Reduced Growth, Increased Vascular Area, and Reduced ...clincancerres.aacrjournals.org/content/clincanres/10/3/1180.full.pdf · gest that CD13 overexpression affects ovarian cancer

Reduced Growth, Increased Vascular Area, and Reduced Responseto Cisplatin in CD13-Overexpressing Human OvarianCancer Xenografts

Yvette van Hensbergen,1 Henk J. Broxterman,1

Sareena Rana,1 Paul J. van Diest,2

Monique C. A. Duyndam,1 Klaas Hoekman,1

Herbert M. Pinedo,1 and Epie Boven1

Departments of 1Medical Oncology and 2Pathology, Vrye UniversiteitMedical Center, Amsterdam, the Netherlands

ABSTRACTPurpose: Expression of aminopeptidase N/CD13 can be

detected in several solid tumor types. Thus far, the role ofCD13 in ovarian cancer has not been studied. We haveinvestigated the expression pattern and biological functionof CD13 in ovarian cancer.

Experimental Design: First, we studied the expression ofCD13 in ovarian cancer tissue of 15 patients representingthree different histological types (5 patients each) by immu-nohistochemistry. We then stably transfected the IGROV-1human ovarian cancer cell line with a CD13 expressionvector and examined the biological effect of CD13 in vitroand in vivo.

Results: The expression of CD13 in ovarian cancer wasassociated with the histological subtype: CD13 expression intumor cells was observed in 80–100% of the patients with aserous or mucinous carcinoma and in only 20% of the clearcell carcinoma patients. In all patients’ tumor samples,CD13-positive blood vessels were present. CD13 overexpres-sion in IGROV-1 cells did not affect in vitro cell growth andsensitivity to doxorubicin, cisplatin, or gemcitabine. CD13overexpression reduced invasion in Matrigel, which ap-peared to be independent of the aminopeptidase activity ofCD13. Furthermore, the growth rate of IGROV-1/CD13xenografts was reduced. The area of the vessel lumens wasenlarged in a small percentage of vessels in the CD13-overexpressing xenografts. In addition, the CD13-overex-pressing tumors were less sensitive to cisplatin.

Conclusions: CD13 is expressed in tumor as well asendothelial cells in human ovarian cancer. Our results sug-

gest that CD13 overexpression affects ovarian cancergrowth, vascular architecture, and response to chemother-apy. Further elucidation of the mechanism of the observedeffects of CD13 is warranted to better understand its role inthe pathophysiology of ovarian cancer.

INTRODUCTIONOvarian cancer is the most important cause of death among

the gynecological malignancies. This is caused mainly by thelate diagnosis of the tumor. The biological behavior of the tumoris associated with clinicopathological parameters, such as Inter-national Federation of Gynecologists and Obstetricians (FIGO)stage, grade, and tumor type (1). Recently, new molecularmarkers with potential diagnostic use in ovarian cancer havebeen identified in patients. CD24, a ligand of P-selectin, as wellas maspin, a noninhibitory member of the serpin family, havebeen identified as adverse prognostic factors (2, 3). Conversely,in a mouse model, dipeptidyl peptidase IV (CD26) expression inSKOV-3 cells significantly decreased dissemination and in-creased survival time (4).

Aminopeptidase N (CD13; EC 3.4.11.2) is a transmem-brane ectopeptidase of Mr 150,000 that is highly expressed oncells of the myeloid lineage and on nonhematopoietic tissues,such as fibroblasts, epithelial cells of the kidney and liver, andpericytes in the brain (5–7). High expression of CD13 has beendetected in various solid tumors (6–8). The expression level ofCD13 was found to correlate with increased malignant behaviorin prostate cancer and colon cancer (9, 10). In contrast, in renalcell cancer, CD13 expression has been reported to decrease withmalignant progression (11). In different studies, a role for CD13in degradation and cellular invasion of the extracellular matrix(ECM) has been established (9, 12, 13). It has been shown formelanoma cells that overexpression of CD13 significantly in-creased invasion in Matrigel, possibly related to an enhanceddegradation of collagen type IV (14). Recently, CD13 has beenassociated with angiogenesis, the formation of new blood ves-sels that is required for tumor growth. CD13 expression in tumormicrovascular endothelial cells was shown to be induced byangiogenic cytokines and hypoxia. Inhibition of CD13 activityreduced endothelial cell tube formation on Matrigel and inhib-ited angiogenesis in various in vivo model systems (9, 15, 16).

We have recently found that the activity of a soluble formof CD13, probably originating from shedding of the membraneprotein, was elevated in malignant ascites from ovarian cancerpatients (17). Given the lack of information on CD13 expressionin human ovarian cancer, we set out to study its expression byimmunohistochemistry in ovarian cancer samples. In addition,as a preclinical model, we developed a CD13-overexpressinghuman ovarian cancer cell line (IGROV-1) to study the effectsof CD13 on tumor biology and vascular development in tumorsgrown in nude mice.

Received 3/31/2003; revised 10/2/2003; accepted 10/16/2003.Grant support: Spinoza Award (to H. M. P.) and Stichting VanderesGrant 65.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 toindicate this fact.Requests for reprints: Henk J. Broxterman, Vrye Universiteit MedicalCenter, Deparment of Medical Oncology, P. O. Box 7057, 1007 MBAmsterdam, the Netherlands. Phone: 31-20-444-2632; Fax: 31-20-444-3844; E-mail: [email protected].

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MATERIALS AND METHODSCells. IGROV-1 human ovarian carcinoma cells (18) and

stably transfected derivatives were cultured in DMEM (Bio-Whittaker) with 20 mM HEPES (Bio-Whittaker), 10% FCS(Invitrogen), 100 units/ml penicillin, and 0.1 mg/ml streptomy-cin.

Plasmid Constructs and Transfection. The pZIP-CD13-SV(x)1 vector and the pZIP-neo-SV(x)1 vector were akind gift of Dr. L. H. Shapiro (University of Connecticut HealthCenter, Farmington, CT). IGROV-1 cells were transfected withpZIP-CD13-SV(x)1 or with control vector pZIP-neo-SV(x)1 bythe calcium phosphate precipitation method (19). After a 6-hincubation period, the cells were washed and left with freshmedium for 16 h. Subsequently, stable transfectants were se-lected and maintained with Geneticin (450 �g/ml; G418; LifeTechnologies, Inc.). Resistant clones were isolated (IGROV-1/neo, IGROV-1/CD13-7, and IGROV-1/CD13-9) and character-ized for CD13 mRNA expression and CD13 protein expressionand activity.

CD13 Detection in IGROV-1 Cells. Expression ofCD13 mRNA was examined by reverse transcription-PCR. To-tal RNA from the cell lines was reversed transcribed to cDNA.For reverse transcription-PCR, the following human-specificprimers were used: for CD13, forward primer 5�-GTAATAC-GACTCACTATAGGGCCAGGGGCCTGTACGTTTTTA-3�and reverse primer 5�-AATTAACCCTCACTAAAGGGCCAC-CAGCTCAGTCTTGTCA-3�; for glyceraldehyde-3-phosphatedehydrogenase (GAPDH), forward primer 5�-ACCACAGTC-CATGCCATCAC-3� and reverse primer 5�-TCCACCACCCT-GTTGCTGTA-3�. The primers were confirmed to be humanspecific by testing mouse fibroblasts, liver, and kidney. Thesamples were denatured for 5 min at 95°C; PCR was performedin 36 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C,followed by 5 min at 72°C.

