Nitric oxide deficiency and endothelial–mesenchymal transition of pulmonary endothelium in the progression of 4T1 metastatic breast cancer in mice

Two hundred and forty female Balb/C mice, 7–11 weeks old, were purchased from Charles River Lab (Germany) and divided into healthy control mice (n = 30) injected orthotopically with Hank’s Balanced Salt Solution (HBSS; IIET, Poland) and mice (n = 210) injected orthotopically with 1 × 10⁴ 4T1 murine breast cancer cells suspended in HBSS. The mice injected with 4T1 cells were euthanized (ketamine and xylazine, 100 and 10 mg/kg, respectively) in the 1st, 2nd, 3rd, 4th, and 5th week after cancer cell injection. Healthy control mice were euthanized concomitantly with mice in the 5th week of the disease. Throughout the experiment, all animals were housed 5–6 mice per cage, in a temperature-controlled environment (22–25 °C), maintained on a 12-h light/day cycle and given unlimited access to food (AIN; Zoolab, Krakow, Poland) and water. Experimental procedures involving animals were accepted by the First Local Ethical Committee on Animal Testing at Jagiellonian University (Krakow, Poland; permit no. 140/2013) and the Second Local Ethical Committee on Animal Testing in the Institute of Pharmacology, Polish Academy of Sciences (Krakow, Poland; permit no. 41/2017).

The mouse mammary adenocarcinoma 4T1 cells were obtained from the American Type Culture Collection (ATCC, USA) and were cultured in RPMI 1640-Glutamax medium (Sigma-Aldrich, Poland) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Poland), 1.0 mM sodium pyruvate (Sigma-Aldrich, Poland), and antibiotic antimycotic solution (100 units/ml penicillin and 100 μg/ml streptomycin, 25 μg/ml amphotericin B) (Sigma-Aldrich, Poland). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. For inoculations, only 4T1 cells at the second passage were used. Prior to the transplantations, 4T1 cells were detached using Accutase solution (Sigma-Aldrich, Poland), centrifuged (300 × g, 4 °C, 5 min), counted, suspended in Hank’s Balanced Salt Solution (HBSS; IIET, Poland) at the appropriate concentration, and inoculated into the mammary gland of female Balb/C mice. All cell cultures were routinely tested for Mycoplasma contamination (MycoAlert Mycoplasma Detection Kit; Lonza).

Body mass was monitored throughout the experiment. To assess the primary tumor growth, the primary tumor volume was measured with calipers each week as described by Kim et al. [27]. After mice euthanasia, primary tumors, lungs, and spleens were excised, weighed, and saved for further analysis. Lungs designated for assessment of metastasis were fixed in formalin and cut into lobes, and the pulmonary metastatic nodules were counted on their surface. After assessment of pulmonary metastasis, lung lobes were paraffin-embedded, cut into 5-μm slices, and stained with hematoxylin and eosin (H&E) to visualize pulmonary metastasis. The lung cross-sections were scanned with a BX51 microscope equipped with the virtual microscopy system dotSlide (objective magnification 20×; Olympus, Japan). To visualize the reorganization of extracellular matrix in the lungs during disease progression, the lung cross-sections were stained with Unna Orcein staining for elastin fibers. Subsequently, randomly chosen visual fields for mice in each experimental group were photographed in such a way that only the lung parenchyma was visible without major pulmonary blood vessels and bronchi. The pictures were subjected to segmentation in Ilastik (developed by the Ilastik team, with partial financial support by the Heidelberg Collaboratory for Image Processing, HHMI Janelia Farm Research Campus and CellNetworks Excellence Cluster), and the relative number of pixels corresponding to elastin fibers in each experimental group was calculated using ImageJ [28].

