selective tumor hypoxia targeting by hypoxia-activated ... · (sigma) in sterile buffered salt...

Cancer Therapy: Preclinical

Selective Tumor Hypoxia Targeting by Hypoxia-ActivatedProdrug TH-302 Inhibits Tumor Growth in PreclinicalModels of Cancer

Jessica D. Sun, Qian Liu, Jingli Wang, Dharmendra Ahluwalia, Damien Ferraro, Yan Wang,Jian-Xin Duan, W. Steve Ammons, John G. Curd, Mark D. Matteucci, and Charles P. Hart

AbstractPurpose: Tumor hypoxia underlies treatment failure and yields a more aggressive, invasive, and

metastatic cancer phenotype. TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug of the

cytotoxin bromo-isophosphoramide mustard (Br-IPM). The purpose of this study is to characterize the

antitumor activity of TH-302 and investigate its selective targeting of the hypoxic cells in human tumor

xenograft models.

Experimental Design: Antitumor efficacy was assessed by tumor growth kinetics or by clonogenic

survival of isolated cells after tumor excision. Hypoxic fractions (HF) were determined by immunohis-

tochemistry and morphometrics of pimonidazole staining. Tumor hypoxia levels were manipulated by

exposing animals to different oxygen concentration breathing conditions. The localization and kinetics of

TH-302 induced DNA damage was determined by gH2AX immunohistochemistry.

Results: TH-302 antitumor activity was dose-dependent and correlated with total drug exposure.

Correlation was found between antitumor activity and tumor HF across 11 xenograft models. Tumor-

bearing animals breathing 95% O2 exhibited attenuated TH-302 efficacy, with whereas those breathing

10% O2 exhibited enhanced TH-302 efficacy, both compared with air (21% O2) breathing. TH-302

treatment resulted in a reduction in the volume of the HF 48 hours after dosing and a corresponding

increase in the necrotic fraction. TH-302 induced DNA damage as measured by gH2AX was initially only

present in the hypoxic regions and then radiated to the entire tumor in a time-dependentmanner, consistent

with TH-302 having a "bystander effect."

Conclusions: The results show that TH-302 has broad antitumor activity and selectively targets hypoxic

tumor tissues. Clin Cancer Res; 18(3); 758–70. �2011 AACR.

Introduction

The existence of hypoxic regions is a common feature oftumors (1, 2). Tumor hypoxia has been characterized clin-ically by a variety of different techniques, including polar-ographic needle-based electrodes, magnetic resonance andpositron emission imaging based approaches (3), immu-nohistochemistry of tumor biopsies using exogenous (e.g.,pimonidazole) or endogenous (e.g., CA-IX) hypoxia bio-markers (4), microRNA biomarkers (5), and circulatingprotein (6). The magnitude and extent of tumor hypoxia

have been shown in numerous studies to correlate directlywith poor clinical prognosis (7). The basis for this correla-tion between tumor hypoxia and prognosis has beenascribed to both therapeutic treatment failure (8–10) andhypoxia-induced prometastatic potential (11–13). Thehypothesis that the efficacy of anticancer therapies is limitedby the presence of hypoxic tumor cells has resulted in avariety of therapeutic approaches designed to eliminatehypoxic areas, to kill hypoxic cells, or to reduce the treat-ment resistance of hypoxic cells (7, 14, 15). The develop-ment of hypoxia-targeted therapies has included bioreduc-tive prodrugs, HIF-1 targeting, and genetic engineering ofanaerobic bacteria (16–18). Among these approaches, hyp-oxia-activated prodrugs (HAPs) are especially promising,because they act via a mechanism involving activation of anontoxic prodrug selectively in the hypoxic regions oftumors. Design criteria for an optimized HAP includepharmacokinetic properties to ensure adequate tumordelivery and penetration; stability to oxygen concentra-tion-independent activating or inactivating reductases; highhypoxia selectivity with activation only in severely hypoxictumor tissues and not moderately hypoxic normal tissues;

Authors' Affiliation: Threshold Pharmaceuticals, South San Francisco,California

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Jessica D. Sun, Threshold Pharmaceuticals, 170Harbor Way, Suite 300, South San Francisco, CA 94080. Phone: 650-474-8246; Fax: 650-474-2529; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-1980

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(3) February 1, 2012758

on December 23, 2019. © 2012 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 19, 2011; DOI: 10.1158/1078-0432.CCR-11-1980

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and a bystander effect, where adjacent tumor cells, nothypoxic enough to activate the prodrug, are nonethelesstargeted by the diffusible effector moiety (19).Early HAP efforts focused on quinone bioreductive drugs

such as porfiromycin, N-oxides such as tirapazamine, andnitroaromatic agents such as CI-1010. The major drawbackof quinone-based prodrugs is that they are often goodsubstrates for oxygen-insensitive 2-electron reductases(20). CI-1010 did not proceed to clinical trials because ofthe irreversible retinal toxicity observed in preclinical test-ing (21). HAPs that have advanced to clinical trials includetirapazamine (22, 23), PR104 (24, 25), AQ4N (26, 27), andTH-302 (28–31). TH-302 (1-methyl-2-nitro-1H-imidazole-5-yl) N, N0-bis (2-bromoethyl) diamidophosphate is a2-nitroimidazole-linked prodrug of a brominated versionof isophosphoramide mustard (Br-IPM; ref. 29). The2-nitroimidazole moiety of TH-302 is a substrate for intra-cellular 1-electron reductases and, when reduced in deeplyhypoxic conditions, releases Br-IPM. In vitro cytotoxicityand clonogenic assays employing human cancer cell linesshow that TH-302 has little cytotoxic activity under nor-moxic conditions and greatly enhanced cytotoxic potencyunder hypoxic conditions (28–30). The clinically validatedwarhead, a bis-alkylator, acts as a DNA cross-linking agentand is able to kill both nondividing and dividing cells.Here we report the in vivo mechanism of action and

antitumor activity of TH-302 with a panel of 11 humantumor xenograft models.

