Oncolytic herpes simplex virus and radiation combination therapy results in a systemic immune response to inhibit neuroblastoma tumor growth

PEDIATRIC SURGERY I lung. We hypothesize that tadalafil, a PDE5 inhibitor, can be administered prenatally to prevent vascular hyperreactivity associated with pulmonary hypertension in fetal lambs with CDH.

Murine endothelium derived microparticle induced early murine lung injury Paul M Jeziorczak, MD, MPH, Sushma Kaul, MS, Kirkwood A Pritchard Jr, PhD, Elizabeth R Jacobs, MD, John C Densmore, MD, FACS Medical College of Wisconsin, Milwaukee, WI, Children’s Hospital of Wisconsin, Milwaukee, WI

METHODS: Diaphragmatic hernias were created in time-dated pregnant ewes at mid-gestation. Postoperatively, pregnant ewes received placebo or tadalafil at 2mg/kg/day until delivery near term gestation. Fourth generation pulmonary arteries were mounted onto a multiwire myograph system (DMT 620M) to assess vessel reactivity. Vessels were passively stretched to a stable resting tone and preconstricted to EC80 (the concentration to achieve 80% of maximal constriction) with norepinephrine. Once the response to norepinephrine reached a plateau, cumulative concentration-response curves to vasodilators acetylcholine, SNAP, and A23187 were obtained.

INTRODUCTION: Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) have 30% mortality and result in 72,000 U.S. deaths annually. Discrete triggers of ALI/ARDS are unknown. Previously we described endothelium-derived microparticles (EMPs) as a novel ALI trigger in a rodent model using intravenous EMPs from HUVECs. Given concern for confounding hyperacute xenograft rejection, we wished to establish a completely murine model and time course of EMP-induced ALI. Thus, we hypothesize that murine-derived EMPs cause increased pulmonary permeability in treated mice at pathophysiologic levels with maximal injury occurring within 12 hours of injection.

RESULTS: Pulmonary arteries had progressive relaxation with increasing concentrations of vasodilators. For normal lambs treated with tadalafil, CDH lambs treated with tadalafil, and untreated CDH lambs respectively, percent of EC80 contraction was 74%, 84% and 93% for acetylcholine at 1⫻10⫺6M, 76%, 77%, and 90% for SNAP at 1⫻10⫺6M, and 66%, 76%, and 88% for A23187 at 1⫻10⫺6M.

METHODS: EMPs were isolated from murine endothelial cell culture (ATCC # CRL-2280) stimulated with murine plasminogen activation inhibitor-1 (PAI-1, 50 ng/mL). 58 C3H/HeOuJ mice (male, 6-8 weeks) were treated with intrajugular PBS, LPS (10 mg/ kg), or EMP (10^6/mL blood) and sacrificed at 2, 4, 6, 12, and 24 hours. Lung permeability was assayed using Evans Blue Albumin (IV, 20 mg/kg ⫹ 40mg/mL BSA) one hour prior to sacrifice. Corrected absorbance values were measured from homogenized lungs. Statistical analysis was performed using a two way ANOVA due to unequal group size.

CONCLUSIONS: Prenatal tadalafil improves endotheliumindependent pulmonary vascular hyperreactivity in fetal lambs with CDH and may be an effective strategy to improve outcomes in CDH.

Oncolytic herpes simplex virus and radiation combination therapy results in a systemic immune response to inhibit neuroblastoma tumor growth Lauren A Gillory, MD, Michael L Megison, MD, Jerry E Stewart, BSc, Hugh C Nabers, BSc, Gregory K Friedman, MD, James M Markert, MD, PhD, FACS, G Yancey Gillespie, PhD, Elizabeth A Beierle, MD, FACS University of Alabama at Birmingham, Birmingham, AL

RESULTS: Mice treated with EMP showed significantly greater pulmonary permeability than PBS controls. Peak injury occurred at 4 hours (treatment p⫽.0324; time p⫽⬍.0001). By 24 hours, permeability between groups was equivalent. CONCLUSIONS: EMP-induced ALI can be consistently modeled without concern for hyperacute xenograft rejection. EMPs are an important early ALI trigger. Abrogating EMP effects in this model will allow us to understand early initiators and therapies in ALI.

