Single-cell Phosphoprotein Assay to Evaluate Brain Tumor Therapeutic Resistance

单细胞磷蛋白测定评估脑肿瘤治疗耐药性

• 批准号: 9927272
• 负责人: Timothy S McConnell
• 金额: $ 99.9万
• 依托单位: ISOPLEXIS, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-12 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9927272
• 关键词: Address; Antibodies; Automation; Automobile Driving; Benchmarking; Biological; Biological Assay; Biopsy Specimen; Brain Neoplasms; Cancer Biology; Cancer Patient; Cell Line; Cells; Cellular Assay; ChIP-on-chip; Clinical; Clinical Research; Clinical Trials; Combination Drug Therapy; Complex; Cytolysis; Data; Detection; Devices; Drug Combinations; Drug Targeting; Drug resistance; Event; Exhibits; Failure; Flow Cytometry; Genetic; Genomics; Glioblastoma; Glioma; Gold; Hour; Human; In Vitro; Individual; Industry; Intracellular Signaling Proteins; Legal patent; Liquid substance; Malignant Neoplasms; Measurement; Measures; Methodology; Modeling; Molecular; Monitor; Nature; Oncogenes; Oncogenic; Pathway Analysis; Pathway interactions; Patients; Performance; Pharmaceutical Preparations; Phase; Phosphoproteins; Population; Proteins; Proteomics; Publications; Publishing; Research Personnel; Resistance; Robot; Sampling; Ships; Signal Pathway; Signal Transduction; Signal Transduction Inhibitor; Small Business Innovation Research Grant; Solid Neoplasm; Stains; Standardization; System; Techniques; Technology; Time; Tumor-Derived; Western Blotting; base; cancer cell; cancer therapy; clinical development; commercialization; cross reactivity; density; design; drug testing; editorial; genomic profiles; improved; inhibitor/antagonist; mouse model; novel; off-patent; phosphoproteomics; pre-clinical; protein metabolite; protein protein interaction; research clinical testing; response; success; targeted treatment; therapy development; therapy resistant; tool

Although signal transduction inhibitors occasionally offer clinical benefit for cancer patients, signal flux emanating from oncogenes is often distributed through multiple pathways, potentially underlying the resistance which causes failure of most such inhibitors. Measuring signal flux through multiple pathways, in response to signal transduction inhibitors, may help uncover network inter- actions that contribute to therapeutic resistance and that are not predicted by analyzing pathways in isolation. Protein–protein interactions within signaling pathways are often elucidated by assessing the levels of relevant pathway proteins in model and tumor-derived cell lines and with various genetic and molecular perturbations. Such interactions, and the implied signaling networks, may also be elucidated via quantitative measurements of multiple pathway-related proteins within single cells. At the single-cell level, inhibitory and activating protein–protein relationships, as well as stochastic (single-cell) fluctuations, are revealed. However, most techniques for profiling signaling pathways require large numbers of cells, and bulk measurements have proven insufficient to detect secondary pathways post resistance. Single- cell immunostaining is promising, and some flow cytometry techniques are relevant, yet limited in finding possible pathways due to intracellular multiplexing limitations. We describe quantitative, multiplex assays of intracellular signaling proteins from single cancer cells using a platform called the single-cell barcode chip (SCBC). The SCBC is simple in concept: A single or defined number of cells is isolated within a microchamber that contains a sensitive antibody array specific for the capture and detection of a panel of proteins. The SCBC design permits lysis of each individual trapped cell. Intracellular staining flow cytometry can assay up to 11 phosphoproteins from single cells. Our SCBC can profile a significantly larger panel (up to 90 different phosphoproteins) with ~2500 single cells per chip for a statistically representative analysis of the sample population. This new high multi-plexed single cell phosphoproteomics analysis tool provides an analytical approach for detecting changes in signal coordination by monitoring phosphoproteins, on a much larger scale. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non- obvious drug combinations. SPECIFIC AIM 1: Develop a robust microchamber array flow cell that can be easily incorporated into larger automated workflow device for analysis of intracellular protein targets. SPECIFIC AIM 2: Double multiplexing capability of high-density barcode SCBC chip by monitoring both intracellular proteins and metabolites simultaneously. Perform single-cell 32-plex measurement for more comprehensive GBM pathway analysis. SPECIFIC AIM 3: Improve consumable to perform “flow cell” in-cartridge lysis, detection and washing capabilities for automation. Develop fully automated device workflow. SPECIFIC AIM 3b: Demonstrate utility of device in patient clinical trials as a commercial tool.

尽管信号转导抑制剂偶尔会为癌症患者提供临床益处，但信号通量散发 来自致癌基因的抗药性通常通过多种途径分布 导致大多数此类抑制剂的失败。测量通过多个途径的信号通量，以响应信号 转导抑制剂，可能有助于发现有助于治疗抗性的网络相互作用，并且 不能通过分别分析途径来预测。信号通路内的蛋白质 - 蛋白质相互作用是 通常通过评估模型和肿瘤细胞系中相关途径蛋白的水平以及 具有各种遗传和分子扰动。这样的相互作用以及隐含的信号网络可能 也可以通过对单个细胞内多个途径相关蛋白的定量测量来阐明。在 单细胞水平，抑制性和激活蛋白质 - 蛋白质关系以及随机（单细胞） 波动已被揭示。但是，大多数用于分析信号通路的技术都需要大量 事实证明，细胞和大量测量不足以检测电阻后的次级途径。单身的- 细胞免疫染色是有希望的，并且某些流式细胞仪技术是相关的，但在发现可能的情况下有限 由于细胞内多路复用限制而引起的途径。 我们描述了使用A的定量，多种测定的细胞内信号传导蛋白 称为单细胞条形码芯片（SCBC）的平台。 SCBC的概念很简单：单个或定义的数字 细胞的细胞被分离在一个微室内，该微型室包含特定于捕获的敏感抗体阵列和 检测一组蛋白质。 SCBC设计允许裂解每个单个被困的细胞。细胞内 染色流式细胞仪可以从单个细胞中测定多达11种磷蛋白。我们的SCBC可以概括 统计学上的面板明显更大的面板（最多90种不同的磷蛋白），每芯片约2500个单元 样本人群的代表分析。这种新的高多重单细胞磷酸蛋白质组学 分析工具提供了一种分析方法，用于通过监视来检测信号协调的变化 磷酸蛋白，大量更大。这种方法可以确定信号协调的可起作用变化 这是适应性抗性的基础，可以通过联合药物治疗来抑制，包括非 - 明显的药物组合。特定目标1：开发一个可以轻松的微型微培训阵列流动池 纳入较大的自动化工作流设备，以分析细胞内蛋白质靶标。具体目标 2：通过监测两个细胞内蛋白的高密度条形码SCBC芯片的双重多路复用能力 和代谢物。对更全面的GBM执行单细胞32-plex测量 途径分析。特定目的3：改善可消耗的方法来执行“流动池”裂解，检测和 自动化的洗涤功能。开发全自动设备工作流程。特定目标3B：演示 设备在患者临床试验中作为商业工具的效用。