Inducible Nitric Oxide Synthase Expression, Apoptosis, andAngiogenesis inin Situ and Invasive Breast Carcinomas1

Merja Vakkala, Katriina Kahlos, Essi Lakari,Paavo Paakko, Vuokko Kinnula, andYlermi Soini2

Departments of Pathology [M. V., P. P., Y. S.] and Internal Medicine[K. K., E. L., V. K.], University of Oulu and Oulu UniversityHospital, FIN-90014, Oulu, Finland

ABSTRACTIn this investigation, we studied the expression of in-

ducible nitric oxide synthase (iNOS) and its association toapoptosis and angiogenesis in 43in situ and 68 invasivebreast carcinomas. Its expression was studied immunohis-tochemically using a polyclonal iNOS antibody, and thestaining was evaluated both in tumor and stromal cells.Apoptosis was detected by 3* end labeling of fragmentedDNA (terminal deoxynucleotidyl transferase-mediated nickend labeling method). Vascularization was detected immu-nohistochemically using an antibody to the FVIII-relatedantigen, and calculated microvessel densities were deter-mined. In addition to strong iNOS expression in stromalcells, iNOS positivity was observed in tumor cells in 46.5%of in situ and 58.8% of invasive carcinomas. In invasivecarcinomas, there were more cases with iNOS positivity bothin tumor and stromal cells compared to in situ carcinomas(0.007). The proportion of cases with iNOS-positive tumorcells increased in in situ carcinomas from grade I to III(20.0%, 46.2%, and 73.3%). In invasive ductal carcinomas,there were more cases with iNOS-positive tumor cells thanwith in situ carcinomas (P5 0.04). Carcinomas with bothiNOS-positive tumor and stromal cells had a higher apo-ptotic index (P 5 0.02) and a higher calculated microvesseldensities index (P5 0.02). A high number of iNOS-positivestromal cells associated with metastatic disease (P 5 0.05).The results show that breast carcinoma cells, in addition tostromal cells, express iNOS and are capable of producingNO. Carcinomas with iNOS-positive tumor and stromal cellshave a higher apoptotic indices and increased vasculariza-tion, suggesting that iNOS contributes to promotion of ap-optosis and angiogenesis in breast carcinoma. The associa-

tion of the number of iNOS-positive stromal cells withmetastatic disease might be attributable to stimulation ofangiogenesis, resulting in a higher vascular density andconsequently a higher probability for tumor cells to invade.

INTRODUCTIONNitric oxide (NO), a diatomic radical, plays a variety of

regulatory functionsin vivo. It has diverse physiological andpathophysiological roles as a vasodilator, neurotransmitter, an-timicrobial effector molecule, and immunomodulator (1). NO issynthesized from the amino acidL-arginine by the NOS3 (2). Asa free radical, NO is highly reactive and reacts in biologicalsystems with other free radicals, molecular oxygen, and heavymetals (3). The biological effects of NO are mainly mediated bythe products of different NO metabolites (3).

There are three isoforms of NOS: iNOS (NOS2), eNOS(NOS3), and nNOS (NOS1; Ref. 1). Each isoform is the productof a distinct gene (4). eNOS and nNOS are constitutive, cal-modulin-dependent enzymes (cNOS; Ref. 4). iNOS is expressedin macrophages, neutrophils, endothelial cells, hepatocytes, car-diac myocytes, chondrocytes, and many other cell types (5). It isinduced most importantly by cytokines and can generate locallyhigh concentrations of NO for prolonged periods of time (4, 6).Calcium independence of iNOS has been questioned with thereport of iNOS enzymatic activity dependent on intracellularfluxes of calcium and binding of calmodulin; but in general,iNOS is calcium-independent (6).

The genotoxicity of NO is attributable to its reaction witheither oxygen or superoxide (7). The intracellular NO quicklyforms nitrite and nitrate,S-nitroso-thiols, or peroxynitrite (3).NO metabolites can mediate genotoxicity and influence theinitiation of cancer by a variety of mechanisms. For instance,NO causes DNA damage by nitrosative deamination, DNAstrand breakage, or DNA modification (e.g., nitration) by per-oxynitrite (3, 7). These reactions may also be associated with theactivation of carcinogenic nitrosamines, initiation of apoptosisand inhibition of DNA repair enzymes, or lipid peroxidation-induced DNA damage (6, 7).

