Tumour-derived and host-derived nitric oxide differentially regulate breast carcinoma metastasis to the lungs
Nolwenn Gauthier1,
Sylvia Lohm2,
Claude Touzery3,
Aurélie Chantôme1,
Bernard Perette3,
Sylvie Reveneau1,
François Brunotte3,
Lucienne Juillerat-Jeanneret2 and
Jean-François Jeannin1,4
1 EPHE/INSERM/Laboratoire d'immunologie et immunothérapie des cancers, Unité 517 et IFR 100, Université de Bourgogne, Dijon, France, 2 Institut Universitaire de Pathologie, CHUV, Lausanne, Switzerland and 3 Service de médecine nucléaire, Centre Georges-François Leclerc, et IFR 100 de l'INSERM, Dijon, France
4 To whom correspondence should be addressed Email: jean-francois.jeannin{at}u-bourgogne.fr
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Abstract
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To study the role of nitric oxide (NO) in lung metastasis of breast carcinoma, we isolated two cell clones (H and J) from the parental EMT-6 murine breast carcinoma cell line, based on their differential NO production. In vitro, EMT-6 J cells, but not EMT-6H cells, constitutively expressed inducible NO synthase (NOS II) and secreted high levels of NO. IL-1ß increased NO production in both clones, and TNF-
had a synergistic effect on IL-1ß-induced NO production, but NO production by EMT-6 J cells was always higher than by EMT-6H cells. Proliferation, survival and adhesion to lung-derived endothelial cells of both clones were similar and were not affected by NO. In vivo, both clones similarly located in the lungs of syngeneic mice 48 h after injection. However, EMT-6H cells were significantly more tumorigenic than EMT-6 J cells as assessed at later time points. Injection of EMT-6 J cells and simultaneous treatment of mice with aminoguanidine (AG), a NOS II inhibitor, significantly increased tumour formation. Injection of EMT-6H and EMT-6 J cells into NOS II-deficient mice resulted in a significant survival increase as compared with wild-type animals. Simultaneous administration of AG increased the death rate of NOS II-deficient mice injected with EMT-6 J cells. These results demonstrate that: (i) NO does not influence the early stages of tumour metastasis to the lungs and (ii) NOS II expression in tumour cells reduces, while NOS II expression in host cells enhances, tumour nodule development. In conclusion, the cellular origin and the local NO production are critical in the metastatic process.
Abbreviations: AG, aminoguanidine; EMEM, Eagle's minimal essential medium; MMP, matrix metalloproteinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NO, nitric oxide; NOS II, inducible nitric oxide synthase
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Introduction
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The metastatic and growth potential of tumours is dependent on the ability of circulating tumour cells to escape immune responses, to adhere to endothelium, to extravasate and to survive and grow in the target tissue. Nitric oxide (NO) is a potent bioactive molecule produced by nitric oxide synthase (NOS) enzymes. NO has been involved in numerous physiological as well as pathological mechanisms such as vasodilatation and inflammation. Demonstration of NO and of inducible NOS (NOS II) expression in various solid tumours and their metastases as well as in tumour cell lines has suggested that this biological mediator may participate in tumour survival and metastatic process. However, conflicting results have been reported that may reflect a more complex effect of NO than anticipated previously. NO has been shown to decrease the expression of adhesion molecules such as VCAM-1, ICAM-1, E-selectin that mediate interactions between tumour cells and host endothelial cells (13). Furthermore, NO could impair tumour cell extravasation and extracellular matrix (ECM) degradation through inhibition of the secretion of matrix metalloproteinases (MMPs) such as MMP-2 (4). Sustained overproduction of NO may also enhance tumour cell susceptibility to apoptosis and death through the expression or activation of cell death effectors such as p53 (5) or caspases and Fas (6). These observations suggest a negative role of NO on tumour progression or metastasis. However, protumorigenic effects of NO have also been reported. NO vasodilatory and angiogenic properties were associated with tumour growth and may participate in tumour-induced immunosuppression by decreasing proliferation and activation of lymphocytes (7). The tumour microenvironment may also influence tumour development as low NO concentrations may favour angiogenesis and tumour growth while higher NO concentrations may be cytostatic (8,9).
