Single-cell immunoblotting resolves estrogen receptor-[alpha] isoforms in breast cancer

• Published in: PloS one 2021-07, Vol.16 (7), p.e0254783
• Main Authors: Kim, John J, Liang, Wenchuan, Kang, Chi-Chih, Pegram, Mark D, Herr, Amy E
• Format: Article
• Language: English
• Subjects: Breast cancer; Estrogen; Genetic aspects; Health aspects; Proteins
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0254783

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-[alpha]66 protein from the activation function-1 deficient ER-[alpha]46 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-[alpha]66, ER- [alpha]46, 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-[alpha]66 from ER-[alpha]46 in each individual cell. We identify ER-[alpha]46 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-[alpha]46: (i) ER-[alpha]46 induces phospho-AKT at Ser473, (ii) S6-ribosomal protein, an upstream ER target, activates both ER-[alpha]66 and ER-[alpha]46 in MCF-7 cells, and (iii) ER-[alpha]46 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.