Collagen g

HPLC grade acetonitrile (MeCN), methanol (MeOH), ethanol (EtOH), dimethylsulfoxide (DMSO), hydrochloric acid (HCl), and filter paper (110 mm pore size) were obtained from VWR International GmbH (Ismaning, Germany), and ultra-pure water (18MΩ.cm⁻¹) from a Millipore S.A.S. Milli-Q Academic system (18,2 MΩ cm⁻¹, Molsheim, France); 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ), potassium phosphate monobasic (KH₂PO₄), potassium phosphate dibasic (K₂HPO₄), ammonium acetate (NH₄AcO), iron(III) chloride (FeCl₃), hydrogen peroxide (30%) were from Merck KGaA (Darmstadt, Germany). Yeast extract peptone and MEM non-essential amino acid solution were obtained from Sigma-Aldrich (Taufkirchen, Germany) and collagen G from Biochrom AG (Berlin, Germany). Trolox was from Enzo Life Sciences GmbH (Lörrach, Germany), 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) and LB Broth Base were from Thermo Fisher (Waltham, MA, USA). DMEM medium was from PAN-Biotech GmbH (Aidenbach, Germany); MEM medium was from PromoCell (Heidelberg, Germany) and HEPES from AppliChem GmbH (Darmstadt, Germany). Fetal calf serum (FCS) was purchased from PAA Laboratories GmbH (Pasching, Austria) and l-glutamine, agar, and glucose were obtained from Carl Roth (Karlsruhe, Germany). Iceberg lettuce and cucumber were bought at a local supermarket and immediately used.

For live-cell analysis of scratch-induced wound closure, 2.5 × 10⁴ MDA-MB-231 and A549 cells per well were seeded in collagen G (40 µg/mL) (Biochrom AG, Germany) coated 96-well plates near confluence and allowed to grow overnight in standard medium (DMEM supplemented with 10% FBS and 1% P/S). At confluence, cells were pretreated 2 h with mitomycin (10 µg/mL) (Medac, Germany) to block cell proliferation and washed with standard media. Afterwards, cells were incubated in the presence of inhibitor at the indicated concentrations or DMSO (0.1% vehicle). Subsequently, a defined scratch (wound width between 642–767 µm) was performed in each well using the certified Essen Bioscience automated 96-wound maker^TM (Essen Biosciences, Hertfordshire, UK) for 96 well-plates. The medium was removed and 100 µL standard medium were added to the wells containing either inhibitor or DMSO. The closure of the wounded area was monitored using the IncuCyte ZOOM system by taking images of each well every 2 h over a period of 24 h. The reduction of wound width was determined over time using the IncuCyte ZOOM microscope software 2014A. Data were expressed as percentage of wound closure after 8 h.

Cells were seeded onto collagen type I-coated (Collagen G, Biochrom AG; final concentration 0.4 mg ml⁻¹) coverslips and cultured for 2 h. The cells were then fixed with 3.5% paraformaldehyde (w/v) in PBS (Dulbecco, Biochrom AG) for 30 min and permeabilized for 25 min in 0.1% (v/v) Triton X-100/TBS in order to ensure that the intracellular F-actin epitopes were accessible to the antibody. After washing with PBS (2×), nonspecific binding sites were blocked with 3% bovine serum albumin (BSA) in PBS (w/v) for 2 h at room temperature. The cells were then stained with Alexa Fluor® 488 Phalloidin (Invitrogen AG; dilution 1:100) for 45 min. Prior to Dako mounting (Dako A/S, Glostrup, Denmark), the cells were washed in PBS once again. Images were taken with a digital camera (Model 9.0, RT-SE-Spot, Visitron Systems, Puchheim, Germany) fitted to an inverted microscope (Axiovert 200, Carl Zeiss AG) and controlled by MetaVue software (Visitron Systems).

Cellfoam matrices (grids, 9 × 1.5 mm; Cytomatrix) were autoclaved and incubated in PBS (1× 10 ml) and collagen G (250 µl, 30 min, room temperature; Biochrom). Punched (4 mm; Kai Europe) skin samples were cut into small pieces (∼1 mm) and transferred onto the grids. The charged grids were transferred into wells of a 24-well plate (Becton Dickinson Labware Europe) containing 2 ml TexMACS medium (1% penicillin/streptomycin) without or with a combination of cytokines (100 U/ml IL-2; PeproTech; 5 ng/ml IL-15/IL-7; Miltenyi Biotec). After 9–14 d of cultivation, cells were harvested and centrifuged (7 min, 4°C) and the supernatant aspirated and frozen. Cell pellets were resuspended in 500 µl PBS and the resulting single-cell suspensions analyzed by flow cytometry. Isolation of cells from cultured skin biopsies was performed as described.

6-well plates were coated with collagen G (extracted from calfskin, consisting of 90% collagen type I and 10% collagen type III; Biochrom, Berlin, Germany; diluted to 400 µg/ml in PBS) overnight. Plastic dishes served as the background control. Plates were washed with 1% BSA (bovine serum albumin) in PBS to block nonspecific cell adhesion. 0.5×10⁶ tumor cells were then added to each well and left for 60 min incubation. Subsequently, non-adherent tumor cells were washed off, the remaining adherent cells were fixed with 1% glutaraldehyde and counted microscopically. The mean cellular adhesion rate, defined by adherent cells_{coated well} − adherent cells_background, was calculated from five different observation fields.

Ewing Sarcoma, neuroblastoma and osteosarcoma cell lines were cultured in RPMI 1640 medium (Biochrom GmbH, Berlin, Germany) supplemented with 10% FCS and 1% glutamine (Gibco, Waltham, MA, USA) using 200µg/mL collagen G (Biochrom, Schaffhausen, Switzerland)-coated flasks at 37 °C, 5% CO₂ for a maximum of 20 passages. For cell volume measurements, cells were incubated in HEPES-buffered RPMI 1640 medium. Table 1 contains a summarized list of cell lines used in this study.