Discovery of a New Immunotherapy Target for Ewing Sarcoma

Background Introduction

Ewing sarcoma (Ewing’s sarcoma, EWS) is a common pediatric bone cancer, accounting for approximately 2% of childhood cancers. Despite significant advances in immune checkpoint inhibitors (ICIs) and CAR-T cell therapy in various cancers, their efficacy in Ewing sarcoma remains unsatisfactory. The survival rate for Ewing sarcoma is low, particularly for patients with metastasis at diagnosis or poor response to chemotherapy, with an overall survival rate of less than 30%. Therefore, identifying new therapeutic targets to improve the treatment outcomes for Ewing sarcoma has become a pressing need.

Ewing sarcoma is characterized by gene fusions involving the FET family of genes and the ETS family of transcription factors (TFs), with the EWSR1-FLI1 gene fusion present in 85% of cases. This gene fusion not only drives tumorigenesis but also influences the efficacy of immunotherapy through the complex tumor immune microenvironment (TIME). Thus, a detailed understanding of the immune regulatory network in Ewing sarcoma, particularly the interactions between tumor cells and the immune microenvironment, is crucial for developing new immunotherapeutic strategies.

Source of the Paper

This paper was completed by a research team led by Fangzhou He, Jiuhui Xu, and Fanwei Zeng from Peking University People’s Hospital and the Beijing Key Laboratory, and was published in the journal Cell Communication and Signaling in 2025. The research team systematically dissected the immune regulatory network of Ewing sarcoma through single-cell RNA sequencing (scRNA-seq), cell function experiments, and humanized models, proposing a new immunotherapy target.

Research Process and Results

1. Single-Cell RNA Sequencing and Data Analysis

The research team first performed single-cell RNA sequencing (scRNA-seq) on 8 tumor samples from 6 Ewing sarcoma patients. These samples included 3 treatment-naïve patients (TN1-3), 2 paired samples from patients who underwent neoadjuvant chemotherapy (NAC) followed by surgical resection (NAC1 paired with TN1, NAC3 paired with TN3), and 3 samples from patients with local recurrence after chemotherapy (RC1-3). Through single-cell sequencing, the team obtained transcriptomic data from 82,127 cells.

For data analysis, the team used the Seurat package for data preprocessing and cell clustering. By inferring copy number variations (CNVs) in malignant cells using the inferCNV algorithm and combining known cell markers, the cells were ultimately classified into 13 main types, including malignant cells, cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), endothelial cells (ECs), and immune cells.

2. Heterogeneity Analysis of Malignant Cells

The research team further analyzed the subpopulations of malignant cells, identifying 9 distinct malignant cell subclusters. These subclusters exhibited different gene expression profiles, such as the MGP subcluster showing mesenchymal characteristics, the FABP7 subcluster exhibiting neural features, and the TOP2A subcluster displaying high proliferative activity. Using the Cytotrace and Monocle 3 algorithms, the team constructed the developmental trajectory of malignant cells, revealing that Ewing sarcoma malignant cells originate from the THY1 subcluster with mesenchymal features and gradually differentiate into the FABP7 subcluster with neural characteristics and the highly proliferative TOP2A subcluster.

3. Dissection of the Immune Microenvironment

The research team conducted a detailed analysis of tumor-infiltrating immune cells (TIICs), finding that the immune microenvironment of Ewing sarcoma is dominated by immunosuppressive myeloid cells, including tumor-associated neutrophils (TANs) and tumor-associated macrophages (TAMs). Notably, MMP8 neutrophils exhibited strong immunosuppressive functions, while CXCL8 neutrophils were associated with tumor stemness and angiogenesis.

Additionally, the team observed a significant increase in the proportion of immune cells, particularly CD8+ T cells and dendritic cells (DCs), in tumors after chemotherapy. Post-chemotherapy macrophages showed enhanced antigen presentation capabilities, and the cytotoxic function of CD8+ T cells was significantly improved.

4. Discovery and Validation of the MIF-CD74 Axis

Through cell-cell interaction analysis, the research team discovered that malignant cells secrete MIF (macrophage migration inhibitory factor), which interacts with the CD74/CXCR4 receptor complex on the surface of macrophages and CD8+ T cells, creating an immunosuppressive microenvironment. Further in vitro experiments confirmed that MIF induces M2 polarization of THP-1-derived macrophages and inhibits the proliferation of CD8+ T cells while promoting their apoptosis.

To validate the therapeutic potential of the MIF-CD74 axis, the team constructed a humanized mouse model and treated it with the MIF inhibitor 4-IPP. The results showed that 4-IPP significantly reduced MIF protein levels in tumors, increased CD8+ T cell infiltration, and decreased the number of M2-type macrophages, thereby inhibiting tumor growth.

Conclusions and Significance

This study systematically dissected the immune microenvironment of Ewing sarcoma through single-cell RNA sequencing and functional experiments, revealing the critical role of the MIF-CD74 axis in tumor immune evasion. The findings suggest that the MIF-CD74 axis is a potential immunotherapy target, and inhibiting MIF can reshape the tumor immune microenvironment, enhancing anti-tumor immune responses.

Research Highlights

1. Single-Cell Resolution Analysis of Tumor Heterogeneity: For the first time, the research team dissected the subpopulations of malignant cells in Ewing sarcoma at single-cell resolution, uncovering the transition of tumor cells from a mesenchymal phenotype to a proliferative phenotype.

2. Detailed Dissection of the Immune Microenvironment: The team identified a significant presence of immunosuppressive myeloid cells in the immune microenvironment of Ewing sarcoma, particularly MMP8 neutrophils and CXCL8 neutrophils, which play important roles in tumor progression.

3. Discovery of the MIF-CD74 Axis: The team proposed the MIF-CD74 axis as a new immunotherapy target for Ewing sarcoma and validated its therapeutic potential through in vitro experiments and a humanized mouse model.

Application Value

This study provides new insights into immunotherapy for Ewing sarcoma, with MIF inhibitors potentially becoming an effective treatment option in the future. Additionally, the single-cell RNA sequencing technology and humanized mouse models used in this research offer valuable references for immunotherapy studies in other types of cancer.

Summary

This study not only revealed the complex immune regulatory network of Ewing sarcoma but also provided important theoretical foundations for developing new immunotherapeutic strategies. By inhibiting the MIF-CD74 axis, the researchers successfully reshaped the tumor immune microenvironment, offering new hope for the treatment of Ewing sarcoma.