Hypoxia is an important feature of tumours, and is associated with aggressive tumour phenotypes and poor patient outcomes [17
]. Tumours can communicate with surrounding tissue to promote tumour progression and invasion through the release of exosomes [5
]. In this study, we investigated the impact of hypoxia, a clinically important feature of tumour progression, on exosome release by breast cancer cells. Here we present evidence that hypoxia enhances the release of exosomes by three different breast cancer cell lines, and that this process may be mediated, at least in part, by the HIF oxygen sensing pathway.
Exosomes were isolated by both ultracentrifugation and ExoquickTM
and were identifiable by their morphology and CD63 immunolabelling. These two purification methods were found to be comparable with regard to nanovesicle size and morphology as determined by NTA and electron microscopy. To our knowledge, this is the first such qualitative comparison of cell culture-derived exosomes currently available for these two methods of exosome isolation, although similar observations have been made in a clinical setting [47
precipitation was found to be significantly more efficient at isolating exosomes, as determined by direct quantitation by Nanosight NTA, supporting previous comparisons using protein quantitation and immunoblotting [28
]. From our experience, the efficiency of ExoquickTM
can allow exosome isolation and detection from as little as 10 μL of conditioned media (data not shown).
There is an increasing body of evidence that tumour cell-derived exosomes play important roles in angiogenesis [5
], cancer cell invasion [45
], metastasis [10
] and immunosuppression [8
] to promote tumour progression. Therefore, understanding the stimuli which promote exosome release by tumour cells is important in understanding tumour development. Here we present evidence that hypoxia promotes the release of exosome-sized nanoparticles. Exposure of breast cancer cells to modest (1%) and severe (0.1%) hypoxia resulted in mean increases of 32.3
4.8% and 90.9
7.1% of exosome-sized nanoparticles harvested from the conditioned media respectively. This is the first report to provide direct exosome quantitation after hypoxic exposure of cells. Previous studies have noted observations of the hypoxic enhancement of secretions of specific proteins, of putative exosomal origin [21
]. Our study offers the possible explanation that the increased concentrations of these proteins may be due to increased release of exosomes under hypoxia. Recent data failed to identify a significant difference in the concentration of exosomes released by hypoxic endothelial cells [36
]. This could be explained by the modest hypoxic exposure (2% O2
) performed, which is consistent with our observation that hypoxic enhancement of exosome release was relative to the severity of the hypoxic treatment. Alternatively, the substantial phenotypic differences between endothelial cells and epithelial tumour cells could also explain a lack of hypoxic enhancement.
HIF induction may play a role in the hypoxic enhancement of exosome release, which was supported here by manipulation of the HIF oxygen sensing pathway using DMOG and siRNA interference. Further circumstantial evidence for this putative role of HIF is provided by HIF-1α dependent secretion of HSP90α by dermal fibroblasts [50
], potentially via exosomes [51
]. However, it is important to recognise that the modest enhancement of exosome release experienced during HIF activation under normoxia (i.e. DMOG treatment) and incomplete abrogation of hypoxic enhancement by HIF siRNA suggests that other hypoxic responses may be involved.
Given the role for exosomes in tumour progression, increased release of exosomes by hypoxic tumour cells could translate to increased tumour invasion and progression during hypoxia. In addition to the effect of increased exosome numbers, hypoxic tumour-derived exosomes contain various pro-angiogenic factors which allow them to promote angiogenesis and endothelial cell activation [20
]. However, these studies did not identify if these proteins were an inherent component of exosomal cargo, or were induced by hypoxic exposure. Recent comparison of normoxic and hypoxic endothelial cell-derived exosomes identified that both protein and mRNA exosomal cargo are affected by hypoxia [36
How exosomal miRNAs might mediate hypoxic signalling requires further investigation. Here we have presented data which suggest that the miRNA miR-210 is elevated in hypoxic exosomes. This could play a role in promoting tumour progression in response to hypoxia, as miR-210 can promote endothelial cell tubulogenesis [52
], as well as repressing DNA repair pathways [53
]. One interesting possibility is that exosomal miRNAs may promote hypoxic signalling, for example miR-424 or miR-31 activation of HIF-1α independent of hypoxia [54
]. Of note, miR-424 is induced by hypoxia [54
], and has been identified in tumour-derived exosomes [38
]. In vivo
treatment with melanoma-derived exosomes promotes HIF-1α mRNA expression in sentinel lymph nodes [10
], highlighting the importance of further studies into exosome-mediated hypoxic signalling.