Simultaneous measurement of ERα, HER2, and PhosphoERK1/2 in breast cancer cell lines by flow cytometry

The activation of human epidermal growth factor receptor-2 (HER2) results in the activation of the mitogen-activated protein kinase (MAPK) cascade that may lead to the resistance to anti-estrogen therapy in estrogen receptor (ER alpha) expressing breast cancer by means of phosphorylation of ER alpha in the N-terminal region by phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and by means of decreasing ER alpha expression. Immunohistochemistry is the most widely used technique for the detection of ER alpha and HER2 in breast cancer specimens, however, is inadequate in its ability to assess the relationship between ER alpha, HER2, and MAPK cascade at the single cell level. To clear this major hurdle, we devised a novel flow cytometric method to quantify the expression of ER alpha, HER2, and the activation of MAPK cascade simultaneously in single cells. The method was validated by concurrent Western blotting in established cell lines: MDA-231 (ER alpha and HER2-negative), MCF-7 (ER alpha-positive, HER2-negative), MCF-7 cells overexpressing ER alpha after long-term incubation in estrogen-free medium, and HER2 transfected MCF7 cells. Using the flow cytometry method, we confirmed the previous finding that ER alpha expression is down-regulated upon epidermal growth factor mediated ERK1/2 phosphorylation in EGFR/MCF-7 cells. To our knowledge, this is the first such assay to incorporate simultaneous single cell measurement for all of these pathways, which may prove useful to determine the intratumoral heterogeneity in breast tumors or the receptor status in circulating tumor cells.