The effects of NO can be tumor promoting or tumorsuppressing. High concentrations of NO can be cytotoxic,whereas low concentration may even protect some cell typesfrom damage and apoptosis (3). During the initiation of tumorgrowth, natural killer cells and macrophages kill tumor cells bya NO-mediated mechanism (5). However, NO may also sup-press the antitumor defense, promote tumor angiogenesis andblood flow in the tumor neovasculature, and enhance tumorgrowth, invasion, and metastasis (5).

Received 11/23/99; revised 2/7/00; accepted 2/29/00.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported by the Finnish Cancer Societies, Ida Montin Foundation,and the Finnish Medical Foundation.2 To whom requests for reprints should be addressed, at Department ofPathology, Box 5000 (Aapistie 5), University of Oulu, FIN-90014, OuluFinland. Phone: 358-8-537-5011; Fax: 358-8-537-5953; E-mail:msoini@cc.oulu.fi.

3 The abbreviations used are: NOS, NO synthase; iNOS, inducible NOS;eNOS, endothelial NOS; nNOS, neuronal NOS; HPF, high power field;CMVD, calculated microvessel density.

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NO exhibits contradictory effects on the regulation ofapoptosis. It has been demonstrated to be both pro- and anti-apoptotic. The proapoptotic effects appear to be linked to thepathophysiological condition where the induction of iNOS isassociated with high concentrations of reactive nitrogen metab-olites (8). Cell protection is associated with the up-regulation ofseveral protective proteins, such as cyclo-oxygenase-2 or heme-ozygenase-1 (9). Typical findings in NO-mediated apoptosisinclude accumulation of tumor suppressor protein p53, caspaseactivation, chromatin condensation, and DNA fragmentation(3, 8, 9).

In human primary breast cancers, relatively high iNOSimmunoreactivity has been noted in stromal cells, and the pres-ence of stromal reactivity appears to correlate with tumor grade(10). However, in another study, iNOS positivity was predom-inantly found in the tumor cells associating positively with thepresence of axillary lymph node metastasis (11). In breast can-cer, a high extent of apoptosis is usually associated with poorprognosis, and more apoptosis is seen in tumors of high grade(12–14). The expression of iNOS in relation of apoptosis has notbeen previously studied in different types of breast cancers. Inthis study, we evaluated the immunohistochemical distributionof iNOS in in situ and invasive breast cancers and its relation tothe apoptotic index, as determined by the terminal deoxynucle-otidyl transferase-mediated nick end labeling method. The sec-tions were also studied immunohistochemically for vasculardensity using FVIII antibody to see whether changes in theexpression of iNOS could influence tumor angiogenesis.

MATERIALS AND METHODSA total of 111 breast lesions consisting of 43in situ ductal

carcinomas, 56 ductal invasive carcinomas, 10 lobular invasivecarcinomas, one mucinous, and one medullary carcinoma werecollected from the files of the Department of Pathology, Uni-versity of Oulu.In situ carcinomas consisted of 15 low-grade (5papillary, 3 solid, and 7 cribriform), 13 intermediate (1 papil-lary, 8 solid, and 4 cribriform), and 15 high-grade (1 cribriformand 14 comedo-type) lesions. In invasive ductal carcinomas,there were 9 well-differentiated (grade I), 24 moderately differ-entiated (grade II), and 23 poorly differentiated (grade III)tumors. The material had been fixed in neutral formalin andembedded in paraffin. The diagnosis of all of the cases werebased on light microscopic examination by the conventionalH&E stain (15, 16). The grading of the ductal invasive carci-nomas were made according to Elston and Ellis (16), and thegrades of thein situ lesions were made by Hollandet al. (17).

The TNM classification was available in 104 cases. Therewere 43 TIS, 45 T1–2, and 16 T3–4 tumors. Thirty-four casescontained nodal metastases (N1–3), and distant metastases (M1)were present in four cases. Mean follow-up time was 5.8 years(range, 0–20 years). In 32 cases, the cancer relapsed. Informa-tion about estrogen and progesterone receptors were availablefor 63 cases. Values.10 fmol were considered positive.

