Abstract 5182: Characterization of miRNA expression pattern from in-vitro obtained exosomes of different urinary bladder cancer cell lines
Authors: Baumgart Sophie , Heinzelmann Joana , Stoeckle Michael , Stampe Ostenfeld Marie , Junker Kerstin 
Published: August 2015

Abstract
Introduction & objectives:
Interaction of tumor cells and the tumor microenvironment (TME) plays an important role in tumor development and progression. It was shown that microRNAs (miRNAs), packed in exosomes, can affect cell-cell communication at the site of origin as well as the TME. The aim of the project is the identification of a specific miRNA expression pattern from in-vitro obtained tumor-derived exosomes of different UBC cell lines in correlation to their malignant potential. Furthermore, we want to analyze the effect of these exosomal miRNAs on tumor-associated fibroblasts (TAFs).
Materials & methods:
Exosomes were isolated from invasive (T-24,253J-BV, J82) and non-invasive (RT-112, 5637) UBC cell lines. The number and size of vesicles were measured by NTA. The vesicles were examined for exosomal and contamination markers by Western blotting. TotalRNA was isolated from the exosomes upon treatment with RNase. MiRNA expression pattern was analyzed from exosomes secreted by invasive and non-invasive UBC cells using miRNA microarray. The validation of significantly differently expressed miRNAs was performed by using qPCR. Exosome-mediated miRNA transfer between cancer cells and TAFs was verified by 1) transfection of donor UBC cells with the C. elegans-specific miRNA, cel-miR-39, 2) Exosome isolation and RNAse treatment, 3) Transfer to recipient TAFs, and 4) miRNA-specific qRT-PCR analysis using totalRNA from the recipient TAFs.
Results:
The isolated exosomes from UBC cells exhibited a high amount of exosomal markers (CD63, CD81, syntenin). 16 miRNAs were identified, which distinguishes invasive UBC cells from non-invasive cells. Exosomes secreted by invasive UBC cells are characterized by a specific miRNA signature of 25 miRNAs compared to exosomes from non-invasive UBC cells. The validation of differently expressed miRNAs is ongoing. After successful transfection of RT-112 and T-24 with cel-miR-39, cel-miR-39 was detected in RT-112 and T-24 exosomes as well as in recipient TAFs cultivated in the presence of these exosomes.
Conclusion:
Exosomes secreted by UBC cells exhibit a specific miRNA signature depending on the invasive potential of the originating cells. We could proof an exosome-mediated transfer of miRNAs between tumor cells and TAF.
These results emphasize the role of exosomal miRNAs for the interaction between tumor cells and the tumor microenvironment. Further studies have to show the functional relevance of selected exosomal miRNAs.
Citation Format: Sophie Baumgart, Joana Heinzelmann, Michael Stoeckle, Marie Stampe Ostenfeld, Kerstin Junker. Characterization of miRNA expression pattern from in-vitro obtained exosomes of different urinary bladder cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5182. doi:10.1158/1538-7445.AM2015-5182