# Characterisation of Bespoke Patient-Derived In Vitro Models of Ewing Sarcoma

## TL;DR

Researchers developed patient-derived Ewing sarcoma models that better reflect the disease and could help identify more effective treatments.

## Contribution

The study introduces and validates patient-derived Ewing sarcoma cells as a novel preclinical model for drug testing.

## Key findings

- Patient-derived Ewing sarcoma cells contain EWSR1 fusions and reflect the tumor's transcriptome and protein profiles.
- These cells respond to treatments similarly to patients, showing resistance to standard chemotherapies and sensitivity to mTKIs and trabectedin.
- Compared to cell lines, patient-derived cells have distinct biological features and drug response profiles.

## Abstract

Ewing sarcoma is a cancer of young people, for whom the outcome and treatment have not changed significantly in the last 30 years. Furthermore, treatment is frequently associated with life-changing side effects. There is therefore a need for more effective targeted treatments or treatment combinations to improve survival and limit treatment-associated morbidities. A paucity of clinically informative preclinical models has hindered the prioritisation of effective treatments for evaluation in clinical trials. To overcome this problem, we propagated and characterised Ewing sarcoma cells recently isolated from tumours. These cells contain EWSR1 fusions pathognomonic of Ewing sarcoma and share the transcriptome and protein profiles of the tumours from which they were derived. Furthermore, the response of these cells to chemotherapy, ionising radiation and investigational drugs reflects activity reported in patients. These cells are therefore a valuable tool, which could be incorporated into the preclinical pipeline to improve the identification of effective drugs for clinical evaluation.

Background/Objectives: Preclinical models that accurately reflect Ewing sarcoma (ES) will enable the prioritisation of clinically active targeted agents from bench to clinic. To expedite this process, we have established and characterised patient-derived ES cultures (PDES) in vitro. Methods: Fluorescence in situ hybridisation, RT-PCR and western blotting were used to examine expression of the pathognomonic EWSR1 fusions. Activation or repression of EWSR1 fusion downstream targets and proliferation was examined by immunofluorescence and immunohistochemistry. Using next-generation sequencing, the DNA and transcriptomic profiles of PDES and cell lines were compared. The response of PDES and cell lines to standard-of-care chemotherapeutics, ionising radiation and investigational drugs was examined. Results: All PDES contain EWSR1 fusion DNA, consistent with a diagnosis of ES. EWSR1 fusion gene RNA and protein were detected in 70% and 21% of PDES, respectively. Markers of proliferation and expression of EWSR1 fusion target genes were consistent with the tumours from which PDES were derived (R2 = 0.74, p < 0.0001) and the paediatric mesenchymal lineage (SBS5 and SBS1, ID1 and ID2). In contrast, the transcriptome of PDES was significantly different from that of cell lines. PDES had a significantly increased doubling time (p < 0.00001), decreased expression of Ki67 (p < 0.0001) and increased migration (p < 0.02) compared to cell lines. Consistent with the longer doubling time, PDES were more resistant to doxorubicin, etoposide and vincristine and ionising radiation (p < 0.0001) than cell lines. PDES were sensitive to mTKIs (cabozantinib, lenvatinib, and regorafenib), and trabectedin. The response of PDES to drugs in vitro reflects the clinical experience of patients. Conclusions: Models incorporating PDES cells may positively contribute to the preclinical pipeline.