{
  "abstract": "Background Ewing Sarcoma (ES) is a rare, aggressive soft tissue and bone sarcoma. While localized disease is treatable, metastatic or relapsed ES remains challenging with the overall survival of <30%. 1 Despite advances in immunotherapy across other cancer types, similar success in solid tumors like ES has been limited, likely due to multifactorial mechanisms that remain poorly understood. Deeper investigation of the immune environment in ES may reveal the blockades for immunotherapy efforts and potential new pathways to be exploited in the future.Methods We have a local biorepository of 37 human ES samples freshly processed to single cell suspension and/or formalin-fixed paraffin embedded (FFPE), plus an additional 80 FFPE samples. We utilized publicly available bulk and single-cell RNA datasets to identify involved cell types and signaling pathways. We then analyzed cases with multiparametric flow cytometry and immunohistochemistry to identify and characterize the immune components of the ES tumor microenvironment (TME).Results Transcriptomic analysis confirmed heterogenous immune cells in the ES TME with a predominance of myeloid cells. Elevated immune signatures correlated with increased lipid metabolism, while low immune signatures aligned with hypoxia and glutamine metabolism, suggesting a relationship between immune infiltration and tumor metabolic state. Single-cell RNA sequencing revealed myeloid populations expressing immunosuppressive genes (e.g., TGFβ1, LGALS9), alongside dendritic cells with high CD86 and HLA-DR and low IL10 expression suggesting a potential pro-inflammatory state in contrast to prior studies. These findings informed IHC marker selection to evaluate spatial architecture of immune cells. We also examined IL1RAP,2 an IL-1 family coreceptor induced by the pathognomonic EWS-FLI1 fusion oncoprotein and implicated in tumor redox homeostasis. IL1RAP was consistently expressed in RNA dataset, immunohistochemical, and flow cytometric analyses and can correlate the expression level with immune cell infiltration.Conclusions ES is considered to have minimal or dysfunctional immune infiltration of the TME, however, the spatial organization has not been evaluated and the phenotypes of these cells have only been investigated recently and have not been confirmed. Our studies provide additional details of the immune milieu in ES, identify potential proinflammatory cell subsets, and correlate their presence with the tumor metabolic state. Further, we included IL1RAP in our analyses to elucidate the role it may play in regulating the TME via redox homeostasis and other signaling pathways, and have laid the groundwork for more detailed studies of this potentially targetable protein.Acknowledgements Thank you to our collaborators, prior laboratory members, and most importantly the patients and their families who have made this work possible.References Miser JS, Goldsby RE, Chen Z, Krailo MD, Tarbell NJ, Link MP, Fryer CJ, Pritchard DJ, Gebhardt MC, Dickman PS, Perlman EJ, Meyers PA, Donaldson SS, Moore SG, Rausen AR, Vietti TJ, Grier HE. Treatment of metastatic Ewing sarcoma/primitive neuroectodermal tumor of bone: evaluation of increasing the dose intensity of chemotherapy--a report from the Children’s Oncology Group. Pediatr Blood Cancer 2007 Dec;49(7):894-900.Zhang HF, Hughes CS, Li W, He JZ, Surdez D, El-Naggar AM, Cheng H, Prudova A, Delaidelli A, Negri GL, Li X, Ørum-Madsen MS, Lizardo MM, Oo HZ, Colborne S, Shyp T, Scopim-Ribeiro R, Hammond CA, Dhez AC, Langman S, Lim JKM, Kung SHY, Li A, Steino A, Daugaard M, Parker SJ, Geltink RIK, Orentas RJ, Xu LY, Morin GB, Delattre O, Dimitrov DS, Sorensen PH. Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma. Cancer Discov. 2021 Nov;11(11):2884-2903.Ethics Approval Human materials for the biobank were collected under a Johns Hopkins IRB-approved protocol and consent was obtained prior to the surgery. Pathology archive human materials were accessed utilizing a Johns Hopkins IRB-approved protocol. All information presented is de-identified.",
  "authors": [
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Katelyn Williams"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Julia Nowik"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Kaushal Sharma"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Aditya A Suru"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "John Gross"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Robert Anders"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Nicolas Llosa"
    }
  ],
  "title": "747 Immune profiling of Ewing Sarcoma reveals a potential pro-inflammatory niche",
  "uid": "795282c0-614d-55b8-ac2f-f3f9f8da4f94"
}