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Int. J. Radiation Oncology Biol. Phys., Vol. 64, No. 1, pp. 38–46, 2006Copyright © 2006 Elsevier Inc.

Printed in the USA. All rights reserved0360-3016/06/$–see front matter

doi:10.1016/j.ijrobp.2005.02.008

TOG 2004

MOLECULAR STRATEGIES TARGETING THE HOST COMPONENT OFCANCER TO ENHANCE TUMOR RESPONSE TO RADIATION THERAPY

DONG WOOK KIM, M.D., PH.D., JESSICA HUAMANI, M.S., ALLIE FU, B.S., AND

DENNIS E. HALLAHAN, M.D.

Department of Radiation Oncology, Vanderbilt Ingram Cancer Center, Nashville, TN

The tumor microenvironment, in particular, the tumor vasculature, as an important target for the cytotoxiceffects of radiation therapy is an established paradigm for cancer therapy. We review the evidence that thephosphoinositide 3-kinase (PI3K)/Akt pathway is activated in endothelial cells exposed to ionizing radiation (IR)and is a molecular target for the development of novel radiation sensitizing agents. On the basis of this premise,several promising preclinical studies that targeted the inhibition of the PI3K/Akt activation as a potential methodof sensitizing the tumor vasculature to the cytotoxic effects of IR have been conducted. An innovative strategy toguide cytotoxic therapy in tumors treated with radiation and PI3K/Akt inhibitors is presented. The evidencesupports a need for further investigation of combined-modality therapy that involves radiation therapy andinhibitors of PI3K/Akt pathway as a promising strategy for improving the treatment of patients with cancer.© 2006 Elsevier Inc.

Akt, Vasculature targeted therapy, Endothelium, Radiation sensitizer, Combined modality therapy.

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INTRODUCTION

raditionally, ionizing radiation (IR) has been accepted as aherapeutic modality that directly targetes the deregulatedancer cells. Therefore, much attention to date has focusedn agents that can lower radiation dose–response thresholdradiosensitizers), such as cisplatin or taxol (1). More re-ently, a new paradigm has been introduced that suggestshat tumor host component (e.g., the tumor microvascula-ure) may also be a target for the cytotoxic effects ofonizing radiation (2–4). In particular, the tumor endothe-ium has received significant attention as a potential targetor radiation sensitization. In fact, several preclinical studiesave indicated the efficacy of combination of antiangio-enic agents and radiation therapy (RT) (5, 6). We and othernvestigators (7, 8) have focused on exploitation of novel

echanism that inhibit the phosphoinositide 3-kinasePI3K)/Akt pathway as molecular targets for radiation sen-itization of the host component of cancer. Endothelial cells,lthough sensitive to IR at higher doses (3, 4), are lessensitive to RT doses in the range of 2 to 3 Gy, in partecause of activation of the PI3K/Akt cell viability signal-ng (9). Targeting this pathway in the tumor endothelium isn effective method of improving tumor response to frac-ionated RT in preclinical studies and is worthy of furtherlinical investigation (9–13). We will briefly introduce annnovative potential strategy for guiding delivery of cyto-

Reprint requests to: Dennis E. Hallahan, M.D., Vanderbilt Univer-ity Medical Center, Department of Radiation Oncology, B-902, 1301

2nd Avenue South, Nashville, TN 37232-5671. Tel: (615) 343-9244; f

38

oxic agents to tumors that have received a combination ofeceptor tyrosine kinase (RTK) inhibitor and RT.