INTRODUCTION: Viral and gene therapy are promising approaches to treating neuroblastoma. An oncolytic herpes simplex virus (oHSV) expressing the cytokine Interleukin-12 (M002) was previously shown to inhibit neuroblastoma xenograft growth. We hypothesized that human neuroblastoma tumors express the viral entry receptor, CD111. Additionally, we hypothesized that M002 would produce a secondary immune response to inhibit murine neuroblastoma growth distant from the treatment site in an immunocompetent model.

The role of prenatal tadalafil in altered relaxation of pulmonary arteries from lambs with surgical diaphragmatic hernia Eveline H Shue, MD, Samuel C Schecter, MB, BS, Wenhui Gong, MD, Michael Johengen, BSc(Hons), William Ferrier, DVM, Jianfeng Wu, BSc(Hons), Mozziyar Etemadi, MS, Peter Oishi, MD, Jeff Fineman, MD, Douglas N Miniati, MD, FACS University of California, San Francisco, San Francisco, CA, University of California, Davis, Davis, CA

METHODS: Formalin-fixed paraffin-embedded human neuroblastoma tumor specimens (IRB-X111123007) were stained for CD111. NXS2 murine neuroblastoma cells (7.5⫻10^5 cells) were injected into bilateral flanks of immunocompetent mice (IACUC120409363). Established right-sided tumors were treated with M002 (1⫻107 pfu) and radiation (3Gy). Half of these mice received a repeat right-sided dose of M002 and radiation. Tumor volumes on both flanks were measured every 2 days. Data were compared with appropriate statistical analysis with significance pⱕ0.05.

INTRODUCTION: Pulmonary hypertension is the main predictor of postnatal survival in congenital diaphragmatic hernia (CDH). Though pulmonary vascular remodeling occurs in utero, there are no prenatal treatments available. Phosphodiesterase type 5 (PDE5) inhibitors are used to treat pulmonary hypertension postnatally, but have not been used prenatally. We have previously shown that tadalafil effectively crosses the placenta and significantly increases cGMP in the fetal lamb

© 2012 by the American College of Surgeons Published by Elsevier Inc.

RESULTS: All 12 human neuroblastoma specimens expressed CD111. In the murine bilateral flank tumor model, repeat treatment of the right flank with M002 and radiation led to significant inhibition of tumor growth in the untreated flank tumor compared to

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Abstracts

animals who received a single dose to the right flank (323.44 ⫾ 215.51 mm3 vs. 955.08 ⫾ 366.54 mm3, pⱕ0.05). CONCLUSIONS: CD111 expression was present in human neuroblastoma specimens, indicating that oHSV would infect these tumors. M002 inhibited progression of murine tumors both locally and distant from the site of therapy, demonstrating a systemic effect. These data support the potential use of oHSV therapy to treat recurrent or metastatic neuroblastoma. Intereleukin (IL)-12 and STAT4 activation leads to decreased neuroblastoma cell survival Michael L Megison, MD, Lauren A Gillory, MD, Jerry E Stewart, BS, Hugh C Nabers, BSc(Hons), Elizabeth A Beierle, MD, FACS University of Alabama at Birmingham, Birmingham, AL INTRODUCTION: Neuroblastoma, a pediatric tumor of neural crest origin, continues to have a poor prognosis and will require novel treatments. Cytokine therapies, such as Interleukin-12 (IL-12), have shown promise in the treatment of some adult tumors. Recent reports show that normal neurons express IL-12 receptor. We therefore hypothesized that neuroblastoma cells would express IL-12 receptor, and that activation of this receptor would lead to downstream STAT4 activation and increased cell death. METHODS: SK-N-AS and SK-N-BE(2) human neuroblastoma cell lines were cultured under standard conditions. RT-PCR and immunoblotting were used to detect IL-12 receptor, and ELISA used to detect IL-12 ligand production. Cells were treated with recombinant human (rh)IL-12 and immunoblotting and immunofluorescence were used to detect STAT4 phosphorylation. Cellular survival was measured with alamarBlue(r) assays. Data were reported as mean ⫾ SEM and compared with Student’s t-test, with p ⱕ 0.05 significant. RESULTS: IL-12 receptor mRNA and protein were seen in both cell lines. Neither cell line produced IL-12. Both cell lines responded to exogenous rhIL-12 by increasing STAT4 phosphorylation (activation). Activated STAT4 migrated into the nucleus in the SK-NBE(2) cells following rhIL-12. Treatment with rhIL-12 significantly decreased survival in SK-N-AS (0.06475 ⫾ 0.0056 vs. 0.03763 ⫾ 0.007, p⫽0.03, control vs. rhIL-12) and SK-N-BE(2) (0.19275 ⫾ 0.0046 vs. 0.13413 ⫾ 0.0117, p⫽0.007, control vs. rhIL-12) cell lines. CONCLUSIONS: The IL-12 receptor was present in human neuroblastoma cells. Treatment with rhIL-12 activated the STAT4 pathway leading to decreased neuroblastoma cell survival. IL-12 cytokine therapy may represent a novel therapeutic strategy for treating neuroblastoma. Chrysin induces growth suppression through apoptosis in neuroblastoma cells Jocelyn F Burke, MD, Logan Schlosser, BA, Herbert Chen, MD, FACS, Muthusamy Kunnimalaiyaan, PhD University of Wisconsin, Madison, WI INTRODUCTION: Neuroblastoma is the most common solid extracranial tumor in children. While surgery offers excellent treatment