Immunohistochemical Stainings. Five-mm paraffin sec-tions were cut from the specimens and placed on SuperFrostPlusglass slides (Menzel-glaser, Germany). Immunostainings withiNOS antibodies were performed as follows. Paraffin sections weresoaked in xylene to remove paraffin and rehydrated in graded

alcohol series. The sections were heated in a microwave oven in 10mM citric acid monohydrate (pH 6.0) for 10 min and then cooledproperly at room temperature. The endogenous peroxidase wasconsumed by immersing the sections in 3% hydrogen peroxide inabsolute methanol for 15 min. Two different primary antibodieswere used: a rabbit polyclonal (dilution, 1:200; Santa Cruz Bio-technology) and a mouse monoclonal (dilution, 1:60; TransductionLaboratories, Lexington) iNOS antibody, which were both incu-bated for 60 min at room temperature. With these two antibodies,Histostain-PLUS BULK KIT (Zymed Laboratories Inc., South SanFrancisco, CA) was used. The color was developed by aminoethylcarbazole substrate solution (Zymed Laboratories Inc.). The sec-tions were counterstained in Meyer’s hematoxylin followed by 2%ammonia water handling, after which the slides were mounted withImmu-Mount (Shandon, Pittsburgh, PA).

Negative control slides were prepared from the same tissueblocks. Instead of using the primary antibody, we used PBS. Inaddition, in seven cases with clear polyclonal iNOS positivity,an absorption test was conducted. Before application to theslides, antibody binding to antigen was neutralized by 2-h pre-absorption at room temperature with a 5-fold excess of blockingpeptide (Santa Cruz Biotechnology, Inc.) to polyclonal iNOSantibody. Macrophages and neutrophils labeled very strongly(1111) in every slide, and they served as an internal positivecontrol for the immunostaining.

The intensity of iNOS immunostainings was evaluated bydividing the cytoplasmic staining reaction in four groups: 15weak; 25 moderate; 35 strong; and 45 very strong cyto-plasmic staining intensity. The quantity of immunostainingswere evaluated as follows: 05 no positive immunostaining, 15,25%; 25 25–50%; 35 50–75%; and 45 .75% of tumorcells showing cytoplasmic positivity. A combined score foriNOS immunostainings, based on both qualitative and quantita-tive immunostaining, was composed by adding the qualitative tothe quantitative score. This sum score was then divided in fivegroups as follows:2 5 0; 1 5 1–2;11 5 3–4;111 5 5–6;and1111 5 7–8.

iNOS staining in stromal cells was evaluated with340objective semiquantitatively and divided in three groups asfollows: weak 15 0–2 positive stromal cells/HPF; moderate2 5 ,10 positive stromal cells/HPF; and strong 35 .10positive stromal cells/HPF.

A combined score for iNOS staining in tumor and stromalcells was also calculated. This sum score was divided in fourgroups as follows: 15 1; 2 5 2–3; 35 4–5; and 45 6–7.

For FVIII-related antigen, the immunostaining was per-formed as follows. The sections were dewaxed in xylene andrehydrated in graded alcohol series. For enzyme predigestion offormalin-fixed tissue, the sections were incubated for 30 min at37°C in 0.04% pepsin (Sigma Chemical Co., St. Louis, MO) in0.01M HCl. The endogenous peroxidase activity was consumedby immersing the sections with 3% hydrogen peroxide in abso-lute methanol. Nonspecific binding was blocked by incubatingthe slides in 20% FCS in PBS for 20 min. The primary poly-clonal antibody for factor VIII (DAKO A/S, Glostrup, Den-mark) was diluted 1:250 in PBS and incubated 30 min at roomtemperature. Then a biotinylated secondary antirabbit antibody(DAKO A/S) diluted 1:300 in PBS was applied on the sectionsfor 30 min, followed by the avidin-biotin-peroxidase complex

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(DAKO A/S). The color was developed by diaminobenzidine,after which the sections were lightly counterstained with hema-toxylin and mounted with Eukitt (Kindler, Freiburg, Germany).

As a positive control, we used slides from a highly vascu-larized tumor. Negative controls consisted of PBS instead of theprimary antibody.

CMVDs were counted from an average of six HPFs with340 objective. Any endothelial-cell cluster consisting of two ormore cells was considered a single, countable microvessel. Ininsitu carcinomas, two distinct vascular patterns could be seen: adiffuse increase of stromal vascularity between ducts and adense rim of microvessels adjacent to ducts. At first, both ofthem were counted together. The mean of six counts was cal-culated and used in statistical analysis. Also, the periductalvessel density (1/mm) was evaluated separately. The periductalvessels from five round neoplastic ducts were calculated. Thissum was then divided by the sum of the measures around theseducts evaluated using the radius of the ducts (2pr), which wasmeasured by an ocular micrometer.

