br between weeks of age In
between 16-20 weeks of age. In some studies, KPf/fC-derived tumor Nocodazole were transplanted into the ßanks of immunocompetent littermates between 5-8 weeks of age. Littermate recipients (WT or REM2-LSL-KrasG12D/+, ; Trp53f/f or Trp53f/f mice) do not develop disease or express Cre. NOD/SCID and NSG mice were enrolled in ßank transplantation studies between 5 to 8 weeks of age; KPf/fC derived cell lines and human FG cells were transplanted subcutaneously for tumor propagation studies in NOD/SCID recipients and patient-derived xenografts and KPf/fC derived cell lines were transplanted subcutaneously in NSG recipients as described in detail below.
Human and mouse pancreatic cancer cell lines
Mouse primary pancreatic cancer cell lines and organoids were established from end-stage, treatment-naive KPR172HC and WT- and REM2-KPf/fC mice as follows: tumors from endpoint mice (10-12 weeks of age for KPf/fC or 16-20 weeks of age for KPR172HC mice)
were isolated and dissociated into single cell suspension as described below. Cells were then either plated in 3D sphere or organoid culture conditions detailed below, or plated in 2D in 1x DMEM containing 10% FBS, 1x pen/strep, and 1x non-essential amino acids. At the Þrst passage in 2D, cells were collected and resuspended in HBSS (GIBCO, Life Technologies) containing 2.5% FBS and 2 mM EDTA, then stained with FC block followed by 0.2 mg/106 cells anti-EpCAM APC (eBioscience). EpCAM+ tumor cells were sorted then re-plated for at least one additional passage. To evaluate any cellular contamination and validate the epithelial nature of these lines, cells were analyzed by ßow cytometry again at the second passage for markers of blood cells (CD45-PeCy7, eBioscience), endothe-
lial cells (CD31-PE, eBioscience), and Þbroblasts (PDGFR-PacBlue, Biolegend). Cell lines were derived from both female and male KPR172HC and WT- and REM2-KPf/fC mice equivalently; both sexes are equally represented in the cell-based studies outlined below.
Functional studies were performed using cell lines between passage 2 and passage 6. Human FG cells were originally derived from a PDAC metastasis and have been previously validated and described (Morgan et al., 1980). Patient-derived xenograft cells and orga-noids were derived from originally-consented (now deceased) PDAC patients and use was approved by UCSDÕs IRB; cells were de-identiÞed and therefore no further information on patient status, treatment or otherwise, is available. FG cell lines were cultured in 2D conditions in 1x DMEM (GIBCO, Life Technologies) containing 10% FBS, 1x pen/strep (GIBCO, Life Technologies), and 1x non-essential amino acids (GIBCO, Life Technologies). 3D in vitro culture conditions for all cells and organoids are detailed below.
Patient cohort for PDAC tissue microarray
The PDAC patient cohort and corresponding TMAs used for RORg immunohistochemical staining and analysis have been reported previously (Wartenberg et al., 2018). Patient characteristics are detailed in Table S6. Brießy, a total of 4 TMAs with 0.6 mm core size was constructed: three TMAs for PDACs, with samples from the tumor center and invasive front (mean number of spots per
patient: 10.5, range: 2-27) and one TMA for matching PanINs (mean number of spots per patient: 3.7, range: 1-6). Tumor samples from 116 patients (53 females and 63 males; mean age: 64.1 years, range: 34-84 years) with a diagnosis of PDAC were included. Matched PanIN samples were available for 69 patients. 99 of these patients received some form of chemotherapy; 14 received radio-therapy. No sexual dimorphism was observed in any of the parameters assessed, including overall survival (p = 0.227), disease-free interval (p = 0.3489) or RORg expression in PDAC (p = 0.9284) or PanINs (p = 0.3579). The creation and use of the TMAs were re-viewed and approved by the Ethics Committee at the University of Athens, Greece, and the University of Bern, Switzerland, and included written informed consent from the patients or their living relatives.