Micro-capsules for versatile multiplexed cytometry

用于多功能多重细胞计数的微胶囊

• 批准号: 10612144
• 负责人: Allon Moshe Klein
• 金额: $ 37.02万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-07 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612144
• 关键词: Address; Adoption; Advanced Development; Animals; Antibodies; Atlases; Benchmarking; Biological; Biological Assay; Buffers; Cell Surface Proteins; Cell surface; Cells; Clinical Research; Complex; Cytolysis; Cytometry; DNA; Development; Enzymes; Evolution; Flow Cytometry; Fluorescence; Future; Gene Expression Profiling; Generations; Genes; Genomics; Goals; Grant; Hour; Image; Immobilization; Individual; Measurement; Measures; Membrane Proteins; Messenger RNA; Methods; Microcapsules drug delivery system; Microfluidics; Molecular Biology; Oils; Patient-Focused Outcomes; Patients; Performance; Periodicity; Permeability; Phenotype; Population; Preparation; Primary Neoplasm; Process; Prognosis; Proteins; Protocols documentation; Publishing; Reaction; Research Personnel; Salts; Sampling; Sensitivity and Specificity; Signal Transduction; Specificity; Speed; Stains; Technology; Testing; Time; Transcript; Tube; Tumor-infiltrating immune cells; Variant; Water; anticancer research; aqueous; capsule; cohort; cost; digital; high throughput screening; improved; microfluidic technology; multiple omics; nanolitre; nanolitre scale; neoplastic cell; new technology; novel; novel therapeutics; pre-clinical research; protein expression; resilience; scale up; screening; single cell analysis; single-cell RNA sequencing; targeted treatment; tumor heterogeneity; tumor microenvironment

Project summary A major goal of cancer research is to define the composition of the tumor micro-environment (TME) across individuals. Once measured, differences in TME composition can be correlated with prognosis, targeted by therapy, and used to test or generate novel therapeutic hypotheses. Our appreciation of TME complexity was significantly advanced with the development of single cell RNA-Sequencing (scRNA-Seq). But scRNA-Seq remains expensive, noisy at the level of single cells, and has a slow turn-around time (typically weeks). scRNA- Seq also typically analyzes only 1000s of cells per sample. As a result, scRNA-Seq is not practical for deep profiling of large patient or animal cohorts, or for routine hypothesis-testing in cancer research. Faster and more scalable alternatives to scRNA-Seq are flow cytometry (FC) and Cytometry by Time of Flight (CyToF) but these methods do not resolve the complexity seen in the TME by scRNA-Seq. Thus, there is an unmet need for rapid, sensitive, highly-multiplexed TME profiling. The focus of this grant is to address this unmet need by advancing a versatile and novel `micro-capsule' technology. Capsules represent an evolution of droplet microfluidics, which is a mature technology for carrying out single cell genomic assays in nanoliter-scale compartments, isolated by oil. Capsules overcome severe technical limitations of water-in-oil droplets: their fragility to handling, and their complete isolation by immiscible oil. By contrast, capsules are resilient, semi-permeable compartments that can be dispersed and processed in any aqueous biological buffer. Prior to this proposal, we optimized capsules to retain cellular mRNA and DNA, while simultaneously enabling rapid exchange of salts, enzymes, primers and probes with the surrounding medium. We have now shown that capsules enable multi-step reactions and serial analyses on single cells and specifically on surface proteins and mRNA molecules. This in turn enables rapid, versatile, highly-multiplexed cytometry. In this R33 we will benchmark and optimize two related capsule-derived methods: the first, “CapFlow”, implements robust multiplexed mRNA flow cytometry with rapid capsule-based signal amplification. The second, “CapCycle”, extends CapFlow to quantifying the abundance of ≥50 gene transcripts and cell surface proteins, by replacing flow cytometry with cyclic imaging of immobilized capsules. With these methods, capsules will enable sensitive, versatile, rapid, low-cost, highly-multiplexed phenotyping of tumor heterogeneity. Thus, this proposal fills an important analytical gap, and develops a versatile microfluidic technology with long-term potential to improve biological assays on single biomolecules and cells.

项目总结 癌症研究的一个主要目标是确定肿瘤微环境(TME)的组成 个人。一旦被测量，TME成分的差异可以与预后相关，目标是 治疗，并用于测试或产生新的治疗假说。我们对TME复杂性的理解是 随着单细胞RNA测序技术(scRNA-Seq)的发展，该技术有了很大的进步。但scRNA-Seq 仍然昂贵，在单个电池的水平上噪音很大，而且周转时间很慢(通常是几周)。单链RNA- SEQ通常也只分析每个样本上千个细胞。因此，scRNA-Seq不适用于深层 大型患者或动物队列的概况分析，或癌症研究中的常规假设检验。速度更快， ScRNA-Seq更具可扩展性的替代方法是流式细胞术(FC)和飞行时间细胞术(CyToF)，但 这些方法不能解决scRNA-Seq在TME中看到的复杂性。因此，有一种未得到满足的需求。 用于快速、灵敏、高度多路传输的TME分析。 这笔赠款的重点是通过开发一种多功能和新颖的微胶囊来解决这一未得到满足的需求。 技术胶囊代表了液滴微流控技术的发展，是一种成熟的携带技术 在纳升规模的隔间中进行单细胞基因组分析，用油分离。胶囊攻克重症 油包水液滴的技术限制：它们对操作的易损性，以及它们通过不相容完全隔离 油。相比之下，胶囊是弹性的、半透气性的隔间，可以分散并在 任何含水的生物缓冲液。在这项提议之前，我们优化了胶囊以保留细胞mRNA和DNA， 同时使盐、酶、引发物和探针能够与周围环境快速交换 5~6成熟。我们现在已经证明，胶囊可以在单个细胞上进行多步反应和系列分析，并 尤其是表面蛋白质和信使核糖核酸分子。这进而实现了快速、多功能、高度多路复用 细胞学。 在本R33中，我们将对两个相关的胶囊派生方法进行基准测试和优化：第一个，“CapFlow”， 通过基于胶囊的快速信号放大，实现强大的多路复用信使核糖核酸流式细胞术。这个 第二，“CapCycle”，将CapFlow扩展到量化≥50基因转录本和细胞表面的丰度 蛋白质，通过用固定化胶囊的循环成像取代流式细胞术。有了这些方法， 胶囊将使敏感、通用、快速、低成本、高度多样化的肿瘤表型成为可能 异质性。因此，这一建议填补了一个重要的分析空白，并开发了一种通用的微流控 具有长期潜力的技术，以改进对单个生物分子和细胞的生物分析。