R E S EARCH ART I C L E

CANCER

Tumor cells, but not endothelial cells, mediateeradication of primary sarcomas by stereotacticbody radiation therapyEverett J. Moding,1 Katherine D. Castle,1 Bradford A. Perez,2 Patrick Oh,2 Hooney D. Min,2

Hannah Norris,2 Yan Ma,2 Diana M. Cardona,3 Chang-Lung Lee,2 David G. Kirsch1,2*

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Cancer clinics currently use high-dose stereotactic body radiation therapy as a curative treatment for severalkinds of cancers. However, the contribution of vascular endothelial cells to tumor response to radiation remainscontroversial. Using dual recombinase technology, we generated primary sarcomas in mice with targeted ge-netic mutations specifically in tumor cells or endothelial cells. We selectively mutated the proapoptotic geneBax or the DNA damage response gene Atm to genetically manipulate the radiosensitivity of endothelial cells inprimary soft tissue sarcomas. Bax deletion from endothelial cells did not affect radiation-induced cell death intumor endothelial cells or sarcoma response to radiation therapy. Although Atm deletion increased endothelialcell death after radiation therapy, deletion of Atm from endothelial cells failed to enhance sarcoma eradication.In contrast, deletion of Atm from tumor cells increased sarcoma eradication by radiation therapy. These resultsdemonstrate that tumor cells, rather than endothelial cells, are critical targets that regulate sarcoma eradica-tion by radiation therapy. Treatment with BEZ235, a small-molecule protein kinase inhibitor, radiosensitizedprimary sarcomas more than the heart. These results suggest that inhibiting ATM kinase during radiation ther-apy is a viable strategy for radiosensitization of some tumors.

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INTRODUCTION

About half of all cancer patients are treated with radiation therapy (1),which may be given with palliative intent in cases where a delay intumor regrowth (growth delay) can be clinically meaningful. However,the majority of cancers treated with radiation therapy are treated withthe intent to cure, where the goal of radiation therapy is to achievecomplete and permanent tumor regression (local control). When pa-tients with cancer are treated with radiation, they usually receive rela-tively small (1.8 to 2.0 Gy) daily fractions for 1 to 2 months. Recently,advances in radiation treatment planning and delivery have made itpossible to safely deliver a small number of high radiation doses (15 to24 Gy), termed stereotactic body radiation therapy (SBRT) or radio-surgery, to improve the local control of some tumors (2).

The tumor microenvironment of human cancers consists of bloodvessels, fibroblasts, and immune cells that modulate cancer develop-ment, progression, and response to therapy (3). However, whether ornot stromal cells, such as endothelial cells, are critical targets of radia-tion therapy remains controversial. Indeed, experiments using trans-planted tumors in mice with radiosensitive stroma have suggested thattumor stromal cells do not contribute to local control of cancer by ra-diation therapy (4).

Recently, endothelial cell apoptosis and microvascular collapse werereported to contribute to the radiation response of transplanted mel-anoma and fibrosarcoma cell lines (5). Endothelial cell apoptosis canoccur because of membrane damage, which triggers rapid ceramide-mediated apoptosis after high doses of radiation exposure (6, 7). As aresult, transplanted tumors with acid sphingomyelinase– or Bcl-2–

1Department of Pharmacology and Cancer Biology, Duke University Medical Center,Durham, NC 27710, USA. 2Department of Radiation Oncology, Duke University MedicalCenter, Durham, NC 27710, USA. 3Department of Pathology, Duke University MedicalCenter, Durham, NC 27710, USA.*Corresponding author. E-mail: david.kirsch@duke.edu

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associated X protein (Bax)–deficient stroma, which have defectiveradiation-induced endothelial cell apoptosis, grow 200 to 400% fasterthan transplanted tumors with wild-type stroma and display a de-creased growth delay after radiation doses of up to 20 Gy (5). Notably,endothelial cell apoptosis has been proposed to occur at a threshold of8 to 10 Gy and to increase up to 20 to 25 Gy (7), suggesting that en-dothelial apoptosis may be contributing to tumor cure by SBRT (8).However, the conclusion that microvascular damage regulates tumorresponse to radiation has been challenged (9, 10), and additionalexperiments using transplanted model systems have failed to resolvethe controversy (11–14).

Unlike transplanted tumor models, which may not fully recapitu-late the vasculature and immune surveillance of autochthonous tumors(15, 16), genetically engineered mouse models (GEMMs) develop tu-mors within the native microenvironment in immunocompetent mice(17) and may more faithfully recapitulate the tumor stroma and mi-croenvironment of human cancers (18). In addition, the response ofthese primary mouse cancer models to therapeutics might mimic theresponse of human cancers in clinical trials (19, 20).

To investigate the contribution of endothelial cells to the radiationresponse of primary sarcomas, we developed the technology to con-temporaneously mutate different genes specifically in the tumor cellsversus the endothelial cells of primary sarcomas (21). In this system,an adenovirus expressing FlpO (adeno-FlpO) activates a conditionalallele of oncogenic Kras (FSF- KrasG12D) and deletes both copies ofa conditional allele of p53 (p53FRT), whereas a tissue-specific Cre drivermutates floxed alleles specifically in endothelial cells. We recently usedthis dual recombinase technology to demonstrate that selectively sen-sitizing endothelial cells to mitotic cell death by deleting the DNA dam-age response gene ataxia telangiectasia mutated (Atm) (22) prolongssarcoma growth delay after SBRT (23). However, an increase in growthdelay does not necessarily translate into improved local control (4, 24, 25).

