ABT-898 Induces Tumor Regression and Prolongs Survival in a Mouse Model of Epithelial ... · VEGF,...

Preclinical Development

ABT-898 Induces Tumor Regression and Prolongs Survivalin a Mouse Model of Epithelial Ovarian Cancer

Nicole Campbell1, James Greenaway1, Jack Henkin2, and Jim Petrik1

AbstractEpithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is often not diagnosed until

late stages due to its asymptomatic nature.Women diagnosedwith EOC typically undergo surgical debulking

followed by chemotherapy; however, disease recurrence often occurs. In this study,we evaluated the ability of

the thrombospondin-1 mimetic peptide, ABT-898, to regress established, late-stage tumors in a mouse model

of human EOC. Ovarian tumors were induced and ABT-898 treatment was initiated at time points that were

representative of late stages of the disease to study tumor regression. ABT-898 induced tumor regression and

reduced the morbidity of treated animals compared with controls. Analysis of tumors from ABT-898–treated

animals showed reduced abnormal tumor vasculature, decreased expression of the proangiogenic compound

VEGF, and reduced tumor tissue hypoxia. ABT-898 treatment initiated at late-stage disease also significantly

prolonged disease-free survival compared with control animals. Results from this study show that ABT-898 is

capable of regressing established ovarian tumors in an animal model of the disease. As most women

are detected at advanced stage EOC, ABT-898 may improve our treatment of ovarian cancer. Mol Cancer

Ther; 10(10); 1876–85. �2011 AACR.

Introduction

Epithelial ovarian cancer (EOC) is the most commonmalignancy of the female reproductive tract and is themost lethal gynecologic cancer. Ovarian cancer isdetected at a late clinical stage in more than 80% of thecases partly due to diffuse clinical signs and the lack ofeffective screening techniques. Current treatment forEOC involves surgical debulking followed by platinum-and taxol-based chemotherapy (1). Patients typically re-spond favorably to this treatment regimen; however, themajority of women will experience disease recurrencecharacterized by chemoresistance (2, 3). There is a needfor improved therapeutic options that will successfullytreat late-stage ovarian tumors and prevent chemoresis-tance, ultimately preventing disease recurrence.

Tumor growth is dependent upon formation of newblood vessels from preexisting vasculature, a processtermed angiogenesis. VEGF is a potent proangiogenicgrowth factor and is highly predictive of poor prognosis

in numerous cancer types (4). In contrast, thrombospon-din (TSP)-1 is an endogenous inhibitor of angiogenesisthat is often inversely expressed with VEGF andincreased TSP-1 expression is associated with a favorableprognosis (5).

Use of compounds that target angiogenic pathways toinhibit growth and metastasis of tumors have had vari-able outcomes (reviewed by A. Sanchez–Munoz; ref. 6).Initially, the goal of antiangiogenic agents was to de-crease tumor vascularity in an effort to impede nutrientdelivery and decrease metastasis, however, numerousstudies have suggested that tumor vasculature under-goes normalization, which involves pruning back theabnormal, tortuous tumor vasculature while leavingthe mature, healthy parental vessels intact (reviewedby R.K. Jain; ref. 7). This results in increased tissueperfusion, which can facilitate increased uptake of cyto-toxic agents when used in combination therapy. We havepreviously reported that the TSP-1 mimetic peptide ABT-510 decreases blood vessel density and increases theproportion of mature blood vessels, allowing for en-hanced tissue uptake of paclitaxel and cisplatin (8).The type I repeat region (TSR) of TSP-1 binds and acti-vates the receptor CD36, which mediates the antiangio-genic effects of TSP-1 such as increased apoptosis andimpaired migration and chemotaxis (9, 10). We haveshown in a mouse model of EOC that this region iscapable of decreasing the size of ovarian tumors andinhibiting the formation of peritoneal lesions and ascitesfluid (8, 11). A second generation TSP-1 mimetic peptide,ABT-898, has been generated that has enhanced stability,an increased half-life of 4 to 5 hours, and hasminimal side

Authors' Affiliations: 1Department of Biomedical Sciences, University ofGuelph, Guelph, Ontario, Canada; and 2Abbott Laboratories, Abbott Park,Illinois

Note:Current address for J. Henkin: Northwestern University, Evanston, IL60208.

