Introduction S. aureus is a major cause of morbidity globally, and in the United States it contributes significantly to both hospital admissions and in-hospital morbidity [1, 2]. The increasing incidence of antibiotic-resistant strains increases the urgency of understanding the mechanisms by which this infection exerts its toxic acute effects, as well as potential longterm impact on infected patients, especially those with comorbid conditions. The major virulent toxin secreted by S. aureus is α-hemolysin (Hla). A Disintegrin And Metalloproteinase domain-containing protein-10 (ADAM10), which is involved in ectodomain shedding, is the eukaryotic receptor for Hla [3-5], and mediates vascular injury caused by Hla [6]. Almost all isolates of S. aureus express Hla, including methicillinresistant strains [7]. Recently, Hla has been shown to mediate VE-cadherin degradation in endothelial cells (EC) via ADAM10, affecting permeability [6]. Importantly, the Notch1 and 2 receptors are known ADAM10 targets [8]. Notch proteins are highly evolutionarily conserved. In mammals, the Notch pathway is comprised of the Jagged and Delta-like ligands, and the receptors Notch1 through Notch4. Both ligands and receptors are membrane-bound: in order for activation to take place, the ligand and receptor must be expressed in adjacent cells. Notch ligands are cleaved at Site 1 (S1) and can be post-translationally modified by glycosyltransferases, such as Fringe. Upon

The family of Notch proteins plays a key role in cell fate determination. Additionally, Notch proteins regulate critical functions of the endothelium, as well as other recruited supporting cells, in concert with other pathways. Despite significant advances in the field and extensive studies focused on elucidating this pathway, many questions remain regarding Notch activation and its upstream/ downstream effects, with vascular biology constituting one area of particular interest. Here, we provide a brief description of the components and functions of the Notch pathway in vasculature, followed by a detailed compilation of recommended methods of evaluation in vitro and in vivo. We provide a rationale for key elements when choosing different approaches and controls, strengths and limitations, and essential considerations when providing a meaningful interpretation of results. Our aim is to describe a careful approach to assessing Notch function in endothelial cells, based on underlying principles, with the overall goal of obtaining physiologically relevant information that will enhance our understanding of this pathway and its role in vascular biology.

Background. Staphylococcus aureus infection is one of the leading causes of morbidity in hospitalized patients in the United States. The secreted agent hemolysin alpha toxin (Hla) requires the receptor A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10) to mediate its toxic effects; ADAM10 in turn activates the Notch pathway. Notch proteins function in developmental and pathological angiogenesis via the modulation of key pathways in endothelial (EC) and perivascular cells. Thus, we hypothesized that Hla would activate Notch in EC in vitro and in vivo. Methods. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant Hla (rHla), Hla-H35L (genetically inactivated Hla), 5mM EDTA (a known Notch activator), or HBSS, and probed with a Luciferase reporter regulated by a Notch promoter. Mice engineered to express yellow fluorescent protein (YFP) upon Notch activation received a non-lethal daily subcutaneous injection of rHla (0.025 µg/5µL) or vehicle (PBS) and their retinal endothelial YFP interrogated at p6. 6 week old male Notch reporter mice received a 1-4 × 107 50 µl subcutaneous inoculation of S. aureus strains USA300/LAC (WT), or its isogenic Δhla mutant lacking Hla, and their skin biopsies were analyzed by histology after 36 hours, 8 and 16 days. Notch activation in endothelial cells of human liver sections from patients whose blood cultures were positive or negative for S. aureus was evaluated by immunohistochemistry. Results. Luciferase assays demonstrated that Hla (0.01 µg/mL) increased Notch activation by 1.75±0.5-fold as compared to HBSS controls (p<0.05) and EDTA (5.4±1.4-fold activation relative to HBSS, p<0.01), whereas Hla-H35L had no effect. Retinal EC in YFP Notch reporter mice revealed significantly greater YFP intensity in EC after Hla injection than controls in two independent litters. Subcutaneous infection of S. aureus in the Notch reporter mice revealed a significant upregulation of Notch activation in response to Hla 36 hours after infection, both in intensity and area (4.2X10-5 ±1.8X10-5 vs. 7.9X10-4 ±6X10-4 mean intensity relative to DAPI area, p=0.008, and 6.7x106 ±3.1x106 vs. 7.7x10-5 ±6x105 YFP area/ DAPI area, p=0.002). Increased EC Notch activation in response to Hla was maintained 8 and 16 days after inoculation. IHC showed EC in human liver had higher Notch expression than controls. In sum, our results demonstrate that the S. aureus toxin Hla can potently activate Notch in endothelial cells, an effect which could have effects in development and pathological angiogenesis such as cancer. Citation Format: Sonia L. Hernandez, Mildred Nelson, Georgia Sampedro, Bianca Lec, Jared Emolo, Naina Bagrodia, Ann M. Defnet, Lydia Wu, Juliane Bubeck-Wardenburg, Jessica Kandel. Staphylococcus aureus alpha toxin activates Notch in endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2055.

