Micro-capsules for versatile multiplexed cytometry

项目摘要 癌症研究的一个主要目标是确定肿瘤微环境（TME）的组成， 个体一旦测量，TME组成的差异可以与预后相关，靶向于 治疗，并用于测试或产生新的治疗假设。我们对TME复杂性的理解是 随着单细胞RNA测序（scRNA-Seq）的发展，这一研究取得了显著进展。但是scRNA-Seq 在单电池的水平上仍然是昂贵的、有噪声的，并且具有缓慢的周转时间（通常为数周）。scRNA- Seq通常每个样品仅分析1000个细胞。因此，scRNA-Seq对于深度测序是不实用的。 分析大型患者或动物队列，或用于癌症研究中的常规假设检验。快更 scRNA-Seq的更可扩展的替代方案是流式细胞术（FC）和飞行时间流式细胞术（CyToF）， 这些方法不能解决通过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和新颖的“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体的发展，这是一种成熟的携带技术， 在纳升级别的隔室中进行单细胞基因组测定，通过油分离。胶囊克服严重 油包水液滴的技术局限性：它们对处理的脆弱性，以及它们被不混溶的 油相比之下，胶囊是弹性的、半渗透的隔室，其可以分散在胶囊中并在胶囊中加工。 任何含水的生物缓冲液。在此之前，我们优化了胶囊以保留细胞mRNA和DNA， 同时使盐、酶、引物和探针能够与周围环境快速交换 介质我们现在已经证明，胶囊能够实现多步反应和对单细胞的系列分析， 特别是表面蛋白和mRNA分子。这反过来又实现了快速、多功能、高度多路复用的 细胞仪 在这个R33中，我们将对两个相关的胶囊衍生方法进行基准测试和优化：第一个是“CapFlow”， 通过基于胶囊的快速信号放大实现稳健的多重mRNA流式细胞术。的 第二，“CapCycle”，将CapFlow扩展到定量≥50个基因转录物和细胞表面的丰度 蛋白质，通过用固定化胶囊的循环成像代替流式细胞术。通过这些方法， 胶囊将使敏感的，多功能的，快速的，低成本的，高度多重的肿瘤表型分析成为可能。 异质性因此，该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体 具有长期潜力的技术，以改善对单个生物分子和细胞的生物测定。

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.