The expression of CD13 protein was examined with fluo-rescence-activated cell-sorting analysis and immunofluores-cence. For fluorescence-activated cell-sorting analysis, cells of70% confluence were harvested by short trypsinization, washed,and suspended in PBS containing 1% BSA. Cells (1 � 105)were incubated at room temperature for 30 min with FITC-conjugated antihuman CD13 monoclonal antibody WM-47 (5�g/ml; DAKO) or FITC-conjugated mouse IgG1 (DAKO). Thecells were washed and analyzed on a FACSCalibur flow cytom-eter (Becton Dickinson).

For immunofluorescence, 2 � 104 cells were plated on acoverslip and allowed to attach for 16 h. Subsequently, the cellswere washed with PBS and incubated with FITC-conjugatedantihuman CD13 monoclonal antibody WM-47 (5 �g/ml;DAKO). Fluorescence was visualized on a confocal laser scanmicroscope (Leica TCS 4D) using a krypton-argon laser (�exc of488 nm and �em of BP-530/30 nm) and a �40 oil lens.

Aminopeptidase Activity Assay. Surface aminopepti-dase activity of intact cells was measured by plating 5000cells/well in a 96-well plate. Cells were allowed to recover for16 h. Subsequently, the cells were washed with PBS and incu-bated with 100 �M L-alanine-4-methyl-7-coumarinylamide trif-luoroacetate (Fluka) in PBS with 0.5% BSA and 20 mM HEPES(pH 7.2). The release of the fluorescent product 7-amido-4-

methylcoumarin was monitored on a Spectrafluor multiplatereader (Tecan; �exc of 360 nm and �em of 465 nm) every 5 min.The aminopeptidase activity was calculated from the slope ofthe fluorescence-time curve, using a calibration curve of 7-amido-4-methylcoumarin (Fluka; Ref. 20). The activity-block-ing antibody WM-15 5 �g/ml; PharMingen; Ref. 21) was usedto determine the CD13-specific aminopeptidase activity.

Cell Doubling Time. Cells growing in log phase wereharvested and plated in duplicate in a 6-well plate at 7.5 � 104

cells/well in DMEM �10% FCS. The number of cells wascounted at 24, 48, 72, and 96 h. The cell doubling time in logphase was determined in three separate experiments.

In Vitro Drug Sensitivity. The antiproliferative effectsof doxorubicin, gemcitabine, and cisplatin were measured in astandard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. Briefly, 3000 cells were plated in a 96-well platein triplicate, and the cells were exposed to a drug concentrationrange for 72 h. The number of viable cells was determined by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideassay and measurement on a Spectrafluor multiplate reader(Tecan; absorption 540 nm). The results were expressed as IC50

values, which indicate the concentrations resulting in a 50%reduction in growth as compared with control cell growth. Theexperiment was repeated three times.

Matrigel Invasion Assay. The Matrigel invasion assaywas performed in a 24-well Transwell system (Falcon) with8-�m pore-size filters (HTS Fluoroblock Insert; Becton Dick-inson) coated with 1% gelatin (Merck) on the lower side andMatrigel on the upper side (1 �g/filter; ECM gel; Sigma). Cellswere harvested and resuspended in DMEM with 0.1% BSA.From each cell line, 2 � 105 cells were added to the uppercompartment (in triplicate), and DMEM plus 10% FCS wasadded to the lower compartment. Bestatin (Sigma) was added toboth compartments. The cells were incubated for 18 h at 37°Cand 5% CO2. Invasion was analyzed by the addition of 5 �M

calcein (Molecular Probes) to the lower compartment during thelast 30 min of the assay. Fluorescence from the released-acetoxymethyl ester calcein was measured in a Spectrafluormultiplate reader (�exc of 492 nm and �em of 535 nm). The totalnumber of invaded cells was calculated using a calibration curveof 100–10,000 cells (for each cell line) incubated for 30 minwith calcein-AM.

Adhesion Assay. A 96-well plate was coated overnightwith 10 �g/ml Matrigel at 4°C. Cells were released from theflask with 5 mM EDTA and resuspended in DMEM �10% FCS.From each cell line, 2 � 105 cells (in triplicate) were allowed toadhere for 1 h at 37°C and 5% CO2. Nonadherent cells wereremoved by washing with PBS. Adherent cells were quantifiedwith the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay.

Real-Time PCR. For the quantitative real-time PCR,total RNA was extracted with RNAzol (Teltest Inc.) from tumorfragments and reverse transcribed into cDNA. For CD13 andGAPDH, the same primer sets were used as described for thereverse transcription-PCR. For vascular endothelial growth fac-tor (VEGF), we used the primer set described previously (22):forward primer 5�-AGCAAGGCCCACAGGGATTT-3�; andreverse primer 5�-ACCGCCTCGGCTTGTCAC-3�. The VEGFprimer set recognizes both human and mouse cDNA. Two �l of

1181Clinical Cancer Research



cDNA (diluted 1:2 with H2O) was amplified with 2 �l ofLightCycler FastStart DNA Master SYBR Green (Roche), 3.3mM MgCl2, and 0.5 mM primers in a total volume of 20 �l. Thereaction was performed in a rapid PCR amplification consistingof an initial denaturation step at 95°C for 10 min, followed by45 cycles of denaturation at 95°C for 0 s, annealing at 54°C for20 s, and amplification at 72°C for 18 s. Specificity of theamplified product was determined from the melting curve of15 s with a target temperature of 50°C (one peak) and byvisualization on gel (one band). The cycling conditions weresimilar for all primer sets. GAPDH was used as internal standardand reference gene.

Relative mRNA expression was calculated by:(E�Cp target gene)/(E�Cp reference gene), in which E � efficiencyand Cp � crossing point. The efficiency (GAPDH, 1.77; CD13,1.89; VEGF, 1.69) was calculated for all target and controlgenes via a cDNA concentration range. Six tumors from the firstand second passage were analyzed at least twice on separateoccasions.

Xenograft Growth. Cells growing in log phase wereharvested by trypsinization. Per cell line, 1 � 107 cells wereinjected s.c. in both flanks of three female nude mice (Harlan,Horst, the Netherlands). On growth, the tumors were measuredin three dimensions, and tumor volume was expressed in mm3 ascalculated by the following formula: length � width � thick-ness � 0.5. The volumes were measured twice weekly startingat a tumor volume of �20 mm3. The mean tumor volumes frompassage 1 were used to draw the growth curves. From the firstpassage, tumor fragments of 2–3 mm were transplanted s.c. inboth flanks of further recipients. The tumors in the secondpassage were measured twice weekly, and tumor volume wasexpressed in mm3 as described above. To determine two volumedoubling times, tumor growth was expressed by use of therelative tumor volumes. To that end, the first tumor volumemeasured was designated V0 on day 0, and the next measure-ments were set as the ratio Vt/V0 in which Vt is the tumor volumeat any time. Two tumor volume doubling times were calculatedfor each individual tumor, defined as the number of days neededfor a tumor to grow from a ratio of 1 to a ratio of 4.

