MP39-02 NEW EXOSOMAL PROTEIN AND MRNA BIOMARKERS FOR RENAL CANCER

Abstract

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-02 NEW EXOSOMAL PROTEIN AND MRNA BIOMARKERS FOR RENAL CANCER Richard Zieren, David Clark, Liang Dong, Leandro Ferreira Moreno, Morgan Kuczler, Kengo Horie, Louis Vermeulen, Hui Zhang, Sarah Amend, Theo de Reijke, and Kenneth Pienta Richard ZierenRichard Zieren More articles by this author , David ClarkDavid Clark More articles by this author , Liang DongLiang Dong More articles by this author , Leandro Ferreira MorenoLeandro Ferreira Moreno More articles by this author , Morgan KuczlerMorgan Kuczler More articles by this author , Kengo HorieKengo Horie More articles by this author , Louis VermeulenLouis Vermeulen More articles by this author , Hui ZhangHui Zhang More articles by this author , Sarah AmendSarah Amend More articles by this author , Theo de ReijkeTheo de Reijke More articles by this author , and Kenneth PientaKenneth Pienta More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal cancer (RCC) accounts for over 73,750 new cases and 14,830 deaths annually in the US. Non-invasive biomarkers are needed to distinguish benign from RCC in small renal masses and to distinguish between clear cell RCC (ccRCC) and papillary RCC (pRCC) subtypes. Extracellular vesicles (EVs), such as exosomes, are a promising source of biomarkers for RCC. Compared with EVs enriched from plasma and urinary, EVs enriched from conditioned cell media (CCM) are readily available and their cellular origin is specific. In this study we aimed to select potential biomarkers by analyzing mRNA and protein cargo of EVs from various RCC and immortalized benign kidney epithelial cell lines and assess biological functions of the EV cargo. METHODS: We used the following cell lines; 786O, 769P, and Caki1 (all ccRCC); ACHN, and Caki2 (all pRCC); and HK2, and RPTEC (benign epithelial kidney cells). CCM EVs were enriched by a combination of differential centrifugation, ultrafiltration, and size exclusion chromatography. All CCM EVs were counted by NanoFCM, negative stained and imaged by TEM, and assessed for EV-markers (CD63, CD81, Flot1) by Western blot. The Qiagen miRNeasy kit was used to extract RNA from CCM EVs, the NanoString nCounter low RNA input kit was used for RNA amplification in combination with the Nanostring nCounter PanCancer Progression assay. CCM EVs were processed by Tandem Mass Tag mass spectrometry (TMT MS). RESULTS: Counts by NanoFCM demonstrated ACHN secreted the most EVs per million cells (1.4×10e7) and Caki-1 the least (5.6×10e4) EVs per million cells. Western blot expression of EV-associated markers CD81, CD63, CD9, and Flot1 was highly correlated with marker expression detected by TMT MS. In mRNA-analyses we found abundance of 74 unique mRNAs in benign EVs, 12 in ccRCC EVs, and 29 in pRCC EVs. By MS analysis we identified 1,726 proteins of which 186 proteins were enriched (> 1.5-fold change) in all types of EVs compared to their parental cell lysates. We found 83-124 proteins with increased abundance in pRCC EVs compared with benign EVs and 83-121 proteins with decreased abundance. In ccRCC EVs we found 95-141 increased abundance compared with benign EVs and 116-130 with decreased abundance. CONCLUSIONS: We compared CCM EVs derived from ccRCC, pRCC, and benign renal cell lines using a multi-omics approach. We used different experimental designs for the mRNA-analysis and mass spectrometry due to technical limitations, yet we were able to analyze exosomal cargos of benign and malign kidney cells. We identified several potential RCC-biomarkers that require further clinical validation. Source of Funding: Stichting Cure for Cancer (Amsterdam) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e704-e704 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Richard Zieren More articles by this author David Clark More articles by this author Liang Dong More articles by this author Leandro Ferreira Moreno More articles by this author Morgan Kuczler More articles by this author Kengo Horie More articles by this author Louis Vermeulen More articles by this author Hui Zhang More articles by this author Sarah Amend More articles by this author Theo de Reijke More articles by this author Kenneth Pienta More articles by this author Expand All Advertisement Loading ...