Cancer Letters

Cancer Letters

Volume 361, Issue 2, 1 June 2015, Pages 233-239
Cancer Letters

Original Articles
Adipose tissue macrophage polarization by intermittent hypoxia in a mouse model of OSA: Effect of tumor microenvironment

https://doi.org/10.1016/j.canlet.2015.03.010Get rights and content

Highlights

  • Sleep apnea is characterized by intermittent hypoxia (IH), and is associated with adverse cancer outcomes.

  • IH-induced changes in TAMs contribute to increased tumor proliferation and invasion in mice.

  • Adipose tissue macrophages show differential polarization under IH, depending on the presence or absence of tumor.

  • Increased Tregs in the ipsilateral adipose tissue to the tumor may contribute to alter polarity of macrophages toward M2 phenotype.

Abstract

Intermittent hypoxia (IH)-induces alterations in tumor-associated macrophages (TAMs) that are associated with adverse cancer outcomes, as reported in patients suffering from sleep apnea. Adipose tissues (AT) and bone-marrow (BM)-derived cells are the inferred sources of macrophages infiltrating malignant tumors. Here, the sources of TAMs and the phenotypic changes induced by IH in the ipsilateral and contralateral AT were investigated by using a syngeneic murine solid tumor model (TC1).

C57/B6 male mice were exposed to either IH or room air (RA) for 6 weeks, with TC1 cells being inoculated in the 2nd week. Macrophage content, phenotype and tissue origin were assessed in tumors, and ipsilateral and contralateral AT.

IH induced a ~2.2-fold increase in TAM tumor infiltration. However, differential responses in the tumor ipsilateral and contralateral AT emerged: IH increased infiltration of preferentially M1 macrophages in contralateral AT, while reductions in macrophages emerged in ipsilateral AT and primarily consisted of the M2 phenotype. These changes were accompanied by reciprocal increases in resident and BM-derived TAMs in the tumor.

IH-induced phenotypic alterations in AT macrophages surrounding the tumor and their increased infiltration within the tumor may contribute to the accelerated tumor progression associated with IH.

Introduction

Chronic intermittent hypoxia during sleep (IH), a hallmark of obstructive sleep apnea (OSA), has now been associated with the majority of OSA-related morbid consequences, including excessive daytime sleepiness, neurocognitive, cardiovascular, metabolic, erectile dysfunction, and more recently, with increased incidence and adverse cancer outcomes [1], [2], [3]. The underlying mechanisms by which IH elicits such deleterious effects appear to involve increased production and propagation of reactive oxygen species (ROS) and the recruitment of inflammatory pathways, both of which have been extensively documented in murine models and in OSA patients [2].

There is now solid emerging evidence implicating the immune system in the adverse cancer outcomes reported in OSA [4], [5], [6]. Concretely, an increased recruitment of macrophages and changes in their polarity and functional properties occur in response to IH in engrafted lung epithelial tumors [4]. Mice exposed to IH exhibited tumors that manifested not only accelerated growth, but also increased invasiveness toward adjacent tissues that were associated with marked abundance of M2-polarity TAMs [4]. Both the adipose tissues (AT) surrounding the tumor and bone-marrow (BM) derived progenitor cells have been suggested as potential sources of macrophages that infiltrate the tumor in normal conditions [7], [8], [9]. The source of increased M2 polarity TAMs in IH is however unclear. On the one hand, we have recently shown that resident macrophages in the AT of mice exposed to IH manifest increased M1 polarization [10]. These findings contrast with the increased M2 polarity of TAMs in tumors exposed to IH, and suggest that the tumor microenvironment may reverse the classical M1 activation of AT macrophages induced by IH in the absence of a tumor. It is possible that either the presence of tumor cells or increased recruitment of immune suppressor cells, such as T cell regulatory lymphocytes (Tregs), may account for the potential shift from the M1 to M2 phenotype within the tumor during IH [4]. AT may act as depots of immune cells whose phenotype can be modified [8]. Furthermore, immune cells within AT can migrate toward the tumor and increase its malignancy [8]. It is therefore possible that interactions between IH and tumor may differentially affect AT macrophage populations when compared to IH alone in the absence of tumor. In addition, IH can mobilize BM-derived monocytes to inflamed tissues and promote their differentiation to macrophages [11]. However, the potential source of TAMs, i.e., AT-resident vs. BM-derived macrophages, in the context of cancer is unclear.

In this study, we hypothesized that IH exerts a differential effect on the immune system contingent on the concurrent presence or absence of a tumor microenvironment. Using a previously described lung epithelial tumor mouse model (TC1 cells), we aimed to assess the potential source and phenotype of macrophages, resident and bone-marrow derived macrophages within the ipsilateral and the contralateral AT to the tumor. In addition, the presence of other potent immune suppressor cells, such as Tregs and adipose and vascular progenitor cells, which could also participate in macrophage polarization and/or support tumor growth and invasion processes, was also examined.

Section snippets

Cells and reagents

Epithelial lung tumor cells TC1 (ATCC, CRL-2785) were purchased from American Type Culture Collection (Manassas, VA). All media and supplements were acquired from Gibco (Grand Island, NY). Tumor cells were cultured in RPMI supplemented with 2 mM L-glutamine, 10 mM HEPES buffer, 1 mM Sodium Pyruvate, 0.1 mM Non-essential amino acids, 100 U/Penicillin/100 µg/mL streptomycin, 10% fetal bovine serum (FBS) and geneticin 0.4 mg/mL. All antibodies were obtained from BioLegend (San Diego, CA): CD45-APC

Intermittent hypoxia increases tumor growth and invasion

Exposures to IH during the usual rest/sleep period of mice led to enhanced tumor growth, as evidenced by the temporal trajectory of tumor volume changes (Fig. 1A). Twenty-eight days after tumor injection, average tumor weights in mice exposed to IH were significantly higher (1.363 ± 0.143 g) when compared to RA-exposed mice (0.677 ± 0.097 g, p < 0.001; Fig. 1B). In addition, IH-induced tumor invasiveness was reflected by the higher proportion of tumors presenting invasion toward adjacent

Discussion

In the present study, we found that the adipose tissues surrounding the tumor appear to serve as a major source of macrophages and other tumor stromal cells to the adjoining tumor. Based on such findings, it is likely that these cells promote tumor growth and invasion, and that such contributions are further enhanced by the concomitant presence of IH mimicking sleep-disordered breathing. The pro-tumoral microenvironment associated with IH was further evidenced by the shift toward the M2

Funding

D.G. was supported by National Institutes of Health grant HL-65270-12 and by the the Herbert T. Abelson Chair in Pediatrics. IA was supported by Beatriu de Pinós fellowship from Generalitat de Catalunya/Marie Curie Actions (2010 BP_A2 00023).

Conflict of interest

None.

Acknowledgements

We thank the technical assistance of Zhuanhong Qiao in the FACS analyses.

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