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Background  Breast cancer is the most common type of cancer among females. Hypoxia mediates cancer hallmarks and results from reduced oxygen level due to irregularities in tumor vascularization or when the tumor size prevents oxygen diffusion and triggers angiogenesis to compensate for low oxygen. Cancer stem cells (CSCs) are a rare subpopulation, able to self-renew and to give rise to tumor-initiating cells. It is proposed that CSCs’ secretions help to recruit endothelial cells via angiogenic factors to establish tumor vascularization. In the tumor microenvironment, the effect of hypoxia on CSCs and the impact of their secretions on triggering angiogenesis and tumor vascularization remain questionable. In this study, three-dimensional (3D) CSCs derived from MCF-7 were directly exposed to repetitive long-term cycles of hypoxia to assess its effect on CSCs and then to evaluate the role of the hypoxic CSCs’ (CSCs HYP ) secretions in angiogenesis using (HUVECs) as a model for tumor neovascularization response.    Methods  CSCs derived from MCF-7 cell-line were expanded under repetitive, strictly optimized, long-term/continuous and intermittent hypoxic shots for almost four months to assess hypoxic effect on CSCs, sorted based on CD44 + /CD24 −  biomarkers. Hypoxic phenotype of CSCs HYP  was evaluated by assessing the acquired chemoresistance using MTT assay and elevated stemness properties were assessed by flow cytometry. To evaluate the effect of the secretions from CSCs HYP  on angiogenesis, HUVECs were exposed to CSCs HYP  conditioned-medium (CdM)—in which CSCs had been previously grown—to mimic the tumor microenvironment and to assess the effect of the secretions from CSCs HYP  on the HUVECs’ capability of tube formation, migration and wound healing. Additionally, co-culture of CSCs HYP  with HUVECs was performed.    Results  CSCs HYP  acquired higher chemoresistance, increased stemness properties and obtained greater propagation, migration, and wound healing capacities, when compared to CSCs in normoxic condition (CSCs NOR ). HUVECs’ tube formation and migration abilities were mediated by hypoxic (CSCs) conditioned media (CdM).    Discussion  This study demonstrates that chemoresistant and migrational properties of CSCs are enhanced under hypoxia to a certain extent. The microenvironment of CSCs HYP  contributes to tumor angiogenesis and migration. Hypoxia is a key player in tumor angiogenesis mediated by CSCs.

peerj

The effect of cycling hypoxia on MCF-7 cancer stem cells and the impact of their microenvironment on angiogenesis using human umbilical vein endothelial cells (HUVECs) as a model