Panc 04

The cell lines PANC-1 (CRL-1469™, pancreatic), PANC0403 (CRL-2555™, pancreatic) and 4T1 (CRL-2539™, breast) were obtained from ATCC®. A375Ppuro and A375Pβ6puro cell lines were created using the human melanoma cell line A375P (CRL-3224™, melanoma), which was infected with pBabe retroviruses encoding puromycin resistance alone or in combination with cDNA for human β6, as previously reported [12] (link). The A375Ppuro and A375Pβ6 cell lines were a kind gift from Prof. John Marshall (QMUL). The A375Pβ6 cell line was subsequently transfected with firefly luciferase (luc) using an SFG retroviral vector whereby luc was co-expressed with dsTomato red fluorescent protein. Transduced cells were then flow sorted for red fluorescence to obtain a pure A375Pβ6-luc cell line [24] (link). All cell lines were maintained at 37 °C, 5% CO₂ and 5% relative humidity. Advanced RPMI (PANC-1, PANC0403, 4T1) or DMEM media (A375Ppuro, A375Pβ6puro) were used, both of these were supplemented with 10% FBS, 1% GlutaMAX™ and 1% Penicillin/Streptomycin.

Human PDA cell lines AsPC-1, BxPC-3, MiaPaCa-2, Panc-1, and Panc-04.03 were purchased from ATCC (Manassas, VA, USA). AsPC-1, BxPC-3, Panc-04.03, and Colo-357 cells were maintained with RPMI-1640 medium. MiaPaCa-2 and Panc-1 cells were maintained with Dulbecco’s modified Eagle’s medium. HPDE cells was maintained with defined keratinocyte serum–free medium (keratinocyte-SMF, Gibco), and the defined Keratinocyte-SFM Growth Supplements, including the bovine pituitary extract (BPE), epidermal growth factor (EGF), and 1% penicillin-streptomycin (P/S, Caisson Labs) to the final concentration of 100 units and 100 μg, respectively. Both the RPMI and DMEM medium were supplemented with 10% fetal bovine serum (FBS, Gibco), and 1% P/S. Panc-04.03 cell culture medium was additionally supplemented with 20 units/ml human recombinant insulin (Sigma). All cells were cultured at 37 °C in a 5% CO₂ chamber.

Pancreatic cancer cell lines (Panc1, Panc0203, Panc0327, Panc0403, Panc0813, Panc1005, AsPc1, BxPc3, MiaPaCa2, PL45, SU8686) were obtained from ATCC (Manassas, VA). They were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and maintained at 5% CO₂ and 37°C. For induction of hypoxia, cells were incubated in temperature-controlled hypoxic culture chamber at 1% O₂, 5% CO₂, and 94% N₂.

The cell lines PANC-1 (CRL-1469™), PANC0403 (CRL-2555™) were obtained from ATCC®. A375Ppuro and A375Pβ6 cell lines were created using the human melanoma cell line A375P (CRL-3224™), which was infected with pBabe retroviruses encoding puromycin resistance alone (A375Ppuro) or in combination with cDNA for human β6 integrin (A375Pβ6), as previously reported [41] (link). The A375Ppuro and A375Pβ6 were a kind gift from Dr. John Marshall (QMUL). All cell lines were maintained at 37 °C, 5% CO₂ and 5% relative humidity. Advanced RPMI or DMEM media were used, both of these were supplemented with 10% FBS, 1% Glutamax and 1% Penicillin/Streptomycin.

HeLa, DU145, COLO205, NIH-OVCAR3, RKO, ES2, SK-OV3, PC-3, SW620, SW480, CAPAN-2, Panc 04.03, and MRC5 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). TCC-PAN2, TYK-nu, and OVTOKO cell lines were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS). DU145, TYK-nu, and MRC5 cells were cultured in Modified Eagle’s Medium supplemented with 10% FBS. COLO205, RKO, NIH-OVCAR3, TCC-PAN2, OVTOKO, and Panc 04.03 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. PC-3 cells were cultured in Ham’s F12 nutrient mixture supplemented with 10% FBS. SKOV3, ES2, and CAPAN-2 cells were cultured in McCoy’s 5a medium supplemented with 10% FBS. SW620 and SW480 cells were cultured in Leibovitz’s L-15 medium supplemented with 10% FBS.

Pancreatic cancer cell line Panc 04.03 was obtained from the American Type Culture Collection. Cells were plated in 96-well plates in a density of 2500 cells/well and cultured in RPMI, DMEM, DMEM + 1 mM proline, with or without 1 mM IPA. All media was supplemented with 2 mM glutamine, 100 U/mL penicillin/streptomycin and 10% dialysed foetal calf serum (3000 Da MWCO) in a humidified incubator at 37 °C, 5% CO₂. Proliferation was measured for 120 h using the IncuCYTE live cell imaging system (Essen Biosciences). Datapoints were taken every 2 h. Growth rates were calculated using the Growthcurver package in R (ver. 0.3.0).