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.

项目概要 癌症研究的一个主要目标是定义肿瘤微环境（TME）的组成 个人。一旦测量，TME 成分的差异可以与预后相关联，目标是 疗法，并用于测试或产生新的治疗假设。我们对 TME 复杂性的认识是 随着单细胞 RNA 测序 (scRNA-Seq) 的发展取得了显着进展。但 scRNA 测序 在单细胞水平上仍然昂贵、嘈杂，并且周转时间很慢（通常为数周）。单链RNA- Seq 通常还只分析每个样本的 1000 个细胞。因此，scRNA-Seq 对于深度分析并不实用。 对大型患者或动物群体进行分析，或用于癌症研究中的常规假设检验。更快并且 scRNA-Seq 更具可扩展性的替代方案是流式细胞术 (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，存在未满足的需求 用于快速、灵敏、高度多重的 TME 分析。 这笔赠款的重点是通过开发一种多功能且新颖的“微胶囊”来解决这一未满足的需求 技术。胶囊代表了液滴微流控的发展，这是一种成熟的运载技术 在纳升规模的隔室中进行单细胞基因组测定，通过油隔离。胶囊克服严重 油包水液滴的技术局限性：处理起来很脆弱，并且通过不混溶完全隔离 油。相比之下，胶囊是有弹性的半渗透性隔室，可以在其中分散和加工。 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， 同时能够与周围环境快速交换盐、酶、引物和探针 中等的。我们现在已经证明，胶囊能够对单细胞进行多步反应和系列分析， 特别是在表面蛋白和 mRNA 分子上。这反过来又实现了快速、多功能、高度多路复用 细胞计数术。 在此 R33 中，我们将对两种相关的胶囊衍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重 mRNA 流式细胞术。这 第二，“CapCycle”，将 CapFlow 扩展到量化 ≥50 个基因转录本和细胞表面的丰度 通过用固定胶囊的循环成像代替流式细胞术来检测蛋白质。通过这些方法， 胶囊将实现灵敏、多功能、快速、低成本、高度多重的肿瘤表型分析 异质性。因此，该提案填补了一个重要的分析空白，并开发了一种多功能的微流体 具有改善单个生物分子和细胞的生物测定的长期潜力的技术。