Chronic intermittent hypoxia, a hallmark of obstructive sleep apnea, promotes 4T1 breast cancer development through endothelin-1 receptors

Drugs and chemicals

Macitentan was graciously provided by Actelion Pharmaceuticals, Allschwil, Switzerland.RPMI-1640 medium, rat tail type I collagen, methycellulose Methocell®MC were obtained from Sigma Aldrich (Saint-Quentin-Fallavier, France). CellTiter96® MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and D-luciferin were purchased from Promega France (Charbonnières-les-Bains, France). Inactivated Fetal Bovine Serum Gibco was obtained from Thermofisher Waltham (Massachusetts, USA). Epidermal growth factor, basic fibroblast growth factor and NeuroBrew-21 were all bought from Miltenyi Biotec (Bergisch Gladbach, Germany). Endothelin-1 ELISA kit was provided by Enzo Life Sciences (Villeurbanne France) and Isorane® by Axience (Pantin, France).

Murine breast cancer cells

Murine 4T1 breast cancer cells were selected to model stage IV human breast cancer with pulmonary metastatic dissemination. 4T1rvluc2 cells (provided by the OPTIMAL platform, Grenoble, France), stably transfected with the firefly luciferase gene in order to perform in vivo bioluminescence imaging, were used in all experiments and will be referred to as 4T1 in the manuscript. They were maintained in RPMI-1640 medium containing 10% FCS and antibiotics.

In vitro intermittent hypoxia exposure

Cultured 4T1 cells were exposed to IH cycles alternating 5 min of normoxia (16% PO2) and 5 min of hypoxia (2% PO2) or to similar normoxia (N) cycles for control experiments, as previously described32. Briefly, this system is adapted from commercial gas-permeable dishes (Zell-Kontakt, Germany) and uses custom-made plate holders (SMTEC, Nyon, Switzerland) connected to gas blenders (Gas Blender 100, MCQ Instruments, Rome, Italy) and hosted in a standard cell culture incubator (SANYO, MCO-15AC). The small volume of air in the holders is rapidly renewed, thus allowing fast cycling.

This system induced oxygen pressure cycles in culture medium ranging from 25 to 120 mmHg during IH32. In accordance with the apnea pattern of OSA patients, IH cycles were applied for 8 h during the animals sleep period followed by 16 h of normoxia. This was repeated daily for up to 4 days according to the experiments.

Wound healing assays

4T1 cells were seeded (7.5 x 103/well) in 24-well semipermeable plates (Zell-Kontakt, Nörten-Hardenberg, Germany) pre-coated with type I collagen and allowed to reach 90% confluence. One day after seeding, wound healings were performed by scratching the wells from top to bottom with a sterile 100-μl cone. Cells were then incubated in medium supplemented or not with 5 µM macitentan and exposed to N or IH for 4 days. Wound repair was assessed daily during 5 consecutive days using a camera-coupled optical microscope (× 10 magnification; Olympus CK2; Olympus, Norfolk, USA). Repaired areas were determined by subtracting cell-free areas at various timepoints from initial cell-free areas (ImageJ software, NIH, Bethesda, USA). Values ​​of filled areas were normalized relative to control areas.

Proliferation assays

For proliferation assays, 4T1 cells (3 x 103) were seeded in 96-well semipermeable plates pre-coated with type I collagen, in medium with or without 5 µM macitentan, and submitted to a 2-day N or IH exposure. Cell viability was then assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) staining.

Transwell migration assays

4T1 cells (106) were seeded in the top chamber of Transwell 8.0 μm pore polycarbonate membrane polycarbonate membrane cell culture inserts (Corning®, CLS3422-48EA, Sigma Aldrich, USA) coated with type I collagen, in serum-free RPMI-1640 medium supplemented or not with 5 µM macitentan. 4T1 cells were loaded in the upper well of each chamber. The bottom chamber was filled with 600 µl of complete RPMI-1640 medium with 10% FBS as chemoattractant. After 2 days of N or IH exposure, the medium was aspirated and cells on the top chamber were gently removed using a cotton swab. Cells attached to the lower layer of the membrane were then fixed with absolute ethanol, stained with 0.2% crystal violet for 10 min at room temperature and imaged using a camera-coupled optical microscope (× 10 magnification; Olympus CK2, Olympus, USA). Migration ability was estimated by OD measurement (595 nm) after solubilization in acetic acid.

Spheroid proliferation and invasiveness

4T1rvluc2 cells were cultured in uncoated 96-well tissue plates with U-shaped bottoms (Greiner Bio-One, Frickenhausen, Germany) to allow the formation of one spheroid/well. Two thousand cells/well were loaded in medium enriched with 10% methylcellulose and the plates were centrifuged at 500×g for 5 min to initiate the process of spheroid formation. Three days later, spheroid formation was assessed by microscopic visualization. Spheroids were isolated and placed in 24-well semipermeable bottom plates pre-coated with type I collagen along with 1 ml of serum-free medium. Serum was replaced by adding 20 ng/mL of epidermal growth factor, 20 ng/mL of basic fibroblast growth factor and 1X NeuroBrew-21.