Colloidal Fe²⁺(DETC)₂ was used for trapping the intracellular NO with EPR detection as described by Cai et al. [29] with minor changes. Briefly, lungs perfused with ice-cold PBS were excised and cut into small pieces and placed into 0.1 ml Krebs Hepes buffer (NaCl 99 mM, KCl 4.7 mM, CaCl₂ 2.5 mM, MgSO₄ 1.2 mM, NaHCO₃ 25 mM, KH₂PO₄ 1.03 mM, glucose 5.6 mM, HEPES 20 mM) on a 24-well plate. The buffer was bubbled for at least 30 min with argon gas on ice to remove oxygen prior to use. Then 2.25 mg of FeSO₄ × 7H₂O/10 ml and 3.6 mg of DETC/10 ml were dissolved separately in argon-bubbled buffer, to obtain final concentrations 0.8 mM and 1.6 mM, respectively, mixed, and immediately added to the tissue samples (0.25 ml per well). The tissues were placed in an incubator at 37 °C and incubated for 90 min in an air atmosphere. Tissue samples were then collected, weighed, introduced into 1-ml insulin syringes, and snap-frozen in liquid nitrogen. Measurements of Fe₂(DETC)₂-NO signals in frozen samples were performed in a finger Dewar using an EMX Plus Bruker spectrometer with the following settings: microwave power, 10 mW; modulation amplitude, 0.8 mT; scan width, 11.5 mT; scan time, 61.44 s; number of scans, 4. The results were collected, and the amplitude of the characteristic NO triplet spectrum was analyzed using Eleana software.

Formalin-fixed and paraffin-embedded lungs were cut into 5-μm slices. Antigen retrieval was performed according to the standard protocol. To visualize expression of Snail, the slices were incubated with primary anti-Snail antibody (ab53519; Abcam) and secondary biotinylated donkey anti-goat antibodies (705-065-147; Jackson ImmunoResearch) concomitantly with ABC vector complex. For each slice, 10 randomly chosen nonobstructed arteries were photographed with a BX51 microscope (objective 20×) equipped with the virtual microscopy system dotSlide (Olympus, Japan) and the length of Snail-positive fragment(s) within the artery was manually measured and expressed as the percentage of the entire circuit of the particular artery. At the same time, the representative images of the investigated arteries were assessed for their patency (i.e., obstruction with blood clot or cancer cells) by a blinded investigator.

Subsequent to anesthesia (100 mg/kg ketamine + 10 mg/kg xylazine, i.p.), mice were injected via the femoral vein with a solution of Evans blue (EB, 60 kDa) dye (Sigma Aldrich) at a dose of 4 ml/kg. Injected dye solution, composed of 2% EB in 0.9% saline, was left to circulate for 10 min, and then the mouse chest was surgically opened and concurrently perfused via left (systemic circulation) and right (pulmonary circulation) ventricles with PBS for 15 min. Lungs were isolated, dry weighed, and homogenized in 200 μl of 50% TCA (dissolved in distillated water). The homogenate was frozen and kept at − 20 °C for EB concentration measurement. Subsequent to thawing, homogenates were centrifuged (at 10625 × g for 12 min at 4 °C), and the supernatant was collected and diluted with 1:3 volumes of 95% ethanol prior to photospectrometric (Synergy 4; Bio-Tek) determination of EB concentration (fluorescence: excitation at 590 nm, emission at 645 nm, absorbance at 620 nm). Results were normalized to the tissue weight.

