Bioluminescent Imaging of HPV-Positive Oral Tumor Growth and … · Therapeutics, Targets, and Chemical Biology Bioluminescent Imaging of HPV-Positive Oral Tumor Growth and Its Response

Therapeutics, Targets, and Chemical Biology

Bioluminescent Imaging of HPV-Positive Oral Tumor Growthand Its Response to Image-Guided Radiotherapy

Rong Zhong, Matt Pytynia, Charles Pelizzari, and Michael Spiotto

AbstractThe treatment paradigms for head and neck squamous cell cancer (HNSCC) are changing due to the emergence

of humanpapillomavirus (HPV)-associated tumors possessing distinctmolecular profiles and responses to therapy.Although patients with HNSCCs are often treated with radiotherapy, preclinical models are limited by the abilityto deliver precise radiation to orthotopic tumors and to monitor treatment responses accordingly. To bettermodel this clinical scenario, we developed a novel autochthonous HPV-positive oral tumor model to track re-sponses to small molecules and image-guided radiation. We used a tamoxifen-regulated Cre recombinase systemto conditionally express the HPV oncogenes E6 and E7 as well as a luciferase reporter (iHPV-Luc) in the epithelialcells of transgenic mice. In the presence of activated Cre recombinase, luciferase activity, and by proxy, HPVoncogenes were induced to 11-fold higher levels. In triple transgenic mice containing the iHPV-Luc, K14-CreERtam,and LSL-Kras transgenes, tamoxifen treatment resulted in oral tumor development with increased bioluminescentactivity within 6 days that reached a maximum of 74.8-fold higher bioluminescence compared with uninducedmice. Oral tumors expressed p16 andMCM7, two biomarkers associatedwithHPV-positive tumors. After treatmentwith rapamycin or image-guided radiotherapy, tumors regressed and possessed decreased bioluminescence.Thus, this novel system enables us to rapidly visualize HPV-positive tumor growth to model existing and newinterventions using clinically relevant drugs and radiotherapy techniques. Cancer Res; 74(7); 2073–81.�2014 AACR.

IntroductionGiven their distinct oncogenic and mutational pathways

(1, 2), head and neck squamous cell cancers (HNSCC) maydifferentially respond to radiation and/or chemotherapy.Existing reports indicate that treatment outcomes depend onthe p53 mutational status and human papillomavirus (HPV)oncogene expression (3–5). To better understand HNSCCbiology, several models exist to study the development ofautochthonous head and neck tumors. Many groups haverelied on chemical carcinogens such as 4-Nitroquinolone1-oxide (4-NQO) to induce cancers with undefined DNAlesions that mimic those caused by cigarette smoking (6, 7).To generate tumors with more homogeneous genetic profiles,other groups have genetically engineered mice to overexpressmutant cellular oncogenes such as Kras (8) or to delete tumorsuppressors such as Trp53 (9–12). Furthermore, groups haveengineered mice to express some of these oncogenes in aspatiotemporal manner using systems such as ligand-regulat-ed Cre recombinases (8, 9, 13).

However, understanding how other oncogenes such as theHPV oncogenes E6 and E7 (E6E7; ref. 14) impact oral tumorresponses to therapy are limited by the availability preclinicalmodels, the accurate delivery of radiotherapy, and the assess-ment of treatment responses. Although several xenotransplantmodels exist for HPV-associated HNSCCs, these tumors weretransplanted into immunodeficient mice and may be biolog-ically distinct from the parental cancer (15–18). Furthermore,oral tumors developed in HPV-transgenic mice treated with 4-NQO (19), but these mice constitutively expressed HPV onco-genes, which may impact immune tolerance and tumor devel-opment. In addition, irradiationof oral tumors has been limitedto 2 to 6 Gy due to the proximity of tumors to the centralnervous system and other vital structures (17). Finally, mon-itoring treatment responses to autochthonous oral tumors hasbeen mostly constrained to crude measurements such assurvival and weight loss. Thus, understanding how the tumorgenotype dictates response to therapy would benefit fromnovel preclinical models that monitor the response of primaryHPV-positive tumors to radiation and other targeted therapies.

