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Formation of hybrid cells by “accidental cell fusion” of normal and neoplastic breast epithelial cells with local tissue‐associated mesenchymal stroma/stem‐like cells (MSC) in an inflammatory microenvironment can generate new cancer cell populations whereby molecular signaling mechanisms of this process remain unclear. Fusions of lentiviral enhanced green fluorescent protein‐labeled MSC with mcherry‐labeled breast epithelial cells were quantified and effects of tumor necrosis factor alpha (TNF‐α) and receptor downstream signaling were investigated. Cocultures of MSC with normal human mammary epithelial cells, with neoplastic MCF10A, or with MDA‐MB‐231 or MCF7 breast cancer cells demonstrated hybrid cell formation between 0.1% and about 2% of the populations within 72 hours, whereby the fusion process occurred in less than 5 minutes. Addition of the pro‐inflammatory cytokine TNF‐α significantly enhanced MCF10A‐MSC cell fusion. Small‐interfering RNA (siRNA) knockdown experiments revealed an involvement of tumor necrosis factor (TNF) receptor‐1 and ‐2 in this process. This was also substantiated by siRNA knockdown of tumor necrosis factor receptor type 1‐associated death domain which abolished TNF‐α‐stimulated fusion. While TNF receptor signaling can be relayed via the Mitogen‐activated protein kinase 8 (MAPK8), NF‐κB or cell death pathways, examination of further downstream signaling exhibited little if any effects of MAPK8 or RelA (p65) on TNF‐α‐mediated cell fusion, respectively. These data suggested that cell fusion between MSC and MCF10A breast epithelial cells can be stimulated by TNF‐α involving TNF receptor‐activated cell death pathways or additional NF‐κB signaling. S tem  C ells   2018;36:977–989

stem cells

In Vitro Fusion of Normal and Neoplastic Breast Epithelial Cells with Human Mesenchymal Stroma/Stem Cells Partially Involves Tumor Necrosis Factor Receptor Signaling