Cell culture and reagents
We purchased human PDAC cell lines ASPC-1, BXPC-3 from the American Type Culture Collection (ATCC). We cultured the cells in complete growth medium according to the manufacturer's instructions. We cultured the cells in an incubator with temperature at 37℃ and concentration of CO2 at 5%. Every 3 months, Mycoplasma-free testing was performed for depollution. (Last verified negative date, May 1, 2020). The experiment used cell lines within ten generations after thawing in this research.
The following reagents were used: We purchased cell culture media RPMI-1640 from Gibco. Fetal bovine serum was obtained from Biological Industries. Pancreatic enzyme and EDTA were acquired from Sigma. Phosphate buffered saline (PBS) were from DHeLix.
We obtained 100 cases of PDAC specimens from patients who underwent pancreatic surgical resection, and they were informed consent before pancreatic operation. None of the patients received radiotherapy or chemotherapy before surgery. Both normal and tumor samples obtained from resected tissues were immediately frozen at -80℃. Both tumor and adjacent nontumor tissues were sampled respectively, with approximate 1 cm3 size of each specimen, and were proved by pathological examination. This study was approved by the Ethics Committee of The Ethics Committee of First Affiliated Hospital of Zhengzhou. Zhengzhou University authorized this study(KY-2021-0382). The study was carried out in compliance with the ARRIVE guidelines.
OSBP2 knockdown and overexpression in PDAC cells
We cloned full-length human OSBP2 cDNA into pcDNA3.1 plasmid (invitrogen Co.,Ltd. China) to induce OSBP2 stable expression. Jima Genomics Co., Ltd designed the siRNAs for OSBP2(si-RNA-OSBP2). In short, 80% confluent cells were seeded at a density of 5×105 cells/well in 6-well plates in medium which contains 10% FBS. When the cells were 80% confluent, they were transfected with 50 nmol/L siRNAs in Lipofectamine 3000 (invitrogen Co., Ltd. China) for 48 hours. Puromycin treatment was used to select stably transfected cells. We used western blot to evaluate the level of OSBP2 expression.
The Laboratory Animal Center of Zhengzhou University provided female BALB/c nude mice and NOD/SCID mice for us. Procedures relating to the care and use of mice in this study were conducted in accordance with National Institute of Health (NIH) guidelines, with the animals housed in an association for assessment and accreditation of Laboratory Animal Care accredited facility. All animal studies were carried out in accordance with approved Institutional Care and Use Committee protocols for Zhengzhou University. And the Zhengzhou university approved the study protocol.
In vivo subcutaneous injection
Female BALB/c nude mice ages 4 to 6 weeks were maintained in a barrier facility on high-efficiency particulate air-filtered racks. Twenty mice were divided into four groups (5 mice/group): one group was injected with mixed populations from OSBP2-interference ASPC-1 cells and the other group with the control ASPC-1 cells; one group was injected with mixed populations from OSBP2-overexpression BXPC-3 and the other group with the control BXPC-3 cells. The number of cells injected in each group was 107.The cells were inoculated subcutaneously on the right posterior back of the nude mice. Tumor nodules were observed weekly, and the length of the tumor were measured. Four weeks later, all tumor nodules were collected and fixed with 4% paraformaldehyde for subsequent experiments. For the sacrifice of the mice,the enthanasia box was used to contain the nude mouse and 20%-30% CO2 was infused into the box. It was ensured that the nude mouse did not move, had no breathing and had dilated pupils. The carbon dioxide was turned off, followed by observation for 2 min to confirm that the nude mouse was dead.
In vivo caudal vein injection
Female NOD/SCID mice ages 3 to 4 weeks were maintained in a barrier facility on high-efficiency particulate air-filtered racks. Twenty mice were divided into four groups (5 mice/group): one group was injected with mixed populations from OSBP2-interference ASPC-1 cells and the other group with the control ASPC-1 cells; one group was injected with mixed populations from OSBP2-overexpression BXPC-3 and the other group with the control BXPC-3 cells. The number of cells injected in each group was 107. The cells were injected into the corresponding groups through tail vein and observed for 8 weeks. Eight weeks later, all of the mice were sacrificed, lung tissues were taken out of each group of mice after death, the wet weight of both lungs was weighed, and fixed with 4% paraformaldehyde for subsequent experiments. For the sacrifice of the mice was as mentioned above
We extracted total cell proteins by using lysis buffer with 1% phenylmethanesulfonyl fluoride and quantified using a Bicinchoninic Acid Protein Assay Kit (wanleibio Co., Ltd., China). We used odium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to denature and separate 20 µg protein. Then, the protein was transfected to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA)., During the course of immunoblotting, the membranes were cultured in Tris-buffered saline-Tween-20 (TBS-T) with 5% skim milk powder (Yili Co., Ltd., China). Then, we incubated it with the corresponding primary antibody overnight at 4˚C. The next day, we rinsed the membrane 4 times with TBS-T for 5 minutes/per time. Then, the membranes were cultured with horseradish peroxidase conjugated secondary antibody for 45 minutes at 37 followed by enhanced chemiluminescent reagent (ECL; EMD Millipore) At last, the cell membranes were cultured at 37 ° C with horseradish peroxidase-bound secondary antibodies for 45 min, followed by enhanced chemiluminescence reagents (ECL; EMD Millipore). The proteins E-Cadherin(cat.no.3195), N-Cadherin (cat.no.13116), Vimentin(cat.no.5741), Snail(cat.no.3897), β-Catenin(cat.no.8480), Slug(cat.no.9585), ZEB1
(cat.no.3396) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). β-actin was purchased from wanleibio. All antibodies were used in accordance with the manufacturer's instructions. The first antibody was diluted at the ratio of 1:1000 and the second antibody was diluted at the ratio of 1:5000 according to the manufacturer's instructions.