PI3K/Akt PATHWAY

argeting radiation inducible cell viability pathwayThe PI3K/Akt pathway is known to be activated by

rowth-factor signaling through receptor tyrosine kinases,hich can lead to activation of prosurvival pathways (14–7). PI3K is composed of heterodimer of a p85 (relativeolecular mass [Mr] (daltons) 85,000) adapter subunit andp110 (Mr 100,000) catalytic subunit (18–21). PI3K can bectivated by a variety of RTKs (22–25). Furthermore, Akt,lso referred to as protein kinase B, is activated when it ishosphorylated by phosphatidylinositide-dependent kinasesand 2 (PDK1 and PDK2) (26, 27), which are downstream

f PI3K. Activated Akt has multiple downstream targets ands involved in regulation of apoptosis, glycolysis, and cellurvival (Fig. 1) (28).

I3K/Akt activated by IR in endothelial cellsPI3K/Akt-regulated signal transduction pathway through

rowth-factor signaling has been characterized as a viabilityathway in many mammalian cells (28–30). Ample evi-ence suggests that radiation could induce tumors to pro-uce angiogenic growth factors, which could be a mecha-

ax: (615) 343-3075; E-mail: dennis.hallahan@vanderbilt.eduReceived Jan 4, 2005 and in revised form Feb 8, 2005. Accepted

or publication Feb 8, 2005.

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39Molecular strategies targeting the host component of cancer ● D. W. KIM et al.

ism of tumor’s paracrine regulation of endothelium’sesistance to radiation (6). However, several investigatorsave now demonstrated that human endothelial cells in vitroemonstrate activation of PI3K/Akt in the absence ofrowth-factor stimulus, which suggests an alternativeethod of this pathway’s activation (9, 31). Our laboratory

as demonstrated that increased doses of ionizing radiationreatment of endothelial cells leads to increased phosphor-lation of Akt, which plateaus at 3 Gy (9). Alternatively,reatment of HUVECs with 3 Gy led to increased Akthosphorylation within 5 minutes and maximally at 30inutes after treatment, without significant changes in the

otal Akt protein levels (9). Similar findings were alsoeported by Zingg et al. (31), who reported that in endothe-ial cells, PI3K/Akt activation occurs upon exposure to IRia upstream RTK activation, even in the absence of itsigand. The exact mechanism of how radiation activates theI3K/Akt pathway in the absence of growth factors inndothelial cell remains to be elucidated. Because endothe-ial cells, although susceptible to apoptosis at high doses (3,), are less sensitive to the lower doses of ionizing radiation2–3 Gy) (9), a reasonable hypothesis is that activation ofI3K/Akt prosurvival pathway by ionizing radiation (IR),ither through paracrine effect, or via mechanism indepen-ent of growth-factor stimulus, is in part responsible forumor vascular radioresistance. This hypothesis has noween validated in several preclinical studies from multipleaboratories that demonstrate inhibition of this pathwayonfers radiation sensitivity of tumor blood vessels even at

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ower doses of fractionated radiation (9–12, 31). The stud- p

es to date suggest at least 2 different methods of PI3K/Aktctivation in endothelial cells in response to ionizing radi-tion, which makes it an attractive target for therapy.

INHIBITION OF RADIATION-INDUCEDACTIVATION OF PI3K/Akt PATHWAY

eceptor tyrosine kinases as upstream targets foradiation sensitization of tumor vasculature

Receptor tyrosine kinases (RTKs) and their ligands par-icipate in angiogenesis, cell proliferation, and survival, andurrent data suggest that they are potential therapeutic tar-ets (32). Split-kinase domain RTKs such as platelet-de-ived growth factor (PDGF) receptor �, Flk-1/KDR, andasic fibroblast growth factor (bFGF) receptor play impor-ant roles in tumor angiogenesis. The antagonism of vascu-ar endothelial growth factor (VEGF) by antibodies (33) andhe use of Flk-1 receptor inhibitors has been shown to haventiangiogenic effects (11). Other RTK ligands, such asGF and PDGF, also appear to contribute to angiogenesisnd tumor growth (34). Moreover, bFGF has been shown tonhibit apoptosis in the microvasculature of mouse lungsxposed to radiation (35). FGF may also indirectly contrib-te to angiogenesis by up-regulation of VEGF (36). PDGFlso increases VEGF secretion in tumor cell lines (37). Thelinical relevance of these findings is that VEGF, FGF, andDGF are all up-regulated in response to radiation (8, 38).RTKs are key elements of the most well-studied families

