Seven- to eight-week-old BALB/c female mice were obtained from Charles River Laboratories Polska -Animal Lab (Poznan, Poland). Mice were housed in specific pathogen-free conditions (SPF) and fed a standard laboratory diet and water ad libitum.
All experimental procedures used in the present study were followed according to the Guidelines for Animal Care and Treatment of the European Communities and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). All procedures were approved by the First Local Ethical Committee on Animal Testing at the Jagiellonian University (Krakow, Poland), permit no: 140/2013.
Tumour cell line
Mouse mammary adenocarcinoma 4 T1 cells were obtained from American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 (Laboratory of Analytical Chemistry, IIET) with Opti-MEM® media (Life Technologies) (1:1 v/v) and 5% fetal bovine serum (HyClone, Thermoscientific), supplemented with 4.5 g/L glucose, 2 mM glutamine, 1.0 mM sodium pyruvate (all from Sigma-Aldrich) and antibiotics (penicillin and streptomycin; Polfa Tarchomin). Cell cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2.
Murine model of metastatic breast cancer
1 × 104 viable 4 T1 tumour cells suspended in 0.05 ml of Hanks Balanced Salt Solution were orthotopically inoculated into the right mammary fat pad of female BALB/c mice. Analyses were conducted at 2, 4, 5 and 6 weeks after 4 T1 cancer cell transplantation. Prior to each analysis, animals were randomly divided into two experimental groups (one group designated for the analysis and the other for further tumour development), so that the mean tumour volumes and tumour volume distributions were similar between experimental groups. Healthy BALB/c mice were used as a control group, and were analysed simultaneously with the tumour-bearing mice at 2, 4, 5 and 6 weeks after 4 T1 cancer cell transplantation.
Animals were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine and xylazine (100 mg ketamine and 10 mg xylazine/kg body weight). Blood samples were collected from the right ventricle of the heart using a syringe containing anticoagulant (EDTA 1.6 mg/ml).
Assessment of the primary tumour and number of metastases in the lungs
Primary tumours were carefully dissected from the surrounding tissues and weighed. Isolated lungs were washed in saline, weighed, and fixed with 4% formalin buffered solution. The number of metastases was macroscopically assessed; metastatic sites visible on the lung surface were visually counted using a magnification glass.
Macroscopic analysis of the lungs was followed by paraffin embedding, histological HE staining and histopathological assessment of the tissue.
Histological and immunohistochemical analysis of the lungs
Lungs were fixed in 4% buffered formalin (for at least 48 h). After macroscopic analysis of the number of metastases, the lungs were prepared using the paraffin method, cut into 6 μm sections on an Accu-Cut® SRM™ 200 Rotary Microtome and stained with hematoxylin and eosin.
Light microscopic examination and photographic documentation were performed using an Olympus BX53F microscope equipped with a digital camera. Pictures were taken under the magnification 20× and 200×.
For immunohistochemical staining of VCAM-1 and vWF in the lung vasculature following deparaffinization, sections were pretreated according to the citrate-based HIER protocol and then preincubated with 5% goat serum (Jackson ImmunoResearch) and 2% dry milk to minimalize non- specific binding of antibodies. Primary rat-anti-mouse VCAM-1 (Chemicon) or rabbit-anti-mouse vWF (Abcam) antibodies were used, followed by Cy3-conjugated goat-anti-rat or Cy3- conjugated goat-anti-rabbit secondary antibodies (Jackson ImmunoResearch), respectively. Images were acquired using the AxioObserver D2 inverted fluorescent microscope (Carl Zeiss) and an AxioCam HRm monochromatic digital camera and stored as TIFF files. Fluorescence intensity was analysed automatically by Columbus software (Perkin Elmer).
Assessment of NO production in the isolated lung preparation
Lungs were isolated from anaesthetized (pentobarbitone, 140 mg/kg, i.p.) animals and perfused at a constant flow of about 1.50 ml/min with low glucose DMEM with 4% albumin and 0.3% HEPES, mounted in a water-jacketed artificial thorax and ventilated with negative pressure at a rate of 90 breaths/min. The end-expiratory pressure in the chamber was set to − 3 cm H2O and inspiratory pressure was adjusted between − 6 and − 10 cm H2O to yield an initial tidal volume (TV) of about 0.2 ml. Every 5 min during the experiment, a deep breath of − 21 cm H2O end-inspiratory pressure was automatically initiated in order to avoid atelectasis. The PAP was set to around 3 cm H2O. Venous pressure was set to 2–5 cm H2O. All lungs preparations were allowed to equilibrate for at least 15 min under perfusion. Nitrate and nitrite concentrations were then measured in the effluent from the isolated lungs perfused with constant, non-recirculating flow. The samples were analysed via sensitive high-pressure liquid chromatography (HPLC) –based techniques (ENO-20 NOx Analyser; EiCom, Kyoto, Japan).
Assessment of NO-dependent endothelial function in isolated rings of mice aorta
Mice aorta preparations and endothelial function assessment of aortic rings were conducted as previously described . Briefly, NO-dependent endothelial function was measured by response to acetylcholine (Ach; 0.01–10 μM) in phenylephrine (Phe; 0.1–1 μM) pre-contracted vessels. Endothelium-independent vasodilatation was determined using sodium nitroprusside (SNP; 0.001–1 μM). To ensure that entire Ach-evoked response was NO-dependent the response to Ach and SNP was also measured in the presence of L-NAME. Responses were recorded using a data acquisition system and recording software (Power Lab, Lab Chart, AD Instruments, Australia).
