TP53 Gene

HEK293T, MIA-PaCa-2, BxPC-3, and PANC-1 cell lines were obtained from the American Type Culture Collection (ATCC). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (#S11150; FBS, Atlanta Biologicals), antibiotics (#P4458; Gibco) and L-glutamine (#17921004; Corning). The murine pancreatic cancer cell line KPC1 was originally described in our recent publication (Parajuli et al., 2018 (link)). The cell line was established from a KP53 mouse, which harbored KrasG12D and one conditional allele of Trp53 (LSL-KrasG12D;LSL-Trp53fl/+;Pdx1-Cre). Freshly isolated specimen from the KP53 mouse with terminal PDAC was gently dissected, minced with scissors, and digested with Dispase II at 2.4 U/ml (#4942078001; Sigma-Aldrich) and Collagenase D at 0.5 mg/ml (#11088858001; Sigma-Aldrich) for 1 h at 37°C in an atmosphere of 5% CO2. Then, cells were washed three times with PBS, suspended in RPMI 1540 containing 20% FCS, and seeded on fibronectin-coated plates. Cell colonies were subsequently passaged by trypsinization, pooled, and propagated in DMEM supplemented with 10% FBS, antibiotics, and L-glutamine. To generate the PANC-1-SMAD4KO and PANC-1-SMAD2/3KO cell lines, cells were transduced with the corresponding lentiCRISPRV2-gRNA lentiviruses, selected with puromycin (for SMAD4) or hygromycin (for SMAD2/3), and all resistant clones were pooled and expanded as a single population. Lentiviruses were produced by transfecting HEK293T cells with lentiviral constructs and the One-Step Lentivirus Packaging System as described by the manufacturer (#631275; Takara). Lentiviral particles in the conditioned media were harvested after a period of 48–72 h. The conditioned media were then cleaned of cell debris by centrifugation at 5,000×g for 15 min, filtered through a 0.45-μm filter, and used immediately for cell transduction.

Freshly resected or thawed cryopreserved (0.5 g tissue as 3-4 mm pieces/cryotube with 1 ml Recovery Cell Culture Freezing Medium (Gibco, Grand Island, NY, US)) tumors were minced with a scalpel and digested with 2 mg/ml collagenase IV and 100 μg/ml DNAse (both from Sigma-Aldrich, St.Louis, MO, USA) dissolved in advanced DMEM/F12 supplemented with Glutamax, HEPES and Penicilin/Streptomycin (concentrations/producers specified in Supplementary Table S1). The digestion was performed at 37°C on rotation for up to 1 h. Where indicated, additional mechanical dissociation using the gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) at “m-imp Tumor 03” settings were applied. The dissociated tissue suspension was diluted with phosphate-buffered saline (PBS)/1% bovine serum albumin (BSA) (both Sigma-Aldrich) and centrifuged at 18g for 4 min. The pellet was re-suspended and centrifuged again first at 32g, then at 200g for 4 min. The cell viability was monitored by staining aliquots with 0.2% trypan blue (NanoEntek, Seoul, Korea). Majority of dead cells remained in the supernatants, while the final pellet consists of a mixture of viable single cells and small non-disrupted tissue fragments. The final pellet was re-suspended in breast cancer organoid medium (OM) described by Sachs et al. (12 (link)) (specified in Supplementary Table S1).
Additional steps to remove normal mouse cells included plating re-suspended pellet in 24-well plates treated with anti-adherence Rinsing Solution (Stemcell Technologies, Cambridge, UK) and culturing in OM supplemented with 5 μM of the MDM2 inhibitor Nutlin-3 (Cayman, Ann Arbor, MI, USA), further called OM+. Nutlin-3 induces death in cells with wild-type TP53 i.e. normal cells, while tumor cells with lost/mutated TP53 stay viable (11 (link)). The PDXs used in this study harbor a mutation of the TP53 gene (18 (link), 19 (link)). Therefore, the tumor tissue can be subjected to Nutlin-3 selection for enrichment of cancer cells. Subsequently, the tissue suspension was filtered through a 100 µm cell strainer to collect fragments below this size that were further sedimented for 2-5 min. The resulting fragment-enriched pellet was used for establishment of PDXCs.