Ht 29

Human colorectal adenocarcinomas (HT29, DSMZ GmbH, Braunschweig, Germany), human fibrosarcoma (HT1080, CLS GmbH, Eppelheim, Germany), and human bladder carcinoma (T24, DSMZ GmbH, Braunschweig, Germany) cells were cultured in DMEM/Ham’s F12 (1:1) with 10% fetal bovine serum (FBS). The human breast adenocarcinoma (MDA-MB-231, CLS GmbH, Eppelheim, Germany) cells were cultured in DMEM/Ham’s F12 (1:1) with 5% FBS, whereas the human pancreas adenocarcinoma (BxPC-3, ATCC, Manassas, VA, USA) cells were grown in RPMI 1640 with 10% FBS and 1 mM sodium-pyruvate. Human colorectal adenocarcinomas (LS174T, CLS GmbH, Eppelheim, Germany) cells were grown in MEM with 10% FBS, 1 mM sodium-pyruvate, and 0.1 mM non-essential amino acid (NEAA). All culture media were purchased from Gibco (Life Technologies Ltd, Paisley, UK). The varying components of each medium and their concentrations are categorized in Table 2. Cells were grown under standard conditions at 37 °C with 5% CO₂ and 95% humidity.

The colon cancer cell lines, HT-29 and CACO-2, were purchased from DSMZ (Braunschweig, Germany). Each cell line was cultured in RPMI-1640 (RPMI; Life Technologies, Carlsbad, CA, USA) supplemented with 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies) with 10% heat-inactivated fetal calf serum (FCS) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 10 mM HEPES. Both cell lines were grown at 37°C in a humidified atmosphere and 5% CO₂.
For all experiments, 29,000 HT-29 cells/well or 19,000 CACO-2 cells/well were seeded onto 24-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 0.5 ml RPMI 10% FCS plus 10 mM HEPES and cultured for 3 days. Thereafter, cells were treated with M1 or M2 macrophage CM (for preparation see below) or 5-FU, alone or in combination, according to the schedule shown in Fig. 1.

HT-29 colon carcinoma cells, MDA-MB-231 breast cancer cells, MCF-7 breast carcinoma cells (all supplied by Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were maintained in Dulbecco's Modified Eagle Medium (4.5 g/L D-glucose, L-glutamine, pyruvate), which was supplemented with gentamycin (50 mg/L) and fetal bovine serum superior, standardized (Biochrom GmbH, Berlin) (10% v/v), and were passaged once a week. RC-124 healthy human kidney cells (supplied by CLS Cell Lines Service GmbH, Eppelheim, Germany) were maintained in McCoy's 5A (modified, with L-glutamine) medium which was supplemented with gentamycin (50 mg/L) and fetal bovine serum superior, standardized (Biochrom GmbH, Berlin) (10% v/v), and were also passaged once a week. For experiments with RC-124 cells, microtiter plates had been pretreated in the following way: 30 μL of a sterilized gelatine solution (1.5% (m/V)) were added to each well of flat bottom 96-well plates, the plates were covered with their lids, incubated for 1h at 37°C, the excess solution was removed, the wells were washed with PBS 7.4 pH, and the new cell-culture medium was added.

The melanoma cell lines (HBL [34 (link),35 (link)] and MM162 [36 (link)]) were established in our laboratory. Multiple myeloma (COLO-677 and EJM), gastroenteropancreatic (GEP) (pancreatic adenocarcinoma, MIA-PACA-2 and colon adenocarcinoma, HT-29) cell lines were purchased from DSMZ (Braunschweig, Germany). HBL and MM162 were cultured in Ham’s F10 (Lonza, Basel, Switzerland); COLO-677 and HT-29 were cultured in RPMI-1640 (Sigma, St. Louis, MO, USA); EJM was cultured in Iscove’s MDM (Gibco, Invitrogen, Waltham, MA, USA) and MIA-PACA-2 was cultured in DMEM (Sigma). Media were supplemented with 10% or 20% (EJM) foetal bovine serum as well as L-glutamine (Sigma), penicillin (Sigma), streptomycin (Gibco, Invitrogen) and kanamycin (Bio Basic, Markham, ON, Canada) at standard concentrations. Cells were grown at 37 °C in a humidified 95% air and 5% CO₂ atmosphere. All cell lines were regularly checked for mycoplasma contamination using a MycoAlert^® Mycoplasma Detection Kit (Lonza). Cell line authentication was performed with a short tandem repeat (STR) test (Eurofins Genomics, Germany). Cell lines were chosen for their SSTR expression [14 (link)] and their range of intrinsic radiosensitivities (to external beam radiation therapy [37 (link)]).

The human colonic epithelial cell lines Caco-2 (ACC 169) and HT-29 (ACC 299) were obtained from DSMZ and grown at 37°C in an incubator under oxic atmosphere with 5% CO₂. Cells were passaged after reaching 80% confluence using HyClone HyQTase (GE Healthcare Life Sciences, USA) to detach the cells. Passage numbers 5–28 were used in the experiments. Caco-2 cells were grown in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with heat-inactivated (30 min at 56°C) fetal bovine serum (FBS, 20%; Integro B.V., Netherlands), non-essential amino acids (1%; Lonza, Belgium), 15 mM HEPES (Lonza, Belgium), 100 U ml^-1 penicillin and streptomycin (PEST; Lonza, Belgium), and 2 mM L-glutamine (Lonza, Belgium). HT-29 cells were cultivated in McCoy 5A (Lonza, Belgium) medium containing 10% FBS and 100 U ml^-1 PEST. For adhesion and ELISA assays, 10,000 Caco-2 or HT-29 cells per well were seeded onto 96-well microplates.

The adherent colon carcinoma cell line HT-29 (ATCC/LGC GmbH, Wesel, Germany) and the non-adherent T cell leukemia cell line Jurkat (ACC 282, DSMZ, Braunschweig, Germany) were used. HT-29 cells were cultured in McCoy’s 5A medium (Gibco^®, Life Technologies GmbH, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS), Jurkat cells were cultivated in RPMI 1640 medium supplemented with 10% FCS, and 1% glutamine (all from Thermo Fisher Scientific, Waltham, MA, USA). All cells were kept under standard cell culture conditions in a humidified incubator (INCOmed, Schwabach, Memmert, Germany) at 37 °C and 5% CO₂. The cells were passaged twice a week and regularly checked for mycoplasma contamination using a PCR kit Venor^®GeM (Minerva Biolabs GmbH, Berlin, Germany). To count cells and determine viability, MUSE^®Cell Analyzer (Merck-Millipore, Billerica, MA, USA) was used.