Promotion of experimental thrombus formation by the procoagulant activity of breast cancer cells - PubMed (original) (raw)

Promotion of experimental thrombus formation by the procoagulant activity of breast cancer cells

M A Berny-Lang et al. Phys Biol. 2011 Feb.

Abstract

The routine observation of tumor emboli in the peripheral blood of patients with carcinomas raises questions about the clinical relevance of these circulating tumor cells. Thrombosis is a common clinical manifestation of cancer, and circulating tumor cells may play a pathogenetic role in this process. The presence of coagulation-associated molecules on cancer cells has been described, but the mechanisms by which circulating tumor cells augment or alter coagulation remains unclear. In this study we utilized suspensions of a metastatic adenocarcinoma cell line, MDA-MB-231, and a non-metastatic breast epithelial cell line, MCF-10A, as models of circulating tumor cells to determine the thrombogenic activity of these blood-foreign cells. In human plasma, both metastatic MDA-MB-231 cells and non-metastatic MCF-10A cells significantly enhanced clotting kinetics. The effect of MDA-MB-231 and MCF-10A cells on clotting times was cell number-dependent and inhibited by a neutralizing antibody to tissue factor (TF) as well as inhibitors of activated factor X and thrombin. Using fluorescence microscopy, we found that both MDA-MB-231 and MCF-10A cells supported the binding of fluorescently labeled thrombin. Furthermore, in a model of thrombus formation under pressure-driven flow, MDA-MB-231 and MCF-10A cells significantly decreased the time to occlusion. Our findings indicate that the presence of breast epithelial cells in blood can stimulate coagulation in a TF-dependent manner, suggesting that tumor cells that enter the circulation may promote the formation of occlusive thrombi under shear flow conditions.

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Figures

Figure 1

Figure 1. Characterization of the procoagulant activity of breast epithelial cells

Human sodium citrate-anticoagulated plasma was incubated with vehicle or suspensions of cultured MDA-MB-231 or MCF-10A cells (1×103 – 2×105/mL) for 3 minutes at 37°C. Coagulation of plasma was initiated by recalcification using 16.7 mM CaCl2 (final concentration) and clotting times were recorded on a coagulometer. Data are reported as mean ± SEM, from 6-8 experiments. In comparison to vehicle, clotting times were significantly shortened at all MDA-MB-231 or MCF-10A cell numbers, #P<0.05. *P<0.05 versus corresponding MDA-MB-231 cell concentration.

Figure 2

Figure 2. Characterization of the procoagulant activity of breast epithelial cells

(a) Cultured MDA-MB-231 or MCF-10A cells (1×106/mL) were labeled with a FITC-conjugated anti-TF antibody (1 μg/mL) and analyzed by flow cytometry. Shaded curves represent background fluorescence of unlabeled cells; white curves represent shift in fluorescence in the presence the anti-TF antibody. Representative curves from two or more independent experiments are shown. (b&c) Human sodium citrate-anticoagulated plasma was pretreated with (b) vehicle; TF (TF,10 pM); the TF pathway inhibitor, FVIIai (20 μg/mL); or a neutralizing antibody to TF (anti-TF, 20 μg/mL) or (c) vehicle; the FXa inhibitor, rivaroxaban (FXa inh, 10 μM); the thrombin inhibitor, hirudin (20 μg/mL); or the phosphatidylserine binding protein, annexin A5 (Ann A5, 10 μg/mL). Cultured MDA-MB-231 or MCF-10A cells were added to treated plasma at 1×104/mL. After 3 minutes of incubation at 37°C, coagulation was initiated by addition of 16.7 mM CaCl2 and clotting times were recorded. Data are reported as mean ± SEM, from 4-8 experiments. If clotting did not occur during 20 minutes of observation, experiments were terminated and a clotting time of 20 minutes was recorded. *P<0.05 versus vehicle treatment.

Figure 3

Figure 3. Cultured breast epithelial cells bind thrombin under procoagulant conditions

Human sodium citrate-anticoagulated plasma was incubated with suspended MDA-MB-231 or MCF-10A cells (2×105/mL) for 3 minutes at 37°C in the presence of OG-488 active-site labeled thrombin (1 μM). Plasma was pretreated with GPRP (10 mM), an inhibitor of fibrin polymerization, to prevent complete gelation. Coagulation was initiated by addition of 16.7 mM CaCl2 and plasma was sampled 5 minutes later. Samples were imaged by DIC and fluorescence microscopy, a representative image of a MDA-MB-231 and MCF-10A cell binding thrombin is shown. OG-488 thrombin fluorescence is indicated in green.

Figure 4

Figure 4. Cultured breast epithelial cells promote TF-dependent occlusive thrombus formation in flowing blood, ex vivo

Human sodium citrate-anticoagulated whole blood was mixed with vehicle, MDA-MB-231 or MCF-10A cells (4×104 or 1×103/mL) for 5 minutes at room temperature. In selected experiments, blood was treated with a neutralizing antibody to TF (anti-TF, 20 μg/mL) or the thrombin inhibitor, hirudin (20 μg/mL), in the presence of MDA-MB-231 or MCF-10A cells. (a) Treated blood was recalcified with CaCl2 and MgCl2 (final concentration 7.5 and 3.75 mM, respectively), added to a reservoir to a set height (hb), and allowed to drain through collagen-coated capillaries into a PBS bath as shown. (b) The time to thrombotic occlusion (time until blood ceased to flow from the capillary) was recorded. The height of blood in the reservoir was maintained at 1.5 cm, yielding an initial shear rate of 285 s-1 in the 0.2 × 2.0 × 50 mm collagen-coated capillary, as described in Materials and Methods. Data are mean ± SEM from 3 or more experiments. *P<0.05 versus vehicle treatment in the absence of cells. #P<0.05 versus vehicle treatment of corresponding cell type at 4×104/mL.

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