Here, we developed a novel head and neck tumor model tomonitor the growth of HPV-positive tumors and their responseto therapy using bioluminescence. We used a ligand-regulatedCre recombinase to induce the HPV oncogenes E6E7 and aluciferase reporter in vitro and in vivo. Oral tumors arose inmicewhen E6E7 andmutantKrasG12D oncogeneswere induced in thebasal epithelial layer. These oral tumors expressed HPV-asso-ciated biomarkers andgrew faster than tumors arising in controlmice harboring only a mutant Kras oncogene. HPV tumorsgained bioluminescence over time, which was modulated by

Authors' Affiliation: Department of Radiation and Cellular Oncology,University of Chicago Medical Center, Chicago, Illinois

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

Corresponding Author: Michael Spiotto, Department of Radiation andCellular Oncology, University of Chicago, KCBD 6142, 900 E. 57th St.,Chicago, IL 60637. Phone: 773-702-2751; Fax: 773-702-1968; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-2993

�2014 American Association for Cancer Research.

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tumoricidal agents, including small- molecule inhibitors andimage-guided radiotherapy (IGRT).

Materials and MethodsGeneration of iHPV-Luc transgenic vector and mice

The pB-actin E6E7 plasmid containing the HPV-16 E6E7wasa generous gift from Karl Munger (Harvard University, Boston,MA; ref. 20) and was obtained from Addgene (plasmid 13712).The E6E7 gene was amplified by the 50 primer 50- TTGAATT-CGCGGCCGCCACCATGCACCAAAAGAGAACTGC-30 and 30

primer 50- TTCTCGAGTTATGGTTTCTGAGAACAGATGG-30.The E6E7 PCR product was digested with Eco RI-Xho I andligated to MSCV IRES Luciferase plasmid, a generous gift ofScott Lowe (Memorial Sloan Kettering Cancer Center, NewYork, NY; Addgene plasmid 18760). An Eco RI-Sal I fragment ofE6E7 IRES Luciferase construct was isolated and ligated to anEco RI-Xho I fragment of pCAGEN, a generous gift of ConnieCepko (Harvard University, Boston, MA; ref. 21) Addgeneplasmid 11160 to generate the HPV-Luc vector. A LoxP EGFPpolyA LoxP PCR fragment was generated by amplifyingpcDNA-EGFP (a generous gift of Doug Golenbock, Universityof Massachusetts, Worchester, MA; Addgene plasmid 13031)with the forward primer 50 TTGAATTCATAACTTCGTATAG-CATACATTATACGAAGTTATTGCCACCATGGTGAGCAAGG-GCGAGGAG-30 and reverse primer 50-TTGCGGCCGCTTATA-ACTTCGTATAATGTATGCTATACGAAGTTATCATAGGGAA-GAAAGCGAAAGGAG-30. This LoxP-EGFP polyA-LoxP frag-ment was digested with Eco RI–Not I and cloned into theHPV-Luc to generate iHPV-Luc. The resulting plasmid waslinearized with SalI-BamHI and transgenic mice were made bymicroinjection into the nuclei of FVB/NJ (The Jackson Labora-tory) zygotes. Mice were maintained on an FVB/N background.

MiceAll mice were maintained under specific pathogen-free

conditions and used according to protocols approved by TheUniversity of Chicago (Chicago, IL) Institutional Animal Careand Use Committee and Institutional Biosafety Committee.B6.129S4-Krastm4Tyj/J (LSL-Kras) mice were purchased fromThe Jackson Laboratory. Transgenic mice STOCK-Tg(KRT14-cre/ERT)20Efu/J (K14-CreERtam) have been described (22).K14-CreERtam mice were backcrossed to the FVB backgroundusing MAX BAX speed congenics Taconic for seven genera-tions and shared 99.74% similarities to FVB mice with 2 non-FVB polymorphisms adjacent to the K14-CreERtam transgene.K14-CreERtammicewere bredwith iHPVmice to generate K14-CreERtam �iHPV-Luc mice (KH mice) on an FVB background.KH mice were then bred with LSL-Kras mice to generate KHRmice or KR mice on a C57BL/6 � FVB F1 background. Miceused for experiments were between 30 and 40 days old.

ReagentsRapamycin was obtained from Sigma Chemical Co. and

reconstituted (0.2% carboxymethylcellulose and 0.25%Tween-80 and injected into mice intraperitoneally; i.p.) at 4mg/kg/d for 3 days. D-Luciferin Potassium Salt was obtainedfrom Gold Biotechnology. Tamoxifen was purchased fromSigma. Tamoxifen was dissolved in sunflower seed oil vehicles

at 20mg/mL and 0.1mLwas injected intraperitoneally daily for5 days. All oligonucleotides were synthesized by IDT.