3* End Labeling of DNA in Apoptotic Cells. To detectapoptotic cells,in situ labeling of the 39ends of the DNAfragments generated by apoptosis associated endonucleases wasused. The 39end labeling of DNA was performed using theApopTag in situ apoptosis detection kit (Oncor, Gaithersburg,MD) with a few modifications as previously described (18, 19).A positive control consisted of a lymph node with follicularhyperplasia. The sections, after been dewaxed in xylene andrehydrated in ethanol, were incubated in 20mg/ml Proteinase K(Boehringer Mannheim GmbH, Mannheim, Germany) at roomtemperature for 15 min. The endogenous peroxidase activitywas blocked by incubating the slides in 3% hydrogen peroxidein PBS (pH 7.2). The slides were then treated with terminaltransferase enzyme and digoxigenin-labeled nucleotides, afterwhich antidigoxigenin-peroxidase solution was applied on theslides. The color was developed with diaminobenzidine, afterwhich the slides were lightly counterstained with hematoxylinand mounted with Eukitt (Kindler).

Cells were defined as apoptotic if the whole nuclear area ofthe cell labeled positively. Apoptotic bodies were defined assmall positively labeled globular bodies in the cytoplasm of thetumor cells, which could be found either singly or in groups. Toestimate the apoptotic index (the percentage of apoptotic eventsin a given area), apoptotic cells and bodies were counted from

10 HPFs with340 objective, and this figure was divided by thenumber of tumor cells in the same HPFs.

Immunoblot Analysis. To test the specificity of the twoiNOS antibodies, immunoblotting analysis using mouse macro-phage lysate (Transduction Laboratories) was performed. Ac-cording to the manufacturer, the lysate was prepared from theRAW 264.7 (ATCC TIB71) cell line. These cells were estab-lished from an ascites tumor derived from a male mouse, whichwas injected with the Abelson leukemia virus. Mouse macro-phage cells were stimulated with IFNg and lipopolysaccharidefor 12 h. The control macrophages were mixed with the elec-trophoresis sample buffer and boiled for 5 min at 95°C. Seventy-five mg of cell protein were applied to a 12% SDS-polyacryl-amide gel (20). The gel was electrophoresed for 2.0 h (80 V) atroom temperature, and the protein was transferred onto Hybondenhanced chemiluminescence nitrocellulose membranes (Amer-sham, Arlington Hights, IL) in a Mini-PROTEAN II Cell (Bio-Rad, Hercules, CA). The blotted membrane was incubated withthe poly- and monoclonal antibodies to iNOS (dilutions, 1:2000for both antibodies) followed by treatment with secondary an-timouse and antirabbit antibodies (dilutions, 1:2000 for bothsecondary antibodies; Jackson Immunoresearch Laboratories)conjugated to horseradish peroxidase. The proteins were de-tected by enhanced chemiluminescence system (Amersham).Cell protein was measured using the Bio-Rad protein assay(Bio-Rad; Ref. 21).

Statistical Analysis. SPSS for Windows (Chicago, IL)was used for statistical analysis. The significance of associationswere determined using Fisher’s exact probability test, correla-tion analysis, and the two-tailedt test. Survival was analyzed byapplying the Kaplan-Meier method with log-rank analysis.Probability values#0.05 were considered significant.

RESULTSiNOS Immunoreactivity. The results of the study are

compiled in Table 1. Strong iNOS expression could be seen instromal macrophages and neutrophils. Also, stromal fibroblastsand endothelial cells often expressed cytoplasmic positivity foriNOS. In benign nonneoplastic breast epithelial cells, inconsis-tent expression of iNOS could be seen both in the ductal andacinar structures (Fig. 1).

In the whole material, 51 (45.9%) cases showed no staining

Table 1 The apoptotic index, vascular density, and iNOS immunohistochemistry in different breast lesions

Diagnosis Apoptosis % CMVD

iNOS in tumor cells iNOS in stromal cells Sum scores for iNOS

0 1 2 3 1 2 3 1 2 3

In situ carcinomas 0.706 0.79 13.16 6.3 23 17 3 0 20 18 5 15 22 6Grade I 0.296 0.23 13.06 6.6 12 3 0 0 9 5 1 8 7 0Grade II 0.856 0.87 10.46 6.1 7 5 1 0 4 6 3 3 7 3Grade III 1.006 0.92 15.66 5.3 4 9 2 0 7 7 1 4 8 3

Invasive carcinomas 1.456 1.24 14.96 7.6 28 32 7 1 21 28 19 9 40 19Ductal grade I 0.716 0.76 14.76 4.6 3 5 0 1 4 4 1 1 6 2Ductal grade II 1.476 1.40 15.26 10.4 9 14 1 0 7 11 6 4 15 6Ductal grade III 2.146 1.07 13.16 4.9 8 10 5 0 4 10 9 2 11 10Lobular 0.586 0.48 19.46 8.4 7 3 0 0 6 2 2 3 7 0Others 0.54 9.7 1 0 1 0 0 1 1 0 1 1