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Here, we irradiated sarcomas with Atm deleted in endothelial cellswith a curative dose of radiation. We also used dual recombinase tech-nology to selectively protect endothelial cells from apoptosis by delet-ing the proapoptotic gene Bax (26). We found that tumor endothelialcells in primary sarcomas did not die via apoptosis within four hoursof SBRT. In addition, endothelial cell death did not contribute to sar-coma eradication by radiation therapy. In contrast, radiosensitizing tu-mor cells by deleting Atm increased local control of primary sarcomasafter radiation therapy. These results demonstrate that tumor cells, butnot endothelial cells, are critical targets of curative radiation therapy inprimary sarcomas. Also, to test whether ATM inhibition can improvethe therapeutic ratio during SBRT, we compared the radiation responseof primary sarcomas and hearts after Atm inhibition with BEZ235 todemonstrate that targeting ATM during radiation therapy might be aviable approach for radiosensitization of tumors at certain anatomic sites.

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RESULTS

Bax and Bak do not regulate endothelial cell death afterSBRT in primary sarcomasBecause apoptosis of tumor endothelial cells is dependent on Bax intransplanted tumor models (5), we used (i) dual recombinase technol-ogy to initiate primary sarcomas in conditional FSF-KrasG12D; p53FRT/FRT

(KPFRT) mice with adeno-FlpO (21) and (ii) VE-Cadherin-Cre (27) todelete Bax floxed alleles (BaxFL) (28) specifically in endothelial cells.We recently demonstrated that this approach efficiently deletes floxedalleles in endothelial cells of primary sarcomas (23). We confirmed byquantitative polymerase chain reaction (qPCR) that VE-Cadherin-Creefficiently deleted Bax in endothelial cells (fig. S1A).

We next investigated the effect of Bax deletion specifically in en-dothelial cells on sarcoma initiation and growth by generating primarysarcomas in KPFRT; VE-Cadherin-Cre; BaxFL/+ (KPFRTVBaxFL/+) andKPFRT; VE-Cadherin-Cre; BaxFL/FL (KPFRTVBaxFL/FL) mice. In contrastto previous reports with tumor cells transplanted into Bax-null mice(5), we observed no change in primary sarcoma initiation or growth inmice with deletion of Bax specifically in endothelial cells (fig. S1, B toD). To determine whether Bax deletion from endothelial cells pro-tected tumor endothelial cells from radiation in an autochthonous modelsystem, we irradiated sarcomas in KPFRTVBaxFL/+ and KPFRTVBaxFL/FL

mice with a single dose of 20 Gy using fluoroscopy-guided radia-tion therapy and examined endothelial cell death through terminaldeoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining at various time points after irradiation.In transplanted tumor models, tumor endothelial cell apoptosis peaks4 to 6 hours after radiation exposure (5). Consistent with our previousresults (23), we did not observe a significant change in apoptotic en-dothelial cell death 4 hours after irradiation of primary sarcomas, butendothelial cell death did increase 48 hours after radiation, likely as aresult of mitotic catastrophe (Fig. 1, A and B). Furthermore, deletionof Bax did not affect endothelial cell death, suggesting that endothelialcells in primary sarcomas do not undergo Bax-mediated apoptosisafter irradiation.

We next evaluated whether Bax in endothelial cells contributed tothe growth delay of primary sarcomas. To this end, we irradiatedsarcomas in KPFRTVBaxFL/+ and KPFRTVBaxFL/FL mice with a singledose of 20 Gy and monitored tumor growth until the sarcomas tripledin size. In contrast to transplanted tumors in Bax-null mice (5),

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primary sarcomas in KPFRTVBaxFL/FL mice displayed a growth delayafter irradiation similar to sarcomas in KPFRTVBaxFL/+mice (Fig. 1, Cand D). To investigate whether there was a difference in local controlin this model, we irradiated primary sarcomas in KPFRTVBaxFL/+ andKPFRTVBaxFL/FL mice with 50 Gy, which we determined to be the max-imally tolerated dose in our system. Although 50 Gy was able to erad-icate a small percentage of primary sarcomas, there was no differencein local control or growth delay between the two genotypes (Fig. 1Eand fig. S2).

