Abstract 4173: Inhibition of EphB4-ephrinB2 signaling increases efficacy of radiation in pancreatic ductal adenocarcinoma
Authors: Lennon Shelby , Bhatia Shilpa , Oweida Ayman , Sharma Jaspreet , Phan Andy , Bukkapatnam Sanjana , Uyanga Nomin , Pitts Todd , Leong Stephen , Goodman Karyn , Raben David , Pasquale Elena , Martinez-Torrecuadrada Jorge , Karam Sana 
Published: August 2018

Abstract
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Survival rates are dismal, at only 8% five year survival. Radiation is one treatment option for PDAC, although historically its efficacy has been controversial. Our research goal is to improve the efficacy of radiation for PDAC patients. An ancillary study of the recent LAP07 trial showed that patients with neutrophilia were more resistant to chemoradiation. Additionally, when patients with systemic inflammation were excluded, one year local control was 80% in the chemoradiation arm compared to 54% in the chemotherapy arm. The EphB4 receptor and ephrinB2 ligand have been shown to play a role in neutrophil mobilization from the bone marrow and infiltration into melanoma tumors. We hypothesize that inhibition of EphB4-ephrinB2 signaling will enhance the efficacy of radiation in PDAC by reducing inflammation by neutrophils.
Materials/Methods: To study tumor growth delay, Athymic nude mice were implanted in the flank with a patient derived xenograft (PDX) model of PDAC. To inhibit EphB4-ephrinB2 signaling, mice were administered with 20mg/kg of B11, a human single chain Fv antibody fragment against ephrinB2. Tumors were irradiated with 12Gy and volume was monitored by caliper. For mechanistic studies, C57/BL6 mice were injected in the flank with 10^6 FC1242 cells, which are derived from the pancreata of KPC mice. To inhibit EphB4-ephrinB2 signaling, mice received a hydrodynamic tail vein injection of 20ug/2mL TNYL-RAW-Fc plasmid. TNYL-RAW is a peptide antagonist of EphB4, and it is fused to human Fc. Tumors were irradiated with 12Gy and harvested four days later for flow cytometry analysis. Blood was also collected at various time points and analyzed by blood film white blood cell differential and flow for neutrophils. Plasmid transduction efficiency was confirmed by analyzing plasma samples collected at different time-points on an Fc protein-based ELISA assay.
Results: We observed a significant tumor growth delay in PDX implanted mice who received B11 and radiation. An ELISA for human Fc confirmed transduction of the TNYL-RAW-Fc plasmid in FC1242 tumor bearing mice. Flow analysis on FC1242 tumors showed a significant decrease in percentage of infiltrating neutrophils when treated with TNYL-RAW and radiation, and blood film white blood cell differential and flow analysis on blood showed a significant decrease in circulating neutrophils in the same mice.
Conclusions: Our findings support the hypothesis that EphB4-ephrinB2 signaling contributes to inflammation by neutrophils in PDAC, and inhibiting this signaling will increase the efficacy of radiation. This data can contribute toward a clinical trial design for combination therapy of EphB4-ephrinB2 inhibition and radiation in patients with pancreatic ductal adenocarcinoma.
Citation Format: Shelby Lennon, Shilpa Bhatia, Ayman Oweida, Jaspreet Sharma, Andy Phan, Sanjana Bukkapatnam, Nomin Uyanga, Todd Pitts, Stephen Leong, Karyn Goodman, David Raben, Elena Pasquale, Jorge Martinez-Torrecuadrada, Sana Karam. Inhibition of EphB4-ephrinB2 signaling increases efficacy of radiation in pancreatic ductal adenocarcinoma [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 4173.