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for iNOS in the tumor cells, 49 (44.1%) stained weakly (1), 10(9.0%) stained moderately (11), and one (0.9%) stainedstrongly (111). None of the breast tumors labeled verystrongly (1111) with the antibody. The number of positivestromal cells was small in 41 (36.9%), moderate in 46 (41.4%),and high in 24 (21.6%) cases. There was a clear associationbetween tumor cell positivity and high number of positivestromal cells (P5 0.017). The sum scores for iNOS immuno-reactivity were 1 in 24 (21.6%), 2 in 62 (55.9%), and 3 in 25(22.5%) cases. None of the cases reached a sum score 4.

The distribution of iNOS expression in tumor and stromalcells of in situ carcinomas can be seen in Table 1 (Fig. 2A). Thenumber of iNOS-positive cases increased from grade I to III(20.0%, 46.2%, and 73.3%). There were significantly lessiNOS-positive cases in grade I than in grade II-IIIin situ lesions(P 5 0.01) and in grade I-II than in grade IIIin situ lesions (P50.01). No significant differences were found when comparingiNOS positivity in stromal cells or the sum scores in differentgrades ofin situ carcinomas (data not shown).

iNOS expression in tumor and stromal cells of invasivecarcinomas are compiled in Table 1 (Fig. 3Aand Fig. 4A). Nosignificant difference was found in iNOS positivity in tumorand/or stromal cells between different grades in invasive ductalcarcinomas (data not shown). Invasive ductal carcinomas con-tained more cases with iNOS-positive tumor cells than invasivelobular carcinomas (64.3% and 30.0%, respectively;P 5 0.04).

iNOS positivity was more often found in tumor cells ofinvasive ductal carcinomas than inin situcarcinomas (P5 0.05;Table 2). Also, the number of positive stromal cells and sumscores were higher in invasive ductal carcinomas (P 5 0.03 andP 5 0.004, respectively). All different invasive carcinomas hadmore cases with a very high number of iNOS-positive stromalcells thanin situ carcinomas (P5 0.03), and they had alsohigher sum scores (P5 0.006).

All PBS control slides were negative. iNOS immunoreac-tivity was completely abolished in all slides where the antibody

was pretreated with the blocking peptide in the absorptionexperiment (Fig. 3Cand Fig. 4C).

Apoptotic Index. The apoptotic indices are shown inTable 1. The mean apoptotic index was 1.166 1.14%, and themedian was 0.63%. Low-gradein situ lesions showed a signif-icantly lower extent of apoptosis (0.296 0.23%) than interme-diate and high-grade lesions (0.936 0.88%;P 5 0.009). Sim-ilarly, low- and intermediate-grade lesions showed a lowerextent of apoptosis (0.556 0.67%) than high-gradein situlesions (1.006 0.92%;P 5 0.07). In different grades of ductalinvasive carcinomas, the apoptotic indices increased with thetumor grade: grade I (0.716 0.76%) and grade II-III (1.8161.28%;P 5 0.02) or grade I-II (1.286 1.30%) and grade III(2.14 6 1.07%;P 5 0.01). There were also significant differ-ences in apoptotic indices between invasive ductal (1.6461.27%) andin situ carcinomas (0.706 0.79%;P , 0.001) andbetweenin situ lesions and all invasive carcinomas (1.4561.24%;P 5 0.001).

Vascular Density. The results of CMVDs detected byFVIII-related antigen are shown in Table 1. The mean CMVDwas 14.26 7.1/HPF, and the median was 12.8/HPF. There wereno significant differences in vascular density betweenin situ orinvasive carcinomas or between different grades of the tumors(data not shown).

In in situ carcinomas, the average periductal microvesseldensity was 10.86 5.8/mm (range, 0.8–31.2/ mm; median,10.15/mm). The densities in different grades ofin situ carcino-mas were: low grade, 9.06 4.9/mm; intermediate, 12.067.5/mm; and high grade, 10.96 4.8/mm. There were no signif-icant differences comparing periductal microvessel densitiesbetween grade I and grades II-III or between grades I-II andgrade III (P5 0.22 andP 5 0.79, respectively).

