Fragmentomics, the investigation of fragmentation patterns of cell-free DNA (cfDNA), has emerged as a promising strategy for the early detection of multiple cancers in the field of liquid biopsy. However, the clinical application of this approach has been hindered by a limited understanding of cfDNA biology. Furthermore, the prevalence of hematopoietic cell-derived cfDNA in plasma complicates the in vivo investigation of tissue-specific cfDNA other than that of hematopoietic origin. While conventional two-dimensional cell lines have contributed to research on cfDNA biology, their limited representation of in vivo tissue contexts underscores the need for more robust models. In this study, we propose three-dimensional organoids as a novel in vitro model for studying cfDNA biology, focusing on multifaceted fragmentomic analyses. We established nine patient-derived organoid lines from normal lung airway, normal gastric, and gastric cancer tissues. We then extracted cfDNA from the culture medium of these organoids in both proliferative and apoptotic states. Using whole-genome sequencing data from cfDNA, we analyzed various fragmentomic features, including fragment size, footprints, end motifs, and repeat types at the end. The distribution of cfDNA fragment sizes in organoids, especially in apoptosis samples, was similar to that found in plasma, implying occupancy by mononucleosomes. The footprints determined by sequencing depth exhibited distinct patterns depending on fragment sizes, reflecting occupancy by a variety of DNA-binding proteins. Notably, we discovered that short fragments (< 118 bp) were exclusively enriched in the proliferative state and exhibited distinct fragmentomic profiles, characterized by 3 bp palindromic end motifs and specific repeats. In conclusion, our results highlight the utility of in vitro organoid models as a valuable tool for studying cfDNA biology and its associated fragmentation patterns. This, in turn, will pave the way for further enhancements in noninvasive cancer detection methodologies based on fragmentomics.

中文翻译：

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对源自患者的类器官释放的无细胞 DNA 进行多维片段组分析

片段组学是对游离 DNA (cfDNA) 片段模式的研究，已成为液体活检领域早期检测多种癌症的一种有前景的策略。然而，由于对 cfDNA 生物学的了解有限，这种方法的临床应用受到了阻碍。此外，血浆中造血细胞来源的 cfDNA 的普遍存在使除造血来源以外的组织特异性 cfDNA 的体内研究变得复杂。虽然传统的二维细胞系为 cfDNA 生物学研究做出了贡献，但它们对体内组织环境的有限代表性强调了对更稳健模型的需求。在这项研究中，我们提出三维类器官作为研究 cfDNA 生物学的新型体外模型，重点关注多方面的片段组分析。我们从正常肺气道、正常胃和胃癌组织中建立了九个源自患者的类器官系。然后，我们从这些处于增殖和凋亡状态的类器官的培养基中提取了 cfDNA。使用 cfDNA 的全基因组测序数据，我们分析了各种片段组特征，包括片段大小、足迹、末端基序和末端的重复类型。类器官（尤其是凋亡样本）中 cfDNA 片段大小的分布与血浆中的分布相似，这意味着被单核小体占据。由测序深度确定的足迹根据片段大小表现出不同的模式，反映了各种 DNA 结合蛋白的占据。值得注意的是，我们发现短片段（< 118 bp）仅在增殖状态下富集，并表现出独特的片段组特征，其特征是 3 bp 回文末端基序和特定重复。总之，我们的结果强调了体外类器官模型作为研究 cfDNA 生物学及其相关碎片模式的宝贵工具的实用性。反过来，这将为进一步增强基于片段组学的非侵入性癌症检测方法铺平道路。