Fibronectin

EMT-6 and 4T1 murine breast carcinoma and MDA-MB-231 human breast carcinoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 1% L-glutamine, 1% sodium-pyruvate and 1% streptomycin. Human umbilical vein endothelial cells (HUVECs) (Lonza, Switzerland) were cultured in plates covered with 10% fibronectin (1 mg/ml Biological Industries, Beit HaEmek, Isreal) following 37°C incubation for 30 min. HUVECs were cultured in M199 medium (Sigma-Aldrich, Rehovot, Israel) supplemented with 20% heat inactivated fetal calf serum (FCS), 50 mg/ml endothelial cell growth supplement (ECGS), 50 mg/ml heparin, 10 mM Hepes, 1% L-glutamine, 1% sodium-pyruvate and 1% streptomycin.

EPCs were isolated and cultured as previously described [20 (link)]. Briefly, human EPCs were isolated from the blood of 18 healthy donors who signed informed consent (Helsinki number 0397-12 RMB). For cell isolation, we collected 50 mL of peripheral blood into sterile heparinized tubes. Blood was diluted 1:1 with phosphate-buffered saline (PBS). Mononuclear cells (MNCs) were isolated using density gradient centrifugation (Lymphoprep, Axis-Shield Diagnostics Ltd., Dundee, Scotland) and pelleted cells resuspended in EGM-2 SingleQuote (Lonza Walkersville Inc. Walkersville, MD, USA), an endothelial growth medium containing 20% fetal bovine serum (FBS) and penicillin-streptomycin (Biological Industries Ltd., Beit Haemek, Israel). Cells were seeded on six-well plates coated with 5 µg/cm² of fibronectin (Biological Industries Ltd.) and grown at 37 °C with humidified 95% air/5% CO₂. After 4 days of culture, nonadherent cells were discarded by gentle washing with PBS, and fresh medium was applied. The attached cells were continuously cultured with EGM-2. Cells were fed three times per week then split when they reached ~80% confluence by brief trypsinization, using 0.5% trypsin in 0.2% ethylenediaminetetraacetic acid (EDTA).

The invasion and migration properties of tumor cells were evaluated in Matrigel- or fibronectin-coated Boyden chambers as previously described [4 (link)]. Briefly, filters (6.5 mm in diameter, 8 μm pore size) were coated with either Matrigel (diluted 1:4 in DMEM, BD Bioscience, USA), or 0.01 μg/μl fibronectin (Biological Industries, Israel) for invasion or migration assays, respectively. Freshly coated filters were left to dry for 2 hours at 37°C. Serum-deprived siIL31 or control MC38 cells were seeded in the upper compartment of the chamber and the lower compartment was filled with serum-free medium. After incubation for 24 hours at 37°C, cells that passed through to the bottom filter were fixed with 4% paraformaldehyde (PFA). Cells were stained with 0.5% Crystal violet (Sigma-Aldrich) and visualized using Leica DMI 6000B microscope (Leica, Wetzlar Germany). All experiments were performed in triplicate.

Jurkat cells, expressing CXCR4 receptors were serum starved in RPMI medium (RPMI 1640, Biological Industries, Kibbutz Beit-Haemek, Israel) for 4 h. Transwell plate membranes (Corning Life Sciences, Tewksbury, MA) were coated with fibronectin (Biological Industries, Kibbutz Beit-Haemek, Israel) for 1 h in 37 degrees. Three hundred and fifty μL of medium with or without the released SDF-1 were added to the bottom chambers of the transwell plates. Three hundred μL of medium containing 1.5 × 10⁵ cells were added to the top chamber and allowed to migrate for 2.5 h in 37 °C, 5% CO2. Medium from the bottom chamber was collected and cell numbers were counted by a hemocytometer (n ≥ 5 in each group).

For colony formation assay, cells were seeded at 500 cells/well of 6-well culture plates in duplicates. For β-catenin inhibitor experiments, 24 h after seeding, cells were treated with PKF118-316 (0.1 µg/ml, Sigma) and 9 days later, the colonies were fixed with 4% formaldehyde, stained with 0.05% crystal violet (Sigma), counted by examination of five microscopic fields and quantified by Image J. For trans-well migration assay, we used modified Boyden chambers with a polycarbonate Nucleopore membrane (Corning, Corning, NY). Filters (6.5 mm in diameter, 8 µm pore size) were coated with fibronectin 10 µg/ml (biological industries, Israel). Cells were maintained in serum-free media for 10 h prior to migration assay. Cells (2 × 10⁵) were then suspended in 200 µl of serum-free medium, seeded in duplicates on the upper part of each chamber, and the lower compartment was filled with 600 µl medium with 10% FCS. After overnight incubation at 37 °C in a 5% CO₂ incubator, non-migrating cells on the upper surface of the filter were wiped with a cotton swab and migrated cells on the lower surface of the filter were fixed with 4% formaldehyde, stained with 0.05% crystal violet (Sigma), and counted by examination of five microscopic fields. The percentage of surface covered by migrated cells was calculated using Image-Pro-Premier (9.3.2).

The substrate chambers were washed in absolute ethanol and sterilized under ultraviolet light. They were fitted into the wells of a 6-well culture plate, and air-plasma treated for 120 seconds at 29.6 W (Harrick Plasma, Ithaca, NY, USA). For electrophysiology studies, a thin layer of patterned substrate less than 1 mm was cut and fitted on glass coverslips in a 24-well plate for culture. The substrates were then washed in absolute ethanol and placed under ultraviolet light for 20 min. After coating with poly-L-ornithine (0.01% in DI H₂O) overnight (Sigma-Aldrich, Missouri, USA), the substrates were washed twice with sterile water and coated with fibronectin (50 µg/ml) (Biological Industries, Israel) and laminin (50 µg/ml) (Life Technologies, California, USA) overnight before cell seeding.