Associations of iNOS with Apoptosis, Vascular Density,TNM Class, Survival, and Estrogen and Progesterone Re-ceptor Status. In the whole material, tumors with high sumscores (.1) for iNOS had a higher apoptotic index ($0.63%;

Fig. 1 iNOS staining in nonneoplastic breast epithelial cells. Clear butweak staining can be observed in acinar cells.

Fig. 2 iNOS staining inin situ carcinoma of the breast. Strong cyto-plasmic staining for polyclonal iNOS antibody can be seen in tumorcells of the neoplastic ducts.

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Fig. 3 iNOS staining in ductal invasive carcinoma of the breast. Strongstaining for polyclonal (A) and monoclonal iNOS (B) can be seen in themajority of the tumor cells. The staining with the monoclonal antibodyis stronger and more granular. In the absorption experiment, the iNOSimmunoreactivity is completely abolished (C).

Fig. 4 iNOS staining in invasive lobular carcinoma of the breast. Also,in this case, staining for polyclonal (A) and monoclonal iNOS (B) can beseen in carcinoma cells. The majority of the neoplastic cells expresspositivity with the monoclonal antibody, whereas staining is weakerwith the polyclonal antibody. In the absorption experiment, the iNOSimmunoreactivity is completely abolished (C).

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Fisher’s exact test,P 5 0.02; Table 3). Moderate or highnumber of iNOS-positive stromal cells associated also withhigher apoptotic index (P5 0.03), but iNOS positivity in tumorcells alone did not (P5 0.12).

There was a significant association between iNOS positiv-ity in tumor cells and a high ($12.8) CMVD value (P5 0.04).Also, a moderate or high number of positive stromal cellsassociated with high CMVD index alone (P 5 0.05) and as thesum with iNOS positivity in tumor cells (P5 0.02; Table 4).The periductal microvessel density tended to be higher in iNOS-positive (12.36 6.3/mm) than in iNOS-negative (9.16 5.0/mm) cases (P5 0.08).

There was no difference in iNOS positivity in tumor cellsbetween invasive (T1–4) and in situ (TIS) carcinomas (P50.14), but in invasive carcinomas, there were more cases with ahigh number of positive stromal cells (P5 0.05). Also, the sumof iNOS positivity in tumor and stromal cells associated clearlywith invasive tumors (P5 0.004). A high number of iNOS-positive stromal cells associated with nodal and/or distant me-tastasis (P5 0.05). The iNOS positivity in tumor cells or thesum of iNOS positivity in tumor and stromal cells did notcorrelate with nodal and/or distant metastases (data not shown).

iNOS positivity in tumor or stromal cells did not associatewith survival, estrogen, or progesterone receptor positivity (datanot shown). However, the sum of iNOS positivity in tumor andstromal cells correlated with the progesterone receptor positivity(P 5 0.02).

Comparison of the Polyclonal with the MonoclonaliNOS Antibody. To study the reliability of polyclonal iNOSantibody, we evaluated iNOS activation with a monoclonaliNOS antibody from 53 samples. According to the results, therewas a strong positive correlation between iNOS positivities withthe polyclonal and monoclonal iNOS antibodies (P 5 0.002;Fig. 3Band Fig. 4B).

Immunoblot Analysis. To further test the reliability ofthe polyclonal and monoclonal iNOS antibodies, an immunoblot

analysis with control macrophages was performed with both ofthem. Positive bands corresponding to theMr 130,000 of iNOSprotein could be detected with both antibodies (Fig. 5).

DISCUSSIONThis study was undertaken to investigate the expression of

iNOS in in situ and invasive carcinomas of the breast. BecauseNO is known to influence apoptosis and angiogenesis, we alsostudied the extent of apoptosis and vascular density in the breastlesions.

A previous study has demonstrated that in human breasttumors, iNOS is mainly expressed in stromal cells and not intumor cells and that its stromal presence correlates with tumorgrade (10). However, a study with ZR-75–1 human breastcancer cells revealed that these cells contain iNOS and sponta-neously produce NO (22). Although we also observed strongexpression of iNOS in stromal macrophages and neutrophils inour study, a proportion of tumor cells also displayed clearpositivity. In fact, 58.8% of all invasive and 46.5% ofin situlesions displayed some iNOS positivity in a proportion of thetumor cells. The results thus suggest that, in addition to stromalmacrophages and neutrophils, breast carcinoma cells also con-tain detectable levels of iNOS and are thus capable of producingNO. In fact, the results of a recent study by Duenas-Gonzalesetal. (11) are in keeping with our results. To substantiate thefindings, we immunostained a part of the lesions with a mono-clonal iNOS antibody. Immunostaining with this antibody alsoshowed the presence of iNOS in breast carcinoma tumor cells,and the results were thus consistent with the results obtained bythe polyclonal iNOS antibody.

