Genomic and phenotypic stability of fusion-driven pediatric 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. However, to what extent these limitations in terms of reproducibility may be generalized to all cancer cell lines remained unexplored. Here, we hypothesized that oligomutated pediatric sarcoma cell lines mainly driven by a fusion transcription factor, 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, together with a simultaneous analysis during 12 months of continuous cell culture showed that fusion-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. Additionally, the analysis of multiple EwS cell lines subjected to long-term continuous culture revealed that variable degrees of genomic/transcriptomic/phenotypic changes among fusion-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. When flasks reached approximately 70% confluency, total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel, Germany) according to the manufacturer’s protocol. RNA quality was verified on a Nanodrop Spectrophotometer ND-1000 (Thermo Fischer) and quantity was measured on a Qubit instrument (Life Technologies). For each sample, 50–100 ng of RNA in three biological and two technical replicates were used as input material and were profiled on an Illumina NextSeq 500 system. Human cell lines were cultured continously over a period of 12 months and subjected to RNA-seq analysis at three different time points (m0=ITP, m6=MTP, m12=FTP)