It has been hypothesized that carcinoma metastasis is initiated by a subpopulation of circulating tumor cells (CTCs) found in the blood of patients. However, although the presence of CTCs is an indicator of poor prognosis in several carcinoma entities, the existence and phenotype of metastasis-initiating cells (MICs) among CTCs has not been experimentally demonstrated. Here we developed a xenograft assay and used it to show that primary human luminal breast cancer CTCs contain MICs that give rise to bone, lung and liver metastases in mice. These MIC-containing CTC populations expressed EPCAM, CD44, CD47 and MET. In a small cohort of patients with metastases, the number of EPCAM(+)CD44(+)CD47(+)MET(+) CTCs, but not of bulk EPCAM(+) CTCs, correlated with lower overall survival and increased number of metastasic sites. These data describe functional circulating MICs and associated markers, which may aid the design of better tools to diagnose and treat metastatic breast cancer.

IntroductionLocal and systemic coagulation activation is a hallmark of advanced malignancies. 1,2 Cancer cell-expressed tissue factor (TF) is a major procoagulant stimulus in cancer-associated thrombosis, and tumor cell TF expression is correlated with tumor progression in several experimental tumor models (reviewed in Schaffner and Ruf 3 ). Although thrombin generated in the course of coagulation supports hematogenous metastasis 4 and regulates angiogenesis and the tumor microenvironment, 5 several lines of pharmacological and genetic evidence show that direct signaling of TF plays a pivotal role in cancer progression and angiogenesis.The TF-VIIa complex cleaves the G protein-coupled protease activated receptor (PAR) 2 6 and triggers breast cancer cells to produce a diverse repertoire of angiogenic regulators and immunemodulatory cytokines. [7][8][9] The cleavage of PAR2 activates G␣q, G␣12/13, and G␣i, and termination of signaling occurs upon internalization of the receptor that is initiated by the binding of ␤-arrestins to phosphorylated residues on the C-terminus of G protein-coupled receptors. ␤-arrestins also promote G-protein independent signaling by serving as a scaffold for activating the extracellular regulated kinases (ERK) pathway. 10 The coupling of ␤-arrestin to PAR2 results in the dephosphorylation of cofilin, which leads to the severing of actin filaments. 11 Thus, the activation of the noncanonical pathway through ␤-arrestin promotes breast cancer motility. [11][12][13][14] The TF-VIIa-signaling complex is associated with integrins and regulates ␣3␤1-dependent migration in a crosstalk that involves the TF cytoplasmic domain in noncancerous epithelial cells. 15 In cancer cells, TF is constitutively associated with ␤1 integrins that regulate TF-VIIa-PAR2-mediated induction of proangiogenic chemokines. 16 Importantly, a unique monoclonal antibody to human TF without significant anticoagulant properties specifically disrupts the interaction of TF with integrins, TF-VIIa-PAR2 cell signaling, and the growth of human breast cancer xenografts in mice, 16 demonstrating that TF-PAR2 signaling is a potential therapeutic target for cancer therapy.Although breast cancer cell PAR1 signaling is deregulated to increase invasiveness 17,18 and is partially overlapping with TF-PAR2 signaling in promoting transcriptional responses, 8 only PAR2 deficiency significantly delays the progression from adenoma to adenocarcinoma in the polyoma middle T (PyMT) model of spontaneous breast cancer development. 19 In this mouse model, the oncogenic middle T-antigen protein is expressed under a mammary-specific promoter, and mice expressing the transgene develop tumors in all mammary glands. Tumor progression resembles the human disease 20 at the molecular level (eg, downregulation of the estrogen and progesterone receptors) and, due to the relatively slow development, allows the study of complex interactions between the host and tumor cells. Because postnatal, hypoxia-induced and transplanted tumor angiogenesis is unaffected in...