MP18-13 IDENTIFICATION OF TUMOR-SPECIFIC MARKERS ON EXTRACELLULAR VESICLES IN PATIENTS WITH RENAL CELL CARCINOMA

Abstract

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II (MP18)1 Apr 2020MP18-13 IDENTIFICATION OF TUMOR-SPECIFIC MARKERS ON EXTRACELLULAR VESICLES IN PATIENTS WITH RENAL CELL CARCINOMA Dirk Himbert, Hiresh Ayoubian, Michael Stockle, Egbert Oosterwijk, and Kerstin Junker* Dirk HimbertDirk Himbert More articles by this author , Hiresh AyoubianHiresh Ayoubian More articles by this author , Michael StockleMichael Stockle More articles by this author , Egbert OosterwijkEgbert Oosterwijk More articles by this author , and Kerstin Junker*Kerstin Junker* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000843.013AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Extracellular vesicles (EVs) are secreted by normal and tumor cells and are involved in cell-cell communication. Their molecular properties reflect in part the parental cells which makes them a promising source of biomarkers from liquid biopsies. Isolation of tumor-released EVs from blood or urine using tumor-specific markers implies potential to discover prognostic and therapeutic markers. The aim of this study is to identify tumor specific EV markers in clear cell renal carcinoma (ccRCC) using cell lines and patient derived tissue samples. METHODS: EVs from RCC cell lines (786-O, RCC53, RCC26, Caki1, Caki2) were isolated using ultracentrifugation (UC). Isolation of EVs from tumor tissue (n=10) was done by UC with three-layer-gradient (0.6 M, 1.3 M, 2.5 M sucrose). Protein expression was analyzed via Westernblot using cell-specific (GM130), exosome-specific (CD9, CD63, CD81) and putative tumor cell-specific (EpCAM, CA9, CD70, CD147 markers). EV imaging was carried out by transmission electron microscopy and Nano Tracking Analysis to prove the quality and quantity of EV isolation and enrichment. RESULTS: EVs from tumor tissues and cell cultures have been isolated with high quantity and purity. Candidate tumor specific markers have demonstrated diverse expression patterns in patient samples. Westernblot imaging exhibit that CD147, CA9 and CD70 are detectable in cells and EVs from tissue, with increased concentration in tumor derived EVs. EpCAM was found to be weakly expressed in cells and EVs from cell cultures, these findings were confirmed by chip-based techniques. In addition, EpCAM was expressed in 5/8 cellular and EV fractions of tissue samples, showing increased occurrence of cleaved fragments in EVs. Expression of CA9 was observed in all cell lines and their EVs as well as in 9/9 primary tumor tissues and EVs. CD70 exhibited different expression patterns in tumor cells and their EVs (10/10) depending on the patient sample, but was not found in EVs from cell lines. CD147 was enriched on EVs compared to the corresponding parental cell lines and also in the cell lysates and EVs in 10/10 primary tumor tissue samples. CONCLUSIONS: We developed an effective technique to isolate EVs directly from human tissue samples with high purity and high concentration. The expression of tumor specific markers reflects the cellular background of EVs. In contrast to EpCAM, CA9, CD70 and CD147 could represent promising tumor specific biomarkers for EVs in RCC. Further investigations will focus on development of bead-based enrichment of tumor specific EVs from blood samples. Source of Funding: Rolf Schwiete Stiftung © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e240-e240 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Dirk Himbert More articles by this author Hiresh Ayoubian More articles by this author Michael Stockle More articles by this author Egbert Oosterwijk More articles by this author Kerstin Junker* More articles by this author Expand All Advertisement PDF downloadLoading ...