Because the proapoptotic genes Bax and Bcl-2 homologous antagonist/killer (Bak) can be redundant for executing programmed cell death insome settings (29), we also deleted the Bax gene from endothelial cellsof Bak-null (Bak−/−) mice (30). Primary sarcomas in KPFRT; VE-Cadherin-Cre; Bak−/−; BaxFL/+ (KPFRTVBak−/−BaxFL/+) and KPFRT;VE- Cadherin-Cre; Bak−/−; BaxFL/FL (KPFRTVBak−/−BaxFL/FL) mice de-veloped at the same time after injection of adeno-FlpO and grew at thesame rate in the absence of radiation (fig. S3). After irradiation with 20 Gy,we observed no difference in endothelial cell death or growth delay forprimary sarcomas in KPFRTVBak−/−BaxFL/+ and KPFRTVBak−/−BaxFL/FL

mice (fig. S4), suggesting that endothelial cell apoptosis does not con-tribute to the radiation response of primary sarcomas. Because previ-ous studies have demonstrated differences between the vasculature oftransplanted and primary cancers (15, 31, 32), our discordant resultson the contribution of endothelial cell death might reflect differencesin the vasculatures of tumors derived from transplanted cell lines ver-sus the autochthonous tumors studied herein. Together, these findingsillustrate the importance of studying the tumor microenvironmentusing multiple complementary models, including GEMMs.

Endothelial cell death does not contribute to eradication ofprimary sarcomas by SBRTWe showed recently that deleting Atm specifically in endothelial cellsradiosensitizes these cells in autochthonous sarcomas, causing them toundergo a delayed mitotic cell death that increases tumor growthdelay after radiation therapy with 20 Gy (23). To determine whetherAtm deletion increased endothelial cell death after a curative dose ofradiation, we treated primary sarcomas in KPFRT; VE-Cadherin-Cre;AtmFL/+ (KPFRTVAtmFL/+) and KPFRT; VE-Cadherin-Cre; AtmFL/FL

(KPFRTVAtmFL/FL) mice with 50 Gy. Consistent with our previousresults, deletion of Atm significantly increased endothelial cell death24 hours after irradiation (Fig. 2, A and B).

To investigate whether endothelial cell death contributes to the lo-cal control of primary sarcomas, we irradiated sarcomas in KPFRTVAtmFL/+

and KPFRTVAtmFL/FL mice with 50 Gy. Although 50 Gy was able toeradicate primary sarcomas in both KPFRTVAtmFL/+ and KPFRTVAtmFL/FL

mice, there was no difference in local control between the two geno-types (Fig. 2C). Moreover, at the curative dose of 50 Gy, there was nodifference in growth delay for primary sarcomas in KPFRTVAtmFL/+

and KPFRTVAtmFL/FL mice (fig. S5, A and B). Because sarcomas inKPFRTVAtmFL/FLmice have a prolonged growth delay after 20 Gy com-pared to sarcomas in KPFRTVAtmFL/+ mice (23), we repeated this ex-periment with four daily fractions of 20 Gy. Similar to a single dose of50 Gy, we observed no difference in local control or growth delay forsarcomas in KPFRTVAtmFL/+ and KPFRTVAtmFL/FL mice after 80 Gy ofradiation was delivered in four 20 Gy fractions (Fig. 2D and fig. S5, Cand D). These results demonstrated that although endothelial celldeath can delay the regrowth of primary sarcomas at noncurative dosesof radiation, endothelial cell death is not rate-limiting for sarcoma

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regrowth after curative doses of radiation and does not contribute tolocal control of primary soft tissue sarcomas.

Tumor cells in primary sarcomas are critical targets thatmediate local control by SBRTFor comparison, we also deleted Atm in tumor cells. First, we injectedthe muscle of LSL-KrasG12D; p53FL/FL; AtmFL/+ (KPloxPAtmFL/+) andLSL-KrasG12D; p53FL/FL; AtmFL/FL (KPloxPAtmFL/FL) mice with adeno-Cre. However, the sarcomas that developed did not reliably deleteboth alleles of Atm (fig. S6A). Therefore, we crossed KPloxPAtmFL/+

and KPloxPAtmFL/FL mice to Pax7-CreER (P7) mice, which express atamoxifen-inducible Cre recombinase in muscle satellite cells (33).P7KPloxPmice develop sarcomas after administration of systemic tamox-ifen (34). In contrast to KPloxPAtmFL/FL mice injected with adeno-Cre,

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intramuscular injection of P7KPloxPAtmFL/FL mice with 4-hydroxytamoxifengenerated primary sarcomas at the site of injection with efficient de-letion of both Atm alleles (fig. S6B).

To determine whether Atm deletion sensitized sarcoma cells to ra-diation, we irradiated cell lines isolated from sarcomas in P7KPloxPAtmFL/+

and P7KPloxPAtmFL/FL mice. Sarcoma cells from P7KPloxPAtmFL/FL

mice were more sensitive to radiation in clonogenic survival assayscompared to sarcoma cells from P7KPloxPAtmFL/+ control mice that re-tained one allele of Atm (Fig. 3A). Next, we irradiated primary sarcomasin P7KPloxPAtmFL/+ and P7KPloxPAtmFL/FL mice with 50 Gy andmonitored the sarcomas for local control. Deletion of Atm in tumorcells significantly improved local control of primary sarcomas (Fig. 3,B and C), suggesting that direct tumor cell death can mediate tumoreradication by SBRT. Before local recurrence, several mice developed