Materials and Methods

CompoundTH-302 (1-methyl-2-nitro-1H-imidazole-5-yl) N, N’-bis

(2-bromoethyl) diamidophosphate, manufactured by Syn-

gene, was used for the studies. TH-302 was formulated insaline (0.9% NaCl) at 10 mg/mL. The solution was filteredthrough 0.2 mm filter before animal dosing.

Cell linesAll cell lines were obtained from the American Type

Culture Collection (ATCC) except for the patient-derivedmelanoma line Stew2 (Dr. Lee Cranmer, University ofArizona Cancer Center, AZ) and have not been reauthenti-cated. Cells were cultured in the ATCC-suggested media(Stew2 in RPMI media) with 10% FBS added and main-tained in a 5% CO2 humidified environment at 37�C.

Xenograft modelsNCI SCID female mice (Taconic) were used for the PLC/

PRF/5, hepatocellular carcoma (HCC) xenograft model.Specific pathogen-free homozygous female nude mice(Nu-Foxn 1nu NU/NU) were used for the other xenograftmodels, except males were used for the prostate cancermodels (Charles River Laboratories). Mice were given foodand water ad libitum and housed in microisolator cages.Four- to 6-week-old animals were tagged with microchips(Locus Technology) for identification. All animal studieswere approved by the Institutional Animal Care and UseCommittee of Threshold Pharmaceuticals, Inc.

Cell suspensions were mixed 1:1 with Matrigel (BDBioscience), except for the H460 non-small-cell lung cancer(NSCLC) cells, which were mixed 2:1 with Matrigel. Cellnumber for inoculation (0.2 mL per mouse to the subcu-taneous area of the flank) was determined by the specifictumor growth kinetics for each xenograftmodel: 1� 106 forH460 and Calu-6 (NSCLC); 3 � 106 for PC-3 (prostatecancer); 5 � 106 for H82 (small cell lung cancer, SCLC),A375 (melanoma), Stew2 (melanoma), 786-O (renal cellcarcinoma, RCC), PLC/PRF/5, Hs766t (pancreatic cancer),BxPC-3 (pancreatic cancer), and SU.86.86 (pancreaticcancer).

For xenograft experiments employing controlled oxygenconcentration breathing chambers, groups of mice wereplaced in a controlled atmospheric chamber and exposed to95%O2 (carbogenwith 5%CO2), 21%O2 (air), or 10%O2,for 30 minutes followed by dosing of TH-302 or vehiclecontrol, and then, the animals remained in the controlledbreathing chambers for 2 additional hours. The chamberswere flushed continuously with gases at 5 L/min.

In vivo antitumor activityTumor growth and body weight were measured twice a

week after cell implantation. Tumor volume was calculatedas (length � width2)/2. When the mean value of tumorvolume was approximately 100 to 150 mm3, mice wererandomized into 10mice per group, and TH-302 or vehiclecontrol treatment started (Day 1). Antitumor activity wasassessed by tumor growth inhibition (TGI) and tumorgrowth delay (TGD). TGI was defined as (1 � DT/DC) �100, where DT/DC is the ratio of the change in mean tumorvolume of the treated group (DT) and of the control group(DC). Animals were called when individual tumor size was

Translational Relevance

The hypoxia-activated prodrug TH-302 is currently inclinical trials for the treatment of cancer, which include arandomized controlled Phase 2 pancreatic cancer trialand a pivotal Phase 3 sarcoma trial. Over 500 patientshave been treated to date.The results described in this paper show the in vivo

proof-of-concept that TH-302 selectively targets the hyp-oxic regions of human xenograft tumors. Multiple com-plementary lines of evidence are presented, which sup-port selective tumor hypoxia targeting by TH-302. Inaddition, the results also show the potential utility forthe diagnostic use of pimonidazole as a hypoxia bio-marker to guide the selection of patients for TH-302treatment. The strong correlation between the magni-tude of tumor hypoxia as measured by pimonidazolestaining and TH-302 efficacy has served as the basis forongoing and new clinical trial proposals that include theanalysis of patient biopsies stained with pimonidazoleand the subsequent comparison with TH-302 efficacy.

Specific Tumor Hypoxia Targeting by TH-302

www.aacrjournals.org Clin Cancer Res; 18(3) February 1, 2012 759




over 2,000 mm3 or mean tumor volume exceeded 1,000mm3 in the group. TGI was determined on the last mea-surement when all the animals in the vehicle group stillsurvived. TGD500 or TGD1,000 was determined as theincreased time (days) for the treated tumor’s size on averageto reach 500 or 1,000 mm3 as compared with the vehiclegroup.

Maximum tolerated dose (MTD) for TH-302 was deter-mined by dose escalations in a small number of nontumorbearing nu/nu mice. The MTD was defined as the highestpossible dose resulting in no animal deaths, less than 20%weight loss for any one animal in an experimental group, nosignificant changes in general clinical signs, and no abnor-mal gross anatomical findings after necropsy. General clin-ical signs included respiratory rate, behavior, and responseto normal stimuli (32). The doses of TH-302 used in allstudies were no higher than MTD.