Lungs were perfused with PBS, excised, rinsed in saline, dried with tissue paper, weighed, cut into small pieces, and snap-frozen in liquid nitrogen; the samples were stored at − 80 °C. For western blot analysis, whole lungs were homogenized and whole lung lysates were used. Lungs were homogenized in the lysis buffer (Thermo Fisher Scientific) for protein extraction with protease and phosphatase inhibitors. Protein concentration was measured with the use of a BCA assay. Subsequently, the samples from at least six mice in each experimental group were pooled together in such a way that an equal amount of protein from each sample was dissolved in an equal volume of the lysis buffer for each mouse in the group to ensure the equal representation of each individual sample in the pooled specimen. After addition of loading buffer, samples were heated at 95 °C for 5 min and then frozen at − 80 °C. Each time, an equal amount of protein from pooled samples was loaded and run on the gel, and then transferred to a nitrocellulose membrane, blocked with 5% dry milk, and incubated with the primary antibodies directed against the following antigens: p(S1177)eNOS (ab195944; Abcam), eNOS (610,296; BD Transduction Laboratories), VEGFA (ab68334; Abcam), VEGFR2 (ab39256; Abcam), Ang-1 (ab8451; Abcam), Ang-2 (PA5-27297; Thermo Fisher Scientific), VCAM-1 (CBL1300; Merck), Slit2 (ab134166; Abcam), ROBO4 (orb101060; Biorbyt), ROBO1 (ab85312; Abcam), TGF-β1 (ab155264; Abcam), VE-CAD (sc-6458; Santa Cruz Biotechnology), CD31 (NBP1-71663H; Novus Biologicals), vWF (ab9378; Abcam), MMP-2 (ab19167; Abcam), MMP-9 (ab19016; Abcam), and MMP-14 (sab4501901; Sigma Aldrich). The appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology (sc-2020, sc-2004, and sc-2005). Equal protein loading was confirmed after transfer onto membranes, as measured by a stain-free technique provided by Bio-Rad [30]. Densitometric assessment of band intensity was performed using ImageJ. The results are presented as the fold change of control corresponding to healthy mice. Total protein was used as a loading control.

Blood samples were collected into a syringe containing 3.8% citrate (blood/citrate at 10:1 (v/v)) from the right heart ventricle. A blood count was performed using the animal blood counter Vet abc (Horiba Medical, France). The samples designated for flow cytometric measurements were diluted with saline and washed with Tyrode buffer. Each sample was double stained with four antibodies that included platelet-specific antigen GpIIbIIIa (CD41/61), either FITC or PE conjugated, for platelet identification and one of four platelet activation markers—PE-conjugated active form of GPIIb/IIIa and P-selectin antibodies, FITC-conjugated fibrinogen, or von Willebrand (vWF) factor—representing platelet binding capacity. Platelets were identified based on their forward-scatter and side-scatter characteristics and were gated on the basis of the expression of platelet-specific antigen CD41/61 (see Additional file 1). Isotype control antibodies, either FITC or PE conjugated, were used to assess nonspecific binding for each individual sample. Basal and ADP-induced (20 μM) activation of circulating platelets was assessed on the basis of the measured expression/binding level of surface membrane antigens expressed as a percentage of all platelets above the isotype control fluorescent signal and the median fluorescence intensity (MFI). Flow cytometric analyses of platelet activation were performed using flow cytometry software (LSRII and FACS/Diva version 6.0, respectively; Becton Dickinson, Oxford, UK). Measurements were made on a logarithmic scale and at least 10,000 events were collected for each sample. Appropriate color compensation was determined in samples singly stained with either FITC-conjugated anti-CD41/61 or PE-conjugated anti-CD41/61.

The first pulmonary metastatic nodules were detected in the 3rd week after breast cancer cell injection (Fig. 1d, g), while the primary tumor became detectable in the 2nd week after cancer cell inoculation (Fig. 1i). Then, both the number of pulmonary metastases and the primary tumor weight and volume increased progressively (Fig. 1). The weight of the lungs was significantly increased only in the 5th week compared to control healthy mice (Table 1), since only at that time was the presence of large metastastic foci in the lungs detected (Fig. 1f, g). The appearance of the first metastatic nodules in the lungs in the 3rd week after breast cancer cell injection correlated with the onset of systemic leukocytosis and increased spleen weight (Table 1), indicating the onset of systemic inflammation. Pulmonary metastasis was also associated with progressive degradation of elastin fibers (Fig. 2a–g) and an increase in the expression of metalloproteinase 2, 9, and 14 (MMP-2, MMP-9, MMP-14) in the lungs 3 and 4 weeks after breast cancer cell injection, respectively (Fig. 2h), being compatible with tissue remodeling accompanying the advanced stage of metastasis progression.

Local NO production in the lungs was impaired already in the 1st week after 4T1 cell injection (Fig. 3a) and remained significantly compromised thereafter in 4T1 breast cancer-bearing mice. The progressive fall in NO production in mice injected with 4T1 cells corresponded with progressive decrease in eNOS phosphorylation of S1177 observed throughout the progression of the disease after 4T1 cancer cell inoculation (Fig. 3b).