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by Bonferroni’s post hoc tests for pairwise comparisons between geno-types showed that the differences were not statistically significant. (C and D)

munofluorescence for CD31 and TUNEL in sarcomas from KPFRTVBaxFL/+

and KPFRTVBaxFL/FL mice 4 and 48 hours after irradiation with 20 Gy. Areasenclosed by dashed lines are shown at higher magnification in the insets.Scale bar, 100 mm. (B) Quantification of CD31 and TUNEL double-positivecells in sarcomas from KPFRTVBaxFL/+ and KPFRTVBaxFL/FL mice at the indicatedtime points after irradiation with 20 Gy (n = 4 mice per group). Two-wayanalysis of variance (ANOVA) for genotype and time interaction followed

Tumor growth curves (C) and time to volume tripling for sarcomas (D) inKPFRTVBaxFL/+ and KPFRTVBaxFL/FL mice after irradiation with 20 Gy (n = 8 miceper group). Two-tailed Student’s t test showed that the differences were notstatistically significant. (E) Kaplan-Meier plot of local sarcoma control definedas the absence of tumor volume tripling for sarcomas in KPFRTVBaxFL/+ andKPFRTVBaxFL/FLmice after irradiation with 50 Gy (n = 18 and 20 mice per group).Log-rank test showed that the differences were not statistically significant.

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second sarcomas at other locations in the body, presumably from sys-temic tamoxifen–mediated Cre recombination. When we analyzed thehistology from the site of irradiated primary sarcomas in nineKPloxPAtmFL/FL

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mice that developed second-site sarcomas, there were no tumor cellspresent in five mice and a few scattered histologically intact tumorcells in three mice (fig. S7).

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mice 24 hours after irradia-tion with 50 Gy. Areas en-closed by dashed lines areshown at higher magnifica-tion in the insets. Scale bar,100 mm. (B) Quantificationof CD31 and TUNEL double-

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positive cells in sarcomas from KPFRTVAtmFL/+ and KPFRTVAtmFL/FL mice 24 hours after irradiation with 50 Gy (n = 6 mice per group). Two-tailed Student’st test, P < 0.05. (C and D) Kaplan-Meier plot of local sarcoma control (defined as the absence of tumor volume tripling) for sarcomas in KPFRTVAtmFL/+

and KPFRTVAtmFL/FL mice after irradiation with 50 Gy (n = 22 and 23 mice per group, respectively) (C) or four daily fractions of 20 Gy (n = 23 and 25 miceper group, respectively) (D). One KPFRTVAtmFL/FL mouse developed an abdominal metastasis 8 weeks after irradiation with 50 Gy, and two KPFRTVAtmFL/+

mice died of unknown causes at 7 and 10 weeks after irradiation with four daily fractions of 20 Gy before sarcoma tripling. Thus, these mice werescored as locally controlled until the time points at which they either developed metastasis or died. Log-rank tests showed that the differences werenot statistically significant. Asterisks represent statistically significant difference between the indicated groups.

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sarcomas generated by intramuscular 4-hydroxytamoxifen injection intoP7KPloxPAtmFL/+ and P7KPloxPAtmFL/FL mice after irradiation with 50 Gy (n =

primary sarcoma cell lines from P7KPloxPAtmFL/+ and P7KPloxPAtmFL/FL mice(n = 3 independent cells lines per genotype). Two-way ANOVA for geno-type and dose interaction (P < 0.05) followed by Bonferroni’s post hoc testsfor pairwise comparisons between genotypes after 2 and 4 Gy (P < 0.05).(B and C) Sarcoma growth curves (B) and Kaplan-Meier plot of local sarcomacontrol (C) defined as the absence of tumor volume tripling for primary

13 mice per group). Several mice were euthanized before sarcoma triplingbecause of the development of second sarcomas at other locations in thebody, presumably from systemic tamoxifen–mediated Cre recombination.These mice were scored as locally controlled until the time point of secondtumor formation. Log-rank test, P < 0.05. Asterisks represent statistically sig-nificant difference between the indicated groups.

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The protein kinase inhibitor BEZ235 preferentiallyradiosensitizes primary sarcomasDeletion of the Atm gene from tumor cells increased the probability oftumor eradication after SBRT, suggesting that therapeutic targeting ofthe ATM protein could improve tumor response to radiation therapy.However, systemic targeting of ATM in normal tissues might also in-crease radiation toxicity. To begin to address whether targeting ATMduring radiation therapy can improve the response of tumors relativeto some normal tissues (that is, to enhance the therapeutic ratio), weshowed recently that deleting Atm preferentially radiosensitizes pro-liferating tumor endothelial cells compared with quiescent heart en-dothelial cells (23). Here, we used a pharmacological approach toinvestigate whether a therapeutic window exists for inhibiting ATMduring radiation therapy. To this end, we treated KPloxP mice thathad primary sarcomas with the phosphoinositide 3-kinase (PI3K)–likekinase (PI3KK) inhibitor BEZ235, which potently inhibits humanATM (35). Pretreatment of the mice with BEZ235 significantly de-creased autophosphorylation of mouse Atm and phosphorylation ofthe Atm target Kap1 (Krüppel-associated box–associated protein 1)in primary sarcomas after irradiation with 20 Gy (fig. S8, A to D).