Xenograft Treatment with Cisplatin. In the secondpassage of xenograft growth, separate tumor-bearing mice wereused for treatment with cisplatin. At a volume of 100–150 mm3

(designated as day 0), treatment started in groups of 3–6 mice(6–12 tumors), whereas the control mice (groups of 3–6 mice;6–12 tumors) did not receive treatment. Cisplatin (Bristol-Myers Squibb, Woerden, the Netherlands) was given at themaximum tolerated dose of 5 mg/kg, i.v., weekly �2 (23). To

determine drug efficacy, mean tumor volumes were used, andthe formula T/C% � [(mean tumor volume of treated tumors)/(mean tumor volume of control tumors)] � 100% was calcu-lated on each day of tumor measurement. Growth inhibition wasexpressed as 100% � T/C%. Mean tumor volumes were alsoused to draw the growth curves.

S-Phase Fraction in Tumor Sections. Four differentxenografts of each cell line out of two independent experimentswere analyzed for S-phase fraction. Frozen tumor tissue wasmanually cut into small pieces in a citrate buffer as describedpreviously (24). After a 10-min centrifugation at 3,000 rpm, thesupernatant was removed, and trypsin was added to the pellet for10 min at room temperature. Subsequently, trypsin inhibitor wasadded for 10 min at room temperature. After filtering the sus-pension, diamidino-phenyl-indole-dihydrochloride and sperm-ine tetra-hydorchloride were added, and the sample was kept inthe dark for at least 15 min. All samples were measured in thesame session on the same day using a Partec PAS mercurylamp-based flow cytometer (Partec, Munster, Germany). Atleast 20,000 events were measured for each sample. Trouterythrocytes were used as calibration cells. The resulting DNAhistograms were exported to ASCII format for semiautomatedanalysis of the S-phase fraction with the MultiCycleR program(Phoenix Flow Systems, San Diego, CA) as described previ-ously (25).

Immunohistochemistry. Frozen tissue of ovarian carci-noma of patients was obtained from the tumor bank of thepathology department. IGROV-1, and IGROV-1/neo, IGROV-1/CD13-7, and IGROV-1/CD13-9 xenografts in the first andsecond passage were removed at a size of 300–500 mm3 andfrozen immediately in liquid nitrogen. Cryostat tissue sections(4 �m) were mounted on SuperFrostPlus slides (Menzel-Glaser)and stored at �20°C. The sections were fixed with acetone andsubsequently incubated with 10% serum of the animal speciesfrom which the secondary antibody was obtained, followed by a60-min incubation with the primary antibodies (for antibodies,see Table 1) and, after that, a 45-min incubation with a biotin-labeled secondary antibody. For color development, the slideswere incubated with peroxidase-labeled streptavidin (DAKO)and the substrate 3-amino-g-ethylcarbazole (DAKO). In be-tween all steps, the slides were washed with PBS. Slides werecounterstained with hematoxylin. In the negative controls, theprimary antibody was replaced with IgG; human placenta wasused as positive control. An experienced pathologist (P. J. v. D.)performed scoring of immunohistochemistry in the patients’tumor samples in a blinded manner. Staining of the xenografts

Table 1 Antibodies used for immunohistochemistry

Antigen AntibodyConcentration

(�g/ml) Speciesa Source

Human CD13 WM-15 10 Mouse(m) PharMingenHuman CD31 JC/70A 5 Mouse(m) DAKOMouse CD31 CD31 (MEC13.3) 5 Rat(m) PharMingenMouse VEGFb VEGF (A20) 5 Rabbit(p) Santa CruzMouse bFGF FGF-2 (147) 5 Rabbit(p) Santa Cruz

a (m), monoclonal; (p), polyclonal.b VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; FGF, fibroblast growth factor.

1182 CD13-Overexpressing Human Ovarian Cancer Xenografts



was semiquantitatively scored as negative, weakly positive, orpositive by two independent observers in a blinded manner.

For counting of the number of blood vessels in xenografttissues, three representative fields (hot spots) per sectionwere selected at �2.5 magnification. Counting of the vesselsin the hot spot was performed at �40 magnification. With amicroscope (Leica TCS 4D; �10 objective) attached to avideocamera (Kappa CF8/1 FMCC), images (5 images/sec-tion) of the blood vessels throughout the whole tumor sectionwere randomly taken. For all of the lumen-containing vesselsin the image, the area was measured and analyzed with LeicaQ500MC/QWin software. Four different xenografts from twoindependent experiments (first passage) of each cell line wereanalyzed.

Statistics. All results are presented as mean SE. Sta-tistical differences between mean values obtained in the in vitroassays and in vivo tumor growth were calculated with paired ttests. The Mann-Whitney test was used for calculation of sta-tistical differences between the blood vessel areas. The statisti-cal difference for the CD13 expression between tumor subtypeswas determined with the Pearson 2 test. Differences wereconsidered significant when P was �0.05.

RESULTSCD13 Expression in Human Ovarian Cancer. Repre-

sentative immunohistochemical stainings of frozen ovarian car-cinomas are shown in Fig. 1. Four of five serous ovariancarcinomas exhibited membranous CD13 antigen expression ontumor cells (80% positive). Of the mucinous type carcinomas,four patients displayed apical CD13 antigen expression, whereas

focal staining of CD13 antigen was observed in one patient(100% positive). Only one of the five clear cell cancers (20%)expressed CD13 antigen on tumor cells. The number of CD13-positive clear cell tumors was significantly lower when com-pared with that in serous and mucinous tumors (P � 0.02). In alltumors, a small number of blood vessels were CD13 positive. Inmost tumors CD13-positive macrophages could be detected(Table 2). This result clearly showed that a high percentage ofovarian carcinomas contain CD13-positive malignant cells.Therefore, we decided to study the effect of CD13 expression ontumor and vascular biology in a human ovarian cancer xenograftmodel.

CD13 mRNA and Protein Expression in IGROV-1Cells. All cell lines were analyzed for CD13 mRNA andCD13 protein expression (Fig. 2, A and B; Table 3) and amino-peptidase activity (Table 3). Expression of CD13 mRNA orCD13 protein could not be detected in IGROV-1/parent andIGROV-1/neo clones. High expression of CD13 mRNA andprotein was present in the CD13-transfected cells. The proteinexpression in IGROV-1/CD13-7 was heterogeneously distrib-uted (small shoulders in the fluorescence histogram were seen),whereas in IGROV-1/CD13-9, it was more homogeneous. Withconfocal laser scan microscopy immunofluorescence, theplasma membrane localization of CD13 in IGROV-1/CD13-7and IGROV-1/CD13-9 was visualized (Fig. 2C). Proper cata-lytic function of the transfected CD13 protein was verified usinga CD13-specific neutral aminopeptidase activity assay (Ref. 20;Table 3). CD13 activity correlated significantly with the CD13expression measured by fluorescence-activated cell sorting (r �0.994; P � 0.006). Taken together, these results demonstrate a

Fig. 1 CD13 expression in human ovarian cancer samples. Frozen tissues of ovarian cancer of patients were stained with an antihuman CD13antibody (WM15; 10 �g/ml). Representative examples of a serous tumor (A), a mucinous tumor (B), and a clear cell tumor (C) are shown (�10magnification; black arrow, tumor cell staining; open arrow, blood vessel staining).

