Please use this identifier to cite or link to this item: https://doi.org/10.3390/cancers13040807
Title: Evaluating the epithelial-mesenchymal program in human breast epithelial cells cultured in soft agar using a novel macromolecule extraction protocol
Authors: Lau, Hiu Yeung
Tang, Jingyi 
Casey, Patrick J. 
Wang, Mei 
Keywords: Constitutively active RAS (CA-RAS)
Epithelial-mesenchymal transition
Human mammary epithelial (HME) cells
Soft agar colony formation assay
SV40 small T antigen
Transformation
Issue Date: 15-Feb-2021
Publisher: MDPI AG
Citation: Lau, Hiu Yeung, Tang, Jingyi, Casey, Patrick J., Wang, Mei (2021-02-15). Evaluating the epithelial-mesenchymal program in human breast epithelial cells cultured in soft agar using a novel macromolecule extraction protocol. Cancers 13 (4) : 1-16. ScholarBank@NUS Repository. https://doi.org/10.3390/cancers13040807
Rights: Attribution 4.0 International
Abstract: The ability to grow in anchorage-independent conditions is an important feature of malignant cells, and it is well-established that cellular phenotypes in adherent cultures can differ widely from phenotypes observed in xenografts and anchorage-independent conditions. The anchorage-independent soft-agar colony formation assay has been widely used as a bridge between adherent cell cultures and animal tumor studies, providing a reliable in vitro tool to predict the tumorigenicity of cancer cells. However, this functional assay is limited in its utility for molecular mechanistic studies, as currently there is no reliable method that allows the extraction of biological macromolecules from cells embedded in soft-agar matrices, especially in experimental conditions where no visible colonies form. We developed a set of new methods that enable the extraction of DNA, RNA and proteins directly from cells embedded in soft agar, allowing for a wide range of molecular signaling analysis. Using the new methods and human mammary epithelial cells (HMECs), we studied the role of epithelial-mesenchymal transition (EMT) in the ability of HMECs to form colonies in soft agar. We found that, when cultured in soft agar instead of in adherent cultures, immortalized non-malignant HME-hTERT cells upregulated the epithelial program, which was noted to be necessary for their survival in this anchorage-independent condition. Overexpression of SV40 small T antigen (ST) or the EMT master-regulator SNAI1 negates this requirement and significantly enhances colony formation in soft agar driven by mutant-RAS. Interestingly, we found that, similar to SNAI1, ST also promotes EMT changes in HMECs, providing further support for EMT as a prerequisite for the efficient anchorage-independent colony formation driven by mutant-RAS in our HMEC model. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
Source Title: Cancers
URI: https://scholarbank.nus.edu.sg/handle/10635/233765
ISSN: 2072-6694
DOI: 10.3390/cancers13040807
Rights: Attribution 4.0 International
Appears in Collections:Staff Publications
Elements

Show full item record
Files in This Item:
File Description SizeFormatAccess SettingsVersion 
10_3390_cancers13040807.pdf4.39 MBAdobe PDF

OPEN

NoneView/Download

Google ScholarTM

Check

Altmetric


This item is licensed under a Creative Commons License Creative Commons