In other types of epithelial tumors, iNOS positivity hasbeen reported in tumor cells of prostate carcinoma (23), gyne-cological carcinoma (24), colon carcinoma (25, 26), and transi-tional cell carcinoma of the bladder (27). Recently, strong iNOSsynthesis was also discovered in malignant mesothelioma.4

These results are consistent with our findings and suggest that inaddition to other neoplasms, breast carcinoma cells are also ableto modulate NO synthesis.

In in situ lesions of the breast, there were significantlymore cases with iNOS-positive tumor cells in high-grade than inlow-grade tumors. iNOS positivity in tumor cells increased in

4 Y. Soini, K. Kahlos, E. Lakari, P. Pa¨akko, and V. Kinnula. Expressionof inducible and endothelial nitric oxide synthase in healthy pleura andin malignant mesothelioma, submitted for publication.

Table 2 iNOS expression in tumor cells ofin situ and invasiveductal carcinomasa

iNOS-negativecases

iNOS-positivecases Total

In situ carcinomas 23 20 43Invasive ductal carcinomas 20 36 56Total 43 56 99

a Invasive carcinomas express significantly more cytoplasmiciNOS positivity thanin situ carcinomas (P5 0.05).

Table 3 Apoptotic index in relation to the iNOS sum score inin situand invasive carcinoma of the breasta

Apoptosis

iNOS

TotalSum score5 1 Sum score.1

Apind ,0.63% 17 39 56Apind $0.63% 7 47 54Total 24 86 110

a The number of cases with a high apoptosis is significantly higheramong cases with a high iNOS sum score (P5 0.02).

Table 4 Vascularization in relation to the iNOS sum score inin situand invasive carcinoma of the breasta

Vascular density

iNOS

TotalSum score5 1 Sum score.1

CMVD ,12.8 16 35 51CMVD $12.8 7 46 53Total 23 81 104

a The number of cases with a high vascular density is significantlyhigher among iNOS-positive cases (P5 0.02).

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ductal lesions fromin situ carcinomas to invasive. Invasivecarcinomas had also more cases with a very high number ofiNOS-positive stromal cells thanin situ carcinomas. Theseresults suggest that there is an up-regulation of iNOS positivityalong with the biological aggressiveness of the breast lesions.Increased iNOS activity has been positively correlated with thedegree of malignancy also in gynecological tumors (24). Incolon carcinomas, the expression of iNOS remains controversialwhile both decreased (25, 28) and increased (26) expression ofiNOS has been reported with increasing tumor stage.

NO has been reported to be both pro- and antiapoptotic. Ithas been shown to inhibit apoptosis in several cell types, in-cluding endothelial cells (29), hepatocytes (30), lymphocytes(31), leukocytes (32), and eosinophils (33). NO induces apo-ptosis in various cells, including macrophages (34), pancreaticb-cells (35), and thymocytes (36). There is also a study sug-gesting that low concentration of NO inhibits apoptosis, but highconcentrations of NO induces apoptosis in human venous en-dothelial cells (37). The role of NO in apoptosis appears to becell-type-specific and depends on the NO concentration beingproduced.

In our material, there was a gradual increase in apoptoticindex from low-gradein situ carcinomas to high-grade invasiveductal carcinomas with a coexistent up-regulation of total iNOS.A consequent increased production of NO might thus be onereason for the accelerated apoptosis. There were significantlymore cases with a high apoptotic index showing iNOS positivityin tumor and stromal cells than in cases with a low apoptoticindex. These results suggests that NO, produced by iNOS inbreast tumor and in stromal cells, could be an additional factorparticipating in the enhancement of apoptosis in them.

There are several reports concerning the role of NO inangiogenesis.In vitro studies have demonstrated that NO donors

increase and iNOS inhibitors attenuate DNA synthesis, prolif-eration, and migration of coronary venular endothelial cells(38). On the other hand, both NO donors and iNOS inhibitorshave no effect on the fibroblast-growth-factor-induced prolifer-ation and migration of endothelial cells (38).In vivo studieshave shown that NO donors potentiate and iNOS inhibitorsattenuate angiogenesis in rabbit (38) and rat cornea (39). Also,iNOS-transfected human colon adenocarcinoma DLD-1 cellshad higher vessel density and growth ratein vivo than parentalcells (40). In the murine mammary tumor model, the datasuggest that NO is a key mediator of C3L5 tumor-inducedangiogenesis being reduced in NOS inhibitor-treated mice (41).However, controversial results are also obtained (42–44). Thesefindings suggest that NO partially mediates angiogenesis andthat the involvement of NO is both tissue- and/or growth stim-uli-dependent.

