Correlation of Transcriptomics and FDG-PET SUVmax Indicates Reciprocal Expression of Stemness-Related Transcription Factor and Neuropeptide Signaling Pathways in Glucose Metabolism of Ewing Sarcoma.

BACKGROUND: In Ewing sarcoma (EwS), long-term treatment effects and poor survival rates for relapsed or metastatic cases require individualization of therapy and the discovery of new treatment methods. Tumor glucose metabolic activity varies significantly between patients, and FDG-PET signals have been proposed as prognostic factors. However, the biological basis for the generally elevated but variable glucose metabolism in EwS is not well understood. METHODS: We retrospectively included 19 EwS samples (17 patients). Affymetrix gene expression was correlated with maximal standardized uptake value (SUVmax) using machine learning, linear regression modelling, and gene set enrichment analyses for functional annotation. RESULTS: Expression of five genes correlated (MYBL2, ELOVL2, NETO2) or anticorrelated (FAXDC2, PLSCR4) significantly with SUVmax (adjusted p-value ≤ 0.05). Additionally, we identified 23 genes with large SUVmax effect size, which were significantly enriched for "neuropeptide Y receptor activity (GO:0004983)" (adjusted p-value = 0.0007). The expression of the members of this signaling pathway (NPY, NPY1R, NPY5R) anticorrelated with SUVmax. In contrast, three transcription factors associated with maintaining stemness displayed enrichment of their target genes with higher SUVmax: RNF2, E2F family, and TCF3. CONCLUSION: Our large-scale analysis examined comprehensively the correlations between transcriptomics and tumor glucose utilization. Based on our findings, we hypothesize that stemness may be associated with increased glucose uptake, whereas neuroectodermal differentiation may anticorrelate with glucose uptake.

Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function.

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100-170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.

MHC Class I-Restricted TCR-Transgenic CD4+ T Cells Against STEAP1 Mediate Local Tumor Control of Ewing Sarcoma In Vivo.

In this study we report the functional comparison of T cell receptor (TCR)-engineered major histocompatibility complex (MHC) class I-restricted CD4+ versus CD8+ T cells targeting a peptide from six transmembrane epithelial antigen of the prostate 1 (STEAP1) in the context of HLA-A*02:01. STEAP1 is a tumor-associated antigen, which is overexpressed in many cancers, including Ewing sarcoma (EwS). Based on previous observations, we postulated strong antitumor potential of tumor-redirected CD4+ T cells transduced with an HLA class I-restricted TCR against a STEAP1-derived peptide. We compared CD4+ T cell populations to their CD8+ counterparts in vitro using impedance-based xCELLigence and cytokine/granzyme release assays. We further compared antitumor activity of STEAP130-TCR transgenic (tg) CD4+ versus CD8+ T cells in tumor-bearing xenografted Rag2-/-gc-/- mice. TCR tgCD4+ T cells showed increased cytotoxic features over time with similar functional avidity compared to tgCD8+ cells after 5-6 weeks of culture. In vivo, local tumor control was equal. Assessing metastatic organotropism of intraveniously (i.v.) injected tumors, only tgCD8+ cells were associated with reduced metastases. In this analysis, EwS-redirected tgCD4+ T cells contribute to local tumor control, but fail to control metastatic outgrowth in a model of xenografted EwS.

The second European interdisciplinary Ewing sarcoma research summit--A joint effort to deconstructing the multiple layers of a complex disease.

Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second "European interdisciplinary Ewing sarcoma research summit" assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNA-sequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intra-tumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits.