3D mammary colony-forming cell assay

The mammary epithelium consists of multiple phenotypically and functionally distinct cell populations, which are organized as a hierarchy of stem cells, progenitors and terminally differentiated cells. Identification of the mechanisms regulating the growth and differentiation of mammary stem and progenitor cells is of great interest not only to better understand the mammary gland development but also to clarify the origins of breast cancer, as these cells seem to be the likely targets of malignant transformation within the mammary epithelium. Hence, a variety of approaches have been developed for quantifying and studying these specific mammary cell subsets. Given their high proliferative capacity, mammary progenitor cells are able to form colonies in vitro in low-density cultures. Here we describe how to perform a three dimensional (3D) Mammary Colony-Forming Cell (Ma-CFC) Assay, an in vitro functional assay suitable for the detection and analysis of mammary progenitor cells in feeder-free culture conditions. Briefly, this protocol involves the seeding of mammary single cells, at clonal density, onto a semi-solid matrix (Matrigel), thus allowing mammary progenitors to proliferate and give rise to discrete 3D colonies. The number and the cell composition of the resulting colonies will vary according to the frequency and the differentiation potential of the progenitors, respectively