Differential effects of anticoagulants on tumor development of mouse cancer cell lines B16, K1735 and CT26 in lung

Three mouse cancer cell lines expressing luciferase were used in experimental models of metastasis in mouse lungs, the B16 mouse melanoma cell line (American Type Culture Collection, Manassas, VA) [29], the K1735 mouse melanoma cell line (kindly provided by Dr. I.J. Fidler, MD Anderson Cancer Institute, Houston, TX), and the CT26 mouse colon carcinoma cell line (also kindly provided by Dr. I.J. Fidler) were used. Preparation of cell cultures, transfection with the luciferase gene and inoculation of the cells in the tail vein of mice have been described previously [30, 31].

TF activity of the three cell lines was measured to test their ability to induce coagulation using a standard procoagulant activity assay (also known as the one-stage clotting assay or recalcification assay) [32]. Cancer cells were suspended in PBS at a concentration of 7.5 x 10⁴ cells/ml and 75 μl of the cell suspensions was added to 75 μl mouse plasma (Sigma, St. Louis, MO) and incubated at 37°C for 2 min. Then, 100 μl of a 25 mM calcium chloride solution was added and the clotting time was measured using a KC-10 coagulometer (Amelung, Lemgo, Germany).

Flow cytometric analysis of the three cancer cell lines was performed to determine the presence of various surface proteins. Cancer cells were incubated in the absence of serum during 24 h before flow cytometric analysis. Cancer cells were centrifuged at 400 g for 5 min. Cells were washed twice with flow-activated cell sorter (FACS)-buffer (PBS supplemented with 0.5% bovine serum albumin, 0.01% NaN₃, and 0.35 mM EDTA) and finally suspended in FACS buffer (4x10⁶ cells/ml). For each analysis, at least 1 × 10⁵ cells were analyzed using a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA). Fc receptors were blocked with purified anti-mouse CD16/32 antibodies (dilution, 1:50; Fcy 111/11 receptor; Pharmingen, BD Biosciences, Breda, The Netherlands). Staining was performed using rat anti-mouse CD24 antibody coupled with phycoerythrin (PE) (dilution, 1:50; Pharmingen, BD Biosciences), goat anti-mouse PAR-4 antibody (dilution, 1:50; M-20 Santa Cruz Biotechnology, Heidelberg, Germany) and rabbit anti-mouse PAR-1 (dilution, 1:50; thrombin R; H-111 Santa Cruz Biotechnology). As secondary antibody anti-rabbit coupled with Alexa fluor 488 (dilution, 1:150; Molecular Probes, Eugene, OR) was used to detect PAR-1 expression and anti-goat coupled with FITC (dilution, 1:200; Dako) was used to detect PAR-4 expression. To correct for nonspecific staining; all analyses were also conducted with cells stained with the appropriate isotype control antibodies. Cancer cells were identified by forward and sideangle light scatter gating. Data are presented as the difference between mean fluorescence intensities of specifically stained and nonspecifically stained cells.

For all experiments, eight weeks-old Balb-/c nude mice with a body weight of 20–27 g were used (Netherlands Cancer Institute, Amsterdam, The Netherlands). Animals were maintained under constant environmental conditions with free access to food and water. All animal experiments were performed in agreement with the Animal Ethics Committee of the Netherlands Cancer Institute.

Thirty mice received 3 × 10⁵ K1735 cells, twenty mice received 3 × 10⁵ B16 cells and twenty mice received 3 × 10⁵ CT26 cells into the tail vein. Half of the mice in each group received 10 mg/kg polyethylene glycol (PEG)-Hirudin (Abbott, Knoll, Ludwigshafen, Germany) subcutaneously before cancer cell inoculation. The other half of the mice received PBS subcutaneously before cancer cell inoculation. Mice were sacrificed at 21 days after cancer cell inoculation and lungs were harvested.

Furthermore, 15 mice received 3 x 10⁵ K1735 cells into the tail vein. Ten mice received 600 anti-Xa (aXa) IU nadroparin (Sanofi-Synthelabo, Berlin, Germany) intraperitoneally per kg body weight prior to cancer cell inoculation. The other mice received PBS intraperitoneally and served as controls. Mice were sacrificed at 21 days after cancer cell inoculation and lungs were harvested.

For all experiments, cancer cell load in lungs was measured at day 1, 7, 15 and 21 after administration of the cancer cells using noninvasive BLI as previously described [30, 31]. To generate BLI by luciferase, its substrate, luciferin (Xenogen, Alameda, CA) was given intraperitoneally to the mice (150 mg/kg body weight). Luciferase converts luciferin into oxyluciferin and the resulting BLI was imaged with the use of a highly sensitive cooled charge-coupled device camera in a lighttight chamber (IVIS Imaging System 100 series; Xenogen). The acquired images were analyzed with the software program Living Image^R 2.11 (Xenogen). The area of the lungs was selected as region of interest (ROI) for the quantification of BLI.

Lungs were analyzed microscopically in order to determine whether LMWH altered fibrin/fibrinogen depositions in the tumors. Cryostat sections (6 μm thick) were cut at a cabinet temperature of −25°C using a motor-driven cryostat (Bright, Huntingdon, UK) and stored at −25°C until used. Cryostat sections were air dried for 60 min at room temp. Sections were fixed in 4% (v/v) formaldehyde in distilled water for 20 min and rinsed 3 times in PBS afterwards. Then, endogenous peroxidase was inhibited by incubating sections in 0.3% (v/v) H₂O₂ and 0.1% (w/v) NaN₃ in distilled water for 20 min at room temp. After rinsing 3 times in PBS, sections were incubated in PBS supplemented with 10% (v/v) normal rabbit serum (Dako, Glostrup, Denmark) for 15 min at room temp as blocking step. This incubation step and all following incubation steps with antibodies were performed in a humidified chamber. A goat monoclonal antibody against mouse fibrinogen that cross reacts with fibrin (Accurate Chemical and Scientific Corp, Westbury, NY) was used as primary antibody. The antibody was diluted 1:500 in PBS in the presence of 1% bovine serum albumin. Sections were incubated for 60 min at room temp. After rinsing 3 times in PBS, the secondary antibody, rabbit anti-goat immunoglobulin coupled to horseradish peroxidase (Dako) diluted 1:100 in PBS in combination with 0.2% (w/v) bovine serum albumin and 5% (v/v) normal mouse serum (Dako) was applied and sections were incubated for 60 min at room temp. Binding of secondary antibodies was visualized using a solution of 10 mM diaminobenzidine (Fluka, Buchs, Switzerland), 10 mM NaN₃ and 4% (v/v) H₂O₂ in 50 mM Tris-HCl buffer, pH 7.6, for 10 min at room temp. Control incubations were performed in the absence of the primary antibody in the incubation medium. Subsequently, the sections were rinsed 3 times in distilled water and counterstained with haematoxylin (‘Z’ stain; Cellpath, Newton Powys, UK) for 5 s. After 10 min of rinsing in tab water, sections were mounted in glycerin-gelatin.