RPMI1640, Dulbecco’s modified Eagle’s medium (DMEM), Endothelial Basal Medium-2 (EBM-2), EGM-2-MV SingleQuots, fetal bovine serum (FBS) and phosphate buffered saline (PBS) were purchased from Lonza (Viviere, Belgium), while the Human mammary epithelial cell (HuMEC) ready medium, Alexa Fluor® 488 goat anti-rabbit IgG (H+L) antibody (A-11008), and SlowFade® Gold antifade reagent w/DAPI (S36938) were from Life Technologies (Carlsbad, CA, USA). The polyclonal rabbit anti-human tissue factor pathway inhibitor antibody (4901/ADG72), monoclonal mouse anti-human tissue factor pathway inhibitor antibody (4903), and monoclonal mouse anti-human tissue factor antibody (ADG4508) were from American Diagnostica (Greenwich, CT, USA), while the rabbit-IgG-UNLB and goat anti-rabbit IgG (H+L)-RPE antibodies were from Southern Biotechnology Associates (Birmingham, AL, USA). The human γ-globulin antibody, PI-PLC, and formalin solution (4% formaldehyde, HT5011) were from Sigma-Aldrich (St. Louis, MO, USA). Heparin was purchased from Leo Pharma (Ballerud, Denmark). The RNaqueous kit (AM1912), High Capacity cDNA Reverse Transcription Kit, TaqMan Gene Expression Master Mix, TaqMan TF assay (Hs00175225_m1), and pre-developed TaqMan Human 18S rRNA assay were all from Applied Biosystems (Life Technologies). Purified bovine FX, FXa, and FXa chromogenic substrate CS-11(22) were from Aniara Diagnostica (Mason, OH, USA), while recombinant FVIIa was from Novo Nordisk AS (Bagsvaerd, Denmark).
The human mammary adenocarcinoma cell lines SK-BR-3 (ATCC HTB-30) and MCF-7 (ATCC HTB-22) and the human mammary ductal carcinoma cell lines ZR-75-1 (ATCC CRL-1500) and BT-474 (ATCC HTB-20) were grown in RPMI1640 with phenol red and 2 mM L-glutamine supplemented with 10% heat inactivated FBS. The intraductal carcinoma cell lines Sum102 and Sum149 and the transformed breast epithelial cell line ME16C2 (hTERT-HME1, ATCC CRL-4010) were grown in HuMEC ready medium containing HuMEC supplements (epidermal growth factor, hydrocortisone, isoprotenerol, transferrin and insulin) and Bovine Pituitary Extract. 5% heat-inactivated FBS was included in the growth medium of Sum149 cells. The human mammary adenocarcinoma cell line MDA-MB-231 (ATCC HTB-26), the human endothelial cell line EA.hy926 (ATCC CRL-2922), and the Chinese hamster ovary cell line CHO-K1 (ATCC CCL-61) were cultured in DMEM containing 2 mM L-glutamine, 4.5 g/L glucose and 10% heat inactivated FBS. The primary human coronary artery endothelial cells HCAEC (#CC-2585, Lonza) were cultivated in modified EBM-2 basal medium supplemented with EGM-2-MV SingleQuots (containing vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor-I, epidermal growth factor, ascorbic acid, and gentamicin), and 10% FBS. All cells were cultured at 37°C in an incubator with a humidified atmosphere and 5% CO2.
Quantitative real-time PCR
Total RNA was isolated from the cells using the RNaqueous kit according to the manufacturer’s protocol, and the concentration of the isolated RNA was assessed by the NanoDrop1 ND-1000 UV–vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). cDNA was synthesized from total RNA using the High Capacity cDNA Reverse Transcription Kit, and 19 ng of cDNA were PCR amplified on the ABI PRISM 7900 Sequence Detection System (Applied Biosystems) according to the protocol, using the TaqMan Gene Expression Master Mix, and the self-designed TFPIα and TFPIβ specific assays  or a Taqman TF assay. All samples were run in triplicate. The relative mRNA expression was calculated using the comparative Ct method, normalizing the Ct values against the endogenous control 18S rRNA, and using HCAEC expression as a normal endothelial reference and CHO expression values as a negative sample. The 18S rRNA showed the least expression variation between the different cell lines of the endogenous controls tested.
TFPI and TF antigen
To determine TFPI and TF protein expression, cells were seeded in six-well trays and grown for three days until ~80% confluence before harvesting. Media were collected and cells were lysed as previously described . The commercial ELISA kits Free and Total TFPI (Asserachrom®, Diagnostica Stago, Asnière, France) were used to measure TFPI antigen in the cell media/lysate, and Zymutest Tissue Factor full length (RK035A, Hyphen BioMed) to measure full length TF in cell lysates, according to the manufacturers’ protocols. The results were corrected against cellular total protein, as measured in cell lysates by the modified Lowry assay (Bio-Rad D
Protein Assay, Bio-Rad Laboratories, Hercules, CA, USA).
