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： 脓毒症很普遍、代价高昂且致命。在美国，脓毒症占住院治疗的 4%，占住院治疗的 13% 医疗保健支出，以及 35% 的院内死亡。虽然脓毒症很常见，但通常很难诊断 并进行有效治疗，因为它是一种综合征，一般定义为因失调而导致的器官功能障碍 对感染的免疫反应。这种宽泛的定义导致疾病的异质性和误诊。临床 因此，试验招募了特征不明的脓毒症患者群体，这些患者的潜在免疫反应各不相同 感染，削弱了新的和其他有前途的疗法的效果，这些疗法可能有益于特定的子集 患者。脓毒症中失调的宿主免疫反应的精确免疫细胞特异性特征是 显然需要。我们的小组是第一个阐明免疫抑制单核细胞亚状态（MS1）扩展 使用单细胞 RNA 测序 (scRNA-seq) 对尿脓毒症患者进行检测 (Reyes et al. Nat Med 2020)， 解决细胞异质性，揭示不明显的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反应，并解释疾病异质性对临床病程、结果的影响， 以及治疗效果。然而，为了便于后续的 scRNA-seq，必须从整个细胞中分离出免疫细胞。 血液样本，目前涉及在临床现场对新鲜血液进行复杂的实时处理，这是一个过程 既不实用，也不具有可重复性，无法在多个临床中心进行部署。 为了满足这一迫切需求，我们试验了一种简单的现场全血冷冻保存方法，该方法仅使用 2 mL 血液，可轻松部署在临床站点，然后在集中中心存储 scRNA-seq 的专业知识。在R21阶段，我们将优化并验证我们的全血冷冻保存方法 与免疫细胞分离的金标准方法相比，在 2 个当地临床中心招募受试者。我们会比较 scRNA-seq 技术质量指标和生物学相关测量，包括 MS1 单核细胞亚型 和其他免疫细胞状态。重要的是，我们将测试冷冻保存细胞的活力和功能 机理研究。在 R33 阶段，我们将把全血冷冻保存方法扩展到 5 个招募 美国各地的临床站点，以证明通过集中式 scRNA-seq 进行多中心收集的可行性 和分析。在这个扩大的脓毒症队列中，我们将进行深度免疫分析并得出 scRNA-seq- 基于内型来表征宿主免疫反应的潜在异质性。我们将比较这些 将内型转化为来自批量 RNA 测序的内型，并将 scRNA-seq 衍生的特征应用到我们自己的特征中 已获得批量RNA测序的临床试验数据。拟议的研究很可能使 提供了一种简单的方法来促进多中心研究中的单细胞免疫分析，从而大大增强我们的研究能力 了解脓毒症中的宿主免疫反应并指导靶向治疗的开发。