Mass cytometric and transcriptomic profiling of epithelial-mesenchymal transitions in human mammary cell lines

Johanna Wagner,Andrea Jacobs,Bernd Bodenmiller,Sujana Sivapatham,Natalie De Souza,Charlotte Soneson,Markus Masek,Mark D Robinson,Nicolas Damond

doi:10.1038/s41597-022-01137-4

Abstract

Epithelial-mesenchymal transition (EMT) equips breast cancer cells for metastasis and treatment resistance. However, detection, inhibition, and elimination of EMT-undergoing cells is challenging due to the intrinsic heterogeneity of cancer cells and the phenotypic diversity of EMT programs. We comprehensively profiled EMT transition phenotypes in four non-cancerous human mammary epithelial cell lines using a flow cytometry surface marker screen, RNA sequencing, and mass cytometry. EMT was induced in the HMLE and MCF10A cell lines and in the HMLE-Twist-ER and HMLE-Snail-ER cell lines by prolonged exposure to TGFβ1 or 4-hydroxytamoxifen, respectively. Each cell line exhibited a spectrum of EMT transition phenotypes, which we compared to the steady-state phenotypes of fifteen luminal, HER2-positive, and basal breast cancer cell lines. Our data provide multiparametric insights at single-cell level into the phenotypic diversity of EMT at different time points and in four human cellular models. These insights are valuable to better understand the complexity of EMT, to compare EMT transitions between the cellular models used here, and for the design of EMT time course experiments.

Highlights

Background & SummaryThe epithelial-mesenchymal transition (EMT) equips epithelial cells with migratory, survival, and plasticity properties upon loss of epithelial hallmark characteristics
EMT was induced in the HMLE and MCF10A cell lines by prolonged exposure to TGFβ19,21 and in the HMLE-Twist-estrogen receptor (ER) (HTER) and HMLE-Snail-ER (HSER) cell lines by treatment with 4-hydroxytamoxifen (4OHT)[9]
The MCF10A human mammary epithelial cell line was obtained from American Type Culture Collection (ATCC) (CRL-10317) and cultured in DMEM F12 Ham medium (Sigma Aldrich) supplemented with 10 μg/ml human insulin (Sigma Aldrich), 20 ng/ml epidermal growth factor (EGF, Peprotech), 500 ng/ml hydrocortisone (Sigma Aldrich), 5% horse serum (Gibco), 100 ng/ml cholera toxin (Sigma Aldrich), and PenStrep (Gibco)[23]

Summary

Background & Summary

The epithelial-mesenchymal transition (EMT) equips epithelial cells with migratory, survival, and plasticity properties upon loss of epithelial hallmark characteristics. There remains the need for a comprehensive analysis of EMT phenotypes at the protein level To address this need, we applied multiplex single-cell mass cytometry[20] to four non-cancerous human mammary epithelial cell lines that serve as widely-used models of EMT. We observed alterations in the surface proteome of EMT-undergoing cells over time and detected distinct gene expression profiles of hybrid epithelial-mesenchymal states compared with epithelial and mesenchymal states. From these analyses, we extracted candidate markers for multiplex mass cytometry, which revealed complex phenotypic transitions in all four EMT models and little phenotypic overlap of EMT states between the cell lines. The data presented here can aid in characterizing the complexity and dynamics of EMT in these widely used in vitro models

Methods

Findings

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