f signaling pathways (39). VEGF receptor (VEGFR) is a

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40 I. J. Radiation Oncology ● Biology ● Physics Volume 64, Number 1, 2006

hosphotyrosines in the RTK insert serve as binding sitesecognized by the SH2 domains of several signaling pro-eins, including PI3K, Grb2, and Shc (40, 41). This local-zed activity increases phosphatidylinositol-3,4,5-triphos-hate (PIP3) concentrations at the cell membrane, thusocally activating Akt. RTK signaling via its downstreamargets, which includes Akt, is thought to play a role inromoting cell proliferation and survival (Fig. 1). Further-ore, RTK signaling of tumor endothelium may contribute

o angiogenesis, maintenance of tumor vascular supply, and,ltimately, to tumor survival and resistance to cytotoxicherapy.

Because radiation activates PI3K/Akt signaling, severalaboratories have investigated the use of RTK inhibitors tolock the radiation induced activation of this pathway in theumor vasculature. Several RTK inhibitors are currently inreclinical and clinical studies, and a number of them havehown efficacy in tumor control in this setting (5). Forxample, treatment of tumors with combination of anti-EGF monoclonal antibody with radiation was based on thebservation that tumors treated with radiation inducedEGF production (8). In these studies, the paracrine up-

egulation of tumor angiogenesis that is induced by ionizingadiation was inhibited by blocking the activity of VEGF byeutralizing antibodies to this ligand. Multiple tumor mod-ls, such as Lewis lung, esophageal, squamous cell, andlioblastoma cells, exhibited greater than additive tumorontrol when anti-VEGF antibody was administered beforeonizing RT (8).

Several investigators have studied direct inhibition of theTK rather than its ligand as a method of improving cyto-

oxic therapy. Much of the effort has been directed atompounds that are either specific or broad-spectrum inhib-tors of VEGFR. Inhibition of these receptors can bechieved in 2 main ways: (1) an antibody directed againsthe extracellular domain of the receptor and (2) small mo-ecular compound specifically targeted for inhibition of theeceptor tyrosine kinases intracellularly (42). Examples ofarrow-spectrum inhibitors of VEGFR include DC101 andU5416. DC101 is a monoclonal antibody specifically di-ected against the murine VEGFR2. Kozin et al. (43) dem-nstrated that with DC101, they were able to lower the dosef radiation necessary to achieve 50% of tumor control, bothn radiation-sensitive (small-cell lung cancer) and in highlyesistant (glioblastoma multiforme) xenograft models.U5416 is a small-molecule receptor tyrosine kinase inhib-

tor. Geng et al. (11) demonstrated similarly the efficacy ofU5416 to revert the resistance to radiation-induced apo-tosis in HUVECs in vitro. Furthermore, combination ther-py with SU5416 and conventional fractionated doses of IRed to destruction of tumor vasculature, which, in turn, ledo significantly improved tumor control in melanoma andlioblastoma multiforme xenografts, which are otherwiseairly resistant to conventional doses of radiation (11).hese findings were further confirmed by Abdollahi et al.

44), who demonstrated that although VEGF and bFGF can

ncrease the radioresistance of endothelial cells in culture, c

U5416, a potential VEGFR RTK inhibitor when combinedith conventional doses (2 Gy) of radiation, led to reduced

ndothelial cell tubule formation and migration and signif-cantly decreased the surviving fraction of HUVECs inlonogenic assay (44). This same group also demonstratedhat triple combination of VEGFR inhibition with SU5416,hemotherapy (premetrexed), and radiation significantly in-reased the inhibition of proliferation, migration, tubuleormation, apoptosis, and clonogenic survival of endothelialells in culture. Interestingly, they demonstrated a link tonhibition of Akt phosphorylation by SU5416 as a potentialechanism of this effect (45). Avastin (Bevacizumab) is aonoclonal antibody against VEGF, a ligand for VEGFR,

