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Superresolution microscopy for human microRNAs

Superresolution microscopy for human microRNAs in metastatic and non- metastatic tumor cells: An interdisciplinary project

Evidence is increasing that microRNAs, secreted by (tumor) cells, can induce sophisticated paracrine interactions between tumor cells and several other stromal cell types, cells of the immune system, and the microenvironment. Specifically, it has been suggested recently that microRNAs which are easily detectable in the blood stream (at least in part, e.g., in exosomes), are potentially able to prime metastatic niches for metastasizing tumor cells. Therefore, it became more and more important to be able to differentiate specific subcellular localizations and dynamics of individual microRNAs and find potential implications for predicting the metastatic phenotype of cancer cells by such investigations. However, so far the detection of small RNAs in biological systems with microscopy methods had been severely limited, in part due to poor target accessibility, low signal-to-background ratio and the theoretical limit of resolution in light microscopy. Existing strategies of visualizing small RNAs and mRNAs with multiple fluorophore labelling allowed for an estimation of relative differences only, and widely used methods like in situ hybridization are generally limited by diffraction (for more details, see our reference.

Differentially compartimentalized microRNAs

Thus, to contribute to identifying metastatically relevant individual cells among heterogeneous tumor cell populations, our group initiated a highly interdisciplinary consortium of collaborators from biophysics and optical physics (Prof. Christoph Cremer, IMB Mainz), light microscopy and bioimaging (Prof. Felix Bestvater, DKFZ Heidelberg), and exosome biology (Prof. Peter Altevogt, DKFZ Heidelberg) to establish the first single-cell, single molecule superresolution microscopy for human microRNAs. The technology we presented recently is able to visualized single microRNA molecules within single fixed cells at a resolution up to 10-15nm. Excitingly, for particular microRNAs we found that they are differentially compartmentalized in metastatic versus non-metastatic tumor cells. Especially, their subcellular localization within exosomes is significantly different between highly metastatic tumor cells and tumor cells with a low metastatic capability. Therefore, we think that our methodology might be able to help to discriminate metastatically relevant cells within a heterogeneous pool of tumor cells in the future, and generally open doors for the single-cell, single-molecule detection and functional analysis of further non-coding RNAs, as well as their detection at the single-molecule level in subcellular compartments of resected human tissues. Ongoing and future activities in our department therefore will promote this technology for advancing differential individualized diagnosis, and collaborate with colleagues in the field of macroscopic imaging and radiomics (e.g., Prof. Stefan Schönberg, M2Olie) to support such technologies in entering the field of innovative imaging technologies for human patients.

Selected publications

Superresolution imaging

  • Oleksiuk O, Abba M, Tezcan KC, Schaufler W, Bestvater F, Patil N, Birk U, Hafner M, Altevogt P, Cremer C, Allgayer H. Single-Molecule Localization Microscopy allows for the analysis of cancer metastasis-specific miRNA distribution on the nanoscale. Oncotarget. 2015 Nov 5.
    http://www.ncbi.nlm.nih.gov/pubmed/26561203
  • Prakash K, Fournier D, Redl S, Best G, Borsos M, Tiwari VK, Tachibana-Konwalski K, Ketting RF, Parekh SH, Cremer C, Birk UJ. Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes. Proc Natl Acad Sci U S A. 2015 Nov 24;112(47):14635-40.
    http://www.ncbi.nlm.nih.gov/pubmed/26561583
  • ?urek-Biesiada D, Szczurek AT, Prakash K, Mohana GK, Lee HK, Roignant JY, Birk U, Dobrucki JW, Cremer C. Localization microscopy of DNA in situ using Vybrant® DyeCycle™ Viole fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution. Exp Cell Res. 2015 Sep 1. pii: S0014-4827(15)30080-X.
    http://www.ncbi.nlm.nih.gov/pubmed/26341267
  • Szczurek AT, Prakash K, Lee HK, Zurek-Biesiada DJ, Best G, Hagmann M, Dobrucki JW, Cremer C, Birk U. Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes. Nucleus. 2014 Jul-Aug;5(4):331-40.
    http://www.ncbi.nlm.nih.gov/pubmed/25482122
  • Cremer C, Kaufmann R, Gunkel M, Pres S, Weiland Y, Müller P, Ruckelshausen T, Lemmer P, Geiger F, Degenhard S, Wege C, Lemmermann NA, Holtappels R, Strickfaden H, Hausmann M. Superresolution imaging of biological nanostructures by spectral precision distance microscopy. Biotechnol J. 2011 Sep;6(9):1037-51. Review.
    http://www.ncbi.nlm.nih.gov/pubmed/21910256

microRNAs

  • Abba M, Mudduluru G, Allgayer H. MicroRNAs in cancer: small molecules, big chances. Anticancer Agents Med Chem. 2012 Sep;12(7):733-43. Review.
    http://www.ncbi.nlm.nih.gov/pubmed/22292749
  • Rusek AM, Abba M, Eljaszewicz A, Moniuszko M, Niklinski J, Allgayer H. MicroRNA modulators of epigenetic regulation, the tumor microenvironment and the immune system in lung cancer. Mol Cancer. 2015 Feb 7;14:34. doi: 10.1186/s12943-015-0302-8. Review.
    http://www.ncbi.nlm.nih.gov/pubmed/25743773
  • Utikal J, Abba M, Novak D, Moniuszko M, Allgayer H Function and significance of MicroRNAs in benign and malignant human stem cells. Semin Cancer Biol. 2015 Dec;35:200-11. doi: 10.1016/j.semcancer.2015.07.001. Epub 2015 Jul 17. Review.
    http://www.ncbi.nlm.nih.gov/pubmed/26192966
  • Abba ML, Patil N, Leupold JH, Allgayer H. MicroRNA Regulation of Epithelial to Mesenchymal Transition. J Clin Med. 2016 Jan 14;5(1). Review.
    http://www.ncbi.nlm.nih.gov/pubmed/26784241

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