To investigate whether BEZ235 can selectively radiosensitize sarco-mas, we collected hearts and primary sarcomas from vehicle- andBEZ235-treated KPloxP mice 24 hours after whole-body irradiationwith 20 Gy. Treatment with BEZ235 significantly increased cell deathin sarcomas but not in hearts (Fig. 4, A and B). Next, we monitored tumorgrowth in KPloxP mice treated with BEZ235 alone or in combinationwith focal 20 Gy irradiation. Although a single dose of BEZ235 alonedid not delay tumor growth (fig. S8, E and F), BEZ235 treatment

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significantly delayed primary sarcoma regrowth after 20 Gy (Fig. 4, Cand D).

We next investigated the effect of BEZ235 treatment on the devel-opment of radiation-induced heart disease. We showed previouslythat deletion of Atm radiosensitizes p53-null cardiac endothelial cellsbut not cardiac endothelial cells with intact p53 (23). Therefore, wetreated VE-Cadherin-Cre; p53FL/+(VPFL/+) mice with one allele of p53deleted in endothelial cells and VE-Cadherin-Cre; p53FL/FL (VPFL/FL)mice with two alleles of p53 deleted in endothelial cells with eithervehicle or BEZ235 2 hours before whole-heart irradiation with 12 Gyand monitored the mice for the development of radiation-induced heartdisease. BEZ235 shifted the myocardial necrosis–free survival curve to theleft in VPFL/FLmice but did not promote the development of radiation-induced myocardial necrosis in VPFL/+ mice (Fig. 4E). Because we usedBEZ235 with a single dose of radiation, we cannot exclude the possi-bility that ATM inhibition has some effect on radiation-induced heartdisease with other radiation doses. However, taken together with thegenetic studies, these experiments with a pharmacological inhibitorof ATM suggest that inhibiting ATM may preferentially radiosensitizesarcomas compared with quiescent normal tissues, such as the heart.

DISCUSSION

Here, we used a new dual recombinase technology to manipulate theradiosensitivity of endothelial cells in primary sarcomas. In contrast toresults with transplanted tumor models (5), we found that tumor en-dothelial cells in primary sarcomas did not die via apoptosis within

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Fig. 4. The PI3KK inhibitor BEZ235 preferentially radiosensitizes primary sarcomascompared to mouse heart tissue. (A and B) Immunofluorescence (A) and quantificationof TUNEL-positive cells (B) in hearts and sarcomas from vehicle- or BEZ235-treated KPloxP

mice 24 hours after irradiation with 20 Gy (n = 5 mice per group). Two-way ANOVA fortissue and treatment interaction followed by Bonferroni’s post hoc tests for pairwise com-parison between treatments showed that the differences were statistically significant insarcomas but not in hearts (P < 0.05). Scale bar, 100 mm. (C and D) Tumor growth curves(C) and time to volume tripling (D) of primary sarcomas in KPloxP mice treated on day 0 withvehicle or a single dose of BEZ235 (50 mg/kg) 2 hours before irradiation with 20 Gy (n = 8mice per group). Two-tailed Student’s t test, P < 0.05. (E) Kaplan-Meier plots of myocardialnecrosis–free survival for mice that lacked p53 in endothelial cells (VPFL/FL) or mice that re-tained p53 in endothelial cells (VPFL/+), both of which were treated with vehicle or BEZ235(50 mg/kg) 2 hours before whole-heart irradiation with 12 Gy (n = 7 to 9 mice per group).Two VPFL/FLmice treated with vehicle were censored because they developed tumors beforedeveloping myocardial necrosis. Log-rank test for VPFL/+ Vehicle versus VPFL/FL Vehicle, VPFL/+

Vehicle versus VPFL/FL BEZ235, VPFL/+ BEZ235 versus VPFL/FL Vehicle, and VPFL/+ BEZ235 ver-sus VPFL/FL BEZ235 (P < 0.05). Asterisks represent statistically significant difference be-tween the indicated groups.

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4 hours after irradiation. Although endothelial cells can contributeto primary sarcoma growth delay after noncurative radiation therapy(23), radiosensitizing endothelial cells did not increase local control. Incontrast, radiosensitization of tumor cells increased the probability ofsarcoma eradication by SBRT. Therefore, tumor cells, but not endothe-lial cells, are critical targets of SBRT that mediate sarcoma eradication.

These results have important clinical implications, suggesting thatincreased endothelial cell death does not contribute to the improvedlocal control of some tumors after SBRT. Instead, the increased effi-cacy of SBRT is likely the result of delivering larger, biologically effec-tive doses than are delivered with standard radiation therapy (36). Ourresults are consistent with the previous observation that local controlis not affected when tumors are transplanted into radiosensitive mice(4) and extend these findings to primary sarcomas. Despite the extensiveendothelial cell death observed after a dose of 50 Gy (Fig. 2, A and B),most of the sarcomas in KPFRTVAtmFL/FLmice recurred (Fig. 2C). Theseresults raise the possibility that the vasculature in recurrent sarcomasmay have come from outside of the irradiation field (37). Although thesource of these newly formed blood vessels remains an area of ongoingdebate (38), targeting vascular recruitment could improve local control.

