PO-369 Effects of cycling hypoxia on the communication between macrophages and endothelial cells in promoting tumour growth and metastasis

Introduction Tumours are not only composed of malignant cells. Indeed, blood vessels, composed of endothelial cells, are required for tumour growth. Interestingly, the induction of angiogenesis requires not only endothelial cells but also immune cells. Among them, tumor-associated macrophages (TAMs) make up to 50% of the tumour immune infiltrate. These TAMs are strong angiogenesis inducer and are in part responsible of the tumour inflammation. Two kinds of macrophages are detected in tumours: pro-inflammatory and anti-tumoral M1 macrophages and anti-inflammatory and pro-tumoral M2 macrophages. Hypoxia is another key feature of tumour microenvironment which induces angiogenesis and enhances tumour inflammation and metastasis. Two types of hypoxia occur in tumour: chronic hypoxia, which impacts cells too far from the blood vessels, and cycling hypoxia (CyH), which causes intermittent oxygenation of malignant and non-malignant cells. We have studied the impact of CyH on macrophage polarisation and activity. Secondly, the impact of CyH on the communication between macrophages and endothelial cells was investigated. Then, the impact of this dialogue on tumour growth and metastasis will be studied. Material and methods THP-1 monocytes are differentiated into M0 macrophages by PMA. M0 macrophages are then polarised into M1 macrophages with LPS and IFNg (24 hour incubation) or into M2 macrophages with IL-4 and IL-13 (48 hour incubation). After exposing these macrophages to normoxia, chronic hypoxia or CyH, the expression and secretion of pro-inflammatory factors were studied by RT-qPCR and ELISA, respectively. To investigate the communication between macrophages and endothelial cells under CyH, endothelial cells were incubated with CyH-exposed macrophage conditioned-medium and the expression of endothelial cell activation markers was analysed by RT-qPCR. Results and discussions CyH induced a pro-inflammatory phenotype in M0 macrophages and enhanced the pro-inflammatory phenotype of M1 macrophages. Indeed, an increased expression of TNFa, IL-6 and IL-1β was observed. These effects depended on NF-kB activation since IKK inhibition prevented these effects. The conditioned-media of M0, M1 and M2 macrophages exposed to CyH induced endothelial cell activation as observed by an increased IL-6, IL-8 and ICAM-1 expression. Conclusion CyH induced a pro-inflammatory phenotype in M0 and M1 macrophages via NF-kB activation. The M0, M1 and M2 macrophages exposed to cycling hypoxia induced endothelial cell activation by a secreted molecule that needs to be identified.