Single-Cell RNA Sequencing of Cardiac Organoids to Determine the Genetic Basis for Cell-Specific Responses to Anticancer Drugs

心脏类器官的单细胞 RNA 测序以确定抗癌药物细胞特异性反应的遗传基础

• 批准号: 10679493
• 负责人: Erik McIntire
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2026-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679493
• 关键词: Acute; Adverse effects; Affect; Alleles; Anthracycline; Antineoplastic Agents; Arrhythmia; Biological Markers; Biological Models; Blood Vessels; Cancer Patient; Cancer Survivor; Candidate Disease Gene; Cardiac; Cardiac Myocytes; Cardiotoxicity; Cardiovascular Physiology; Cardiovascular system; Catalogs; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cells; Cessation of life; Classification; Data; Disease; Doxorubicin; Drug usage; Endoderm; Endothelial Cells; Environment; Fibroblasts; Fluorouracil; Functional disorder; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genotype; Heart; Heart failure; Heritability; Hypertension; Individual; Intuition; Long-Term Effects; Malignant Neoplasms; Maps; Measures; Mediating; Mesoderm Cell; Modeling; Morbidity - disease rate; Neural Crest Cell; Oncology; Organoids; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacogenomics; Pharmacotherapy; Predisposition; Primitive foregut structure; Procedures; Reporting; Reproducibility; Risk; Risk Factors; Sample Size; Sampling; Supporting Cell; Symptoms; System; Testing; Time; Tissues; Toxic effect; Transcript; Treatment Protocols; Variant; Vascular Endothelial Cell; Vascular System; Work; anticancer treatment; bevacizumab; cancer therapy; cardiovascular risk factor; cell type; clinical phenotype; differentiation protocol; disorder risk; effective therapy; experimental study; genetic architecture; genetic variant; genotyped patients; improved; individual patient; induced pluripotent stem cell; inter-individual variation; mortality; novel; personalized medicine; premature; response; side effect; single-cell RNA sequencing; stem cells; trait; transcriptomics

PROJECT SUMMARY Anticancer drug-induced cardiovascular toxicity (CT) is a major side effect for many patients undergoing treatment for oncological disorders. CT symptoms vary widely across individuals, both in presentation and in time to onset. Risk of CT complicates treatment protocols and places cancer patients under additional duress. Genetic background is broadly understood to be a component of CT susceptibility, but specific variants and mechanisms remain largely unknown. To understand the genetic basis for drug-induced CT, we need to understand how anticancer drugs stimulate transcriptomic responses in multiple cardiovascular cell types from individuals of different genetic backgrounds. Inter-individual variability in response to anticancer drugs is mediated by genetic variants that affect gene regulation in a drug-dependent manner (response eQTLs). In other words, genetic variants respond to anticancer drugs by regulating the activity of specific genes. Notably, different cell types can vary in their response eQTLs to anticancer drugs. I propose to determine the genetic basis for transcriptomic responses to the anticancer drugs doxorubicin (DOX), 5-fluorouracil (5-FU), and bevacizumab (BVC) in multiple cardiac cell types from a genetically diverse panel of 70 individuals. Identification of CT-associated response eQTLs necessitates a high-throughput model system comprised of multiple cardiac cell types. For Aim 1 of my proposal, I have developed a culture environment and guided differentiation protocol conducive to cardiac lineage and supporting cell types. This procedure reproducibly transforms induced pluripotent stem cell (iPSC)-derived embryoid bodies (EBs) into cardiac organoids. Preliminary data demonstrate that the cardiac organoids harbor cardiomyocytes, fibroblasts, vascular endothelial cells, and other mesodermal cell types. In Aim 2, I will perform single-cell RNA sequencing (scRNA-seq) on a panel of 70 cardiac organoids cultured in control and drug-treated conditions. Repeating this experiment across multiple individuals will allow me to identify the response eQTLs that regulate how different cardiovascular cell types respond to each drug. In Aim 3, I will quantify gene expression levels and identify response eQTLs that regulate transcriptional changes to each anticancer drug in cardiovascular cell types. Response eQTLs (which are anchored by genotype) provide a catalog of loci that interact either directly or indirectly with the treatment. These response eQTLs may reveal specific genes and pathways important for normal cardiovascular function. Elucidating the genetic architecture underlying CT risk will provide intuition on cardiotoxic mechanisms and associated genes and inform future studies that aim to classify individual patient susceptibility.

项目摘要 抗癌药物诱导的心血管毒性（CT）是许多正在接受的患者的主要副作用 治疗肿瘤疾病。 CT症状在演示和在 是时候发作了。 CT的风险使治疗方案复杂化，并使癌症患者受到额外的胁迫。 遗传背景广泛理解为CT易感性的组成部分，但特定的变体和 机制在很大程度上仍然未知。要了解药物引起的CT的遗传基础，我们需要 了解抗癌药物如何刺激多种心血管细胞类型中的转录组反应 具有不同遗传背景的个体。响应抗癌药物的个体间变异性是 由以药物依赖性方式影响基因调节的遗传变异（反应eqtls）介导。在 换句话说，遗传变异通过调节特定基因的活性来对抗癌药物做出反应。尤其， 不同的细胞类型的反应EQTL对抗癌药物的反应EQTL可能有所不同。我建议确定遗传 对抗癌药阿霉素（DOX），5-氟尿嘧啶（5-FU）的转录组反应的基础 来自70个个体的遗传多样化面板的多种心脏细胞类型中的贝伐单抗（BVC）。 CT相关响应eqtls的识别需要一个由高通量模型系统组成的 多种心脏细胞类型。对于我的提议的目标1，我已经建立了一种文化环境并指导了 分化方案有利于心脏谱系和支持细胞类型。此过程可重复 转化诱导的多能干细胞（IPSC）衍生的胚胎体（EB）成心脏器官。 初步数据表明心脏器官携带心肌细胞，成纤维细胞，血管 内皮细胞和其他中胚层细胞类型。在AIM 2中，我将执行单细胞RNA测序 （SCRNA-SEQ）在对照和药物治疗条件下培养的70个心脏器官面板上。重复 跨多个人的实验将使我能够确定调节如何调节的响应eqtl 不同的心血管细胞类型对每种药物有反应。在AIM 3中，我将量化基因表达水平和 确定调节心血管细胞中每种抗癌药物转录变化的响应eqtls 类型。响应eqtls（由基因型锚定）提供了一个基因座的目录，直接相互作用 或间接处理治疗。这些响应EQTL可能揭示特定基因和途径 正常的心血管功能。阐明CT风险的遗传结构将提供直觉 心脏毒性机制和相关基因，并告知未来的研究，旨在对患者进行分类 敏感性。