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Abstract Objective: Ewing sarcoma is an aggressive cancer of adolescents. Patients with metastatic Ewing sarcoma often receive radiation as part of their treatment protocol. We have demonstrated that both Ewing tumor cells and T cells infiltrating Ewing tumors express Galectin-3 (Gal3), an immunosuppressive protein in the tumor microenvironment. Here, we utilize an immunocompetent, humanized mouse model of Ewing sarcoma to investigate the role of Galectin-3 in the Ewing tumor immune microenvironment both at baseline and following radiation. Specifically, we sought to determine the influence of Galectin-3 on post-radiation inflammation in Ewing sarcoma. Methods: Human Ewing cells -/+ Gal3 expression were generated using CRISPR/Cas 9 technology. Cells -/+ Gal3 were orthotopically injected into humanized mice and allowed to established tumors over ~3 weeks. Tumors were then treated with radiation (or no radiation controls). To also address immune cell expression of Gal3, galectin inhibitors were utilized. Tumors were analyzed by bulk RNA sequencing and flow cytometry analysis (Cytek Aurora) for human CD45, CD3, CD4, CD20, CD8, CD56, CD14, CD16, etc. Peripheral blood controls were also collected and analyzed to ensure consistent baseline immune reconstitution. Results: CHLA10 and TC32 Ewing sarcoma cell lines both demonstrate Gal3 expression at baseline by western blot. CRISPR/Cas 9 knock-out of Gal3 in both cell lines showed successful knock down. Impact on growth and sensitivity to radiation in vitro was assessed by live cell Incucyte assays. Tumors in humanized mice were established utilizing these cells (or Gal3 expressing non-targeting controls) and changes in the post-radiation transcriptional profiles were assessed. Immune cells infiltrating these tumors were also quantified. Ongoing work focuses on additional characterization of these tumor infiltrating immune cell populations following radiation therapy and the impact of broader galectin inhibition on this effect. Conclusion: This work contributes to the understanding of radiation-induced tumor inflammatory responses in Ewing sarcoma by defining the role of tumor and T cell galectin-3 expression inn Ewing immunobiology. Next steps will explore translational opportunities based on these findings. Citation Format: Yunash Maharjan, Sreya Dey, Elina Mukherjee, Jessica Daley, Kelly M. Bailey. Galectin-3 expression in Ewing sarcoma: Modulation of the immune microenvironment in an immunocompetent model abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 5464.
Maharjan et al. (Fri,) studied this question.