Panc 1

Established PDAC cell lines PANC-1, MIA Paca-2, and BxPC-3, as well as cells isolated from pancreatic cancer patient-derived xenografts (4666, 5160, 6052, 4911, 4833, 5641, 6105, 4535, 6164, and 4041) were used in this study. PANC-1 (CRL-1469), MIA Paca-2 (CRL-1420), and BxPC-3 (CRL-1687) were purchased from ATCC (Manassas, VA, USA). PDX-derived PDAC cell lines were provided by the Pancreatic Cancer SPORE Tissue and Cell repository (Mayo Clinic, Minnesota). The procedure for isolation of these cells from PDAC-derived xenografts has been described elsewhere [82 ,83 (link),85 (link),86 (link),105 (link)]. PANC-1, MIA Paca-2, and BxPC-3 cells were routinely expanded in Improved Minimum Essential Medium (IMEM, Gibco, Waltham, MA) supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 1 % L-glutamine, 1 % sodium pyruvate, 1 % penicillin-streptomycin. BxPC-3 cells were maintained in RPMI1640 medium supplemented with 10 % FBS and 1 % L-glutamine (Gibco, Waltham, MA). PDX-derived PDAC cell lines were cultured in DMEM/F12 medium supplemented with 10 % FBS and 1 % L-glutamine (Gibco, Waltham, MA).
Cells were grown at 37 °C in a humidified atmosphere containing 5 % CO₂. Cells grown to 70–80 % confluence were used at the beginning of all the experiments. Cell lines were authenticated by STR profiling, both performed by the manufacturer and confirmed in-house at the time of purchase according to ATCC guidelines. Cells were passaged by starting a low-passage cell stock every month until to 2–3 months after resuscitation. Cell lines were screened for mycoplasma contamination using a MycoAlert Mycoplasma Detection ELISA (Lonza, Basel Tower, Switzerland) prior to experimentation and were intermittently tested thereafter.

BxPC-3, CFPAC-1, HPAF-II, MIA PaCa-2, and PANC-1 were purchased from ATCC (Manassas, VA). AsPC-1 and Capan-2 were generously provided by Dr. Martin Fernandez-Zapico (Mayo Clinic, Rochester, MN). Pan02 was obtained from the Division of Cancer Treatment and Diagnosis Tumor Repository of the National Cancer Institute, was STR profiled and tested negative for Mycoplasma. Cells were maintained in appropriate cell culture media (AsPC-1 and BxPC-3 – RPMI, CFPAC-1 – IMDM, HPAF-II – EMEM, MIA PaCa-2 – DMEM +2.5% Horse Serum, PANC-1 – DMEM) containing 10% fetal bovine serum (FBS) (R&D Systems; Minneapolis, MN) and 1% penicillin-streptomycin (Thermo Fisher; Waltham, MA) in a 37°C, 5% CO₂ incubator (HeraCell; Thermo Fisher). Experiments were performed on cells having undergone less than 20 passages. PDX lines (6164, 6741, 6413, 6182) were a generous gift from Dr. Debabrata (Dev) Mukhopadhyay (Mayo Clinic Jacksonville, FL). Lines were maintained in DMEM-F12 (Gibco) containing 20% FBS (R&D Systems, Minneapolis, MN) and 1% penicillin-streptomycin (Thermo Fisher, Waltham, MA) in a 37°C, 5% CO₂ incubator (HeraCell, Thermo Fisher). PDX cells were not passaged more than six times prior to experimental use.

The following human PDAC cell lines were used: CFPAC1 (KRAS G12V; ATCC CRL-1918) and PANC1 (KRAS G12D; ATCC CRL-1469), were used for the following experiments. CFPAC1 cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM; Euroclone) + 10% fetal bovine serum (FBS) while PANC1 were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Euroclone) + 10% fetal bovine serum (FBS). Media were all supplemented with 2 mM L-glutamine. The cell line was authenticated by the IEO Tissue Culture Facility using the GenePrint10 System (Promega) and was routinely screened for Mycoplasma contamination.Irinotecan (Sigma-Aldrich, I1406) and Etoposide (Sigma-Aldrich, E1383) were diluted in DMSO and used at 3 different concentrate ions for the cell viability experiments. No commonly misidentified cell lines were used in the study.

Human pancreatic cancer cell lines, including PANC-1, MiaPaCa-2, AsPC-1, T3M4, Patu8988, and normal pancreatic duct cells (hTERT-HPNE), were purchased from the American Type Culture Collection (ATCC). All cells were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin solution (Gibco, USA). The cells were maintained under normoxic conditions in a cell culture incubator (ThermoFisher, USA) with 5% CO2 and 20% O2. To construct DNASE1L3 overexpression stable cell lines, overexpression lentivirus and a negative control were acquired from Genechem (Shanghai, China). The DNASE1L3 gene (NM_004944.4) was sourced from Genechem’s cDNA library. The lentiviral vector plasmid GV492 (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin), along with the DNASE1L3 gene sequence, was digested using AgeI and BamHI restriction enzymes, followed by cloning through the In-fusion recombination method. The recombinant vector was confirmed by DNA sequencing.
Lentivirus production involved transfecting the viral vectors and two helper plasmids, psPAX2 and pMD2.G, into 293T cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Seventy-two hours post-transfection, infectious lentiviruses were harvested, centrifuged to remove cell debris, and filtered through 0.45 μm cellulose acetate filters. The virus titer, determined by fluorescence-activated cell sorting of GFP-positive 293T cells, was approximately 2.5 × 10^8 transducing units (TU)/mL, and stored at -80 °C for further use.