Cancer Biology

Cell surface protrusions include F-actin rich, wave-like ruffles that are erected transiently in response to stimuli and during cell migration. Macrophages are innate immune cells that ruffle constitutively and more dramatically in cells activated by pathogens. Dorsal ruffles and their resulting macropinosomes are key sites for environmental sampling, pathogen detection and immune signaling. Quantitative assessment of ruffling is important for assessing pathogen responses in macrophages and for analysis of growth factor responses in other cell types but automated and quantitative methods are lacking, and rely on manual and qualitative assessments. Here we present an automated ImageJ macro for quantifying dorsal cell surface protrusions from 3D microscope images. The assay presented here is suitable for high-throughput screening applications to detect drug, pathogen, or growth factor induced changes in cell ruffling by measuring ruffle area and intensity and providing normalized values in an easy to read combined spreadsheet.

MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify critical cell pathways that couple epithelial structure to individual cell based responses such as cell cycle exit and apoptosis. These studies will help to interrogate genetic changes critical for early breast tumorigenesis. The protocol describes a library of lentiviral shRNA constructs designed to target epithelial integrity and a highly efficient method for lentiviral transduction of suspension MCF10A cultures. Furthermore, protocols are provided for setting up MCF10A 3D cultures in Matrigel for morphometric and cellular response studies via structured illumination and confocal microscopy analysis of immunostained 3D structures.

Cellular transformation is a widely used method to artificially induce cells to form tumours in vivo. Here, we describe the methodology for malignant transformation of mouse embryonic fibroblasts (MEFs) for transplantation into immunodeficient nude mice, as used in Leong et al. (2013). The two-step process involves: 1) down-regulation of Trp53 expression using a short hairpin RNA (shRNA); and 2) overexpression of the oncogenic HRas^V12 protein. Reduction of Trp53 expression leads to cell immortalisation, and the subsequent overexpression of oncogenic HRas^V12 results in malignant transformation of a cell.