The Pancreatic Ductal Adenocarcinoma cell lines T3M-4 and CD18/HPAF cells were a generous obtained from Dr. Hollinsworth and Dr. Batra’s laboratory respectively, University of Nebraska Medical Center; UNMC, Omaha, NE, USA. Use of these cell lines did not require any additional ethics approval. We maintained these cell lines as an adherent monolayer in Dulbecco’s Modified Eagle Medium (Hyclone Laboratories, Logan, UT). The medium was supplemented with 5% fetal bovine serum (Sigma, St. Louis, MO), 1% of 100X MEM vitamin solution (Mediatech, Herdon, VA), 1% of 200 mM L-glutamine and 0.02% gentamycin (Invitrogen, Carlsbad, CA). We tested the cell lines for mycoplasma using MycoAlert Plus Mycoplasma Detection kit (Lonza). For cell line authentication, Human DNA Identification Laboratory, University of Nebraska Medical Center, Omaha, NE, USA performed the short tandem repeat (STR) tests. They evaluated fifteen STR markers (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA) and the gender marker Amelogenin.
Cloning of SEMA5A shRNA oligos
SEMA5A specific (5’-GCG GAT TTC TCG CAG TTA A-3′) oligonucleotide was selected using OligoEngine software (OligoEngine Inc., Seattle, WA) and inserted downstream of the start codon in the pSUPER.neo vector, as instructed by the manufacturer. The oligonucleotide was annealed and cloned into the BglII and HindIII restriction site using enzyme digested pSUPER.neo vector. We used vector pSUPER.neo without insert as a negative control for SEMA5A knockdown and verified the sequences.
Transfection of PC cells
We transfected T3M-4 and CD18/HPAF cells with pSUPER.neo-shSEMA5A or control pSUPER.neo vector using LipofectAMINE Plus reagent (Invitrogen, Carlsbad, CA) according to the protocol provided by the manufacturer. Stably transfected cells were selected and maintained in 1000 μg/mL and 600 μg/mL neomycin sulfate (G418, Mediatech) containing media for T3M-4 cells and CD18/HPAF cells respectively. Similarly, shSEMA5A and Control (empty vector) T3M-4 cells were transiently transfected with FLAG-tagged cDNA containing full-length mouse Sema5A in pBK-CMV vector (a generous gift from Dr. Andreas W. Püschel, Westfälische Wilhelms-Universität Münster, Germany).
Total cellular RNA was isolated from cells using TRIzol® reagent (Invitrogen, Carlsbad, CA). Cells were washed three times with Phosphate Buffer Saline (PBS) made in DEPC-treated water. Cells were scraped using one mL of Trizol and RNA isolated using manufacturer’s instruction. RNA was resuspended in 20 μL DEPC containing water, quantified, and checked for the quality of the RNA obtained. The cDNA was synthesized using two μg of the obtained RNA for a 20 μL reaction using iScript™ Reverse Transcription Supermix for RT-qPCR (BIO-RAD, Hercules, CA, USA). qRT-PCR reactions were prepared using FastStart SYBR Green Master Mix (Roche; Indianapolis IN, USA) and performed using the BIO-RAD Connect machine. BIO-RAD RFX Manager 3.1 software was used to run the analysis. We have listed the details of primer sets used for the study in Additional file 1. For real-time PCR, mean Ct values of the target genes were normalized to mean Ct values of the endogenous control, Hypoxanthine-guanine phosphoribosyltransferase(HPRT); [−∆Ct = Ct (HPRT) – Ct (target gene)]. We calculated the ratio of mRNA expression of target genes versus HPRT (2(−∆Ct)). Melting curve analysis was performed to check the specificity of the amplified product. We resolved the amplified cDNA on EtBr containing 1% agarose gels.
