To the Editors:
Obstructive sleep apnoea (OSA) is a very prevalent syndrome that induces or aggravates cardiovascular, metabolic and neurocognitive disorders. Among the various challenges imposed on a patient by OSA as a consequence of apnoeas (sleep disruption, increased inspiratory efforts and recurrent hypoxaemia), intermittent hypoxia plays a major role in the pathophysiology of this sleep breathing disorder.
It has been well established that hypoxia plays an important role in regulating the various stages of tumour formation and progression [1]. Accordingly, the aim of the experimental study reported here was to test the hypothesis that high-rate intermittent hypoxia with a time course similar to that found in OSA (one or more hypoxic event per minute) enhances tumour growth. To avoid the interaction of any comorbidity, this investigation was carried out in a well-controlled animal model where the main variable under study was intermittent hypoxia.
This study, which was approved by the Ethical Committee for Animal Research of the University of Barcelona (Barcelona, Spain), was conducted in 15 pathogen-free male C57BL/6J mice (25–30 g) using a conventional murine melanoma model consisting of tumour induction by subcutaneous injection of 106 B16F10 melanoma cells (ATCC-CRL-6475; American Type Culture Collection, Manassas, VA, USA) in the left flank region of the mouse [2]. This is a widely used cancer model in experimental research [3, 4], which has a response to hypoxia that is representative of a variety of cancer types [5, 6].
Application of chronic intermittent hypoxia (60 events·h−1 consisting of 20 s of 5% O2 followed by 40 s of room air) started on the same day as the injection of cancer cells. Briefly, a continuous flow of gas was circulated through a box (26 cm long, 18 cm wide and 6 cm high) by means of a distribution system based on multiple orifices. A pneumatic valve placed near the inlet of the box cyclically switched from the room air entrance (40 s) to a gas reservoir of hypoxic air at an oxygen fraction of 5% (20 s). Seven mice were placed in the intermittent hypoxia box, with food and water available, for 6 h·day−1 during the light period (10:00–16:00 h) for 14 consecutive days. A normoxia control group of eight mice was placed in an identical system with the reservoir of 5% O2 air replaced by room air. Accordingly, both groups of melanoma-injected mice were subjected to exactly the same protocol, the only difference being the breathing of normoxic or intermittently hypoxic air.
The growth of the melanoma tumour was assessed at days 8, 11 and 14 after the injection of cancer cells by externally measuring the tumour’s dimensions with a calliper and estimating tumour volume as W2×L/2, where W is tumour width and L is tumour length [2]. At day 14, the mice were sacrificed and the tumours were excised and weighed to directly measure cancer growth. Figure 1 shows that intermittent hypoxia resulted in a two-fold increase in the growth of the melanoma tumours.
Tumour growth in both groups of mice. At day 14, tumour volume in the animals subjected to intermittent hypoxia (n=7) was significantly greater (p<0.001 by two-way ANOVA with Holm-Sidak post-test) than that of the normoxia group (n=8). Tumour weight at day 14 was significantly greater (p=0.012 by unpaired t-test) in the intermittent hypoxia group than in the normoxia group. #: p=0.012; ***: p<0.001.
The excised tumours were fixed in formalin, embedded in paraffin blocks, cut into 4-μm thick sections and stained with haematoxylin–eosin. Necrotic cells were identified by a loss of cell borders and the presence of cellular debris (fig. 2) [4]. Blind microscopic examination of a section from the central part of each animal's tumour showed a two-fold increase (p=0.080 by unpaired t-test) in the percentage of necrotic tumour area in the animals subjected to intermittent hypoxia (mean±se 29.1±6.2%), compared with controls (14.5±4.7%). Whereas in the normoxic animals, tumour necrosis tended to be more peripheral (12.3±4.6%) than central (2.2±0.7%) (p=0.07), in the animals subjected to intermittent hypoxia, this difference decreased (17.7±7.3% and 11.3±4.9% for peripheral and central, respectively; p=0.58).
Representative tumour tissue preparations stained with haematoxylin–eosin corresponding to a, b) the normoxic group and c, d) animals subjected to intermittent hypoxia. a, c) whole section of the tumour, where viable tumour areas appear darker than necrotic areas. Areas marked within a square are enlarged in b) and d), respectively. a, c) Scale bars=2 mm; b, d) scale bars=200 μm.
The potential mechanisms involved in the enhancement of cancer progression by hypoxia have been identified [1]. It is also well known that tumour growth can be modulated by the relatively slow changes (lasting from tens of minutes to hours) in the level of tissue oxygenation caused by progressive angiogenesis and irregular blood flow inside a tumour during its growth [7]. The rate of tumour growth is boosted by increased angiogenesis into the tumoural tissue and hence by augmented perfusion [8]. There is evidence that over-expression of hypoxia-inducible factor-1α caused by tissue hypoxia triggers upregulation of proangiogenic mediators, such as vascular endothelial growth factor, in tumour cells [8]. Moreover, tumour progression could be enhanced by external growth factors reaching the cancer cells, in particular those systemically induced by hypoxia and hence circulating in the blood. This pathway could be of particular relevance in OSA since patients with this sleep breathing disorder exhibit increased proangiogenic factors in blood.
Although angiogenesis is augmented by hypoxia, it is known that the newly formed vascular network could present structural and functional abnormalities that lead to reduced perfusion and delivery of oxygen to the tumour tissue [9], thereby promoting tumour necrosis [10]. In fact, a higher percentage of necrotic tissue in the tumour, as we observed in the animals subjected to intermittent hypoxia, is a predictor of an aggressive cancer phenotype and a feature of tumour progression and poor prognosis [10]. However, a more detailed histological characterisation of tumour vascularisation and/or inflammation induced by intermittent hypoxia remains to be completed.
To our knowledge, the study reported here is the first one providing experimental evidence that a high-rate intermittent hypoxia mimicking the one experienced by OSA patients enhances tumour growth. Ascertaining whether there is a relationship between cancer incidence or mortality and the severity of OSA, measured by the apnoea/hypopnoea index and independent of all other relevant variables (e.g. age, obesity, smoking habits or alcohol intake), would require future studies on cohorts with a high number of patients over an extended time period. The present animal study, carried out under well-controlled conditions to investigate the effect of intermittent hypoxia, provides support for a hypothesis of a causal link between OSA and cancer.
Acknowledgments
The authors thank Esteve-Teijin (Barcelona, Spain) for kindly providing the gas concentrators used to prepare the gas mixtures. The authors wish to thank R. Nieto (CIBER Enfermedades Respiratorias, Bunyola, Baleares, Spain) and M.A. Rodríguez (Universitat de Barcelona, Barcelona, Spain) for their excellent technical assistance.
Footnotes
Support Statement
This work was supported in part by Ministerio de Ciencia e Innovación (grants SAF2009-02991, PI081908 and PI080277) and SEPAR.
Statement of Interest
A statement of interest for the study itself can be found at www.erj.ersjournals.com/site/misc/statements.xhtml
- ©ERS 2012
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