pproved for treatment of metastatic colorectal cancer pa-ients when combined with chemotherapy (46). Phase Itudy of Avastin with concurrent radiotherapy and capecit-bine in locally advanced pancreatic cancer has recentlyeen reported, and final publications of these results areending (47).Broad-spectrum RTK inhibitors have the advantage of

argeting multiple RTKs that may be present in the tumor,ts microenvironment, or both. One such small molecule isTK787 (Novartis). PTK787 belongs to chemical class ofminophthalazines and is an oral inhibitor of the VEGFR2/DR/flk-1 tyrosine kinase (IC50 � 37 nM/L). It also exhib-

ts activity against flt-1, PDGFR-�, and c-Kit (IC50 � 770,80, and 730 nM/L, respectively) (48). It is not activegainst EGFR, FGFR, c-Met, Tie-2, c-Src, or c-Abl (48).TK787 in combination with radiation induced a dose-ependent reduction of proliferation of HUVECs but did notause these cells to undergo apoptosis. Treatment of p53-ysfunctional SW480 xenografts in vivo with PTK787 (4ays at 100 mg/kg/day) and radiation (4 days � 3 Gy) ledo increased tumor-growth delay compared with treatmentith either agent alone. Independently, each treatment mo-ality had minimal effect on tumor size and neovascular-zation. Significant reduction in microvessel density of theseumors was noted, which suggested that the combined ther-py was effectively targeting the tumor vasculature (49).imilarly, 2 broad-spectrum compounds from SUGENnow Pfizer), SU11248 and SU6668, have demonstratedignificant radiation sensitization effects in preclinical mod-ls. SU6668 was designed to inhibit kinase activity of FGF,DGF, and VEGF receptors (50, 51). Griffin et al. (52)emonstrated that combination of SU6668 with a singleigh dose (15 Gy) of radiation was significantly more ef-ective at controlling tumor growth compared with eitherodality alone. This observation was primarily attributed to

he increased radiosensitivity of tumor blood vessels. Fur-hermore, Lu et al. (53) demonstrated that concurrent ad-

inistration of SU6668 with fractionated conventionaloses of IR (3 Gy � 7 fractions) led to improved tumor-rowth delay compared with either treatment alone in LLCnd GL261 xenograft models. Akt phosphorylation inducedy IR was inhibited in endothelial cells that were pretreatedith SU6668 in vitro (53). Unfortunately, because of unac-

eptable toxicity profiles in clinical studies, SU6668 and

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41Molecular strategies targeting the host component of cancer ● D. W. KIM et al.

U5416 are no longer being investigated in the clinicaletting (32). However, SU11248, another broadspectrumTK inhibitor from SUGEN/Pfizer, is still under active

nvestigation. SU11248 is an orally available indolinone-ased synthetic molecule, which was identified as a low-nMelective inhibitor of the angiogenic receptor tyrosine ki-ases Flk-1/KDR and PDGFR in both biochemical andellular assays (54). SU11248 was also found to inhibitellular signaling via Kit and FLT3. SU11248 exhibitedroad and potent antitumor activity in a wide range ofuman tumor xenografts in mice (A431 human epidermoidumors, Colo205 human colon tumors, H460 human lungumors, MDA-MB-435 human breast tumors, PC3 humanrostate tumors, SF763T human glioma) and in C6 ratlioma xenografts in mice (32, 54). Recent work from ouraboratory has demonstrated that SU11248 enhances radia-ion-induced endothelial cytotoxicity (13). Part of this re-ponse was the result of increased apoptosis in endothelialells treated with combination of SU11248 and RT. Thisondition was reflected by destruction of tumor vasculaturen a tumor vascular window model. We have further dem-nstrated that this tumor blood flow destruction in tumorenografts treated with conventional fractionated radiationoses (3 Gy � 5) and SU11248 can be assessed noninva-ively via imaging technology such as amplitude-modulatedoppler sonography (Fig. 2). Noninvasive imaging has thebvious advantage of being able to follow the same animalith serial measurements. For example, in Fig. 2, we dem-nstrate that by calculating power-weighted pixel density (aeasurement of vascularity), a marked reduction of vascu-

