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Cited by 37Cancer Cells and MetastasisMelanoma and MAPK PathwaysProtein Kinase Regulation and GTPase Signaling

A novel MCF-10A line allowing conditional oncogene expression in 3D culture

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Herr, Ricarda, Wöhrle, Franziska U, Danke, Christina, Berens, Christian, Brummer, Tilman·2011·Cell Communication and Signaling
DOI 10.1186/1478-811x-9-17PMC PMC3163222
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Abstract

Introduction: Non-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene. Methods: MCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HA BRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-Raf V600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out. Results: MCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-Raf V600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression. Conclusions: Taken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance.

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  • While ensuring controlled and efficient transfection, inducible systems are hardly compatible with high-throughput analysis.

    “Nonetheless, while ensuring a controlled and efficient transfection, this system is hardly compatible with high-throughput analysis.”

    From Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens (2018) · cites this paper as [41]

    The Results section describes tightly controlled, efficient gene expression, and Methods (Cell lines and tissue culture; page unavailable) reports improved transfection efficiency with nucleofection. The paper does not say the system is hardly compatible with high-throughput analysis; the Discussion instead says its protocol can generate several sublines in parallel. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • A viable strategy to transfect ECM-embedded organoids is to use inducible systems.

    “A viable strategy to transfect ECM-embedded organoids is to use inducible systems (41).”

    From Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens (2018) · cites this paper as [41]

    The Results describe transfecting MCF-10A cells with a tetracycline-inducible system and inducing transgene expression after the cells formed 3D acini. This supports inducible expression in embedded structures, but not transfection of organoids while they are ECM-embedded; the cells were transfected before 3D culture. The indexed passages provide section labels but no page numbers. (LLM, conf=0.88)

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Authors
Herr, R., Wöhrle, F. U., Danke, C., Berens, C., & Brummer, T.
Affiliations
  • University of Freiburg (DE)
  • Friedrich-Alexander-Universität Erlangen-Nürnberg (DE)
Corresponding author
Brummer, Tilman
ORCID
Venue
Cell Communication and Signaling
Publisher
Springer Science and Business Media LLC
Published
2011-07-13
Volume
9
Issue
1
Pages
17-17
ISSN
1478-811X (ISSN-L)
DOI
10.1186/1478-811x-9-17
PMC
PMC3163222
OpenAlex
W1996398546
Cited by
37
References
1
Citations by year
2025: 1 · 2024: 2 · 2023: 2 · 2021: 1 · 2020: 1 · 2019: 4 · 2018: 2 · 2017: 4 · 2016: 1 · 2015: 6 · 2014: 6 · 2013: 2 · 2012: 4
FWCI
2.05
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Cell Communication and Signaling (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OPUS FAU (Kooperativer Bibliotheksverbund Berlin-Brandenburg (KOBV), on behalf of the Universitätsbibliothek Erlangen-Nürnberg) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • FreiDok plus (Universitätsbibliothek Freiburg) (submittedVersion)
Funders
  • Deutsche Forschungsgemeinschaft (GSC‐4, SFB 473)
  • Université de Fribourg
Topics
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Melanoma and MAPK Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
Keywords
MCF10A cells, transcriptional silencer, BrafV600E, mammary epithelial cells, epithelial-mesenchymal transition, tumor invasion, early carcinogenesis

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