Mammary stem cells (MaSCs) are enriched and localized to the leading edge of elongating outgrowths and side branches. a Two representative immunofluorescence images of SLUG (green), SOX9 (red), and actin (purple) expression in day 10 outgrowths. Scale bar represents 200 μm. b
Top: Immunofluorescence of SLUG, SOX9, and DAPI at the growing tips of ducts. Arrows indicate the direction of growth. Scale bars represent 50 μm. Bottom: High-magnification images reveal that new side branches are enriched for SLUG+/SOX9+ double-positive cells. Scale bar represents 200 μm. c Photomicrograph of the 3D printed organoid outgrowth from (a). Leader cells could be visualized and located at the leading edge of many outgrowths (black arrowheads). Immunofluorescence images depict SLUG (green), SOX9 (red), and DAPI (blue) in representative outgrowths with long (left), intermediate (top right), and short (bottom right) ductal elongation. d Quantification of the number of SLUG+/SOX9+ cells in relation to duct length revealed a significant anticorrelation between the number of double-positive cells and duct length (p < 0.01). e Quantification of the fraction of SLUG+ and SOX9+ cells in the indicated regions of the organoid outgrowths in (a). Every pairwise comparison was statistically significant (p < 0.001). N indicates the number of cells quantified.