Tissue samples and cell lines
This study was approved by the academic committee at the First Hospital of China Medical University. Written informed consent has been obtained from each patient. Eight-one pancreatic ductal adenocarcinoma (PDAC) tissues were procured from surgical resection specimens collected by the Department of Gastrointestinal Surgery at the First Hospital, China Medical University.
Human Capan-2 PC cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). SW1990 human PC cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured with recommended growth media with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA).
Fluo-3 assay
Thapsigargin (TG, Sigma, St Louis, MO, USA) is one of the key stimulators that cause acute ERS via specific inhibiting sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCAs), resulting in an increase of cytoplasmic Ca2+ concentration13. The intracellular free Ca2+ concentration was measured using Fluo-3 AM (Beyotime, Shanghai, China), according to the manufacturer’s instructions. Briefly, transfected PC cells were pretreated with 200 nM TG and 1% DMSO (control) for 4 h. Cells without or with TG treatment were subsequently loaded with 2 µM Fluo-3 AM for 30 min at 37 °C and then washed with Hanks' Balanced Salt Solution (HBSS, Beyotime) for 3 times. Kept incubating with HBSS for 20 min, the fluorescence was visualized on a confocal microscopy (Leica Tcs Sp5 II, Leica, Heidelberg, Germany) at an excitation wavelength of 488 nm with an emission wavelength of 525 nm.
In addition, cells without or with TG stimuli were harvested by pancreatic enzymes without EDTA, washed by HBSS for 3 times, and then submitted to analysis by flow cytometry. Image analysis was performed using the Image J software. Each experiment was repeated 3 times.
Immunohistochemistry (IHC) assay
As described previously4,14, 4-µm sections were covered with 0.3% H2O2, subjected to high pressure, added with goat serum, and then incubated with primary antibodies: CRT (Abcam, Cambridge, UK) and IRE1α (Cell Signaling Technology, CST, Beverly, MA, USA). Then the slices were incubated with the secondary antibodies, treated with streptavidin–peroxidase reagent, visualized with DAB, counterstained with hematoxylin and finally evaluated under microscope. The location of CRT and IRE1α in cytoplasm were considered for scoring. Staining intensity was scored as 0-3 (negative, weak, medium and strong). Extent of staining was scored as 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%) according to the positive staining areas to the whole carcinoma. The final scores were calculated by 3 pathologists. We used the same scoring method to evaluate the IHC assay in vivo and in human PDAC specimens.
Immunofluorescence (IF) staining
Capan-2 and SW1990 cell lines were implanted into 24-well culture plates covered with slices, fixed in 4% paraformal dehyde, permeabilized with Triton X-100 (0.5%) and incubated with 5% BSA. Then plates were incubated with the primary antibodies overnight: CRT (Abcam) and IRE1α (Cell signaling technology). The secondary antibodies (Proteintech, Chicago, IL, USA) were conjugated with FITC for CRT and TRITC for IRE1α. Hoechest33258 were used for nuclear visualizing.
Western blot (WB) assay
Whole protein lysates were prepared from transfected PC cells. Samples were loaded onto 10% SDS-polyacrylamide gels, transferred to PVDF membranes and incubated with primary antibodies: CRT (Abcam), IRE1α (CST), PREK (CST), phosphorylation PKR-like endoplasmic reticulum kinase (p-PERK, CST), ATF-6 (CST), ZO-1 (Proteintech), ZEB1 (Proteintech), N-cadherin (Proteintech), E-cadherin (Proteintech), Vimentin (Proteintech), phosphorylation extracellular regulated protein kinases (pERK, CST), extracellular regulated protein kinases (ERK, CST), X-box-binding protein 1 (XBP1, Proteintech), Snai1 (Proteintech), Slug (CST), Cavelino-1 (Proteintech), GAPDH (Proteintech) and β-actin (Proteintech) antibodies overnight at 4°C. Then, membranes were incubated with secondary antibodies (Santa Cruz, CA, UK) and finally detected with an ECL detection kit (Thermo Scientific, Rockford, IL, USA). The experiments were repeated for 3 times.
Coimmunoprecipitation (CoIP) assay
CoIP was performed as before4,14. Briefly, PC cells were lysed in lysis buffer and the soluble supernatants were isolated. Magnetic beads (Bio-Rad, California, USA) were preincubated with primary CRT (Abcam), IRE1α (CST) or IgG (Santa Cruz) antibodies at 4°C for 4 h with rotation. Then antibody-beads complexes were incubated with soluble supernatants at 4°C overnight. Immunoprecipitated proteins were analyzed by WB with a variety of antibody.
CRISPR/Cas9 and siRNA mediated silencing of CRT and IRE1α
Lentiviruses were synthesized by Genechem (Shanghai, China). PC cells were transfected with lenti-cas9 or lenti-sgRNA as described previously4,14, and then screened using puromycin (Sigma). The stable sub-lines were subsequently transfected with sg1-CRT or sg2-CRT to specifically silence the target gene or an sgRNA control (scramble).
IRE1α siRNA and siRNA control were synthesized from GenePharma (Shanghai, China). Cells were transiently transfected with siRNA (20 µM) using oligofectamine3000 (Invitrogen, Carlsbad, CA, USA) as described by the protocol. All target sequences mentioned above were shown in Supplemental Material Table 1.
TG induced EMT construction
Stable transfected PC cells were treated with 200 nM TG or 1% DMSO (as a control) for 4 h. The EMT construction was verified by EMT-enhanced cell invasion and migration and EMT-induced changes in key proteins involving in EMT signaling.
Invasion and migration assays
Briefly, transfected PC cells (pretreated with TG or co-transfected with IRE1α) were plated in inserts that coated with matrigel (BD Biosciences, Sparks, MD, USA) in 24 well plates with FBS-free growth media. Growth media with 10% FBS was added to the bottom wells to generate a serum gradient. After 24 h, cells that had migrated to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma). The migratory cells were counted in five random fields per well. The migration assay was done in a similar fashion without matrigel. Each experiment was repeated 3 times.
In vivo xenograft model
All animal work was performed in accordance with protocols approved by the Animal Care Committee of China Medical University. Total 15 nude mice (BALB/c-nu) were used. Transfected Capan-2 cells (1 × 106) were respectively injected into bilateral axillae of 5 nude mice to construct subcutaneous tumor formation. Tumor volumes were calculated by the following formula: length × width × height × 0.52 in cm. Besides, transfected SW1990 cells (1 × 106) were injected into the spleen of 10 nude mice to construct distant liver metastasis model, which were assessed by the number of liver metastases. These nude mice were killed 30 days later, and samples were extracted and fixed for hematoxylin and eosin (HE), and IHC staining.
Statistical analysis
Statistical analysis was performed using SPSS software 21.0 (Chicago, IL, USA). Continuous variables were expressed as the mean ± SD. The differences in intracellular free Ca2+ concentration, WB assay, cell migration and invasion assays and the number of liver metastases were compared through Student’s t-test. The differences of orthotopic tumor volumes were compared with paired sample t-test. Non-parametric and spearman correlation tests were analyzed for IHC assays in vivo and human PC samples. The association of target proteins expression with clinicopathological data was analyzed by Chi-squared. The Kaplan-Meier curve was used to estimate survival, and differences were analyzed by the log-rank test. P <0.05 or P <0.01 was considered significant.