Simulating cell-free chromatin using preclinical cancer models for liquid biopsy applications [ATAC-Seq]

Cell-free DNA circulates in blood bound to nucleosomes, forming cell-free chromatin (cfChromatin) that retains epigenetic features, including nucleosome positioning and histone modifications. cfChromatin provides a rich source of cancer biomarkers; however, low abundance of tumor-derived cfChromatin and limited availability of clinical samples pose challenges for liquid biopsy research. To address this, we developed a framework to simulate cfChromatin nucleosomal distributions using nuclease-treated conditioned media from tissue cultures. Whole-genome sequencing confirmed that inferred nucleosome positioning reflected cell-type-specific gene expression and chromatin accessibility patterns, and comparisons with plasma cfChromatin from xenografted mice revealed concordant nucleosome profiles. Notably, simulated cfChromatin displayed stronger tumor-specific nucleosome profiles than patient plasma, where hematopoietic-derived cfChromatin dilutes signal. We further leveraged simulated cfChromatin to advance cell-free chromatin immunoprecipitation and sequencing methods, identifying repressive and bivalent chromatin domains predictive of transcriptional activity. Altogether, our results demonstrate utility of simulated cfChromatin as a scalable preclinical tool for liquid biopsy research. Overall design: We performed open chromatin profiling by the assay for transposase-accessible chromatin with sequencing (ATAC-Seq) of cellular chromatin from CAMA-1 cells. ATAC-seq from CAMA-1 cells was performed with biological replicates.无细胞DNA（cell-free DNA, cfDNA）在血液中与核小体结合循环，形成保留表观遗传特征的无细胞染色质（cell-free chromatin, cfChromatin），其包含的表观遗传特征包括核小体定位与组蛋白修饰。无细胞染色质是癌症生物标志物的丰富来源，但肿瘤源性无细胞染色质丰度较低，且临床样本可及性有限，这给液体活检研究带来了挑战。为解决这一问题，我们利用组织培养经核酸酶处理的条件培养基，开发了一套模拟无细胞染色质核小体分布的研究框架。全基因组测序结果证实，推测得到的核小体定位模式能够反映细胞类型特异性基因表达与染色质可及性特征；与异种移植小鼠血浆中的无细胞染色质进行比对后，发现二者核小体谱具有一致性。值得注意的是，模拟得到的无细胞染色质比患者血浆样本展现出更强的肿瘤特异性核小体谱——患者血浆中造血细胞源性无细胞染色质会稀释肿瘤信号。我们进一步利用模拟无细胞染色质优化了无细胞染色质免疫共沉淀测序技术，成功鉴定出可预测转录活性的抑制性染色质结构域与二价染色质结构域。综上，本研究结果证实，模拟无细胞染色质可作为一种可规模化应用的临床前工具，用于液体活检相关研究。 实验设计：我们通过转座酶可及性染色质测序（assay for transposase-accessible chromatin with sequencing, ATAC-Seq）技术，对CAMA-1细胞的细胞染色质进行开放染色质谱分析；该实验设置了生物学重复。