Ethics Statement
The study was approved by the Institutional Review Board for Studies in Humans at Osaka University (approval number: 15144-6). ). All assays were performed in accordance with
the committee’s guidelines and regulations.
Cell lines and culture conditions.
The cell culture and quality maintenance techniques have been previously described.13,14 The human pancreatic cancer cell line Panc-1 was obtained from American Type Culture Collection (Manassas, VA). Cell lines were cultured in Dulbecco's modified Eagle’s medium (DMEM, 08456–36; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA ). 100 U/mL penicillin, and 100 mg/mL streptomycin at 37C in a humidified incubator with 5% CO2. All experiments were performed with cells passaged <8 times.
Transduction of the degron reporter into pancreatic cancer cells.
The low-proteasome activity cell (LPAC) isolation system was established by engineering cells that stably expressed ZsGreen fused to the carboxyl terminal degron of ornithine decarboxylase (ODC), as previously described.15 The degron sequence of ODC is directly degraded by proteasomes. Consequently, cells with low proteasome activity accumulate the fluorescent fusion protein and can be detected by fluorescent microscopy or flow cytometry (FITC channel).
Flow cytometry.
We used FACSAria II (BD Biosciences) for cell sorting and FACSDiva software (BD Biosciences) for analysis. Cells were washed with PBS containing 2% FBS, and then incubated with the primary antibody (Ab), anti-CD44v9 (Cosmo Bio, Tokyo, Japan) at 4 °C for 20 min. To detect CD44v9, PE mouse anti-rat IgG2a (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used as a secondary Ab. Degron+, cells were detected using Cell Sorter SH800Z (Sony Biotechnology Inc. San Jose, CA). The cell population with high fluorescence intensity was collected by the EGFP channel as we previously described.12,16
Clinical tissue samples.
PDAC tissue samples (n = 75) were collected during surgeries performed between 2007-2012, at the Department of Gastroenterological Surgery, Osaka University. All patients had clear diagnoses with PDAC, according to the clinicopathological criteria described by the Japanese Society for pancreatic cancer. Samples were fixed in buffered formalin at 4℃ overnight, processed through graded ethanol solutions, and embedded in paraffin. The specimens were appropriately used, with approval by the Ethics Committee at the Graduate School of Medicine, Osaka University.
Construction of the LY6D‑shRNA lentivirus.
We used two short hairpin RNA (shRNA) sequences specifically targeting LY6D (GeneBank ID:8581; 5'-ATCTGGTGAAGAAGGACTGTG-3' and 5'-CCAGCAACTGCAAGCATTCTG-3'). Control cells were generated by transfecting cell with empty vector. The LY6D gene was cloned into the enhanced green fluorescent protein (eGFP) containing pReceiver‑Lv193x lentivector (iGene Biotechnology Co., Ltd.) using FastDigest KpnI (cat. no. FD0524) and XhoI (cat. no. FD0694) restriction endonucleases (both Thermo Fisher Scientific, Inc.) to produce plasmids termed LY6D-shRNA-1, and LY6D-shRNA-2.
Cell transfection.
We purchased pCMV6-XL5-LY6D and pCMV-XL5-vector (empty vector) from ORIGENE (USA). For transfection experiments, lipofectamine 3000 (Invitrogen, USA) was used to transfect LY6D or empty vector into Panc-1 cells. Transduction efficiency was analyzed by PCR and western blot.
Quantitative real-time reverse transcriptase–polymerase chain reaction.
Total RNA was extracted from cultured cells using TRIzolR RNA Isolation Reagents (Thermo Fisher Scientific) as previously described.17 Complementary DNA (cDNA) was synthesized from 10 ng of total RNA using a High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Polymerase chain reaction (PCR) was performed in a Light CyclerTM 2.0 System (Roche Applied Science) using the Thunderbird® SYBR® quantitative PCR (qPCR) mix (Toyobo Life Science, Osaka, Japan). For each experiment, data were normalized to the expression of a control gene (GAPDH). The following primers were used: LY6D: forward, 5′-CATTGCTGCTCCTTGCAG-3′ and reverse, 5′ -ATGCTTGCTTGCAGTTGCTGGAG-3′ ; GAPDH: forward: 5′ -AGCCACATCGCTCAGACAC-3′ and reverse, 5′ -GCCCAATACGACCAAATCC-3′.
Immunohistochemical staining.
Immunohistochemical staining was performed as previously described.18 Slides were incubated with the anti-LY6D rabbit antibody (1:500 dilution, HPA024755; Sigma-Aldrich, Tokyo, Japan), overnight at 4℃. As a positive control for LY6D, we used human esophageal squamous tissue, based on the database of the Human Protein Atlas (https://www.proteinatlas.org). Groups that were slightly stained were considered positive, and those that were not stained at all were considered negative.