arity is noted in animals treated with SU11248 � RT for 5ays compared with either agent alone. We have furtheremonstrated that treatment of glioma xenografts withU11248 also leads to inhibition of PDGFR� phosphory-

ation, which suggests that the efficacy of this compound atumor control may also be the result of specific inhibition ofhe tumor cells that express PDGFR�. Furthermore, inhibi-ion of PDGFR� in the tumor cells may also cause increasedensitivity to the cytotoxic effects of IR directly in theseumors.

Thus, we have strong preclinical evidence that targetinghe upstream regulators of PI3K/Akt pathway in the tumorasculature is a potentially effective method of improvingumor therapy. This method is particularly attractive in theurrent clinical setting, as demonstrated by the numerousEGFR targeting agents being developed by the pharma-

eutical industry, which provide a potentially fertile area ofuture clinical trials that involve RT.

I3K as a direct target for radiation sensitization ofumor vasculature

Although upstream effectors such as RTKs are attractiveargets for therapy, the mechanism by which PI3K/Aktathway is activated in tumor vasculature exposed to con-entional doses of radiation is not fully understood. There-ore, other redundant pathways, such as VEGFR may pos-

ibly activate PI3K independent of RTKs. This prospect h

eads to the possibility that direct inhibition of PI3K activitys an even more efficacious method of sensitizing the tumorasculature to the cytotoxic effects of ionizing radiation.Targeted therapy by exploiting this pathway has been

emonstrated to increase radiation sensitivity of the endo-helial cells in culture and in xenograft models in vivo. Ouraboratory has demonstrated that inhibition of PI3K by usef extremely potent compounds such as wortmannin orY294002 (IC501.9 and 1.4 nM, respectively) when com-ined with conventional doses of IR led to effective destruc-ion of tumor vasculature in vivo and increased apoptosis ofndothelial cells in vitro (10). This process occurred even atelatively low concentrations of these compounds (4 nM forortmannin, and 2 �M for LY294002), which suggests that

t was likely through inhibition of PI3K, rather thanNA-PK or other potential targets of these compounds.owever, the potential effects that these compounds could

ig. 2. Tumor blood-flow analysis. Amplitude-modulated doppleronography was used to quantify microscopic blood flow in LLCumors implanted into the hind limb of C57BL6 mice. Shown areepresentative images of intensity of blood flow in tumors at Day0 of treatment. Tumors were imaged on Days 1 and 5 of treat-ent. Main indicates tumors that were treated with maintenanceU11248 alone without irradiation. The bar graphs represent theverage blood flow to the peripheral portion of the tumor graftsreated as described above. Bars indicate standard error. Asterisks*) indicate p � 0.05. PWPD � Power-weighted pixel density.rom Schuenemann AJ, Himmelfarb E, Geng L, et al. SU11248aintenance therapy prevents tumor regrowth after fractionated

rradiation of murine tumor models. Cancer Research 2003;63:009–4016, reproduced with permission.)

ave on other kinases, such as DNA-PK or CK2, were a

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42 I. J. Radiation Oncology ● Biology ● Physics Volume 64, Number 1, 2006

otential limitation of this study. These compounds can alsonhibit most isoforms of PI3K (18). Furthermore, studiesave indicated that radiation sensitization can also occur inumor cells when these compounds are used (55–58). There-ore, the lack of selectivity of these compounds, the lack oftability of wortmannin, and the lack of solubility ofY294002 are some reasons that have impeded furtherlinical studies of these agents.

