Abstract 2305: Hypoxia modulates tumor exosomes, which function as mediators of angiogenesis.
Authors: Umezu Tomohiro , Tadokoro Hiroko , Ohyashiki Kazuma , Ohyashiki Junko H. 
Published: August 2014

Background and aim. We have recently shown tumor cell to endothelial cell communication and the induction of endothelial cell migration via exosomes (Umezu et al., Oncogene 2012). The aim of this study was to clarify whether hypoxia promotes angiogenic activity through tumor exosomes.

Experimental procedures. We used the human leukemia cell line K562 as exosome-generating cells and human umbilical vein endothelial cells (HUVECs) as exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) condition for 24h were isolated using Exoquick (System Bioscience) and quantitated by nano-particle analysis as well as immunoblotting for the exosomal protein CD63. These exosomes were added to HUVECs cultured on Matrigel for the validation of angiogenic activity, such as tube formation assay. Cellular and exosomal miRNAs responding to hypoxia were analyzed using TaqMan low-density miRNA array (TLDA, Applied Biosystems).

Results. The exosomes derived from K562 cells cultured under hypoxic condition enhanced tube formation of HUVECs. Culture under hypoxic condition for 24h affected neither the tumor cell growth nor the amount of exosome secreted into the culture medium, while culture under the same condition induced the up-regulation of HIF-1α expression. The exosomes derived from K562 cells cultured under hypoxic condition for 24h (K562-1%O2-exosomes) markedly enhanced the tube formation of HUVECs compared with the exosomes obtained from K562 cells cultured under normoxic condition (K562-20%O2-exosomes), indicating that K562-1%O2-exosomes potentially included angiogenic factors. Cellular and exosomal miRNA profiling of K562 cells cultured under hypoxic condition. Using TLDA, we profiled the expression of cellular and exosomal miRNAs and observed a fold change of &gt;1.5 under hypoxic condition compared with normoxic condition. We found that a subset of miRNAs, including miR-210, was significantly elevated under hypoxic condition in both cells and exosomes. The exosomal miRNAs derived from K562 cells cultured under hypoxic condition reduced the expression of Ephrin-A3 in HUVECs. We next examined the expression of Ephrin-A3 (EFNA3), an antiangiogenic factor, since miR-210 targets EFNA3. We demonstrated that EFNA3 expression was inhibited when HUVECs were exposed to hypoxic condition for 24h. Similarly, EFNA3 expression was drastically reduced by the addition of K562-1%O2-exosomes into HUVECs.

Conclusions. We demonstrated that tumor cells and their exosomes have altered miRNA profiles under hypoxic condition. Although exosomes contain various molecular constituents, such as proteins and mRNAs, altered exosomal compartments under hypoxic condition, including miR-210, affect antiangiogenic factors. Our results suggest the possibility that exosomal miRNA derived from tumor cells cultured under hypoxic condition may partly affect angiogenic activity in endothelial cells.

Citation Format: Tomohiro Umezu, Hiroko Tadokoro, Kazuma Ohyashiki, Junko H. Ohyashiki. Hypoxia modulates tumor exosomes, which function as mediators of angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2305. doi:10.1158/1538-7445.AM2013-2305