Tissue factor (TF) is the primary cellular initiator of blood coagulation and a modulator of angiogenesis and metastasis in cancer. Indeed, systemic hypercoagulability in patients with cancer and TF overexpression by cancer cells are both closely associated with tumor progression, but their causes have been elusive. We now report that in human colorectal cancer cells, TF expression is under control of 2 major transforming events driving disease progression (activation of K-ras oncogene and inactivation of the p53 tumor suppressor), in a manner dependent on MEK/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K). Furthermore, the levels of cell-associated as well as circulating (microvesicle-associated) TF activity are linked to the genetic status of cancer cells. Finally, RNA interference experiments suggest that TF expression is an important effector of the K-ras-dependent tumorigenic and angiogenic phenotype in vivo. Thus, this study establishes a causal link between cancer coagulopathy, angiogenesis, and genetic tumor progression. ( IntroductionCancer is believed to arise and progress toward increasing malignancy as a result of cumulative genetic "hits" sustained by the tumor cell genome. Paradigmatic in this regard is the development of colorectal carcinoma (CRC), where sequential transition through clinical stages of the disease is paralleled by a series of well-characterized alterations in proto-oncogenes and tumor suppressor genes. 1 In this tumor type, activation of mutant K-ras and subsequent inactivation/loss of p53 are key changes, which drive many interrelated aspects of the malignant phenotype including aberrant mitogenesis and survival. 2 Moreover, both of these genetic alterations are thought to contribute to proangiogenic properties of affected cancer cells, 3,4 and thereby enable them to exploit the host vascular system to advance malignant growth and metastasize in vivo. 5 The involvement of the vascular system in malignancy encompasses not only angiogenesis but also systemic hypercoagulability. Blood clotting abnormalities are detected in up to 90% of patients with metastatic disease, and thrombosis represents the second most frequent cause of cancer-related mortality. 6 Cancer coagulopathy is often linked to up-regulation of tissue factor (TF), the primary cellular initiator of the blood coagulation cascade. 7,8 Interaction of coagulation factor VIIa with TF on the cell surface leads to activation of factor X and generation of thrombin, with subsequent involvement of platelets and formation of a fibrin clot. 9 Remarkably, as a member of the class II cytokine receptor family, TF is also capable of transducing intracellular signals and regulating gene expression. 10,11 Interestingly, elements of the coagulation/fibrinolytic system in general, 12 and TF in particular, have been implicated in regulation of angiogenesis, 13,14 as well as tumor growth 15 and metastasis 16 in various experimental settings. This is consistent with the observed up-regulation of TF in huma...

Recently activated, but not resting, CD4(+) T cells express CD154, providing costimulatory signals to B cells and antigen-presenting cells (APCs). Therefore, de novo CD154 expression after stimulation identifies antigen-specific CD4(+) T cells. Previous assays were limited by the transient nature of surface CD154 expression; we overcame this by including fluorescently conjugated CD154-specific antibody during stimulation. Our assay is fully compatible with intracellular cytokine staining, and can be used for stimulations as long as 24 h. Notably, it is nonlethal, providing a means to purify viable antigen-specific CD4(+) T cells for further analysis. Using this assay, we found that stimulated cells expressing tumor necrosis factor (TNF)-alpha, interleukin (IL)-2 or interferon (IFN)-gamma were predominantly CD154(+). Furthermore, some cells expressing none of these cytokines also expressed CD154, suggesting that CD154 marks cells with other effector functions. For vaccine- or pathogen-specific responses, we found substantial heterogeneity in expression of CD154 and cytokines, suggesting previously unrecognized diversity in abilities of responding cells to stimulate APCs through CD40.