Determining mammosphere-forming potential: application of the limiting dilution analysis

Abstract

Originally adapted from the neurosphere assay, the nonadherent mammosphere assay has been utilized to assess early progenitor/stem cell frequency in a given population of mammary epithelial cells. This method has also been used to measure the frequency of tumorsphere initiating cells in both primary mammary tumors as well as in tumor cell lines. Although, the mammosphere assay has been used extensively in the mammary gland field, a standard method of quantifying and analyzing sphere growth in this assay has remained undefined. Here, we discuss the use and benefit of using a limiting dilution analysis to quantify sphere-forming frequency in primary mammary epithelial cells grown in nonadherent conditions.

Figure 1

Schematic showing strategy for limiting dilution plating of dissociated epithelial cells in 96 well plate for SLDA. The “+” refers to a well containing one or more spheres and a “–” refers to a well without any spheres. The figure shows the cells being plated in a limiting dilution fashion across a 96-well plate. The amount of wells negative for sphere formation will increase as the plating density decreases as depicted

Figure 2

SLDA comparing stem cell frequency in tertiary spheres between dnhIGF-1R and FVB (WT) primary mammary epithelial cells grown in PRO-N media in 96 well ultra-low adherent plates (Corning). Plating density ranges from 1000 to 0.00012 cells/well, which was converted to cells/cm² in this graph. Tertiary sphere forming frequency for dn-IGF-1R is 1 in 526 cells versus 1 in 127 cells for the wild type control. PRO-N media contained EGF/bFGF (20 ng/ml), bovine Insulin 25 μg/ml (Sigma), d-Biotin 10 ng/ml (Sigma), Progesterone 20 nM (Sigma), Putrescine 100 μM (Sigma), Selenium 5 ng/ml (Sigma), Apo-transferrin 50 μg/ml (Sigma), Gentamycin 50 μg/ml (Invitrogen-Gibco), Hydrocortisone 0.5 μg/ml (Sigma). Linear (fvb) and Linear (dn) indicate best fit line for linear regression analysis