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

Ewing sarcoma is an aggressive cancer diagnosed in adolescents and young adults. Inhibition of TGFβ is being tested in limited clinical trials for relapsed Ewing sarcoma. 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. In this study, we use single-cell RNA sequencing 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 Ewing sarcoma to demonstrate that TME signatures in these models differ significantly from Ewing sarcoma tumors developed in immunodeficient mice. Using this humanized model, we investigate the effect of TGFβ inhibition on the Ewing sarcoma TME during radiotherapy, a treatment that is commonly used to treat unresectable, metastatic, and relapsed/refractory Ewing sarcoma that is known to enhance TGFβ activation in multiple cancers. Utilizing a trivalent ligand TGFβ trap to inhibit TGFβ, we demonstrate that in combination with radiotherapy, TGFβ inhibition both increases Ewing sarcoma immune cell infiltration and decreases lung metastatic burden in vivo. These data demonstrate the value of immunocompetent models to address immune-biological preclinical questions in Ewing sarcoma and demonstrate that TGFβ inhibition during radiotherapy is a promising strategy to enhance antitumor immune response and improve treatment efficacy for metastatic Ewing sarcoma. SIGNIFICANCE: This work demonstrates the importance of disrupting immunosuppression during radiotherapy to reduce lung metastatic potential in Ewing sarcoma. Humanized mouse models of Ewing sarcoma are also established as an immunocompetent preclinical tool to ask therapeutic questions about the Ewing TME.