Establishment and Characterization of PDAC Cell Lines

Low-passage patient-derived PDAC cell lines (fewer than 35 passages from patient tumors) and pancreatic cancer-associated fibroblasts used in this project were received from Dr. Anirban Maitra (The Johns Hopkins University) and maintained as previously described^{15 (link),22 (link),38 (link),39 (link)}. Cells were submitted for STR analysis (CellCheck with IDEXX BioResearch) and were regularly confirmed to be free of mycoplasma contamination. Cell lines were passaged fewer than 12 times before resuscitating fresh stocks. Hypoxic conditions in monolayer experiments were generated in a Ruskinn Invivo₂ 200 hypoxia work station (Baker Ruskinn; Sanford, ME) at 0.2% oxygen. Cell proliferation and viability in monolayer cultures was measured with alamarBlue assay as previously described^{15 (link),39 (link)}. Growth of 3-dimensional tumor spheroid cultures was performed and quantified as described previously using tumor cells stably transduced with TdTomato (red), and CAFs stably transduced with EGFP (green) to differentiate the two cell types in 3D co-cultures and track the growth of each over time^{15 (link),40 (link),41 (link)}.