Abstract 342: Profiling mRNAs of parental prostate cancer cells with different phenotypes and their daughter extracellular vesicles using the NanoString low RNA input nCounter assay
Authors: Dong Liang , Huang Chung-Ying , Zieren Richard , Horie Kengo , Amend Sarah R. , Pienta Kenneth J. 
Published: September 2020

Abstract
Introduction and objective: Cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (EMT) and MET is critical in the metastatic cascade. Extracellular vesicles (EVs) may play an important role in this balance by shuttling molecular cargos into recipient cells. This study aims to evaluate the feasibility of profiling mRNAs of parental prostate cancer (PCa) cells with different phenotypes and their daughter EVs using the NanoString low RNA input nCounter assay.
Methods: PC3-Epi and PC3-EMT cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original PC3 cell line. The cell culture supernatant was collected after 48 h of serum starvation of cells and processed by differential ultracentrifugation. The supernatant was first pre-cleared for any dead cells and debris by centrifugation at 2000×g for 20 min. Without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at 10,000×g for 20 min at 4 °C. The remaining SN was then centrifuged to isolate the EVs at 100,000×g for 120 min at 4 °C. The EVs pellet was further washed in 1× PBS followed by a second centrifugation at 100,000×g for 120 min at 4 °C. The final EVs pellet was resuspended in 1×PBS for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and Western blot) and nCounter assays. The total RNA of cells and their daughter EVs were assayed by the nCounter PanCancer Progression Panel to determine expression of 770 selected mRNAs. The NanoString nCounter Low RNA Input Kit with the multiplex 770-gene primer pool was used for the pre-amplification of mRNA and overnight hybridization with the PanCancer Progression panel. Each sample type was submitted to the assay in biological triplicate.
Results: When comparing all 12 samples, Eisen Cluster analysis separated all the cells and all EVs into two groups, regardless of their phenotypes. In subgroup analysis, the expression patterns between PC3-Epi and PC3-EMT cells were significantly different. CLEC2B, KDR, CRIP2, IL13RA2, CC2D1B were significantly upregulated in PC3-EMT cells, while CXCL8, EPCAM, LAD1, SERPINH1, ESRP1, CLDN7, CLDN1, TACSTD2, TGFB2, TMEM30B, CDH1, S100A14, ST14, NOX5, OVOL2 were significantly downregulated in PC3-EMT cells. The expression patterns between PC3-Epi and PC3-EMT cells were also significantly different. TBX1, CAV1, COL4A1, SLC35A3, MYC, ITGB2, TIMP4, CAMK2B, PTGDS, P3H2, PECAM1, CXCL13, CNN1, TFPI2, MTDH, STAB2, ITGB6, VIM, GTF2I, ZNF143, STAT3 were all significantly downregulated in PC3-EMT cell derived EVs.
Conclusions: The NanoString low RNA input nCounter assay can provide reliable mRNA expression profiling of EVs. The mRNA expression patterns are very different between cells and their daughter EVs. Both cells and EVs with different phenotypes have different gene expressions.
Citation Format: Liang Dong, Chung-Ying Huang, Richard Zieren, Kengo Horie, Sarah R. Amend, Kenneth J. Pienta. Profiling mRNAs of parental prostate cancer cells with different phenotypes and their daughter extracellular vesicles using the NanoString low RNA input nCounter assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 342.