Tumorspheres derived from prostate cancer cells possess chemoresistant and cancer stem cell properties

Abstract

Purpose: Prostate cancer (PCa) becomes lethal when cancer cells develop into castration-resistant PCa, which remains incurable because of the poor understanding of their cell origin and characteristics. We aim to investigate the potential role of cancer stem cells (CSCs) in PCa progression.

Methods: Human PCa cell lines (LNCaP, 22RV1, DU145 and PC-3) were plated in serum-free suspension culture system allowed for tumorsphere forming. To evaluate the CSC characteristics of tumorspheres, the self-renewal, chemoresistance, tumorigenicity of the PCa tumorsphere cells, and the expression levels of stemness-related proteins in the PCa tumorsphere cells were assessed, comparing with the parental adherent cells.

Results: Tumorsphere cells from PCa cell lines displayed enhanced self-renewal, chemoresistance and tumor-initiating capacity when compared with the adherent cells. Additionally, these cells overexpressed CSC marker CD44. Also, the tumorsphere cells expressed high levels of "stemness" genes Gli1, ABCG2 and Bmi-1.

Conclusions: Collectively, these data demonstrated that tumorspheres derived from PCa cells possess chemoresistant and CSC properties. Our study suggests that the identification of PCa CSCs could provide new insight into the lethal phenotype of PCa and therapeutic implications.

Fig. 1

PCa cells can form the self-renewal tumorspheres. a Schematic diagram of expanding the self-renewal population of PCa tumorspheres. b Typical PCa tumorspheres generated from a single cell after 7 and 14 days under non-adherent culture condition. Scale bar = 100 μm. c PCa tumorsphere formation number at the initial passage (P0). *P < 0.05 versus LNCaP cells

Fig. 2

PCa tumorsphere cells have a higher self-renewal capacity in vitro. a Tumorspheres from adherent cells and tumorsphere cells were photographed at day 14. Scale bar = 100 μm. b The number of tumorspheres was counted under microscope. *P < 0.05 versus adherent cells. c Percentage of tumorspheres with diameters <100, 100–150 or >150 μm. ACs, adherent cells; TCs, tumorsphere cells

Fig. 3

PCa TCs display increased colony-forming capacity in vitro. a 2,000 cells were plated onto 60-mm dishes. After 14 days, cells were stained by crystal violet and colony was counted. The representative photography was shown. b The number of colonies was counted and plotted. *P < 0.05 versus ACs. c Clonogenic assays. The single cells were plated at a density of 1,000 cells/well in 6-well plate. Photographs were taken at day 14 after plating. Scale bar = 100 μm. d The number of colonies was calculated and plotted. *P < 0.05 versus ACs

Fig. 4

PCa TCs exhibit higher tumor-initiating capacity in vivo. a Tumors derived from DU145 cells grown in nude mice 40 days after S.C. injection. Each group has 3 nude mice. b Size of S.C. tumor following injection of DU145 cells. ^#Tumor latency time for TCs is 10 days and that for ACs is 25 days. c Tumorspheres derived from DU145 TCs xenograft. Scale bar = 100 μm. d S.C. tumor growth in nude mice at day 25 after injection of TCs (1 × 10⁴) derived from DU145 TCs xenograft

Fig. 5

PCa TCs display higher resistance to chemotherapeutic drugs. a The cells were plated then incubated with 20 ng/ml doxorubicin, 10 μg/ml docetaxel or 5 nM vinblastine for 48 h. The proportion of viable cells relative to the control (non-treatment, NT) was shown. *P < 0.05 versus ACs. b ABCG2 expression was determined by Western blotting. ABCG2 levels from each cell were normalized with β-actin levels. *P < 0.05 versus ACs

Fig. 6

PCa TCs overexpress CD44. a Cell cytometrical analysis of CD44 expression in PCa cells. The respective isotype control was shown in dotted line. b CD44 expression was confirmed by Western blotting. Protein levels from each cell were normalized with β-actin levels. *P < 0.05 versus ACs. c The tumorsphere-forming assay. FACS was used to sort DU145 cells into CD44-positive and CD44-negative subpopulation. The number of tumorspheres was counted and plotted. *P < 0.05 versus CD44⁻ DU145 cells. Scale bar = 100 μm

Fig. 7

PCa TCs overexpress “stemness” genes. a The “stemness” genes Gli1 and Bmi-1 were analyzed by Western blotting. b Protein levels from each cell were normalized with β-actin levels. *P < 0.05 versus ACs

Fig. 8

Specific blockage of Gli1 eliminates the self-renewal capacity of PCa TCs. a Gli1 expression was determined by Western blotting. Protein levels from each cell were normalized with β-actin levels. b Tumorsphere-forming assay. The cells were treated with 10 μM GANT61 for 7 days. The representative photographs were shown. The number of tumorspheres was counted and plotted.*P < 0.05 versus the control. c Clonal assay. The cells were treated with 10 μM GANT61 for 14 days. The representative photographs were shown. The number of clones was counted and plotted. *P < 0.05 versus the control