Abstract 3743: Neutrophil extracellular traps and their implication with radioresistance in muscle invasive bladder cancer
Authors: Shinde-Jadhav Surashri , Mansure Jose Joao , Rayes Roni , Ayoub Mina , Spicer Jonathan , Kassouf Wassim 
Published: August 2019

Abstract
PURPOSE: Radiotherapy modifies diverse components of the tumor microenvironment and inflammation plays a pivotal role in modulating radiation responsiveness of tumors. Neutrophils are one of the first-line responders during the acute phase of inflammation and are increasingly being recognized as drivers of tumor progression. One mechanism by which neutrophils play a role in tumor progression is through the formation of neutrophil extracellular traps (NETs). NETs are web-like structures expelled by the neutrophil composed of DNA studded with various proteins. Initially, this was described as a mechanism of antimicrobial defense but lately NETs have been associated with a variety of adverse effects, such as pathogenesis of autoimmune diseases, surgical stress, tumor progression, and metastasis. Recent studies show that the protein High Mobility Group Box-1 (HMGB1), a key player in radioresistance can in fact promote NETs. Importantly, the impact of NETs has not yet been explored in the context of radiation, so we sought to explore this further.
METHODS: In vitro: a) Human neutrophils isolated from healthy donors were stimulated with 50ng of rHMGB1 for 4 hours. NETs were quantified through Sytox green fluorescence. b) Neutrophils were co-cultured with irradiated or non-irradiated conditioned media from UM-UC3 human bladder cancer cell line in combination with glycyrrhizin (GLZ), a natural inhibitor of HMGB1. In vivo: Murine bladder cancer cell line (MB49) was subcutaneously implanted into flanks of C57BL/6 and NETosis deficient PAD4-/- mice. Tumors were irradiated (2x5Gy) using the XRAD Smart Irradiator. Intraperitoneal injections of GLZ were used to modulate HMGB1 and intramuscular injections of DNAse were used to deplete NETs. Tumor volumes were measured using a digital caliper till endpoint.
RESULTS: Our in vitro results demonstrate incubation of neutrophils with 50ng rHMGB1 significantly induced NETs formation compared to controls (p&lt;0.0001) and this was reversed through addition of GLZ (p&lt;0.0001). Similarly, co-culture of neutrophils with irradiated MB49 conditioned media induced NETs formation (p=0.01) and this effect was reversed with GLZ (p=0.009). Our in vivo results demonstrate that NETosis deficient mice treated with an HMGB1 inhibitor significantly improves response to radiation therapy. PAD4-/- mice treated with GLZ showed delayed tumor growth kinetics (p=0.023) and increased overall survival post radiation (p=0.0231) compared to all other irradiated arms: C57BL/6, PAD4-/-, C57BL/6 + DNAse and C57BL/6 + GLZ. Similarly, C57BL/6 mice treated with DNAse and GLZ also showed a delay in tumor growth kinetics post radiation (p&lt;0.0001).
CONCLUSION: NETs may induce radioresistance through interactions with HMGB1. Highlighting the role of HMGB1 in NET formation will provide valuable information on the responses that occur in the tumor microenvironment post radiation therapy.
Citation Format: Surashri Shinde-Jadhav, Jose Joao Mansure, Roni Rayes, Mina Ayoub, Jonathan Spicer, Wassim Kassouf. Neutrophil extracellular traps and their implication with radioresistance in muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3743.