Our results do not exclude a contribution from other stromal cells to tu-mor eradicationwith SBRT. Indeed, recent studies suggest that the recruit-ment of macrophages from outside the radiation field through a stromalcell–derived factor 1 (SDF-1)–C-X-C chemokine receptor 4 (CXCR4) axismight participate in tumor cell repopulation after radiotherapy (37). In ad-dition, a growing body of data suggests that the immune system can con-tribute to the killing of tumor cells after radiation therapy (39, 40), and thisresponse may be greater after SBRT than after standard radiation therapy.Furthermore, our experiments focused on soft tissue sarcomas, and addi-tional experiments are needed to determine whether endothelial celldeath contributes to the radiation response of other tumor types. In thefuture, dual recombinase technology can be applied to other primary tu-mormodel systems and stromal cell populations to further explore howthe tumormicroenvironment contributes to curative radiation therapy.

Although most patients are treated with curative intent, for patientsreceiving palliative radiation therapy, an increased growth delay can beclinically meaningful. For example, radiation therapy is the standard ofcare for children with diffuse intrinsic brainstem gliomas and providesmany months of relief from severe neurological symptoms (41). Target-ing tumor endothelial cells during radiation therapy could prolong thetime that these patients remainneurologically intact.However, our resultssuggest that this approach may not improve tumor eradication. Instead,targeting tumor cells with radiosensitizers, such as inhibitors of ATM,might be a more promising treatment approach to achieve local control.

MATERIALS AND METHODS

Study designThe goal of this controlled laboratory experiment was to determinethe relative contribution of endothelial cells and tumor cells to pri-mary sarcoma response to radiation therapy. We used genetically en-gineered mice and observed histological, growth delay, and localcontrol endpoints. Sample sizes were selected before initiating thestudy on the basis of prior primary sarcoma radiation response datafrom the laboratory and power calculations performed as describedpreviously (42); data collection was stopped if a smaller sample sizeachieved statistical significance. Outliers were defined before initiating

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the study as falling greater than 2 SD from the mean. No outliers wereexcluded from this study. Sarcoma eradication was assumed if a sar-coma failed to triple in size 18 weeks after radiation treatment. Assum-ing a constant sarcoma doubling time of 4 days and tumor cell diameterof 25 mm (volume = 8.18 × 10−6 mm3), it would take a single tumor cell100 days (~14 weeks) to reach 300 mm3. Mice that died before sarcomatripling as a result of metastasis or sarcoma development at a distant sitewere censored at the time of death and were scored as locally controlleduntil this point. The mice in this study were not randomized to theirtreatments and were selected based on availability. The investigatorswere not blinded when performing sarcomameasurements. Histologicalquantification was performed by an observer blinded to treatment andgenotype. Histological assessment of residual sarcoma cells in the legs ofirradiated mice was assessed by a sarcoma pathologist (D.M.C.) whowas blinded to treatment and genotype.

Mouse strains and sarcoma inductionAll animal studies were performed in accordance with protocols ap-proved by the Duke University Institutional Animal Care and Use Com-mittee. All of the mouse strains used in this study have been describedpreviously, including LSL-KrasG12D, p53FL, FSF-KrasG12D, p53FRT,VE-Cadherin-Cre, AtmFL, BaxFL, Bak−/−, and Pax7-CreER (21, 27, 28, 30, 33, 43–46).LSL-KrasG12D and FSF-KrasG12D mice were provided by T. Jacks,AtmFL mice were provided by S. Zha and F. Alt, BaxFL and Bak−/−

mice were provided by S. Korsmeyer, p53FL mice were provided byA. Berns, and Pax7-CreER mice were provided by C.-M. Fan. VE-Cadherin-Cre mice were obtained from The Jackson Laboratory.Primary sarcomas were generated in the right hind leg of KPloxP

or KPFRT mice between 6 and 10 weeks of age as described previous-ly (21, 47). Tamoxifen-induced primary sarcomas were generated byinjecting 50 ml of 4-hydroxytamoxifen (5 mg/ml; Sigma-Aldrich) indimethyl sulfoxide into the right hind leg of P7KPloxP mice (48).All mice were on a mixed genetic background. To minimize the effectof genetic background, age-matched littermate controls were usedfor every experiment so that potential genetic modifiers would berandomly distributed between the experimental and control groups.

Radiation treatmentSarcoma and whole-heart irradiations were performed using the X-RAD225Cx small animal image-guided irradiator (Precision X-Ray).The irradiation field was centered on the target via fluoroscopy with40 kilovolt peak (kVp), 2.5 mA x-rays using a 2-mm aluminum filter.Sarcomas were irradiated with parallel-opposed anterior and posteriorfields with an average dose rate of 300 cGy/min prescribed to mid-plane with 225 kVp, 13 mA x-rays using a 0.3-mm copper filter anda collimator with a 40 × 40 mm2 radiation field at treatment isocenter.