PI-PLC and heparin treatment
Confluent cells in six-wells trays were washed three times with PBS and serum starved for one hour before treated with PI-PLC (1 U/mL), or heparin (5 U/mL) for two hours, or with PI-PLC for two hours followed by removal of the supernatant and subsequent treatment with heparin for additional two hours, at 37°C. Serum-free media (SFM) was used as a control for the treatments. A second control containing SFM and glycerol from a 55% solution with 0.05% NaN3 to mimic the PI-PLC buffer was also tested but showed no differences from SFM alone. The supernatants were collected and analyzed for TFPI antigen, while the cells were washed twice with PBS and collected for flow cytometry analysis or lysed as previously described  for antigen detection.
Immediately after treatment, Sum 102, MDA-MB-231, and HCAEC cells were detached using 5 mM EDTA and transferred to Eppendorf tubes. Cells were washed twice in cold PBS before blocked with human γ-globulin (100 μg/mL) in dilution buffer (PBS with 1% BSA and 12.5 mM Na-Azide) for 15–20 min. Cells were incubated with a rabbit anti-human TFPI specific antibody or an irrelevant rabbit antibody at a final concentration of 40 ng/μl for 30 min at 4°C. Cells were washed twice in cold PBS and stained with RPE linked goat anti-rabbit antibody for 30 min at 4°C, before pelleted cells were resuspended in dilution buffer. Flow cytometry analysis was performed on the FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany), and the CellQuest 3.3 software (BD Biosciences) was used for data collection. Flow data were analyzed and presented using Flow Jo 7.6.4 (Tree Star, Inc., Ashland, OR).
Sum102 cells (1.0⋅105/well) were seeded the day before in 4-wells glass chamber slides (Lab Tek II, Thermo Fisher Scientific, Waltham, MA), serum-starved for 1 hour and treated with SFM containing glycerol from a 55% solution with 0.05% NaN3 (denoted untreated) or PI-PLC (1 U/mL) for 2 hours at 37°C. After treatment, the cells were washed once in PBS, fixed with formalin for 20 min on ice, washed once with PBS (with 1% FBS) and incubated 20 min at RT with blocking buffer (PBS with 5% BSA and 1% Tween 20). After blocking, the cells were washed once in dye buffer (PBS with 1% BSA and 1% Tween 20) and incubated with primary rabbit anti-human TFPI and mouse anti-human TF specific antibodies for 45 min at RT. Cells were washed three times with dye buffer and stained with fluorescent goat anti-rabbit and donkey anti-mouse secondary antibodies for 45 min at RT. The cells were washed three times with dye buffer, the chambers were released from the slides, and two drops of antifade solution were added and the slides sealed with cover glass. Stained cells were visualized using a fluorescence Nikon eclipse E400 inverted microscope with a Plan Fluor 40x/0.75 DIC M objective (Nikon, Tokyo, Japan) and the appropriate filter. Images were captured using a Nikon digital sight DS-Fi1 Camera system.
Sum102 (1.5⋅105/well), MDA-MB-231 (0.5⋅105/well), and HCAEC (0.7⋅105/well) cells were seeded the day prior to the experiment, serum-starved for 1 hour and treated with SFM (denoted untreated), PI-PLC (1 U/mL), TFPI blocking antibody (10 μg/mL), or TF blocking antibody (10/20 μg/mL) for 2 hours at 37°C. After treatment, the cells were washed twice in wash solution (10 mM HEPES, 150 mM NaCl, 4 mM KCl, and 11 mM Glucose, pH 7.5) and incubated for 1 hour at 37°C in reaction solution (wash buffer with 5 mg/mL BSA and 5 mM CaCl2, pH 7.5) containing 10 nM FVIIa and 175 nM FX. Following incubation, 50 μL were transferred to 100 μL stop solution (50 mM Tris HCl, 150 mM NaCl, 25 mM EDTA, and 1 mg/mL BSA, pH 7.5) on ice before incubated with 50 μL CS-11(22) substrate. The absorbance at 405 nm was recorded at 37°C for 45 min at 15 sec intervals using a Spectra Max Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The maximum velocities (Vmax) in mU/min were used to calculate the amount of FXa generated, using a standard curve obtained with known concentrations of FXa.
Supernatants from Sum102 cells treated with SFM containing glycerol from a 55% solution with 0.05% NaN3 (denoted untreated) or PI-PLC were collected and deglycosylated using the Enzymatic Protein Deglycosylation Kit (Sigma-Aldrich) following the manufacturer’s instruction, combined with loading buffer (Bio-Rad Laboratories Hercules, CA) containing 5% β-mercaptoethanol and denatured for 5 min at 97°C. The concentrated supernatants were separated on a 10% SDS-polyacrylamide gel (Bio-Rad), before proteins were transferred to a PVDF membrane (Bio-Rad), blocked in 5% BSA, and incubated with a primary anti-human TFPI specific antibody over night at 4°C under constant agitation. After incubation with the appropriate secondary HRP-linked antibody for 1 hour at 20°C, proteins were visualized using the ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). Images were produced using the Luminescent Image Analyzer LAS-4000 mini (Fujifilm, Tokyo, Japan).
The associations between mRNA and protein levels were evaluated using Pearsons correlation test, while significant differences between treated samples and controls were calculated using the Student’s t or one-way ANOVA (Bonferroni corrected) tests in GraphPad Prism 5.0 (Graphpad, San Diego, CA, USA), and a P-value of <.05 was considered statistically significant. * = p<.05, ** = p<.01, and *** = p<.001.