Cytoplasmic-only Expression of Maspin Predicts Unfavorable Prognosis in Patients With Pancreatic Ductal Adenocarcinoma

Patients and Methods

Patients and tumor specimens. A total of 92 consecutive patients who underwent radical surgery between May 2006 and March 2017 at Tottori University Hospital (Yonago, Japan) for PDAC were included in the analysis. The protocol was approved by the Ethics Committee of Tottori University (approval number: 18A148), and all patients provided written informed consent for pathological analysis. All clinicopathological parameters and laboratory data of all patients were extracted from their electronic medical records. Patients’ characteristics, including surgical and peri-operative parameters, were retrospectively analyzed. Pathological findings were classified according to the 7^th edition of TNM classification.

Immunohistochemistry. All specimens were fixed in 10% neutral buffered formalin and embedded in paraffin, and a representative block was selected from each case. Sections (4 μm-thick) were deparaffinized, blocked to inhibit endogenous peroxidase activity, and then pre-treated in citrate buffer (0.01 M, pH 6.0) using a microwave oven for 20 min. We then performed immunohistochemical examination using a mouse monoclonal antibody for human maspin (clone G167-70, 1:500 dilution; BD Pharmingen, NJ, USA), as described elsewhere (15).

Evaluation of immunohistochemical findings. Cells were defined as positive when strong cytoplasmic-only staining was identified. Strong staining was defined as a staining intensity equal to that in myoepithelial cells in normal breast tissue. Cases with positive cells accounting for more than 10% of the total tumor were considered maspin-positive, as previously described (16). The subcellular localization of maspin was classified into four categories, cytoplasmic-only, pancellular (combined nuclear and cytoplasmic), nuclear-only, and no staining. All slides were evaluated independently by E.U. and Y.U., who were blinded to the patient’ clinicopathological data.

Cell culture. PANC-1, MIA PaCa-2, and BxPC-3 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). PANC-1 and MIA PaCa-2 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂, whereas AsPC-1 and BxPC-3 cells were cultured in RPMI 1640 (Nissui Pharmaceutical) supplemented with 10% FBS at 37°C with 5% CO₂.

RNA extraction and quantitative polymerase chain reaction. Total RNA from PANC-1, MIA PaCa-2, AsPC-1 and BxPC-3 cells was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and reverse-transcribed into complementary DNA (cDNA) using a High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Gene expression levels were measured using the TaqMan Gene Expression Assays (Thermo Fisher Scientific) with the following gene-specific primers: GAPDH, human GAPDH endogenous control (Hs99999905_m1, Thermo Fisher Scientific) and maspin, TaqMan Gene Expression Assays (Hs00985285_m1, Thermo Fisher Scientific).

Western blot analysis. Cells were lysed in protein extraction buffer (cat. no. 28941279; GE Healthcare, Buckinghamshire, UK) containing protease inhibitors (cat. no. 80650123; GE Healthcare). Protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.2-μm polyvinylidene difluoride membranes (cat. no. 1704156; Bio-Rad Laboratories). Following incubation in 5% skim milk, the membranes were reacted with the primary antibodies. The primary antibodies used were mouse monoclonal anti-human maspin antibody (clone G167-70, 1:500 dilution; BD Pharmingen), rabbit monoclonal anti-GAPDH antibody (clone D16H11, 1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-HDAC1 antibody (clone 10E2, 1:1,000 dilution; Cell Signaling Technology) and anti-HSP90 antibody (clone C45G5, 1:1,000 dilution; Cell Signaling Technology). Then, horseradish peroxidase (HRP) conjugated anti-mouse IgG (cat. no. NA931; 1:3,000 dilution; GE Healthcare) or HRP-conjugated anti-rabbit IgG (cat. no. NA934, 1:4,000 dilution; GE Healthcare) were added to 5% skim milk. For AsPC-1 and BxPC-3 cells, subcellular protein fractionation was performed using a subcellular protein fractionation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The signals were visualized using the Immobilon Western chemiluminescent substrate (Millipore, Billerica, MA, USA) and quantified using the Image Quant LAS 4000 mini (GE Healthcare).

Immunofluorescence analysis. AsPC-1 and BxPC-3 cells were incubated for 24 h in 8-well chamber slides (Nalgen Nunc International, Rochester, NY, USA). After incubation, cells were fixed in 4% paraformaldehyde and permeabilized with methanol at 20°C for 5 min. Samples were blocked by 5% blocking buffer (BLOCK ACE; Megmilk Snow Brand, Sapporo, Japan) for 60 min at room temperature. Immunofluorescence analysis was performed using an anti-maspin primary antibody (clone G167-70, 1:200 dilution; BD Pharmingen) as described elsewhere (15).

Plasmid and siRNA transfection. AsPC-1 and BxPC-3 cells were seeded into 6-well plates and allowed to adhere overnight. For each well, 10 nM siRNA targeting maspin (Silencer Select siRNA, s10466, Thermo Fisher Scientific), siRNA targeting maspin (Silencer Select siRNA, s10468, Thermo Fisher Scientific), or control siRNA (Silencer Negative Control #1; Thermo Fisher Scientific) was used to transiently transfect cells using 9 μl Lipofectamine RNAiMAX (cat. no. 13778030, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Cell invasion assay. Cell invasion assays were performed using a BioCoat Matrigel invasion chamber (BD Biosciences, Bedford, MA, USA) according to the manufacturer’s protocol. Cells (AsPC-1: 2×10⁵; BxPC-3: 1×10⁵) transfected with siRNA were starved in serum-free medium for 24 h and seeded into the upper chambers of transwells on an 8.0 μm pore size membrane in 24-well plates. The lower chambers were filled with medium containing 10% FBS. After 24 h, non-invading cells were removed from the top of the filter with a cotton swab. The invading cells at the bottom of the filter were fixed with methanol for 10 min, stained with 0.2% crystal violet, and counted in five different fields of view using a microscope (ECLIPSE Ts2; Nikon, Tokyo, Japan) (magnification, 200×). The mean values of three independent experiments were used for statistical analysis.

Statistical analysis. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics; IBM Corporation, Armonk, NY, USA). Overall survival (OS) and recurrence-free survival (RFS) rates were calculated using the Kaplan–Meier method and compared using log-rank tests. OS was calculated from the date of surgery to the date of death or last visit. RFS was calculated from the date of surgery to the date of disease recurrence or last visit. Relative maspin mRNA expression was compared using one-way analysis of variance and the Tukey honest significant difference test. Differences in proliferation and invasion were evaluated using Student’s t-tests. All tests were considered significantly different when the p-value was less than 0.05. All continuous values are presented as means±standard deviations.