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Quantification of cell fusion events human breast cancer cells and breast epithelial cells using a Cre-LoxP-based double fluorescence reporter system

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Abstract

The biological phenomenon of cell fusion plays an important role in several physiological processes, like fertilization, placentation, or wound healing/tissue regeneration, as well as pathophysiological processes, such as cancer. Despite this fact, considerably less is still known about the factors and conditions that will induce the merging of two plasma membranes. Inflammation and proliferation has been suggested as a positive trigger for cell fusion, but it remains unclear, which of the factor(s) of the inflamed microenvironment are being involved. To clarify this we developed a reliable assay to quantify the in vitro fusion frequency of cells using a fluorescence double reporter vector (pFDR) containing a LoxP-flanked HcRed/DsRed expression cassette followed by an EGFP expression cassette. Because cell fusion has been implicated in cancer progression four human breast cancer cell lines were stably transfected with a pFDR vector and were co-cultured with the stably Cre-expressing human breast epithelial cell line. Cell fusion is associated with a Cre-mediated recombination resulting in induction of EGFP expression in hybrid cells, which can be quantified by flow cytometry. By testing a panel of different cytokines, chemokines, growth factors and other compounds, including exosomes, under normoxic and hypoxic conditions our data indicate that the proinflammatory cytokine TNF-α together with hypoxia is a strong inducer of cell fusion in human MDA-MB-435 and MDA-MB-231 breast cancer cells.

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Acknowledgments

We would like to thank Dr. Oliver Thamm (Clinic for Plastic- and Reconstructive Surgery, Handsurgery, Burn Care Center, University of Witten/Herdecke, Cologne-Merheim Medical Center, Cologne, Germany) for providing us with chronic wound fluid (CWF). This work was supported by the Fritz-Bender-Foundation, Munich, Germany.

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Authors and Affiliations

  1. Institute of Immunology and Experimental Oncology, Center for Biomedical Education and Research, Witten/Herdecke University, Stockumer Str. 10, 58448, Witten, Germany

    Marieke Mohr, Songül Tosun, Kurt S. Zänker & Thomas Dittmar

  2. Department of Biological and Material Sciences in Dentistry, School of Dentistry, Faculty of Health, Witten/Herdecke University, Witten, Germany

    Wolfgang H. Arnold

  3. Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology, Julius-Maximilians-University Würzburg, Würzburg, Germany

    Frank Edenhofer

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  1. Marieke Mohr
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  2. Songül Tosun
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  3. Wolfgang H. Arnold
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  4. Frank Edenhofer
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  5. Kurt S. Zänker
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Correspondence to Thomas Dittmar.

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Supplementary data 1

These tables summarize the tested compounds, including concentration, and conditions, used in this study for each cell line. The relative fold change was calculated as described in the Material & Methods section. Shown are the mean ± STD of at least three independent experiments (DOC 335 kb)

Supplementary data 2

In this figure the relative fold changes in the amount of cell debris, also containing apoptotic bodies, of the compounds and conditions, which led to an increased number of green fluorescing cells are summarized. The amount of cell debris/apoptotic bodies was determined by gating the population located in the lower left corner of a FSC-H/SSC-H dot plot diagram. Shown are the mean ± STD of at least three independent experiments (TIFF 633 kb)

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Mohr, M., Tosun, S., Arnold, W.H. et al. Quantification of cell fusion events human breast cancer cells and breast epithelial cells using a Cre-LoxP-based double fluorescence reporter system. Cell. Mol. Life Sci. 72, 3769–3782 (2015). https://doi.org/10.1007/s00018-015-1910-6

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  • DOI: https://doi.org/10.1007/s00018-015-1910-6

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