Expression of Snail in the lungs of mice orthotopically injected with 4T1 breast cancer cells, compared with healthy control mice, was higher in the endothelial layer of small arteries as soon as 1 week after cancer cell injection and stayed elevated throughout the entire progression of the disease, except for the terminal time point 5 weeks after 4T1 breast cancer cell inoculation (Fig. 4a–g). TGF-β1 expression in the lungs was slightly increased only in the 1st week after 4T1 breast cancer cell inoculation (Fig. 4h). The phenotypic change of pulmonary endothelium compatible with endothelial–mesenchymal transition, evidenced by downregulation of endothelium-specific proteins such as VE-CAD, CD31, vWF, or VEGFR2, seemed to be evident 3 weeks after cancer cell inoculation (Fig. 4i), concomitant with the early phase of metastasis (Fig. 1g).

Increased permeability of pulmonary endothelium expressed as increased deposition of Evans blue (EB) in the lungs of 4T1 breast cancer-bearing mice was found only in the 3rd week after cancer cell inoculation (Fig. 5a), indicating an evident increase in endothelial permeability at the early phase of metastasis. Then, EB leakage from the circulation into the lungs started to decrease and, finally, it was lower than in healthy control mice in the 5th week after cancer cell inoculation. Decreasing EB penetration into the lungs of mice in the 4th and 5th weeks of the disease appeared to be due to the occlusion of pulmonary vessels by cancer cells proliferating in their lumen (Fig. 5b–d), rather than due to changes in pulmonary endothelial permeability itself. Increased permeability of the pulmonary endothelium in the 3rd week after cancer cell inoculation correlated with a transient increase in VEGFA and VCAM-1 expression in the lungs (Fig. 5e).

Slit2 maintains endothelium integrity via interaction of its full-length or N-terminal part with endothelium-specific ROBO4 via ROBO1 [19, 31, 32]. Full-length Slit2 was undetectable in lungs homogenates of both control and 4T1 breast cancer-bearing mice, while its N-terminal fragment was found both in healthy control and breast-cancer bearing mice (Fig. 6). Upregulation of both receptors ROBO4 and ROBO1 was detected only in the 1st week after 4T1 cell injection when the level of N-terminal Slit2 binding ROBO receptors was still preserved. Starting from the 2nd week after cancer cell inoculation, the Slit2–ROBO4–ROBO1 protective signaling pathway was gradually downregulated in the lungs of 4T1 breast cancer-bearing mice.

Basal platelet activation and ADP-induced reactivity were both assessed as the percentage of platelets expressing P-selectin, active form of GPIIb/IIIa, vWF, and fibrinogen bound to platelet surface in their entire population (Fig. 7) as well as median expression of these antigens on the platelet surface (Fig. 8). The signs for activation of platelets in the early phase of metastasis and loss of reactivity to stimuli afterward were detected. The percentage of platelets expressing P-selectin and active form of GPIIb/IIIa peptide in basal condition or upon ADP stimulation were not different between healthy control and 4T1 breast cancer-bearing mice (Fig. 7a, b). However, fibrinogen binding in basal conditions was increased in 4T1 breast cancer-bearing mice as compared with control mice 1–3 weeks after 4T1 cancer cell inoculation, reaching significance 2 weeks after cancer cell inoculation. Similarly, vWF binding to platelets was also increased 1–3 weeks after cancer cell inoculation (not significantly). In contrast, 5 weeks after 4T1 breast cancer cell inoculation, the capacity of platelets to bind fibrinogen and vWF was significantly diminished in basal conditions as well as after ADP stimulation (Fig. 7c, d). As shown in Fig. 8, median expression of P-selectin, active form GPIIb/IIIa, vWF, and bound fibrinogen on the unstimulated platelet surface was not altered in 4T1 breast cancer-bearing mice as compared with controls. However, after ADP stimulation, platelet surface expression of vWF and fibrinogen but not P-selectin and active form of GPIIb/IIIa was increased to a lesser extent in 4T1 breast cancer-bearing mice as compared with healthy controls (Fig. 8c, d).