Spheroid diameter was measured using a graduated scale placed into the objective of a camera-coupled optical microscope and pictures were taken twice daily (× 10 magnification Axiovert 25; Carl Zeiss, Hombrechtikon, Switzerland).

Measurement of ET-1 levels in culture media

ET-1 levels in culture media of 4T1rvluc2 cells were assessed using ELISA kit according to the supplier’s recommendations.

Murine orthotopic breast cancer model

Animal experiments were performed according to the Declaration of Helsinki conventions for the Use and Care of Animals and approved by the Institutional Ethics Committee for Animal Research on August 28th 2018 (agreement No. 2016012818193391). The study was conducted on 5-week-old female NMRI nude mice (Janvier Labs, Le Genest-Saint-Isle, France) weighing 20 to 25 g. Animals were housed in controlled light and temperature conditions with food and tap water ad libitum and were acclimated for 1 week prior to the beginning of the experiments.

In vivo intermittent hypoxia exposure

Four independent experiments were performed with different numbers of mice as summarized in Fig. 1. Altogether seventy-one female NMRI nude mice were used in this study and were divided in N (n = 35) and IH (n = 36) groups.

Figure 1

Four independent experiments were conducted. Mice were divided into normoxic and hypoxic groups for experiments 1, 2 and 3. In experiment 4, mice were splittd into 4 groups to assess the both effects of exposure to IH and macitentan. For all groups (n = 71 mice), the experiment consists of a 28-day exposure to HI or normoxia with implantation of 4T1rvluc tumor cells after fourteen days (D0). Quantification of tumor growth by bioluminescence imaging is performed at 7 days and 14 days post-implantation in all groups. In experiment 4, mice received treatment (macitentan or control) every day from implantation. Regular follow-up of tumour volume was performed by caliper measurement.

Evaluation of the effects of intermittent hypoxia was repeated 4 times through 4 independent experiments. The effect of macitentan treatment was evaluated in experiment 4. Mice were exposed daily during 28 days: 14 days before (D-14) and 14 days after (D 14) tumor implantation (D0). Intermittent hypoxia (IH) exposure consisted of 8 consecutive hours of 1-min IH cycles, comprising 30 s at 21% FiO2 and 30 s at 5% FiO2, or to similar air-air cycles for normoxic (N) exposure. After 4T1 cell implantation at D0, the animals were treated or not with oral macitentan throughout the remaining 14 days of N or IH exposure.

As previously described8mice were exposed daily to 8 consecutive hours of 1-min IH cycles (30 s at 21% FiO2 and 30 s at 5% FiO2) or to similar air-air cycles. FiO2 level in housing cages was monitored throughout the experiments (ML206 gas analyzer; ADInstruments, UK). After 14 days of N or IH exposure, mice were injected with 5 x 105 4T1 cells into the mammary fat pad33 and then re-exposed for 14 additional days before primary tumor resection. Fourteen of the mice were submitted to the same N (n = 7) or IH (n = 7) protocol but were treated daily, following tumor implantation, with 30 mg/kg macitentan added to their food. Tumor growth (volume = 0.5 x length x width2) was monitored thrice a week using an electronic caliper.

Bioluminescence imaging

Proliferation and thoracic dissemination of 4T1 cells were quantified by in vivo bioluminescence imaging (IVIS Kinetic, PerkinElmer, Villebon-sur-Yvette, France) on 7 and 14 days following implantation. Five minutes before imaging, vigil mice received an intraperitoneal injection of 150 µg/g of d-luciferin. They were then anesthetized (isoflurane 4% for induction and 1.5% thereafter) and placed in the BLI system (IVIS Kinetic, PerkinElmer, Villebon-sur-Yvette, France), in supine position.

Signal quantifications were carried out by drawing regions of interest on the primary tumor and thorax areas (LivingImage software; PerkinElmer, Villebon-sur-Yvette, France). Results were expressed as photons per second (ph/s).

Statistical analysis

Comparison between two groups was performed by t-test or Mann–Whitney test, while comparisons of several groups were performed by a two-way ANOVA or Kruskal–wallis test followed by the appropriate post hoc tests. Time-series effects were analyzed by repeated-measures analyzes of variance. The choice of parametric or nonparametric tests depended on the normal distribution of values ​​and on the equality of variances. Frequencies were analyzed by Chi-squared tests. In cell culture experiments, to take into account the fact that several duplicate wells were usually done in a single independent experiment, the experiment was included as a random factor. A two-tailed p-value < 0.05 was considered significant. Continuous data were represented as mean ± standard error of mean or median ± 10%–90% range, depending on the normality of values.

A meta-analytical method was performed on repeated independent in vivo experiments to evaluate the global Hedge’s g effect size (see supplementary methods for details)34. By convention, overall effect sizes of 0.2, 0.5 and 0.8 standard deviations are considered as “small”, “medium” and “large”, respectively.

Statistical analysis and meta-analysis were performed using SPSS Statistics (version 20.0, IBM, Armonk, USA) and RStudio (version 1.2.1335, RStudio, Boston, USA) software, respectively.

Ethics approval

Animal experiments were performed according to the Declaration of Helsinki conventions for the Use and Care of Animals and approved by the Institutional Ethics Committee for Animal Research on August 28th 2018 (agreement No. 2016012818193391).