Although metabolic adaptation is a cornerstone of the hypoxia response, very little is known about hypoxia-induced metabolic changes in neuroblastoma or how the metabolome influences phenotype. Identifying genes that regulate neuroblastoma metabolism and also influence clinical behavior may provide clues for novel therapeutic targets. Mixed linear models were used to identify differentially expressed genes between neuroblastoma cells grown in hypoxia or normoxia in two independent experiments (GEO accessions GSE17714 and GSE55391). Linear models were also used in to identify differentially expressed genes from tumors of two independent cohorts of neuroblastoma patients (GEO accession GSE16254 and EMBL accession E-MTAB-179) who survived compared to those that did not. We utilized a false discovery rate of 0.01 and fold change in the top or bottom 10% of all genes as a significance cutoff. qPCR was used to validate expression differences in multiple neuroblastoma cell lines. 157 genes, significantly exceeding chance (p < 1x10-6) on permutation testing, were present in both neuroblastoma cell line data sets, all but five of which showed consistent directionality of expression change from normoxia to hypoxia, including 44 involved in metabolism. These genes were enriched for hypoxia and metabolic pathways: HIF-1α transcription factor network (p = 1.3x10-11), glycolysis (p = 1.9x10-8) and fructose metabolism (p = 1.0x10-3). 826 genes, significantly exceeding chance (p < 1x10-6) were differentially expressed in both patient cohorts of 478 and 88 patients. These genes were enriched for cell cycle pathways (p = 4.5x10-7). Of these genes, nine of them were also differentially expressed in hypoxia compared to normoxia with consistent directionality, again more than expected by chance (p < 1x10-6). High expression in eight of the nine genes was also significantly associated with poor outcome in a Kaplan-Meier analysis of both of the patient cohorts evaluated. Three of these genes are part of the glycolytic pathway and three more are directly involved in cellular metabolism. In the SK-N-BE2 cell line, all eight of our identified genes are up-regulated in hypoxia (p < 0.05). Analysis of the LAN-5, La1-55n, SK-N-DZ cell lines and show similar results. MTT assays of proliferation show the expected decreased proliferation for all of these cell lines in 48 hours of hypoxia compared to normoxia (p < 0.05). Analysis of cell cycle by flow cytometry in SK-N-BE2 cells shows an increase of cells in G0/G1 in normoxia compared to hypoxia (58.3% vs. 71.5% p = 0.04) and decrease of cells in S phase (24.5% vs. 13.6%, p = 0.001). We have identified eight genes with increased expression in hypoxia and are associated with poor survival in patients. Efforts to use shRNA to alter cellular phenotype are ongoing. Citation Format: Mark A. Aplebaum, Aashish Jha, Alexandre Chlenski, Christopher Mariani, Clara Kao, Mildred Nelson, Kyle Hernandez, Helen Salwen, Marija Dobratic, Kevin White, Barbara Stranger, Susan L. Cohn. Evaluation of hypoxia adaptation in neuroblastoma identifies reproducible transcriptional and phenotypic responses. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A01.