Tumor excision assays with clonogenic survivalendpoints

Twenty-four hours post TH-302 treatment, tumors wereexcised, weighed, minced, and then digested with anenzyme cocktail containing 0.2 mg/mL each of collage-nase (Sigma-Aldrich), DNaseI (Sigma), and pronase(Sigma) in sterile buffered salt solution (Sigma) for 15minutes at 37�C with stirring, and the resulting cellsuspension counted and plated to determine clonogeni-city fractions. Cell colonies (>50 cells per colony) werecounted, and the surviving fraction (SF) was calculated asthe plating efficacy (PE) of treated divided by the PE ofvehicle control.

Histology and immunohistochemistryTo determine the magnitude of the hypoxic fraction

(HF) for each of the different xenograft tumor models,when tumor size reached 400 to 600 mm3 pimonidazole(Natural Pharmacia International) at 60 mg/kg was intra-peritoneally (i.p.) injected 1 hour before animal waskilled. To label regions of blood perfusion, Hoechst33342 (Invitrogen) at 10 mg/kg was administered viatail vein injection 1 minute before animal was killed(33). Tumors were recovered immediately and eitherembedded and frozen in OCT or fixed in 10% neutralbuffered formalin and embedded in paraffin. A total of8-mm-thick frozen tissue sections or 5-mm-thick paraffinsections were cut and adhered to poly-L-lysine-coatedglass microscope slides. Frozen sections were storedat �80�C, and paraffin sections were placed at roomtemperature (RT) until use.

Hematoxylin and eosin (H&E) staining was conductedfor tumor morphology and to quantify tumor necroticfraction. Cryostat sections were air dried and fixed in coldacetone for 10 minutes. After washing with PBS and incu-bation with Peroxidazed 1 (Biocare Medical) to quench theendogenous peroxidase, sectionswere incubatedwith Back-ground Sniper (Biocare Medical) at RT for 15 minutes toblock the nonspecific binding site. Sections were thenincubated 1 hour at RT with the appropriate dilutions of

primary antibodies: anti-pimonidazole rabbit polyclonal at1:500 (Natural Pharmacia International), or anti-CD31 ratpolyclonal at 1:250 (Biocare Medical). HRP-conjugatedgoat anti-rabbit IgG (Epitomics) served as secondary anti-body for pimonidazole adducts, and Rat Probe HRP Poly-mer Detection Kit (Biocare Medical) was used for CD31.The colorimetric reaction was developed by DAB chromo-gen (Biocare Medical) system with hematoxylin used as acounter stain. Hoechst 33342 was observed under UV lightwith blue 465/30 nmol/L band-pass filter on cryostat sec-tions before immunostaining. Hypoxia (by pimonidazole),and TH-302 induced DNA damage (by gH2AX immunos-taining), were characterized in consecutive paraffin sec-tions. For pimonidazole staining, sections were incubatedwith Pronase Reagent (GeneTex) at 37�C for 40 minutes toretrieve the antigen. For gH2AX (1:2,000, rabbit monoclo-nal, Epitomics) detection, antigen retrieval was conductedby incubating the slides with Heat Induced Epitope Retriev-al Buffer (Biocare Medical) in a Decloaker Chamber (Bio-care Medical).

Image analysisAt 20� magnification, digital images of H&E stained

or pimonidazole stained sections were captured andassembled to compose a whole tumor image. Pimonida-zole-positive and necrotic regions were extracted usingImage-Pro Plus v6.0 (MediaCybernetics). The necroticfraction (NF) in the individual tumor was calculated asthe percentage of the necrotic area of the tumor tissue inthe whole tumor. The HF was evaluated in the viabletissue with the necrotic area excluded. To compare thevolume of the hypoxic compartment in the tumors afterTH-302 treatment, pimonidazole-positive hypoxic areasin the whole tumor were analyzed as well. Hoechst 33342positivity was only evaluated in the viable tissue bysubtracting the necrotic area in the section. At 200�magnification, 10 to 15 images per section were randomlyselected. All images were captured under consistent illu-mination and exposure for their respective stains. Noimage postprocessing was carried out. Scripts were devel-oped in Image Pro-Plus to analyze the target signals usingcolor and morphologic segmentation tools. For semi-quantification of CD31-positive areas and microvesseldensity, point counting was carried out. A total of 15 to20 fields at 400� magnification, per section, werecounted in each animal on a 1-cm2 eyepiece graticulewith 10 equidistant grid lines. The percentage of fraction-al area (percentage of positive area per total area countedof the section) was calculated using the following formu-la: percentage of positive fractional area ¼ number of gridintersections with positive staining/total number of gridintersections multiplied � 100%. gH2AX positive cellswere counted at 400� magnification. Five fields persection were used. Normoxic compartment was identifiedby cell morphology, and hypoxic compartment wasidentified by pimonidazole staining in serial sections.The percentage of gH2AX positive cells in each com-partment was calculated as number of gH2AX positive

Sun et al.

Clin Cancer Res; 18(3) February 1, 2012 Clinical Cancer Research760




cells per number of total cells in the compartment �100%.

Statistical analysisData are expressed as the mean � SEM. A Student t test

was used to find the significance between 2 groups.Comparison of the multiple groups was done usingOne-way analysis of variance with Dunnett test (Graph-Pad PRISM 4). Correlation was calculated by linear regres-sion. A P level less than 0.05 was considered statisticallysignificant.

