Journal: British Journal of Cancer

Article Title: Insights into homeobox B9: a propeller for metastasis in dormant prostate cancer progenitor cells

doi: 10.1038/s41416-021-01482-y

Figure Lengend Snippet: The endogenous expression of HOXB9 protein was detected in para-carcinoma and PCa tissues with Gleason scores 7–9 from 32 patients with primary PCa, as well as prostate tissues from patients with benign prostate hyperplasia, using immunofluorescent staining ( a , a1–a5), immunohistochemical staining ( a , a6–a10) and western blot assay ( b ), respectively. a Morphological staining for HOXB9. a1–a5, Immunofluorescence study on HOXB9 (red) with nucleus counterstained by DAPI (blue); a6–a10, Immunohistochemical staining for HOXB9 (pink colour in the cytoplasm), as well as P63 (brown colour in the nucleus) and cytokeratin 18 (brown colour in the cytoplasm), with nucleus counterstained by hematoxylin (blue). a1–a10, representative images derived from para-carcinoma tissue (a1, a6), benign hyperplasia (a2, a7), prostate cancer tissue at Gleason score 7 (a3, a8), 8 (a4, a9) and 9 (a5, a10), respectively. Scale bars, 20 µm for a1–a5, 100 µm for a6–a10. b Western blotting for HOXB9 in tissues as indicated (upper panel) and quantitative analysis (lower panel, n = 8 for each group). The positive control refers to breast cancer tissue derived from a breast cancer patient at the clinical stage of T4N2M0. c mRNA expression of HOXB9 was found to positively correlate with the Gleason score and negatively correlate with survival years after operation. Correlation analysis between HOXB9 expression levels and developmental stages of prostate cancer as well as prognosis suggested that HOXB9 mRNA levels were positively correlated with prostate cancer Gleason scores ( n = 12, r = 0.918; P < 0.01), and negatively correlated with prognostic survival years after radical prostatectomy ( n = 12, r = -0.917; P < 0.01). d Western blotting analysis on HOXB9, indicating the expression of HOXB9 is upregulated in the orthotopic vs subcutaneous tumours originated by LNCaP, DU154, LAPC4 and LAPC9 cells at 12 weeks post injection. Negative ctrl, negative controls (in which lane-specific antibodies were replaced with saline); positive ctrl, positive controls derived from human prostate cancer tissue of Gleason 8 after prostatectomy.

Article Snippet: The vectors expressing HOXB9 shRNA (5′-CCC TTC AAT TTG TAG ACT CTT-3′ and 5′-CTC CTC AAT CTG AGT GAG AGA-3′; ThermoFisher Scientific) and CD44 (5′-GAC CTC TGC AAG GCT TTC AAT-3′ and 5′-ATT GAA AGC CTT GCA GAG GTC-3′; Santa Cruz Biotechnology) were transduced into Du-145 cells using FuGENE 6 (Roche Applied Science, Indianapolis, IN, USA) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), respectively.

Techniques: Expressing, Staining, Immunohistochemical staining, Western Blot, Immunofluorescence, Derivative Assay, Positive Control, Injection, Saline

Journal: British Journal of Cancer

Article Title: Insights into homeobox B9: a propeller for metastasis in dormant prostate cancer progenitor cells

doi: 10.1038/s41416-021-01482-y

Figure Lengend Snippet: Du-145-GFP cells were stably transfected with myc-coupled vectors expressing HOXB9. a Semi-quantitative PCR was carried out to determine the mRNA expression of HOXB9, TGFβ2, CD44, MMP9, CD24, TGFβ1, Smad1, Smad2, osteopontin (SPP1) and CD133. GAPDH was used as an internal control. b Western blot analysis was performed to determine the protein levels of total Smad1, phospho (p)-Smad1, Smad2, p-Smad2, HOXB9, SPP1 and MMP9 in HOXB9-overexpressed Du-145-GFP cells, in the absence or presence of TGFβ inhibitor SD208 (5 μM) for 2 h. β-actin was used as an internal control. c HOXB9, TGFβ2, CD44, CD24 and CD133 protein levels were determined in Du-145-GFP cells with HOXB9 knockdown. d HOXB9, TGFβ2, total Smad2, p-Smad2 and CD44 protein levels were determined in Du-145-GFP cells with CD44 knockdown. An empty vector was used as a negative control.

Article Snippet: The vectors expressing HOXB9 shRNA (5′-CCC TTC AAT TTG TAG ACT CTT-3′ and 5′-CTC CTC AAT CTG AGT GAG AGA-3′; ThermoFisher Scientific) and CD44 (5′-GAC CTC TGC AAG GCT TTC AAT-3′ and 5′-ATT GAA AGC CTT GCA GAG GTC-3′; Santa Cruz Biotechnology) were transduced into Du-145 cells using FuGENE 6 (Roche Applied Science, Indianapolis, IN, USA) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), respectively.

Techniques: Stable Transfection, Transfection, Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Knockdown, Plasmid Preparation, Negative Control

Journal: British Journal of Cancer

Article Title: Insights into homeobox B9: a propeller for metastasis in dormant prostate cancer progenitor cells

doi: 10.1038/s41416-021-01482-y

Figure Lengend Snippet: 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 × 10 4 cells/well) and treated with different anti-androgens (as indicated) and chemotherapeutic agents, with 0.2% DMSO and 0.5% H 2 O 2 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 × 10 4 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 × 10 6 (together with 1 × 10 6 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 β 1 + - 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). Δ P < 0.05 vs. ALDH + CD44 + CXCR4 + CD24 + cells-based implantation.

Article Snippet: The vectors expressing HOXB9 shRNA (5′-CCC TTC AAT TTG TAG ACT CTT-3′ and 5′-CTC CTC AAT CTG AGT GAG AGA-3′; ThermoFisher Scientific) and CD44 (5′-GAC CTC TGC AAG GCT TTC AAT-3′ and 5′-ATT GAA AGC CTT GCA GAG GTC-3′; Santa Cruz Biotechnology) were transduced into Du-145 cells using FuGENE 6 (Roche Applied Science, Indianapolis, IN, USA) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), respectively.

Techniques: Western Blot, Derivative Assay, Biomarker Discovery, Control, Incubation, Proliferation Assay, Isolation, Knockdown, Injection, Expressing