Single-cell Phosphoprotein Assay to Evaluate Brain Tumor Therapeutic Resistance

尽管信号转导抑制剂偶尔会对癌症患者产生临床益处，但信号通量 通常通过多条途径分布，可能是耐药的潜在基础 导致大多数此类抑制剂失效。测量通过多个路径的信号流量，以响应信号 转导抑制剂，可能有助于揭示导致治疗耐药的网络相互作用 并不是通过孤立地分析通路来预测的。信号通路中的蛋白质-蛋白质相互作用是 通常通过评估模型和肿瘤衍生细胞系中相关途径蛋白的水平来阐明 有各种遗传和分子上的扰动。这种交互以及隐含的信令网络可以 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、抑制和激活蛋白质-蛋白质关系以及随机(单细胞) 波动，被揭示出来。然而，大多数分析信号通路的技术都需要大量的 细胞，和大量测量已被证明不足以检测抗药后的次级通路。单人- 细胞免疫染色是有希望的，一些流式细胞仪技术是相关的，但在寻找可能的可能性方面存在局限性。 由于细胞内多路传输的限制而导致的通路。 我们描述了来自单个癌细胞的细胞内信号蛋白的定量、多重分析。 该平台称为单细胞条形码芯片(SCBC)。SCBC在概念上很简单：单个或定义的数字 的细胞被隔离在一个微室中，该微室包含专用于捕获和 检测一组蛋白质。SCBC的设计允许裂解每一个捕获的细胞。细胞内 染色流式细胞术可以从单个细胞中检测多达11个磷酸蛋白。我们的SCBC可以配置一个 更大的面板(多达90种不同的磷蛋白)，每个芯片约有2500个单细胞，从统计上讲 样本人群的代表性分析。这种新的高度复合的单细胞磷酸蛋白质组学 分析工具提供了一种分析方法，用于通过监控检测信号协调的变化 磷蛋白，在更大的范围内。这种方法可以识别信号协调中的可操作改变 这是适应性耐药的基础，可以通过联合药物治疗来抑制，包括非 明显的药物组合。具体目标1：研制一种坚固耐用的微腔阵列流动池 集成到更大的自动化工作流设备中，用于分析细胞内蛋白质目标。特定目标 2：高密度条形码SCBC芯片通过监测两种细胞内蛋白质的双路复用能力 和代谢物同时存在。执行单单元32路测量，以实现更全面的GBM 通径分析。具体目标3：改进消耗品，以执行筒内的“流动单元”裂解、检测和 用于自动化的洗涤功能。开发完全自动化的设备工作流程。特定目标3b：演示 作为商业工具的设备在患者临床试验中的应用。

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.