Results

Antitumor activity of TH-302 as a single agentUsing theH460NSCLC xenograftmodel, we investigated

the dose-dependent antitumor activity of TH-302. WhenTH-302 was administered at 6.25, 12.5, 25, or 50 mg/kg,QD � 5/wk � 2 wks (once a day for 5 days per week for 2weeks) i.p., the TGI at Day 22 was 43%, 51%, 75%, and89%, respectively (Table 1). A dose of 50 mg/kg yieldedmodest body weight loss of 2.9% on average. With thisdosing regimen, 50 mg/kg was considered as MTD, as

Table 1. Comparison of different doses and regimens of TH-302 on tumor growth in the H460 humanNSCLC xenograft model

Dose,mg/kg Regimen

Drug exposure,mg/kg TGI% TGD500, d TGD1,000, d

Max. BW loss%(day "x")

6.25 QD � 5/wk � 2 wks 62.5 43 4.5 6 1.5 (4)12.5 QD � 5/wk � 2 wks 125 51 7 8.5 1.7 (4)25 QD � 5/wk � 2 wks 250 75 11 12 2.5 (4)50 QD � 5/wk � 2 wks 500 89 18 19 2.9 (4)100 Q7D � 2 200 72 10 10 0100 Q3D � 5 500 89 16 16 2.3 (9)150 Once 150 58 8 10 1.9 (4)

NOTE: TH-302 was administered intraperitoneally. TGI, tumor growth inhibition was defined as (1 � DT/DC) � 100, where DT/DCpresented the ratio of the change in mean tumor volume of the treated group and of the vehicle group. TGI of all the groups werecalculated at Day 22. TGD500 or TGD1,000 was determined as the increased time (days) for the treated tumors' size on average to reach500or 1,000mm3as comparedwith the vehicle groupusing the tumor growth curves.Datawereobtained from10miceper group;Drugexposure (in mg/kg)¼ dose (in mg/kg)� number of doses; Max. BW loss%, percentage of mean maximal body weight lost comparedwith pretreatment with the time point (day "x") when reached.

Table 2. TH-302 exhibits broad antitumor activity in a panel of xenograft tumors

Cell lineCancertype Dose

Drugexposure TGD500 TGD1,000

TGI%(day "x")

P value vs.vehicle

Maximal BWloss% (day "x") HF% (n)

H460 NSCLC 50 500 18 19 89 (22) <0.001 2.9 (4) 16.3 (8)Calu-6 NSCLC 50 500 11 12 51 (33) <0.01 1.2 (11) 10.2 (6)H82 SCLC 50 500 4 5 50 (15) <0.01 0.1 (4) 6.9 (7)Stew2 Melanoma 50 500 14 NA 31 (39) <0.05 0 6.4 (6)A375 Melanoma 50 500 5 7 49 (18) <0.05 0 5.0 (5)PLC/PRF/5 HCC 50 500 5 NA 29 (39) >0.05 5.6 (22) 4.7 (7)PC-3 Prostate 50 500 13 11 47 (36) <0.01 1.2 (11) 4.2 (8)786-O RCC 50 500 10 NA 10 (64) >0.05 0.1 (8) 1.4 (8)Hs766t� Pancreas 75 375 16.5 >25 75 (11) <0.01 3.4 (11) 14.8 (9)BxPC-3� Pancreas 75 375 1 6 40 (15) <0.01 1 (8) 7.2 (11)SU.86.86� Pancreas 75 375 1 3.5 28 (18) >0.05 0 4.8 (8)

NOTE: TH-302was administered intraperitoneally. and the regimens were QD� 5/wk� 2wks except �, Q3Dx5 in Hs766t, BxPc3, andSU.86.86; Drug exposure (in mg/kg) ¼ dose (in mg/kg) � number of doses. TGI was obtained from 10 animals per group. Data arepresentedasmeanvalue for eachgroup.P valueof tumorgrowthbetweenTH-302–treatedandvehicle-treatedgroupswasanalyzedbyt test when TGIwas calculated. The hypoxic fraction (HF) was evaluated in the viable tumor tissuewith necrotic area excluded. day "x",time points at which TGI and weight loss was determined. n, number of tumors samples analyzed; NA, not available before the studywas terminated.






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Figure 1. A, representative images of tumor hypoxia detected by pimonidazole binding and antitumor activity of TH-302 in the 8 xenograft tumor models. TH-302 was dosed at 50 mg/kg, QD � 5/wk � 2 wks, intraperitoneally (i.p.) in all the models. Dotted lines with open circles are the vehicle-treated groups, andsolid lines with closed circles are the TH-302–treated groups. Data are presented as mean � SEM of 10 animals in each group; �, P < 0.05 or ��, P < 0.01versus vehicle-treatedgroupby t test. B, correlation between hypoxic fraction (HF) in the 8 xenograft tumormodels and the corresponding antitumor activity ofTH-302 expressed as tumor growth inhibition (TGI). HF was obtained as mean � SEM from 5 to 11 animals for each tumor type, TGI was obtainedas mean � SEM from 10 animals of each tumor type.

Sun et al.





75 mg/kg resulted in severe body weight loss (>20%) in 1of 10 animals and led to a maximal body weight loss onaverage 9.3%. TH-302 was also tested at the higher doses of100 and 150 mg/kg with a less frequent dosing regimen(once, i.p.) or 100 mg/kg with an intermittent regimen(Q3D � 5, i.p.). Under all these regimens, there was noorgan abnormalities noted after necropsy at treatment end.Furthermore, hematologic toxicity of TH-302 was investi-gated in the immunocompetent CD-1 animals 3 or 7 daysafter TH-302 treatment. As was observed in the nude mice,there was no significant body weight loss observed amongthe groups. TH-302 50 mg/kg, QD� 5/wk� 2 wks did notshow any hematologic toxicity as evaluated by white bloodcell, neutrophil, or lymphocyte counts on either 3 or 7 daysafter treatment end. TH-302 at 100 mg/kg showed adecrease in blood cell counts 3 days after treatment end,but was totally recovered 7 days posttreatment (Supple-mentary Fig. S1). TH-302 under all tested regimens exhi-bited efficacy metrics ranging from 58% to 89% TGI(Table 1). In the H460 NSCLC model, when TH-302 wastested at no higher thanMTDwith different regimens, therewas a total drug exposure (¼ dose � number of injections)

dependent TGD to 500 or 1,000 mm3. In addition, signi-ficant correlation between antitumor activity, evaluated asTGI and total drug exposure was observed, R2 ¼ 0.80, P <0.01. As TH-302 50 mg/kg QD � 5/wk� 2 wks i.p. yieldedthe highest drug exposure, exhibited minor body weightloss, and showed a safe hematologic profile, this regimenwas employed in many of the preclinical models tested.

