RIPA cracking liquid kits were obtained from Beyotime Biotechnology (Shanghai China). The DMEM culture medium and fetal calf serum were purchased from the Hyclone (USA). Transwell chambers were purchased from Millipore (Shanghai, China). Matrigel and One-Step RT-PCR kit were obtained from BD Biosciences (NJ, USA). E-cadherin, vimentin, Gli1, Snail and β-actin antibodies were purchased from the Santa Cruz Biotechnology (Santa Cruz, CA, USA). PTCH specific blocker were purchased from the Sigma Co. Ltd., Shanghai, China.

The human pancreatic cancer cell lines (BxPc-3, Panc-1; obtained from the American Tissue Type Collection, USA) were maintained in Dulbecco's modified Eagle's medium (Gibco, USA) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 0.1 mM non-essential amino acids, 0.2 mM glutamine, 1 mM pyruvate, and 10% heat-inactivated fetal bovine serum and incubated in 5% CO₂ humidified atmosphere at 37°C. Cells were grown to 80% confluency. In the invasion and migration experiments, the cells were cultured in Dulbecco's modified Eagle's medium without fetal bovine serum.

Normal fibroblast (NF), CAF and hepatic stellate (HSF) cells were derived from patients with pancreatic cancer and hepatic trauma from the Second Affiliated Hospital of Xi'an Jiaotong University. All patients were newly diagnosed and had not received any relevant treatment prior to surgery. Informed consent was obtained from all patients before taking specimens. Fibroblast isolation was conducted as described previously (10). First, the tissue was trimmed to 1×1×1 mm and washed gently with PBS three times (5 min each). Next, the tissues were washed once with the medium and placed in fresh cell cultural medium containing 15% fetal calf serum, 2 mM L-glutamine, and 10% double-antibody. The tissue was cut with a sterile scalpel blade and sections of cells were gently scraped with a blunt blade. The cells were cultured in an incubator for 3–5 days at 37°C and 5% CO₂. The medium was replaced once and every 3 days afterwards; after 14 days, the cells fully covered the petri dish. When the cell density reached 80–90%, the cells were digested with trypsin and regenerated at a rate of 1:3. The CAFs and NFs used in the experiment were the 3rd and 5th generations of cells cultured in vitro, respectively, and showed no obvious aging phenotype.

CAFs were added to 6-well plates at a density of 1.5×10⁵/ml and rinsed with PBS after 24 h. The medium was replaced with serum-free medium and cultured for 48 h, after which the culture broth was collected, centrifuged to remove the cells and debris, and the supernatant obtained was the CAF conditioned medium. These samples were stored at 4°C. The conditioned medium of NFs and HSFs was collected in the same manner.

The pancreatic cancer cells BxPc-3 and Panc-1 were added to Petri dishes at a density of 1.5×10⁵/ml; after 24 h, CAF-CM was added, and the cells were cultured for 48 h. Cells in PBS or serum-free medium were used as controls. An inverted phase contrast microscope was used to observe the morphology and growth of pancreatic cancer cells in each Petri dish. Proteins were extracted from the cells. The co-culture of HSFs, NFs and pancreatic cancer cells was performed in the same manner.

Cell migration capability was evaluated in a scratch test. BxPc-3 (10×10⁵) and Panc-1 cells were seeded in 1.5 ml media in each well into a 24-well plate. The cells were grown to a confluent layer (48 h), and then a scratch was made in each well by using a pipette tip. Subsequently, the cells were washed gently with PBS, and then culture broth of CAF, NF and F were added to the wells. Starting picture was taken at time-point 0. The cells were then incubated at 37°C in a 5% CO₂ atmosphere, and new pictures were taken after 24 h. The 24 h time-point was chosen to decrease the potential impact of proliferation on the closing of the scratch. Image-Pro Plus 5.0 from NIH was used to standardize and present the results.

