Fig. 6: The ALDH⁺CD44⁺CXCR4⁺CD24⁺ subpopulation in human PCa tissues.

a Western blot analysis was performed to determine the protein expressions of PSA, HOXB9, ALDH, CD44, CXCR4 and CD24 in the controls (PCa tissue with Gleason score 6), para-carcinoma (2 mm away from PCa tissue), initial PCa tissue (derived from PCa at first diagnosis via radical prostatectomy) and refractory PCa tissue (derived after recurrence), respectively. β-actin was used as an internal control. b Quantification of (a). *P < 0.05 vs. initial PCa tissues. (n = 6). c Human PCa tissue was subcutaneously implanted into NOD-SCID mice to establish a patient-derived xenograft (PDX) model. Subsets of cells (as indicated) were derived from the PDX model and seeded in 96-well plates (1 × 10⁴ cells/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. d Subsets of cells (as indicated) were derived from the PDX model and seeded in 96-well plates (1 × 10⁴ cells/well). Cells were treated with different chemotherapeutic drugs, as indicated, for 72 h. Then, a WST-1 proliferation assay was performed. The absorbance was measured at 450 nm using a microplate reader. ^#P < 0.05 vs. bulk cells, as well as ALDH^–CD44^–CXCR4^–CD24^– -cells (n = 12)^{; Δ}P < 0.05 vs. ALDH⁺CD44⁺CXCR4⁺CD24⁺-cells (n = 12). e ALDH⁺ CD44⁺ CXCR4⁺ CD24⁺-cells were isolated from PDX tumours at 8 weeks post implantation, before they were subject to HOXB9 knockdown. Then cells at 1 × 10⁶ (together with 1 × 10⁶ HS-5 cells to facilitate tumour formation) were subcutaneously injected into the NOD/SCID mouse. Derived tumours at 12 weeks post injection were subject to Western blotting analysis of the expression of APLN, HIF-1α, MSH6, GSTT2, metallothionein, ABCG2 and Bcl-2). GAPDH was used as an internal control. f Western blotting analysis of the expression of epithelial–mesenchymal transition-associated genes (Slug, Vimentin and E-cadherin). β-actin was used as an internal control. (G-I) CD44⁺-, CD44⁺ α2β1⁺-, ALDH⁺ CD44⁺ α2^β1⁺- and ALDH⁺ CD44⁺ CXCR4⁺ CD24^+-PCa cells were obtained from orthotopic CWR22 tumours by FACS using the respective antibodies, whereas HOXB9-silenced ALDH⁺ CD44⁺ CXCR4⁺ CD24⁺-PCa cells were derived from ALDH⁺ CD44⁺ CXCR4⁺ CD24⁺-cells. Orthotopic tumour models were established using these subsets of cells, respectively. Mice were sacrificed at week 14 after inoculation. The time for developing a palpable tumour (g), tumour weights (h) and the number of metastatic foci (i) were recorded (n = 12). ^ΔP < 0.05 vs. ALDH⁺ CD44⁺ CXCR4⁺ CD24⁺ cells-based implantation.