Fig. 6

Analysis of sphere formation capacity and expression of prostate cancer stem-like cell (PCSC)-associated biomarkers in three-dimensional culture prostatospheres in serial passages on agar-coated dishes. a Representative images of DU145 cells grown under the adherent two-dimensional (2D) culture condition, their corresponding prostatospheres formed upon serial passages P1–P3 on the same agar-coated dishes and 2D culture of re-adherent cells from the last P3-passage prostatospheres. Bars: 200 μm. b Sphere formation assay of DU145-derived prostatospheres in serial passages P1–P3. Single-cell suspensions (500 cells) of P1–P3 prostatospheres were seeded in Matrigel-coated 48-well plates and grown for 1–3 weeks followed by enumeration of spheres. Results showed that there was a significant increase of the number of spheres scored in late passages as compared with early passage. c Reverse transcription quantitative polymerase chain reaction analysis of PCSC-associated markers expressed in serial passaged DU145 spheres and their corresponding adherent 2D cultured cells. Results showed that the DU145-derived prostatospheres in serial passages expressed significantly higher levels of PCSC-associated markers but lower levels of CD24 as compared with 2D culture adherent cells and re-adherent cells from the last-passage spheres. *P <0.05, compared with corresponding adherent cells. d Fluorescent (upper) and fluorescent overlay-phase contrast (lower) images of DU145-SORE6 spheres regrown under adherent 2D culture conditions for 48 h. Bars: 200 μm