Corresponding Author: Jim Petrik, Department of Biomedical Sciences,University of Guelph, 50 Stone Rd, University of Guelph, Guelph, N1G 2W1Canada. Phone: 519-824-4120-54921; Fax: 519-767-1450; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-0402

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 10(10) October 20111876

on July 28, 2020. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 15, 2011; DOI: 10.1158/1535-7163.MCT-11-0402

http://mct.aacrjournals.org/

effects or toxicities (12, 13). In this study, we used ABT-898 to determine its effects on late-stage epithelial ovariantumors and its ability to induce tumor regression andprolong disease-free survival.

Materials and Methods

Reagents and cell linesWe evaluated the effect of the TSP-1 mimetic peptide

ABT-898 (Abbott Labs) on murine and human endothe-lial and ovarian epithelial cells. Murine microvascularendothelial cells (mEC) were obtained from AmericanType Culture Collection (ATCC) and were cultured inDulbecco’s Modified Eagle’s Medium (DMEM) with 10%FBS and 1% ABAM, and human umbilical vein endothe-lial cells (HUVEC) were cultured in F-12K (ATCC)supplemented with 0.1 mg/mL heparin (Sigma), 0.03mg/mL ECGS (Sigma), 10% FBS, and 1% ABAM. Thefollowing epithelial cell lines were also cultured with theappropriate media: ID8 cells (DMEM), normal humanovarian surface epithelial cells [NOSE; generously donat-ed by Dr. Jinsong Liu (MD Anderson Cancer Center,Houston, TX)], OVCAR (RPMI with 20% FBS) andSKOV3 (McCoys 5A, supplemented with 10% FBS and1% ABAM). All cell lines were immediately frozen afteracquisition. Once thawed, cells were tested for morphol-ogy and growth; absence of Mycoplasma was confirmed,and the cells were used within 3 months. Phenotype ofendothelial and epithelial cells was confirmed withexpression of cell-specific markers with immunocyto-chemistry and Western blotting.

Mouse modelMice were purchased from Charles River Laboratories

andmaintained in accordance with the Canadian Councilon Animal Care. To evaluate the preclinical efficacy ofABT-898, we used an orthotopic, syngeneic mouse modelof EOC described previously (14). Briefly, transformedmurine ovarian surface epithelial cells from C57b/6 mice(ID8; 1.0 � 106) were injected directly under the ovarianbursa of syngeneicmice. In thismodel, 60 days posttumorinduction, mice form primary ovarian masses and by90 days posttumor induction, there are large ovariantumors, numerous secondary peritoneal lesions, and ab-dominal ascites. The ability of ABT-898 to induce regres-sion of established epithelial ovarian tumors wasassessed in our animal model. Tumors were allowed todevelop for 60 or 80 days posttumor induction, at whichtime treatmentwas initiatedwith i.p. once daily injectionsof ABT-898 (25 mg/kg) or D5W vehicle control (200 mL).ABT-898 has not shown any toxicity or immune responsein this syngeneic mouse model. Mice were euthanized90 days posttumor induction, which corresponded to30 days of treatment in the 60-day posttumor inductiongroup and 10 days of treatment in the 80-day posttumorinduction group. Primary tumors were collected andperitoneal tumors were assessed for metastatic spreadon the basis of a lesions scoring system in which mice

were categorized as having no observable abdominallesions, 1 to 2 lesions, between 3 to 10 lesions, or greaterthan 10 lesions as a way to quantify the extent of abdom-inal disease, as we have done previously (8).

SurvivalIn a second cohort of mice, daily treatments with ABT-

898 were initiated at 60 or 80 days posttumor induction(n ¼ 12 animals per group) and animals continued toreceive treatment until they became moribund, whichwas assessed on the basis of noticeable ascites and anincreased weight gain of 20% of their pretumor inductionbody weight. All animals were euthanized at 150 daysposttumor induction and those that were free of morbid-ity (had not developed ascites fluid) were recordedaccordingly. The ovaries of these animals were assessedhistologically to ensure that the surgical injection ofID8 cells was successful and created the presence of afocal necrotic region from which regressed tumors hadoriginated.

