Abstract 455: Identification of exosomal surface marker derived from Ewing sarcoma cells
Authors: Yoshida Aki , Fujiwara Tomohiro , Uotani Koji , Yoshioka Yusuke , Ueda Koji , Morita Takuya , Takeda Ken , Kunisada Toshiyuki , Nezu Yutaka , Kawashima Hiroyuki , Ochiya Takahiro , Ozaki Toshifumi 
Published: July 2016

Abstract
Introduction: Exosomes are small vesicles that are secreted by a multitude cell types. Recent studies have identified that circulating exosomes play an important role in tumor develompment and provide perspectives in novel diagnostic and therapeutic challenge. In this study, we investigated the surface protein on the tumor-derived exosomes by proteome analysis to utilize for a new biomarker of Ewing sarcoma.
Methods: Exosomes was collected from the culture medium of two Ewing sarcoma cells (SKES1, RDES) and human mesenchymal stem cells (hMSC) by gel filtration (EVsecond®) and ultracentrifugation. Exosomes extracted from each cell line were checked by transmission electron microscopy (TEM) and Nanosight®, a nanoparticle-tracking system. Global analysis of exosomal surface proteins were performed by liquid chromatography and mass spectrometry (LC/MS), then ES specific protein candidates were validated by immunoblot and ExoScreen for exosomes extracted by ultracentrifugation, and enzyme-linked immunosorbent assay (ELISA) for exosomes extracted by EVsecond®.
Results: Extracellular vesicles from ES cells were obtained by ultracentrifugation and EVSecond®. TEM and Nanosight® showed that the size of extracted exosomes derived from ES cells were 40-100 nm in diameter. Proteomic analysis detected 287 proteins from SKES-derived exosomes, 256 proteins from RDES-derived exosomes, and 133 proteins from hMSC-derived exosomes by LC/MS. We identified CD99 as entrapped protein on the exosomes derived from ES cells. For validation study, we performed immunoblot analysis that CD9, CD63, CD81 and CD99 were detected in ES-derived exosomes. Furthermore, immunogold TEM detected CD81 and CD99 on the exosomal surface. Next, we performed ELISA to detect the CD9 and CD99 on the surface of ES-derived exosomes purified by EVsecond®. Finally, we identified that ExoScreen, that has been clinically useful, was able to detect ES-derived exosomes, which expressed CD9, CD63 and CD99 on their surface.
Discussion and conclusion: Although the EWS-FLI1 oncogenic activity is increased by CD99 in ES, no study, to date, has reported that CD99 is on the tumor-derived exosomes and circulating in the patients’ serum. This study demonstrates that exosomes derived from ES cells can be detected by the CD99 surface marker, providing the possibility of the exosome as a novel biomarker for liquid biopsy.
Citation Format: Aki Yoshida, Tomohiro Fujiwara, Koji Uotani, Yusuke Yoshioka, Koji Ueda, Takuya Morita, Ken Takeda, Toshiyuki Kunisada, Yutaka Nezu, Hiroyuki Kawashima, Takahiro Ochiya, Toshifumi Ozaki. Identification of exosomal surface marker derived from Ewing sarcoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 455.