基于游离 DNA (cfDNA) 分析的液体活检作为实体恶性肿瘤的诊断、基因分型和监测的微创方法具有重大前景。人类肿瘤通过细胞死亡、主动和被动释放等一系列事件在血流中释放 cfDNA。然而，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会受到多种因素的困扰，例如肿瘤负荷程度、解剖学和脉管系统障碍以及正常细胞的核酸释放。在这项工作中，我们利用癌症模型来剖析 DNA 释放的基本机制。我们测量了代表分子的结直肠癌 (CRC) 细胞系集合 (N = 76) 上清液中的细胞损失率、倍增时间和 cfDNA 释放。先前在癌症患者中发现的亚型。对 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评估。进行功能实验来测试调节 DNA 甲基化对 cfDNA 释放的影响。上清液 cfDNA 水平较高与细胞周期减慢和细胞死亡增加显着相关。此外，在非 CpG 岛甲基化表型 (CIMP) 模型中发现了更高的 cfDNA 脱落。这些结果表明较低的甲基化与增加的 cfDNA 水平之间存在正相关。为了进一步探索这一点，我们利用甲基化微阵列来识别与 cfDNA 脱落显着相关的探针子集，并得出能够区分高和低 cfDNA 释放剂的甲基化特征。我们将这一特征应用于一组独立的 176 个 CRC 细胞系和患者来源的类器官，以选择 14 个预测为低或高释放剂的模型。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 甲基转移酶的基因消除增加了染色质的可及性和 DNA 片段化，导致同基因 CRC 细胞系中 cfDNA 的释放增加。此外，用去甲基化剂对五种低释放CRC细胞进行体外处理能够诱导cfDNA脱落显着增加。癌细胞系的甲基化状态导致体外cfDNA脱落的变异性。甲基化模式的变化与 cfDNA 释放水平相关，可用于提高液体活检检测的灵敏度。© 2024。作者。

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.© 2024. The Author(s).