Docetaxel

Doxorubicin (Sigma, St. Louis, USA), 5-aza-2′-deoxycytidine (decitabine/5-AZA-CdR, Sigma), azacitidine (Celgene, Summit, NJ, USA), cisplatin (Sigma), gemcitabine (Sigma), etoposide (Sigma), docetaxel (Sigma), paclitaxel (Sigma), vinblastine (Sigma), panobinostat (Sigma), vorinostat (Sigma) and bortezomib (Sigma) were added from a stock solution to the 10% serum-containing RPMI medium (Gibco, Waltham, MA, USA). Doxorubicin, etoposide, docetaxel, paclitaxel, vinblastine, panobinostat, vorinostat and bortezomib were dissolved in dimethyl sulfoxide (DMSO; Sigma), cisplatin was dissolved in 10% DMSO (Sigma) in 10% serum-containing RPMI, and 5-AZA-CdR, azacitidine and gemcitabine were dissolved in distilled water. Dose calculation was based on human dosages and previous publications [10 (link)].

Tumor tissues were collected at room temperature and cut in ~1 mm³ pieces with forceps and scissors. Next, the tissue slices were placed on nitrocellulose filter inserts (6- well filter inserts, pore size of 4 μm, Corning Costar) positioned in 6-well culture plates. The culture plates were filled with 1 ml of culture medium (13 (link)). Prostate cancer tissues were cultured in DMEM Glutamax, 4.5 g/L D-Glucose with Pyruvate (Gibco) supplemented with 10% FCII or 10% FCS and 1% penicillin-streptomycin. Bladder cancer tissues were maintained in EMEM (ATCC) with 10% FCS and 1% penicillin-streptomycin.
The tissue slices were cultured in an oxygenated and sealed container system containing 95% O₂ in the presence of docetaxel, gemcitabine, or vehicle solution. Previous in vitro viability assays have indicated the sensitivity of PC-3M-Pro4 and UM-UC3 cells for docetaxel (IC₅₀ = 1.9 nM) and gemcitabine (IC₅₀ = 17.7 nM), respectively (Supplementary Figures 1A,B). The tissue slices were treated with 0.3 and 3 nM docetaxel (Sigma-Aldrich), 10 and 100 nM gemcitabine (kindly provided by the Leiden University Medical Center's pharmacy) or vehicle solution (100% ethanol 3300 × diluted in medium for docetaxel, 5000 × diluted in medium for gemcitabine). After culturing the tissues, the tissue was harvested and processed for histological analyses (Figure 1A).

To evaluate the immune phenotype, PC3 and PC3RR cells were detached with 0.25% trypsin EDTA (Sigma), washed with PBS and incubated with bv421-conjugated Integrin alpha-2 antibody (BD Bioscience, San Jose, CA, USA) in PBS/0.1% BSA (Sigma) for 30 min on ice prior to flow cytometric analysis. Propidium iodide (PI) solution (Sigma-Aldrich P4864) was added to exclude dead cells. After washing, cells were assayed using a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using FCS5 Express Software (De Novo Software, Glendale, CA, USA). For cell cycle analysis, PCa and PCaRR cells were fixed with 70% ethanol, washed three times with PBS and stained for 3 h at room temperature with PI, then analyzed using flow cytometry.
To assess the effects of Docetaxel treatment, PC3, PC3RR, DU-145 and DU-145RR cells were seeded 9 × 10⁴/well and 6 × 10⁴/well, respectively, in 12-well plates, then treated with different concentrations (from 10 nM to 100 nM) of Docetaxel (Sigma-Aldrich) for 24 and 48 h. Then, apoptosis/necrosis were assayed by using PI/Annexin Pacific Blue staining (Thermo Fisher Scientific, Rockford, IL, USA) and evaluated using flow cytometry.

HEK293T, human prostate adenocarcinoma LNCaP, Du145 and PC3 cells lines were purchased from the American Type Culture Collection, Manassas, VA, USA. The Docetaxel resistant cell lines Du145 and PC3 were developed as previously described [31 (link)]. HEK293T were cultured in DMEM (BioWest, Nuaillé, France), Docetaxel sentitive and resistant Du145 cells and LNCaP cells were cultured in RPMI-1640 (BioWest) and PC3 DS and PC3 DR in F12K nutrient mixture medium (Thermo Fisher Scientific, Waltham, MA, USA). Culture media were supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 0.1 mM non-essential amino acids (all from BioWest). Docetaxel resistant cell lines were maintained with 2.5 nM of Docetaxel (Sigma-Aldrich, St. Louis, MO). Antibody to PTOV1 was produced and purified as previously described [8 (link), 9 (link)]. Additional antibodies were obtained from: goat anti-actin (I-19) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-HA.11 (16B12) (Covance, MA); mouse anti-Cyclin B1(V152) (Abcam, Cambridge, MA); rabbit anti-PARP1 (H-250) (Santa Cruz Biotechnology). Lentiviral vectors carrying short-hairpin RNA (shRNA, TRCN0000143905, TRCN0000140104 and TRCN0000139737) to PTOV1 were obtained from Sigma.

Human PCa 22Rv1 cells were obtained from the American Type Cell Culture Collection (Manassas, VA, USA). The cells were authenticated with short-tandem repeat analysis (Bio-Synthesis, Lewisville, TX, USA). The docetaxel-resistant subline of 22Rv1 cell (22Rv1-DR), which has a characteristic of androgen- and ligand-independent growth [20 (link), 21 (link)], was established in the presence of increasing concentrations of docetaxel (Sigma-Aldrich, St. Louis, MO, USA) up to the final concentration of 5 nM, which is the IC₅₀ concentration in our pilot study. Development of the drug-resistant cell line took ≥4 months and further studies using sublines cultured for ≥4 months that were based on the results of MDR1 expression in the cells were performed. The cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) in a 5% CO₂ humidified incubator at 37 °C and passaged for 3–4 days in a fresh medium to achieve approximately 80% confluency. SiYAP1 (SI02662954) and negative control siRNA (AllStars Negative Control siRNA) were purchased from Qiagen (Valencia, CA). Transfections of siRNAs were performed by using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s procedure.