Breast tumours mainly metastasize to the lungs and bones (10). In the lungs, mammary tumour cells are retained in capillaries (11) where they adhere to endothelial cells through specific adhesion molecules such as integrins, selectins and N-cadherin. Tumour cells may then invade the lung parenchyma following proteolysis of the ECM through secretion of proteolytic enzymes such as MMP-2 (12). In previous work we had observed high levels of NOS II reactivity in malignant human breast cancer, which negatively correlated with proliferation and tumour grade (13). In order to define in more detail the role of NO in the metastatic process, we developed an experimental model consisting of high and low NO-producing murine breast carcinoma cell clones derived from the parental mammary EMT-6 tumour cell line (14). The in vitro proliferation and adhesive properties to murine lung-derived endothelial cells were determined. The implication of tumour-derived and of host-derived NO in the metastatic potential of both clones to the lungs was further investigated in syngeneic NOS II-deficient mice. How the cellular origin of NO may differentially regulate tumour metastasis is discussed.
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Materials and methods
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Cell selection
The cell clones EMT-6H and EMT-6 J, syngeneic to Balb/c mice, were obtained by limiting dilution from the EMT-6 parental breast tumour cell line (kindly provided by Dr M.Lepoivre, CNRS, Orsay, France). They were selected based on NO production: EMT-6 J released significant levels of NO while EMT-6H did not. Cells were grown as monolayers at 37°C in Eagle's minimal essential medium (EMEM, Eurobio, Les Ulis, France) supplemented with 10% fetal calf serum (FCS Hyclone, Biovalley, Marne la Vallée, France) and 2 mM L-glutamine (Biowhittaker, Fontenay sous Bois, France), in a humidified atmosphere (5% CO2 and 95% air). Cultures were tested monthly for Mycoplasma contamination with the mycotect kit (Life Technologies, Cergy Pontoise, France) and before each in vivo experiment.
Cell culture protocols
For cytokine-stimulated NO production, EMT-6H and EMT-6 J cells (5 x 104 cells/well) were grown for 24 h in 96-well plates then treated for another 24 h with the cytokines IL-1ß (Sanofi, Labège, France), TNF-
and IFN-
(Insight Products, UK). When indicated, cells were grown for 24 h with 0.5 mM aminoguanidine (AG) (Sigma-Aldrich, Lyon, France). For proliferation assay, EMT-6H and EMT-6 J cells (5 x 103 cells/well) were grown in 96-well plates for 48 h in the presence or not of 0.5 mM AG. Proliferation was assessed after 48 h by incorporation of [3H]thymidine (1 µCi/well, Amersham Pharmacia, Buckinghamshire, UK) for the last 2 h of incubation. The cells were washed twice with PBS and adherent cells were lysed in 1 N NaOH for 2 h at 60°C. Radioactivity was determined by liquid scintillation spectrometry (LKB Rackbeta LKB Wallac, Turku, Finland). Spontaneous cell death was measured as described previously (15). Briefly, 2.5 x 105 EMT-6H or EMT-6 J cells were seeded in 6-well plates and incubated overnight at 37°C with 1 µCi [3H]thymidine. The labelled cells were detached and seeded (25 x 103 cells/ml, 200 µl/well) in a 96-well plate for 48 h. Cells were lysed and cell-associated radioactivity determined as above. For the determination of cellular ability to adhere to plastic (15,16), EMT-6H or EMT-6 J cells (104 cells/well) were seeded in 96-well plates and incubated at 37°C for 48 h. Cells were washed with PBS, fixed in methanol, stained with 1% methylene blue for 30 min, dissolved in 0.1 N HCl and absorbance was measured at 620 nm in a multi-well microtitre plate reader (Anthos, Labtech Instruments, Emerainville, France). Alternatively EMT-6H or EMT-6 J cells (5 x 103 cells/well) were seeded in 96-well plates and incubated at 37°C for 48 h. Cells were washed with 0.9% NaCl, stained with neutral red for 2 h, fixed in a 0.4% formalin1% CaCl2 solution and extracted in 1% acetic acid in 48% ethanol. Absorbance was measured at 540 nm in a multi-well microtitre plate reader. Cellular survival was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (17). Briefly, EMT-6H or EMT-6 J cells (5 x 103/well) were seeded in 96-well plates and incubated at 37°C for 48 h. Then 10 µl of 5 mg/ml MTT in PBS were added for 2 h. Formazan crystals were dissolved in 200 µl DMSO and absorbance was measured at 570 nm in a multi-well microtitre plate reader.