The cells of various treatment groups (ASPC-1，OSBP2-interference ASPC-1; BXPC-3,OSBP2-overexpression BXPC-3) were cultured until the density was about 90%，we washed them once with PBS (DHeLix Co., Ltd., China) and disposed of the supernatant. Then, we added 0.25% appropriate volume trypsin (Sigma Co., Ltd., USA) digested cells, and when the cells became round, adding complete medium to terminate the reaction. Next, we blow all the cells in the cell culture plate with the tip of a 5ml gun, and collected the mixture into a 15ml test tube, and centrifuged at 88g for 3min. The supernatant was removed, 1ml of complete medium was added, and the cells were counted. Cells were seeded into 96-well plates according to experimental groups, and each well was seeded with 5×103 cells. Then, CCK-8 test was performed after cultured in an incubator at 37℃ and 5% CO2 for 0h, 24h, 48h and 72h, respectively. The supernatant was discarded, then added 100 μL complete medium and 10 μL CCK-8 to each well, then cultured in incubator at 37℃ and 5% CO2 for 2h. The optical density value at 450nm was measured on an enzyme standard instrument (BIOTEK Co., Ltd., USA).
Cells were seeded at low density (5000 cells per well) in 96-well plates and treated with different concentrations (0-2000 μmol/L) of 5-FU or gemcitabine, respectively. After 72 hours incubation, we used CCK-8 test to measure the optical density value at 450nm, as mentioned above.
We used transwell chambers (Corning Inc. USA) to evaluate cell migration and invasion. Matrigel was applied in the invasion assay. Cells were suspended in medium without fetal bovine serum (FBS). Then we added 6x103 cells of various treatment groups respectively to the upper chamber pre-coated with Matrigel (Corning Inc. USA), and added 800 μl medium with 30% FBS to the lower chamber. The transwell chamber was washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 20min, stained with 0.5% crystal violet solution for 5min, and rinsed with distilled water. The cells migrated to the sublayer of the microporous membrane were counted under an inverted microscope (200×) (Motic Electric Group Co., Ltd., Xiamen, China).
Wound healing assay
The cells of various treatment groups (ASPC-1, OSBP2-interference ASPC-1; BXPC-3, OSBP2-overexpression BXPC-3) were cultured until the density was fused. Before the experiment, the medium was changed to serum-free medium and 1μg/ mL mitomycin C was added to treat for 1h. Cells in each group were scratched by 200 μL pipette tip, washed cell surface with serum-free medium once, removed cellular debris, observed the cells under the microscope and took photos in groups, noted down the positions of the cells in the photos, and prepared them for subsequent photos. The culture medium without serum was used instead. Cells in each group were placed in an incubator at 37℃ and 5%CO2 for 48h and then photographed.
The Annexin V-FITC/PI Apoptosis Detection kit (BD Biosciences, San Jose, CA, USA) was used to access cell apoptosis. We carried out the test according to the manufacturer's instructions. In brief, we used ice-cold PBS to wash these cells. Then, Annexin V-FITC and PI solutions was used to culture them for 15 min in the dark. Next, we used FACScan flow cytometry to study them for apoptosis. At last, we summarized the data as the mean ± standard error (SD).
The fixed tissue was sectioned into 5 µm‑thick sections. After heating to 60˚C for 2 h, the specimens were deparaffinized using xylene at room temperature and rehydrated in a descending graded series of ethanol (95, 85 and 75%). For IHC staining, endogenous peroxidases were inactivated by 3% hydrogen peroxide for 15 minutes. Tissues were stained with primary antibodies for 12 hours at 4℃ according to the manufacturer’s instructions, then, stained with secondary antibodies. The staining results were observed and imaged through microscopy (OLUMPUS Co., Ltd., Japan) at a magnification of 400.
The fixed tissue was sectioned into 5 µm‑thick sections. After heating to 60˚C for 1 h, the specimens were deparaffinized using xylene at room temperature and rehydrated in a descending graded series of ethanol (95,85 and 75%). Subsequently, the samples were stained with hematoxylin for 5 min and eosin for 3 min both at room temperature. Then, after dehydration, transparent and sealing piece, images were captured using a microscope (x200; OLUMPUS Co., Ltd., Japan).
PDAC tissues and adjacent para-cancer tissues for the tissue microarray (TMA) were derived from the First Affiliated Hospital of Zhengzhou University. Firstly, an experienced pathologist is responsible for selecting the 1 mm core area, then, we used a TMA Grand master machine (3DHISTECH) to drill and place onrecipient blocks. Each primary tumor included three cores and metastatic lymph nodes contained 1–3 cores. We coded and placed the cores on the recipient randomly. Relevant clinical information comes from hospital cases. To compare staining intensity and survival, at least two histological core scores are required per patient. In the process of tissue staining analysis, the clinical information was blinded.
We used SPSS16.0(IBM) to conduct statistical analysis in this study. Mean±SD was used to represent the data. All in vitro experiments were conducted independently in triplicate. Comparisons between treatment groups were done by Student t test. Statistical analysis was conducted with either Microsoft Excel or Origin Labs. The Kaplan–Meier method was used to compare OS among patients in different groups. The log-rank test was used to estimate differences in survival. Univariate and multivariate analyses were based on the Cox proportional hazards regression model. P < 0.05 was considered statistically significant.