Intermittent hypoxia increases melanoma metastasis to the lung in a mouse model of sleep apnea

https://doi.org/10.1016/j.resp.2013.03.001Get rights and content

Highlights

  • Obstructive sleep apnea (OSA) is characterized by a high rate intermittent hypoxia.

  • Intermittent hypoxia mimicking OSA increases induced melanoma lung metastasis.

  • Intermittent hypoxia increases spontaneous lung metastasis from a primary tumor.

  • Intermittent hypoxia could contribute to enhance cancer progress in OSA patients.

Abstract

Obstructive sleep apnea (OSA) has recently been associated with an increased risk of cancer incidence and mortality in humans. Experimental data in mice have also shown that intermittent hypoxia similar to that observed in OSA patients enhances tumor growth. The aim of this study was to test the hypothesis that intermittent hypoxia mimicking OSA enhances lung metastasis. A total of 75 C57BL/6J male mice (10-week-old) were subjected to either spontaneous or induced melanoma lung metastasis. Normoxic animals breathed room air and intermittent hypoxic animals were subjected to cycles of 20 s of 5% O2 followed by 40 s of room air for 6 h/day. Spontaneous and induced lung metastases were studied after subcutaneous and intravenous injection of B16F10 melanoma cells, respectively. Compared with normoxia, intermittent hypoxia induced a significant increase in melanoma lung metastasis. These animal model results suggest that intermittent hypoxia could contribute to cancer metastasis in patients with OSA.

Introduction

Obstructive sleep apnea (OSA) is a breathing disorder characterized by recurrent obstructions of the upper airway associated with increased inspiratory efforts, intermittent hypoxemia and sleep fragmentation. Extensive research in animal models and humans indicates that these chronic insults contribute to the well characterized mid- and long-term cardiovascular, cognitive and metabolic consequences of OSA. Very recent experimental and clinical data suggest that cancer could be another pathology enhanced by this sleep breathing disorder. Specifically, it has been shown that intermittent hypoxia with a time pattern mimicking OSA increases the growth rate of melanoma tumors in mice (Almendros et al., 2012a, Almendros et al., 2012b). Moreover, a study in a cohort of 1522 subjects from the general population over the course of 22 years has shown an increase in cancer mortality in patients with OSA (Nieto et al., 2012). It has also been reported in a series of 4910 patients with suspected OSA followed for 4.5 years that cancer incidence and mortality was greater in patients with diagnosed OSA (Campos-Rodriguez et al., 2013, Martinez-Garcia et al., 2012). Interestingly, in these two human cohort studies the risk of cancer incidence and mortality increased in parallel with the severity of OSA, as measured by an overnight hypoxia index.

Cancer progression can be caused by an increase in the rate of tumor growth and also by an enhancement of the metastatic dissemination and homing in of malignant cells from a primary tumor on to a secondary organ. Whereas evidence is available on tumor growth rates (Almendros et al., 2012a, Almendros et al., 2012b), we lack data indicating that an intermittent hypoxia pattern realistically reproducing OSA also increases tumor dissemination in vivo. Indeed, the few studies that have addressed metastasis and changes in oxygenation are not directly applicable to OSA since they do not reproduce the fast and pronounced changes characteristic of oxygen desaturations in this sleep breathing disorder (up to 60 events/h). Some authors (Cairns et al., 2001, Rofstad et al., 2010) have used a timing of intermittent hypoxia (20 min periods) that simulated the time course of spontaneous tumor oxygenation oscillations caused by progressive angiogenesis and irregular blood flow inside a tumor during its growth (Toffoli and Michiels, 2008). More recently, other authors (Karoor et al., 2012) have used a slightly faster period of intermittent hypoxia (4 min), but with a relatively mild level of hypoxia (10% O2). Although these results were not conclusive, they suggest that periodically modifying oxygenation could increase tumor dissemination, mainly via the up-regulation of pro-angiogenic and pro-inflammatory factors (Toffoli and Michiels, 2008). The aim of the present work was therefore to test the hypothesis that a pattern of intermittent hypoxia realistically mimicking OSA increases lung metastasis. Specifically, the study was conducted with melanoma mouse models of spontaneous and induced metastasis.

Section snippets

Animals

This study was carried out on 75 pathogen-free C57BL/6 10-week-old male mice (Charles River Laboratories, Saint Germain sur L’arbresle, France). The protocol used was approved by the Ethical Committee for Animal Research of the University of Barcelona. All the mice were housed in standard cages, were fed and had tap water ad libitum.

Melanoma cells

Murine melanoma cells B16F10 cells were obtained from American Type Culture Collection, Manassas, VA). These cells were maintained in high glucose Dulbecco's

Results

The application of intermittent hypoxia in mice produced oxygen desaturation in the periphery of the tumor tissue, as shown in Fig. 2. On average, the basal values of PtO2 obtained from 5 mice was ~45 mmHg and the application of a recurrent pattern of intermittent hypoxia promoted reductions with minima of ~8 mmHg.

In the spontaneous metastasis model, 13 (out of 18) and 15 (out of 18) mice from the intermittent hypoxia and the normoxia groups, respectively, survived up to day 30. The number of

Discussion

This study shows that a fast-rate intermittent hypoxia reproducing the time pattern typically found in OSA patients enhanced melanoma lung metastasis in mice.

The experimental setting used in this investigation was particularly suitable for mimicking intermittent hypoxia in OSA. In contrast with most of the experimental systems previously used to study intermittent hypoxia in rodents – where the duration of the hypoxic and normoxic phases was relatively long – we applied a controlled pattern of

Conflict of interest

None declared.

Acknowledgements

The authors wish to thank to Miguel A. Rodríguez and Rocío Nieto for their excellent technical assistance. This work was partially supported by the Ministerio de Economía y Competitividad (SAF2011-22576, FIS-PI11/00089, FIS-PI11/01892), SEPAR and FUCAP.

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