In vitro endothelial and epithelial cell deathABT-898–induced apoptosis in endothelial and epithe-

lial cells was evaluated with a terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay(Roche) and by immunofluorescence staining for cas-pase-3. Endothelial and ovarian tumor cells were cul-tured in serum-free media alone or with ABT-898 (50nmol/L) or vehicle control (5% dextrose in PBS) for 24hours and were then fixed in 10% buffered formalin for 1hour and permeabilized with 0.2% Triton-X for 5 min-utes. For detection of apoptosis, cells were washed withPBS and incubated with the TUNEL reaction mixture(label solution and enzyme solution) for 60 minutes at37�C in the dark. Cells were rinsed with PBS, nuclei werestained with 40,6-diamidino-2-phenylindole (DAPI), andcoverslips were mounted onto slides with Prolong Goldantifade (Invitrogen) and allowed to dry overnight. Neg-ative and positive controls were generated according tothe kit instructions. All slides were visualized using anOlympus BX-61 fluorescent microscope, and quantifica-tion of the percent immunopositive cells was done by ablinded individual using integrated morphometry soft-ware (Metamorph). For fluorescence detection of cas-pase-3, cells were incubated in the presence of a rabbitanti-human caspase-3 antibody (Cell Signaling; 1:200dilution) overnight at 4�C, then rinsed and incubatedwith rabbit Alexa Fluor 488 secondary antibody (Molec-ular Probes; 1:100 dilution) for 2 hours at room temper-ature. Brightfield and fluorescence images were capturedwith anOlympus phase contrast fluorescencemicroscopeand overlayed using integrated morphometry software(Metamorph).

ImmunohistochemistryImmunohistochemistry was done to quantify expres-

sion of the endothelial cell marker CD31 and a markerof hypoxia, carbonic anhydrase. At 90 days posttumor

ABT-898 Induces Tumor Regression in Ovarian Cancer

www.aacrjournals.org Mol Cancer Ther; 10(10) October 2011 1877




induction, following 30 days of treatment with ABT-898,ovarian tissue was harvested from animals and immedi-ately formalin fixed. Five micrometer sections were cutand mounted onto slide, deparaffinized, rehydrated,and incubated with 1% hydrogen peroxide for 10 min-utes at room temperature. Antigen retrieval was con-ducted by immersing sections in 10 mmol/L citratebuffer at 90�C for 12 minutes. Tissues were blockedfor 10 minutes at room temperature with 5% normalserum and incubated with anti-CD31 (BD BiosciencesPharmingen) or carbonic anhydrase (Abcam) antibodiesovernight at 4�C in a humidity chamber. Slides wereincubated with anti-mouse biotinylated secondaryantibody (Sigma-Aldrich) for 2 hours at room temper-ature followed by ExtrAvidin (Sigma-Aldrich) for 1hour at room temperature. Antigens were visualizedusing DAB (Sigma-Aldrich) and counterstained withCarazzi’s hematoxylin. After mounting, slides wereimaged by a blinded individual using brightfieldmicroscopy.

Evaluation of microvessel density and vesselmaturity

Microvessel density was evaluated in tissue sectionsthat were immunostained for CD31 as mentionedabove. Images were obtained at �200 magnificationand 4 fields of view per section were used. Microvesseldensity (number of vessels per field and blood vesselarea) was quantified using Metamorph integrated mor-phometry software (Molecular Devices). To evaluatevessel maturity, a marker for mature vessels was colo-calized with endothelial cells using immunofluores-cence in tumor sections. Immunostaining was donewith CD31 and alpha smooth muscle actin (a-SMA),a pericyte marker. Briefly, slides were processed asmentioned above and the CD31 antibody was incubatedfor 1 hour at room temperature. Tumor sections werewashed with PBS and antimouse secondary antibodyconjugated to Alexa Fluor 594 (Invitrogen) was appliedfor 1 hour at room temperature. Slides were then incu-bated for 1 hour at room temperature with anti-SMAprimary antibody (Fitzgerald Industries International)and secondary antibody conjugated to Alexa Fluor 488(Invitrogen) was applied for 1 hour. Tissues were coun-terstained with DAPI and mounted using Prolong Gold(Invitrogen). SMA-positive vessels indicated matureblood vessels and they were quantified as a percentageof total CD31-positive vessels. Images were captured bya blinded individual with an Olympus BX-61 micro-scope at room temperature using integrated morphom-etry software (Metamorph). SMA-positive vesselsindicated mature blood vessels and they were quanti-fied as a percentage of total CD31-positive vessels.

