Abstract B048: Galectin-3 regulation of immunosuppression in Ewing sarcoma

Abstract Objective: Ewing sarcoma is a fusion oncoprotein-driven cancer most commonly diagnosed in adolescents. Utilizing single-cell RNAseq of human Ewing tumors, we have previously demonstrated that Galectin-3, an immunosuppressive lectin family member protein, is expressed in both Ewing tumor cells and in CD8 T cells in the Ewing tumor microenvironment, a finding not noted in CD8 T cells infiltrating osteosarcomas. Galectin-3 serves to modulate tumor immunobiology through multiple mechanisms, including interactions with the immune checkpoint protein LAG3. In addition to Galectin-3, Galectin-3BP (binding protein) is also expressed in Ewing sarcoma and can bind to and modulate Galectin-3 function. Here, we aim to determine the role of Galectin-3 and Galectin-3BP on Ewing tumor immunosuppression. Methods: CHLA10 and TC32 human Ewing sarcoma tumor cells -/+ LGALS3 (gene encoding galectin-3) expression were generated using CRISPR/Cas 9 technology. T cells primed again human Ewing tumor cells were generated. The galectin-3 inhibitor TD-139 was also utilized. In vitro IncuCyte live cell monitoring assays, ELISA, and co-culture apoptosis assays were conducted. In vivo analyses of tumor growth, metastasis, and immune cell infiltration were conducted on Ewing tumors developed in immunocompetent, humanized mice and immunodeficient NSG mice. Peripheral blood and spleen controls were also analyzed. Results: CRISPR/Cas 9 knock-out of LGALS3 in both CHAL10 and TC32 cell lines was successful. GSEA of RNAseq data demonstrate distinct developmental signatures and flow cytometry analyses demonstrate altered immune cell signatures in LGALS3 KO Ewing tumors versus controls developed in humanized mice. ELISA demonstrates Galectin-3 is secreted by some Ewing tumors, the functional implications of which are under ongoing investigation. Expression of Galectin-3 and Galectin-3BP alone versus co-expression impacts the net effect of on Ewing tumor behavior. Conclusion: This work contributes to the understanding of galectin-3 on inflammatory responses and tumor progression in Ewing sarcoma. Understanding the complex relationship of Galectin-3 and Galectin-3BP on immunobiology provides valuable clues to best disrupt Galectin-3 mediated immunosuppression and promote enhanced tumor control. Citation Format: Yunash Maharjan, Adriana C. Tufino, Sreya Dey, Elina Mukherjee, Kelly M. Bailey. Galectin-3 regulation of immunosuppression in Ewing sarcoma abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B048.