Determination of NO production
NO production was determined as nitrite and nitrate accumulation in cell culture media using the Griess microassay (18). Briefly, nitrates were reduced to nitrites using nitrate reductase (Diffchamb, France) and 100 µl of reduced supernatants were added to 100 µl Griess reagent (0.5% sulfanilamide and 0.05% naphthylethylenediamine in 2.5% phosphoric acid). Absorbance was measured at 540 nm in a multi-well microtitre plate reader and nitrite concentration was calculated by comparison with a standard sodium nitrite solution.
NOS II RTPCR analysis
Total RNA was prepared using the Nucleospin® RNA II kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer's protocol. 550 ng RNA were used for NOS II amplification and 125 ng for GAPDH amplification. RTPCR was performed using the two-enzyme Access RTPCR system (Promega, Charbonnière, France). First strand cDNA synthesis was carried out at 48°C for 45 min, then the AMV reverse transcriptase was inactivated and the RNAcDNA hybrid denatured by a 2-min incubation at 94°C. Amplifications were performed using 25 cycles as follows: 94°C/30 s, 63°C/30 s, 68°C/1 min. The sequences of NOS II RNA, located between +1379 and +1877, and GAPDH RNA, located between +256 and +750, were amplified using the following sense and antisense primers: 5'-CCAGTGTCTGGGAGCATCACCCCTG-3' and 5'-GAACTGAGGGTACATGCTGGAGCC-3' (NOS II) and 5'-GCCCATCACCATCTTCCAGGAGCG-3' and 5'-GGGGTAGGAACACGGAAGGCCATGC-3' (GAPDH). The PCR products migrated as a single band of 498 bp for NOS II and of 504 bp for GADPH.
In vitro adhesion of tumour cell clones to lung-derived endothelial cells
The End p23 murine pulmonary endothelial cell line and the adhesion assay have been described previously (19,20). Briefly, sub-confluent End p23 cells were cultured for 24 h in 48-well microtitre plates and washed twice with PBS immediately before the adhesion assay. EMT-6H and EMT-6 J tumour cells were labelled with 0.4 µCi/ml of [3H]thymidine for 16 h and added (105 cells/well) to confluent End p23 monolayers in Dulbecco's Modified Eagle's medium (DMEM) without FCS. After 30, 60 or 180 min incubation at 37°C non-adherent cells were removed by washing three times with DMEM. Adherent cells were dissolved overnight in 0.1% SDS/0.1 M NaOH and radioactivity was quantified by liquid scintillation spectrometry. Adhesion (%) was determined as the ratio of the c.p.m. of adherent cells to the c.p.m. of total (adherent and non-adherent) cells x 100.