TH-302 antitumor activity was then evaluated in 11different ectopic xenograft models, including NSCLC,SCLC, melanoma, prostate cancer, HCC, RCC, and pancre-atic cancer (Table 2). The samedose and regimenof TH-302,50 mg/kg, QD � 5/wk � 2 wks, i.p. was used in thesexenografts, except the pancreatic cancer models (75 mg/kg,Q3D � 5, i.p.). Under the same drug exposure, all theanimals tolerated TH-302 well, and there was no apparentdrug induced toxicity determined by body weight change.TGIs across all models ranged from approximately 10% toapproximately 89%. Antitumor activity was reflected inTGD as well, with from 1 day to more than 25 days delayobserved. Compared with vehicle control–treated groups,TH-302 significantly inhibited tumor growth in 8 of 11xenograft tumor models examined.

Figure 1. (Continued ) C,representative images of tumorhypoxia detected by pimonidazolebinding and antitumor activity ofTH-302 in 3 pancreatic xenografttumor models Hs766t, BxPC-3, andSU.86.86. TH-302 was dosed at 75mg/kg, Q3Dx5, i.p. in all the models.Dotted lines with open circles are thevehicle-treated groups, and solidlines with closed circles are theTH-302–treated groups.D, correlation between HF in the 3pancreatic xenograft tumors andantitumor activity of TH-302, whichwas expressed as TGI. HF wasobtained as mean � SEM from 8 to11 animals in each tumor type, andTGI was obtained as mean � SEMfrom 10 animals of each tumor type.

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Antitumor activity of TH-302 as a function of tumorhypoxia

Hypoxic regions in the tumors were detected by pimo-nidazole immunostaining. Representative pimonidazolestaining images in 8 xenograft tumors are presentedin Fig. 1A. Semiquantitative morphometric analysis of thehypoxic compartments was conducted with tumor samplesfrom 5 to 8 animals per xenograft tumormodel. TheHFwasdetermined in the viable area only for the tumor modelsprofiled (Table 2 and Fig. 1A). HF varied in the models,from16.3% (in theH460 xenografts) to 1.4% (in the 786-Oxenografts). The corresponding tumor growth kinetics ineach xenograft model, and the antitumor activity of TH-302as a monotherapy is presented in Fig. 1A. Under the samedose and regimen of TH-302 the HF and TH-302 efficacy(TGI) in 8 xenograft models showed a good correlation(R2 ¼ 0.77, P ¼ 0.004) by linear regression (Fig. 1B).Hypoxia regions were detected in the 3 pancreatic xenografttumors as well (Fig. 1C). The Hs766t, BxPc3, and SU.86.86models showed hypoxia levels ranging from high to low,with HF of 14.8%, 7.2%, and 4.8%, respectively (Fig. 1C).The antitumor activity of TH-302 significantly correlatedwith HF in the 3 pancreatic models (R2 ¼ 1.0, P ¼0.001; Fig. 1D). In the PLC/PRF/5 HCC, 786-O RCC, andSU.86.86 pancreatic cancer xenograft models, where theantitumor effect did not achieve statistical difference com-pared with the vehicle treatment, the pimonidazole-positivity was relatively low, with HF less than 5%.

Antitumor activity of TH-302 as a function of breathingoxygen concentration

To further investigate the relationship between tumorhypoxia and TH-302 activity, we modified tumor hypoxiaby exposingH460NSCLC tumor-bearinganimals exposed todifferent oxygen concentration levels in controlled atmo-spheric breathing chambers gassed with 95%, 21%, and10%O2. Antitumor activitywas assessed by clonogenic assayof tumors excised 24hours after a single doseof TH-302 at 50mg/kg, i.v. TheSFof individual tumors is presented inFig.2A,where the mean SFs were 50.4%, 5.8%, and 2.6% for 95%,21%, and 10%O2, respectively. The results showed that TH-302 induced cell killing was breathing oxygen concentrationdependent, with the greatest cytotoxicity occurring when thetumor-bearing mice were exposed to low oxygen concentra-tions. On the basis of these results, we further evaluateddifferential TH-302 antitumor activity with multiple dosingunder varied breathing oxygen concentration conditionsusing TGI and TGD as read-outs. H460 tumor-bearing ani-mals were treated with vehicle or TH-302 at 50 mg/kg, i.p.,QD� 5/wk� 2wks,when tumor size reached 100mm3.Ontreatmentdays, allmicewere exposed to thedifferent levelsofoxygen in the controlledatmosphere chamber for30minutesbefore and 2 hours after each dose. As shown in Fig. 2B,tumor growth rates in the vehicle control group were notaffected by the different oxygen level breathing conditions.However, after TH-302 treatment, TGIwas clearly dependenton oxygen level breathing conditions, with superior efficacyachieved in the lower breathing oxygen concentration

groups. Tumor growth was significantly reduced by TH-302 in animals breathing 10% O2 compared with 95% O2

breathing. The TGIs on day 29 were 90% in 10% O2 ascompared with 50% in the 95%O2 group, P < 0.05. TGD500

was 20, 16, and 7 days in animals breathing 10%, 21%, and95% O2, respectively. Animal body weight was not affected

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Figure 2. TH-302 exhibits controlled oxygen concentration breathingcondition-dependent antitumor activity. A, clonogenic assay of H460cells isolated from excised tumors grown in nude mice and treated with50 mg/kg of TH-302, intravenously (i.v.) for 24 hours in animals exposedto varying breathing oxygen concentration levels. Hypoxia enhancedantitumor activity (B) but not toxicity (C) of TH-302 in H460 xenografttumor-bearing mice. Animals were treated with TH-302 50 mg/kg, QD �5/wk � 2 wks with different atmospheric O2 concentrations (10%, 21%,and95%)breathing conditions at dosing. Tumor volumeandbodyweightwere measured twice a week. Data are expressed as the mean� SEM of10 animals per group.

