Congratulations to the winners of the 2013 Spring Conference Stipend! We are excited to have research performed in our Perfecta3D Hanging Drop Plates represented at three prestigious and diverse conferences. The quality and breadth of the work represented here demonstrates the potential impact of 3D cell culture! The three winners are: TD139, a galectin-1 and galectin-3- specific inhibitor, mitigates VEGF-A-induced Angiogenesis Authors: Wei-Sheng Chen1, Hakon Leffler3, Ulf J Nilsson4 , Noorjahan Panjwani1,2 Institutions: 1Sackler School of Graduate Biomedical Sciences, 2New England Eye Center/Department of Ophthalmology, Tufts University, Boston, MA, 3Department of Laboratory Medicine, 4Department of Organic Chemistry, University of Lund, Lund, Sweden Conference: Experimental Biology 2013 Presentation information: Poster presentation Monday April 22, 12:25-1:55, Poster C343; Oral presentation Tuesday April 23, 4:15-4:30, Room 235A Abstract: Both galectin-1 (Gal-1) and galectin-3 (Gal-3) are vital modulators for vascular endothelial growth factors receptor-2 (VEGFR-2) signaling pathway. To date, TD139 is the only compound that specifically targets both galectins with high affinity. Our goal is to study TD139’s efficacy against VEGF-A-mediated pathological angiogenesis. Here we showed that Gal-1 and Gal-3 interact with VEGFR-2 in a carbohydrate-dependent manner and that TD139: (i) reduces cautery-induced corneal neovascularization in vivo, (ii) reduces VEGF-A-induced sprouting of human umbilical vein endothelial cells (HUVECs) as assessed by a three-dimensional sprouting assay, (iii) efficiently abolishes VEGF-A-induced HUVEC chemotaxis, (iv) has little effect on HUVEC viability, and (v) does not affect total protein expression of VEGFR-2 in HUVECs. Our data provide proof of principle that targeting Gal-1 and Gal-3 by the novel, small molecule inhibitor, TD139, ameliorates pathological angiogenesis. The findings provide a new therapeutic strategy for various disease conditions, including inflammation, diabetes, cancer metastasis, age-related macular degeneration and corneal neovascularization. This work is supported by the NIH Grant R01EY007088 to N. Panjwani. Tumor Histoids: Scalable Production of Living Human Mini-tumors For High Throughput Drug Screening Authors: M. Ingram1, G.B. Techy1, S.A. Imam1, J.R. Nolan2, and B.R. Ward1 Institutions: 1Huntington Medical Research Institutes, Pasadena, CA, 2La Jolla Bioengineering Research Institute Conference: Cyto2013 Presentation information: Poster B189, May 20-22, Author present May 20 5:15-6:45 and May 22 3:00-4:00 Abstract: TUMOR HISTOIDS (TH) are living three-dimensional human mini-tumors consisting of stroma invaded by tumor cells. They can be produced in quantity and format suitable for high throughput screening. Stromal-epithelial interactions occur spontaneously during TH culture and model those that activate pathways controlling tumor growth, malignant progression and response to therapies in vivo. This report describes: 1. adaptation of the original TH production method1 to production in Perfecta™ hanging drop plates, 2. an experiment comparing cisplatin effect on TH, tumor cell spheroids and monolayers. METHODS. TH are cultured in DMEM/F12 containing FBS, DNAse, fibronectin (1st day only) and PSA. TH are cultured in 384 well Perfecta™ hanging drops plates in a humidified incubator, 37°C. A Biomek™ 4000 robotic system facilitates fluid transfers. The simplest TH contain normal human foreskin fibroblasts and human tumor cells (various ATCC cell lines). TH are cultured in two stages. In stage one, 7300 fibroblasts in 15μl of medium are introduced via the accession ports above each well to form hanging drops; cultured for two days. Stage 2: 200 tumor cells in 10µl of medium are added to each hanging drop, bringing droplet volumes to 25μl. The culture is maintained for 7-10 days (feeding every 2-3 days). Droplet volume in 96 well plates is increased to 50μl and cell numbers adjusted proportionately. Monolayers are grown in standard 96 well plates seeded with 2500 cells/well; cultured to 50-70% confluence. TH are either harvested and processed for histology or analyzed using a Synergy™ plate reader (530 excitation/590 emission). A preliminary trial comparing cisplatin effect on TH with its effect on tumor cell spheroids and monolayers employed the resazurin ratiometric assay to estimate viability following exposure to various concentrations of cisplatin. RESULTS.1. Adapting TH culture to hanging drop culture in 384 well Perfecta™ plates yields highly uniform TH that can be analyzed spectrophotometrically within the droplets. Uniform size and cellularity makes TH well suited for high throughput screening. Although TH need not be harvested for analysis, harvesting them for analyses such as COPAS™ flow cytometry is entirely feasible. TH produced using the protocol presented above, for example, were successfully analyzed by COPAS™ flow cytometry to estimate size. Using a protocol still under development, TH with a vascular component in their stroma were also produced in 384 hanging drop cultures. Guardian angels in the Grave Yard: B1 B cells promote thymic Treg survival Authors: Xuemei Zhong*, Danielle Alaimo§, Hung Vo§, Matthew Hall§, John Lovell§, Chunyan Bai, Stanley Lau, Wenda Gao *Corresponding author, §Contributed equally Institution: Boston University Medical Center Conference: Immunology 2013 AAI Annual Meeting Presentation information: May 6, 11:45-1:00 Abstract: The thymus is the primary site for T cell development. Within the medullar of the thymus, negative selection of immature T cells causes a large number of cells to undergo apoptosis, appropriately deeming this thymic region a “grave yard.” In observing the thymus medulla of BXSB mice, we note a significant increase of B cells in lupus-symptomatic males as compared to control, lupus-asymptomatic females. Composition analysis of these thymic B cells reveals that both B1 and B2 cells are present. Immunohistochemistry analysis suggests possible chemo-attraction for B cells in the medullar of the thymus due to the expression of CXCL13 in lupus male mice, but not in control female mice. In vivo tracking of peritoneal B cells over a two-week time period indicates that some of the thymic B cells originate from the peritoneal cavity. To test the possible influence of B cells on thymic T cell development, we co-cultured peritoneal and spleen B cells with thymocytes in a 3D hanging drop plate. The results suggest a “guardian angel” effect by B1 B cells, depicting an enhanced central tolerance by promoting regulatory T cell survival.