Ewing sarcoma is infiltrated by immunosuppressive macrophages and functionally impaired APCs. A, UMAP and dotplot of the myeloid compartment, showing conventional dendritic cells type 1 and 2 (cDC1, cDC2), undifferentiated macrophages (Mo), and differentiated macrophage populations (Mφ) and a selection of their marker genes. B, Macrophage-specific scores for proinflammatory and anti-inflammatory signatures as well as M1 and M2 signatures. C, Antigen presentation and costimulatory capacity score as constructed with genes shown in Supplementary Fig. S7D. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus Mo. D, Barplot showing percentage of costimulatory gene-expressing Mo, Mφ, and cDCs. Neuroblastoma (NBL; Kildisiute and colleagues) data included as comparative group. Mean percentages and error bars are plotted for individual samples. Samples with <5 cells per group were excluded. *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, Violin plot depicting DC maturation score for Ewing sarcoma and NBL cDCs. Z-scores were calculated using a curated MSigDB gene set (“LINDSTEDT_DENDRITIC_CELL_MATURATION_B”). This gene set includes upregulated genes both at 8 and 48 hours in response to inflammatory stimuli. ***, P < 0.001. F, Violin plots showing the STAT3 and NFKB1 gene expression levels of Ewing sarcoma and NBL cDCs. ***, P < 0.001 with Bonferroni correction.
ARTICLE ABSTRACT
Novel therapeutic strategies are urgently needed for patients with high-risk Ewing sarcoma and for the reduction of severe side effects for all patients. Immunotherapy may fill this need, but its successful application has been hampered by a lack of knowledge on the composition and function of the Ewing sarcoma immune microenvironment. Here, we explore the immune microenvironment of Ewing sarcoma, by single-cell RNA sequencing of 18 Ewing sarcoma primary tissue samples. Ewing sarcoma is infiltrated by natural killer, T, and B cells, dendritic cells, and immunosuppressive macrophages. Ewing sarcoma–associated T cells show various degrees of dysfunction. The antigen-presenting cells found in Ewing sarcoma lack costimulatory gene expression, implying functional impairment. Interaction analysis reveals a clear role for Ewing sarcoma tumor cells in turning the Ewing sarcoma immune microenvironment into an immunosuppressive niche. These results provide novel insights into the functional state of immune cells in the Ewing sarcoma tumor microenvironment and suggest mechanisms by which Ewing sarcoma tumor cells interact with, and shape, the immune microenvironment.
This study is the first presenting a detailed analysis of the Ewing sarcoma microenvironment using single-cell RNA sequencing. We provide novel insight into the functional state of immune cells and suggests mechanisms by which Ewing tumor cells interact with, and shape, their immune microenvironment. These insights provide help in understanding the failures and successes of immunotherapy in Ewing sarcoma and may guide novel targeted (immuno) therapeutic approaches.
