Chemicals and solutions
TaqMan gene expression assays, MTT 3-(4,5-dimethylthianol-2-yl)-2,5-diphenyltetrazolium bromid, siRNAs for MUC4 (Cat. No.: AM 16708), and MUC17 (Cat. No. : AM 16709), an oligofectamine transfection kit, and the rabbit MUC17 polyclonal antibody (Cat #PA5-56805) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Mouse MUC4 monoclonal IgG1 antibody was ordered from Santa Cruz Biotechnology (Cat. No.: sc-33654; Dallas, TX, USA). A Technical Manual Cell Counting Kit-8 was ordered from Dojindo Molecular Technologies (Rockville, USA). All other laboratory chemicals were ordered from Sigma-Aldrich Kft. (Budapest, Hungary).
The clinical part of the study was carried out with the approval of the Ethics Committee of the University of Szeged (No. : 4714), followed by the EU Member States’ Directive 2004/23/EC on presumed consent practice for tissue collection, the guidelines of the Helsinki Declaration, and the General Data Protection Regulation (EU) 2016/679. Written informed consent was obtained from all patients and healthy volunteers for sample and data collection.
Pathological characterization of the patients
Immunohistochemistry (IHC) was performed on pancreatic samples obtained from 52 patients and classified into the following groups: 1) PDAC (average age: 63.78 ± 2.8; male/female ratio: 12/9), 2) PDAC + OJ (average age: 69.7 ± 1.8; male/female ratio: 11/9), 3) neuroendocrine tumor (NET) (average age: 72.5 ± 2.05; male/female ratio: 6:1), and 4) control group (average age: 62.75 ± 3.3; male/female ratio: 1:3). All of the samples were obtained from surgical resection or biopsy. Table 1 shows the pathological characterization of the patients.
Cell line and treatment
Capan-1, a human PDAC cell line, was obtained from American Type Culture Collection and cultured in RPMI-1640 media supplemented with 15% fetal bovine serum; 1% L-glutamine, and 2% antimycotic/antibiotic. The medium was replaced every second day, and the cells were seeded at 100% confluency. The cells were used between passages 30–35. The cells were seeded into 25-cm2 tissue culture flasks or 96-well tissue plates 2 days before the BAs or serum treatment. The BAs treatment was performed with six different types of BAs [glycocholic acid (GCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), and taurochenodeoxycholic acid (TCDCA)] at two different concentrations (100 and 500 µM), for 24, 48, and 72 h. The serum treatment was performed with serum obtained from PDAC patients with OJ (average age: 72.6 ± 9.8; male/female ratio: 5/5) or without OJ (average age: 80 ± 2.5; male/female ratio: 2/3) and from healthy volunteers (average age: 40.9 ± 18.77; male/female ratio: 6/8) (Table 2) at 50X dilution, for 24, 48, and 72 h. The degree of dilution was determined based on our previous results 6.
Tissue plates (96 wells) were coated with 40 µg/ml type 1 collagen from rat-tail in phosphate-buffered saline (PBS) at 4ºC, then 100 µl of the cell suspension (105 cells/ml) was added to each of the coated wells and incubated for 20 min. After washing, the cells were incubated with BAs. After the treatments, 10 µl of MTT substrate was added to each well, and the cells were incubated for an additional 3 h. The MTT-treated cells were then lysed in DMSO, and a FLUOstar OPTIMA Spectrophotometer (BMG Labtech, Ortenberg, Germany) at 560 nm, with background subtraction at 620 nm, was used to measure absorbance.
Cytotoxicity assay was performed as previously described 6. Briefly, 100 µl of cell suspension was seeded into a 96-well plate (2 × 104 cells/well) and allowed to adhere overnight. On the following day, the cells were incubated with BAs, and then 100 µl of supernatant from each of the wells was carefully transferred to a new 96-well plate containing 100 µl of reaction mixture. We then used a FLUOstar OPTIMA Spectrophotometer (BMG Labtech, Ortenberg, Germany) to measure lactate dehydrogenase (LDH) activity at 490 nm. Calculated the percentage of cytotoxicity using the following formula: Cytotoxicity (%) = (exp. value–low control/high control-low control)*100. Low control determines the LDH activity released from the untreated normal cells (spontaneous LDH release), whereas high control determines the maximum releasable LDH activity in the cells (maximum LDH release). As a positive control, 0.1% Triton-X-100 was used.
For proliferation, 100 µl of cell suspension was seeded into a 96-well plate (5 × 103 cells/well), and then the cells were incubated with BAs. After the treatments, 10 µl of a CCK8 solution was added to each well, and the cells were incubated for an additional 3 h. We used a FLUOstar OPTIMA Spectrophotometer (BMG Labtech, Ortenberg, Germany) to measure absorbance at 450 nm.
Wound healing assay
Cells were seeded onto 24-well cell culture plates at a 2.5 × 105 cell density and allowed to adhere overnight. On the following day, P2 tips were used to gently scratch the confluent monolayer, and the cells were incubated with BAs. Automated time-lapse imaging was performed with an Olympus IX83 inverted microscope and the Olympus ScanR screening platform (Olympus, Japan) upgraded with an OKOLAB incubator system (with a gas, temperature, and humidity controller; Pozzuoli, NA, Italy). Image J software (free) was used to analyze the digital images.
Capan-1 cells (103 cells/well) were seeded onto 6-well cell culture plates and allowed to adhere overnight. On the following day, the cells were treated with BAs, and then the normal media was given back. The cells were allowed to grow until day 9 after which the media was removed, and the cells were washed with PBS, fixed with methanol–ethanol solutions (3:1 dilution) and then stained with Giemsa. An Olympus IX83 microscope-based screening platform was used for image acquisition, and the Olympus Cellsense Dimension software was used for automated object detection, classification, and measurement to enumerate colonies organized by the treated and untreated cells.
MUC4- and MUC17-targeted siRNA oligonucleotides were used to perform MUC4 and MUC17 silencing. The Oligofectamine™ Transfection Reagent was used to perform transfection following the manufacturer’s instructions. Then, 2 × 105 cells per well were seeded onto 6-well plates 1 day before the transfection. MUC4 and/or MUC17-targeted siRNAs were transfected into the cells at 40–50% confluency and incubated for 72 h.
A NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany) was used to isolate the total RNA from the cells. A High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA) was used for reverse transcription of 2 µg of RNA. TaqMan RT-PCR assays were used to perform teal-time PCR reactions of samples as previously described 6.
Immunocytochemistry (ICC) was performed on cytospin preparations as previously described 6. For the staining procedure, the slides were incubated with MUC17 (1:100 dilutions) or MUC4 (1:100) primary antibodies for 30 min. After incubation, the slides were washed and incubated with secondary antibody (EnVision™ Flex/HPR anti-mouse/rabbit) for 30 min. For visualization, an Ultra View Universal diaminobenzidine (DAB) Detection Kit (EnVision™ Flex DAB) was used, and for nuclear staining, EnVision™ Flex Hematoxylin was used. The Olympus IX83-based system was used to scan all specimens. ImageJ was used to further analyze the images, and the intensities of the pixels of the DAB staining were quantified. In the human pancreatic samples, formalin-fixed and paraffin-embedded tissue specimens were used to analyze for MUC4 and − 17 expressions as previously described 6.
Quantitative variables were described as means ± SE. Significant differences between groups were assessed by performing ANOVA, and p ≤ 0.05 was accepted as indicating statistical significance. The Kaplan–Meier method was used to prepare survival curves, and differences in survival were studied by performing the Log-rank test.