Fluorescence cytometryA total of 2 � 105 293 HEK cells (American Type Culture

Collection; ATCC) were transfected with iE6E7 vector withor without PGK-Cre-bpA vector, a gift of Klaus Rajewski(Harvard University, Boston, MA; Addgene plasmid 11543)using Fugene HD (Promega). Cells were trypsinized after 48hours and EGFP fluorescence was assessed. Peripheral bloodlymphocytes were isolated by retroorbital bleeding and redblood cells were lysed with red blood cell lysis buffer(eBioscience). Cells were analyzed by FACscan and datawere analyzed by FlowJo software.

Western blot analysisA total of 106 293 HEK cells were transfected with iHPV-Luc

vectorwith orwithout iCre vector using FugeneHD (Promega).Forty-eight hours later, cells were trypsinized and lysed with amodified radioimmunoprecipitation assay buffer [50 mmol/LTris-HCl (pH 7.4), 150mmol/L NaCl, 1 mmol/L EDTA, 1%NP40,1 mg/mL aprotinin, 1 mg/mL leupeptin, and 10 mmol/L phe-nylmethylsulfonylfluoride) for 30minutes on ice. Samples weresubjected to SDS-PAGE using a 4% stacking gel and a 10%resolving gel. The protein gel was then transferred to nitro-cellulose, blocked, and probed using a 1:200 dilution of primaryantibody and subsequently a 1:1000 dilution of a secondaryhorseradish peroxidase-conjugated goat anti-rabbit immuno-globulin G antibody (Pierce). The membrane was developedusing enhanced chemiluminescence (Amersham).

Luciferase assaysA total of 2 � 105 293 HEK cells (ATCC) were transfected

with iE6E7 vector with orwithout PGK-Cre-bpA vector, a gift ofKlaus Rajewski using Fugene HD (Promega). Forty-eight hourslater, cells were lysed in Bright Glo lysis buffer (Promega) for 20minutes and luciferase activity was assessed using a Turner TD20/20 Luminometer (Turner Designs) and luciferase activitywas normalized to protein concentration of the sample.

In vivo imagingLuciferase activity was measured noninvasively using the

IVIS-200 imaging system (Caliper LifeSciences). Mice wereinjected intraperitoneally with luciferin (300 mg/kg dissolvedin PBS; Caliper) and anesthetized via inhaled 3% isoflurane(Abbott Laboratories Ltd.). Exposure time for all images rangedfrom 1 to 5 seconds. All images were analyzed using LivingImage software (Caliper) with a binning of 8. In vivo biolumi-nescent signal was quantified by taking the total photon countsfor each region of interest.

HistologyTumors were isolated and placed into 4% paraformaldy-

hyde solution for 24 hours and then dehydraded with 70%ethanol. Tissues were embedded in paraffin, sectioned andstained with hematoxyalin and eosin or the indicated anti-bodies at the University of Chicago Immunohistochemistrycore facility.

Zhong et al.

Cancer Res; 74(7) April 1, 2014 Cancer Research2074




Excision KRAS-specific PCRFor detection of iE6E7 recombination, tumors were digested

in lysis buffer supplemented with proteinase K and genomicDNA was isolated from tumors using ethanol precipitation.The iE6E7 gene was amplified by PCR using the primers:forward primer: 50-CAACGTGGTTATTGTGCTG-30 corre-sponding to the 30 end of the CAG promoter and the reverseprimer: 50-GGTAACTTTCTGGGTCGCTCCT-30 correspondingthe 50 region of the E6E7 gene with a 58�C annealing temper-ature and 72�C extension temperature repeated over 33 cycles.The 1,629 bp amplicon represents the unexcised HPV vectorand the 388 bp amplicon represents the excised iE6E7 gene.For detection of the LSL-Kras recombination, gDNA was

amplified by PCR using the forward primer: GTCTTTCCCCAG-CACAGTGC and the reverse primer: GGATGGCATCTTG-GACCTTA that flank the floxed stop gene cassette. The result-ing amplicon was digested with HindIII that cuts the uniquerestriction site engineered into the second exon of the mutantallele specific. The undigested 947 bp amplicon represents thewild-type allele and the digested 323 bp and 624 bp ampliconsrepresent the mutant allele.