Table 2 CD13 expression in ovarian carcinoma samples of patients

Tumor type Tumor cell Pattern Vesselsa Stromal cellsb

Serous 80%c Plasma membrane (4) 100% 40%Mucinous 100%c Plasma membrane (5); apical (4)/focal (1) 100% 40%Clear cell 20% Plasma membrane (1) 100% 20%

a In 100% of the tumors, �1 CD13-positive blood vessels were observed.b In 20–40% of the tumors, CD13-positive stromal cells were observed.c Significantly different CD13 expression as compared with the clear cell subtype (P � 0.02).




high overexpression of active membrane-bound CD13 inIGROV-1 cells.

High CD13 Expression Does Not Affect Proliferationand Drug Sensitivity in Vitro. For characterization of thetransfected cells, cell proliferation was measured by calculatingthe doubling time of the cells in vitro. As described in Table 4,no alterations in doubling time were observed. This was con-firmed by the cell cycle distribution pattern, as measured withpropidium iodide, which was similar for all cell lines (data notshown). Additionally, we determined the sensitivity of the cellsfor doxorubicin, cisplatin, and gemcitabine. No significant dif-ferences in in vitro sensitivity were observed for the tested drugsamong the cell lines (Table 4).

Tumor Cell Invasion Through Matrigel Is Reduced byHigh CD13 Expression. In a Matrigel invasion assay (Tran-swell system), the effect of CD13 on the invasive capacity of the

cells was determined. Without the chemoattractant FCS, all cellsinvaded similarly (909 101 cells). In the presence of FCS,invasion was stimulated 5.6 0.2-fold for IGROV-1/parent and4.6 0.3-fold for IGROV-1/neo (P � nonsignificant comparedwith IGROV-1/parent; Fig. 3). The CD13-overexpressing cell linesresponded less to FCS. FCS stimulated the invasion of IGROV-1/CD13-7 2.7 0.4-fold (P � 0.03 compared with IGROV-1/parent) and only 1.7 0.1-fold for IGROV-1/CD13-9 (P � 0.01compared with IGROV-1/parent). The addition of 250 �M bestatin,a synthetic inhibitor of CD13 activity that inhibits aminopeptidaseactivity by 90% at 15 �M (under our experimental conditions), didnot affect invasion of the IGROV-1/parent (121 14% of FCS-stimulated invasion) or that of the CD13-overexpressing cells(109 5% of FCS-stimulated invasion). This suggests that thecatalytic activity of CD13 is not responsible for the decreasedinvasion of the CD13-overexpressing cells.

Fig. 2 CD13 mRNA and protein in IGROV-1and IGROV-1 transfectants. A, a representativeillustration of mRNA expression of CD13 andglyceraldehyde-3-phosphate dehydrogenase (GAPDH)as determined by reverse transcription-PCR in IGROV-1/parent (1), IGROV-1/CD13-9 (2), IGROV-1/CD13-7(3), and IGROV-1/neo (4) cells (M, marker).GAPDH is used as a reference gene. B, a repre-sentative illustration of CD13 by fluorescence-activated cell sorting. The CD13-7 and CD13-9clones show high expression of CD13. In IGROV-1/parent and IGROV-1/neo, the intensity of CD13-FITC (thick black line) overlapped with IgG-FITC(thin black line), demonstrating the absence ofCD13 in these cells. C, cells were stained withFITC-labeled CD13 antibody, and fluorescent(a�d) and transmission (e�h) images were takenwith confocal laser scan microscopy. No fluores-cence was observed in IGROV-1/parent (a) andIGROV-1/neo (b), whereas a clear plasma mem-brane staining was observed in IGROV-1/CD13-7(c) and IGROV-1/CD13-9 (d).




High CD13 Expression Slightly Reduces Adhesion toMatrigel. The adhesion of the cell lines to Matrigel wascomparable for IGROV-1/parent (0.20 0.02), IGROV-1/neo(0.21 0.02), and IGROV-1/CD13-7 (0.19 0.03). In contrast,the adhesion of IGROV-1/CD13-9 was slightly reduced [0.15 0.02; a 27% reduction (P � 0.04) compared with IGROV-1/parent; Fig. 4]. The adhesion to defined ECM components(vitronectin, fibronectin, and collagen IV) was similar for allcell lines (data not shown).

CD13 Expression Reduces IGROV-1 XenograftGrowth. To examine the effect of CD13 overexpression ontumor growth, all cell lines were injected s.c. into nude mice,and tumor growth was measured in the first and second passage.Fig. 5 depicts the growth of the tumors in the first passage,starting at the inoculation of the tumor cells (thus including thelag phase of the tumors). At day 15 after inoculation, theIGROV-1/CD13-overexpressing xenografts were significantlysmaller than the IGROV-1/parent or IGROV-1/neo xenografts.Mean tumor volumes of 122 18 (IGROV-1/CD13-7) and352 31 mm3 (IGROV-1/CD13-9) for the CD13-overexpress-ing xenografts and 868 162 and 686 91 mm3 for IGROV-1/parent and IGROV-1/neo, respectively, were calculated. Be-cause the tumor growth rate appeared less variable betweenindividual tumors in the second passage, two volume doublingtimes of the tumors were calculated in this passage. Similardoubling times (of at least six independent tumors) were deter-mined for the IGROV-1/parent (5.7 0.7 days) and IGROV-1/neo (4.4 0.6 days), whereas the doubling times of theCD13-overexpressing clones were significantly prolonged[IGROV-1/CD13-7, 15.0 2.9 days (P � 0.023 compared withIGROV-1/parent); IGROV-1/CD13-9, 12.2 0.8 days (P �0.001 compared with IGROV-1/parent)]. The volume doublingtimes of the two CD13-overexpressing clones were not signif-icantly different.

In addition to the volume doubling times, the S-phase

fraction, which is a measurement of the number of cells that areactively dividing, of the xenografts was determined. The per-centage of cells in S phase was similar in the IGROV-1/parentand IGROV-1/neo xenografts (21.5 1.5% and 22.1 1.3%,respectively). A slightly lower percentage of cells in S phasewas detected for the IGROV-1/CD13-7 (18.3 1.4%) and theIGROV-1/CD13-9 xenografts (18.4 1.0%). The differencewas of borderline significance toward the control transfectants(P � 0.04 for IGROV-1/CD13-9 and P � 0.06 for IGROV-1/CD13-7, unpaired t test).

CD13 Expression in IGROV-1 Tumors. Real-timePCR with human-specific primers showed that CD13 mRNAwas virtually absent in the xenografts of the IGROV-1/parentand IGROV-1/neo cells [relative mRNA in arbitrary units(�10�4), 2.2 1.0 and 1.9 0.4, respectively], whereas highmRNA expression was detected in the IGROV-1/CD13-7(738 152) and IGROV-1/CD13-9 xenografts (4875 1085).Similarly, immunohistochemical staining revealed prominentheterogeneous expression of CD13 protein in IGROV-1/CD13-7 tumors and homogeneous expression in IGROV-1/CD13-9 tumors (Fig. 6, A�D).

IGROV-1/CD13-9 Is Less Sensitive to Cisplatin in Vivo.Because cisplatin is an important drug for treatment of ovariancancer, we studied the sensitivity of the IGROV-1 xenografts inthe second passage. We measured a maximum inhibition of

Fig. 3 Expression of CD13 on IGROV-1 cells reduces invasionthrough Matrigel. Cellular invasion was measured in a Transwell systemwith 10 �g/ml Matrigel (18 h). The FCS-stimulated invasion wassignificantly less for the CD13-transfected cells as compared with thecontrol cells (�, P � 0.05; ��, P � 0.01). Results are given as themean SE of three experiments.

