Mia paca 2

The human mammary epithelial cell line MCF10A; breast cancer cell lines MDA-MB-436, MDA-MB-231, MDA-MB-453, BT-20, HCC1937, SKBR3, T47D, and HEK293; normal HPDE cells; and human PDAC cell lines PANC1, BxPC-3, MiaPaCa-2, and Capan-2 were purchased from the American Type Culture Collection (Manassas, VA). All breast cancer cells and PANC1 and MiaPaCa-2 cells were cultured in DMEM/F12 (Sigma, St. Louis, MO). BxPC-3 and Capan-2 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and a 100-U/mL penicillin-streptomycin solution (Sigma). All media were supplemented with 10% FBS and a 100-U/mL penicillin-streptomycin solution. MCF-10A cells were maintained in a nutrient mixture consisting of DMEM/F12 supplemented with 5% horse serum, epidermal growth factor, hydrocortisone, insulin, and cholera toxin. All cultured cells were incubated at 37°C in a water-saturated 95% air-5% CO₂ atmosphere.

MIA PaCa-2 and PANC-1 were purchased from ATCC. Human pancreatic ductal epithelial cells (HPDE) and human pancreatic stellate cells (hPSC) were a kind gift from Diane Simeone (University of Michigan) and David Yule (University of Rochester), respectively. MIA PaCa-2, PANC-1, and hPSC were cultured in DMEM media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma–Aldrich, Gillingham, UK). HPDE were cultured in Keratinocyte-SFM media (Fisher Scientific, Loughborough, UK). All cells were cultured under 5% CO₂ (g), at 37 °C. Mycoplasma contaminations were screened by DAPI/Hoechst 33,342 staining [81 (link)] using a Zeiss Axioimager.D2 upright microscope using a 100×/1.4 Plan Apochromat (Oil) on a DAPI bandpass filter set (Carl Zeiss Ltd., Oberkochen, Germany) or submitted to the University of Manchester FBMH Media Order services for PCR-based detection method.

Human PDAC cell lines MIA PaCa-2 and Capan-1 were purchased from ATCC (Manassas, VA, USA). Normal fibroblasts (NFs, id = 6 and 11) were obtained from patients undergoing surgery for PDAC as described previously [9 (link)]. Three pancreatic fibroblasts (PFs, id = 10295, 14289 and 14358) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) (Catalog #3830).
Capan-1 and MIA PaCa-2 were cultured in a Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Palo Alto, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in 5% CO₂. PFs were maintained in a Stellate Cell Medium (ScienCell Research Laboratories, Carlsbad, CA, USA) at 37 °C in 5% CO², while NFs were maintained in an MF-medium (Toyobo, Tokyo, Japan).

The human pancreatic cancer cell lines AsPC-1, BxPC-3, Panc-1 and MiaPaCa-2 were obtained from ATCC (Manassas, VA, USA). AsPC-1 was maintained in RPMI-1640 culture medium (Sigma-Aldrich, Tokyo, Japan) and BxPC-3, Panc-1 and MiaPaCa-2 were maintained in DMEM culture medium (Sigma-Aldrich). Each culture medium was supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were cultured in a humidified incubator containing 5% CO₂ in air at 37°C.

6 human cancer cell lines were selected to be investigated in vitro; H460 (large cell lung cancer), MiaPaCa2 (pancreatic carcinoma), DU145 (prostate carcinoma), MCF7 (breast adenocarcinoma), U87 (brain glioblastoma) and HEPG2 (liver carcinoma). All cell lines were acquired from the American Type Culture Collection (ATCC), and were routinely tested for mycoplasma. All cell lines have also been authenticated by ATCC using Short Tandem Repeat (STR) Screening. Cells were incubated at 37 °C in 5% CO₂, and all tissue culture was performed in a Class II laminar flow cabinet (Thermo, US). All cell lines, except for MiaPaCa2, were cultured in Dulbecco’s Modified Eagle Media (DMEM) (Sigma, US), supplemented with 10% Foetal Bovine Serum (FBS) (Sigma, US) and 1% Penicillin Streptomycin (Pen-Strep) (Sigma, US). MiaPaCa2 cells were incubated in High Glucose DMEM media, supplemented with 10% FBS, 1% Pen-Strep, and 0.2% Sodium Pyruvate. Cells were passaged every 2–3 days to maintain exponential grown.
NPs used for this study were SPIONs with a 5 nm diameter, acquired from Sigma, US (product number 725331), suspended in H₂O at a concentration of 5 mg/ml, with the molecular weight of Fe₃O₄ being 231.53 g/mol. For the purpose of this study, all assays were performed at a SPION concentration of 23.5 μg/ml, which was determined as being within the range investigated by Kirakli et al. [56 (link)].

The human pancreatic cell lines, PANC-1, MIA PaCa-2, Capan-1, AsPC-1, CFPAC-1, and Hs766T, were obtained from the American Type Culture Collection (Manassas, VA, U.S.A) The PANC-1 and MIA PaCa-2 in Dulbecco’s Modified Eagle Medium (DMEM: Sigma-Aldrich, St Louis, MO, USA) with 10% fetal bovine serum (FBS) and antibiotics (1% penicillin and streptomycin), CFPAC-1 and Capan-2 in Iscove’s Modified Dulbecco’s Medium (IMDM: Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS and antibiotics, and AsPC-1 cells in RPMI-1640 medium (Thermo Fisher Scientific) with 10% FBS and antibiotics. Mouse primary pancreatic cells were cultured and maintained as described previously [26 (link)]. Murine PDAC (KPC1) and paired metastases (KPC1Liv) cell lines were provided by Dr. Sunil Hingorani (University of Washington). In brief, KPC1 cell lines were established from primary PDAC of a genetically engineered mouse model of PDAC (LSL-Kras^G12D/+;p53^R172H/+;Pdx1-cre) [32 (link)], whereas the KPC1Liv cell lines were isolated from paired liver metastases arising in KPC1 mice. KPCY cells are derived from a Pdx1-cre;LSL-Kras^{G12D/+;p53fl/+};R26^YFP mouse (KPCY mice) and were provided by Dr. Andrew D. Rhim (The University of Texas MD Anderson Cancer Center).