J Am Coll Surg

for patients with low grade tumors, therapeutic options for high grade tumors are more limited. Neuroblastomas arise from sympathetic neuronal precursor cells, and thus share many characteristics with other neuroendocrine tumors. Chrysin (5,7-dihydroxyflavone) is a flavonoid currently used in many oral vitamin supplements that we have shown to have anti-tumor effects in neuroendocrine tumors. We hypothesized that chrysin would similarly limit tumor growth in neuroblastoma tumor cells. METHODS: Two neuroblastoma cell lines, NGP and SK-N-AS, were treated with varying concentrations of chrysin (0-100 micromolar) in standard cell media. Cell viability in response to chrysin treatment was measured by MTT colorimetric assay. Protein lysates were isolated after 2 days of treatment, and the mechanism of growth suppression was investigated using Western blot analysis for common apoptotic markers. RESULTS: Chrysin significantly suppressed growth in both cell lines; at 50 micromolar, growth was reduced by 39% on day 2 and 62% on day 4 in NGP cells, and by 21% on day 2 and 55% on day 4 in SK-N-AS cells. By Western analysis, apoptosis markers cleaved PARP and cleaved Caspase 3 increased with chrysin treatment at 50 micromolar in both cell lines, with pro-survival proteins XIAP and Survivin concurrently decreasing. CONCLUSIONS: Chrysin suppresses tumor growth by inducing apoptosis in neuroblastoma cells. Since this naturally occurring flavonoid has been shown to be safe in humans, clinical trials with chrysin in patients with unresectable neuroblastoma are warranted.

Inhibition of host Notch function disrupts hepatic vasculature, and promotes tumor growth Alejandro Garcia, MD*, Debarshi Banerjee, PhD, Sonia Hernandez, PhD, Roderick Alfonso, BS, Arul Thirumoorthi, MD, John Andrews, BA, Emily Sbiroli, BA, Angela Kadenhe-Chiweshe, MD, Darrell J Yamashiro, MD, PhD, Jessica J Kandel, MD Columbia University Medical Center, New York, NY INTRODUCTION: Notch signaling regulates the vascular endothelial growth factor (VEGF) pathway, mediating endothelial-stromal cell interactions. Notch inhibition has consequently been proposed as an anti-angiogenic therapy for cancer to complement VEGF blockade. However, in the liver Notch can promote vascular integrity. We hypothesized that disrupting Notch signaling would paradoxically increase primary and metastatic hepatic tumor growth. METHODS: 10[6] NGP-luc neuroblastoma cells engineered to express a soluble Notch1-decoy construct (N1D) or Fc control were implanted intrarenally in immunodeficient mice, and spontaneous hepatic metastases evaluated, both during VEGF blockade (bevacizumab 10mg/kg intraperitoneally biweekly) and vehicle treatment (N⫽30 each). To study tumor growth in the liver directly, NGP Notch1-knockdown construct (N1KD), NGP-N1D, or control cells were implanted intrahepatically (N⫽7, 6, 6, respectively). Tumor growth was quantified by bioluminescence. To examine the role of Notch1 in hepatic vasculature, we generated novel Notch1-KO het-