Figure 1

breast cancer cells form cancer stem cell-enriched mammospheres under serum-free culture conditions. A) Alterations in number, size and shape of mammospheres was observed over the course of seven days. Photographs were taken at Day 1 (i), Day 3 (ii), Day5 (iii) and Day 7 (iv). Images are representative of at least three randomly selected fields. Insets show specific structural features of MCF-7 mammospheres at various stages of their development. Images were captured under a light microscope at 100× magnification using a Nikon Coolpix 990 camera. Scale bars representative of 100 μ m. Note: Insets not set to scale. B) Sphere forming efficiency SFE was calculated as the number of mammospheres/spheres (average diameter = 100 μm) formed in 96 wells plated with a single cell divided by the original number of single cells seeded and expressed as a percentage. Error bars represent the standard error of the mean where n = 3. C) Flow cytometry histogram showing an increase in the CD44^high/CD24^low cell surface marker-bearing subpopulation between adherent and mammosphere-derived (anchorage-independent) cells generated using FlowJo software (Tree Star Inc.). D) Confocal microscopic analysis of anchorage-independent cells where Hoechst 33342 was used to stain nuclei (blue) while CD44 was stained with an allophycocyanin-conjugated antibody (purple). Images were captured using a Zeiss LSM510 Meta confocal microscope. Scale bars represent 20 μ m. E) Western analysis to assess Oct3/4 expression in mammospheres-derived cells compared to the bulk MCF-7 population. Western analyses were carried out on a total of 30 μg of protein derived from whole cell lysates after cultivation in either regular adherent or serum-free anchorage-independent growth conditions. In each case, lysates were probed for histone H3 as a loading control.