Maintenance and genotyping of NOS II-deficient mice
C57BL/6x129SvEv mice with a targeted disruption of the NOS II gene (NOS II/) (21) were obtained from Dr Y.Courtois (INSERM U450, Paris, France) and mated in our animal facility with Balb/c wild type mice (+/+) (Charles River Laboratories, l'Arbresle, France) for nine generations to produce heterozygous NOS II-deficient mice (+/). Heterozygous NOS II-deficient mice were mated to produce NOS II / mice. Genotyping to confirm the presence or absence of the NOS II gene was performed using PCR of DNA from tail biopsies. Samples were incubated overnight at 54°C in lysis buffer [100 mM Tris, pH 8.5, 5 mM ethylene diamine tetraacetic acid (EDTA), 200 mM NaCl, 0.2% SDS and 100 µl proteinase K (10 mg/ml)]. After centrifugation, the DNA was purified by two cycles of isopropanol precipitation and phenolchloroform extraction, precipitated and washed in 70% cold ethanol and resuspended in 100 µl of TrisEDTA (10 mM Tris, pH 7.5, 1 mM EDTA, pH 8). DNA products were amplified by 30 cycles of PCR (94°C/30 s, 60°C/30 s and 72°C/45 s) using Taq DNA polymerase (Eurobio, Les Ulis, France). The sequence of the NOS II gene located between 499 and +161 was amplified using the following sense and antisense primers: 5'-GCACAGCCCATCCACTATTCT-3' and 5'-GAGTGAACAAGACCCAAGCGT-3'. To identify the NOS II +/ and / mice, the amplification of the neo resistance gene was performed using the sense and antisense oligonucleotides: 5'-ATCGACAAGACCGGCTTCCATCCGA-3' and 5'-TCAGCGCAGGGGCGCCCGGTCTTT-3', which amplify a 475 bp PCR product. The amplified products were electrophoresed in 1% agarose gel containing ethidium bromide.
Tumour nodule induction in lungs
Animal experiments were performed using 89-week-old female wild-type mice and NOS II-deficient (NOS II KO) female Balb/c mice. All experimental protocols had been approved by the Ethics Committee of the University of Dijon, France. EMT-6H or EMT-6 J cells in exponential growth phase were harvested, dissociated to produce single-cell suspensions, washed and suspended in culture medium. Cell viability was determined by trypan blue exclusion and only cell suspensions with >90% viability were used for animal experimentation. 100 000 cells in 0.1 ml of EMEM were injected into the lateral tail vein of non-anaesthetized mice. The mice were killed after 35 days. Killed and spontaneously dead mice were autopsied and pulmonary nodules were counted. AG-treated mice received intraperitoneal (i.p.) injections of 50 mg/kg AG, an inhibitor of NOS II activity (22), twice a day, throughout the experiment.
111Indium oxine labelling procedure and location of 111In-labelled cells in mice
EMT-6H or EMT-6 J cells [2 x 106 cells in 0.5 ml EMEMHEPES (Biowhittaker, Fontenay sous Bois, France)] were incubated with 85 µCi of 111indium oxine (Mallinckrodt, Les Ulis, France) for 15 min at 37°C as described previously (23). Cells were washed three times with EMEMHEPES to remove free 111In. Cell-bound 111In radioactivity was measured using a gamma counter (MR 480 Packard, Packard Bioscience, Meriden, CT). Labelled cells (105 cells) were injected in the tail vein of mice. Radioactivity location was determined in anaesthetized animals using a single-head Basicam gamma camera (Siemens Medical System, Erlangen, Germany) for image acquisition consisting of 30 images of 1 min each. The images were analysed with the SMV-GE Vision processing system (Sopha Medical Vision-General Electric, Buc, France). Thorax and abdomen were manually defined and the radioactivity in the two regions was calculated based on a summed image. Mice were killed at different time points after injection, and heart, liver and lungs were collected, washed with 0.9% NaCl and their 111In content was determined in an automatic well counter (MR 480 Packard, Packard Bioscience).
