Figure 3.

MECs from SPI-fed Wnt1-Tg mice have lower repopulating activity and mammosphere-forming ability. (a) Whole mounts depicting mammary outgrowths from transplantation of freshly isolated MECs (10,000 cells) from hyperplastic mammary glands of Wnt1-Tg mice fed CAS (6/8 outgrowths) or SPI (2/8 outgrowths). Scale bar, 1000 µm. Eight independent experiments were conducted, with each experiment representing an individual mouse for each donor and recipient. (b) MECs from SPI-fed Wnt1-Tg mice displayed lower ability to form mammospheres (from secondary passage, P2), quantified as mammosphere-forming units (MFU). Representative mammospheres are shown at day 7 of secondary passage (P2). Scale bar, 100 µm. Data represent the mean % MFUs ± SEM of seven independent experiments, where each experiment utilized mammary epithelial cells isolated from a different mouse per diet group and performed in 8–16 replicates. * P≤ 0.001 relative to CAS. (c) Schematic diagram for in vitro analysis of FACS-sorted MECs from CAS- and SPI-fed Wnt1-Tg mice. Basal (CD29^hiCD24⁺) and luminal (CD29^loCD24⁺) epithelial subpopulations were subjected to colony-forming and mammosphere-forming assays. (d) Diet did not influence the colony-forming ability of luminal epithelial subpopulations. Data (mean ± SEM) are from three independent experiments, with each experiment representing an individual mouse and carried out in triplicates. (e) Dietary exposure to SPI enhanced the clonogenic activity of MaSC-enriched (CD29^hiCD24⁺Lin) epithelial subpopulation. Data (mean ± SEM) are from three independent experiments, with each experiment representing an individual mouse and carried out in triplicates. *P≤0.05 by t-test. (f) Representative colonies formed in matrigel (CFU) and mammospheres formed under non-adherent conditions (MFU) for FACS-sorted MaSC-enriched (CD29^hiCD24⁺Lin) epithelial subpopulation from CAS-and SPI-fed Wnt1-Tg mice are shown. Scale bar, 250 µm. (g) Mammosphere-forming ability of MaSC-enriched (CD29^hiCD24⁺Lin) epithelial subpopulation is reduced with dietary SPI exposure. Data (mean ± SEM) are from three independent experiments, with each experiment representing an individual mouse and carried out in triplicates. * P≤ 0.05, by t-test.