Sun et al.





by different breathing conditions. All vehicle and TH-302–treated groups showed less than 3% bodyweight loss duringthe study.

TH-302 reduces the volume of the hypoxiccompartmentTo determine TH-302 effects on the tumor microenvi-

ronment, vascular, hypoxic, and necrotic staining of H82SLCLC xenograft tumor sections was employed at variousposttreatment times after TH-302 dosing. Representativeimages are shown in Fig. 3A, and morphometric analysisresults are shown in Fig. 3B–E. CD31 endothelial cellstaining was used to identify blood vessels, and Hoechst33342was used to detect functional blood perfusion. In thevehicle-treated control tumors, blood vessels were evenlydistributed in the tumor and appeared as distinct clustersof endothelial cells with intact lumen. After 150 mg/kgTH-302 i.p. treatment, there was no apparent loss ofvessel integrity or any evidence of hemorrhaging or loss ofCD31 staining. By semiquantitative morphometric analy-sis, tumor vessel density (Fig. 3B) and vascular perfusion(Fig. 3C) in the viable tumor tissue showed no significantdifference between vehicle and TH-302–treated tumors atany of the time points examined. The HF in the tumors wasassessed by pimonidazole immunostaining (Fig. 3D). AfterTH-302 treatment, the pimonidazole-positive area was sig-nificantly decreased at 48 hours after dosing (6.3 � 1.2%in vehicle vs. 1.8 � 1.1% in the TH-302 treatment group,P < 0.05). The percentage of pimonidazole-positive area inthe whole tumor remained at this reduced level at 72 hoursafter dosing. The tumor necrotic fraction (NF, Fig. 3E)exhibited a corresponding time-dependent increase, wherethe NF was increased from 7.3% in vehicle to 37.9% afterTH-302 treatment. In addition, 48 hours after TH-302treatment at 150 mg/kg, i.p., pimonidazole-positive area inthe whole tumor was also investigated in the H460(NSCLC), Hs766t (pancreatic), and A375 (melanoma)xenograft tumors. Representative images are shown in Fig.3F, and themorphometric analysis showed significantpimo-nidazole-positivity reduction in all 3 models (Fig. 3G). Thereduced pimonidazole-positive area in H460 xenografttumors was recovered and was back to normal 7 days afterTH-302 150mg/kg, i.p., treatment (Supplementary Fig. S2).

Intratumoral distribution of TH-302 induced DNAdamagegH2AX foci formation was employed to assay TH-302

induced DNA damage. Animals bearing H460 tumorswere treatedwith TH-302 at 100 or 25mg/kg, i.p., or vehiclewhen the tumor size reached 400 to 600 mm3. Tumorswere excised at 6 or 24 hours later, and gH2AX and pimo-nidazole immunostaining was carried out on consecutiveformalin-fixed, paraffin-embedded sections (Fig. 4A). Inthe vehicle-treated H460 xenografts, few gH2AX positivecells were present and were mainly located near thenecrotic regions. Six hours after 100 mg/kg of TH-302,i.p., treatment, there was a significant increase of gH2AX-positive cells. Those gH2AX-positive cells were predomi-

nantly found in the pimonidazole-positive hypoxic areas(47% vs. 11% in vehicle in the hypoxic compartment [HC],P < 0.001; 19% vs. 2%, P < 0.001 in the oxic compartment[OC], Fig. 4B). Twenty-four hours after TH-302 treatment,gH2AX positive cells were significantly increased in the OC,which were evenly distributed; however, the percentage ofpositive cells was reduced in the HC. Six hours after a lowerdose of TH-302 (25 mg/kg), there was a significant increaseof gH2AX-positive cells in both the OC (4%) and the HC(19%), P < 0.05 versus vehicle. Twenty-four hours aftertreatment, the percentage of gH2AX-positive cells in OCwas5%,but therewas a significant reduction in theHC.Theotherexogenous hypoxia biomarker marker employed, F6, wasadministered sequentially with pimonidazole to identifyregions with changes in hypoxia. Immunostaining of pimo-nidazole (PIMO), F6, gH2AX, and Ki-67 (a marker of pro-liferation)were carried out in consecutive sections. PIMO(þ)/F6(þ) cells were scored as chronically hypoxic cells, whichwere predominant in this model. PIMO(þ)/F6(�) orPIMO(�)/F6(þ) cells were scored as acutely hypoxic cells, andwere usually found proximal to blood vessels. Six hours afterTH-302 (100 mg/kg) treatment, gH2AX-positive cells weresignificantly increased. gH2AX-positive cells were observedin both PIMO(þ)/F6(þ) (chronic) and PIMO(þ)/F6(�) (acute)hypoxia regions. TheDNAdamagewas predominantly locat-ed in the PIMO-dark and Ki-67 negative areas. Ki-67 positiveproliferating cells were dramatically decreased in both acuteand chronic hypoxic regions (Supplementary Fig. S3).

