Micro-capsules for versatile multiplexed cytometry
用于多功能多重细胞计数的微胶囊
基本信息
- 批准号:10612144
- 负责人:
- 金额:$ 37.02万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AddressAdoptionAdvanced DevelopmentAnimalsAntibodiesAtlasesBenchmarkingBiologicalBiological AssayBuffersCell Surface ProteinsCell surfaceCellsClinical ResearchComplexCytolysisCytometryDNADevelopmentEnzymesEvolutionFlow CytometryFluorescenceFutureGene Expression ProfilingGenerationsGenesGenomicsGoalsGrantHourImageImmobilizationIndividualMeasurementMeasuresMembrane ProteinsMessenger RNAMethodsMicrocapsules drug delivery systemMicrofluidicsMolecular BiologyOilsPatient-Focused OutcomesPatientsPerformancePeriodicityPermeabilityPhenotypePopulationPreparationPrimary NeoplasmProcessPrognosisProteinsProtocols documentationPublishingReactionResearch PersonnelSaltsSamplingSensitivity and SpecificitySignal TransductionSpecificitySpeedStainsTechnologyTestingTimeTranscriptTubeTumor-infiltrating immune cellsVariantWateranticancer researchaqueouscapsulecohortcostdigitalhigh throughput screeningimprovedmicrofluidic technologymultiple omicsnanolitrenanolitre scaleneoplastic cellnew technologynovelnovel therapeuticspre-clinical researchprotein expressionresiliencescale upscreeningsingle cell analysissingle-cell RNA sequencingtargeted treatmenttumor heterogeneitytumor microenvironment
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Allon Moshe Klein其他文献
The Inflammatory Cytokine IL17 Tunes and Amplifies the Erythropoietic Response to Erythropoietin <em>In Vivo</em>
- DOI:
10.1182/blood-2024-211825 - 发表时间:
2024-11-05 - 期刊:
- 影响因子:
- 作者:
Qiu Chang Wu;Ashley Winward;Aishwarya Swaminathan;Logan Lalonde;Merav Socolovsky;Allon Moshe Klein - 通讯作者:
Allon Moshe Klein
The Inflammatory Cytokine IL17 Tunes and Amplifies the Erythropoietic Response to Erythropoietin emIn Vivo/em
炎症细胞因子IL17在体内调节并增强红细胞生成对促红细胞生成素的反应
- DOI:
10.1182/blood-2024-211825 - 发表时间:
2024-11-05 - 期刊:
- 影响因子:23.100
- 作者:
Qiu Chang Wu;Ashley Winward;Aishwarya Swaminathan;Logan Lalonde;Merav Socolovsky;Allon Moshe Klein - 通讯作者:
Allon Moshe Klein
Allon Moshe Klein的其他文献
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{{ truncateString('Allon Moshe Klein', 18)}}的其他基金
Semi-permeable capsules for high-throughput single cell multi-omics
用于高通量单细胞多组学的半透胶囊
- 批准号:
10698044 - 财政年份:2022
- 资助金额:
$ 37.02万 - 项目类别:
Semi-permeable capsules for high-throughput single cell multi-omics
用于高通量单细胞多组学的半透胶囊
- 批准号:
10569373 - 财政年份:2022
- 资助金额:
$ 37.02万 - 项目类别:
Mapping the signaling landscape of vertebrate development at single cell resolution
以单细胞分辨率绘制脊椎动物发育的信号图谱
- 批准号:
9912795 - 财政年份:2018
- 资助金额:
$ 37.02万 - 项目类别:
Single Cell Genome-Wide Myeloid Response Profiling in Immunotherapy
免疫治疗中的单细胞全基因组骨髓反应分析
- 批准号:
10442529 - 财政年份:2018
- 资助金额:
$ 37.02万 - 项目类别:
Mapping the signaling landscape of vertebrate development at single cell resolution
以单细胞分辨率绘制脊椎动物发育的信号图谱
- 批准号:
10392393 - 财政年份:2018
- 资助金额:
$ 37.02万 - 项目类别:
Mapping the signaling landscape of vertebrate development at single cell resolution
以单细胞分辨率绘制脊椎动物发育的信号图谱
- 批准号:
9766326 - 财政年份:2018
- 资助金额:
$ 37.02万 - 项目类别:
Single Cell Genome-Wide Myeloid Response Profiling in Immunotherapy
免疫治疗中的单细胞全基因组骨髓反应分析
- 批准号:
10183187 - 财政年份:2018
- 资助金额:
$ 37.02万 - 项目类别:
Novel Growth Factor Regulators of Early Erythropoieisis
早期红细胞生成的新型生长因子调节剂
- 批准号:
9916811 - 财政年份:2018
- 资助金额:
$ 37.02万 - 项目类别:
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