136 EC = endothelial cell; IBC = inflammatory breast cancer; MRA = magnetic resonance angiography; VEGF = vascular endothelial growth factor; VM = vasculogenic mimicry. Breast Cancer Research Vol 5 No 3 Shirakawa et al. IntroductionGrowth, proliferation, and metastasis of breast cancer, and of most other tumors, have been thought to be angiogenesis-dependent processes [1]. However, a non-angiogenesis-dependent pathway, in which tumors can feed themselves using alternative pathways, has also been reported [2][3][4][5][6][7][8][9][10][11][12]. Previously, we and others described the presence of vasculogenic mimicry (VM, a condition in which tumors [inflammatory breast cancer {IBC} and melanoma] feed themselves using alternative pathways without the participation of endothelial cells [ECs]) in the tumor-bearing state. In the present study, we established a new human IBC xenograft (WIBC-9) in BALB/c nude mice and investigated the hemodynamics of VM and angiogenesis of IBC, using WIBC-9 xenografts and dynamic micro-magnetic resonance angiography (micro-MRA) analysis. The unique patterns characteristic of VM and its hemodynamics provide a framework for the design of non-invasive imaging techniques for detecting IBC and its metastases. Method Morphological and chromosomal analysisThe animal protocols for all experiments were approved by the Animal Use Committee of the National Cancer Center. Hematoxylin-eosin and Giemsa staining of paraffin-embedded specimens were performed, as were electron microscopic examinations following a conventional method. For karyotype studies of the xenograft, the Giemsa G banding method was performed after 6 and 12 passages. Dynamic micro-MRA with an intravascular contrast agentWe performed dynamic micro-MRA analysis, using our newly developed intravascular macromolecular contrast agent for magnetic resonance imaging, which consistently showed no significant leakage through the vascular wall AbstractWe recently established a new human inflammatory breast cancer (IBC) xenograft (WIBC-9) originating from a patient with IBC. The original tumor and WIBC-9 revealed invasive ductal carcinoma with a hypervascular structure of solid nests and marked lymphatic permeation in the overlying dermis. In the central part of the solid nests, vasculogenic mimicry, which showed an absence of endothelial cells, was observed. Comparison of WIBC-9 with an established non-IBC xenograft (MC-5), using time-course dynamic micro-magnetic resonance angiography analysis (with a newly developed intravascular macromolecular contrast agent for magnetic resonance imaging) demonstrated that the WIBC-9 tumor had blood flow and a vascular mimicry-angiogenesis junction.

Tumors require a blood supply for growth and hematogenous metastases. Until recently, most research in this area has focused on the role of angiogenesis, the recruitment of new vessels into a tumor from preexisting vessels. Previously, in a study of breast cancer (IBC), in which we used estab-

Inflammatory breast cancer (IBC) isInflammatory breast cancer (IBC) is clinically characterized by rapid tumor enlargement with skin erythema. The disease tends to invade locally and metastasize systemically and the histopathologic features are consistent with an extremely aggressive phenotype. 1 Despite recent advances in strategies for multimodal treatment of patients with IBC, this disease is still considered incurable and there is a great need for further improvement of treatment strategies to combat this breast cancer variant. 2 With most solid tumors, growth, proliferation and metastasis are thought to be dependent on angiogenesis, a process in which vessels grow, in part, by incorporating circulating endothelial cells (ECs) and endothelial precursor cells (EPCs). [3][4][5][6][7][8][9][10] Studies have shown that soluble Flt-1 (sFlt-1) can inhibit vascular endothelial growth factor (VEGF) activity not only by sequestering VEGF but also apparently via a dominant negative mechanism in which it forms heterodimers with the full-length membrane-spanning receptors Flt-1 and KDR and thereby prevents VEGF-dependent transphosphorylation and activation of these receptors. 11-15 Soluble Tie2 (sTie2), an artificial form of Tie2 (a full-length membrane-spanning receptor), is a heparin-binding protein that complexes with angiopoietin (Ang) with the same affinity and specificity as Tie2. 16 -18 In the present study, we transfected tumor cells with genes for sFlt-1 and sTie2, assessed the effects of these genes in association with tumor-infiltrating (TI) ECs/EPCs in IBC xenografts and found that VEGF and Ang pathways are crucial in IBC angiogenesis and metastasis. The present results help to clarify the mechanism of induced migration of ECs/EPCs into IBC nests and the effects on IBC of blocking VEGF and Ang pathways.

Recently, there have been reports of postnatal vasculogenesis in cases of ischaemia models. The aim of the present study is to provide evidence of postnatal vasculogenesis in breast-cancer -bearing mice. Based on cell surface antigen expression, we isolated endothelial precursor cells from bone marrow, peripheral blood and tumour-infiltrating cells from mice that had received six human breast cancer xenografts. In all three areas (bone marrow, peripheral blood and tumour-infiltrating cells), endothelial precursor cell population was elevated in all transplanted mice. Differentiation and migration activities of endothelial precursor cells were measured by comparing levels of the endothelial precursor cell maturation markers Flk-1, Flt-1, Tie2, VE-cadherin and CD31 among these three areas. The endothelial precursor cell population was 14% or greater in the gated lymphocyte-size fraction of the inflammatory breast cancer xenograft named WIBC-9, which exhibits a hypervascular structure and de novo formation of vascular channels, namely vasculogenic mimicry (Shirakawa et al, 2001). In vitro, bone marrow-derived endothelial precursor cells from four human breast cancer xenografts proliferated and formed multiple clusters of spindle-shaped attaching cells on a vitronectin-coated dish. The attaching cells, which incorporated DiI-labelled acetylated low-density lipoprotein (DiI-acLDL) and were negative for Mac-1. The putative bone marrow derived endothelial precursor cell subset, which was double positive of CD34 and Flk-1, and comparative bone marrow derived CD34 positive with Flk-1 negative subset were cultured. The former subset incorporated DiI-acLDL and were integrated with HUVECs. Furthermore, they demonstrated significantly higher levels of murine vascular endothelial growth factor and interleukin-8 in culture supernatant on time course by enzyme-linked immunosorbent assay. These findings constitute direct evidence that breast cancer induces postnatal vasculogenesis in vivo.

The results suggest that synchronous determination of the levels of MMP-9 in portal and peripheral blood would be useful for selecting colorectal cancer patients at high risk of hepatic recurrence.