Image-guided radiotherapyMice bearing 14 to 21-day-old tumors were irradiated using

an XRAD 225Cx (Precision Xray) small animal image-guidedirradiator. Anesthetized mice were immobilized in the supineposition and a cone beam computed tomography (CT) wasobtained using 40 kVp and 250 mA. For each mouse, theisocenter was placed in themidpoint of the tumor and anteriorto the mouth to cover the oral tumor using half of a 1.5-cmcollimator. A radiation dosimetry plan was generated usingopposed lateralfields.Micewere treatedwith 225 kVpX-rays at13 mA to a dose of 20 Gy.

In vivo tumor growthTumors were measured every 3 days. Given the ellipsoid

growth of oral tumors, we reasoned that the oral tumorvolume would approximate a solid ellipsoid minus a cylin-drical volume to account for space of the oral aperture. Tomeasure the tumor volume, we measured the intercommis-sural distance (a) as well as two additional orthogonalmeasurements (b and c). In addition, the lip thickness wasmeasured on each side (d and e) to calculate the diameter ofthe oral aperture. The oral tumor volume (mm3) was thencalculated as: (1/6�p�a�b�c) � p((1/2(a-d-e))2)�c. We illus-trate this method in Supplementary Fig. S1.

Statistical analysisTwo-tailed independent Student t tests were done to analyze

the results of in vitro luciferase assays, in vivo bioluminescence,or tumor growth time points.

ResultsGeneration of a Cre-loxP regulated inducible HPVtransgenic mouseCurrent HPV transgenic mouse models constitutively

expressed E6 and/or E7 and, therefore, do not recapitulate

the HPV infections that occur in young adults who are firstexposed to exogenous HPV oncogenes. To generate an induc-ible HPVmousemodel that better reflects the clinical scenario,we generated a transgenic vector where E6E7 expression wasdependent on Cre recombinase (iHPV-Luc; Fig. 1A). To mon-itor oncogene activation and tumor development, the E6E7cassette was followed by an internal ribosomal entry site(IRES)-luciferase gene that enabled bicistronic expression ofthe E6E7 oncogenes and the luciferase reporter. iHPV-Luccontained a floxed EGFP gene that inhibited expression of theE6E7-IRES-luciferase gene cassette expression. In the absenceof Cre recombinase, cells transfected with iHPV-Luc hadhigher levels of EGFP and lower levels of E6 expression andluciferase activity (Fig. 1B–D). In the presence of Cre recom-binase, cells expressed lower levels of EGFP and 10.1-foldhigher levels of luciferase activity and E6 expression consistentwith the Cre-mediated recombination of the floxed EGFPcassette and induction of the E6E7 genes and the luciferasereporter.

We next generated transgenic mice containing this iHPVconstruct. Of the 49 founder mice, 24 mice contained theiHPV-Luc transgene. In transgene-positive mice, we thenassessed EGFP expression in the peripheral blood leukocytesthat acted as a surrogate for the potential level of oncogeneinduction. We selected one mouse that expressed EGFP 290-fold above background (Fig. 2A). To assess the induction ofthe transgene, we bred iHPV-Luc mice to Rosa-CreERtam

mice to generate HRosa mice. Treatment with tamoxifenactivated the CreERtam fusion protein that was ubiquitouslyexpressed by all tissues. After a 5-day course of tamoxifentreatment, HRosa mice had increased bioluminescence inthe non-fur bearing skin that was 11-fold above background(tamoxifen treated: 10.3 � 1.2-fold vs. vehicle treated: 0.9 �0.2-fold; Fig. 2B and C).

Generation of an autochthonous oral HPV tumor modelWe next generated mice that developed autochthonous

HPV-positive oral tumors. On the basis of previous reports,mice induced to express the KrasG12D mutant in the basalepithelial layer developed oral papillomas over the course of 2months (8, 9). Similarly, we first bred K14-CreERtam mice toiHPV-Luc mice to generate KH mice (Fig. 3A). The K14-CreERtam transgene expresses the ligand-regulated Cre recom-binase driven by the basal keratinocyte promoter, K14. KHmice were then bred to LSL-Kras mice to generate KR micecontaining the K14-CreERtam and LSL-Kras transgene or KHRmice containing all three transgenes (K14-CreERtam �LSL-Kras �iHPV-Luc; Fig 3A). Tamoxifen treatment of KH, KR,or KHR mice resulted in excision of the respective LSL-Krasand/or iHPV-Luc transgenes (Fig. 3B).