Cell invasion was examined using Transwell assays, Following incubation for 48 h, 3×10⁴ cells were transferred to the top of the Matrigel-coated invasion chambers (BD Biosciences, San Jose, CA, USA) in serum-free DMEM. DMEM containing 10% FBS was added to the lower chamber. After 24 h, the non-invading cells were removed, and the invading cells were fixed using 95% ethanol, stained with 0.1% crystal violet and images were captured at ×100 magnification under an inverted phase contrast microscope (Olympus CKX31/41; Olympus, Tokyo, Japan). The experiments were repeated three times independently.

Total RNA was extracted from the cells using TRIzol reagent. A total of 2 µg RNA was reversed transcribed into first-strand cDNA using the Revert Aid First Strand cDNA Synthesis kit. PCR primer sequences were as follows: E-cadherin forward, 5′-CAATGGTGTCCATGTGAACA-3′; reverse, 5′-CCTCCTACCCTCCTGTTCG-3′. Vimentin forward, 5′-CGCTTCGCCAACTACAT-3′; reverse, 5′-AGGGCATCCACTTCACAG-3′. β-actin forward, 5′-ATCGTGCGTGACATTAAGGAGAAG-3′; reverse, 5′-AGGAAGGAAGGCTGGAAGAGTG-3′. Gli1 forward, 5′-ATAGTGAGCCATGCTGTCTCC-3′; reverse, 5′-TCTCTCTGGCTGCTCCATAACC-3′. Snail1 forward, 5′-AAGGATCTCCAGGCTCGAAAG-3′; reverse, 5′-GCTTCGGATGTGCATCTTGA-3′. The PCR conditions were as follows: an initial reaction at 42°C for 1 h was used for cDNA synthesis, followed by denaturation at 94°C for 5 min and 22 cycles of the following reactions: 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec. After the last cycle, the reaction was amplified at 72°C for 10 min. The housekeeping gene β-actin was used as an internal reference.

A total of 5×10⁵ cells in the logarithmic growth phase was added to 0.5 ml of pre-chilled cell lysis buffer and incubated on ice for 30 min. After centrifugation, the supernatant was collected and the protein contents were measured. The proteins were separated by 10% SDS-PAGE and blotted onto a nitrocellulose membrane by semi-dry transfer. Next, the membrane was immersed in TBST containing 5% skim milk for blocking followed by overnight incubation with the primary antibody at 4°C. The following day, the membrane was incubated with the secondary antibody conjugated to horseradish peroxidase at 1:2,000 dilution (Santa Cruz) at room temperature for 2 h, and then an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Amersham, UK) was used for staining. The membrane was photographed and the results were analyzed.

Each experiment was repeated at least three times. The data were expressed as mean ± SD and analyzed using the Student's t-test and one-way ANOVA. P<0.05 indicates a statistically significant difference.

After 10 days, cell morphology was observed using an inverted phase contrast microscope, HSFs, NFs and CAFs showed a spindle shape (Fig. 1). The CAFs were spindle- or fusiform-shaped, had inconsistent sizes, showed dense growth, and were disorderly arranged. For NFs and HSFs, the cells showed multiple flat stellate shapes with similar cell sizes, were arranged in the same direction, and had a radially outward appearance (Fig. 1). TEM observations showed that CAFs had large cell nuclei, were evenly colored, and contained one or two nucleoli. In the cytoplasm, a large number of rough endoplasmic reticulum, mitochondria, and bundles of parallel subcapsular filaments were observed. For NFs and HSFs, the cells had an irregular cell nucleus, were rich in rough endoplasmic reticulum in the cytoplasm, and had no filaments (Fig. 1). Immunohistochemical results of CAFs showed that vimentin and α-smooth muscle actin expression was positive. In NFs and HSFs, vimentin showed positive expression, while α-smooth muscle actin expression was negative (Fig. 1C and D).

