Figure 1. Ascites enriches for CSCs.

A. ID8 cells were cultured for the indicated times in medium or 50% acellular ascites from ID8 bearing mice. 6×10³ cells/well were seeded in a primary sphere assay, and then harvested, trypsinized and passaged to form secondary spheres. Y axis represents spheres ≥ 50µm +/− SD. B. Competition between ascites pre-treated and untreated ID8-GFP cells in sphere formation. Left: ID8-GFP cells were pre-treated with acellular ascites derived from tumor-bearing mice for 7 days and then mixed 1:1 with untreated ID8 cells. The mixture was seeded into a sphere assay (primary spheres). On day 7, primary spheres were trypsinized and seeded into a sphere assay (secondary spheres). Pictures were taken from 5 random fields. A representative field is shown for primary spheres at 2.5×, secondary spheres at 2.5×, primary spheres at 20× and secondary spheres at 20×. Right: Quantification of % ID8-GFP and ID8 cells in 5 fields at 20× magnification from primary and secondary sphere assays. Red arrows indicate GFP+ spheres and white arrows indicate GFP-spheres. C. Culturing in vivo ascites cells in vitro for 7 days (re-cultured) (left panel) or re-culturing ID8 cells pre-treated with ascites for 7 days (ascites treated 7 days) in culture for 9 days (ascites off 9 days) (right panel) decreases their sphere-forming ability. Error bars represent SD from 3 trials in triplicate. D. After 7 day ascites treatment, 34.5% of ID8 cells became Annexin V positive, while 7.7% ID8 cells were positive in normal culture. E. ID8 cells were labeled with DiD on day 0 and split into two groups, receiving either medium or 50% ascites for 7 days. The majority of ascites treated ID8 cells maintained DiD label on day 7, while most ID8 cells in medium lost the dye. F–G. OvCar3 or ES2 cells were pre-treated with 50% ascites from either of two ovarian cancer patients (Ov476, Ov480) for 7 days and sphere number was counted. Error bars represent SD from 3 different trials in triplicate for this figure.