We studied tumor angiogenesis with an antibody to FVIII-related antigen and compared vascular density with iNOS ex-pression. Tumors with iNOS positivity in tumor and/or stromalcells had increased vascular densities in the whole material.Also, in in situ carcinomas where vascular densities could alsobe determined in the vicinity of neoplastic ducts, the vasculardensities tended to be higher in iNOS-positive cases. Theseresults suggest that local NO production by iNOS in breastcarcinoma cells are able to modulate angiogenesis. NO produc-tion by stromal cells enhance this effect even more.

iNOS expression may play a role in human cancer progres-sion. Although a few reports indicate that the presence of NO intumor cells or their microenvironment is detrimental for tumor-cell survival, a large body of evidence suggests that NO pro-motes tumor progression (6, 45, 46). In murine mammary ade-nocarcinoma, increased NO production has been shown topromote tumor-cell invasiveness (47). We tested whether theiNOS positivity in breast cancer influences tumor growth meas-ured by TNM status or patient survival. There were no associ-ations between iNOS positivity in breast tumor cells alone andTNM class. However, there were more cases with iNOS positivetumor and stromal cells within invasive tumors (T1–4) comparedto in situ tumors (TIS). Also, nodal and distant metastasesincreased in cases with iNOS-positive stromal cells. These re-sults indicate that in breast tumors NO produced by stromal cellsenhance tumor growth, invasiveness, and metastatic ability. Apart of this effect might be mediated through an increasedangiogenesis caused by iNOS, which, on one hand, wouldenhance the nutrition of the tumor cells, and on the other hand,through increased vessel density, would make more blood ves-sels available for tumor cells to invade.

There are no previousin vivo or in vitro studies on iNOSexpression and hormone receptors in breast cancer. However,eNOS and nNOS have been shown to be expressed only inestrogen-receptor-positive breast cancer cell lines (48). Studieswith other tissues or cell lines have revealed that high amountsof estradiol induce iNOS production in rat aortas (49) and inhuman umbilical vein endothelial cells (50). However, physio-logical concentrations of 17b-estradiol inhibit iNOS productionin the murine macrophage cell line (51). Also, progesterone hasbeen shown to inhibit iNOS production in murine macrophages(52). In our material, there were no associations between iNOSpositivity and estrogen receptor positivity. However, the sum of

Fig. 5 Western blots with mouse macrophage lysate using polyclonaland monoclonal iNOS antibodies. In both cases, aMr 130,000 bandcorresponding to the known molecular weight of iNOS can be seen.

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iNOS positivity in tumor and stromal cells correlated with theprogesterone receptor positivity. The results indirectly suggestthat hormone receptor status and hormonal stimulus may influ-ence iNOS expression in breast carcinoma cells. What effectcertain hormone stimulus eventually has seems to be dependenton cell type and hormone concentration.

At the present time, the role of NO in tumor biology is stillpoorly understood. According to previous reports, NO seems tohave a double-edged role in tumor progression, apoptosis, andangiogenesis. iNOS response to hormone stimulus varies alsodepending on cell type and activity. The effect of iNOS dependson the concentrations of NO being produced and the localenvironment in different tumor and cell types. Our results showthat in addition to stromal cells, iNOS is expressed in neoplasticcells of breast carcinoma. In ductal lesions, the iNOS positivityin tumor cells increased fromin situ carcinomas to invasivecarcinomas. Invasive carcinomas had also more cases with avery high number of iNOS-positive stromal cells thanin situcarcinomas. The results thus show that there is an up-regulationof iNOS positivity along with the biological aggressiveness ofthe breast lesions. NO produced by iNOS in breast tumor andstromal cells seems to enhance apoptosis. On the other hand,NO production by tumor cells and stromal cells increases tumorvascularization and possibly through this effect also enhancetumor growth and metastatic ability.

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2416iNOS in Breast Carcinoma

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2000;6:2408-2416. Clin Cancer Res   Merja Vakkala, Katriina Kahlos, Essi Lakari, et al.  

and Invasive Breast Carcinomasin SituAngiogenesis in Inducible Nitric Oxide Synthase Expression, Apoptosis, and

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