Abstract A004: Radiation-induced changes to the immune microenvironment in an immunocompetent mouse model of Ewing sarcoma
Authors: Daley Jessica D. , Mukherjee Elina M. , Cillo Anthony R. , Tufino Adriana C. , Bailey Nathanael G. , Bruno Tullia C. , McAllister-Lucas Linda M. , Vignali Dario A. , Bailey Kelly M. 
Published: September 2022

Background: Ewing sarcoma is a rare, aggressive, fusion oncoprotein-driven pediatric cancer. Patients with upfront metastatic or surgically unresectable disease commonly undergo radiation as part of standard of care therapy. Currently, little is known about the effect of radiation specifically on the immune microenvironment of Ewing tumors, as no routine biopsies or other clinical samples are acquired during radiation. Further, historically, the field has lacked an immunocompetent mouse model of Ewing sarcoma in which to study Ewing tumor-immune cell interactions. Given our interest in understanding the anti-tumor immune response specifically during times of DNA damage, we sought to model Ewing sarcoma in a humanized mouse model (where human immune cell interactions with human tumor cells can be examined) in order to examine the Ewing tumor immune microenvironment (TIME) during radiation. Here, we determine the likeness of our established humanized mouse model of Ewing sarcoma to that of human Ewing tumors at baseline and examine changes in the Ewing TIME upon delivery of radiation. Methods: Blood from humanized NSG mice was analyzed by flow cytometry to confirm human immune cell reconstitution. Human Ewing tumor cells were injected into humanized mice and allowed to grow for approximately three weeks. Tumors were treated with radiation (either single dose or fractioned doses x 5 days) using the MultiRad350 Precision irradiator and lead shields were used to isolate the tumor area in the radiation field. Immune cells infiltrating tumors with/without radiation exposure were subsequently analyzed by multiplexed immunohistochemistry, flow cytometry, PCR, and RNAseq analysis. Ewing tumor immune infiltrates (as baseline/without radiation) from humanized mice models were compared to that of patient Ewing tumors. Results: Ewing tumors from both established and primary cell lines were successfully established in humanized mice. Similar to patient primary Ewing tumor samples, local Ewing tumors in humanized mice demonstrate a proportion of T-cell infiltration, although the overall number of infiltrating immune cells is low. Macrophage populations in Ewing sarcoma are not as predominant as in tumors such as osteosarcoma and are represented in this model. Analyses detailing the spatial localization and transcriptional profiles of Ewing tumor immune infiltrates following radiation are ongoing. Conclusions: In the absence of syngeneic and transgenic models of Ewing sarcoma, the use of humanized mouse models is a feasible alternative to address specific questions regarding the Ewing sarcoma TIME. Understanding the Ewing immune microenvironment during radiation therapy, a commonly used treatment modality in Ewing sarcoma, provides clues as to promising agents that may be worthy of preclinical testing to enhance the anti-tumor immune response during radiation in high-risk patients.

Citation Format: Jessica D. Daley, Elina M. Mukherjee, Anthony R. Cillo, Adriana C. Tufino, Nathanael G. Bailey, Tullia C. Bruno, Linda M. McAllister-Lucas, Dario A. Vignali, Kelly M. Bailey. Radiation-induced changes to the immune microenvironment in an immunocompetent mouse model of Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A004.