In vivo determination of epithelial and vascularendothelial cell apoptosis

Following treatment with D5WorABT-898 in vivo from60 days posttumor induction, tissues were collected at

90 days posttumor induction and processed for immu-nofluorescence and apoptosis. For determination ofepithelial tumor cell apoptosis, tissues from D5W- orABT-898–treated animals were analyzed using theTUNEL assay according to manufacturer’s instructions(Roche). For evaluation of vascular endothelial cell apo-ptosis, 2 adjacent tissue sections were placed on eachslide, and 1 was immunofluorescently stained for CD31,whereas the other was probed for a-SMA as above.Following incubation with Alexa Fluor 594–conjugatedsecondary antibodies, tissues were rinsed and subjectedto TUNEL analysis as described above. Following theTUNEL procedure, images of CD31, a-SMA, and TUNELwere collected, and images of adjacent sections wereoriented and overlaid using image processing software.This approach allowed us to determine the incidence ofapoptosis in the endothelium of vessels with and withoutpericyte coverage.

Expression of VEGFProtein levels of proangiogenic VEGF was determined

in vitro and in vivo using Western blot analysis. In vitro,ID8 cells were cultured in the presence of 50 mmol/LABT-898 in serum-free DMEM, supplemented with 1%antibiotic/antimycotic (Gibco) for 24 hours. For in vivoexperiments, tumor tissue was collected at 90 days post-tumor induction from mice treated with ABT-898 for10 days (80-day posttumor induction group) or 30 days(60-day posttumor induction group) and was flash fro-zen. Cells were lysed and tumor tissues were homoge-nized in radioimmunoprecipitation assay buffer withprotease and phosphatase inhibitors, and protein con-centrations were determined using a DC protein assay(Bio-Rad Laboratories). All Western blots were doneusing an XCell II Blot Module System (Invitrogen). Sam-ples (20 mg of total protein) were reduced and subjectedto SDS-PAGE. Proteins were transferred to polyvinyli-dene difluoride membranes (Bio-Rad Laboratories) andblocked at room temperature for 1 hour in 5% skim milkwith TBST. Membranes were probed for overnight at 4�Cwith primary antibody against VEGF (Santa Cruz). Fol-lowing washes with TBST, membranes were incubatedfor 1 hour at room temperature with antirabbit IgGhorseradish peroxidase–linked secondary antibodies(Cell Signaling Technology, Inc.). Expression of VEGFwas detected using Western Lightning Chemilumines-cence Reagent Plus (PerkinElmer BioSignal, Inc.) andvisualized using medical X-ray film (Konica MinoltaMedical Imaging Inc.). Computer assisted densitometrywas done using AlphaEase FC software (AlphaInnotech)and results were quantified and reported as integrateddensitometry values relative to b-actin.

Statistical analysisAll in vitro experiments contained 3 replicates and all

in vivo experiments consisted of at least 6 animals pergroup. Results from cell apoptosis, ovarian tumorweight,blood vessel density, and maturity were evaluated by

Campbell et al.

Mol Cancer Ther; 10(10) October 2011 Molecular Cancer Therapeutics1878




analysis of variance with a Fisher’s post hoc test. Toanalyze secondary lesions and ascites fluid, a c2 testwas conducted. To evaluate survival in our model, aKaplan–Meier estimator was done and significancewas determined using the log-rank test. Significancethreshold was set at 0.05 and the error bars within thegraphs are based on SD of the mean.

Results

ABT-898 induces apoptosis in endothelial andepithelial cells and significantly reduces the size ofepithelial ovarian tumorsIn this study, animals were allowed to develop epi-

thelial ovarian tumors for 60 or 80 days, which repli-cates late-stage human disease, before being treatedwith ABT-898. We evaluated the apoptotic inducingeffect of ABT-898 on cells comprising the vascularand epithelial compartments of ovarian tumors. Variousendothelial and epithelial cell lines that were treatedwith ABT-898 in culture for 24 hours exhibited a sig-nificant increase in cell death compared with untreatedcontrols (Fig. 1A and B). In human and murine endo-thelial and epithelial cancer cells, treatment with50 nmol/L ABT-898 for 24 hours resulted in approxi-mately a 5-fold increase in apoptosis. Cancer cellsshowed an increase in expression of caspase-3 followingtreatment with the peptide (Fig. 1C).Animals that received daily i.p. injections of ABT-898

for 30 days (60 days posttumor induction) or 10 days (80days posttumor induction) had a significant reduction in

tumor size compared with the size of the tumors at thestart of treatment and in comparison with the vehicle(D5W) treatment controls also collected at 90 days post-tumor induction (Fig. 2).