More specific inhibition of this pathway, as a proof of theossibility, was achieved by use of adenovirus-mediatedverexpression of �85, a dominant negative mutant of p85ubunit of PI3K in endothelial cells (9). The transduction ofdenovirus (Ad) �85 leads to attenuation of the downstreamignaling of PI3K by preventing the recruitment of the p110omponent, which, thereby, inhibits PI3K function. Inter-stingly, Ad �85 transduction attenuated the radiation-in-uced phosphorylation of Akt. Furthermore, inhibition ofI3K in this manner led to increased endothelial cell apo-tosis when treated with 3 Gy of IR, with release of cyto-hrome C and caspase 3 and 9 cleaved products (9). Al-hough these studies demonstrated the importance ofctivation of PI3K/Akt activity in conferring endothelialell’s resistance to the cytotoxic effects of IR, the limitationf this study was that adenoviral delivery of dominantegative constructs are as of yet difficult to achieve in alinical setting.

Clinically relevant methods of inhibiting PI3K pathwayy use of pharmacological compounds are difficult tochieve because of the ubiquitous presence of PI3K in manyammalian cells. Such compounds, therefore, may render

ignificant toxicity. Most recently, ICOS Corporation hasntroduced a number of compounds that target specificsoforms of PI3K, in an attempt to confer improved selec-ivity of the compound for appropriate cellular targets.C486068 from ICOS targets the p110 � isoform of theI3K proteins. P110 � was found to be expressed in HL60

eukemia cells and in endothelial cells, which suggests someevel of cell-type specificity (12). Furthermore, this com-ound demonstrated no inhibitory effect of DNA-PK (59),nlike other nonspecific inhibitors of PI3K, such as wort-annin or LY294002. Most importantly, this compoundas highly effective at inhibiting PI3K/Akt pathway in

itro. Combination therapy of IC486068 with conventionalractionated doses of IR led to increased apoptosis, de-reased clonogenicity, and decreased migration of humanndothelial cells in vitro and led to tumor vasculature de-truction in vivo (12). This finding translated to increasedumor growth delay in xenograft models of GL261 and LLCn vivo (12).

ownstream target for radiation sensitization ofumor vasculature

A significant number of downstream effectors of PI3K/kt pathway have been identified and include pathways that

nvolve glycogen synthase kinase (GSK) 3�, mammalianarget of rapamycin (mTOR), FKHR, MDM2, BAD, and

F-�B (18). A number of these targets have molecular r

ompounds under preclinical and clinical investigations.or example, mTOR inhibitors under investigation includeapamycin and its derivatives (Rad001, CCI-779, andP23573) (60). Exposure of HUVECs to conventionaloses of IR has been demonstrated to activate mTOR sig-aling, presumably through the PI3K/Akt pathway (B. Lu,ersonal communication). Furthermore, mTOR inhibitorsAD001 and rapamycin were potent radiosensitizers ofndothelial cells in vitro and led to improved tumor-growthelay of glioma xenografts in vivo (B. Lu, personal com-unication). GSK-3� is another downstream target of theI3K/Akt pathway, and studies from our laboratory dem-nstrate that in endothelial cells, radiation leads to increasedhosphorylation of GSK-3�, which is mediated by PI3K.hese studies support the idea that downstream targets ofI3K/Akt pathway are relevant areas of investigation for

mproving the cytotoxic effects of radiation on the endothe-ium.

