DNA去甲基化触发结直肠癌细胞中游离DNA的释放

基于游离DNA（cfDNA）分析的液体活检作为一种微创诊断、基因分型和监测实体瘤的手段具有重要潜力。人类肿瘤通过细胞死亡、主动和被动释放等机制在血流中释放cfDNA。然而，cfDNA释出的具体机制尚待阐明。研究中，由于肿瘤负荷、解剖和血管屏障以及正常细胞核酸的释放等因素，患者中的机制研究受到干扰。本研究利用癌症模型，解析DNA释放的基础机制。我们测量了结直肠癌（CRC）细胞系集（N=76）中的细胞丢失率、倍增时间和上清液中的cfDNA释放，代表了先前在癌症患者中鉴定的分子亚型。通过相关分析，评估了cfDNA释放的定量参数与细胞增殖及分子特征的关系。还通过功能实验，测试了DNA甲基化调控对cfDNA释放的影响。较高的上清液cfDNA水平与细胞周期变慢和细胞死亡增加显著相关。此外，在非-CpG岛甲基化型（CIMP）模型中发现更高的cfDNA释出。这些结果表明，甲基化降低与cfDNA水平升高呈正相关。为进一步探索，我们利用甲基化微阵列鉴定出一组与cfDNA释出显著相关的探针，建立了能区分高低cfDNA释出者的甲基化特征。将该特征应用于另一组176个CRC细胞系和患者源性类器官，筛选出14个预测为低或高释出者的模型。该甲基化谱成功预测了上清液中cfDNA的释放量。在功能层面，敲除DNA甲基转移酶增加了染色质的可及性与DNA断裂，从而在等源CRC细胞系中增加了cfDNA的释放。此外，体外用去甲基化药物处理五个低释出CRC细胞系显著增加了cfDNA的释出量。癌细胞系的甲基化状态影响体外cfDNA释出的变异，甲基化模式的变化与cfDNA水平相关，或可用于提高液体活检的敏感性。

Liquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release.We measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release.Higher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding.Methylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.