Silencing of Neuropilins and GIPC1 in pancreatic ductal adenocarcinoma exerts multiple cellular and molecular antitumor effects

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality, with new treatment options urgently needed. Neuropilins-1/-2 (NRP1, NRP2) are receptors for semaphorins and angiogenic growth factors, while the GAIP interacting protein C-terminus 1 (GIPC1, aka Synectin) interacts with the neuropilins. They are overexpressed in PDAC and associated with poor survival as well as tumor-promoting activities. Thus, neuropilin and/or GIPC1 silencing may inhibit PDAC growth. In this study, we directly compare the various tumor-inhibitory effects of transient RNAi-mediated depletion of NRP1, NRP2 and GIPC1, alone or in combination, in a set of cell lines with different expression levels. Inhibition of anchorage-dependent and –independent proliferation, colony formation and cell migration, alterations of 3D-spheroid size and shape as well as retardation of cell cycle and induction of apoptosis have been analyzed and found to vary between cell lines. The observed effects are independent of initial expression levels. Knocking down NRP1, NRP2, and GIPC1 alone demonstrates significant effects. Only small additive effects upon combined knockdown and no counter-upregulation of the respective other genes could be detected. Making the study more translational, we show that systemic treatment of PDAC xenograft-bearing mice with polymeric nanoparticles for delivery of specific siRNAs results in tumor inhibition, reduces proliferation, and induces apoptosis. In conclusion, NRP and GIPC1 inhibition emerges as a promising avenue in PDAC treatment due to pleiotropic tumor-inhibitory effects.


Soft agar assay
For soft agar assays, cells were seeded in 12 well plates 24 h prior to transfection. 48 h after transfection, cells were trypsinized (Trypsin-EDTA; Biowest, Nuaillé, France) to obtain a single cell suspension and counted. For soft agar preparation, an autoclaved 2.4% agar solution (Carl Roth, Karlsruhe, Germany) and cell culture media were placed in a water bath to reach 42°C. A mixture of 12.5 mL agar solution, 1.25 mL MEM (10x) and 37.5 mL RPMI (10% FCS) were pipetted as bottom layer into a 6-well plate. For the top layer, 1 mL of a 6 x 10 4 cells/mL single cell suspension was mixed with 1.5 mL of the agar mixture, and 750 µL/well of this mixture was directly pipetted onto the bottom agar. Plates were incubated for 2 -4 weeks and colonies > 50 µm in diameter were counted by 2 blinded investigators.

Colony formation assay
For colony formation assays, 1 x 10 3 cells in 2 ml culture medium were seeded in triplicates in a 6well plate and cultivated for 7 -9 days. Subsequently, cell culture medium was removed and cells were washed with 1 mL PBS, prior to adding 1 mL clonogenic reagent (0.5% methylene blue (w/v) in 50:50 (v/v) ethanol/water) to each well and incubating at room temperature for 45 min. Cells were washed at least 3 times with water prior to image acquisition under the microscope. Images were analyzed with the ImageJ software for quantitation of blue stained cells.

Scratch Assay
3.5 x 10 5 Panc89 cells / well were seeded in a 6-well plate 24 h prior to transfection. After transfection with 10 nM siRNA, the cells were further cultivated under standard conditions, and when they reached 100% confluency after 2 days, a scratch in the cell layer was introduced using a 200 µL tip. The cell layer was washed twice with PBS and once with cell culture medium to remove floating cells, prior to adding 2 mL cell culture medium to each well. The plate was placed in a microscopic chamber set to 37°C and 5% CO2, and cell invasion into the scratches was documented automatically over 24 h at three different positions per scratch via a microscope, with pictures taken every 5 min. The percentage of scratch closure over time was quantitated using the ImageJ software and calculated relatively to t=0.