Discussion

Preclinical mouse models of cancer have been shown torecapitulate the architecture of the tumor microenviron-ment and the prevalence of subregional hypoxia. The use ofhuman tumor xenografts in immunocompromisedmice forthe preclinical evaluation of new anticancer drugs is anintegral part of drug discovery and optimization strategiesin which specific characteristics of tumor lines can be usedto orient future clinical development (34, 35). Selectivetargeting of hypoxic tumor regions with HAPs designed toactivate or release a cytostatic or cytotoxic effector selectivelyin hypoxic tumor regions is a promising therapeutic strategyfor the treatment of cancer. To show the hypoxic selectivityof TH-302 in vivo, we utilized a panel of 11 human tumorxenografts and characterized TH-302 activity with differentmechanistic antitumor read-outs.

Employing the H460 NSCLC xenograft model, we haveshowed that the antitumor activity of TH-302 is solelydependent on total drug exposure regardless of how fre-quently the drug was administrated (Table 1). The ability ofthe TH-302 toxin (Br-IPM) and other DNA cross-linkingagents to yield long-lived DNA lesions and exhibit cell cycleindependent cytotoxicity underlies their utility in killing thehypoxic cells in tumors. Thismechanism of action feature isconsistent with our finding of the relative regimen inde-pendence of TH-302 as monotherapy. TH-302 show anti-tumor activity as a single agent across a broad panel ofdifferent tumor type xenograft models. Significant TGI was






observed in 8 of 11 xenograft models examined, The lowactivity of TH-302 observed in the 786-O RCC, PLC/PRF/5HCC, and SU.86.86 pancreatic xenograft models was con-

sistent with their tumor microenvironment phenotypesexhibiting little hypoxia as measured by pimonidazolestaining. A key finding described here is the correlation

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Figure 3. Tumor microenvironmentchanges after TH-302 treatment inH82SCLCxenograftmodel. TH-302was administered as 150 mg/kg, i.p.Tumors were recovered 24, 48, and72 hours after the treatment andcompared with vehicle (V). A,representative images of CD31immunostaining, Hoechst 33342perfusion, pimonidazole staining,and H&E in vehicle, 24-, 48-, and72-hour treatment groups.Morphometric analysis of (B)microvessel density in the viabletissue of the tumor by CD31immunostaining, (C) percentage ofblood perfusion in the viable tissueof the tumor by Hoechst 33342staining, (D) percentage ofpimonidazole-positive area in thewhole tumor by pimonidazolestaining, and (E) percentage ofnecrosis in the whole tumor by H&Estaining. Each bar represents mean� SEM of 5 to 8 animals per group.�P < 0.05 versus vehicle group.

Sun et al.





between TH-302 antitumor activity and the correspondingmagnitude of tumor hypoxia present in a xenograft model(Fig. 1). Significant correlation was found between therelative volume of hypoxia and TH-302 activity in the 8xenografts at equal total drug exposure. A higher HF usuallyyielded greater antitumor effect of TH-302. The HAPtirapazamine also show a correlation between hypoxicvolume (as measured by EF5 staining) and DNA damage(as measured by comet assay) between individual SCCVIItumor-bearing animals (22). However, other cell auton-omous factors may also be involved in the antitumoractivity of TH-302. For example, although the xenograftmodels, Calu-6 (NSCLC) and H82 (SCLC), exhibiteddifferent HFs (10.2% in Calu-6 vs. 6.9% in H82), theyexhibited very similar TH-302 antitumor activity profiles,with TGIs of 50%, after the same TH-302 treatmentregimen (50 mg/kg, daily). Factors affecting TH-302 anti-tumor activity could be the difference in DNA repairmachinery; for example, H82 has wild-type p53 (36) andCalu-6 has mutant p53 (37). Others have shown mutantp53 elevated DNA damage repair led to drug resistance(38). Another factor responsible for the different antitu-mor activity could be differential prodrug activatingreductase expression (39).To extend the observation of a significant correlation

between the volume of the HF in xenografts and the corre-sponding TH-302 antitumor activity, we examined whetherlower oxygen level breathing conditions (10% O2 breath-ing) would yield greater efficacy, becausemore of the tumorwould be hypoxic and more TH-302 would be activated,and conversely, whether higher oxygen concentrationbreathing conditions (95%) would yield less efficacy forTH-302. Previous published studies have showed the effectof modified oxygen concentration breathing conditions ontheHFof xenograft tumors (40–43) and differential efficacyof an HAP (43). Both acute (single dose) and chronic (daily

dosing for 2 weeks) TH-302 dosing regimens wereemployed. In both acute and chronic breathing conditionand dosing regimen models, the results show TH-302induced cytotoxicity in an oxygen concentration breathinglevel dependent manner.

We investigated the spatial distribution of TH-302induced changes in tumor microenvironment architecturewith a focus on characterizing selective effects on the hyp-oxic compartment. In the 4 xenograft tumor models exam-ined (H82 SCLC, H460 NSCLC, Hs766t pancreatic cancer,and A375 melanoma), the volume of the hypoxic regionswas significantly reduced after TH-302 treatment. We havealso shown the reduction of hypoxic regions after TH-302treatment was not because of the disruption of tumor vesselperfusion that prevented the pimonidazole hypoxia probefrom reaching the hypoxic areas. In contrast to the reducedhypoxia region, the volume of the necrotic compartmentwas correspondingly increased. Given that the necroticfraction increased to over 30% of the tumor volume in theH82 model (compared with 7% in the vehicle-treatedtumors), whereas the HF at the time of dosing was usuallyless than 8% in this tumor type, the relative change in the 2compartments (the hypoxic compartment decreasing from6% to 2% and necrotic compartment increasing from 8% to30%) is consistent with a bystander effect occurring, whereadjacent normoxic cells are also killed by the releasedcytotoxic warhead, although the possibility that normoxicactivation of TH-302was high enough to kill normoxic cellscannot be excluded under the test conditions employed.