Data analysis
Data are presented as mean ± SD. Statistical differences were determined by unpaired Student's t test or
2 test. Survival rates were calculated using the KaplanMeier method, according to tumour cell clone or treatment. Curves were compared using the log rank test. Survival rates were computed using the Stata package (Stata 1999, College Station, TX). Differences were considered significant for P
0.05.
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Results
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Two cell clones were isolated by limiting dilution from the parental EMT-6 murine mammary carcinoma cell line and selected from their production of NO (measured as the accumulation of nitrites plus nitrates in the culture supernatant). EMT-6H cells produced NO levels below the detection threshold whereas EMT-6 J cells released up to 5 µM NO2 + NO3/48 h/5 x 104 cells (Table I). EMT-6 J cells, but not EMT-6H cells, expressed NOS II mRNA (Figure 1) as detected by RTPCR amplification. IL-1ß increased NO production while TNF-
and IFN-
had no effect (Table I). Synergistic effect was observed with IL-1ß plus TNF-
but not with IL-1ß plus IFN-
or TNF-
plus IFN-
(Table I). In all treatments, NO released in response to cytokines by EMT-6 J cells was always higher than that of EMT-6H cells (P < 0.001, Table I).

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Fig. 1. EMT-6 J cells but not EMT-6H cells express NOS II mRNA Cells were grown to sub-confluence then total RNA was prepared for RTPCR amplification. The results of one representative experiment out of three are shown.
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Spontaneous as well as induced NO production was inhibited by 0.5 mM AG (Table I). In vitro proliferation, spontaneous cell death, cellular ability to adhere to plastic and cellular metabolic activities were similar for both cell clones and were not affected by AG. These results excluded a direct role of NO on the growth and survival of EMT-6H or EMT-6 J tumour cells in vitro. In vitro proliferation was measured by [3H]thymidine incorporation, EMT-6H cells with or without AG: 266 x 103 ± 13 x 103 and 234 x 103 ± 24 x 103 c.p.m., respectively, and EMT-6 J cells with or without AG: 253 x 103 ± 32 x 103 and 239 x 103 ± 14 x 103 c.p.m., respectively. Spontaneous cell death was measured by [3H]-thymidine release, EMT-6H cells with or without AG: 100 x 103 ± 7 x 103 and 96 x 103 ± 9 x 103 c.p.m., respectively, and EMT-6 J cells with or without AG: 112 x 103 ± 15 x 103 and 134 x 103 ± 55 x 103 c.p.m., respectively. Cellular ability to adhere to plastic was determined by methylene blue assay, absorbance at 620 nm, EMT-6H cells with or without AG: 1.63 ± 0.21 and 1.46 ± 0.17, respectively, and EMT-6 J cells with or without AG: 1.80 ± 0.09 and 1.77 ± 0.08, respectively. Cellular metabolic activities were assessed by neutral red exclusion and MTT reduction. Neutral red exclusion, absorbance at 540 nm, EMT-6H cells with or without AG: 0.82 ± 0.05 and 0.79 ± 0.04, respectively, and EMT-6 J cells with or without AG: 0.82 ± 0.1 and 0.80 ± 0.04, respectively. MTT reduction, absorbance at 570 nm, EMT-6H cells with or without AG: 0.71 ± 0.09 and 0.78 ± 0.06, respectively, and EMT-6 J cells with or without AG: 0.65 ± 0.05 and 0.77 ± 0.07, respectively.
For tumour cell location in organs after i.v. injection, the tumour cells were labelled with 111indium oxine. In vitro proliferation and survival of labelled EMT-6H and EMT-6 J cells were similar for both clones, as >80% of labelled H or J cells were alive after 48 h culture. 100 000 of 111In-labelled cells were injected in the tail vein of syngeneic mice and tumour cell location was evaluated by whole mice scintigraphy images, within 48 h, both clones located mainly in the thorax. As a control, injection of cell-free 111indium oxine in mice resulted in a homogenous distribution over the whole mice (data not shown). The organs of mice injected with 111In-labelled EMT-6H or EMT-6 J cells were excised at different times and radioactivity was quantified. Three independent experiments showed that 48 h after tumour cells injection
40% of total injected radioactivity was retained in the lungs for both cell clones. The radioactivity ratio (mice injected with EMT-6H to EMT-6 J cells) measured in the lungs was stable for 48 h (Figure 2), indicating that short-term tumour cell location in the lungs was independent of clones. Radioactivity was also detected in the liver (results not shown), but as tumour nodules were never observed in this organ, liver-associated radioactivity was attributed to accumulated dead cells.

