br METHOD DETAILS br In vitro and in vivo experimental
In vitro and in vivo experimental strategies
Tissue dissociation, cell isolation, and FACS analysis
Mouse pancreatic tumors were washed in MEM (GIBCO, Life Technologies) and cut into 1-2 mm pieces immediately following resec-tion. Tumor pieces were collected into a 50 mL Falcon tube containing 10 mL GeyÕs balanced salt solution (Sigma), 5 mg Collagenase P (Roche), 2 mg Pronase (Roche), and 0.2 mg DNase I (Roche). Samples were incubated for 20 minutes at 37 C, then pipetted up and down 10 times and returned to 37 C. After 15 more minutes, samples were pipetted up and down 5 times, then passaged through a 100 mm nylon mesh (Corning). Red blood cells were lysed using RBC Lysis Buffer (eBioscience) and the remaining tumor cells were washed, then resuspended in HBSS (GIBCO, Life Technologies) containing 2.5% FBS and 2 mM EDTA for staining, FACS analysis, and cell sorting. Analysis and cell sorting were carried out on a FACSAria III machine (Becton Dickinson), and data were analyzed with FlowJo software (Tree Star). For analysis of cell surface markers by ßow cytometry, 5x105 cells were resuspended in HBSS contain-ing 2.5% FBS and 2 mM EDTA, then stained with FC block followed by 0.5 mL of each antibody. For intracellular staining, cells were Þxed and permeabilized using the BrdU ßow cytometry kit (BD Biosciences); Annexin V apoptosis kit was used for analysis of apoptotic cells (eBioscience). The following rat Pimozide were used: anti-mouse EpCAM-APC (eBioscience), anti-mouse CD133-PE (eBioscience), anti-mouse CD45-PE and PE/Cy7 (eBioscience), anti-mouse CD31-PE (BD Bioscience), anti-mouse Gr-1-FITC (eBioscience), anti-mouse F4/80-PE (Invitrogen), anti-mouse CD11b-APC (Affymetrix), anti-mouse CD11c-BV421 (Bio-legend), anti-mouse CD4-FITC (eBioscience) and CD4-PaciÞc blue (Bioglegend), anti-mouse CD8-PE (eBioscience), anti-mouse IL-17-APC (Biolegend), anti-mouse BrdU-APC (BD Biosciences), and anti-mouse Annexin-V-APC (eBioscience). Propidium-iodide (Life Technologies) was used to stain for dead cells.
In vitro growth assays
We describe below the distinct growth assays used for pancreatic cancer cells. Colony formation is an assay in Matrigel (thus
adherent/semi-adherent conditions), while tumorsphere formation is an assay in non-adherent conditions. We have found that cell types from different sources grow better in different conditions. For example, the murine KPR172H/+C and the human FG cell lines grow much better in Matrigel, while KPf/fC cell lines often grow well in non-adherent, sphere conditions (though they can also grow in Matrigel).
Pancreatic tumorsphere formation assay
Pancreatic tumorsphere formation assays were performed and modiÞed from Rovira et al. (2010). Brießy, low-passage (< 6 passages) WT or REM2-KPf/fC cell lines were infected with lentiviral particles containing shRNAs; positively infected (red) cells were sorted 72 hours after transduction. 100-300 infected cells were suspended in tumorsphere media: 100 mL DMEM F-12 (GIBCO, Life Technol-ogies) containing 1x B-27 supplement (GIBCO, Life Technologies), 3% FBS, 100 mM B-mercaptoethanol (GIBCO, Life Technologies), 1x non-essential amino acids (GIBCO, Life Technologies), 1x N2 supplement (GIBCO, Life Technologies), 20 ng/ml EGF (GIBCO, Life Technologies), 20 ng/ml bFGF2 (GIBCO, Life Technologies), and 10 ng/ml ESGRO mLIF (Thermo Fisher). Cells in media were plated in 96-well ultra-low adhesion culture plates (Costar) and incubated at 37 C for 7 days. KPf/fC in vitro tumorsphere formation studies were conducted at a minimum of n = 3 independent wells per cell line across two independent shRNA of n = 3 wells; however, the majority of these experiments were additionally completed in > 1 independently-derived cell lines n = 3, at n = 3 wells per shRNA. shRNA sequences and average knockdown efÞciencies are available in Table S7.