Genomic and phenotypic stability of fusion-driven pediatric Ewing sarcoma cell lines

Human cancer cell lines are the mainstay of cancer research. Recent reports showed that highly mutated adult carcinoma cell lines (mainly HeLa and MCF-7) present striking diversity across laboratories and that long-term continuous culturing results in genomic/transcriptomic heterogeneity with strong phenotypical implications. This highlighted how despite human cell line models being powerful tools for cancer research, the findings derived from their use may present limitations in terms of reproducibility. However, to what extent these conclusions can be generalized to the majority of cancer cell lines remained unexplored. Here, we hypothesized that oligomutated pediatric sarcoma cell lines driven by a chimeric oncogenic transcription factor (COTF), such as Ewing sarcoma (EwS), were genetically and phenotypically more stable than the previously investigated (adult) carcinoma cell lines. A comprehensive molecular and phenotypic characterization of multiple EwS cell line strains in direct comparison to the HeLa and MCF-7 cell lines, together with a simultaneous analysis during 12 months of continuous cell culture showed that COTF-driven pediatric sarcoma cell line strains are genomically more stable than adult carcinoma strains, display remarkably stable and homogenous transcriptomes, and exhibit uniform and stable drug response. The analysis of multiple EwS cell lines subjected to long-term continuous culture conditions revealed that variable degrees of genomic/transcriptomic/phenotypic may be observed among COTF-driven cell lines, further exemplifying that the potential for reproducibility of in vitro scientific results may be rather understood as a spectrum, even within the same tumor entity.