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Fig. 2. 111Indium-labelled EMT-6H or EMT-6 J tumour cells location in the lungs was independent of clones EMT-6H and EMT-6 J cells were labelled with 111indium oxine (111In) and injected in the tail vein of syngeneic mice. The results from one representative experiment out of four are shown. Radioactivity measured in the excised lungs of mice at different times after i.v. injection of 105 111In-labelled EMT-6H (clear bar) or EMT-6 J cells (black bar).
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As in vivo experiments showed that both tumour cell clones located in the lungs within 48 h, we evaluated the in vitro adhesive properties of EMT-6H and EMT-6 J cells on murine lung-derived p23 endothelial cells. Short-term (30 or 60 min) and long-term (3 h) adhesion assays were performed (Figure 3) and showed that both EMT-6H and EMT-6 J cells adhered similarly to lung-derived endothelial cells in vitro. Taken together these results suggest that tumour-derived NO was not critical in one of the first steps of tumour nodule formation, the initial arrest of breast tumour cells in the pulmonary endothelium.

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Fig. 3. Adhesion of EMT-6H and EMT-6 J cells to murine lung-derived endothelial cells. Adhesion of [3H]thymidine-labelled EMT-6H cells or EMT-6 J cells to endothelial cells was measured in vitro after either 30 (shaded bar), 60 (clear bar) or 180 min (black bar). Results are expressed in per cent of adherent cells and are the means of triplicate wells ± SD. Three independent experiments were performed. No statistically significant difference was found.
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However, long-term survival experiments showed a significant difference between mice injected with EMT-6H or EMT-6 J cells. Twenty-seven out of twenty-nine mice (93%) injected with EMT-6H cells died within 35 days, while only eight out of 29 mice (28%) injected with EMT-6 J cells died before day 35 (P < 0.0001, Figure 4). In contrast, 19 out of 30 mice (63%) injected with EMT-6 J cells and receiving AG (50 mg/kg i.p. injected twice a day for 35 days), an inhibitor of NOS II activity, died within 35 days (Figure 4, P = 0.0071, mice treated with AG compared with untreated mice). On the other hand, the life span of mice injected with EMT-6H cells did not change when mice were treated with AG. EMT-6H cells induced the formation of numerous tumour nodules in the lungs of all the injected mice (Table II). In contrast, the number of mice with pulmonary tumour nodules was lower and the number of nodules per lung was lower (P < 0.001) in the EMT-6 J group (Table II). AG treatment increased the number of tumour nodule-bearing mice and decreased the number of tumour nodules per lung in mice injected with EMT-6 J cells (P < 0.01) but had no significant effect in mice injected with EMT-6H cells (Table II). Therefore, there was a relationship between the life span of mice and the number of tumour nodules they bore.

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Fig. 4. Survival of mice injected with EMT-6H cells or EMT-6 J cells. Mice were injected in the tail vein with 105 EMT-6H cells or EMT-6 J cells and mice survival was determined. AG was injected i.p. twice a day. Surviving mice were killed 35 days after the injection of tumour cells.
