Abstract
BackgroundThe interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis. Invasion and intravasation, two critical steps in the metastatic process, are influenced by these interactions. Macrophages are of particular interest when it comes to studying tumor cell invasiveness. Previous studies have shown that there is paracrine loop signaling between breast cancer cells and macrophages involving colony stimulating factor 1 (CSF-1) produced by tumor cells and epidermal growth factor (EGF) production by macrophages. In this paper, we identify a novel paracrine loop between tumor cells and macrophages involving neuregulin (NRG1) and notch signaling.MethodsThe aim of this study was to determine the role of NRG1, a ligand of the ErbB3 receptor, in macrophage stimulation of tumor cell transendothelial migration and intravasation. We used fluorescence-activated cell sorting (FACS) and western blot to determine ErbB3 and NRG1 expression, respectively. An in vitro transendothelial migration (iTEM) assay was used to examine the effects of short hairpin (sh)RNA targeting NRG1 in tumor cells and clustered regularly interspaced short palindromic repeats (CRISPR) knockout of jagged 1 (JAG1) in macrophages. Orthotopic xenograft injections in mice were used to confirm results in vivo.ResultsIn our system, macrophages were the primary cells showing expression of ErbB3, and a blocking antibody against ErbB3 resulted in a significant decrease in macrophage-induced transendothelial migration of breast cancer cells. Stimulation of macrophages with NRG1 upregulated mRNA and protein expression of JAG1, a ligand of the Notch receptor, and JAG1 production by macrophages was important for transendothelial migration of tumor cells.ConclusionsThis study demonstrates that stimulation of macrophages by tumor cell NRG1 can enhance transendothelial migration and intravasation. We also demonstrate that this effect is due to induction of macrophage JAG1, an important ligand of the Notch signaling pathway.
Highlights
The interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis
ErbB3 is expressed on macrophages and NRG1 protein is expressed by tumor cells In order to determine surface expression levels of ErbB3, macrophages (BAC), MDA-MB 231 breast cancer cells
NRG1 protein expression was evaluated in the three different cell types: western blot analysis of cell lysates showed strong NRG1 protein expression in the MDA-MB 231 cells, and a second breast cancer triple negative cell line BT549, showed less expression in the Human umbilical vein endothelial cells (HUVEC) and macrophages (Fig. 1c)
Summary
The interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis. Macrophages are of particular interest when it comes to studying tumor cell invasiveness. Previous studies have shown that there is paracrine loop signaling between breast cancer cells and macrophages involving colony stimulating factor 1 (CSF-1) produced by tumor cells and epidermal growth factor (EGF) production by macrophages. We identify a novel paracrine loop between tumor cells and macrophages involving neuregulin (NRG1) and notch signaling. Cancer cells have their own inherent invasive properties, interaction with other cell types in the tumor microenvironment can facilitate metastasis. Tumor associated macrophages (TAMs) can play a role in breast cancer metastasis, and higher TAM density has been associated with worse prognosis [3, 4]. In particular a subset of TAMs form the tumor microenvironment of metastasis
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