Caspase Assay and Annexin V staining
For caspase assays, cells were seeded and transfected as described above, and after 48 -96 h 50 µL of the caspase-3/7-Glo reagent, diluted 1:10 or 1:5 in serum free medium depending on the cell line, was added to each well. After 1 h of incubation at room temperature in the dark, luminescence was measured by using a Fluostar Optima reader (BMG Labtec, Ortenberg, Germany). To normalize for different cell densities upon transfection, a WST-1 assay for quantitating viable cells was run in parallel on the same plate. WST-1 was diluted 1:10 in serum free medium and 50 µL of this mixture was added to each well. After incubation at 37°C and 5% CO2 for 1 h, the plates were measured at 450 nm in a DigiScan plate reader (Asys Hitech, Eugendorf, Austria). By calculating the ratio caspase activity / WST-1 signal, the caspase signal was adjusted to the cell density.
For Annexin V staining, cells were transfected as described above and harvested at different time points (48 -96 h), prior to washing twice in ice cold PBS, resuspending at 1 x 10 6 cells/mL in 1x annexin binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4) and addition of 5 µL of annexin V conjugate to each 100 µL cell suspension. During a 15 min incubation at room temperature, 5 µL propidium iodide (PI) solution (1 mg/mL) was diluted in 395 µL 1x annexin binding buffer per sample. 400 µL PI solution was added to each reaction tube and the cell suspension was incubated for further 5 min on ice in the dark. Samples were kept on ice until measurement in an Attune ® Acoustic Focusing Cytometer (Thermo Scientific, Schwerte, Germany).

Cell Cycle Analysis
At 2 -3 days post transfection, cells were treated for 14 -20 h, depending on the cell line, with 25 ng/mL nocodazole in standard cell culture medium. Cells were harvested, washed once with PBS and fixed with 1 mL cold 70% ethanol for at least 1 h at -20°C. After fixation, the cells were spun down (1,000 rpm, 5 min) and washed once with ice-cold PBS, prior to resuspension in 50 µg/mL RNaseI solution in PBS and incubation for 1 h at 37°C. During incubation, 5 µL propidium iodide (PI; 1 mg/mL) were diluted in 345 µL PBS for each sample. The PI solution was added to the reaction tube and the cell suspension was incubated for further 5 min on ice in the dark. Samples were kept on ice until measured in an Attune® Acoustic Focusing Cytometer. The cell cycle distribution was determined using the Attune® Cytometric Software.

Western blotting
Lysates were prepared from cells in 6-well plates by adding 100 µl RIPA buffer and were homogenized by drawing the lysate up and down through a needle, using a 1 mL syringe. After centrifugation at 10,000 rpm for 10 min at 4°C, the supernatant was transferred to a new tube. Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's protocol and calculated from a bovine serum albumin standard curve.
For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), samples containing 40µg protein were mixed with NuPAGE LDS sample buffer (Thermo Scientific) containing 5% βmercaptoethanol (Sigma-Aldrich), heat denatured and loaded onto a precast NuPAGE 4-12% Bis-Tris gradient gel (Life Technologies) placed in an XCell SureLock Mini-Cell system (Invitrogen). After electrophoresis in NuPage MOPS buffer (Life Technologies) at 4°C for 3 hours at 120V, bands were electroblotted onto a methanol activated PVDF membrane (Bio-Rad, Munich), using a PROTEAN Tetra Cell transfer system (Bio-Rad) for wet blotting, with NuPage blotting buffer (Life Technologies) containing 20% methanol. The blotting was performed at 80 V for 3 h or at 20 V overnight at 4°C.
After blotting, the membrane was washed once with TBS-T (Tris-buffered saline (pH 7.4) + 0,05% Tween-20) and incubated for 1 h in 5 % milk powder in TBS for blocking of non-specific binding sites. Primary antibodies were diluted in 5 mL blocking solution as detailed in Suppl. Table 3 and used for membrane overnight incubation at 4°C. After washing three times with TBS-T, membranes were incubated in the secondary antibody dilution for 2 h at 4°C, followed by three additional washing steps. ECL substrate or, for higher sensitivity, Dura substrate (both Thermo Scientific) were used for visualization of protein bands. After exposure of X-ray films (GE Healthcare), bands were quantitated using the ImageJ software. Attached also scans of original full immunoblots.