Cls3471 24ea

Pancreatic tumors were dissected with a scalper, cut into small pieces and incubated with collagenase D/HBSS (5mg/ml) (Roche #11088866001) for half an hour at 37°C. Collagenase D was then deactivated using culture medium DMEM (Gibco #41965-039), containing 10% fetal bovine serum (FBS) (Gibco #10270106). The cell suspension was successively applied to strainers of 100μm, 70μm and 40μm pores diameter. Then, the suspension was centrifuged, the pellet was resuspended in culture medium DMEM/F12 containing GlutaMAX (Gibco #31331028) and B-27 supplement (Gibco #17504-044) and finally seeded on ultra-low attachment plates (MilliporeSigma #CLS347124EA). Three days later, the cells were seeded to cell culture dishes (Greiner #664160) with DMEM (GIBCΟ #41965-039) containing 10% FBS and 1% L-glutamine (Gibco #25030-024). FibrOut 0.2% (VWR #10786-022) was added to the cell cultures for 6 days. Cells were then cultured for a maximum of 3 passages and were used for downstream applications.

Pancreatic cancer tissue was dissected into small pieces with a scalper and incubated in collagenase D/HBSS (5 mg/mL) (Roche #11088866001) for 30 min at 37 °C. Collagenase D was deactivated after addition of culture medium DMEM (Gibco #41965-039) containing 10% fetal bovine serum (FBS) (Gibco #10270106). Cell suspension was applied successively to cell strainers with 100, 70 and 40 μm pore diameters. Cells were then centrifuged, resuspended in culture medium DMEM/F12 containing GlutaMAX (Gibco #31331028) and B-27 supplement (Gibco #17504-044) and seeded on ultra-low attachment plates (MilliporeSigma #CLS347124EA). After 3 days, cells were harvested and seeded to cell culture dishes (Greiner #664160) with culture medium DMEM (GIBCO #41965-039) containing 10% FBS and 1% L-glutamine (Gibco #25030-024). FibrOut (VWR #10786-022) at a concentration of 0.2% was added to the culture media for 6 days. Cells were cultured for a maximum of 3 passages and were then either harvested for protein isolation or used for invasion and migration assays.

Neural progenitor cells were seeded at 250 cells/ml in neural stem cell medium in ultra-low attachment 6-well plate (CLS3471-24EA, Sigma). 25ng/ml PTN, 10ng/ml PDGF or the combination of PTN and PDGF were added every second day. On day 6, microscopic images of all spheres were taken using a phase contrast microscope to allow size quantification. Then the spheres were fixed with 4% PFA on ice for 10 min, and embedded in OCT medium (Tissue-Tek Sakura) for frozen section. Immunostaining was performed on 7 μm sections. Slides were blocked with 3% bovine serum albumin (Roche Diagnostics) in phosphate-bufferd saline (PBS) and incubated with primary antibody (Table S4) diluted in blocking solution for 2 hours, followed by secondary antibody and nuclear staining with Hoechst 33342 (2μg/ml; Sigma).