Whole-heart irradiation was performed using a collimator to pro-duce a 15-mm circular radiation field at treatment isocenter. Doserates were measured with an ion chamber by the Radiation Safety Di-vision at Duke University. Sarcomas were irradiated at ~250 mm3 bycaliper measurement for growth delay and histological endpoints and~100 mm3 for local control endpoints. After sarcoma irradiation,sarcomas were measured three times per week for growth delay andonce per week for local control experiments until they tripled in sizefrom the initial volume measured at the time of radiation. Afterwhole-heart irradiation, mice were monitored daily for symptoms ofheart disease. Myocardial necrosis was confirmed histologically byhematoxylin and eosin (H&E) staining of heart sections.

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Histological analysisH&E was performed on paraffin-embedded tissue sections. Tissuespecimens were fixed in 10% neutralized formalin overnight and pre-served in 70% ethanol until paraffin embedding. Five micron sectionswere deparaffinized with xylene and rehydrated with a graded series ofethanol and water washes before performing H&E staining. Sarcomaand heart immunohistochemistry was performed on frozen tissue sec-tions. Specimens were embedded directly in optimal cutting tempera-ture compound (Sakura Fintek) by snap freezing in a dry ice/isopentaneslurry and stored at −80°C until sectioning. Ten-micrometer sectionswere fixed in 4% paraformaldehyde before immunofluorescence staining.The primary antibodies were rat anti-mouse CD31 (1:250; BD Pharmingen,#553370), rabbit anti-mouse Ser824-phosporylated KAP1 (1:250; BethylLaboratories, #A300-767A), and polyclonal rabbit anti-mouse Ser1987-phosphorylated ATM [diluted 1:500; provided by M. Kastan (49)].The secondary antibodies were Alexa Fluor 488–conjugated donkeyanti-rat immunoglobulin G (1:500; Invitrogen, #A21208) and Alexa Fluor555–conjugated goat anti-rabbit immunoglobulin G (1:250; Invitrogen,#A21429). Nuclear staining was performed using Hoechst 33342 (10 mM;Sigma-Aldrich). TUNEL staining was performed with the In Situ CellDeath Detection Kit, TMR red (Roche) according to the manufacturer’sinstructions. Pictures were acquired with a Leica DFC340 FX fluo-rescence microscope (Leica Microsystems) using Leica Suite software(Leica Microsystems). Quantification was performed using ImageJ[National Institutes of Health (NIH)]. Each data point represents theaverage of eight randomly selected 200× fields per sample.

Flow sorting of endothelial cellsLungs were dissected, washed in phosphate-buffered saline, homogen-ized, and digested in type I collagenase (0.8 mg/ml; WorthingtonBiochemical Corp.) for 1 hour at 37°C. Digested tissues were filtered,and red blood cells were lysed with ACK lysing buffer (Lonza Group).Total number of cells was counted by Coulter counter (BeckmanCoulter Inc.). Three million cells were stained with phycoerythrin(PE)–conjugated anti-mouse CD31 (BioLegend, #102407), PE-Cy5–conjugated anti-mouse CD45 (eBioscience, #15-0451), and eFluor660–conjugated anti-mouse CD34 (eBioscience, #50-0341) antibodies.Dead cells were excluded by staining with 7-aminoactinomycin D (BDPharmingen). Viable CD45-negative and CD31 and CD34 double-positive cells were sorted by FACSVantage (BD Pharmingen) andused for RNA isolation.

Quantitative reverse transcription PCR analysisTotal RNA was extracted from sorted lung and tumor endothelial cellswith the RNAqueous-Micro Kit (Ambion), and reverse transcriptionwas performed with the iScript complementary DNA (cDNA) Synthe-sis Kit (Bio-Rad). Quantitative reverse transcription PCR was per-formed using TaqMan Universal PCR Master Mix (Applied Biosystems)and TaqMan Gene Expression Assay Mix (Applied Biosystems) for Bax(Mm00432050_m1) or Hprt (Mm0446968_m1). Hprt was used as aninternal control to correct for the concentration of cDNA in differentsamples. Each experiment was performed with three replicates fromeach sample, and the results were averaged.

Cell line experiments and clonogenic survival assaysCell lines were isolated by digestion of primary sarcomas with trypsin,type IV collagenase, and dispase (Invitrogen). Cells were cultured inDulbecco’s modified Eagle’s medium with high glucose and pyruvate

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(Gibco) supplemented with 10% fetal bovine serum. Cells were pas-saged five times to deplete stromal cells before isolation of genomicDNA for PCR or clonogenic survival assays. Deletion of Atm was verifiedby PCR using primers flanking the 3′ loxP site (sense, 5′- GGGCTAC-GAAATGAGACACACAC-3′; antisense 5′-CTTCCCCTGTTCAA-AAGCCACTC-3′) and primers flanking the recombined loxP site (sense,5′-TGAGTTCAAATCCCAGGAGCCAG-3′; antisense, 5′- CTTCCCCT-GTTCAAAAGCCACTC-3′). For clonogenic survival assays, cells wereplated in triplicate and allowed to adhere overnight before irradiationwith an X-RAD 320 Biological Irradiator (Precision X-Ray). Cells wereplaced 50 cm from the radiation source and were irradiated with a doserate of 161 cGy/min using 320 kVp, 10 mA x-rays and a 2-mm alu-minum filter. After development of colonies, cells were fixed with 70%ethanol, stained with Coomassie Brilliant Blue (Bio-Rad), rinsed withdeionized water, and dried. A population of more than 50 cells wascounted as one colony, and surviving fractions were calculated relativeto unirradiated controls.