Western blot analysis
We carried out Western Blot analysis as described previously . Membranes were incubated with following primary antibodies for overnight at 4 °C: SEMA5A (Invitrogen; 1:1000), anti-E-cadherin (E-cad) (HECD1; 1:100), anti-N-cadherin (N-cad) (1:100), anti-β-catenin (1:200), anti-SNAIL (Abcam; 1:500), anti-GAPDH (Santa Cruz, 1:1000) and anti-β-actin (Sigma, 1;1000). PI3K (Sigma, 1:1000), p-PI3K (p85Tyr458-p55Tyr199, Cell Signaling, 1;750), AKT (Cell Signaling 1:1000), p-AKT (Ser473, Cell Signaling, 1:1000), GSK3β (Cell Signaling, 1:1000) p-GSK3β (Ser9, Cell Signaling 1:1000). Anti-E-cad, anti-N-cad, and anti-β-catenin were generous gift from Dr. Keith Johnson, University of Nebraska Medical Center; UNMC, Omaha, NE, USA. The membrane was incubated with secondary horseradish peroxidase antibody (mouse (Sigma), 1:5000, rabbit (Thermo Scientific); 1:5000) for an hour at room temperature. We utilized NIH Image J Software for quantification of the intensity of the bands, of our protein of interest and their respective loading control. We also normalized the bands to the Control cells used in the study.
We plated cells (5 × 105) on 22 × 22 mm sized coverslips (Fisherbrand ®, Pittsburgh, PA) placed in 6-well plates and the protocol is described previously in detail . We used primary antibodies like, 1:25; E-cad, 1:5; N-cad, 1:5; β-catenin, 1:10; Fascin (Developmental Studies Hybridoma Bank) 1:20; and phalloidin (Texas Red, Molecular Probes) 1:50, and Secondary antibodies conjugated with either mouse or rabbit Cy3 (Jackson Immuno Research). Immunofluorescence localization was determined using either a Nikon Eclipse E800 Fluorescent microscope or LSM 710 Zeiss Confocal Microscope.
We used the same number of cells as described above for quantification of Lamellipodium and Filopodium; actin cytoskeleton was stained using Texas Red Phalloidin for 20 min. We quantified the number of peripheral cells in a colony, Lamellipodia, and Filopodia with a free edge in 5 different random fields in the same coverslip at 630× resolution using LSM 710 Zeiss Confocal Microscope for T3M-4-Control and T3M-4-shSEMA5A cells. Similarly, for CD18/HPAF-Control and SEMA5A knockdown cells, we have used Nikon Eclipse E800 fluorescent microscope at 400× resolution and counted different colonies with a number of cells at periphery and lamellipodia in seven different random fields. We determined the average number of Lamellipodia per cell.
Cell motility assays
We analyzed cellular motility using Wound Healing assay and Transwell Migration assay. For Wound Healing assay, cells (0.2 × 106 for T3M-4-cells and 0.5 × 106 for CD18/HPAF cells) were plated per well in 12-well plate. The following day, the cells had reached 90–95% confluency. A wound was generated using a one mL pipette tip. Cells were washed with HBSS and incubated with a fresh serum-free medium for 24 h. Cells were photographed under an inverted TS100 microscope (Nikon, Melville, NY) at a 40× magnification at time T = 0 h and T = 24 h. The distance was evaluated at time 0 h (T0) and 24 h (T24) using Image J software (NIH, Bethesda, MD). The percent of distance migrated was calculated using the formula: 100 x[(T0)–(T24)]/ (T0).
For Transwell Migration assay, we plated 0.2 × 106 cells/well in the top chamber of noncoated polyethylene terephthalate membranes (six-well insert, 8 μm pore size; Becton Dickinson, Franklin Lakes, NJ) with the bottom chamber containing 1.0 ml of serum-free media. The cells were incubated overnight at 37 °C. After removal of non-migrated cells, membrane pores were stained using Hema 3 kit (Fisher Scientific Company L.L.C., Kalamazoo, MI) as per the manufacturer’s instructions. Cells were counted in ten random fields (100x) and expressed as the average number of cells per field of view. Representative data is the average of independent experiments.