To determine whether co-culture of pancreatic cancer cells with CAF promotes EMT, we first observed their morphology under conventional light microscopy. We found that after indirect co-culture with CAF, pancreatic cancer cells changed significantly from rounded to spindle-shaped or fusiform cells with a chaotic, non-directional arrangement, and radial appearance (Fig. 2A). Confocal laser scanning microscopy was carried out to assess the fluorescent intensity of Panc-1 cells. The results showed that pancreatic cancer cells co-cultured with CAF had a higher vimentin fluorescent intensity than the NF/HSF co-culture group and single culture group, whereas the E-cadherin fluorescent intensity was lower than the NF/HSF co-culture group and single culture group (Fig. 2B). Western blotting results showed that pancreatic cancer cells in the CAF co-culture group had a higher protein expression of vimentin than the NF/HSF co-culture group, whereas the protein expression of E-cadherin was lower than that in the NF/HSF co-culture group (Fig. 2C and D). The detection results of BxPc-3 cells were consistent with Panc-1 cells, suggesting that activated CAF can enhance EMT changes in pancreatic cancer cells, while EMT could not occur after co-culturing with quiescent HSF.

To clarify whether CAF-induced partial EMT in pancreatic cancer cells can promote the pancreatic tumor migration, an indirect co-culture model was employed, where BxPc-3 and Panc-1 cells were treated with CAF-conditioned medium (CM) for 48 h, followed by a scratch assay to examine the effects of CAF on the migration of pancreatic cancer cells. After 48 h of treatment, single culture or NF/HSF co-cultured BxPc-3 and Panc-1 cells exhibited weaker migratory capacity than the CAF co-culture group (Fig. 3). Results showed that CAF-induced partial EMT in pancreatic cancer cells enhancing pancreatic cancer cell migration; however, co-culture with quiescent HSF was unable to promote the migration of cancer cells.

To determine whether CAF-induced partial EMT in pancreatic cancer cells was able to promote the invasive capacity of pancreatic cancer cells, an indirect co-culture model was employed, followed by a Transwell migration assay using an NF-CM induction group and PBS alone as a control. Results showed that the CAF-CM treatment group had a significantly higher number of migrated cells than the PBS or NF/HSF-CM induction group (Fig. 4). This suggests that CAF-induced partial EMT in pancreatic cancer cells enhancing the invasive capacity of pancreatic cancer cells, whereas co-culture with quiescent HSF was unable to promote the invasive capacity.

Snail has been reported to regulate EMT marker, E-cadherin, which is a transcription factor upstream of vimentin expression (11). In order to clarify whether pancreatic cancer cells co-cultured with CAF affected Snail expression, RT-PCR and western blotting were used to determine Snail mRNA and protein expressions, respectively. PCR results showed that the CAF co-culture group had a significantly higher level of Snail mRNA than the NF/HSF co-culture group or single culture group (P<0.05) (Fig. 5). Western blotting results showed that the level of Snail protein expression was significantly upregulated as compared with the NF/HSF co-culture group and single culture group (P<0.05) (Fig. 5). These results further confirm that Snail expression in pancreatic cancer cells was enhanced after co-culture with CAF.

Gli1 have been reported to be terminal transcription regulators of the HH pathway, and play a very important role in the activation of CAF (12). Activated CAF can promote tumor progression via paracrine action. In order to clarify whether the upregulation of Snail in pancreatic cancer cells after co-culture is related to the HH pathway in CAF, changes of Gli1 immunofluorescence in CAF were determined using confocal laser scanning microscopy. Results showed that CAF had a significantly higher Gli1 fluorescent intensity than the NF group (Fig. 6). Western blot analysis showed that Gli1 protein levels were significantly upregulated as compared with the NF group (P<0.05) (Fig. 6). These results further confirm that the HH pathway was activated in CAF. To verify whether the HH pathway in CAF was related to EMT in pancreatic cancer cells, CAF was pretreated with an HH-specific blocker (cyclopamine) for 24 h; the supernatant was then obtained and cultured with pancreatic cancer cells. Then, Snail protein expression was determined by western blotting. The results showed that Snail protein expression in the cyclopamine group was significantly decreased as compared with the CAF group, and the corresponding expression of E-cadherin and vimentin showed significant differences as compared with the CAF group. The above results confirm that activating the HH signaling pathway in CAF may trigger Snail transcription factors in pancreatic cancer cells via paracrine action, thereby enhancing EMT in pancreatic cancer cells.