Area A: In-Depth Proteome Mapping of the Tumor Microenvironment with Single- Cell Resolution

项目摘要/摘要 开发有效的癌症治疗方法需要对肿瘤有深入的分子理解。 通过先进的组学技术和空间分辨率测量实现异质性。不幸的是，有一些 在敏感性方面，现有能力存在很大差距。例如，至少有数千个 细胞是对生物样本中的蛋白质进行深入分析所必需的。我们最近开发了一种 突破性技术，称为纳米POTS(痕量样品一锅纳米液滴制备)， 当与超灵敏的液-质联用(LC-MS)联用时，能够有效地分析 只有10个哺乳动物细胞，覆盖了3000种已识别的蛋白质。我们假设纳米POTS Platform将成为一项能够表征肿瘤异质性的分子基础的技术，通过 创建人类肿瘤的3D蛋白质组图。我们研究的总体目标是扩展和验证这一点 分析平台，支持高通量的单细胞分辨率测量(每天100个样本)和 应用该平台创建人体肿瘤的三维蛋白质组图。目标1的研究将侧重于优化和 验证超灵敏的纳米POTS蛋白质组工作流程，以实现≥3,000蛋白质的强大蛋白质组图谱 通过流式细胞仪和激光捕获显微切割(LCM)获得的单个人类细胞分组。目标 2将评估和比较将测量吞吐量提高到约100的两种互补技术 细胞数/天，对蛋白质组覆盖率的影响最小。我们将确定是否通过应用程序进行多路复用 用于混合分析的等压标记，或快速LC与超高分辨率离子迁移率光谱联用-MS 以所需的吞吐量为单细胞蛋白质组学提供最大的覆盖率、定量和重复性。 在目标3中，我们将应用优化的平台来创建人类胰腺导管的深入蛋白质组图谱 单细胞分辨率的腺癌(PDA)。在对感兴趣的特定细胞进行初步靶向研究后 在切片的组织中，我们将使用冷冻切割、LCM和优化的纳米POTS的组合 分析肿瘤微环境中单个细胞的平台。这些深度、空间上的3D重建 解析的蛋白质组学分析将在单细胞分辨率下提供第一个全球蛋白质组肿瘤图谱。这 该项目不仅将建立一种创新的测量能力，将广泛推进癌症研究， 但也将提供与PDA病理相关的细胞异质性的独特分子洞察力。这个 由此产生的平台将通过出版和商业化相结合的方式进行传播。

PROJECT SUMMARY/ABSTRACT The development of effective therapies for cancer requires a deep molecular understanding of tumor heterogeneity by advanced omics technologies with spatially resolved measurements. Unfortunately, there are substantial gaps in existing capabilities in terms of sensitivity. For example, a minimum of many thousands of cells is required for in-depth profiling of proteins in a biological sample. We have recently developed a breakthrough technology, termed nanoPOTS (Nanodroplet Preparation in One pot for Trace Samples) which, when coupled to ultrasensitive liquid chromatography-mass spectrometry (LC-MS), enables effective analysis of as few as 10 mammalian cells with a coverage of >3000 identified proteins. We hypothesize that the nanoPOTS platform will be an enabling technology to characterize the molecular underpinnings of tumor heterogeneity by creating 3D proteome maps of human tumors. The overall objective of our study is to extend and validate this analysis platform to enable single-cell resolution measurements at high throughput (>100 samples per day) and apply the platform to create 3D proteome maps of human tumors. Studies in Aim 1 will focus on optimizing and validating the ultrasensitive nanoPOTS proteomic workflow to enable robust proteome profiling of ≥3,000 protein groups from single human cells obtained by both flow cytometry and laser capture microdissection (LCM). Aim 2 will evaluate and compare two complementary technologies for increasing measurement throughput to ~100 cells/day with minimal impact on proteome coverage. We will determine whether multiplexing through application of isobaric labels for pooled analysis, or rapid LC coupled with ultrahigh resolution ion mobility spectrometry-MS provides greatest coverage, quantitation and reproducibility for single cell proteomics at the desired throughput. In Aim 3, we will apply the optimized platform to create in-depth proteome maps for human pancreatic ductal adenocarcinomas (PDAs) at single cell resolution. Following initial targeted studies of specific cells of interest within sectioned tissues, we will use a combination of cryosectioning, LCM and the optimized nanoPOTS platform to analyze single cells within the tumor microenvironment. 3D reconstruction of these in-depth, spatially resolved proteomic analyses will provide the first global proteomic tumor maps at single-cell resolution. This project will not only establish an innovative measurement capability that will broadly advance cancer research, but will also provide unique molecular insights into cellular heterogeneity relevant to PDA pathology. The resulting platform will be disseminated through a combination of publication and commercialization.