Clonal heterogeneity of PC3 cells. A. Morphologies of holoclones, meroclones and paraclones isolated from the prostate cancer cell line of PC3. Additional examples are shown in Additional file 2. B. The frequency of each type of cell clone as determined in three replicate 96-well experiments (mean ± SD) based on a total of 68 single cell-derived colonies. C. PC3-derived holoclones showed chemoresistance to 4OOH-cyclophosphamide when compared to PC3-derived paraclones. Cells were treated with 4-OOH-CPA at the indicated concentrations for 4 hr, and cell survival was determined after continued culture for 4 days in drug-free medium. Cell survival data shown are mean ± SE values for a set of 4 holoclones and 3 paraclones. D. Self-renewal spheroid formation assay. Parental PC3 cells were seeded at low density in serum-free medium on low attachment plates (a), and a small number of cells formed individual spheres by day 7 (b), which grew in size by day 12 (c). Holoclones grew well and formed spheres with a 3-dimensional structure, as shown here for holoclones 2H10 and 2G7, on day 12 (d, e). Meroclones formed spheres with lower efficiency, shown for meroclones 2G5 and 1E5 (f, g). Paraclones cannot grow and form spheres, as shown for paraclones 2B6 and 1C4 (h, i).