Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.
PLoS One
; 16(7): e0254783, 2021.
Article
em En
| MEDLINE
| ID: mdl-34314438
ABSTRACT
An array of isoforms of the nuclear estrogen receptor alpha (ER-α) protein contribute to heterogeneous response in breast cancer (BCa); yet, a single-cell analysis tool that distinguishes the full-length ER-α66 protein from the activation function-1 deficient ER-α46 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-α66, ER- α46, 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-α66 from ER-α46 in each individual cell. We identify ER-α46 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-α46 (i) ER-α46 induces phospho-AKT at Ser473, (ii) S6-ribosomal protein, an upstream ER target, activates both ER-α66 and ER-α46 in MCF-7 cells, and (iii) ER-α46 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-α protein.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Receptor alfa de Estrogênio
/
Análise de Célula Única
Limite:
Female
/
Humans
Idioma:
En
Revista:
PLoS One
Assunto da revista:
CIENCIA
/
MEDICINA
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos