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...

Mycobacterium tuberculosis (M.tb), a bacterial pathogen that causes tuberculosis disease (TB), exerts an extensive burden on global health. The complex nature of M.tb, coupled with different TB disease stages, has made identifying immune correlates of protection challenging and subsequently slowing vaccine candidate progress. In this work, we leveraged two delivery platforms as prophylactic vaccines to assess immunity and subsequent efficacy against low-dose and ultra-low-dose aerosol challenges with M.tb H37Rv in C57BL/6 mice. Our second-generation TB vaccine candidate ID91 was produced as a fusion protein formulated with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion) or as a novel replicating-RNA (repRNA) formulated in a nanostructured lipid carrier. Protein subunit- and RNA-based vaccines preferentially elicit cellular immune responses to different ID91 epitopes. In a single prophylactic immunization screen, both platforms reduced pulmonary bacterial burden compared to the controls. Excitingly, in prime-boost strategies, the groups that received heterologous RNA-prime, protein-boost or combination immunizations demonstrated the greatest reduction in bacterial burden and a unique humoral and cellular immune response profile. These data are the first to report that repRNA platforms are a viable system for TB vaccines and should be pursued with high-priority M.tb antigens containing CD4+ and CD8+ T-cell epitopes.

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Epithelial to mesenchymal transition (EMT) is a pivotal mechanism contributing to cancer metastasis and has been associated with resistance to multiple cancer therapies including targeted kinase inhibitors (TKIs). EMT has been associated with an acquired resistance to the EGFR TKI erlotinib independent of known resistance mechanisms such as secondary mutations T790M or MET amplification. RTK-addicted cancer cell lines that are induced to undergo EMT with exogenous TGF-β become resistant to their appropriate TKI. In vitro-based assays determined that the resistance is not due to exogenous TGF-β, drug efflux, or autocrine/paracrine growth factor signaling. To identify potential therapeutic vulnerabilities specifically within the mesenchymal, TKI resistant cells, we performed a small molecule screen of approximately 200 agents using the EGFR mutant non-small cell lung cancer (NSCLC) HCC827 cell line and its corresponding TGF-β-induced mesenchymal derivative line. This screen revealed that the mesenchymal cells are cross-resistant to a variety of small molecules, including TKIs and chemotherapeutic agents. However, a small number of agents showed selective growth inhibitory activity against the mesenchymal cells, with the most potent being the dual Brc-Abl and Src inhibitor, dasatinib. Within the parental HCC827 cell line, we detected a non-proliferating vimentin positive sub-population of cells. Interestingly, when the parental cell line was co-treated with erlotinib and dasatinib the entire cell population was killed and drug-resistant clones failed to emerge after prolonged treatment. To determine the mechanism underlying the resistance observed within the mesenchymal cells a proteomic analysis was undertaken, which demonstrated that the focal adhesion kinase, FAK, is the most abundant differentially phosphorylated protein in the resistant mesenchymal cells that is specifically suppressed by dasatinib. However, specific gene knock-down of FAK was not sufficient to kill the mesenchymal cells or revert their sensitivity to Erlotinib suggesting that FAK alone is not required to maintain resistance upon EMT. These findings identify dasatinib as a potential therapeutic for treatment of erlotinib resistance associated with EMT. Citation Format: Catherine R. Wilson, Katrina Nicholes, Daisy Bustos, Eva Lin, Lily Shi, Donald Kirkpatrick, Jeffrey Settleman. Resistance to selective kinase inhibitors upon epithelial to mesenchymal transition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4462. doi:10.1158/1538-7445.AM2013-4462