Priming of the organ-specific premetastatic sites is thought to be an important yet incompletely understood step during metastasis. In this study, we show that the metastatic tumors we examined overexpress granulocyte-colony stimulating factor (G-CSF), which expands and mobilizes Ly6G+Ly6C+ granulocytes and facilitates their subsequent homing at distant organs even before the arrival of tumor cells. Moreover, G-CSF-mobilized Ly6G+Ly6C+ cells produce the Bv8 protein, which has been implicated in angiogenesis and mobilization of myeloid cells. Anti-G-CSF or anti-Bv8 antibodies significantly reduced lung metastasis. Transplantation of Bv8 null fetal liver cells into lethally irradiated hosts also reduced metastasis. We identified an unexpected role for Bv8: the ability to stimulate tumor cell migration through activation of one of the Bv8 receptors, prokineticin receptor (PKR)-1. Finally, we show that administration of recombinant G-CSF is sufficient to increase the numbers of Ly6G+ Ly6C+ cells in organ-specific metastatic sites and results in enhanced metastatic ability of several tumors.breast cancer | myeloid | CSF3 | prokineticin 2

In response to inflammatory stimuli, dendritic cells (DCs) trigger the process of maturation, a terminal differentiation program required to initiate T-lymphocyte responses. A hallmark of maturation is downregulation of endocytosis, which is widely assumed to restrict the ability of mature DCs to capture and present antigens encountered after the initial stimulus. We found that mature DCs continue to accumulate antigens, especially by receptor-mediated endocytosis and phagocytosis. Internalized antigens are transported normally to late endosomes and lysosomes, loaded onto MHC class II molecules (MHCII), and then presented efficiently to T cells. This occurs despite the fact that maturation results in the general depletion of MHCII from late endocytic compartments, with MHCII enrichment being typically thought to be a required feature of antigen processing and peptide loading compartments. Internalized antigens can also be cross-presented on MHC class I molecules, without any reduction in efficiency relative to immature DCs. Thus, although mature DCs markedly downregulate their capacity for macropinocytosis, they continue to capture, process, and present antigens internalized via endocytic receptors, suggesting that they may continuously initiate responses to newly encountered antigens during the course of an infection. . They are localized to both peripheral and lymphatic tissues and sample their surroundings, internalizing, processing, and presenting captured antigens to T cells on MHC class I molecules (MHCI) and MHC class II molecules (MHCII). They distinguish between self-and foreign antigens using receptors of the innate immune system [e.g., Toll-like receptors (TLRs)], inducing immunity when antigen is captured in the presence of microbial products or inflammatory stimuli but tolerance in the absence of these signals (3). DCs exhibit dramatic functional and morphological changes, termed maturation, that maximize antigen presentation to T cells in response to such stimuli (1, 2). These changes involve acidification of the lysosomal compartment to optimize antigen processing, up-regulation of costimulatory molecules, and reorganization of MHCII from the late endocytic compartments to the plasma membrane for recognition by T cells (4, 5).Modulation of endocytosis also occurs during maturation. Immature DCs endocytose avidly through a variety of mechanisms, including "nonspecific" uptake by constitutive macropinocytosis and "specific" uptake via receptor-mediated endocytosis and phagocytosis (2). Macropinocytosis is transiently up-regulated immediately on the receipt of an inflammatory signal (6), but this is followed by its dramatic down-regulation, partly mediated by a reduction of the active form of the Rho GTPase, Cdc42 (7). Most studies of endocytosis in DCs have involved exposing cells to a high concentration of antigens or endocytic tracers. Although such assays mainly measure macropinocytosis, it is generally presumed that all forms of endocytosis are down-regulated in mature DCs. The idea seems to fit...

The hepatocyte growth factor (HGF) and its receptor, c-Met, have been implicated in driving proliferation, invasion, and poor prognosis in pancreatic cancer. Here, we investigated the expression of HGF and c-Met in primary pancreatic cancers and described in vitro and in vivo models in which MetMAb, a monovalent antibody against c-Met, was evaluated. First, expression of HGF and MET mRNA was analyzed in 59 primary pancreatic cancers and 51 normal samples, showing that both factors are highly expressed in pancreatic cancer. We next examined HGF responsiveness in pancreatic cancer lines to select lines that proliferate in response to HGF. Based on these studies, two lines were selected for further in vivo model development: BxPC-3 (c-Met + , HGF À ) and KP4 (c-Met + , HGF + ) cells. As BxPC-3 cells are responsive to exogenous HGF, s.c. tumor xenografts were grown in a paracrine manner with purified human HGF provided by osmotic pumps, wherein MetMAb treatment significantly inhibited tumor growth. KP4 cells are autocrine for HGF and c-Met, and MetMAb strongly inhibited s.c. tumor growth. To better model pancreatic cancer and to enable long-term survival studies, an orthotopic model of KP4 was established. MetMAb significantly inhibited orthotopic KP4 tumor growth in 4-week studies monitored by ultrasound and also improved survival in 90-day studies. MetMAb significantly reduced c-Met phosphorylation in orthotopic KP4 tumors with a concomitant decrease in Ki-67 staining. These data suggest that the HGF/c-Met axis plays an important role in the progression of pancreatic cancer and that targeting c-Met therein may have therapeutic value.

BackgroundHeparan sulfate proteoglycans (HSPGs) use highly sulfated polysaccharide side-chains to interact with several key growth factors and morphogens, thereby regulating their accessibility and biological activity. Various sulfotransferases and sulfatases with differing specificities control the pattern of HSPG sulfation, which is functionally critical. Among these enzymes in the mouse are two secreted 6-O-endosulfatases, Sulf1 and Sulf2, which modify HSPGs in the extracellular matrix and on the cell surface. The roles of Sulf1 and Sulf2 during normal development are not well understood.Methods/ResultsTo investigate the importance of Sulf1 and Sulf2 for embryonic development, we generated mice genetically deficient in these genes and assessed the phenotypes of the resulting secreted sulfatase-deficient mice. Surprisingly, despite the established crucial role of HSPG interactions during development, neither Sulf1- nor Sulf2-deficient mice showed significant developmental flaws. In contrast, mice deficient in both Sulf1and Sulf2 exhibited highly penetrant neonatal lethality. Loss of viability was associated with multiple, although subtle, developmental defects, including skeletal and renal abnormalities.ConclusionsThese results show that Sulf1 and Sulf2 play overlapping yet critical roles in mouse development and are redundant and essential for neonatal survival.

Magnetic resonance microscopy (microMRI) is becoming an important tool for non-destructive analysis of fixed brain tissue. However, unlike MRI, X-ray computed tomography (CT) scans show little native soft tissue contrast. In this paper, we explored the use of contrast enhanced (brains immersion stained in iodinated CT contrast media) micro-CT (microCT) for high resolution 3D imaging of fixed normal and pathological brains, compared to microMRI and standard histopathology. An optimum iodine concentration of 0.27 M resulted in excellent contrast between gray and white matter in normal brain and a wide range of anatomical structures were identified. In glioma bearing mouse brains, there was clear deliniation of tumor margin which closely matched that seen on histopathology sections. microCT tumor volume was strongly correlated with histopathology volume. Our data suggests that microCT image contrast in the immersion-stained brains is related to axonal density and myelin content. Compared to traditional histopathology, our microCT approach is relatively rapid and less labor intensive. In addition, compared to microMRI, microCT is robust and requires much lower equipment and maintenance costs. For simple measurements, such as tumor volume and non-destructive postmortem brain screening, microCT may prove to be a valuable alternative to standard histopathology or microMRI.