Single-cell Phosphoprotein Assay to Evaluate Brain Tumor Therapeutic Resistance

虽然信号转导抑制剂偶尔为癌症患者提供临床益处，但信号通量的释放可能会导致癌症患者的死亡。 通常通过多种途径分布，可能是耐药性的基础， 导致大多数此类抑制剂失效。响应于信号，测量通过多个路径的信号通量 转导抑制剂，可能有助于揭示网络的相互作用，有助于治疗耐药性， 是无法通过孤立地分析途径来预测的。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， 各种遗传和分子干扰这样的交互以及隐含的信令网络可以 也可以通过单细胞内多种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，已经显露出来。然而，大多数用于分析信号通路的技术需要大量的 细胞和整体测量已证明不足以检测耐药后的次级途径。单身- 细胞免疫染色是有希望的，一些流式细胞术技术是相关的，但在发现可能的 由于细胞内多路复用的限制。 我们描述了使用流式细胞仪对单个癌细胞的细胞内信号蛋白进行定量、多重测定 一种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 在含有对捕获特异性的敏感抗体阵列的微室内分离细胞， 检测一组蛋白质。SCBC设计允许每个单独捕获的细胞裂解。细胞内 染色流式细胞术可以测定来自单细胞的多达11种磷蛋白。我们的SCBC可以分析 显著更大的面板（多达90种不同的磷蛋白），每个芯片约2500个单细胞，用于统计学分析。 对样本人群的代表性分析。这种新的高多重单细胞磷酸化蛋白质组学 分析工具提供了一种分析方法，用于通过监控来检测信号协调中的变化 磷蛋白，在更大的范围内。该方法可以识别信号协调中的可操作的改变 这是适应性耐药性的基础，可以通过联合药物治疗来抑制，包括非 明显的药物组合具体目标1：开发一种坚固的微室阵列流动池， 将其并入更大的自动化工作流程装置中用于分析细胞内蛋白质靶。具体目标 2：通过监测两种细胞内蛋白质，高密度条形码SCBC芯片的双重多重能力 和代谢物。执行单细胞32重测量，实现更全面的GBM 路径分析具体目标3：改进耗材，以执行“流动池”盒内裂解、检测和 自动化的洗涤能力。开发完全自动化的设备工作流程。具体目标3b：展示 器械在患者临床试验中作为商业工具的实用性。

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.