G1 to S phase2 resulting from E2F-mediatednegative regulation where E2F-RB inhibitory complexes bind to thepromoter during early G1. In vivo, however, the E2F1 promoter hasbeen shown to mediate tumor-selective transgene expression unre-lated to normal mitotic activity3, probably as a result of loss of RBpathway control in the tumor cells. Thus, bioluminescence from theEf-luc transgenic mouse is expected to correlate not only with thenumber of tumor cells but also with the loss of RB pathway necessaryfor transformation activity and the proliferative capacity of tumorcells. We have developed this reporter line for use in mouse gliomamodels and report findings with this tumor type.

Bioluminescence imaging of PDGF-induced gliomagenesisTo demonstrate that the Ef-luc mouse could be used for BLI oftumors, we crossbred the Ef-luc transgenic mouse line with the N–tv-a mouse strain allowing BLI in the well-characterized RCAS/TV-Amouse model system of brain tumors9 (Supplementary Fig. 1bonline). The N–tv-a transgenic mouse expresses the viral receptor tv-a from the nestin promoter, allowing retroviral transduction to glialprogenitor cells using replication-competent ASLV long terminalrepeat with a splice acceptor (RCAS) vectors. The double transgenicEf-luc N–tv-a mice were infected with the avian leukosis virus–basedRCAS-PDGFB vector that induces oligodendrogliomas in N–tv-amice10. First, we determined whether BLI could be used to detectbrain tumors induced with the RCAS/TV-A model system. Miceinjected with RCAS-PDGFB were imaged by BLI weekly. We foundthat all mice with the Ef-luc transgene showed a background lightproduction, especially over the regions of skin not covered by fur(Supplementary Fig. 2 online). Mice found to emit additional lightbetween the ears (Supplementary Fig. 2 online and Fig. 1a) were ana-lyzed and the emitted light could be quantified using Living Imagesoftware. We confirmed the presence of gliomas in these mice by his-tologic analysis of the brains following imaging (Fig. 1b). For tumorsof the same proliferation index, the amount of light productionroughly correlated with the size of the tumor (Fig. 1).

1Uppsala University, Department of Genetics and Pathology, Rudbeck Laboratory, SE-75185 Uppsala, Sweden. 2Memorial Sloan-Kettering Cancer Center,Departments of Surgery (Neurosurgery), Neurology and Cancer Biology and Genetics, RRL 917B, 1275 York Avenue, New York, New York 10021, USA. 3These authors contributed equally to this work. Correspondence should be addressed to E.C.H. (hollande@mskcc.org).

Published online 24 October 2004; doi:10.1038/nm1120

Dissecting tumor maintenance requirements usingbioluminescence imaging of cell proliferation in amouse glioma modelLene Uhrbom1,2,3, Edward Nerio2,3 & Eric C Holland2

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Development of gliomas over timeNext we performed a longitudinal study by screening Ef-luc N–tv-amice that had been injected with RCAS-PDGFB every third day withBLI in order to follow tumor development. A representative graph ofone such mouse that developed a brain tumor is shown (Fig. 2a). Thetime-dependent increase in light production represents the sum ofthe tumor cells’ capacity to proliferate and the overall size of thetumor. In a subsequent study we imaged five mice daily that had beenidentified to harbor gliomas. These mice showed a variable increase inlight production, indicating the baseline for glioma growth in thismodel (Fig. 2b).

Bioluminescence imaging of therapeutic efficacyWe then used the model in two longitudinal preclinical studies aimedat determining the requirements for maintenance of tumor-cell pro-liferation in this PDGF-driven glioma model. First, we used BLI tofollow PDGFB-induced glioma-bearing mice that were treated withan inhibitor of the PDGF receptor, PTK787/ZK222584 (ref. 10). Theresult of this analysis for one representativemouse showing substantial decrease in lightproduction over time is shown in Figure 3a.We compared five glioma-bearing micetreated with PTK787/ZK222584 to micetreated with buffer only. We imaged these twocohorts of mice daily and found that,whereas the buffer-treated mice showed acontinued increase in photon emission fromthe brain area over 5 d (Fig. 3b), the micetreated with PTK787/ZK222584 showed aclear reduction in light emission (Fig. 3b). Allof these mice were killed and their brainsanalyzed for evidence of proliferation usingimmunohistochemical staining for PCNA,Ki-67 and phosphohistone H3. Consistentwith our previous data11, thePTK787/ZK222584-treated tumors showed

minimal staining for these markers (less than 5% of tumor cells) (Fig. 3b) whereas the buffer-treated (and untreated) tumors showed ahigh level of PCNA staining (greater than 50% of tumor cells) (Fig. 3b). The Ki-67 and phosphohistone H3 immunostainings wereconsistent with the PCNA results (data not shown). In all cases therewas minimal evidence of apoptosis, as determined by TUNEL andcaspase 3 staining (data not shown), also consistent with our earlierfindings11. These data indicate that the light production from thesetumors in an Ef-luc N–tv-a transgenic background correlates withother indicators of cell cycle progression and proliferation.