Table 3 CD13 expression and activity in IGROV-1/parent andIGROV-1 transfected cells

Values are the mean of three experiments SE.

CD13 expression(mean fluorescence)

CD13 activity(fmol/104 cells/min)

IGROV-1/parent 3.2 0.1a 526 354IGROV-1/neo 3.6 0.3a 142 270IGROV-1/CD13-7 662.7 57.2b 5689 428b

IGROV-1/CD13-9 867.0 108.2b 8587 1069b

a The isotype IgG antibody displayed a mean fluorescence of 3.1 0.1 and 3.5 0.2, respectively, for IGROV-1/parent and IGROV-1/neo.

b Significantly different from the IGROV-1/parent (P � 0.001).

Table 4 In vitro doubling time and the sensitivity for drugs (expressed in IC50) of IGROV-1 and IGROV-1 transfected cellsValues are mean of three experiments SE.

Doubling time (h) Doxorubicin (nM) Cisplatin (�M) Gemcitabine (nM)

IGROV-1/parent 20.0 3.5 41.3 5.9 1.2 0.1 4.1 0.1IGROV-1/neo 20.2 3.8 44.8 6.0 1.2 0.4 4.1 0.1IGROV-1/CD13-7 21.5 2.9 33.8 4.9 1.5 0.4 4.4 0.2IGROV-1/CD13-9 20.5 4.2 33.6 3.9 2.0 0.5 4.2 0.1




tumor growth with cisplatin treatment of 67% (day 14; n � 6)for IGROV-1/parent and 61% (day 14; n � 6) for IGROV-1/neo, whereas cisplatin was not effective in the IGROV-1/CD13-9 (maximum growth inhibition of 23% at day 12; n � 12;Fig. 7).

CD13 Expression Affects the Mean Vessel Area but notVessel Density. Because it has been reported that CD13 ac-tivity, when expressed in endothelial cells, affects microvesselformation in tumors (16), we analyzed whether a more gener-alized overexpression of CD13 in tumor tissue has effects onmicrovessel formation. No significant differences were ob-served in microvessel density for all tumors (calculated fromCD31 staining; data not shown). By visual examination, how-ever, some of the microvessels in the IGROV-1/CD13-9 tumorsexhibited a clearly enlarged lumen. To a much lesser extent,these were also observed in IGROV-1/CD13-7, IGROV-1/par-ent, or IGROV-1/neo xenografts (Fig. 6, E�H). Fig. 6H illus-trates a representative example of an enlarged vessel in IGROV-1/CD13-9. Quantification of the vessel area revealed asignificant increase in vessel area of the IGROV-1/CD13-9tumors (mean area of the lumen was 7.87 1.59 �m2; P �0.0005 compared with IGROV-1/parent), whereas no differencein vessel area was observed for IGROV-1/CD13-7 (3.51 0.50�m2), IGROV-1/parent (3.79 0.46 �m2), and IGROV-1/neo(2.47 0.26 �m2) tumors (Fig. 8). Calculation of the percent-age of blood vessels with a large lumen area (�15 �m2) furtherillustrated the increase in vessel area; 12 3% of the bloodvessels of the IGROV-1/CD13-9 were �15 �m2, whereas thisvalue was only 1–2% for the xenografts derived from the othercell lines.

VEGF and basic fibroblast growth factor are not induced inCD13-overexpressing xenografts. VEGF mRNA real-time PCRin six xenografts revealed a nonsignificant trend toward a higherVEGF mRNA expression in CD13-overexpressing tumors (rel-

ative mRNA in arbitrary units: CD13-7, 0.36 0.05; CD13-9,0.40 0.10) when compared with expression in the parental(0.26 0.06) and neo tumors (0.34 0.11; P � nonsignifi-cant). Immunohistochemical staining for VEGF and basic fibro-blast growth factor protein did not reveal any obvious differencein VEGF or basic fibroblast growth factor expression among thetumors.

DISCUSSIONIn this study, we demonstrated that CD13 is highly ex-

pressed in tumor cells of the most frequently occurring humanovarian cancers. We showed in a human ovarian cancer xe-nograft model that CD13 overexpression reduced the growth ofthe xenografts. In addition, the vascular architecture and re-sponse to chemotherapy were altered in the CD13-overexpress-ing xenografts.

The expression of CD13 antigen in ovarian cancer cellswas associated with the most common subtypes (serous andmucinous). Importantly, CD13-positive tumor cells displayed avery prominent plasma membrane staining comparable with thestaining observed in the CD13-overexpressing xenografts. Thusfar, the biological or pathophysiological role of CD13 expres-sion in ovarian cancer is unknown. No peptides relevant for theprogression of ovarian cancer have been identified as a substratefor CD13, but these might be sought in growth-regulating pep-tide growth factors (described below). In line with recent reportsshowing an up-regulation of CD13 antigen in tumor angiogenicvessels (16), we found in all tumors a small number of CD13-positive blood vessels that were not specifically associated withone tumor type or with CD13 expression in tumor cells.

To obtain data on the possible pathophysiological role(s) ofCD13 in ovarian cancer, we have generated a CD13-overex-pressing IGROV-1 ovarian cancer cell line (18) and studiedsome important cell biological properties. An important obser-vation was the significantly reduced invasion of CD13-overex-

Fig. 4 Adhesion of IGROV-1 to Matrigel is reduced by high expres-sion of CD13. Wells of a 96-well plate were coated with Matrigelovernight, and cells were allowed to adhere for 1 h. Adhesion wasquantified with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide assay. The adhesion to Matrigel was slightly but signifi-cantly reduced for IGROV-1/CD13-9 (27% reduction when comparedwith adhesion of IGROV-1 cells; �, P � 0.05). Results are given as themean SE of at least three experiments.

Fig. 5 In vivo tumor growth rate is reduced by high expression ofCD13. Per cell line, 1 � 107 cells were injected s.c. in both flanks ofthree mice, and tumor growth was monitored. High expression of CD13significantly reduced the growth rate of the tumors (CD13-7, P � 0.001;CD13-9, P � 0.014). Results are given as mean tumor volume SE (12tumors from two separate experiments).