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As i.p. injection of AG would result in inhibition of NOS II activity both in tumour cells and in host cells, these experiments did not allow to evaluate the contribution of host-derived NO in the formation of tumour nodules in the lungs. To further investigate the respective roles of tumour cell-derived or host-derived NO, we used NOS II-deficient mice. Twenty-four out of twenty-seven (90%) wild-type mice injected with EMT-6H cells died within 35 days, while 16 out of 27 (60%) NOS II-deficient mice (Figure 5A, P = 0.0006) died within the same time. Seven out of twenty-eight (25%) wild-type mice injected with EMT-6 J cells, while two out of 30 (7%) NOS II-deficient mice (Figure 5B, P = 0.06) died within 35 days. The number of tumour nodules in the lungs of NOS II-deficient mice injected with EMT-6H was lower than in wild-type mice (P = 0.016). In mice injected with EMT-6 J cells, the number of tumour nodule-bearing mice and the number of tumour nodules per lung were lower in NOS II-deficient mice than in wild-type mice (P < 0.01) (Table III). Taken together, these results demonstrated that tumour growth and mice survival was increased in NOS II-deficient mice independently of the cell clone injected.

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Fig. 5. Survival of wild-type (WT) mice (circles) and NOS II KO mice (diamonds) injected with EMT-6H cells or EMT-6 J cells. Mice were injected in the tail vein with 105 EMT-6H (A) or EMT-6 J (B) cells and mice survival was determined. Surviving mice were killed 35 days after the injection of tumour cells.
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Table III. Counting of tumour nodules in the lungs of wild-type and NOS II-deficient mice injected either with EMT-6H or EMT-6 J cells
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None of the nine NOS II-deficient mice injected with EMT-6 J cells died within 35 days while three out of the nine mice receiving AG died within the same time (P < 0.05, Figure 6). The number of tumour nodule-bearing mice and the number of tumour nodules per lung increased in AG-treated mice (data not presented because of marginal statistical significance). Therefore, NO production by tumour cells prevented tumour progression.

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Fig. 6. Survival of NOS II KO mice injected with EMT-6 J cells. Mice were injected in the tail vein with 105 EMT-6 J cells and mice survival was determined. AG was injected i.p. twice a day or not. Surviving mice were killed 35 days after the injection of tumour cells.
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Discussion
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It was shown previously that NOS II is heterogenously expressed in human breast cancer (24). NOS II expression has been demonstrated using immunohistochemistry in mammary tumour cells (13,2426), or in cancer stroma (25,27). Moreover, the evaluation of NOS II activity in human breast cancer gave inconsistent results, being either negatively correlated (13,25), positively correlated (26) or not correlated (24) to tumour grade. The role of NO in breast tumour progression is also controversial: NO may either favour metastasis by promoting cancer cell migration, invasiveness and angiogenesis (2830,34), or antagonize metastasis by decreasing the expression of adhesion molecules (13).
To investigate whether the cellular origin of NO may affect the metastatic potential to the lungs of breast tumour cells we investigated first the role of tumour-derived NO. Toward this goal, we subcloned low and high NO-producing murine breast carcinoma cell lines from the parental EMT-6 cells (14). The results indicated that NO produced by tumour cell was not implicated in vitro in tumour cell proliferation, survival or adhesion to murine lung-derived endothelial cells. Thus, both EMT-6H and EMT-6 J cells are NO resistant. In vivo, tumour cell-derived NO was not involved in the localization of tumour cells in the lungs of syngeneic mice within 48 h following i.v. injection. These data indicated that NO produced from tumour cells was not a determining factor in the initial arrest of tumour cells in the lung capillaries. However, in vivo, in the long term, the low NO-producing tumour cells (EMT-6H cells) developed more tumour nodules in the lungs than the high NO-producing tumour cells (EMT-6 J cells). The death rate of mice injected with low NO-producing cells was also higher than that of mice injected with high NO-producing cells. These results suggested that NO may be protective at later metastatic stages than the initial tumour cells arrest in the lungs. In long-term in vivo experiments, inhibition of NOS II activity by AG (22) in tumour cells and in animals increased the death rate, the number of tumour-bearing mice and the number of nodules in mice injected with EMT-6 J cells, but not in mice injected with EMT-6H cells, which rules out a toxic effect of AG. Thus, in NOS II-expressing mice, NO was protective against metastasis. In NOS II-deficient mice, the development in the lungs of tumour nodules was decreased and mice survival increased, whatever the tumour cell clone. These results indicate that NOS II expression by the host cells favours metastasis. Thus, in our model, NO had opposite effects depending on whether it was tumour cell-derived or host-derived. In agreement with our results, it was shown that NOS II disruption in mice enhanced metastasis of NO-sensitive murine ovarian sarcoma cells (31). And NOS II disruption in mice suppressed metastasis of NO-resistant murine melanoma cells, which was dependent on down-regulation of VEGF in melanoma metastases (32).