ABT-898 reduces the number of peritoneal lesionsand prevents formation of ascites fluid

The number of peritoneal lesions and the presence ofascites fluid were determined in animals followingtreatment of ABT-898 as described above (n ¼ 6 miceper treatment group). Four of 7 animals that receivedtreatment at 80 days posttumor induction had less than2 visible lesions in their peritoneal cavity comparedwith 1 of 6 animals that exhibited a similar frequencyof lesion formation in the D5W control group (Fig. 3).Examination of the 60-day posttumor induction grouprevealed that treatment of ABT-898 significantly low-ered the abdominal or extraovarian lesion formationcompared with controls. Five of 6 animals had 0 to 2detectable metastatic lesions and 3 had no lesionswhereas among controls, 5 of 6 animals had more than2 lesions and none of the controls was lesion free. All 6animals that received the vehicle control developedascites fluid during the study; however, treatment withABT-898 reduced the number of animals that developedascites. In the 80-day posttumor induction group, 2 of 6animals had no ascites, a significant reduction com-pared with controls. Most interestingly, there was acomplete absence of ascites in mice whose treatmentbegan at 60 days posttumor induction (30 days of ABT-898 treatment; Fig. 3).

Figure 1. ABT-898 inducesapoptosis in endothelial andepithelial cells. Murine and humancell lines were treated with 25nmol/L ABT-898 for 24 hours andapoptosis was measured viaTUNEL. Treatment with ABT-898increased the percentage ofapoptotic cells in both mEC andHUVEC (A). Transformed murinesurface epithelial (ID8), humanNOSE, and human ovariancarcinoma cell lines (OVCAR-3and SKOV-3) were also treatedwith ABT-898. All of these cellslines showed an increase inapoptosis following treatment withABT-898 compared with serum-free or vehicle controls (B). Reliefcontrast imaging showed anincrease in expression ofcaspase-3 following 24 hours oftreatment with ABT-898 in murineand human EOC cells (C). In vitroexperiments were conducted intriplicate and the assays wererepeated 3 times. *, P < 0.01.

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ABT-898 remodels the vasculature and reducestumor tissue hypoxia

The effect of ABT-898 on tissue hypoxia and tumorvasculature was evaluated in vivo. ABT-898 treatmentreduced the levels of tumor tissue hypoxia comparedwith controls, as indicated by the proportion of tissuepositive for carbonic anhydrase (Fig. 4A). Blood vesselmaturity was measured as the percentage of blood ves-sels associated with a-SMA–positive pericytes. Immuno-fluorescence colocalization of CD31 and a-SMA showedthat in late-stage disease, ABT-898 increased the propor-tion of mature vessels compared with vehicle-treatedcontrols (Fig. 4B). ABT-898 treatment resulted in a sig-

nificant decrease in tumor vessel area and blood vesseldensity. Treatment for 30 days resulted in statisticallysignificant reduction in vessel area compared with bothcontrols (P < 0.05) and 10-day treatment (P < 0.05; Fig. 4C).

ABT-898 reduces expression of VEGF in vitro andin vivo

ID8 cells treated with 50 mmol/L ABT-898 for 24 hoursexpressed lower levels of VEGF compared withthe untreated controls (Fig. 4D). Protein was collectedfrom whole tumor tissue homogenates from mice whoreceived ABT-898 for 10 days (80-day posttumor induc-tion group) or 30 days (60-day posttumor inductiongroup) or who received D5W vehicle control. Westernblot analysis showed a decrease in VEGF protein levelsafter 10 days of treatment with ABT-898 and a further

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Figure 2. Treatment with ABT-898 induces regression of epithelial ovariantumors. Tumors were allowed to progress without intervention for 60 or80 days posttumor induction. Animals were treated with 25 mg/kg/d ofABT-898 or the vehicle control D5W resulting in treatment for 30 or 10days, respectively. At 90 days posttumor induction, ABT-898–treatedtumors were significantly smaller compared with pretreatment or D5Wtreatment (n¼ 6 animals per group; *, P < 0.05; **, P < 0.01). PTI, posttumorinduction.