ole of adjuvant or maintenance therapyAlthough inhibition of PI3K/Akt pathway in combination

ith RT clearly leads to improved tumor control, we ob-erved that tumors can rapidly resume growth in test ani-als when combined therapy with SU11248 and IR are

iscontinued (13). Interestingly, persistent tumor controlas achieved by adjuvant/maintenance therapy withU11248 (13), compared with animals that did not receiveurther maintenance therapy. Similar results were seen inenograft models treated with antiangiogenic agents alone61). A potential advantage of maintenance therapy withhese agents is that resistance to this form of therapy doesot seem to develop, contrary to traditional chemotherapygents for which target cancer cells are prone to developultidrug resistance (61). Implications are that maintenance

herapy with antiangiogenic agents such as SU11248 mayead to reduced likelihood of early recurrence of cancer afteronclusion of definitive therapy. Furthermore, if tumor re-rowth is seen, salvage therapy by use of these agents maytill be an option, because the endothelium is unlikely toevelop resistance to these compounds.

NOVEL THERAPEUTIC STRATEGY:IDENTIFICATION OF NEOANTIGENS INDUCEDN THE TUMOR OR ITS MICROENVIRONMENT

IN RESPONSE TO COMBINED RTKINHIBITOR � RADIATION THERAPY

One of the most desirable attributes in targeted therapy ishe concept of specific targeting of tumors and relativeparing of normal tissues. In fact, many of these highlyffective compounds in preclinical studies fail to progressn to clinical studies because of the unacceptable normal-issue toxicity that can occur in patients. Although most ofhese toxicities are introduced early in the course of therapy,hronic adverse effects could also occur and, thereby, limithe potential use of these compounds. For example, the

ecent reports of increased cardiovascular toxicity of

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43Molecular strategies targeting the host component of cancer ● D. W. KIM et al.

OX-2 inhibitors will lead to questions about the future ofhis compound in treatment of patients with or without RT.

A novel strategy introduced initially by Arap, Pasqualini,nd Rouslahti (62, 63) and Pasqualini, Koivunen, and Rous-ahti (64) involves use of phage display libraries to isolateeptides that home specifically to tumor blood vessels. Theyave identified several peptides that target the �v integrineceptor (64), and coupling doxorubicin to these peptidesed to remarkably improved tumor control, with reducedoxicity in preclinical setting (63). We have used a similarpproach to successfully identify peptide sequences thatind to irradiated tumor vasculature in xenograft models65, 66). These results are the impetus for development of aadiation-guided drug delivery system to tumors and itsicroenvironment as a novel paradigm for targeted drug

elivery.We have demonstrated that combination therapy of

I3K/Akt inhibitors (PAIs) and a clinically relevant dosef radiation leads to significant disruption of the tumornd its microenvironment. We define PAIs as compoundsr drugs that lead to eventual downstream down-regula-ion of the PI3K/Akt pathway, including the RTKs weave described above, and, thereby, lead to increasedadiosensitivity. We make this distinction because weypothesize that the neoantigens produced by this ap-roach with any of the variety of PAIs (including RTKs,irect inhibitors of PI3K, or downstream effectors suchs rapamycin) and RT should have similar applicability

(A) RT (B) CONTROL

Fig. 3. Mice treated with SU11248�RT shows increasenude mice with D54 human glioblastoma cells implanteGy), (B) untreated, (C) 80 mg/kg SU11248 � RT (3 Gyafter therapy, the animals received intravenous injectioHVGGSV. Animals were imaged periodically (from 2 tocan detect the fluorescence emitted by the Cy-7 fluoroch24 hours. A representative image is shown. Tumors

Xenogen-IVIS as indicated (A–D).

or our targeted drug delivery approach. In our initialtudies, we have demonstrated increased apoptosis ofumor endothelial cells within 24 hours of a single dosef SU11248 � 3-Gy radiation (13). Therefore, we hy-othesized that this mode of therapy would have a highikelihood of induction of neoantigens within the tumorr its microenvironment. To begin to test this hypothesis,e selected peptides that bind preferentially to tumors

