Decoding the Tissue of Origin of Cellular Damage from Cell-free DNA in Liquid Biopsies

摘要 液体活检应用正在迅速崛起，成为一种微创的方法来收集全系统的 用于生理和疾病相关变化的基因组监测的代表性分析物。垂死的细胞释放 碎片DNA进入循环，称为无细胞DNA(CfDNA)。破译cfDNA的细胞起源 随着时间的推移，可以揭示细胞成分的改变，反映了纵向组织损伤的动态变化 学习。在这里，我将集中讨论肝移植后细胞和组织动态平衡的变化 CfDNA的分析。将使用细胞类型特定的甲基化模式来追踪cfDNA的细胞起源 分子。此外，实体器官移植将一套与供体器官分开的DNA植入体内。 使来自同种异体移植物的cfDNA分子能够通过基因分型得到鉴定和验证。 使用来自捐赠者的SNPs。在这项提案中，我的目标是跟踪细胞损伤后不断变化的成分- 并利用这些信息改进移植物功能障碍的诊断和管理(目标1)。 在移植过程中，组织驻留的免疫细胞与供体器官同时转移。 组织。我将使用免疫细胞特异性DNA甲基化模式和捐赠者SNP分析来 区分移植后和移植过程中宿主和供体组织驻留免疫细胞的变化 免疫抑制治疗(目标2)。 这项拟议的研究将评估一种创新的方法，以深入了解宿主细胞的反应， 供体器官细胞，加上宿主和供体免疫细胞，与不同的移植结果相关。一系列的证据- 将肝移植作为一种理想的移植方案，引入了一种具有 不同的基因组在特定的时间点，在该时间点将引起细胞内稳态的变化 同种异体移植物中的细胞以及宿主。除了移植结果分析，cfDNA方法 根据这一提议建立的可以扩展以确定细胞对组织损伤的贡献 任何场景。细胞类型特定的甲基化模式是通用的标记，可用于追踪受损的 CfDNA的细胞来源，而不考虑损伤的原因。肝脏中的细胞损伤可能是由于靶向 治疗、化疗、免疫抑制或其他干预措施，启动或复发原发性肝脏 在新冠肺炎大流行期间观察到的恶性肿瘤、癌症转移播种或器官损害。我们建议 从不同类型的损伤中可以观察到不同的细胞cfDNA特征。此外，还有一个未满足的问题 需要在新治疗方法的开发过程中深入了解组织损伤，并了解 与治疗效果相关的不良事件的细胞基础。这项提议的全球影响将是 将循环中的cfDNA与它们的细胞起源联系起来，从而揭示病理生理学的驱动因素。

ABSTRACT Liquid biopsy applications are rapidly emerging as a minimally invasive approach to collect system-wide representative analytes for genomic monitoring of physiologic and disease-related changes. Dying cells release fragmented DNA into the circulation, referred to as cell-free DNA (cfDNA). Decoding the cellular origins of cfDNA over time can reveal altered cellular contributions reflective of dynamic changes to tissue damages in longitudinal studies. Here, I will focus on the changes in cellular and tissue homeostasis post liver transplant using molecular analyses of cfDNA. Cell-type specific methylation patterns will be used to trace the cellular origins of cfDNA molecules. In addition, solid organ transplant place a separate DNA set with the donor organ into the body of the host, allowing cfDNA molecules from the allograft to be identified and validated through genotyping using donor-derived SNPs. In this proposal, I aim to track the changing composition of cellular damage post- liver transplant and use this information to improve diagnosis and management of graft dysfunction (Aim 1). During transplant there is simultaneous transfer of tissue-resident immune cells along with the donor organ tissue. I will use immune cell-specific DNA methylation patterns together with the donor SNP analysis to distinguish the host and donor tissue-resident immune cell changes after transplant and during immunosuppressive treatment (Aim 2). The proposed research will evaluate an innovative approach to gain insights into the reaction of host cells, donor organ cells, plus host and donor-immune cells relative to different transplant outcomes. A series of proof- of-principle studies are outlined using liver transplantation as an ideal setup that introduces an organ with a distinct genome at a specific timepoint where there will be induced changes in cell homeostasis to a range of cells in the allografts as well as the host. Beyond the transplant outcome analysis, the cfDNA approach established under this proposal can be expanded to determine the cellular contributions to tissue damages in any setting. Cell type-specific methylation patterns are universal markers that can be used to trace the damaged cell origin of cfDNA irrespective of the cause of damage. Cellular damage in the liver can be due to targeted therapy, chemotherapy, immunosuppression or other interventions, initiation or recurrence of primary liver malignancy, cancer metastatic seeding or organ damage observed during the COVID-19 pandemic. We propose that distinct cellular cfDNA signatures will be observed from different types of injury. Also, there is an unmet need to gain insights into tissue damage during the development of new treatments and understand the cellular basis of adverse events relative to therapeutic efficacy. The global impact of this proposal will be to link cfDNAs in the circulation to their cellular origins and thus reveal drivers of pathophysiology.