携带致癌基因的外显子循环DNA的存在与癌症发生和患者生存率下降强烈相关。肿瘤活检和体外癌细胞系常被用作研究外显子循环DNA在癌症中的作用的模型。然而，外显子循环DNA在癌细胞系的体外增殖中经常丢失，这对于利用癌细胞系模型进行研究的可重复性提出了质疑。在这里，我们对七个癌细胞系（MCA3D，PDV，HaCa4，CarC，MIA-PaCa-2，AsPC-1，PC-3）进行了外显子循环DNA变异的综合分析。我们比较了每个细胞系的三重复样本中独特外显子循环DNA的含量，发现独特外显子循环DNA的数量对每个细胞系是特异性的，而外显子循环DNA序列内容在三重复样本中存在显著变异（约0-1%的外显子循环DNA坐标相似性）。在PC-3细胞系中，我们发现大的携带MYC的外显子循环DNA在NCI细胞系分离物中存在高拷贝数，而在ATCC分离物中不存在。综上所述，这些结果揭示了癌细胞系中外显子循环DNA的序列内容是高度可变的。这凸显了在使用之前测试癌细胞系的重要性，并且为那些希望研究外显子循环DNA在癌症中作用的细胞系中进行亚克隆富集，以获取相对纯净的细胞群体。© 2023作者。由Research Network of Computational and Structural Biotechnology代表Elsevier B.V.出版。

The presence of oncogene carrying eccDNAs is strongly associated with carcinogenesis and poor patient survival. Tumour biopsies and in vitro cancer cell lines are frequently utilized as models to investigate the role of eccDNA in cancer. However, eccDNAs are often lost during the in vitro growth of cancer cell lines, questioning the reproducibility of studies utilizing cancer cell line models. Here, we conducted a comprehensive analysis of eccDNA variability in seven cancer cell lines (MCA3D, PDV, HaCa4, CarC, MIA-PaCa-2, AsPC-1, and PC-3). We compared the content of unique eccDNAs between triplicates of each cell line and found that the number of unique eccDNA is specific to each cell line, while the eccDNA sequence content varied greatly among triplicates (∼ 0-1% eccDNA coordinate commonality). In the PC-3 cell line, we found that the large eccDNA (ecDNA) with MYC is present in high-copy number in an NCI cell line isolate but not present in ATCC isolates. Together, these results reveal that the sequence content of eccDNA is highly variable in cancer cell lines. This highlights the importance of testing cancer cell lines before use, and to enrich for subclones in cell lines with the desired eccDNA to get relatively pure population for studying the role of eccDNA in cancer.© 2023 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.