Cell proliferation assay.
We counted the number of living cells using Cell Counting Kit8 (DOJINDO) according to the manufacturer's instructions. After a 2-hour preincubation in the assay solution, we determined the viable cell number in each well, based on the absorbance at 450 nm (OD 450) as measured by a microplate reader (BIO-RAD Model 680 XR).
Scratch wound healing assay.
Cells were seeded at a density of 5×105 cells per well in 6-well plates, and grown to confluence under standard conditions. The scratch assay was performed by running a 200μL pipette tip though the well and then containing to culture the cells under standard conditions, except with DMEM containing 1% FBS, to prevent proliferation. Next, the plates were washed with fresh DMEM with 1% FBS, to remove non-adherent cells before each photography session. Cell migration was evaluated by measuring the average distances between the wound edges at 9 random areas.
Invasion assay.
The cell invasion assay was performed in a 24-well Corning Matrigel invasion chamber (Corning, NY) with 8-micron pores. To the top chamber, we added 200 μL of cell suspension (5 × 104 cells/mL) in serum-free media. Next, to the lower chamber, we added 500 μL of FBS-supplemented media to serve as a chemoattractant. After 72 h, the supernatant was discarded, the cells in the upper chamber were removed using a cotton swab, and the cells on the lower surface were fixed and stained with hematoxylin eosin. The number of invaded cells was counted under a microscope, assessing six high-power fields per group. Cells were counted using ImageJ software.
Sphere formation assay.
The sphere formation assay was performed as previously described.12 Briefly, sorted cells were plated in 96-well ultralow attachment plates (Corning Inc.) at a density of 100 cells per well, These cells were grown in tumorspheric culture medium (DMEM/F-12) supplemented with 20 ng/mL human platelet growth factor, 20 ng/mL epidermal growth factor, G418, and 1% antibiotic–antimycotic solution, at 37℃ in a humidified atmosphere of 95% air and 5% CO2. We counted the numbers of spheres in all wells, and evaluated differences in the average number per well.
Drug sensitivity assay.
LP/CD44+ Panc-1, cells were seeded at a density of 2.0 × 103 cells/well in 96-well plates, and pre–cultured for 24 hours. The cells were exposed to various concentrations of oxaliplatin and gemcitabine, and the cytotoxic effects were evaluated using a Cell Counting Kit-8 (Dojindo), according to the manufacturer’s protocol.
Animal experiments.
Animal experiments were performed in 8-week-old male BALB/cAJcl-nu/nu immunodeficient mice (CLEAJapan, Tokyo). To produce tumorsin vivo, cells were mixed with Matrigel (BD Biosciences) and medium at a 1:1 ratio (vol:vol). Mice were subcutaneously injected with approximately 1.0 × 103 cells in 100 μL medium/Matrigel solution, in both sides of the lower back regions. On week 6, the mice were sacrificed and the tumors were excised. Collected tissues were homogenized using a TissueLyser II (QiagenInc, Valencia, CA).
Western blotting.
Total protein (60 µg) was extracted from cultured cells using RIPA buffer containing protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific). Protein was electrophoresed on 10 % SDS-PAGE gels and electroblotted onto PVDF membranes (Merck Millipore, Darmstadt, Germany) at 100V for 90 min. As a loading control, we used β-actin, and β-actin antibodies (A2066; Sigma-Aldrich). After blocking with 5 % skim milk for 1 h, these membranes were incubated with primary antibodies at the appropriate dilutions (1:1000 for LY6D, 1:1000 for CD24 and 1:2000 for β-actin) overnight at 4℃. After incubation with secondary antibodies, the protein bands were detected with the Amersham ECL Detection System (Amersham Biosciences, Piscataway, NJ).
Prognostic analysis of LY6D.
To examine LY6D expression in normal and tumor tissues, we used clinical information about tumors downloaded from the TCGA database.19 We analyzed the correlation between LY6D expression and PDCA prognosis, using overall survival (OS).
Gene mutation analysis of LY6D.
We analyzed mutations of the LY6D gene in all tumor tissues using the cBioPortal platform.20,21 The identified alterations included mutations, amplifications, deep deletions, and multiple alterations
Statistical analysis.
Statistical analyses were performed using JMP Pro 16.0.0 (SAS Institute Inc., Cary, NC, USA). We conducted an overall survival analysis, including all patients (n = 75), using the Kaplan–Meier method. The log-rank test was used to test differences between the survival curves. Data are reported as mean ± standard error of the mean (SEM). Variables that were significantly correlated with survival in univariate analysis were entered into a Cox proportional hazards regression model for multivariate analysis. A P value of < 0.05 was considered to indicate a statistically significant difference.
Data availability
Raw data of RNA-Seq are available and ready to be uploaded to the public structured data depository. The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.