We next monitored tumor growth in KR and KHR mice.Although oral tumors formed in KR and KHR mice, KHRtumors grew faster than in KR tumors (Fig. 3C). Histologicanalysis of KHR tumors revealed papillomas that expressedthe HPV-biomarkers p16 and MCM7 (Fig. 3D). Comparedwith oral tumors in KR mice, oral tumors developing inKHR mice had increased MCM7 expression and similar p16expression.

In Vivo Imaging of Inducible HPV Oral Tumors

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KHR tumor growth was associated with increasedbioluminescence

We then assessed the kinetics of bioluminescence after tamox-ifen treatment (Fig. 4A). Within 3 days of initiating tamoxifentreatment, mice had increased bioluminescence compared withuntreatedmice. By 24 days after tamoxifen treatment, KHRmicehad 65.7-fold higher bioluminescent signal compared withuntreated controls (Fig. 4B). KHR mice that developed oraltumors had progressively increasing oral bioluminescence thatcorrelated with tumor growth (P < 0.0001, r ¼ 0.88; Fig. 4C). In

contrast, bioluminescence around the oral cavity plateaued inKH mice within 6 days at 7.4-fold above background. KR micetreated with tamoxifen developed oral tumors that did notpossess bioluminescence signal above background.

In vivo bioluminescent monitoring of response totumoricidal agents

Because bioluminescent signal correlated with tumor vol-ume, we tested the extent to which bioluminescent signal inautochthonous tumors would enable monitoring response to

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Figure 2. Generation of an iHPV-Luc transgenic mouse line. A, EGFP expression in the peripheral blood leukocytes from a founder FVB/N mousecontaining the iHPV-Luc transgene. Foundermice containing the iHPV-Luc transgeneswere screened for EGFPexpressionusing fluorescencecytometry.Weselected the founder linewith the highest level of EGFP expression. B, global induction of bioluminescence in iHPV-Luc�Rosa-CreERtammice (HRosamice).Micewere treatedwith vehicle or 2mg/day tamoxifen (Tam) i.p. for 5 days and bioluminescencewas assessed 7 days later. Tamoxifen-treatedmice displayedhigher levels of bioluminescence in the non-fur bearing areas. The red arrows indicate non-fur bearing skin having increased bioluminescent signal. C,quantitation of the bioluminescence from the paws of vehicle or tamoxifen-treatedmice in B. Results were derived from 6mice from the vehicle-treated groupand 10 mice from the tamoxifen-treated group. B and C were representative from two similar experiments.

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Figure 1. Development of an iHPV-Luc transgene. A, scheme foriHPV-Luc transgene. A floxedEGFP reporter was placeddownstream from the CAGpromoter to inhibit expression ofthe E6E7-IRES-Luc bicistroniccassette. Excision of the floxedEGFP cassette resulted ininduction of the E6E7-IRES-Lucgene cassette. B and D, Crerecombination of iE6E7 causeddecreased EGFP (grayhistograms; B) and increased E6expression (C) and luciferaseactivity (D). The iHPV-Lucconstruct was transfected intoHEK cells with or without a Crerecombinase expression vector.Transfected cells were assessedfor EGFP fluorescence using flowcytometry, E6 expression byWestern blot analysis, andluciferase activity. Data, twoseparate experiments performedin duplicate.

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agents that impacted tumor growth (9). Rapamycin has beenshown to impact the growth in autochthonous oral tumormodels driven by a mutant Kras, in transplanted head andneck squamous cell carcinoma models and in clinical trials.Fourteen days after tamoxifen treatment, we treated KHRmicewith rapamycin for 3 days and monitored tumor growth.Compared with the vehicle-treated controls, mice treated withrapamycin had a transient decrease in tumor growth withregrowth after rapamycin was discontinued (Fig. 5A). Com-pared with untreated tumors, tumors treated with rapamycindisplayed 3.3-fold decreased bioluminescent signal at thecompletion of treatment (Fig. 5B and C).Because oral tumors in KHR mice possessed decreased

bioluminescence that correlated with rapamycin treatment,we next tested the extent to which IGRT impacted KHR tumorgrowth and bioluminescence (Fig. 6). IGRT is rapidly beingimplemented in the clinic to treat patients with HNSCC giventhe increased ability to target tumors (23). As in patients, conebeam CT images were obtained for each tumor and theisocenter was placed according to the tumor extent (Fig.6A). We developed individual radiotherapy plans usingopposed lateral radiation beams to treat mice to 20 Gy.Fourteen days after tamoxifen treatment, mice were irradiatedwith 20 Gy and tumors regressed (Fig. 6B), whereas unirradi-ated tumors grew progressively. The final oral tumor volumewas significantly smaller for irradiated KHR mice (31.63 �22.03 mm3; n¼ 9) compared with unirradiated mice (414.01�80.96mm3;n¼ 10;P¼ 4.96� 10�9). Six days after radiotherapy,

irradiated tumors had 3-fold less bioluminescence comparedwith nonirradiated controls consistent with tumor regression.Therefore, bioluminescent signal was a surrogate for responseto different targeted therapies in autochthonous oral tumors.

