TGFβ Inhibition during Radiotherapy Enhances Immune Cell Infiltration and Decreases Metastases in Ewing Sarcoma

Ewing sarcoma (EwS) is an aggressive cancer diagnosed in adolescents and young adults. Inhibition of TGFβ is being tested in limited clinical trials for relapsed EwS. TGFβ is an immunosuppressive cytokine that exists in latent and active states. The functional impact of TGFβ inhibition on the Ewing tumor microenvironment (TME) and on Ewing tumor behavior remains largely unknown. Here, we use single-cell RNAseq analysis of human Ewing tumors to demonstrate that immune cells are the largest contributors of TGFB1 expression in the human Ewing TME. We utilize a humanized mouse model of EwS to demonstrate that TME signatures in these models differ significantly from EwS tumors developed in immunodeficient mice. Using this humanized model, we investigate the effect of TGFβ inhibition on the EwS TME during radiation therapy, a treatment that is commonly used to treat unresectable, metastatic, and relapsed/refractory EwS that is known to enhance TGFβ activation in multiple cancers. Utilizing a trivalent ligand TGFβ trap to inhibit TGF, we demonstrate that in combination with radiation therapy, TGFβ inhibition both increases EwS immune cell infiltration and decreases lung metastatic burden in vivo. These data demonstrate the value of immunocompetent models to address immune-biologic preclinical questions in EwS and demonstrate that TGFβ inhibition during radiation therapy is a promising strategy to enhance anti-tumor immune response and improve treatment efficacy for metastatic EwS.