title: Quantification of cell fusion events human breast cancer cells and breast epithelial cells using a Cre-LoxP-based double fluorescence reporter system.
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.
authors.affiliation: Institute of Immunology and Experimental Oncology, Center for Biomedical Education and Research, Witten/Herdecke University, Stockumer Str. 10, 58448, Witten, Germany.
authors.forename: Marieke
authors.initials: M
authors.lastname: Mohr
authors.affiliation: Institute of Immunology and Experimental Oncology, Center for Biomedical Education and Research, Witten/Herdecke University, Stockumer Str. 10, 58448, Witten, Germany.
authors.forename: Songül
authors.initials: S
authors.lastname: Tosun
authors.affiliation: Department of Biological and Material Sciences in Dentistry, School of Dentistry, Faculty of Health, Witten/Herdecke University, Witten, Germany.
authors.forename: Wolfgang H
authors.initials: WH
authors.lastname: Arnold
authors.affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology, Julius-Maximilians-University Würzburg, Würzburg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Edenhofer
authors.affiliation: Institute of Immunology and Experimental Oncology, Center for Biomedical Education and Research, Witten/Herdecke University, Stockumer Str. 10, 58448, Witten, Germany.
authors.forename: Kurt S
authors.initials: KS
authors.lastname: Zänker
authors.affiliation: Institute of Immunology and Experimental Oncology, Center for Biomedical Education and Research, Witten/Herdecke University, Stockumer Str. 10, 58448, Witten, Germany. thomas.dittmar@uni-wh.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dittmar
mesh_terms: D015153:Blotting, Western; D001943:Breast Neoplasms / Q000503:physiopathology*; D002459:Cell Fusion / Q000379:methods*; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D019426:Integrases; D042361:Mammary Glands, Human / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D014409:Tumor Necrosis Factor-alpha; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases
keywords: Breast cancer; Cell fusion; Cre-LoxP recombination; Flow cytometry