In the H82 SCLC xenograft model, TH-302 treatment didnot exhibit any apparent effect on the microvasculature orblood perfusion during the 72-hour observation period.There was no decrease of microvessel density in the viablezone as assessed by endothelial cell surface marker CD31staining. Moreover, there was no significant reduction offunctional blood perfusion detected by Hoechst 33342

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Figure 3. (Continued ) TH-302 treatment reduces pimonidazole-positive area in the H460 NSCLC, Hs766t pancreatic cancer and A375 melanoma xenografttumors. TH-302 was administered as 150 mg/kg, i.p. Tumors were harvested 48 hours after the treatment compared with vehicle. F, representative images;G, morphometric analysis of percentage of PIMO-positive area in the whole tumor by pimonidazole staining. Each bar represents mean � SEM of 5 to 11animals per group. �P < 0.05 versus vehicle group.






staining, which suggested little or no vessel disruption aftertreatment. Similar results were obtained in the H460NSCLC xenograft model. It has been previously shownthat tirapazamine causes extensive, central vascular dys-function in HCT-116 xenograft tumor within 24 hours ofdosing (44). Given that the xenograft models are differ-ent, it is not clear whether these different effects on thevasculature of these 2 different HAPs indicate a functionaldifference between the HAPs. One possible mechanismunderlying tirapazamine as a vascular disrupting agent isactivity on moderately hypoxic endothelial cells or peri-vascular cells (45). Tirapazamine is activated at relativelyhigher oxygen concentration compared with TH-302(28). Therefore, only severely hypoxic tumor cells maybe targeted by TH-302 but not the moderately hypoxicmicrovessel cells in the tumor. This feature of TH-302exhibiting little effect on tumor microvasculature mayfacilitate the delivery of both TH-302 itself and a com-panion coadministered conventional chemotherapeuticdrug when used in combination with TH-302 and, there-fore, maximize antitumor activity.

The phosphorylation of the variant histone H2AX atSer139 (gH2AX) is a sensitive marker of DNA damageresponses (46). In vehicle control–treated H460 tumors,therewere low levels of gH2AXpositive cells,mainly locatedin the areas adjacent to the necrotic areas. This observationis consistentwith previous published studies (47). Six hoursafter TH-302 treatment, gH2AX-positive cells were dramat-ically increased in the hypoxic regions of the tumors in adose-dependent manner. Using 2 different exogenous mar-kers of hypoxia administered at different times showincreased gH2AX in both double stained (chronic hypoxia)and single stained (acute hypoxia) regions. This pattern ofDNA damage by TH-302 treatment was consistent with thepreferential toxicity of TH-302 for hypoxic cells. A similarpattern has also been reported after HAP tirapazaminetreatment in SiHa xenograft tumors (48). At 24 hours afterTH-302 treatment, however, DNA damage had radiatedthroughout the tumor, consistent with TH-302 having abystander effect. A bystander effect could also be responsi-ble for the high antitumor efficacy of TH-302 as a singleagent in the broad panel of xenograft models.

Taken together, the results presented here on (1) thecorrelation of HF and TH-302 antitumor activity; (2) theenhanced antitumor activity in tumor-bearing animalsafter breathing lower oxygen concentration atmospheres;(3) the selective reduction in the volume of the hypoxiccompartments in xenograft tumors and correspondingincrease in the necrotic compartments volumes in xenografttumors; and (4) the colocalization of gH2AX positiveimmunostaining in the pimonidazole-positive hypoxicregions of xenograft tumors, are all consistent with theselective targeting of the hypoxic compartments of tumorsby TH-302. The extension of TH-302 induced DNA damageto the normoxic regions indicates an accompanyingbystander effect. TH-302 is currently in clinical trials forthe treatment of cancer (31). The results described heresupport thehypoxic selectivity of TH-302 in vivo, and the use

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Figure 4. Representative images of immunostaining of pimonidazole andgH2AX on consecutive 10% formalin-fixed paraffin-embedded sections.A total of 60 mg/kg of pimonidazole was i.p. injected 1 hour beforeTH-302was administeredat 100or 25mg/kg, i.p. Tumorswere recovered6 and 24 hours after the TH-302 treatment compared with vehicle.C, capillary; O, oxic (normoxic) compartment; H, hypoxic compartment;andN, necrotic region.Magnification scale bar, 100mm.B,morphometricanalysis of percentage of gH2AX positive cells in oxic and hypoxiccompartments. Each bar represents mean � SEM of 3 to 4 animals pergroup. �,P < 0.05 versus vehicle group; #,P < 0.05 versus TH-302 6-hour–treated group.

Sun et al.





of hypoxia biomarkers to profile patient biopsies and guidepatient selection for therapy with TH-302.

Disclosure of Potential Conflicts of Interest

All authors are current or former employees and stockholders of Thresh-old Pharmaceuticals, Inc.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 3, 2011; revised December 9, 2011; accepted December12, 2011; published OnlineFirst December 19, 2011.

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Sun et al.




2012;18:758-770. Published OnlineFirst December 19, 2011.Clin Cancer Res Jessica D. Sun, Qian Liu, Jingli Wang, et al. TH-302 Inhibits Tumor Growth in Preclinical Models of CancerSelective Tumor Hypoxia Targeting by Hypoxia-Activated Prodrug

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selective tumor hypoxia targeting by hypoxia-activated ... · (sigma) in sterile buffered salt...

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