Fig. 6 Immunohistochemical staining of CD13 and CD31/platelet/endothelial cell adhesion molecule 1 in xenografts. Frozen sections fromxenografts were stained for CD13 (WM15; 10 �g/ml) and CD31/platelet/endothelial cell adhesion molecule 1 (MEC13.3; 5 �g/ml). CD13 expressionin tumor cells was only observed in the CD13-transfected tumors (�10 objective; inset, �40 objective; A�D). The insets in C and D show membranestaining of CD13. In the CD13-overexpressing tumors, some extremely large vessels were visible that were hardly observed in the parent or the neocontrol tumors (�10 objective; E�H). A representative example of an enlarged vessel in IGROV-1/CD13-9 is shown (H).




pressing cells into Matrigel as compared with that of the non-CD13-transfected cells. This is in contrast to data published byFujii et al. (14), who have reported an enhanced migration byCD13-overexpressing A375M melanoma cells through Matrigel(14). This discrepancy suggests that the balance of proteolyticand/or adhesive activities, which determine the invasive poten-tial of these cell lines, differs qualitatively or quantitatively withregard to the effect of CD13 activity. Moreover, a high concen-tration of the aminopeptidase activity inhibitor bestatin did notreverse the effect of CD13 overexpression on invasion ofIGROV-1 cells. It is unlikely that the absence of an effect ofbestatin is due to its inability to completely inhibit the high

levels of CD13 activity in the transfected cells. In our previouswork with the HT-1080 fibrosarcoma cell line, which alsoexpresses high levels of CD13, we have shown that bestatin caninhibit �80% of CD13 enzymatic activity. Moreover, the inva-sion of HT-1080 cells was inhibited up to 50% on bestatintreatment (20). Therefore, we conclude that it is not likely thatthe CD13 catalytic activity is directly responsible for the reduc-tion of the FCS-stimulated invasion of the IGROV-1 cells. Inthis respect, our results strongly resemble those of a recent studyby Kajiyama et al. (4), who have shown a decreased migrationof dipeptidyl peptidase IV/CD26-overexpressing ovarian cancercells through Matrigel that was also independent of aminopep-tidase activity (4). An explanation for the reduced invasion ofCD13-overexpressing cells might be a decreased adhesion of thecells, which is required for proper invasion in the ECM (Ref. 26;also reviewed by Skubitz (27)]. Because we found only amoderate reduction in adhesion to Matrigel (30% reduction), itis questionable whether this explains the less invasive pheno-type.

The fact that overexpression of an aminopeptidase resultsin a decrease of the invasion, rather than an increase, is appar-ently paradoxical. Such conflicting data, however, have alsobeen reported for SPARC, a matrix glycoprotein that modulatescellular interaction with the ECM (28). SPARC has been linkedto an invasive phenotype when expressed by melanoma andglioma cells, whereas expression of SPARC in ovarian cancercells was inversely correlated with tumor growth (28).

It is possible that cooperation of CD13 with other amin-opeptidases, as has been proposed by Riemann et al. (29), mayaffect intracellular signal transduction pathways involved inadhesion and motility of the cells. The possibility of such acooperation is based on, among others, the finding that CD13and other peptidases such as CD10 and CD26 colocalize in

Fig. 7 Sensitivity for cisplatin is reduced by high expression of CD13.Tumor fragments (3 mm) were transplanted s.c. in both flanks of themice. At a volume of 100–150 mm3, one group was treated withcisplatin (5 mg/kg, weekly � 2, i.v.), and one group served as control.A significant inhibition in growth was calculated for IGROV-1/parent(n � 6; A) and IGROV-1/neo (n � 6; B) when treated with cisplatin,whereas no significant reduction was calculated for IGROV-1/CD13-9(n � 12; C). Results are given as mean tumor volume SE.

Fig. 8 High CD13 expression increases the diameter of the bloodvessels. A microscope attached to a video camera was used to takeimages (�10 objective, 5 images/tumor section). The area of the vessels(expressed in �m2) was measured using Quantimax software. The boxrepresents the range that contains 50% of the values; the black line in thebox illustrates the median value. The bars indicate the highest and thelowest values, excluding outliers. A significant increase in vessel areawas calculated for the IGROV-1/CD13-9 tumors as compared with theparent or neo control tumors (��, P � 0.0005).




membrane caveolae in different cell types (30, 31). Furthermore,CD13 (as well as other ectopeptidases) may associate with otherproteins into specific membrane regions (e.g., with certain in-tegrins), thereby enabling CD13 to affect signaling processes,such as cell adhesion or apoptosis (32). Similar mechanismshave been described for many non-receptor tyrosine kinasespresent in caveolae (33). Alternative explanations might besought in CD13-induced up- or down-regulation of adhesionproteins and/or (metallo)proteases resulting in a decreased in-vasive capacity. Interestingly, such a mechanism has been de-scribed very recently for CD26; overexpression of CD26 in theovarian cancer cell line SKOV-3 was related to an increasedexpression of E-cadherin and �-catenin and/or decreased ex-pression of certain matrix metalloproteinases (34). It will bevery interesting to study the effects of introducing CD13 inSKOV-3 or, vice versa, CD26 in IGROV-1 to evaluate in moredetail the significance of these aminopeptidases for ovariancancer in general.

One of the most remarkable findings of this study was thesignificantly reduced tumor growth rate of the CD13-overex-pressing clones in vivo. The IGROV-1/CD13-7 xenografts,which have a heterogeneous CD13 expression, grew moreslowly than IGROV-1/CD13-9 xenografts in the first passage. Inthe second passage, however, the number of days to double involume did not differ significantly between IGROV-1/CD13-9and IGROV-1/CD13-7 tumors. A slower tumor growth ofCD13-overexpressing xenografts may seem inconsistent withthe expression data from the patient tumors because, in general,serous tumors (high CD13 expression) grow faster than clearcell tumors (low CD13 expression). However, because in thepresent study we did not analyze other factors that may affectovarian cancer growth, more extensive patient studies will berequired to answer this type of question. Because there was nodirect effect of CD13 expression on the growth of the cells in amonolayer in vitro, CD13 overexpression apparently indirectlyaffects the growth and/or differentiation properties of theIGROV-1 cells in an in vivo environment. Because of the highCD13 expression in the transfected tumors, it is reasonable toassume that the aminopeptidase activity is elevated in vivo aswell. As has been suggested for the role of CD13 in normalovarian cells, active CD13 may catabolize biologically activepeptides adjacent to the cell surface, such as tyrosine kinase-activating growth factors (epidermal growth factor and hepato-cyte growth factor) and cytokines [interleukin 8 (35, 36)]. Thus,CD13 might contribute to local regulation of the concentrationand/or activity of one or more of these peptides [reviewed byFujiwara et al. (37)] and, in this way, may affect the growth rateof the tumors (38, 39). Additional studies should include trans-fection of CD13 in other ovarian cancer cell lines as well asstudies with catalytic inactive CD13. The search for in vivoalterations in signal transduction pathways involved in cellularmigration, invasion, and cell growth is an additional way toproceed with further elucidation of the mechanisms involved.

The IGROV-1/CD13-9 xenografts exhibited a reduced sen-sitivity for cisplatin in vivo. Because we did not observe achange in the sensitivity to cisplatin or other cytotoxic agents invitro, it is unlikely that CD13 directly induced resistance againstcisplatin in the IGROV-1 cells. Instead, the reduced sensitivityfor cisplatin may be sought in the slower growth of these tumor

cells when grown in vivo. Based on the results of Kolfschoten etal. (23), who have studied the relationship between the S-phasefraction and cisplatin sensitivity in a panel of 14 ovarian xe-nografts, the small reduction in S-phase fraction observed in ourCD13-overexpressing xenografts is at most partly responsiblefor the resistance against cisplatin. Other drug resistance fea-tures, such as impaired drug delivery to the tumors (e.g., due toaltered vessel structures), have to be considered. It will be quiteinteresting to investigate the in vivo sensitivity to other cytotoxicagents to elucidate whether the observed resistance is drugspecific, or whether this is a more generalized property ofCD13-overexpressing ovarian xenografts.

