Immunocompetent murine model of Ewing sarcoma reveals role for TGFβ inhibition to enhance immune infiltrates in Ewing tumors during radiation

Ewing sarcoma (ES) is an aggressive cancer diagnosed in adolescents and young adults. The fusion oncoprotein (EWSR1::FLI1) that drives Ewing sarcoma is known to downregulate TGFBR2 expression (part of the TGFβ receptor). Because TGFBR2 is downregulated, it was thought that TGFβ likely plays an inconsequential role in Ewing biology. However, the expression of TGFβ in the Ewing tumor immune microenvironment (TIME) and functional impact of TGFβ in the TIME remains largely unknown given the historical lack of immunocompetent preclinical models. Here, we use single-cell RNAseq analysis of human Ewing tumors to show that immune cells, such as NK cells, are the largest source of TGFβ production in human Ewing tumors. We develop a humanized (immunocompetent) mouse model of ES and demonstrate distinct TME signatures and metastatic potential in these models as compared to tumors developed in immunodeficient mice. Using this humanized model, we study the effect of TGFβ inhibition on the Ewing TME during radiation therapy, a treatment that both enhances TGFβ activation and is used to treat aggressive ES. Utilizing a trivalent ligand TGFβ TRAP to inhibit TGFβ, we demonstrate that in combination with radiation, TGFβ inhibition both increases ES immune cell infiltration and decreases lung metastatic burden in vivo . The culmination of these data demonstrates the value of humanized models to address immunobiologic preclinical questions in Ewing sarcoma and suggests TGFβ inhibition as a promising intervention during radiation therapy to promote metastatic tumor control. ### Competing Interest Statement Consultant for Galvanize Therapeutics (IF). Consultant AboundBio (ARC). Scientific Advisory Board Walking Fish Therapeutics, BeSpoke Therapeutics, Kalivir Therapeutics (TB). All other authors declare no potential conflicts of interest.