DiscussionHere, we developed a preclinical model to mimic the devel-

opment and treatment of autochthonous HPV-positive oraltumors. Similar to patients who are first infected with HPV asyoung adults, we generated an inducible HPV oral tumormodel to control HPV oncogene expression in a spatiotem-poral manner. Furthermore, compared with previous models,this bioluminescent signal, linked to HPV oncogene expres-sion, provided a noninvasive method to monitor the growth ofintraoral tumors that were otherwise difficult to assess. Finally,we used this model to track responses to IGRT and smallmolecules, two interventions currently being used in the clinic.Therefore, we intend our novel model to study how HPVoncogenes alone or in cooperation with other genotypesimpact tumor growth and response to therapy.

We recognize the limitations for our model to accuratelyreflect the clinical scenario of patients with HNSCC. First, micedeveloping HPV-positive tumors also required mutant Kras fortumor development. Although mutations in the ras familyaccount for approximately 5% of HNSCCs (1, 2), several groupshave shown that the ras–MAPK pathway was aberrantly acti-vated inHNSCCsaswell as in otherHPV-positive tumors (24–27).

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Figure 3. Generation of KHR micethat recombine the iHPV-Luc andLSL-Kras genes upon tamoxifen(Tam) treatment. KHmice andLSL-Kras transgenic mice were bred togenerate offspring containing allcombinations of the transgenes. A,PCR confirmed the genotype ofKHR mice containing the K14-CreERtam � LSL-Kras � iHPV-Luctransgene. Transgenic mice: KH:K14-CreERtam � iHPV-Luc; R:LSL-Kras; KR: K14-CreERtam �LSL-Kras; KHR: K14-CreERtam �iHPV-Luc � LSL-Kras. B,treatment of these offspring withtamoxifen resulted in excision ofthe iHPV-Luc and LSL-Krastransgenes. C, after tamoxifentreatment, oral tumors grew fasterin KHR mice than in KR mice.Similar results were observed intwo independent experiments.Symbols, individual mice. Theasterisks denote significantdifferences between KHR and KRmice (�, P < 0.05; ��, P < 0.001). D,oral tumors arising in KHR micewere positive for p16 and MCM7,two biomarkers for HPV positivity.






Second, HPV oncogene expression was driven by a cytomegalo-virus promoter, which may express oncogenes at artificial levelsthat do not reflect human HPV-positive tumors. Nevertheless,most, if not all, HPV-positive cancers have viral sequences thatnonspecifically integrated throughout the host genome, likelyresulting in a range of expression levels (28). In addition, HPVoncogene expression accelerated oral papilloma growth but we

did not find any obvious invasive cancer. Still, given the accel-erated tumor growth, ourmodelmaynothavehad sufficient timeto develop invasive disease. Finally, HPV-positivemicedevelopedoral tumors and not oropharyngeal cancers that are seen inpatients with HNSCC. Still, these oral tumors occurred atmurineanatomic sites with epithelial transitional zones that are classi-cally affected by HPV oncogenes (29).

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Figure 4. The increase inbioluminescence correlated withtumor growth in KHRmice. A, timecourse for bioluminescence ofKHR mice treated with vehicle ortamoxifen. Mice were imagedevery 3 days after the initiation oftreatment. B, kinetics ofbioluminescence in KHR (n ¼ 7),KR (n ¼ 2), and KH (n ¼ 4) micetreated with tamoxifen (Tam) orKHR mice (n ¼ 5) treated withvehicle. �, P < 0.05, significantdifferences between vehicle-treated or tamoxifen-treated KHRmice. Error bars, �1 SD. C,correlation of tumor volume withbioluminescence.

