Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.

IL-17 is a proinflammatory cytokine, and its in vivo expression induces neutrophilia in mice. IL-17E is a recently described member of an emerging family of IL-17-related cytokines. IL-17E has been shown to bind IL-17Rh1, a protein distantly related to the IL-17R, suggesting that IL-17E probably possesses unique biological functions. In this study, we have identified the murine ortholog of IL-17E and developed transgenic mice to characterize its actions in vivo. Biological consequences of overexpression of murine (m)IL-17E, both unique to IL-17E and similar to IL-17, were revealed. Exposure to mIL-17E resulted in a Th2-biased response, characterized by eosinophilia, increased serum IgE and IgG1, and a Th2 cytokine profile including elevated serum levels of IL-13 and IL-5 and elevated gene expression of IL-4, IL-5, IL-10, and IL-13 was observed in many tissues. Increased gene expression of IFN-γ in several tissues and elevated serum TNF-α were also noted. In addition, IL-17E induces G-CSF production in vitro and mIL-17E-transgenic mice had increased serum G-CSF and exhibit neutrophilia, a property shared by IL-17. Moreover, exposure to mIL-17E elicited pathological changes in multiple tissues, particularly liver, heart, and lungs, characterized by mixed inflammatory cell infiltration, epithelial hyperplasia, and hypertrophy. Taken together, these findings suggest that IL-17E is a unique pleiotropic cytokine and may be an important mediator of inflammatory and immune responses.

Most mouse models of hepatocellular carcinoma have expressed growth factors and oncogenes under the control of a liver-specific promoter. In contrast, we describe here the formation of liver tumors in transgenic mice overexpressing human fibroblast growth factor 19 (FGF19) in skeletal muscle. FGF19 transgenic mice had elevated hepatic alpha-fetoprotein mRNA as early as 2 months of age, and hepatocellular carcinomas were evident by 10 months of age. Increased proliferation of pericentral hepatocytes was demonstrated by 5-bromo-2'-deoxyuridine incorporation in the FGF19 transgenic mice before tumor formation and in nontransgenic mice injected with recombinant FGF19 protein. Areas of small cell dysplasia were initially evident pericentrally, and dysplastic/neoplastic foci throughout the hepatic lobule were glutamine synthetase-positive, suggestive of a pericentral origin. Consistent with chronic activation of the Wingless/Wnt pathway, 44% of the hepatocellular tumors from FGF19 transgenic mice had nuclear staining for beta-catenin. Sequencing of the tumor DNA encoding beta-catenin revealed point mutations that resulted in amino acid substitutions. These findings suggest a previously unknown role for FGF19 in hepatocellular carcinomas.

Epithelial to mesenchymal transition (EMT) is a key process in embryonic development and has been associated with cancer metastasis and drug resistance. For example, in EGFR mutated non-small cell lung cancers (NSCLC), EMT has been associated with acquired resistance to the EGFR inhibitor erlotinib. Moreover, “EGFR-addicted” cancer cell lines induced to undergo EMT become erlotinib-resistant in vitro. To identify potential therapeutic vulnerabilities specifically within these mesenchymal, erlotinib-resistant cells, we performed a small molecule screen of ~200 established anti-cancer agents using the EGFR mutant NSCLC HCC827 cell line and a corresponding mesenchymal derivative line. The mesenchymal cells were more resistant to most tested agents; however, a small number of agents showed selective growth inhibitory activity against the mesenchymal cells, with the most potent being the Abl/Src inhibitor, dasatinib. Analysis of the tyrosine phospho-proteome revealed several Src/FAK pathway kinases that were differentially phosphorylated in the mesenchymal cells, and RNAi depletion of the core Src/FAK pathway components in these mesenchymal cells caused apoptosis. These findings reveal a novel role for Src/FAK pathway kinases in drug resistance and identify dasatinib as a potential therapeutic for treatment of erlotinib resistance associated with EMT.

• The DropArray technology is compatible with the retention of suspension cells in multistep procedures thus enabling novel assay methods.• This technology enabled visualization and quantification of specific killing events triggered by bispecific antibodies engaging T cells. IntroductionThe demonstration that single cells could be grown in vitro 1 combined with the development of specific growth media 2 and later the establishment of the first cell lines [3][4][5][6] definitively marks the birth of cell culture as a critical research tool. Since then, investigators have developed a myriad of in vitro cellular models to further the understanding of various normal and pathologic cellular processes as well as to screen and characterize potential therapeutic modalities. All this progress occurred in concert with the development of many technologies that have impacted the investigator's ability to establish specific culture conditions and measure or visualize different cellular signals. As a result, cell-based assays are today almost universally used in biology research laboratories.During the past decade, cell-based assays have become fundamental and irreplaceable tools in the drug discovery and development industry. This trend was driven mainly by 2 rationales. First, the completion of the human genome, 7 combined with advances in functional genomics and proteomics, 8 has led to the need to evaluate thousand of potential new targets. Second, the high attrition rate of therapeutic candidates at the preclinical and clinical stages generated the need for more biologically relevant highthroughput screening approaches, bringing cell-based assay technologies to the forefront of the drug-discovery strategy. As a result, investigators' ability to develop rapid, flexible, robust, and costeffective high-throughput cell-based assays became of paramount importance.Despite significant technological progress in enabling technologies such as molecular labeling and the advent of high content screening approaches, cell-based assays, in part because of their well-plate format, continue to have major limitations. Although wells are an efficient and simple strategy to segregate experimental conditions in formats from 6-to 1536-well plates, they have major restrictions when suspension, loosely adherent, and in some cases fully adherent cells are used, especially with high-throughput formats such as 96-, 384-, and 1536-well plates. Microwell plates not only limit the use of certain cells but also significantly reduce the spectrum of experimental procedures that can be implemented in high-throughput cell-based screenings. Because the addition and removal of reagents to the wells could definitively compromise the cells, various technologies have been developed to circumvent these limitations. The homogeneous assay, developed in recent years, provides a convenient "add, mix, and read" approach to explore a broad spectrum of biologic events from cell viability/ Submitted July 31, 2012; accepted November 24, 2012. Prepublished online as Bl...