Evaluating the Epithelial-Mesenchymal Program in Human Breast Epithelial Cells Cultured in Soft Agar Using a Novel Macromolecule Extraction Protocol

SIMPLE SUMMARY: Anchorage-independent soft agar colony formation assays have been widely used as an in vitro surrogate for in vivo tumour formation in xenograft studies, and has found much utility in studies such as cancer drug development. However, molecular characterisation of cells grown in soft agar has proven difficult and sometimes even impossible. We developed a set of new methods that allow DNA, RNA and proteins (including phosphoproteins) to be extracted from cells grown in soft agar, even without visible colony formation. We used these methods to demonstrate the role of the epithelial-mesenchymal program in the malignant transformation of a classical human mammary epithelial cell model. 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.