Vitronectin

Laminin, collagen, plasma and cellular fibronectin, elastin, vitronectin, and the control proteins fetuin and BSA were purchased from Sigma—Aldrich. (St. Louis, Mo., USA). Laminin-1 and collagen type IV were derived from the basement membrane of Engelbreth-Holm-Swarm mouse sarcoma; cellular fibronectin was derived from human foreskin fibroblasts; plasma fibronectin, vitronectin, and human complement serum were isolated from human plasma; elastin was derived from human aorta and collagen type I was isolated from rat tail. Native PLG, purified from plasma human, and factor H were purchased from EMD Chemicals, Inc. (San Diego, CA, USA). C4BP, isolated from normal human serum, was purchased from Complement Technology, INC. (Tyler, TX, USA).

Cancer cell adhesion capacity to vitronectin was assessed using 96-well plate coated with vitronectin. Briefly, 96-well plate was coated with 10 μg/ml vitronectin (Sigma-Aldrich) in a final volume of 75 μl/well and blocked using 1% BSA for 2 h and then washed 3 times in sterile HBSS buffer and stored at 4°C until use. CT-26 cells were plated and stimulated with either PMA-induced NET conditioned media or CXCL2-induced NET conditioned media and co-incubated with the CXCR2 antagonist SB225002, anti-CD51 antibody, and DNase I. Cancer cells were then lysed in lysis buffer and stained with the fluorescent CyQuant^® GR Dye (Cell Biolabs, San Diego, CA). Cancer cell adhesion was measured by fluorometer (Tecan's Infinite M200, Mannedorf, Switzerland) at 480 nm/520 nm and at least three times of experiments were performed. The adhesion index was then calculated as the ratio of the number of adhered cells in all groups divided by the number of adhered cells in the control group and expressed as a percentage of adhesion.

Integrin expression was assessed by flow cytometry using CD49a-phycoerythrin (CD49a-PE), CD49b-PE, CD49c-PE, CD49d-PE, CD49e-PE, CD49f-PE, CD29-allophycocyanin (APC) monoclonal antibodies (Pharmingen, San Diego, CA, USA) following manufacturer’s instructions. Nonspecific IgG of the corresponding class were used as isotype controls. ECM proteins fibronectin, from human plasma, collagen type IV, laminin, and vitronectin were from Sigma (St Louis, MO, USA). Matrigel was from BD Biosciences (San Jose, CA, USA). Native SPARC was purified from A375N human melanoma cells conditioned media.

The sera collected on day 42 for the determination of humoral responses were also used for the inhibition of the vitronectin binding assay, which is a method to assess the functionality of anti-PE antibodies. A pool of all serum samples within each group was made. The vitronectin binding assay was carried out in microtiter plates. The plates were coated with PE (5 μg/ml in PBS) for 2 h at 37°C. After washing, saturation of the nonspecific binding sites was done by incubation with PBS–1% bovine serum albumin (BSA), and then 2-fold serial dilutions of heat-inactivated immune murine sera (in PBS-T–0.02% BSA) were added to the wells for overnight incubation at 4°C. After washing, vitronectin (4 μg/ml; catalog number SRP3186; Sigma-Aldrich) was added and the plates were incubated for 1 h at 37°C. Finally, after another washing step, bound vitronectin was detected by the addition of horseradish peroxidase-conjugated sheep antivitronectin antibodies (1/1,000 in PBS-T for 30 min at 37°C; catalog number L12050350 C12120412; U.S. Biological), followed by o-phenylenediamine dihydrochloride as described in the previous paragraph. The midpoint titer (corresponding to the first dilution of murine sera able to inhibit 50% of binding) of each tested pool was determined.

Laminin and collagen IV, both at 20 µg/mL, and vitronectin at 5 µg/mL (Sigma Aldrich) were added to 96 well plates and left for 1 h at room temperature. The content was then drained, and wells were blocked with 1% BSA for 30 min at 37°C. The plates were washed with sterile PBS, and 2 × 10⁴ cells of U-251 MG, C-, C12, C15 or C23 cells were added to the wells and incubated for 2 or 4 h. The cells were then washed twice with PBS, fixed with methanol, stained with crystal violet, and destained with type I water. After drying, the wells were imaged, and the dye was solubilized with 10% acetic acid for absorbance quantification at 570 nm.

Migration and invasion assays were run in triplicate porous (8-μm pore size) Transwell migration chambers (BD Biosciences, Bedford, MA, USA) as described previously (Chia et al., 2007 (link); Kusuma et al., 2012 (link); Sloan et al., 2006 (link)). Transwells were coated with ECM proteins overnight at 4°C (Chia et al., 2007 (link)). Recombinant human laminin-511 (alpha5beta1gamma1) was isolated as previously described (Doi et al., 2002 (link)), and vitronectin was obtained from Sigma. For migration assays, cells (2×10⁵/200 μl) in serum-free medium (SFM) were seeded into the top chamber of the Transwell. For invasion assays, a cell suspension of 1×10⁵ cells in 50 μl of SFM was mixed with 50 μl Matrigel (BD Biosciences). 80 μl of the mixture was placed in the Transwell and allowed to set for 30 minutes, followed by addition of 100 μl of SFM. Cells were allowed to migrate for 4–5 hours or invade for 18 hours. The data represent the mean number of migrated or invaded cells ± s.d. of a representative experiment (n=3).