reated with PAI (in this case an RTK SU11248) �onventional-dose RT by use of a phage display libraryngineered to express 106 to 108 unique peptide se-uences on its capsid. Phages that bind to the treatedumors were recovered, and this process was repeatedhrough 6 passages to enrich for the specifically bindinghages. Phages bearing the peptide sequence HVGGSVere recovered from tumors treated with SU11248� RTut not from untreated control tumors. To directly testhether this peptide binds to tumors that are pretreatedith SU11248/RT combination therapy, we performed an

n vivo imaging experiment. Phages bearing the peptideequence HVGGSV were labeled with Cy-7 fluoro-hrome. These labeled phages were injected into miceearing tumor xenografts 6 hours after treatment withT, vehicle, SU11248�RT, or SU11248 alone as indi-ated (Fig. 3A–3D). The animals were then imaged undernesthesia by a Xenogen-IVIS System. Cy-7 emits atear-infrared wavelengths that can be visualized by theenogen-IVIS system. Preliminary studies indicate that

C) SU11248 + RT D) SU11248

ing of phage that express peptide HVGGSV. Athymictheir hindlimbs bilaterally were treated with (A) RT (3D) 80 mg/kg of SU11248 alone as indicated. Six hours10 to 1011 Cy-7–labeled phage that expressed peptideurs) on Xenogen-IVIS infrared imaging system, whichaximal binding of the Cy-7–labeled phage occurred at

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44 I. J. Radiation Oncology ● Biology ● Physics Volume 64, Number 1, 2006

his peptide sequence binds with higher affinity to tumorsreated with SU11248�RT (Fig. 3C) compared with tu-ors that were treated with either agent alone (Fig. 3A

nd 3D) or untreated (Fig. 3B). Because of the potentialor artifactual fluorescence attenuation from the skin ofhe mice, we excised the tumors from the animals andmaged and quantitated them directly in the Xenogen-VIS system (Fig. 3A–3D). The tumors treated withU11248 � RT maintained a higher level of binding of

he Cy-7–labeled phage peptide HVGGSV, which furtheronfirmed our in vivo observation. On the basis of thisncouraging preliminary data, our laboratory is in therocess of identifying other peptide sequences that bindo the treated tumors, and, more importantly, we areorking on identification of inducible neoantigens that

re binding to these peptides. We are encouraged by ouresults, which demonstrate that even within the samenimal that received systemic SU11248, increased bindingccurs only in the presence of RT (Fig 3C). This resulttrongly suggests that the combination therapy is importantor this response. We do realize that potential confoundingactors exists, such as the reported abscopal effects, whichan occur in the contralateral untreated tumors (67). There-ore, we are pursuing our current studies with single-tumor–

earing animals to confirm our findings. This novel method w

REFEREN

0. Edwards E, Geng L, Tan J, et al. Phosphatidylinositol 3-ki-

1

1

1

1

1

1

1

1

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f targeted drug delivery system can target cells that haveeen pretreated with minimal doses of radiation � RTKnhibitors. As an example, we linked cytotoxic agents toigands that bind to the neoantigens induced by PAI � RT68). The goal of this approach of guided delivery of drugss to reduce normal-tissue toxicity and increase bioavailabil-ty of the cytotoxic agents to the tumor.

CONCLUSION

Radiation therapy as a modality continues to evolve. Recentiologic discoveries have led to the concept that tumor micro-nvironment is an effective target for cancer therapy, includingT. However, the inherent resistance of the tumor vasculature

o the cytotoxic effects of RT at conventional fractionatedoses (2–3 Gy) needs to be overcome before this mode ofherapy can become a reality. PI3K/Akt pathway activation isn important factor that contributes to the relative radioresis-ance of the tumor vasculature. Timely development of com-ounds that inhibit this pathway, rapid preclinical assessmentf these compounds, and quick translation of these findings inell-designed clinical studies will lead to validation of target-

ng RT not only to the tumor but also to its vasculature.urthermore, innovative strategies to improve targeted deliveryf cytotoxic therapy to the tumor and its microenvironment

ill lead to improved therapeutic index.

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