Hct116

The human colon cancer cell line HCT116 was purchased from the European Collection of Cell Cultures (Salisbury, UK). From this cell line, a 5-Fluorouracil (5-FU) resistant cell line (HCT116R) was generated as previously described [32 (link)]. Cell culture was performed in tissue culture flasks in growth medium and in a humidified incubator at 37 °C in an atmosphere of 95% air and 5% CO₂ and the medium was changed every two to three days. Cells were passaged at 80–90% confluency using trypsin/EDTA.

A human breast-cancer cell line (MDA-MB 231) and colon-cancer cell line (HCT-116) were obtained from the European Collection of Cell Cultures (ECACC-HPA cultures, Salisbury, UK). Cells were cultured in 75 cm² flasks (5520200, Orange Scientific) at 37 °C in a 5% CO₂ atmosphere, in the recommended media supplemented with the appropriate amount of heat-inactivated fetal bovine serum (10106-169, Invitrogen, NY, USA) and 2 mM l-glutamine (G5792, Sigma-Aldrich, Munich, Germany). Approximately, 3,750,000 cells were isolated and divided into five 15 ml Falcon tubes each containing 1.5 ml of blood from healthy individuals, with EDTA anticoagulant (with a total of ~ 750,000 cancer cells in each tube).

The colon cancer cell lines HCT116 and SW480 were obtained from the European Collection of Cell Cultures (Salisbury, UK). The colon cancer cell line RKO was from the American Type Culture Collection. Cells were cultured in a humidified incubator (37 °C, 5% CO₂) with normal cell culture growth medium containing 10% FCS, as described in detail before [33 (link)].

DLD-1, LOVO, HT29, SW480, SW1116, WiDR, and HCT116 cells were purchased from the European Collection of Cell Cultures (Salisbury, UK), and SW620 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). COLO205, HCT-15, and LS174T cells were provided by the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). COLO201 cells were provided by the Japanese Collection of Research Bioresource (Tokyo, Japan), and COLO-320 cells were provided by RIKEN Bio-Resource (Tsukuba, Japan). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37°C in humidified air containing 5% CO₂. Cells were used when in the exponential growth phase.

The human colorectal carcinoma cell lines LS180 (Deutsche Krebsforschungzentrum, Heidelberg, Germany), HCT116, and LoVo (European Collection of Cell Cultures) were cultivated in MEM-α, McCoy′s 5A, and Ham’s Nutrient Mixture F12 (all from Sigma), respectively. Media were supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM glutamine (Sigma), and antibiotics (10.000 U/ml penicillin, 10 mg/ml streptomycin, 25 µg/ml amphotericin B) (Gibco). Mouse preadipocyte 3T3-L1 cells (ATCC) were cultured in DMEM medium containing 4.5 g/l glucose and 1.5 g/l NaHCO₃ (IITD PAN, Wroclaw, Poland) supplemented with 10% bovine calf serum (BCS) (Thermo Fisher Scientific), 2 mM glutamine, and antibiotics (10 000 U/ml penicillin, 10 mg/ml streptomycin, 25 µg/ml amphotericin B). All cells were grown at 37 °C in 5% CO₂ in a humidified atmosphere and passaged using 0.25% trypsin/0.05% EDTA solution (IITD PAN, Wroclaw, Poland).

Human colorectal adenocarcinoma cell lines HT29 and HCT116 (both European Collection of Cell Cultures through Sigma-Aldrich, Dorset, UK) were grown in Dulbecco’s Modified Eagle Medium (DMEM) at 1000 mg/L glucose (Sigma-Aldrich, Dorset, UK). Human adult-donor dermal fibroblasts (HDF) (Promocell, Heidelberg, Germany) were grown in 4500 mg/L glucose DMEM (Sigma-Aldrich, Dorset, UK). Human umbilical vein endothelial cells (HUVEC) were grown in Endothelial Cell Growth Medium (both Promocell, Heidelberg, Germany). After isolation, CAF cells were cultured using fibroblast growth medium 2 (Promocell, Heidelberg, Germany). All media were supplemented with 10% foetal calf serum (FCS) (First Link, Birmingham, UK) as well as 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco^TM through Thermo Fisher Scientific, Loughborough, UK). All cell types were cultured at 5% carbon dioxide (CO₂) atmospheric pressure at 37 °C, and routinely passaged in 2D monolayers. HDFs and HUVECs were used at passage ≤5.

Established human colon cancer cell lines (LS180, HCT116, and HT29) were purchased from European Collection of Cell Cultures (ECACC) and immediately stored in liquid nitrogen. Cells used for specific investigations were recovered from cryopreserved aliquots and cultured for a maximum of 10 passages. LS180 and HCT116 were maintained in RPMI-1640 supplemented with 10% FBS, GlutaMAX-I, nonessential amino acids (NEAA), 100 mM sodium pyruvate, 10 mM HEPES (all from GIBCO), and 50 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), whereas HT29 was maintained in McCoy’s 5A medium (Sigma) supplemented with 10% FBS and GlutaMAX-I.
Absence of mycoplasma contamination in cultured cells was verified by PCR testing prior to investigation. In specific experiments, LS180 cells co-expressing green fluorescent protein (GFP) and firefly luciferase (GFP/Luc-LS180 cells) [27 (link)] were also used.