Cellular and Molecular Life Sciences

, Volume 72, Issue 19, pp 3769–3782 | Cite as

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

  • Marieke Mohr
  • Songül Tosun
  • Wolfgang H. Arnold
  • Frank Edenhofer
  • Kurt S. Zänker
  • Thomas DittmarEmail author
Research Article


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.


Cell fusion Breast cancer Cre-LoxP recombination Flow cytometry 



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.

Supplementary material

18_2015_1910_MOESM1_ESM.doc (335 kb)
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)
18_2015_1910_MOESM2_ESM.tif (634 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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Copyright information

© Springer Basel 2015

Authors and Affiliations

  • Marieke Mohr
    • 1
  • Songül Tosun
    • 1
  • Wolfgang H. Arnold
    • 2
  • Frank Edenhofer
    • 3
  • Kurt S. Zänker
    • 1
  • Thomas Dittmar
    • 1
    Email author
  1. 1.Institute of Immunology and Experimental Oncology, Center for Biomedical Education and ResearchWitten/Herdecke UniversityWittenGermany
  2. 2.Department of Biological and Material Sciences in Dentistry, School of Dentistry, Faculty of HealthWitten/Herdecke UniversityWittenGermany
  3. 3.Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell BiologyJulius-Maximilians-University WürzburgWürzburgGermany

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