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Figure 3. ABT-898 reduces the number of secondary lesions and preventsformation of ascites fluid. The number of secondary lesions and thepresence of ascites fluid was assessed in the animals at 90 daysposttumor induction following treatment with ABT-898. Mice treated for10 days (80 days posttumor induction) and 30 days (60 days posttumorinduction) showed a significant reduction in the number of secondarylesions compared with D5W-treated controls. A significant reduction wasalso observed between the 60- and 80-day posttumor induction groups(A). All of the animals treated with the vehicle control (D5W) had developedascites fluid at 90 days posttumor induction. There was a significantreduction in the number of mice with ascites following treatment withABT-898. Approximately 40% of mice treated for 10 days (80 daysposttumor induction) did not present with ascites fluid. Animals treated at60 days posttumor induction did not show any signs of ascites fluidproduction, showing the ability of ABT-898 to inhibit its formation (B). n¼ 6animals per group; in A, *, P < 0.05 compared with D5W control; in B, barswith different symbols are statistically different, P < 0.05.

Campbell et al.





reduction in VEGF protein in the 60-day posttumorinduction group (Fig. 4D).

ABT-898 induces apoptosis in endothelial cells ofimmature but not mature blood vesselsDouble-labeling immunofluorescence, coupled with

TUNEL analysis allowed us to view endothelial cell

apoptosis in immature (without pericyte coverage)and mature (pericyte covered) tumor vessels. ABT-898 induced apoptosis predominantly in vessels thatwere void of pericytes, as indicated by a lack of a-SMAcostaining (Fig. 5). Endothelial cells from mature, peri-cyte-covered vessels were typically void of apoptosis(Fig. 5).

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Figure 4. Treatment with ABT-898 reduces tumor tissue hypoxia, alters the vascular profile, and decreases VEGF protein levels in vitro and in vivo.Ovaries harvested from mice treated with ABT-898 for 30 days (ABT-898 60 d posttumor induction group) were immunostained for carbonic anhydraseto evaluate tumor hypoxia (A). There was a significant reduction in the percent of immunopositive staining in tumors from animals that were treateddaily with ABT-898 compared with controls (B). Colocalization experiments conducted with CD31 (red) and SMA (green) revealed that following treatment withABT-898, there was an increase in the proportion of pericyte-covered mature blood vessels at 60 and 80 days posttumor induction (C). n ¼ 6 animalsper group. Bars, A, 150 mm; B, 120 mm. Ovaries were collected from animals treated with ABT-898 for 30 (60 days posttumor induction) and 10 (80 daysposttumor induction) days and immunohistochemical analysis investigated vasculature parameters. Treatment with ABT-898 significantly (P < 0.05)reduced the blood vessel density (top) and tumor vessel area (bottom) at both time points. A further significant reduction in tumor vessel area was noted in the60-day posttumor induction group compared with 80 days posttumor induction (C). n ¼ 6 animals per group; bars with different symbols are statisticallydifferent, P < 0.05. ABT-898 reduced VEGF protein levels in vitro and in vivo. ID8 cells were either untreated (Cntrl) or treated with 50 nmol/L ABT-898for 24 hours in serum-free media (D). Ovarian tumor tissue was collected at 90 days posttumor induction from untreated mice (Cntrl) or mice in whichABT-898 (25 mg/kg) treatment was initiated at 60 or 80 days posttumor induction (D). In vitro experiments were conducted in triplicate and repeated 3 times,whereas in vivo experiments included 6mice per treatment group. The above blots depict an average representative fromeach group. PTI, posttumor induction.






ABT-898 prolongs survival in a mouse model ofepithelial ovarian cancer

ABT-898 treatment caused a significant reduction inthe size of ovarian tumors (Fig. 2). To determine whetherthis treatment had an impact on survival, we continuedtreatment and assessed for signs of morbidity. Treatmentbegan at 60 or 80 days posttumor induction and contin-ued until animals became moribund, which was definedas abdominal distension due to ascites fluid accumula-

tion. Animals whose treatment began at 80 and 60 daysposttumor induction had a significantly longer survivalcompared with controls. Mice that received ABT-898 at80 days had a mean survival of 108 days posttumorinduction compared with 98 days for controls. Treatmentthat started at 60 days posttumor induction resulted inthe animals living on average 130 days compared with94 days posttumor induction. The experiment was ter-minated at 150 days posttumor induction and all animalswere euthanized independent of any signs of morbidity.Approximately 27% of mice with treatment started at80 days and 46% of mice starting at 60 days posttumorinduction had no sign of disease such as abdominaldistension, coat condition deterioration, or lethargyand at 150 days posttumor induction had no grosslyobservable ovarian tumors at euthanasia (Fig. 6A and B).

