Related Experiment Video
Updated: Sep 8, 2025
Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells
Published on: March 7, 2025
SARC028 Samples Reveal an Interplay between TGF-β, IFN Signaling, and Low HLA Class I Expression as Contributors to
Jessica D Daley1, Elina Mukherjee2, David Ferraro2
1University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Purpose:
Ewing sarcoma, in contrast to some adult sarcoma subtypes, generally does not respond to single-agent immunotherapy targeting PD1. The features of Ewing sarcoma that preclude the effectiveness of immunotherapy remain largely unknown. To address this question, we utilized biopsies from patients with Ewing sarcoma obtained before and after pembrolizumab (anti-PD1) therapy from the phase II clinical trial SARC028 to interrogate the Ewing tumor microenvironment and features associated with resistance to checkpoint inhibition.
Experimental Design:
We utilized multiplexed immunofluorescence, spatial proteomics, and spatial transcriptomics to analyze paired pretreatment and 8-week posttreatment biopsy specimens from patients with Ewing sarcoma enrolled in SARC028.
Results:
Pembrolizumab therapy did not alter the quantity of immune cell infiltration in Ewing tumor biopsies. Analysis of tumor-associated protein markers revealed increased immunoregulatory markers after pembrolizumab. Spatial transcriptomics identified 10 cellular neighborhoods (CN) across patients consisting of specific cell subsets. CN10 was consistently observed across patients with a poor response. This CN was enriched for a tumor subpopulation with a high TGF-β response, low IFN response, and low HLA class I expression. IFN response, HLA class I expression, and overall immune infiltration were correlated.
Conclusions:
Analyses of paired Ewing sarcoma tumor samples from SARC028 reveal an immunosuppressive triad, the disruption of which should be pursued to improve antitumor immunity. This work highlights the unique insight that can be gained from the analysis of paired patient Ewing sarcoma tumor biopsy samples from clinical trials.
Insights
Ewing sarcoma resists PD1 immunotherapy due to an immunosuppressive tumor microenvironment. This study identified a specific cellular neighborhood associated with poor response, offering targets for improved Ewing sarcoma treatments.
Area of Science:
- Oncology
- Immunology
- Cancer Biology
Background:
- Ewing sarcoma exhibits limited response to PD1-targeted immunotherapy, unlike some adult sarcomas.
- The underlying mechanisms of immunotherapy resistance in Ewing sarcoma are not well understood.
Purpose of the Study:
- To investigate the Ewing sarcoma tumor microenvironment and identify features associated with resistance to PD1 checkpoint inhibition.
- To analyze changes in tumor biopsies before and after pembrolizumab therapy in a phase II clinical trial.
Main Methods:
- Utilized multiplexed immunofluorescence, spatial proteomics, and spatial transcriptomics.
- Analyzed paired pretreatment and posttreatment biopsy specimens from Ewing sarcoma patients in the SARC028 trial.
Main Results:
- Pembrolizumab therapy did not change immune cell infiltration levels but increased immunoregulatory markers.
- Spatial transcriptomics revealed a distinct cellular neighborhood (CN10) linked to poor treatment response.
- CN10 featured tumor cells with high TGF-β response, low IFN response, and low HLA class I expression, correlating with immune infiltration.
Conclusions:
- Identified an immunosuppressive triad in Ewing sarcoma that hinders antitumor immunity.
- Disrupting this triad is a potential strategy to enhance immunotherapy effectiveness in Ewing sarcoma.
- Analysis of paired tumor biopsies from clinical trials provides valuable insights into treatment resistance.
