Figure 2. Responses of AnD3 and AnD5 cells to fulvestrant and CAF-CM.

(A) Growth activities of AnD3, AnD5 and MCF-7 cells in the presence of different concentrations of fulvestrant or in the absence of fulvestrant. Cell mass was determined after six days of growth by a luciferase-based assay from at least three independent experiments. For statistical analyses by student t-test, growth of AnD3 or AnD5 cells was compared with growth of MCF-7 parental cells for each condition. (B) Colony growth assay. Growth assay was done at low density which forces cells to form individual colonies. After six days of growth, colonies sizes were measured and, for each cell line, the relative average colony size (in the presence vs. absence of fulvestrant) was determined from three independent experiments. (C) Effect of 3-day-exposure of MCF-7, AnD3 and AnD5 cells to CAF-CM (20%) on the expression of selected proteins as determined by Western blot analysis. Protein loading was checked by Commassie Blue. PM, CE and NE denote plasma membrane, cytosolic and nuclear extract, respectively. (D) Effect of 6-day-exposure of CAF-CM (20%) on colony size of MCF-7, AnD3 and AnD5 cells in the presence or absence of 1µM fulvestrant. For each condition, the sizes of 50 colonies were measured. Statistical analyses were performed by Mann-Whitney-U-test. (E) Effect of increasing concentrations of CAF-CM (given as percentage added to the medium) on cell aggregation after three days of incubation in 3D suspension culture. (F) Wound healing assay in the presence and absence of 20% CAF-CM in the medium. Gap closure was measured on day 1, 2 and 3 after the wound had been set.