BEZ235 treatmentMice were treated by oral gavage with a single dose of BEZ235 (50 mg/kg;Novartis) dissolved in 10% N-methyl-2-pyrrolidone/90% polyethyleneglycol 300 (Sigma-Aldrich) 2 hours before radiation therapy.

StatisticsResults are presented as means ± SEM. Two-tailed Student’s t test wasperformed to compare the means of two groups. Two-way ANOVAwas performed to examine the interaction between genotypes andtreatments followed by Bonferroni’s post hoc tests for pairwise com-parisons of individual treatments or genotypes. Non-normally distrib-uted data were log-transformed before applying statistical tests. Forsurvival studies, Kaplan-Meier analysis was performed followed bylog-rank test for statistical significance. Significance was assumed atP < 0.05. All calculations were performed using Prism 5 (GraphPad).

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/278/278ra34/DC1Fig. S1. Deletion of Bax in primary tumor endothelial cells does not affect sarcoma initiation orgrowth.Fig. S2. Deletion of Bax in endothelial cells does not affect sarcoma growth delay after a curativedose of irradiation.Fig. S3. Loss of Bak and Bax in endothelial cells does not affect primary soft tissue sarcomainitiation or growth.Fig. S4. Deletion of Bak and Bax in endothelial cells does not affect primary sarcoma responseto radiation therapy.Fig. S5. Deletion of Atm in endothelial cells does not affect sarcoma growth delay after curativedoses of irradiation.Fig. S6. Pax7-CreER, but not adeno-Cre, efficiently recombines both AtmFL alleles in primarysarcomas in vivo.Fig. S7. Spectrum of histological responses for primary sarcomas in KPloxPAtmFL/FL mice afterirradiation with 50 Gy.Fig. S8. BEZ235 inhibits Atm in primary sarcomas.

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Acknowledgments: We thank S. Zha and F. Alt for providing the AtmFL mice, T. Jacks forproviding the LSL-KrasG12D and FSF-KrasG12D mice, A. Berns for providing the p53FL mice,S. Korsmeyer for providing the BaxFL and Bak−/− mice, and C.-M. Fan for providing thePax7-CreER mice. We also thank Novartis for providing BEZ235 to complete this work, M. Kastanfor providing the antibody against pATM, L. Woodlief and L. Luo for their help in caring forthe mice, and C.-Y. Li and J. Chute for critical reading of the manuscript. Funding: This workwas supported by the National Cancer Institute of the U.S. NIH under award numbers F30CA177220 (E.J.M.) and R21 CA175839 (D.G.K.) and by Susan G. Komen under award numberIIR13263571 (D.G.K.). Author contributions: E.J.M., C.-L.L., and D.G.K. conceived and designedthe experiments. E.J.M, K.D.C., B.A.P., P.O., and C.-L.L. performed the experiments. E.J.M. and D.G.K.analyzed the data. H.D.M. and H.N. irradiated the mice and measured the sarcomas. Y.M. per-

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formed the histology. D.M.C. reviewed the histopathology. E.J.M. and D.G.K. wrote the manu-script. Competing interests: The authors declare that they have no competing interests.

Submitted 3 December 2014Accepted 20 February 2015Published 11 March 201510.1126/scitranslmed.aaa4214

Citation: E. J. Moding, K. D. Castle, B. A. Perez, P. Oh, H. D. Min, H. Norris, Y. Ma, D. M. Cardona,C.-L. Lee, D. G. Kirsch, Tumor cells, but not endothelial cells, mediate eradication of primarysarcomas by stereotactic body radiation therapy. Sci. Transl. Med. 7, 278ra34 (2015).

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stereotactic body radiation therapyTumor cells, but not endothelial cells, mediate eradication of primary sarcomas by

M. Cardona, Chang-Lung Lee and David G. KirschEverett J. Moding, Katherine D. Castle, Bradford A. Perez, Patrick Oh, Hooney D. Min, Hannah Norris, Yan Ma, Diana

DOI: 10.1126/scitranslmed.aaa4214, 278ra34278ra34.7Sci Transl Med

inhibition of a DNA-damage response enzyme can enhance radiosensitization of some tumors.endothelial, cells that mediate primary sarcoma shrinkage by radiation therapy and that selective small-moleculegenetic mutations that modulate radiation sensitivity. The authors found that it was the tumor, rather than

cells,earlier findings in studies conducted with primary sarcomas in mice that carried, in either tumor or endothelial challenge theet al.which comprises blood vessels and various cell types that influence tumor growth. Now Moding

suggest that radiation targets, not only the tumor cells themselves, but also components of the surrounding milieu, hasthrough the safe delivery of high doses of radiation. Previous research with transplanted tumor models in mice

sometimes achieving complete tumor regression−−of the treatment regimens of nearly half of all cancer patients Personalized cancer therapies dominate the news. But radiation therapy continues to be an essential part

Not all cells are eradicated equally

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