Human laminin

Invasion assays were performed by using transwells with 8.0 μm porous membrane coated with an invasion matrix containing Type IV Collagen (#C6745-1ML, Sigma Aldrich), Human Laminin (# l6274), and Gelatin diluted in 1X PBS. HCT116 cells were transfected with siRNAs against N-BLR (N-BLR siRNAs1 + 3 pool) and control siRNAs at a final concentration of 100 nM for 48 h and then 300,000 cells were plated on the top of the transwell. The same number of cells was also plated in a separate culture well for normalization purposes (total cells). Each experiment was performed in triplicate. The same experiments were performed also with HCT116 stable shRNA N-BLR expressing clones #3-1 and #4-7, stable WT and pyk90 DEL N-BLR variants overexpressing clones, and the empty vector clone. The invasion assay was stopped after 36 h and cells were fixed and stained with HEMA 3. For each well, ten random fields were counted and the average number of cells was determined. For transient transfection of RKO and HCT116 cells, we followed the same protocol as for the migration assay. The invasion results were normalized by the total number of cells to minimize the effect of proliferation/viability.

MIN6 pseudo-islets were resuspended in a sufficient amount of 5% w/v poly(ethylene glycol diacrylate) (PEGDA) with a molecular weight of 5000 (Laysan) in PBS (Gibco). A 10% w/v solution of Irgacure 2959 (Basf) in 70% Ethanol was prepared and 10 µl added to 1 ml of PEGDA solution prior to crosslink. Each sample was prepared using 50 µl of the precursor PEGDA, additioned with the photocrosslinker, and contained on average about 1400 MIN6 pseudo-islets.
For samples containing human collagen IV (Merck Millipore) or human laminin (Sigma-aldrich), a 20 µg/ml stock solution of protein was prepared in sterile PBS (Gibco) and diluted to the working concentration of 10 µg/ml by using 10% w/v concentrated PEGDA solution prior the addition of Irgacure (Basf). MIN6 pseudo-islets were resuspended in the solution, 50 µl samples were pipetted in a 96 well plate, and exposed to 356 nm UV light for 10 min at 5 cm distance from the light source with an intensity of 8.2 mW/cm².
In case a bi-layered construct was prepared, the 50 µl PEGDA sample containing aggregates was crosslinked on top of a pre-gelled 50 µl vascularization gel.

Breast cancer cells were plated onto sterile 96-well non-tissue culture plates (Titertek, Flow Laboratories Inc.; McLean, VA, USA) that had been treated with one of: 20 μg/mL of human laminin (Sigma; SUM159 cells), 5 μg/mL of human vitronectin (Sigma; MDA-MB-468 cells), or PBS (negative control), using 1 × 10⁴ cells/well (n = 3) for each cell population. Laminin and vitronectin were chosen based on previous experiments in our laboratory that have demonstrated that SUM159 and MDA-MB-468 cells differentially express integrin receptors for vitronectin and laminin respectively [48 (link),49 ]. Cells were allowed to adhere for 5 h, after which non-adhered cells were rinsed away. Adhered cells were fixed with 2% gluteraldehyde and stained using Harris’ hematoxylin. Five high powered fields (HPF) (200×) were counted for each well, and mean numbers of adhered cells/field were calculated and normalized to control cell populations.

We assessed the cells’ adhesiveness to the extracellular membrane in vitro. We pre‐coated 96‐well plates overnight with 1 μg/ml human laminin (Millipore), which were washed in PBS prior to cell plating. We plated 3,000 cells from single‐cell suspensions per well, and the average number of viable cells adhering to the plate after three hours across the replicates was compared against total cell counts of viable cells after 14 h, using the CellTiter‐Glo Luminescent Cell Viability Assay (Promega). We calculated all P‐values using a two‐sided t‐test.

Human fibronectin and vitronectin were purchased from R&D Systems (Minneapolis, MN), human laminin from Millipore (Temecula, CA), β-1,6-glucanase from Takara Bio Inc. (Otsu, Shiga, Japan), β-1,3-glucanase (lyticase) from Sigma and α1-2,3 mannosidase and α1-6 mannosidase from New England Biolabs (Ipswich, MA). Proteinase K and bovine serum albumin (BSA) were obtained from BioShop Canada Inc. (Burlington, Ontario, Canada), trypsin from Promega (Madison, WI) or Biocentrum (Krakow, Poland) and horseradish peroxidase-conjugated streptavidin solution (SA-HRP) from MP Biomedicals (Solon, OH).