In the second preclinical study, we tested the effect of mTOR inhi-bition by using the rapamycin analog CCI-779 (ref. 12) in the PDGF-induced gliomas. We treated 5 tumor-bearing mice with CCI-779 andimaged them over 5 d. Inhibition of mTOR by treatment with CCI-779 reduced the light production from PDGF-induced gliomas to anextent similar to that seen with PDGF receptor inhibition (Fig. 3b).Furthermore, when we analyzed the brains from these mice withimmunohistochemical markers for proliferation, the results showedessentially the same effect when inhibiting mTOR as seen for PDGFreceptor inhibition (Fig. 3b and data not shown). Notably, there wasminimal staining for TUNEL in these sections, indicating that block-ade of mTOR does not induce apoptosis in this glioma model. Takentogether, these data imply that the activities of both PDGF receptorsand mTOR are essential for the proliferation of PDGF-inducedgliomas in mice.

DISCUSSIONExperimental data generated in vitro does not always correlate withwhat is seen in vivo. Preclinical trials need to be performed in order tounderstand the rules that govern the maintenance of tumor cells in aliving organism in vivo. Previous reports have described BLI ofxenograft gliomas in which luciferase was driven by a constitutivepromoter and the light output was a measure of cell number13–16. Inthis study we show that the Ef-luc mouse can be used in such studies,as it generates light that is proportional to both cell number and pro-liferation index. In the therapeutic studies presented here, apoptoticcell loss appears not to occur. Therefore, the reduction in light pro-duction seen after treatment with either PTK787/ZK222584 or CCI-779 is predominantly an effect of inhibition of cell proliferation, anotion concordant with the results from the immunohistochemicalanalysis. By contrast, during the development of a tumor where bothcell number and proliferation rates are changing over time, this wouldnot be the case. Also, light is lost during penetration through tissue,

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Figure 1 Approximate correlation between BLI output and tumor size. (a) Ef-luc BLI of PDGF-induced oligodendrogliomas. Luciferase activity intumor-bearing Ef-luc N–tv-a transgenic mice. (b) Whole mount histologicanalysis of the brains from the same mice as were imaged in a. Tumor sizeroughly correlates with the amount of emitted light.

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making comparisons between mice difficult because of the variabledepth of each tumor. But using each mouse as its own control nor-malizes these variations and reduces the number of mice required togenerate compelling results in preclinical trials.

The Ef-luc model makes it possible to investigate the importance ofdownstream signaling pathways in glioma maintenance. Previousresults have shown that the Akt activity in our PDGF-driven gliomasis quite low17 and therefore one might have guessed a priori thatmTOR activity would not be important for the proliferation of thesetumor cells. Notably, however, our present data show that this appearsnot to be the case. It is possible that the mTOR dependence of thesetumors may be trivial, having to do with the nutrient status of thecells18. An alternative explanation for the substantial effect of mTORinhibition would be that it interferes with the recruitment of onco-genic mRNAs into polysomes. Recent data have shown that this is oneof the mechanisms of Ras- and Akt-driven gliomagenesis19. Althoughit is possible that the growth-inhibitory effect seen with CCI-779results from effects on proteins other than mTOR, there are no otherknown targets of the rapamycin analogs, and it seems unlikely that anunidentified target of this family of molecules would happen to beessential for PDGF-induced glioma proliferation. Assuming the effectis a result of mTOR inhibition, these data imply that pathways notactivated by oncogenic stimulation may still be essential for tumormaintenance and therefore valid therapeutic targets.

In conclusion, these studies make it clear that qualified guessingat the importance of signaling pathways in tumors in vivo cannotbe substituted for experiments that directly test the hypothesis. Thedevelopment of the Ef-luc mouse can be of great help in thisprocess and will be a valuable tool in such in vivo explorations inthe future.

