Fig. 5: Castration resistance of ALDH⁺CD44⁺CXCR4⁺CD24⁺-PCa cells.

a A microarray analysis was performed to identify the DEGs (P < 0.05, fold change ≥2) between ALDH⁺CD44⁺CXCR4⁺CD24⁺- and ALDH⁻CD44⁻CXCR4⁻CD24⁻-CWR22 cells. The pie chart illustrates how the DEGs were classified. b A heatmap obtained by hierarchical clustering shows the altered genes (>2.0-fold) between these two subsets of cells. Purified ALDH⁺CD44⁺CXCR4⁺CD24⁺- and ALDH⁻CD44⁻CXCR4⁻CD24⁻-CWR22 cells were subcutaneously implanted into male NOD-SCID mice. When tumours became palpable, the mice were treated with castration and bicalutamide, in combination. Tumour weights (c), tumour volumes (d) and serum PSA levels (e) were determined at different time points as indicated, respectively. *P < 0.05 vs. ALDH⁺ CD44⁺ CXCR4⁺ CD24⁺ (control); ^#P < 0^.05 vs. ALDH⁻ CD44⁻ CXCR4⁻ CD24⁻ (control). f CWR22 xenograft^-derived bulk cells, ALDH⁻CD44⁻CXCR4⁻CD24⁻-, ALDH⁺CD44⁺-, and ALDH⁺CD44⁺CXCR4⁺CD24⁺-cells were plated in 96-well plates at a density of 1 × 10⁴/well and treated with different anti-androgens (as indicated) and chemotherapeutic agents, with 0.2% DMSO and 0.5% H₂O₂ were used as negative and positive controls, respectively. After 48 h of treatment, cells were incubated with alamarBlue solution for 4 h, and cell viability was measured with excitation wavelength at 530–560 nm and emission wavelength at 590 nm using a TECAN Infinite 200 PRO microplate reader. *P < 0.05 vs. ALDH⁻ CD44⁻ CXCR4⁻ CD24⁻ cells as well as bulk culture. g Western blot analysis was conducted to show time-dependent changes in phenotypic molecules and epithelial–mesenchymal transition-associated genes in ALDH⁻ CD44⁻ CXCR4⁻ CD24⁻ cells derived from CWR22 xenograft in the presence of CDSS and bicalutamide (10 µM).