Thomsen et al. (33) have reported that NOS II inhibition by 1400W reduced the size of subcutaneous (s.c.) EMT-6 tumours. Thus, NO functions in the growth of s.c. tumour are different from its role in tumour metastasis to the lungs. Another explanation of this disagreement is certainly the heterogeneity of the EMT-6 parental cells. Using two mammary tumour cell lines differing in their metastatic phenotype, Orucevic et al. (34) showed that NO promoted tumour cell invasiveness. This metastasis-promoting effect of NO released by tumour cell was attributed to NOS III, which produces only low amounts of NO, comparable with the levels secreted by EMT-6H cells. Our present results demonstrated that low levels of tumour-derived NO promoted tumour development. Thus, it appears that not only the cellular origin of NO, but also the level of NO secretion is crucial for its biological effects in cancer.
The mechanisms involved in NO-dependent reduction of the development of tumour nodules in the lungs are not yet elucidated. NO has been shown to decrease leukocyte adhesion to endothelial cells (3537) through inhibition of the expression of adhesion molecules, either on endothelial cells (1) (such as VCAM-1) or on leukocytes (38) (such as E-selectin or ICAM-1). In vitro NO was shown to up-regulate the expression of ICAM-1 on tumour cells (3) and to increase the adhesion of tumour cells to endothelial cells (39). However, in vivo only few data are available. Our in vitro and short-term in vivo experiments did not support a NO-dependent adhesion of breast cancer cells to pulmonary endothelial cells. NO was also shown to inhibit platelet aggregation (40), which promotes metastasis. The regulating role of NO in inflammatory response could also be involved. In support of this possibility, we have shown previously that NO sensitizes colon tumour cells to Fas-mediated cell death in vitro (6) implying the involvement of NK or T lymphocytes. However, our studies do not exclude the possibility that phenotypic differences between the EMT-6H and EMT-6 J cells other than NO-producing ability, e.g. the degree of MMP expression, may have added to the differences in their metastatic ability or mortality in tumour-bearing hosts.
In conclusion, our results demonstrated: (i) that NOS II expression by breast tumour cells reduces, while NOS II expression by host cells enhances, tumour nodule development in the lungs of syngeneic mice; (ii) that local NO concentration is important in this process; and (iii) that NO is not decisive in the initial arrest of breast tumour cells in lung capillaries, but is critical in later stages of nodule development.
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Acknowledgments
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We thank François Clinard (Faculty of Pharmacy) for his help in statistical calculation and Valérie Saint-Giorgio (INSERM) for her help in animal manipulations. This work was supported by grants from the Ligue contre le Cancer de Bourgogne, the Ligue contre le Cancer du Jura et de Haute-Marne, OM-Pharma and the Conseil Régional de Bourgogne and grants from the Swiss League and Research against Cancer Foundation (grants SKL 353-9-1996, KFS 947-09-1999 and KFS 1070-09-2000) and from Ministère des Affaires Etrangères (PAI, Germaine de Stael: 05556QM).
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Received May 17, 2003;
revised March 24, 2004;
accepted March 25, 2004.