An intriguing observation was the enlarged vascular area inthe IGROV-1/CD13-9 xenografts. As can be observed in Fig.6G, which is a representative picture of IGROV-1/CD13-7xenograft tissue, differences in vascular area are present in thetumors of this clone, but it has to be noted that overall, nosignificant difference was found for the specific parameter cal-culation as used here when compared with the vasculature of theIGROV-1/parent. Given these considerations, it is likely thatCD13 overexpression in IGROV-1 has effects on the develop-ment of tumor vasculature. Again, these results display similar-ity to those of the SPARC study mentioned earlier. Brekken etal. (28) have also described that the vessel area was enlarged inSPARC�/� mice compared with SPARC�/� mice, without af-fecting the microvessel count.

There was no change in the expression level of angiogenicfactors (VEGF and basic fibroblast growth factor) in theIGROV-1/CD13-9 tumors that could account for the change invessel morphology. Several groups have suggested factors thatcan attribute to the enlargement of the vessel lumen, such as (a)a compensation for a local reduction in the number of bloodvessels and blood volume (40), (b) a higher blood flow rate ina specific area that increases the diameter of the vessel (41, 42),and (c) a reduction of the interstitial pressure as well as areduction in tumor cell density, leading to decompression of thevessels (43, 44). To evaluate whether any of these alternativesapply to CD13-overexpressing tumors and whether differencesin vascular function occur in these tumors, additional studies areneeded. Treatment of mice bearing CD13-overexpressing tu-mors with bestatin or other aminopeptidase inhibitors may givemore insight into the biological and vascular effects of CD13.

Given that CD13 is abundantly expressed in tumor andendothelial cells in human ovarian cancer and our findings thatCD13 overexpression affects the growth of ovarian cancer xe-nografts, the vascular architecture, and response to chemother-apy, further elucidation of the biochemical effects of CD13 andits role in the pathophysiology and treatment of ovarian canceris required.

ACKNOWLEDGMENTSWe thank Henk Dekker, Caroline Erkelens, and Mark Broeckaert

for technical assistance.

REFERENCES1. Serov, S. F., Scully, R. E., and Sobin, L. H. Histological Typing ofOvarian Tumours. International Histological Classification of Tumours,9th ed., pp. 17–54. Geneva: WHO, 2003.




2. Kristiansen, G., Denkert, C., Schluns, K., Dahl, E., Pilarsky, C., andHauptmann, S. CD24 is expressed in ovarian cancer and is a newindependent prognostic marker of patient survival. Am. J. Pathol., 161:1215–1221, 2002.

3. Sood, A. K., Fletcher, M. S., Gruman, L. M., Coffin, J. E., Jabbari,S., Khalkhali-Ellis, Z., Arbour, N., Seftor, E. A., and Hendrix, M. J. Theparadoxical expression of maspin in ovarian carcinoma. Clin. CancerRes., 8: 2924–2932, 2002.

4. Kajiyama, H., Kikkawa, F., Suzuki, T., Shibata, K., Ino, K., andMizutani, S. Prolonged survival and decreased invasive activity attrib-utable to dipeptidyl peptidase IV overexpression in ovarian carcinoma.Cancer Res., 62: 2753–2757, 2002.

5. Bordessoule, D., Jones, M., Gatter, K. C., and Mason, D. Y. Immu-nohistological patterns of myeloid antigens: tissue distribution of CD13,CD14, CD16, CD31, CD36, CD65, CD66, and CD67. Br. J. Haematol.,83: 370–383, 1993.

6. Mechtersheimer, G., and Moller, P. Expression of aminopeptidase N(CD13) in mesenchymal tumors. Am. J. Pathol., 137: 1215–1222, 1990.

7. Alliot, F., Rutin, J., Leenen, P. J., and Pessac, B. Pericytes andperiendothelial cells of brain parenchyma vessels co-express aminopep-tidase N, aminopeptidase A, and nestin. J. Neurosci. Res., 58: 367–378,1999.

8. Dixon, J., Kaklamanis, L., Turley, H., Hickson, I. D., Leek, R. D.,Harris, A. L., and Gatter, K. C. Expression of aminopeptidase-N (CD13) in normal tissues and malignant neoplasms of epithelial and lymph-oid origin. J. Clin. Pathol., 47: 43–47, 1994.

9. Hashida, H., Takabayashi, A., Kanai, M., Adachi, M., Kondo, K.,Kohno, N., Yamaoka, Y., and Miyake, M. Aminopeptidase N is in-volved in cell motility and angiogenesis: its clinical significance inhuman colon cancer. Gastroenterology, 122: 376–386, 2002.

10. Ishii, K., Usui, S., Sugimura, Y., Yoshida, S., Hioki, T., Tatematsu,M., Yamamoto, H., and Hirano, K. Aminopeptidase N regulated by zincin human prostate participates in tumor cell invasion. Int. J. Cancer, 92:49–54, 2001.

11. Ishii, K., Usui, S., Yamamoto, H., Sugimura, Y., Tatematsu, M.,and Hirano, K. Decreases of metallothionein and aminopeptidase N inrenal cancer tissues. J. Biochem., 129: 253–258, 2001.

12. Fujii, H., Nakajima, M., Aoyagi, T., and Tsuruo, T. Inhibition oftumor cell invasion and matrix degradation by aminopeptidase inhibi-tors. Biol. Pharm. Bull., 19: 6–10, 1996.

13. Saiki, I., Fujii, H., Yoneda, J., Abe, F., Nakajima, M., Tsuruo, T.,and Azuma, I. Role of aminopeptidase N (CD13) in tumor-cell invasionand extracellular matrix degradation. Int. J. Cancer, 54: 137–143, 1993.

14. Fujii, H., Nakajima, M., Saiki, I., Yoneda, J., Azuma, I., andTsuruo, T. Human melanoma invasion and metastasis enhancement byhigh expression of aminopeptidase N/CD13. Clin. Exp. Metastasis, 13:337–344, 1995.

15. Bhagwat, S. V., Lahdenranta, J., Giordano, R., Arap, W., Pas-qualini, R., and Shapiro, L. H. CD13/APN is activated by angiogenicsignals and is essential for capillary tube formation. Blood, 97: 652–659, 2001.

16. Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto,M., Stryhn, A., Ashmun, R. A., Shapiro, L. H., Arap, W., and Ruoslahti,E. Aminopeptidase N is a receptor for tumor-homing peptides and atarget for inhibiting angiogenesis. Cancer Res., 60: 722–727, 2000.

17. van Hensbergen, Y., Broxterman, H. J., Hanemaaijer, R., Jorna,A. S., Van Lent, N. A., Verheul, H. M., Pinedo, H. M., and Hoekman,K. Soluble aminopeptidase N/CD13 in malignant and nonmalignanteffusions and intratumoral fluid. Clin. Cancer Res., 8: 3747–3754, 2002.

18. Benard, J., Da Silva, J., De Blois, M. C., Boyer, P., Duvillard, P.,Chiric, E., and Riou, G. Characterization of a human ovarian adenocar-cinoma line, IGROV1, in tissue culture and in nude mice. Cancer Res.,45: 4970–4979, 1985.

19. Graham, F. L., and van der Eb, A. J. A new technique for the assayof infectivity of human adenovirus 5 DNA. Virology, 52: 456–467,1973.

20. van Hensbergen, Y., Broxterman, H. J., Elderkamp, Y. W.,Lankelma, J., Beers, J. C., Heijn, M., Boven, E., Hoekman, K., andPinedo, H. M. A doxorubicin-CNGRC-peptide conjugate with prodrugproperties. Biochem. Pharmacol., 63: 897–908, 2002.

