Pdx-derived ewing’s sarcoma cells retain high viability and disease phenotype in alginate encapsulated spheroid cultures

Ewing’s Sarcoma (ES) is the second most frequent malignant bone tumour in children and young adults and currently only untargeted chemotherapeutic approaches and surgery are available as treatment, although clinical trials are on-going for recently developed ES-targeted therapies. To study ES pathobiology and develop novel drugs, established cell lines and patient-derived xenografts (PDX) are the most employed experimental models. Nevertheless, the establishment of ES cell lines is difficult and the extensive use of PDX raises economic/ethical concerns. There is a growing consensus regarding the use of 3D cell culture to recapitulate physiological and pathophysiological features of human tissues, including drug sensitivity. Herein, we implemented a 3D cell culture methodology based on encapsulation of PDX-derived ES cell spheroids in alginate and maintenance in agitation-based culture systems. Under these conditions, ES cells displayed high proliferative and metabolic activity, while retaining the typical EWSR1-FLI1 chromosomal translocation. Importantly, 3D cultures presented reduced mouse PDX cell contamination compared to 2D cultures. Finally, we show that these 3D cultures can be employed in drug sensitivity assays, with results similar to those reported for the PDX of origin. In conclusion, this novel 3D cell culture method involving ES-PDX-derived cells is a suitable model to study ES pathobiology and can assist in the development of novel drugs against this disease, complementing PDX studies.