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.

项目摘要 癌症研究的一个主要目的是定义整个肿瘤微环境（TME）的组成 个人。一旦测量，TME组合物的差异就可以与提示相关联，针对 治疗，用于检验或产生新的治疗假设。我们对TME复杂性的欣赏是 随着单细胞RNA - 序列（SCRNA-SEQ）的发展，显着发展。但是scrna-seq 保持昂贵，在单个单元格的水平上噪音，并且转弯时间缓慢（通常是几周）。 scr SEQ通常也只能分析每个样品的1000个单元。结果，Scrna-seq对于深 大型患者或动物队列的分析，或用于癌症研究中的常规假设测试。更快和 SCRNA-SEQ的更可扩展的替代方法是流式细胞术（FC）和通过飞行时间（Cytof）的细胞仪，但 这些方法无法解析SCRNA-SEQ在TME中看到的复杂性。那是一个未满足的需求 用于快速，敏感，高度多样化的TME分析。 这笔赠款的重点是通过推进一种多功能且新颖的“微包”来满足这种未满足的需求 技术。胶囊代表了液滴微流体的演变，这是一种携带的成熟技术 在纳米尺度室中排出单细胞基因组测定，并通过油分离。胶囊克服了严重的 水滴水的技术局限性：它们对处理的脆弱性，并通过不可分割地隔离 油。相比之下，胶囊是可以分散和处理的弹性，半渗透室 任何水性生物缓冲液。在此提案之前，我们优化了胶囊以保留细胞mRNA和DNA， 同时可以快速交换销售，酶，底漆和周围的问题 中等的。现在，我们已经表明，胶囊可以在单细胞和 特别是在表面蛋白和mRNA分子上。这反过 细胞仪。 在此R33中，我们将基准并优化两个相关胶囊衍生的方法：第一种“ Capflow”， 通过基于快速胶囊的信号扩增，实现可靠的多重mRNA流式细胞仪。这 其次，“ capcycle”扩展了capflow，以量化≥50个基因转录本和细胞表面的抽象 蛋白质，通过用固定化胶囊的环状成像代替流式细胞仪。使用这些方法， 胶囊将使肿瘤的敏感，多功能，快速，低成本，高成本表型 异质性。这是填补重要的分析差距，并发展了多功能的微流体 具有长期潜力来改善单个生物分子和细胞生物学测定的技术。