Long-term treatment of ABT-898 significantlyreduces the number of peritoneal lesions

Mice involved in the survival study were scored for thepresence of peritoneal lesions at the time of necropsy.Mice, in which treatment was initiated at either 60 or80 days posttumor induction, continued to receive treat-ment until they became moribund (or until the experi-ment was terminated at 150 days posttumor induction).Animals that received treatment had significantly fewerlesions when sacrificed compared with untreated con-trols. The 80 days posttumor induction group had areduction in the number of lesions, whereas the 60 daysposttumor induction group had greater than 50%, withno signs of peritoneal disease (Fig. 6C and D).

Discussion

We have shown that the TSP-1 mimetic, ABT-898, caninduce regression of advanced-stage EOC and that itinduces apoptosis of tumor cells and endothelial cells ofimmature tumor blood vessels. Although several studieshave targeted the VEGF family in ovarian cancer(15–18), another effective approach for the treatment ofvarious cancers are peptides derived from the TSR anti-angiogenic domain of TSP-1 (19). Numerous studies haveshown the antiangiogenic and antitumor effects of 3TSR(20–22) and ABT-510 (11, 23, 24) and have shown safetyand efficacy in animal and human studies (12, 13, 25, 26).WehavepreviouslyevaluatedABT-510 in the treatmentofEOC and reported decreased tumor burden as well as anincrease in theuptakeof the chemotherapydrugs cisplatinand paclitaxel through vascular normalization (8).

Due to the relatively asymptomatic nature of EOC,approximately two-thirds of women are not diagnoseduntil advanced stages (stage III, IV; ref. 27), at which timedisease has spread throughout the peritoneum and the5-year survival rate is only approximately 37% (28, 29). Inour animal model, we can replicate late-stage diseasebefore beginning treatment, which mimics the currentclinical situation. In this study, we evaluated the abilityof ABT-898 to regress established ovarian tumors and

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Figure 5. ABT-898 induces apoptosis in tumor cells and in endothelialcells from immature blood vessels. AT 90 days posttumor induction,tissues from control, and ABT-898–treated mice were subjected tobrightfield TUNEL analysis (A) or immunofluorescence colocalization ofmarkers for endothelial cells (CD31), pericytes (a-SMA), and apoptosis(TUNEL; B). ABT-898 treatment caused a significant increase in tumorcell apoptosis. Endothelial cell death typically was observed in bloodvessels that were not associated with pericytes and ABT-898 treatmentresulted in fewer blood vessels with apoptotic endothelial cells.Bar, 120 mm. n ¼ 6 mice per group; *, statistically significant differencebetween groups (P < 0.05).

Campbell et al.





prolong disease-free survival. ABT-898 induced apopto-sis in both vascular endothelial and tumor cells, resultingin reduced tumor size and decreased tumor microvesseldensity. This combined antiangiogenic and antitumoreffect may explain the potent regression of advancedtumors following relatively short treatment periods of10 or 30 days. Recently, ABT-898 has been shown to havea similar combined antiangiogenic and proapoptotic in-fluence on ovarian cells, in which it inhibits follicularangiogenesis and concomitantly induces atresia of antralfollicles (30). Antiangiogenic therapies may act to prunetumor vessels, removing the immature and abnormaltumor vessels resulting in vessel normalization and anincreased perfusion pressure of the residual tumor tissue(31, 32). In our study, ABT-898 specifically inducedendothelial cell death in immature tumor vessels thatwere not associated with pericytes, and followingtreatment, the remodeled blood vessels were morefunctional with enhanced vascular supply. Vesselpruning by ABT-898 is supported by other studies thathave shown a proapoptotic effect of TSP-1 on endothelial

cells in vitro and in vivo (20, 33–35). By specifically target-ing immature tumor vessels, ABT-898 seems to havelimited impact on established vasculature and mayexplain why we have not seen changes in the vasculaturein other peritoneal organs that are exposed to this TSP-1mimetic. ABT-898 also exerts a proapoptotic effect on theepithelial cell compartment, which likely contributed tothe significant decrease in tumor mass seen at both the60- and 80-day posttumor induction groups. TSP-1 hasalso been shown by us and others to induce apoptosis innonendothelial leukemias and ovarian cancer cells,which may provide an important contribution to itsantitumor activity in addition to its antiangiogenic effects(8, 11, 36, 37). Tumor vasculature exhibits inefficient flowcausing numerous hypoxic areas with limited bloodsupply (38, 39) resulting in suboptimal drug deliverythat can facilitate drug resistance, allowing the tumorto persist and grow (40, 41). By increasing vascularsupply to a smaller tumor, ABT-898 may increase thedelivery and uptake of chemotherapy drugs, enhancingtheir effectiveness and reducing drug resistance.

