DNA脱甲基触发大肠癌细胞中无细胞DNA释放

基于无细胞DNA（CFDNA）分析的液体活检作为诊断，基因分型和监测固体恶性肿瘤的最低侵入性方法具有重要的希望。人类肿瘤通过包括细胞死亡，主动和被动释放在内的事件结合在血液中释放cfDNA。但是，导致CFDNA脱落的确切机制仍有待表征。在患者中解决这个问题的几个因素，例如肿瘤负担范围，解剖和脉管障碍以及从正常细胞中释放核酸。在这项工作中，我们利用了癌症模型来剖析DNA释放的基本机制。我们测量了细胞损耗比，倍增时间和CFDNA释放的结直肠癌（CRC）细胞系收集（n = 76）代表的分子亚型的代表性（n = 76）。评估了CFDNA释放，细胞增殖和分子特征的定量参数之间的关联分析。进行了功能实验，以测试调节DNA甲基化对CFDNA释放的影响。上清液的较高水平与细胞循环较慢和细胞死亡增加显着相关。此外，在非CPG岛甲基表型（CIMP）模型中发现了较高的CFDNA脱落。这些结果表明较低的甲基化与CFDNA水平升高之间存在正相关。为了进一步探讨这一点，我们利用了甲基化微阵列，以确定与CFDNA脱落显着相关的探针的子集，并得出能够将高CFDNA释放剂区分开的甲基化特征。我们将此签名应用于一组独立的176个CRC细胞系和患者衍生的类器官，以选择14个模型，预计为低或高释放器。甲基化谱成功预测了上清液中释放的CfDNA量。在功能水平上，DNA甲基转移酶的遗传消融增加了染色质的可及性和DNA碎片化，从而导致等源性CRC细胞系中的CfDNA释放增加。此外，用脱甲基化剂对五个低释放酶CRC细胞的体外处理能够诱导CfDNA脱落的显着增加。癌细胞系的甲基化状态有助于体外CFDNA脱落的变异性。甲基化模式的变化与CFDNA释放水平有关，可能会利用以提高液体活检测定的敏感性。

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.