METHODSGeneration of Ef-luc and Ef-luc N–tv-a transgenic mice. A 1714-bp SmaI-XbaI fragment of the pGL3-Basic vector encoding a modified fireflyluciferase (Promega) was cloned into a plasmid behind a 273-bp PCR-gener-ated fragment of the human E2F1 promoter. The PCR primers used wereE2F1-1 (5′-GGAATTCCATCCGGACAAAGCCTGCGCGC-3′) and E2F1-2(5′-GGAATTCAGGCCTCGGCGAGGGCTCGAT-3′); we added an EcoRI siteat each end of the E2F1 promoter. The gene encoding luciferase was followed

by a polyadenylation sequence. We generated chimeric founder mice bypronuclear microinjection of the linearized Ef-luc construct into fertilizedFVB oocytes. Genotyping of transgenic mice was done by PCR using primersE2F1-1 and EFLUC-1 (5′-TGCGGGAGTTTCACGCCACCA-3′), yielding aproduct of 335 bp. We screened EFLUC-positive mouse lines for correctexpression of the transgene in vitro. We identified 10 chimera founders thatlead to 8 founders with germline transmission; three of these showed tightcell-cycle regulation of expression in vitro. The luciferase activity was meas-ured in primary cultured cells obtained from the brains of newborn mice asdescribed previously10. Equal numbers of cells were cultured in the presence of10% fetal bovine serum for 1 d or in 0.5% fetal bovine serum for 4 d beforelysis. Luciferase activity was measured using the Luciferase Assay System(Promega) according to the manufacturer’s protocol.

Generation of mouse brain tumors. Double-transgenic neonatal Ef-luc N–tv-a mice were injected intracranially with 1 µl DF-1 cells producing RCAS-PDGFB retrovirus as described previously11. Mice were monitored carefullyfor symptoms of tumor development (hydrocephalus, lethargy). All injectedmice were routinely screened with BLI, and image-positive mice were followedover time, treated and followed over time, or killed.

BLI of Ef-luc N–tv-a mice. Mice were anesthetized with 3% isofluorane beforeretro-orbital injection with 75 mg/kg body weight n-Luciferin (Xenogen).Three minutes after injection of the n-Luciferin, images were acquired for 2 min with the Xenogen IVIS system (Xenogen) using Living Image analysisand acquisition software (Xenogen). A photographic image was taken, ontowhich the pseudocolor image representing the spatial distribution of photoncounts was projected. We defined a circular region between the ears and used itas a standard in all experiments. From this region the photon counts werecompared between different mice.

Drug treatment of tumor bearing Ef-luc N–tv-a mice. Image-positive Ef-luc N–tv-a mice were treated daily with PTK787/ZK222584 at 100 mg/kg body weight, CCI-779 at 40 mg/kg body weight, or buffer onlyfor the indicated number of days. All doses were administered throughintraperitoneal injection. We took transgenic images 24–144 h after initiation of treatment.

The resuspension solution for PTK787/ZK222584 consists of 5% DMSOand 1% Tween-80 in distilled water stored at 4 0C. This solution was usedwhen treating the buffer-treated mice. We prepared PTK787/ZK222584 at 10 mg/ml and stored it at –20 °C. PTK787/ZK222584 does not dissolve in solu-tion and therefore we inverted the tubes before each use. CCI-779 was pre-

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Figure 3 Preclinical trials of PDGFB-induced glioma-bearing Ef-luc N–tv-a mice. (a) Longitudinal imaging of one tumor-bearing mouse treated withPTK787/ZK222584 daily for 6 d. (b) Longitudinal study with five Ef-luc N–tv-a tumor-bearing mice in each cohort: untreated (left panel) or treated dailywith PTK787/ZK222584 (middle panel) or CCI-779 (right panel). Upper panels show photon counts of emitted light and lower panels showimmunohistochemical staining for PCNA as a measure of cell proliferation.

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pared at 2 mg/ml in a resuspension buffer containing 5% Tween-80 and 5%PEG400, and stored at –20 °C.

Histological analysis, immunohistochemistry and TUNEL analysis. Weremoved the brains of killed mice, fixed the brains in formalin and embeddedthem in paraffin. Immunohistochemical stainings were performed using anti-bodies for PCNA (Oncogene), caspase-3 (Cell Signaling), Ki-67 (Novacastra),and histone H3 (Upstate). TUNEL analysis (Roche) was performed accordingto the manufacturer’s protocol.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTSThe authors would like to thank C. Glaster for preparation of this manuscript,C. Discafani (Wyeth Research) for the CCI-779 and J. Wood (NovartisPharmaceuticals) for the PTK787/ZK222584. This work was supported by the Tow,Seroussi, Bressler and Kirby Foundations.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 24 March 2004; accepted 7 July 2004Published online at http://www.nature.com/naturemedicine/

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