21. Favaloro, E. J. CD-13 (gp150; aminopeptidase-N): co-expressionon endothelial and haemopoietic cells with conservation of functionalactivity. Immunol. Cell Biol., 69: 253–260, 1991.

22. Wellmann, S., Taube, T., Paal, K., Graf, V. E., Geilen, W.,Seifert, G., Eckert, C., Henze, G., and Seeger, K. Specific reversetranscription-PCR quantification of vascular endothelial growth fac-tor (VEGF) splice variants by LightCycler technology. Clin. Chem.,47: 654 – 660, 2001.

23. Kolfschoten, G. M., Hulscher, T. M., Pinedo, H. M., and Boven, E.Drug resistance features and S-phase fraction as possible determinantsfor drug response in a panel of human ovarian cancer xenografts. Br. J.Cancer, 83: 921–927, 2000.

24. Bergers, E., van Diest, P. J., and Baak, J. P. Tumour heterogeneityof DNA cell cycle variables in breast cancer measured by flow cytom-etry. J. Clin. Pathol., 49: 931–937, 1996.

25. Bergers, E., Baak, J. P., van Diest, P. J., Willig, A. J., Los, J.,Peterse, J. L., Ruitenberg, H. M., Schapers, R. F., Somsen, J. G., vanBeek, M. W., Bellot, S. M., Fijnheer, J., and van Gorp, L. H. Prognosticvalue of DNA ploidy using flow cytometry in 1301 breast cancerpatients: results of the prospective Multicenter Morphometric MammaryCarcinoma Project. Mod. Pathol., 10: 762–768, 1997.

26. Carreiras, F., Thiebot, B., Leroy-Dudal, J., Maubant, S., Breton,M. F., and Darbeida, H. Involvement of v�3 integrin and disruption ofendothelial fibronectin network during the adhesion of the human ovar-ian adenocarcinoma cell line IGROV1 on the human umbilical vein cellextracellular matrix. Int. J. Cancer, 99: 800–808, 2002.

27. Skubitz, A. P. Adhesion molecules. Cancer Treat. Res., 107: 305–329, 2002.

28. Brekken, R. A., Puolakkainen, P., Graves, D. C., Workman, G.,Lubkin, S. R., and Sage, E. H. Enhanced growth of tumors in SPARCnull mice is associated with changes in the ECM. J. Clin. Investig., 111:487–495, 2003.

29. Riemann, D., Kehlen, A., and Langner, J. CD13: not just a markerin leukemia typing. Immunol. Today, 20: 83–88, 1999.

30. Boonacker, E., and Van Noorden, C. J. The multifunctional ormoonlighting protein CD26/DPPIV. Eur. J. Cell Biol., 82: 53–73, 2003.

31. Navarrete, S. A., Roentsch, J., Danielsen, E. M., Langner, J., andRiemann, D. Aminopeptidase N/CD13 is associated with raft membranemicrodomains in monocytes. Biochem. Biophys. Res. Commun., 269:143–148, 2000.

32. Antczak, C., De, M. I., and Bauvois, B. Ectopeptidases in patho-physiology. Bioessays, 23: 251–260, 2001.

33. Frank, P. G., Woodman, S. E., Park, D. S., and Lisanti, M. P.Caveolin, caveolae, and endothelial cell function. Arterioscler. Thromb.Vasc. Biol., 23: 1161–1168, 2003.

34. Kajiyama, H., Kikkawa, F., Khin, E., Shibata, K., Ino, K., andMizutani, S. Dipeptidyl peptidase IV overexpression induces up-regu-lation of E-cadherin and tissue inhibitors of matrix metalloproteinases,resulting in decreased invasive potential in ovarian carcinoma cells.Cancer Res., 63: 2278–2283, 2003.

35. Kohn, E. C., Fidler, I. J., Fishman, D., Jaffe, R., Liotta, L. A., vanTrappen, P., Mills, G. B., and Trope, C. Discussion: metastasis andangiogenesis in epithelial ovarian cancer. Gynecol. Oncol., 88: S37–S42, 2003.

36. Mills, G. B., Fang, X., Lu, Y., Hasegawa, Y., Eder, A., Tanyi, J.,Tabassam, F. H., Mao, M., Wang, H., Cheng, K. W., Nakayama, Y.,Kuo, W., Erickson, J., Gershenson, D., Kohn, E. C., Jaffe, R., Bast,R. C., Jr., and Gray, J. Specific keynote: molecular therapeutics inovarian cancer. Gynecol. Oncol., 88: S88–S92, 2003.

37. Fujiwara, H., Imai, K., Inoue, T., Maeda, M., and Fujii, S. Mem-brane-bound cell surface peptidases in reproductive organs. Endocr. J.,46: 11–25, 1999.




38. Hoffmann, T., Faust, J., Neubert, K., and Ansorge, S. Dipeptidylpeptidase IV (CD 26) and aminopeptidase N (CD 13) catalyzed hydrol-ysis of cytokines and peptides with N-terminal cytokine sequences.FEBS Lett., 336: 61–64, 1993.39. Rosenzwajg, M., Tailleux, L., and Gluckman, J. C. CD13/N-aminopeptidase is involved in the development of dendritic cells andmacrophages from cord blood CD34(�) cells. Blood, 95: 453–460, 2000.40. Drevs, J., Muller-Driver, R., Wittig, C., Fuxius, S., Esser, N.,Hugenschmidt, H., Konerding, M. A., Allegrini, P. R., Wood, J., Hen-nig, J., Unger, C., and Marme, D. PTK787/ZK 222584, a specificvascular endothelial growth factor-receptor tyrosine kinase inhibitor,affects the anatomy of the tumor vascular bed and the functionalvascular properties as detected by dynamic enhanced magnetic reso-nance imaging. Cancer Res., 62: 4015–4022, 2002.

41. Bevan, J. A., and Laher, I. Pressure and flow-dependent vasculartone. FASEB J., 5: 2267–2273, 1991.

42. Loufrani, L., Levy, B. I., and Henrion, D. Defect in microvascularadaptation to chronic changes in blood flow in mice lacking the geneencoding for dystrophin. Circ. Res., 91: 1183–1189, 2002.

43. Griffon-Etienne, G., Boucher, Y., Brekken, C., Suit, H. D., and Jain,R. K. Taxane-induced apoptosis decompresses blood vessels and lowersinterstitial fluid pressure in solid tumors: clinical implications. CancerRes., 59: 3776–3782, 1999.

44. Netti, P. A., Roberge, S., Boucher, Y., Baxter, L. T., and Jain, R. K.Effect of transvascular fluid exchange on pressure-flow relationship intumors: a proposed mechanism for tumor blood flow heterogeneity.Microvasc. Res., 52: 27–46, 1996.




2004;10:1180-1191. Clin Cancer Res Yvette van Hensbergen, Henk J. Broxterman, Sareena Rana, et al. Ovarian Cancer XenograftsResponse to Cisplatin in CD13-Overexpressing Human Reduced Growth, Increased Vascular Area, and Reduced

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Reduced Growth, Increased Vascular Area, and Reduced ...clincancerres.aacrjournals.org/content/clincanres/10/3/1180.full.pdf · gest that CD13 overexpression affects ovarian cancer