Bxpc 3

Human pancreatic cancer cell lines BxPC3, HPAF2, Panc1, human colon adenocarcinoma cell lines Caco2, LS174T, and human lung adenocarcinoma cell line A427, NCI-H292 were obtained from the American Type Culture Collection. HPAF2, LS174T, and Caco2 cells were cultured in Eagle's MEM (Sigma, MO, USA), PANC1 and A427 cells were cultured in DMEM (Sigma, MO, USA), and BxPC3 and NCI-H292 cells were cultured in RPMI 1640 (Sigma, MO, USA). The media was supplemented with 10% fetal bovine serum (Invitrogen, Tokyo, Japan) and 100 U/mL of penicillin and 100 μg/mL streptomycin (Sigma, MO, USA). Hypoxic culture conditions were achieved with a multi-gas incubator containing a gas mixture of 94% N₂, 5% CO₂ and 1% O₂ (ASTEC, Fukuoka, Japan).

Established PDAC cell lines, BxPC-3 and Panc-1, were obtained from American Type Culture Collection (Manassas, VA) and were grown in RPMI-1640 (Life Technologies, Grand Island, NY) and high glucose DMEM (Hyclone, Logan, UT), respectively. Low-passage patient-derived PDAC cells, 10.05, as well as CAFs were grown in high glucose DMEM without sodium pyruvate (Life Technologies)^{54 (link),87 (link)}. All medium was supplemented with 10% heat-inactivated fetal bovine serum (HI FBS; Life Technologies). Medium for BxPC-3 and Panc-1 was also supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma Aldrich, St. Louis, MO), while 10.05 and CAFs were cultured in absence of antibiotics. Cells were maintained in a humidified environment of 5% CO₂ in air at 37 °C. All cells were passaged at 70–90% confluency; established PDAC lines and CAFs were used below passage 20. Patient-derived PDAC cells were used below passage 10, were authenticated by STR analysis (CellCheck with IDEXX BioResearch) and were tested regularly for mycoplasma contamination.

Four pancreatic cancer cell lines were used: BxPC-3, CFPAC-1, Panc-1, and AsPC-1 (ATCC, Wesel, Germany) (Table 1).
BxPC-3 and AsPC-1 were grown in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1% HEPES and 1% sodium pyruvate. CFPAC-1 was grown in Iscoves Modified Dulbecco’s Medium (IMDM) (Sigma-Aldrich, St. Louis, MO, USA), Panc-1 in Dulbecco´s Modified Eagle Medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) with the addition of 1% L-glutamine. All of the media were additionally supplemented with 10% FBS and 1% PEST (Sigma-Aldrich, St. Louis, MO, USA). PCR was used for testing the cell lines for mycoplasma infection (LookOut Mycoplasma PCR Detection Kit #MP0035, Sigma-Aldrich, St. Louis, MO, USA).

Human breast (MCF-7), colon (HCT-15), lung (A549) and pancreatic (BxPC3) carcinoma cell lines together with melanoma (A375) cells were obtained from American Type Culture Collection (ATCC, Rockville, MD). The human ovarian cancer cell line 2008 and its cisplatin resistant variant, C13*, were kindly provided by Prof. G. Marverti (Department of Biomedical Science of Modena University, Italy). Human cervical carcinoma (A431) cells were kindly provided by Prof. F. Zunino (Division of Experimental Oncology B, Istituto Nazionale dei Tumori, Milan, Italy). Cell lines were maintained in the logarithmic phase at 37 °C in a 5% carbon dioxide atmosphere using the following culture media containing 10% fetal calf serum (Euroclone, Milan, Italy), antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin) and 2 mM L-glutamine: (i) RPMI-1640 medium (Euroclone) for 2008, C13*, MCF-7, HCT-15, A431 and BxPC3 cells, (ii) F-12 HAM’S (Sigma Chemical Co.) for A549 cells, iii) DMEM (Sigma Chemical Co.) for A375 cells.