Human breast cancer is extensively infiltrated by mast cells that contain powerful anticoagulants such as heparin, tryptase and chymase. To determine if human breast cancer is associated with mast cell activation, we measured the levels of mast cell tryptase (an indicator of mast cell activation) in the blood of 20 women with varying stages of breast cancer. The mean level of tryptase in women with breast cancer (10.3 ؎ 4.2 g/L) was significantly higher than in 50 normal healthy women without breast cancer (3.0 ؎ 2.5 g/L, p < 0.05 by two-tailed t-test). To explore the role of mast cells in breast cancer in more detail, we then carried out experiments that were aimed at determining if an inhibitor of mast cell function, sodium cromolyn, could increase blood clotting and hypoxia within subcutaneous implants of the 4T1 mammary adenocarcinoma cell line in mice. We treated tumor-bearing mice with 5 consecutive daily doses of sodium cromolyn (10 mg/kg, i.p.). An average of 30% of the periphery of the tumors from the 5 drug-treated mice contained large lakes of clotted blood that were not evident in any of the tumors from the control (untreated) mice. By computerized image analysis of tumors immunostained for a hypoxia marker (pimonidazole), the tumors from the treated mice had significantly more hypoxia (35 ؎12 % hypoxic regions, n ؍ 5) than the tumors from untreated (control) mice (16 ؎ 7%, n ؍ 5). We conclude that sodium cromolyn enhanced peri-tumoral blood clotting and intratumoral hypoxia. These results suggest that mast cells may play an important role in regulating blood clotting and hypoxia within breast cancer.
Hypereosinophilic syndromes are often associated with thrombosis through unclear mechanisms, and mastocytosis has been associated with a variety of bleeding disorders. The present studies were aimed at defining the roles and interactions of eosinophil and mast cell constituents on the kinetics of blood clotting as measured by thromboelastograms. Eosinophil granule proteins and purified eosinophil peroxidase markedly reduced the anticoagulant properties of the mast cell tryptase/heparin complex. Moreover, eosinophil peroxidase by itself functioned as a powerful procoagulant and also inhibited the anticoagulant actions of heparin in a chromogenic assay for antithrombin III/factor Xa activity. The anticoagulant activity of the tryptase/heparin complex was attributable exclusively to the associated heparin and not to the intrinsic enzymatic activity of tryptase. Eosinophil granule proteins also strongly inhibited the enzymatic activity of tryptase in the presence of hydrogen peroxide, thus implicating a critical role for eosinophil peroxidase. We conclude that eosinophil granule proteins and eosinophil peroxidase both function as powerful procoagulants and also inhibit the anticoagulant and enzymatic activities of mast cell tryptase. The present results thus provide a mechanistic rationale for the well-established link between certain eosinophilic inflammatory disorders and hypercoagulant states. They also suggest that eosinophils may play an important role in neutralizing the anticoagulant activity of mast cell tryptase/heparin in various diseases.
Imatinib mesylate (Gleevec™) inhibits the BCR-ABL tyrosine kinase in chronic granulocytic leukemia. Previous studies have demonstrated that imatinib mesylate also inhibits the survival and functions of normal mast cells by interfering with the receptor tyrosine kinase for stem cell factor (SCF), c-kit, which is expressed by mast cells. Because mast cells extensively surround many types of cancer and contain powerful anticoagulants such as heparin, we investigated the effects of imatinib mesylate on blood clotting and tumor growth within subcutaneous implants of a mammary adenocarcinoma cell line (4T1) in BALB/c mice. After 5 days of oral treatment with 10 mg/kg of the drug, the average mass of the tumors in treated mice (198 ؎ 42 mg, n ؍ 5) was significantly (p < 0.05) greater than the average mass of the tumors from untreated (control) mice (60 ؎ 23 mg, n ؍ 5). Moreover, the tumors in the treated mice were frequently surrounded by large lakes of clotted blood that were not evident in tumors from the control mice. Accelerated growth and blood clotting were also observed in tumor-bearing mice treated with heparinase I enzyme to destroy endogenous mast cell heparin and in NDST-2 knockout mice in which there is a targeted disruption in the gene coding for mast cell heparin synthesis. We conclude that imatinib mesylate accelerated the growth and peri-tumoral blood clotting of implants of mammary adenocarcinoma in mice. These results suggest that imatinib mesylate may have significant effects on mast cells infiltrating tumors, in addition to its other biologic activities. Our results also indicate that the mechanism of this effect may be related to the anticoagulant properties of mast cell heparin.
An unexplained paradox of malignant melanoma is the apparent failure of the blood within the tumor to clot despite the presence of multiple factors that should promote blood clotting. Here we present histochemical evidence that human and murine melanomas are extensively infiltrated by abundant mast cells. Because mast cells contain the natural anticoagulant heparin, the present studies were aimed at defining the role of mast cell heparin in preventing the blood from clotting within B16 melanoma grafts in C57BL/6 J mice. Mice bearing B16 melanoma grafts were treated with non-specific or specific inhibitors of mast cell heparin (protamine or heparinase, respectively). After the drug treatment there was histologic and functional evidence of selective thrombosis of the blood vessels within the protamine and heparinase treated melanoma grafts. A similar, high degree of thrombosis was also observed in B16 tumors grown in transgenic NDST-2 knockout mice bearing a targeted disruption in the gene coding for mast cell heparin synthesis. The tumors grown in the protamine-treated animals were significantly smaller than the tumors from control (untreated mice). By contrast, the tumors treated with heparinase or grown in the NDST-2 knockout mice were significantly larger than the tumors from control (untreated) mice. We conclude that the intrinsic procoagulant properties of malignant melanoma are neutralized in vivo by the anticoagulant properties of endogenous heparin produced by mast cells that naturally infiltrate the tumor. Our results also suggest that thrombosis and hemostasis within melanoma may play a complex role in modulating the growth of the tumor.
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