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Figure 5. Bioluminescence tracked oral tumor response to small-molecule therapy. A, oral tumors in tamoxifen (Tam)-treated KHR mice regressed afterrapamycin treatment. Mice bearing 14-day-old oral tumors were treated with 4 mg/kg of rapamycin for 3 days (n¼ 3) or vehicle (n¼ 5) and tumor growth wasmonitored every 3 days. Similar results were observed in two independent experiments. �, P < 0.05, significant differences between vehicle-treated orrapamycin-treated KHR mice. Error bars, �1 SD. Arrow, rapamycin (Rap) treatment. B, five days after rapamycin treatment, oral tumors displayed lowerbioluminescence levels. C, quantitation of bioluminescence in KHR mice bearing oral tumors treated with rapamycin (n ¼ 3) or vehicle (n ¼ 3).

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As in previous models (8, 9), we used a tamoxifen-regulatedCre recombinase expressed in the basal epithelial layer toinduce HPV-positive oral tumors. Previous models of autoch-thonous oral tumors have relied on chemical carcinogens (6) orgenetically engineered transgenes (8, 9, 13). These geneticallydefined models expressed mutant Kras and/or mutant Trp53as well as deleted Notch1 (12) and/or Tgfbr1 (11). In addition,mouse models with inducible promoters have been used todrive oncogenes in the basal epithelial layer, resulting insquamous tumors (30, 31). Finally, other groups have directlyfused oncogenes to hormonal receptors that spatially seques-ters oncogene activity within the cell (32). Upon treatmentwithestrogen or other ligands, these mice developed squamouspapillomas and invasive cancers. However, these induciblepromoters and oncogene fusion proteins act only transientlywhile the ligand is present, which may impact tumorigenesis.Therefore, in our model, Cre-mediated recombination enabledsustained tissue-specific expression of HPV-oncogenes.In our study, HPV oncogene expression had functional

consequences as HPV-positive oral tumors grew faster thanHPV-negative tumors and gained expression of MCM7, aknown HPV biomarker. These results are consistent withprevious studies demonstrating that the ras–MAPK pathwaycooperated with HPV oncogenes in vitro and in vivo to increasetumor aggressiveness through mechanisms such as dysregula-tion of the cell cycle and/or cell invasiveness (29, 33). As thecooperation of HPV and Ras oncogenes have been widelystudied, our model will enable us to further examine how HPVoncogenes altered the biology of oral tumors.Our system mimics the HPV's life cycle where HPV onco-

genes expression was induced in the basal epithelial layer and

continued as cells differentiated into the suprabasal layers. Incontrast, previousmousemodels constitutively expressedHPVoncogenes using tissue-specific promoters including keratin 10and keratin 14 that restricted oncogene expression based onthe differentiation state of the cell (34–37). In addition, theinduction of these oncogenesmayminimize immune tolerancethat occurs with constitutively expressed HPV antigens tobetter mimic immune responses to exogenous viral proteins(38). Thus, our inducible model may more physiologicallyreflect viral oncogene expression to enhance our understand-ing of HPV oncogenesis and response to therapy.

Here, we applied a bioluminescent signal to monitor thedevelopment of autochthonous HPV-positive tumors. Biolumi-nescent systems have overcome the difficulties in monitoringthe growth of tumors transplanted in orthotopic sites such asthe oral cavity, which is difficult to assess (39, 40). However,orthotopic transplants were often studied in immunodeficienthosts that may not recapitulate the heterogeneous tumorbiology seen in primary tumors (18). Other groups have adaptedin vivo imaging technologies to monitor the development ofautochthonous tumors including prostate (41, 42), pancreas(42), lymphoma (43), and others. However, in the majority ofthese models, the reporter transgenes were not linked toinitiating oncogenic events and, therefore, it remained unclearwhether regional differences existed between oncogene expres-sion and reporter activity. In addition, others have linkedreporter genes to tissue-specific promoters such as prostate-specific antigen and CD19 (43, 44) that are expressed in bothmalignant and nonmalignant parenchyma. Yet, these tissue-specific promoters may not be active in poorly differentiatedtumors and, therefore, may not adequately track tumor growth

KHR+ 0 Gy

KHR+ 20 Gy

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C

Vehicle IGRT

KHR mice bearing oral tumors treated with:

* *

Figure 6. IGRT caused tumorregression and decreasedbioluminescence. A, IGRTplanning for KHRmice bearing oraltumors. First, a cone beam CTscan was obtained. Then radiationbeam angles were designed togenerate a dosimetry plan. Reddose cloud represented the 100%isodosecloud.B, fourteen-day-oldprimary tumors regressed aftertreatment with 20 Gy. Error bars,�1 SD. Results wererepresentative of three similarexperiments using 2 to 5 mice pergroup. Arrow, IGRT (XRT)treatment. �, P < 0.001, significantdifferences between unirradiatedor irradiated KHRmice. C, six daysafter irradiation, oral tumorsdisplayed lower bioluminescencelevels. D, quantitation ofbioluminescence in KHR micebearing oral tumors receiving20 Gy (n ¼ 3) or nonirradiatedmice (n ¼ 3). Results wererepresentative of two separateexperiments.