Assessment of nitrite production in isolated aorta rings
Basal NO production by the aorta was estimated using measurements of nitrite. Segments from the aortic arch were longitudinally opened, placed in a 96-well plate with endothelium facing up, and incubated for 1 h in 120 μl K-H buffer at 37 °C using a specially-designed closed chamber (BIO-V (Noxygen)) that was equilibrated with carbogen gas mixture (95% O2, 5% CO2). Samples from the incubation buffer were put on ice and used for measurement of nitrite with reductive gas-phase chemiluminescence in 1% wt/vol KI in acetic acid using Sievers* Nitric Oxide Analyzer NOA 280i, according to the manufacturer’s instructions. The averaged blank signal (without aortic rings) for a given set of experiments was subtracted as a background signal, to account for nitrite contamination in the buffer and/or laboratory atmosphere. Nitrite concentration was expressed as ng/ml/mg of dry weight of aortic rings.
Assessment of PGI2 production in isolated rings of mice aorta
PGI2 production by aortic rings was quantified on the basis of the formation of 6-keto PGF1α, a stable metabolite of PGI2, in the supernatant of the aortic rings.
Aortic rings were incubated on a thermoblock (Liebisch Labortechnik) at 37 °C, in 1 ml of K-H buffer equilibrated with carbogen gas mixture (95% O2, 5% CO2), either in the absence or presence of selective COX-2 inhibitor DuP-697 (1 μM) or nonselective COX-1/COX-2 inhibitor indomethacin (5 μM). Both DuP-697 and indomethacin were dissolved in DMSO. Control rings were incubated with DMSO (1 μl/ml).
After equilibration and pre-incubation in the presence or absence of inhibitors, the aortic rings were placed in fresh buffer and incubated with or without inhibitors for 30 min. Effluent samples were taken after 3 (initial) and 30 (final) minutes of incubation. PGI2 production was calculated as the difference between final and initial 6-keto PGF1α concentrations and was expressed as pg/ml/mg of dry weight of aortic rings. 6-keto-PGF-1α, concentration was measured using an EIA kit (Enzo, Life Technologies).
Immunohistochemistry of aortic endothelium
Aortic rings were placed perpendicularly in OCT compound (Thermo) and then snap-frozen at − 80 °C. The blocks were mounted on the cryostat holder and cut into 10-μm-thick cross-sectional slides using the Leica CM1950 automatic cryostat. The sections were placed on polylisine-covered (Sigma-Aldrich) microscopic slides (Super Frost, Mentzel Gläser), and then acetone fixed (10 min). Pre-incubation with 2.5% horse serum (Vector Labs) and 2% dry milk was performed to minimize non-specific binding of antibodies. For indirect immunohistochemical detection of von Willebrand factor in endothelium, sections were incubated inside humid chambers with polyclonal rabbit anti-mouse vWF Ig (Abcam) following rinsing in PBS secondary biotinylated horse anti-rabbit Ig (Vector Labs). After another rinse in PBS, sections were incubated with Cy3-conjugated streptavidin (Jackson ImmunoResearch), and then mounted in glycerol-PBS. Images of the immunostained sections were acquired using the AxioObserver D2 inverted fluorescent microscope (Carl Zeiss) connected to a AxioCam HRm monochromatic digital camera, and stored as TIFF files. Fluorescence parameters were analysed automatically by Columbus software (Perkin Elmer).
Assessment of nitrite in plasma and NOHb in erythrocytes
Blood samples were centrifuged (1000 x g, 5 min, 4 °C) to isolate plasma and erythrocytes. 50 μl plasma was mixed with 100 μl of cold ethanol and kept on ice for protein precipitation (30 min), centrifuged (14,000 x g, 5 min.) and the resulting supernatant was used immediately after sample preparation to determine nitrite concentration using reductive chemiluminescence analysis (Sievers* Nitric Oxide Analyzer NOA 280i), as described above.
EPR spectra of the isolated erythrocytes were used for the detection of nitrosylhemoglobin (NOHb), as described previously [14, 15]. Briefly, erythrocytes were snap-frozen in insulin syringes and EPR measurements were performed in in liquid nitrogen (77 K) using a Bruker EMX Plus spectrometer. Nitrosylhaemoglobin levels were expressed as the EPR amplitude of the second hyperfine line of the NOHb spectra in arbitrary units, and normalized to sample weight.
Blood count and cytokine analysis in plasma
Blood count was performed immediately after blood collection using a blood counter (abc Vet, HORIBA). To obtain blood plasma, samples were centrifuged for 7 min (1000 x g). Acute-phase protein concentration in plasma was measured using magnetic-beads-based immunoassay (Multiplex, Millipore). SAA and IL-6 plasma concentrations were measured with ELISA kits (Invitrogen and R&D Systems, respectively).
For statistical analysis, STATISTICA 10 software (StatSoft, Inc.) and OriginPro 9 software (OriginLab, Northampton, MA) were used. The Shapiro-Wilk test was used to verify whether the data were normally distributed. Levene’s test was used to determine homogeneity of variances. The significance of between-group differences was evaluated by the Kruskal-Wallis test or ANOVA with post-hoc Tukey multiple comparisons test or by Mann-Whitney U test, depending on the data distribution. Spearman or Pearson’s correlation coefficient test was used to assess dependence between two parameters. Results are presented as mean ± SEM. Differences between means were considered significant if p < 0.05.