An array of isoforms of the nuclear estrogen receptor alpha (ER-alpha) protein contribute to heterogeneous response in breast cancer (BCa); yet, a single-cell analysis tool that distinguishes the full-length ER-alpha66 protein from the activation function-1 deficient ER-alpha46 isoform has not been reported. Specific detection of protein isoforms is a gap in single-cell analysis tools, as the de facto standard immunoassay requires isoform-specific antibody probes. Consequently, to scrutinize hormone response heterogeneity among BCa tumor cells, we develop a precision tool to specifically measure ER-alpha66, ER- alpha46, and eight ER-signaling proteins with single-cell resolution in the highly hetero-clonal MCF-7 BCa cell line. With a literature-validated pan-ER immunoprobe, we distinguish ER-alpha66 from ER-alpha46 in each individual cell. We identify ER-alpha46 in 5.5% of hormone-sensitive (MCF-7) and 4.2% of hormone-insensitive (MDA-MB-231) BCa cell lines. To examine whether the single-cell immunoblotting can capture cellular responses to hormones, we treat cells with tamoxifen and identify different sub-populations of ER-alpha46: (i) ER-alpha46 induces phospho-AKT at Ser473, (ii) S6-ribosomal protein, an upstream ER target, activates both ER-alpha66 and ER-alpha46 in MCF-7 cells, and (iii) ER-alpha46 partitions MDA-MB-231 subpopulations, which are responsive to tamoxifen. Unlike other single-cell immunoassays, multiplexed single-cell immunoblotting reports-in the same cell-tamoxifen effects on ER signaling proteins and on distinct isoforms of the ER-alpha protein.
View details for DOI 10.1371/journal.pone.0254783
View details for PubMedID 34314438