(44). Thus, genetically linking oncogene expression to reporteractivity more faithfully reflects tumor growth.

Finally, we have used our novel autochthonous HPV tumormodel to monitor responses to IGRT and small molecules thatare currently used in the clinic. Although previous studiesdemonstrated that rapamycin prevented the outgrowth ofautochthonous HPV-negative oral tumors (9), we observedthat that rapamycin also caused regression of HPV-positivetumors. Because rapamycin inhibitsmTOR, our results suggestthat the mTOR–PI3K pathway was active in our tumor modeland are consistent with HNSCC sequencing data describingmutations in PIK3CA and PTEN, leading to the activation of themTOR pathway (1, 2, 45). As the phosphoinositide 3-kinase(PI3K) pathway is often mutated in HNSCCs, we are applyingforward and reverse genetic approaches to study the mTOR–PI3K pathway in our HPV mouse model (M. Spiotto; unpub-lished observations). Paralleling the clinical setting, IGRTinvolves tumor localization with a cone beam CT, real-timedosimetry planning, and subsequent dose delivery (46, 47).Recently, several groups have treated autochthonous sarcomasand non–small cell lung cancers as well as normal tissues(8, 48–50). However, irradiation of these tumors at mostresulted in growth arrest, whereas, in our model, we observedtumor regression. Although previous studies treated the headand neck region with low doses of radiation, we were able todeliver therapeutic doses of radiation using IGRT. Further-more, IGRT will enable us to test how the tumor genotypeimpacts responses to biologically relevant doses deliveredusing different fractionation schemes (15). Thus, IGRT willmore accurately target autochthonous HPV-positive tumorsand enable a better understanding for how the tumor genotypeimpacts the radiation response.

In conclusion, we have developed a novel model to monitorthe growth of HPV-positive oral tumors and their response toclinically relevant treatments. This model induced HPV onco-genes in the basal epithelial layer, mimicking the naturalhistory of this disease. Furthermore, the bioluminescent signal

from these oral tumors acted as a surrogate for tumor growthand response to therapy. Finally, we have validated this modelto monitor responses to small molecules and to IGRT, twomodalities that are currently being used in patients. Weanticipate this system to serve as a platform to better testpreclinical concepts in vivo as well as to address underlyingmechanisms for how the genotype of autochthonous tumorsimpacts responses to existing and novel therapies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. SpiottoDevelopment of methodology: R. Zhong, C. Pelizzari, M. SpiottoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Zhong, M. Pytynia, C. Pelizzari, M. SpiottoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. SpiottoWriting, review, and/or revision of the manuscript: R. Zhong, C. Pelizzari,M. SpiottoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Pytynia, M. SpiottoStudy supervision: M. Spiotto

AcknowledgmentsThe authors thank Dr. Linda Degenstein and the University of Chicago

Transgenic Core Facility for expert help in producing the iHPV transgenicmouse, the University of Chicago Cytometry Core Facility, Dr. Lara Leoniand the University of Chicago Optical Imaging Facility for expert adviceand assistance, and Terry Li and Dr. Mark Lingen of the Universityof Chicago Human Tissue Resource Center for expert assistance withimmunohistochemistry.

Grant SupportThis work was supported by the Burroughs Wellcome Career Award for

Medical Scientists and the Fanconi Anemia Research Fund (M. Spiotto). Acqui-sition of the image-guided animal irradiator was supported by NIH SharedInstrumentation Grant 1S10RR026747-01.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 18, 2013; revised January 14, 2014; accepted January 15, 2014;published OnlineFirst February 13, 2014.

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2014;74:2073-2081. Published OnlineFirst February 13, 2014.Cancer Res Rong Zhong, Matt Pytynia, Charles Pelizzari, et al. Response to Image-Guided RadiotherapyBioluminescent Imaging of HPV-Positive Oral Tumor Growth and Its

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