Cell viability assay
We seeded cells in 96-well plates at low density (2000–4000 cells/well). Following overnight adherence, we incubated the cells with media alone or medium containing different serum concentrations (0–5%) for 24 or 48 h. We determined cell viability using MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, EMD Millipore Corp, Burlington, MA) reagent as described previously in . Data presented as absorbance at 570 nm.
Propidium iodide staining
Seeded 1X106 cells in a 6-well plate for overnight. Following morning, cells were harvested, washed in PBS and fixed in cold 70% ethanol. Cells were added drop-wise to the 70% ethanol solution while vortexing to ensure homogenous fixation with minimizing clumping of cells. Fixed the cells for 30 min at 4 °C. Cells were washed twice in PBS and centrifuged at a speed of 850 g. We resuspended the cells in 500 μl Propidium Iodide/Triton X-100 staining solution. (Freshly prepared 5 mL of 0.1% Triton X-100 (Sigma), 1 mg DNAse free RNase A (Sigma) and 0.2 mL of 50 μg/mL stock solution of Propidium Iodide (Roche). Incubated cells for 30 min at room temperature protected from light. Data were acquired using FACSCalibur.
We performed this procedure as described . We plated T3M-4- shSEMA5A and -Control cells in 12-well dishes. The following day, we transiently transfected the cells with the TOP-flash or FOP-flash vector (0.5 μg/well) and a renilla vector as a transfection control (0.1 μg/well), n = 6 per cell line and vector. We obtained vectors from Promega (Promega, Madison, WI). Two days later, we assayed the cells for firefly/renilla luciferase using the Dual-Glo reagent (Promega), per the manufacturer’s instruction.
Tumorigenic and metastasis assays
We purchased female athymic BALB/c nude mice (NCI-nu, 6–8 weeks old) from the National Cancer Institute. The Institutional Animal Care and Use Committee of the University of Nebraska Medical Center approved all the animal experiments performed in this. Cells were trypsinized and harvested for preparation of in vivo injection. We tested the viability of cells using trypan blue exclusion assay and used single cell suspensions of > 95% viability for injection.
Mice were injected subcutaneously into the lateral flank with 106 cells suspended in 0.05 mL of HBSS for tumorigenic assay. We monitored tumor growth and euthanized animals when they were 10-weeks old. Tumors were measured with calipers twice a week. We calculated tumor volume (V) by taking a square of short diameter (W) multiplied by half of the long diameter (L) of the tumor (V = W2*L/2). Tumor tissues were harvested and processed for further analysis.
For studying tumor growth as well as metastasis, tumor cells were injected orthotopically into the pancreas of the mice. Mice were anesthetized, and we created a small incision for the injection of pancreatic tumor cells. We injected T3M-4 (5 X 105 cells/0.05 mL HBSS) and CD18/HPAF (2.5 X 105/0.05 mL HBSS) cells into the anterior lobe of the pancreas using a 1/2-cc U-100 insulin disposable syringe (EXEL International Inc., Los Angeles, CA). The appearance of a fluid bleb indicated a successful subcapsular intrapancreatic injection of tumor cells without leakage. The abdominal muscular layer was closed using 4–0 or 5–0 vicryl or polydioxanone (PDS), and the outer skin layer was closed with stainless steel wound clips. The animals tolerated the surgical procedure well, and no anesthesia-related deaths occurred. We removed wound clips at approximately 14 days post-surgical procedure. Mice were monitored for tumor growth and killed after 21 days post-injection. Primary tumors and metastases were resected and analyzed.
We performed immunohistochemistry as described previously . In brief, we incubated slides with αPCNA, (1:100; Santa Cruz, CA); or αSEMA5A  (1:50) primary antibodies in antibody diluent overnight at 4 °C. Next morning, we washed the slides and incubated with biotinylated anti-rabbit or anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) for 45 min. We quantitated the cell number for a particular stain by counting at least five different random fields in the same section at high resolution using a Nikon microscope.
The significance of the data was determined by the Student’s t-test (two-tailed) for all in vitro studies. Comparisons between different mice groups were evaluated using Mann-Whitney U-test. The p < 0.05 was deemed significant. All statistical analyses were done using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA).