Mia paca 2

Normal and PDAC tissues were collected and obtained from the Department of Histopathology, Royal London Hospital (London, UK), Department of Pathology and Forensic Medicine (Osijek, Croatia) and from European Pancreas Centre Research Laboratory at the University of Heidelberg (Heidelberg, Germany). All tissues were obtained with full ethical approval from the respective Institutional Boards and used in accordance with the Human Tissue Act 2004.
PDAC cell lines (MiaPaca2, BxPC3, and Capan1) were obtained from Cancer Research UK Tissue Culture Service and routinely cultured in Dulbecco's modified Eagle's medium, DMEM (Invitrogen, Paisley, UK) supplemented with 10% heat‐inactivated fetal bovine serum (FBS) (Autogen Bioclear, Wiltshire, UK) and penicillin/streptomycin at 37 °C in 5% CO₂. PC12 cells, a rat pheochromocytoma cell line, were a kind gift from Dr Lesley Robson (Blizard Institute, QMUL London) and were grown in poly‐d‐lysine hydrobromide (PDL; MW 30 000–70 000, Sigma, Dorset, UK P7886) coated tissue culture flasks in RPMI media supplemented with 10% heat‐inactivated horse serum, 5% heat‐inactivated FBS, and penicillin/streptomycin at 37 °C in 5% CO₂. The identity of the cell lines utilized was confirmed by STR profiling.

Porcine kidney tubular epithelium nucleoside transporter deficient cells (PK15NTD) were kindly donated by Dr. Chung-Ming Tse (Johns Hopkins University, Baltimore, MD). Human pancreatic cancer Panc-1, HPAC II and MIA PaCa-2 cell lines were purchased from ATCC (Manassas, VA). Cells were maintained in Eagle’s minimal essential medium/Earle’s Balanced Salt Solution with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (PK15NTD), Dolbeco’s minimum essential medium (Panc-1 and MIA PaCa-2) and 10% fetal bovine serum (Invitrogen, Grand Island, NY) and 2.5% horse serum additionally for MIA PaCa-2), Dulbecco’s modified Eagle’s medium and Ham’s F12 medium containing 1.2 g/l sodium bicarbonate, 2.5 mM l-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate supplemented with 0.002 mg/ml insulin, 0.005 mg/ml transferrin, 40 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor and 5% fetal bovine serum (HPACII) at 37 °C in a humidified atmosphere of a mixture of 5% CO₂ and 95% air.

Cell lines KATO III (signet ring diffuse cell type gastric carcinoma), HT-29, DLD-1, COLO 205, HCT-15 (colon carcinoma), BxPC-3(_luc2), PANC-1, MIA PaCa-2 (pancreatic carcinoma), and CHO (Chinese hamster ovary) were obtained from ATCC, except for BxPC-3_luc2 which was purchased from PerkinElmer (Waltham, MA, USA). KATO III, HT-29, DLD-1, COLO 205, HCT-15, and BxPC-3(_luc2) cells were cultured in RPMI 1640 cell culture medium (Gibco, Invitrogen, Carlsbad, CA, USA). PANC-1, MIA PaCa-2, and CHO cells were cultured in DMEM + GlutaMAX™ cell culture medium (Gibco, Invitrogen). Both media were supplemented by l-glutamine, 25 mM HEPES, 10 % fetal bovine serum (FBS; Hyclone, Thermo Scientific, Rockford, Il, USA), and penicillin/streptomycin (both 100 IU/ml; Invitrogen). Absence of mycoplasma was confirmed using polymerase chain reaction. Cells were grown to 90 % confluence in a humidified incubator at 37 °C (5 % CO₂) and detached with trypsin/EDTA. Viability was assessed using Trypan Blue staining in 0.4 % solution (Invitrogen).

HEK293, CFPAC1, Capan-1, HPAF-II, SW1990, HUPT3, MiaPaCa-2, and Hs766T cells were obtained from the American Type Culture Collection. P411T1 were generated from PDAC PDXs^{63 (link)}. OCIP.236, OCIP.347, and PPTO.93 were generated by the Princess Margaret Living Biobank Organoid core facility (https://www.livingbiobank.ca, Toronto, Canada). HEK293, MiaPaCa-2, and Hs766T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Wisent) at 37 °C in a humidified 5% CO₂ atmosphere. CFPAC1, Capan-1, HPAF-II, SW1990, HPAC, and HUPT3 cells were maintained similarly in RPMI-1640 (Wisent) medium supplemented with 10% (v/v) FBS. P411T1 cells were maintained in DMEM/F12 medium (Thermo Fisher Scientific, 11330-032) supplemented with 5 ng/ml EGF (R&D Systems, 236-EG-01M), 10 μg/ml insulin (Thermo Fisher Scientific, 12585-014), and 10% (v/v) FBS.

The human pancreatic cancer cell line Mia-paca-2, Panc-1 and human embryonic kidney cell line 293T (293T) were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Mia-paca-2 and Panc-1 cells were cultured in DMEM (Invitrogen). All medium was supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Invitrogen). KLK8 overexpression was performed using a lentiviral packaging system. To construct overexpressing exogenous KLK8 cell lines, full-length KLK8 (NM_144505) was cloned into the expression vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (Shanghai Genechem Co. Shanghai, China) and transfected into Mia-paca-2 and Panc-1 cell lines according to the manufacturer’s instructions. Briefly, Mia-paca-2 and Panc-1 cells were placed in 6-well plates at a density of 1 × 10^5cells/well the day before infection. The next day lentivirus were added in cell culture medium. Viruses were removed 24 h after infection and fresh cell culture medium was added. 72h after transfection, puromycin (2 µg/ml; Roche, USA) was added into the cell culture medium to generate stable KLK8-overexpression cell line four weeks later. Antibiotic-resistant cells were pooled for subsequent analysis.