Optimizing methods of clinical sample processing for scRNA-seq and mechanistic studies in sepsis to enable reliable, reproducible, and high-yield multi-center collection efforts

优化脓毒症 scRNA-seq 和机制研究的临床样本处理方法，以实现可靠、可重复和高产的多中心采集工作

• 批准号: 10571958
• 负责人: ROBY PAUL BHATTACHARYYA
• 金额: $ 21.82万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10571958
• 关键词: Address; B-Lymphocytes; Biological; Blood; Blood Cells; Blood specimen; Cell Separation; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Cessation of life; Clinical; Clinical Data; Clinical Trials; Collection; Complex; Coupled; Cryopreservation; Cryopreserved Cell; Data; Development; Diagnosis; Disease; Enrollment; Evolution; Functional disorder; Gene Expression; Genes; Genetic Transcription; Genomics; Goals; Health Expenditures; Heterogeneity; Hospitalization; Hospitals; Human; Immune; Immune response; Immunologic Stimulation; Immunologics; Immunology; Immunotherapy; In Vitro; Individual; Infection; Institutional Review Boards; Kinetics; Maps; Measures; Methods; Morbidity - disease rate; Multicenter Studies; Neurosciences; Oncology; Organ; Organ failure; Outcome; Pathway interactions; Patients; Phase; Population; Population Heterogeneity; Procedures; Process; RNA; Recovery; Reproducibility; Sampling; Scientist; Sepsis; Site; Standardization; Stimulus; Syndrome; T-Lymphocyte; Techniques; Testing; Therapeutic Trials; Time; Whole Blood; adjudication; cell type; clinical center; clinical investigation; clinical research site; cohort; cost; disease heterogeneity; insight; monocyte; mortality; novel; patient population; patient subsets; preservation; programs; repository; response; septic patients; single-cell RNA sequencing; targeted treatment; transcriptome sequencing; treatment effect

Project summary: Sepsis is prevalent, costly, and deadly. In the U.S, sepsis accounts for 4% of hospitalizations, 13% of in-hospital healthcare expenditures, and 35% of in-hospital deaths. Although common, sepsis is often difficult to diagnose and treat effectively, since it is a syndrome defined generically by organ dysfunction resulting from a dysregulated immune response to infection. This broad definition leads to heterogeneity of disease and misdiagnosis. Clinical trials thus enroll ill-characterized populations of sepsis patients with variable underlying immune responses to infection, diluting the effects of novel and otherwise promising therapies that might benefit a defined subset of patients. Precision immune cell-specific characterization of the dysregulated host immune response in sepsis is clearly needed. Our group was the first to elucidate an immunosuppressive monocyte substate (MS1) expanded in patients with urosepsis (Reyes et al. Nat Med 2020) using single cell RNA sequencing (scRNA-seq), which resolves cellular heterogeneity, revealing rich signatures of immune cell-specific gene expression not evident from standard immune profiling techniques. Clinical investigation in sepsis would greatly benefit from the scalable deployment of scRNA-seq across multicenter studies and clinical trials to enable robust characterization of the host immune response in sepsis, and to explain the effect of disease heterogeneity on clinical course, outcomes, and treatment effects. Yet in order to facilitate subsequent scRNA-seq, immune cells must be isolated from whole blood samples, which currently involves complex real-time processing of fresh blood at clinical sites, a process neither practical nor reproducible enough to allow deployment across multiple clinical centers. To address this critical need, we piloted a simple method of onsite whole blood cryopreservation that uses only 2 mL of blood and is easily deployable at clinical sites, followed by storage for scRNA-seq at a centralized center of expertise. In the R21 phase, we will optimize and validate our method of whole blood cryopreservation on enrolled subjects at 2 local clinical sites versus gold standard methods for immune cell isolation. We will compare scRNA-seq technical quality metrics and biologically-relevant measures including the MS1 monocyte subtype and other immune cell states. Importantly, we will test the viability and function of cryopreserved cells in mechanistic studies. In the R33 phase, we will scale our whole blood cryopreservation method to 5 enrolling clinical sites around the U.S. to demonstrate feasibility of a multicenter collection with centralized scRNA-seq and analysis. In this expanded sepsis cohort, we will perform deep immune profiling and derive scRNA-seq- based endotypes to characterize underlying heterogeneity of host immune responses. We will compare these endotypes to those derived from bulk RNA sequencing and apply scRNA-seq-derived signatures to our own clinical trials data that have obtained bulk RNA sequencing. The proposed studies are highly likely to make available a simple method to facilitate single-cell immune profiling in multicenter studies to greatly enhance our understanding of the host immune response in sepsis and guide the development of targeted therapies.

项目概要： 败血症是普遍的，昂贵的，和致命的。在美国，脓毒症占住院治疗的4%，占住院治疗的13%。 医疗支出，35%的住院死亡。虽然常见，败血症往往很难诊断 并有效地治疗，因为它是一种综合征，一般定义为由失调引起的器官功能障碍， 对感染的免疫反应。这种宽泛的定义导致了疾病的异质性和误诊。临床 因此，试验招募了特征不佳的脓毒症患者群体，这些患者具有不同的潜在免疫反应， 感染，稀释新的和其他有前途的治疗，可能有利于一个确定的子集的影响， 患者败血症中失调的宿主免疫应答的精确免疫细胞特异性表征是 显然需要。我们的小组是第一个阐明免疫抑制单核细胞亚群（MS 1）扩增的。 在患有尿脓毒症的患者中（Reyes等人，Nat Med 2020），使用单细胞RNA测序（scRNA-seq）， 解决了细胞异质性，揭示了免疫细胞特异性基因表达的丰富特征， 来自标准免疫分析技术。脓毒症的临床研究将大大受益于可扩展的 在多中心研究和临床试验中部署scRNA-seq，以实现对 脓毒症中的宿主免疫应答，并解释疾病异质性对临床病程，结局， 和治疗效果。然而，为了促进随后的scRNA-seq，免疫细胞必须从整个细胞中分离。 血液样本，目前涉及在临床站点对新鲜血液进行复杂的实时处理， 既不实用也不可重复，不足以允许在多个临床中心部署。 为了满足这一关键需求，我们试验了一种简单的现场全血冷冻保存方法， 2 mL血液，可轻松部署在临床研究中心，然后在集中中心储存scRNA-seq 专业知识。在R21阶段，我们将优化和验证我们的全血冷冻保存方法， 在2个当地临床研究中心招募受试者，对比金标准方法进行免疫细胞分离。我们将比较 scRNA-seq技术质量指标和生物学相关指标，包括MS 1单核细胞亚型 和其他免疫细胞状态。重要的是，我们将测试冻存细胞的活力和功能， 机械研究。在R33阶段，我们将把我们的全血冷冻保存方法扩大到5例入组 美国各地的临床研究中心，以证明使用集中scRNA-seq进行多中心收集的可行性 和分析在这个扩展的脓毒症队列中，我们将进行深度免疫分析并推导scRNA-seq- 基于内型来表征宿主免疫应答的潜在异质性。我们将比较这些 将scRNA-seq衍生的特征应用于我们自己的 已获得批量RNA测序的临床试验数据。拟议的研究极有可能使 提供一种简单的方法，以促进单细胞免疫分析在多中心研究，大大提高我们的 了解脓毒症中的宿主免疫反应，并指导靶向治疗的发展。