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Figure 6. ABT-898 prolongs disease-free survival and reduces the number of secondary lesions. In a separate cohort of mice, animals continued toreceive daily i.p. injections of ABT-898 until they became moribund. Animals treated at 80 days posttumor induction had a significant increase in survivalcompared with D5W controls. The mean survival in this group was 108 days compared with 98 days for the controls. At 150 days posttumor induction,27% of the mice did not exhibit signs of disease (A). There was also an overall increase in survival in animals treated at 60 days posttumor induction.These mice were disease free for 130 days on average compared with 94 days posttumor induction for the controls. At the duration of the study, 46% ofthe mice in this group did not display signs of disease (B). n ¼ 15 animals per group. Animals that commenced treatment at 80 days posttumor inductionhad a significant reduction in the number of secondary lesions compared with controls. On average, treated mice had a score of 2 to 10 lesions comparedwith more than 10 lesions that the controls exhibited (C). There was also a significant reduction in the number of secondary lesions in the 60-dayposttumor induction group. More than 50% of treated mice did not have any lesions compared with controls that had around 10 lesions (D). n ¼ 15 animalsper group; *, P < 0.05.






During ovarian tumor growth, tumor cells typicallybegin to shed from the surface, a process known astranscoelomic metastasis (42). These cells will then seedthroughout the peritoneal cavity and attach to the abdom-inal wall, the surfaces of peritoneal organs, and mesen-teries. The cells will coalesce and formwell-differentiatedsecondary tumors with recruitment of an independentblood supply. The proangiogenic drive from the primarytumor, along with the multiple secondary peritoneallesions, often present/generate significantly increasedexpression of proangiogenic VEGF (43–45). In additionto its proangiogenic effect, VEGF is a potent stimulator ofvessel permeability (46, 47). It is thought that increasedtumor vessel permeability contributes to fluid extravasa-tion in primary and secondary ovarian tumors, and,ultimately, the accumulation of abdominal ascites. Inour study, ABT-898 reduced VEGF protein levels in ID8cells cultured in vitro and in collected tumor tissue, whichwould contribute to the reduced blood vessel density andincreased tumor cell apoptosis observed in this model.Mice that were treated with ABT-898 at 60 and 80 daysposttumor induction had a significant reduction in thenumber of secondary lesions comparedwith controls. Thereduced number of peritoneal lesions was also associatedwith reduced ascites following treatment. Most notablewas the fact that the animals in the 60-day posttumorinduction group had a complete absence of ascites fluid.

Treatment with ABT-898 significantly prolonged dis-ease-free survival in both groups and a number of ani-mals were completely void of any signs of disease at 150days. Of the animals that did present with morbiditybefore 150 days, survival may have been enhanced byperiodic draining of abdominal ascites or a combinationaltherapy including chemotherapy drugs such as cisplatinor taxol, which are procedures carried out clinically.

Despite some of the mice in the 60-day posttumor induc-tion treatment group having to be euthanized early due toaccumulation of ascites fluid, more than half of theseanimals did not display peritoneal lesions. It seems thatABT-898 treatment in these animals eliminated the peri-toneal lesions, but ascites were still being generated fromthe residual primary tumor. In these mice, treatment wasencouraging as most women succumb to EOC due toperitoneal disease rather than primary tumor burden.

Currently, the standard of care for patients with EOCinvolves cytoreductive surgery followed by chemother-apy. Over the past few decades, optimization of thistreatment regime has resulted in a slight overall increasein survival (2); however, the long-term survival rateremains low. Results from this study suggest that ABT-898 may be beneficial in the treatment of late-stage EOCand may improve survival for women diagnosed withthis disease.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowlegments

We thank Michelle Ross for technical assistance.

Grant Support

This work was supported by a research grant from the Canadian Institutes ofHealth Research (596213).

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 indicate thisfact.

Received June 3, 2011; revised August 5, 2011; accepted August 10, 2011;published OnlineFirst August 15, 2011.

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2011;10:1876-1885. Published OnlineFirst August 15, 2011.Mol Cancer Ther Nicole Campbell, James Greenaway, Jack Henkin, et al. Mouse Model of Epithelial Ovarian CancerABT-898 Induces Tumor Regression and Prolongs Survival in a

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