Carcinoma-associated fibroblast-like tumor cells remodel the Ewing sarcoma tumor microenvironment

Abstract

Tumor heterogeneity is a major driver of cancer progression. In epithelial-derived malignancies, carcinoma-associated fibroblasts (CAFs) contribute to tumor heterogeneity by depositing extracellular matrix (ECM) proteins that dynamically remodel the tumor microenvironment (TME). Ewing sarcomas (EwS) are histologically monomorphous, mesenchyme-derived tumors that are devoid of CAFs. Here we identify a previously uncharacterized subpopulation of transcriptionally distinct EwS tumor cells that deposit pro-tumorigenic ECM. Single cell analyses revealed that these CAF-like cells differ from bulk EwS cells by their upregulation of a matrisome-rich gene signature that is normally repressed by EWS::FLI1, the oncogenic fusion transcription factor that underlies EwS pathogenesis. Further, our studies showed that ECM-depositing tumor cells express the cell surface marker CD73, allowing for their isolation ex vivo and detection in situ. Spatial profiling of tumor xenografts and patient biopsies demonstrated that CD73+ EwS cells and tumor cell-derived ECM are prevalent along tumor borders and invasive fronts. Importantly, despite loss of EWS::FLI1-mediated gene repression, CD73+ EwS cells retain expression of EWS::FLI1 and the fusion-activated gene signature, as well as tumorigenic and proliferative capacities. Thus, EwS tumor cells can be reprogrammed to adopt CAF-like properties and these transcriptionally and phenotypically distinct cell subpopulations contribute to tumor heterogeneity by remodeling the TME.

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

“…A total of 2112 differentially expressed genes (fold change >1.5, p value <0.05) were identified in tumors developed in a humanized model versus NSG model (Fig 2i). Upregulated gene transcripts in Ewing tumors developed in the hu-CD34+ models compared to the NSG model include Wnt3, Hox gene family members, and collagen family genes, some of which have been previously reported to be pro-tumorigenic in Ewing sarcoma (14). Tumors grown in hu-CD34+ mice demonstrate upregulation of pathways driving collagen biosynthesis and extracellular matrix (ECM) remodeling (Supp.…”

“…TGFβ is an immunosuppressive cytokine that can prevent effective anti-tumor immune responses and remodel the extra-cellular matrix toward a pro-tumor phenotype (13, 14). Increased TGFβ pathway signaling is noted in Ewing tumors that demonstrate a pro-metastatic, aggressive phenotype (26).…”

“…Recent studies of the TME of Ewing sarcoma, an aggressive fusion oncoprotein (EWSR1::FLI1)-driven primary bone tumor of adolescents and young adults, (11–13) revealed the importance of extra-cellular matrix (ECM) remodeling (14) and hypoxia (15, 16) in promoting Ewing tumor cell survival and metastatic potential. An aspect of the Ewing TME that continues to be poorly understood --and not well represented in in vivo studies of Ewing sarcoma--is Ewing immunobiology.…”

TCR-transgenic T cells and YB-1-based oncolytic virotherapy improve survival in a preclinical Ewing sarcoma xenograft mouse model

“…Hence, further immunological insight is urgently needed. Cellular immunotherapy approaches have to address a multitude of obstacles: physical barriers, low to absent MHC class I expression, immunosuppressive innate cells of myelomonocytic origin, cancerassociated fibroblast-like tumor cells, chemo-and cytokines as well non-classical MHCs in the TME preventing an impactful T cell attack (8,(50)(51)(52)(53)(54). They additionally have to include properties to revert immunosuppressive systemic factors, such as fibroblastic/ myeloid-derived suppressor cells, chronic inflammation or tumorderived extracellular vesicles hampering proper antigen presentation and activation of DC to enable formation of antitumor T cell immunity (1,2,55).…”