Cell lines

Human PC cell lines LNCaP and PC-3 obtained from JCRB Cell Bank (Osaka, Japan), and 22Rv1 purchased from The European Collection of Authenticated Cell Cultures were maintained in RPMI-1640 (FUJIFILM Wako Pure Chemical, Osaka, Japan) containing penicillin, streptomycin and 10% fetal bovine serum (Equitech-Bio, Kerrville, TX). The docetaxel-resistant 22Rv1 cell line, 22Rv1DR, was previously described18. All cell culture experiments were performed using cells within less than 20 passages except for PC-3PRF cells stably transfected with Tet-on tetracycline-inducible perforin expression vector and docetaxel-resistant 22Rv1DR cells.

Docetaxel was purchased from Selleckchem (Houston, TX, USA).

The perforin expression vector for Tet-On system (pT-Rex-DEST30-perforin) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The pcDNA6/TR regulatory vector (Thermo Fisher Scientific) and pT-Rex-DEST30-perforin vector were transfected to PC-3 cells using Lipofectamine 2000 (Thermo Fisher Scientific). Transfected cells were selected under 500g/ml G418 and 10g/ml Blasticidin (Thermo Fisher Scientific). Perforin was induced by 1g/ml of tetracycline. The human PSA promoter-driven perforin expression vector (pDRIVEperforin-psa-hpsa) was purchased from InvivoGen (San Diego, CA, USA).

Whole cell lysates were harvested and lysed in RIPA buffer containing the protease inhibitor cocktail (Sigma-Aldrich St. Louis, MO, USA). Western blot analysis was performed as described previously19. The anti-PSA and anti--actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The immunoreactive proteins were detected using horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signaling Technology) and ImmunoStar (FUJIFILM Wako Pure Chemical).

Perforin expression in conditioned medium, mouse serum and harvested xenograft tumors was measured by human perforin ELISA kit according to manufacturers instruction (Abcam, Cambridge, UK).

SS-cleavable and pH-activated lipid-like material (ssPalmM), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) were purchased from NOF Corporation (Tokyo, Japan). Encapsulation of plasmid vector in lipid nano-particles was conducted according to a previous report20. First, plasmid DNA and protamine solutions (0.3mg/mL and 0.144mg/mL) were prepared in 10mM HEPES buffer (pH5.3). Plasmid DNA/protamine core particle was prepared by the drop-wise addition of 1mL of the protamine solution into the 1mL of the DNA solution with vortexing. Liposome was composed of ssPalmM, DOPE, cholesterol and DMG-PEG2000 in a molar ratio of 3:4:3:0.5. Lipids (3.3mol of total lipids) were dissolved in 2mL of ethanol and the lipid solution (2mL) was rapidly diluted with an equal volume of the plasmid DNA/protamine core particle suspension with vortexing. The solution was further diluted with 36mL of 10mM HEPES (pH 5.3) to obtain 5% ethanol(v/v) concentration. The diluted solutions were concentrated to ten times by Amicon 8400 ultrafiltration stirred cell with a Biomax membrane (Merck Millipore, Allen, TX, USA) following further serial ultrafiltration with 100mM HEPES (pH 7.4) and 10mM HEPES (pH 7.4) using Amicon 8050 ultrafiltration stirred cell with a Biomax membrane. Finally, the liposome solution was filtrated by 0.45m of pore size Millex HV (Merck Millipore).

Liposome concentration was measured as total cholesterol concentration in the presence of sodium dodecyl sulfate using a Cholesterol E test Wako (FUJIFILM Wako Pure Chemical) and the total amount of fatty acids was calculated based of the molar ratio of each lipid. DNA concentration in liposomes was determined using Quant-iT Picogreen dsDNA Assay Kit (Thermo Fisher Scientific) in the presence of Triton X-100. Particle size and -potential were measured at 25C using Zetasizer Nano-S90 (Malvern Panalytical, Worcestershire, UK) after 50 times dilution of samples with distilled water.

This study was approved by the Medical Review Board of Gifu University, Graduate School of Medicine (No. 2018219). A written informed consent was obtained from participants and blood was collected from male volunteers without clinically detectable cancer. All methods were performed in accordance with the relevant guidelines and regulations in compliance with the Declaration of Helsinki. Human PBMCs were isolated by Ficoll-Paque density gradient centrifugation according to the manufacturers instructions (Amersham Biosciences, Piscataway, NJ).

Cells were seeded on 96-well plates. Twenty-four h after seeding, agents with or without PBMCs were added. Cell viability was determined using WST-1 assay kit (Roche Diagnostics, Mannheim, Germany). The mean value obtained from PBMCs alone was deducted from the values obtained from co-culture of prostate cancer cells and PBMCs.

All animal experiments were approved by the Gifu University Animal Experiment Approval Committee (No. 2019116) and carried out in accordance with the approved guidelines. This study is compliant with the ARRIVE guidelines. Six-week-old male athymic nude mice (BALB/cSlc-nu/nu) were purchased from Japan SLC, Inc. (Shizuoka, Japan). A suspension of 22Rv1DR cells (1107) cells in PBS was mixed with Matrigel (1:1) in a final volume of 0.2mL. The mixture was subcutaneously injected to generate tumors. Two weeks after the injection, tumor volume was measured and mice were randomly assigned to 2 groups (n=5). Agents were intravenously administrated via tale vein. The tumor volume and body weight were monitored and measured once a week. Four weeks after treatment, mice were sacrificed and the resected tumors were weighed.

Statistical analysis was performed using Graph Pad Prism 7 version 7.03 (Graph Pad Software, CA, USA). Comparison of 2 groups was made using t-test or MannWhitney U test. Comparison among 4 groups was made using one-way ANOVA with Tukeys post hoc for multiple comparisons. Differences were considered significant if p<0.05.

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