title: Single-Molecule Localization Microscopy allows for the analysis of cancer metastasis-specific miRNA distribution on the nanoscale.
abstract: We describe a novel approach for the detection of small non-coding RNAs in single cells by Single-Molecule Localization Microscopy (SMLM). We used a modified SMLM-setup and applied this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA (miR)-molecules in fixed cells with a localization accuracy of 10-15 nm, and is able to quantify and analyse clustering and localization in particular subcellular sites, including exosomes. We compared the metastasis-site derived (SW620) and primary site derived (SW480) human colorectal cancer (CRC) cell lines, and (as a proof of principle) evaluated the metastasis relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in both cell lines was very heterogeneous with the largest subpopulation of optically acquired weakly metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells. Furthermore, the highly metastatic cells had significantly more miR-31-molecules in the extracellular space, which were visualized to co-localize with exosomes in significantly higher numbers. From this study, we conclude that miRs are not only aberrantly expressed and regulated, but also differentially compartmentalized in cells with different metastatic potential. Taken together, this novel approach, by providing single molecule images of miRNAs in cellulo can be used as a powerful supplementary tool in the analysis of miRNA function and behaviour and has far reaching potential in defining metastasis-critical subpopulations within a given heterogeneous cancer cell population.
authors.affiliation: Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany.|Centre for Biomedicine and Medical Technology Mannheim, University of Heidelberg, Heidelberg, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Oleksiuk
authors.affiliation: Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany.|Centre for Biomedicine and Medical Technology Mannheim, University of Heidelberg, Heidelberg, Germany.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Abba
authors.affiliation: Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany.|Centre for Biomedicine and Medical Technology Mannheim, University of Heidelberg, Heidelberg, Germany.
authors.forename: Kerem Can
authors.initials: KC
authors.lastname: Tezcan
authors.affiliation: Light Microscopy Facility, German Cancer Research Centre (DKFZ), Heidelberg, Germany.|Karlsruhe Institute of Technology, Karlsruhe University, Karlsruhe, Germany.
authors.forename: Wladimir
authors.initials: W
authors.lastname: Schaufler
authors.affiliation: Light Microscopy Facility, German Cancer Research Centre (DKFZ), Heidelberg, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Bestvater
authors.affiliation: Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany.|Centre for Biomedicine and Medical Technology Mannheim, University of Heidelberg, Heidelberg, Germany.
authors.forename: Nitin
authors.initials: N
authors.lastname: Patil
authors.affiliation: Institute of Molecular Biology (IMB), Mainz, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Birk
authors.affiliation: Institute for Molecular and Cellular Biology, Mannheim University of Applied Sciences, Mannheim, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Hafner
authors.affiliation: Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg and Dept. of Dermatology, Venereology and Allergology, UMM, University of Heidelberg, Heidelberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Altevogt
authors.affiliation: Institute of Molecular Biology (IMB), Mainz, Germany.|Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Cremer
authors.affiliation: Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany.|Centre for Biomedicine and Medical Technology Mannheim, University of Heidelberg, Heidelberg, Germany.
authors.forename: Heike
authors.initials: H
authors.lastname: Allgayer
mesh_terms: D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D055354:Exosomes / Q000235:genetics / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008207:Lymphatic Metastasis; D035683:MicroRNAs / Q000235:genetics*; D008853:Microscopy / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D010641:Phenotype; D014162:Transfection
chemical_list: C534612:MIRN31 microRNA, human; D035683:MicroRNAs
keywords: localization microscopy; metastasis; miR-31; microRNAs; super-resolution