Spatially Resolved Metagenomics to Explore Tumor-Microbiome Interactions in Human Colorectal Cancer

项目摘要 微生物越来越被认为是癌症肿瘤微环境的关键组成部分， 出现在上皮屏障表面，如人结肠直肠癌（CRC）。空间相互作用 微生物之间以及微生物与宿主组织之间的相互作用，是微生物群驱动 CRC的致癌作用，但这些相互作用仍然缺乏研究。这种知识的缺乏在很大程度上是由于 研究微生物和微生物组的工具的基本局限性。微生物研究主要 依赖于鸟枪DNA测序，这破坏了关于微生物及其空间环境的所有信息。 功能相互作用，或成像方法，仅限于识别少数生物体 使用一般的物种标记标签。 在这个项目中，我们将发明和应用空间分辨宏基因组学（SRM），一个革命性的 分子分析技术，能够创建数千个位置的微尺度地图， 在密集的微生物群落中有不同的细菌种类。SRM利用光学条形码， 基于光谱成像的条形码解码，能够通过其独特的16S 核糖体RNA，甚至宿主基因表达的定量。SRM是一种灵活且廉价的技术 这增加了独特的微生物物种的数量，可以通过至少 两个数量级，并得到试验数据的充分支持。我们将探讨三个目标。首先，我们将细化 一系列创新技术共同奠定了SRM的基础，包括但不限于软件 用于杂交探针的自动化设计、光谱成像程序和用于自动化 图像的注释。第二，我们将设计和构建一个定制的宽波长共聚焦显微镜， 这将使SRM的多路复用性和速度提高一个数量级，这反过来又将 扩大SRM的可能应用范围。第三，我们将对SRM进行严格的验证， 这些实验解决了人类CRC中高度及时的问题。促癌的功能作用 微生物在CRC中的作用，生物膜形成作为CRC发展中的一个重要的早期事件， CRC肿瘤中存在持久性微生物组，都是最近的里程碑式发现，我们将 能够研究前所未有的空间和系统发育的分辨率，利用SRM的功能。 SRM不仅可以调查谁在那里，还可以调查谁在谁旁边，谁在什么旁边，因此 提供了一个强大的，新的手段来研究微生物群在启动和发展的功能作用， CRC是一种在美国每年导致5万多人死亡的疾病。

PROJECT SUMMARY Microbes are increasingly recognized as a critical component of the tumor microenvironment of cancers that arise at epithelial barrier surfaces, such as human colorectal cancer (CRC). Spatial interactions between microbes and between microbes and host tissues, are fundamental to the mechanisms by which microbiota drive carcinogenesis in CRC, yet these interactions remain poorly studied. This lack of knowledge is in large part due to fundamental limitations of the tools available to study microbes and microbiomes. Microbiome studies primarily rely on shotgun DNA sequencing, which destroys all information about the spatial context of microbes and their functional interactions, or imaging methodologies that are limited to identifying a small number of organisms using general species marker tags. In this project, we will invent and apply spatially resolved metagenomics (SRM), a revolutionary molecular analysis technology that enables to create micro-scale maps of the locations of thousands of different bacterial species in dense microbial communities. SRM takes advantage of optical barcoding and spectral imaging-based barcode decoding, enabling the identification of bacterial species by their unique 16S ribosomal RNAs, and even quantification of host gene expression. SRM is a flexible and inexpensive technology that increases the number of unique microbial species that can be identified over existing methods by at least two orders of magnitude and is well supported by pilot data. We will investigate three aims. First, we will refine a host of innovative technologies that together lay the foundation for SRM, including but not limited to software for the automated design of hybridization probes, spectral imaging procedures and software for the automated annotation of images. Second, we will design and construct a custom broad-wavelength confocal microscope, that will improve the multiplexity, and speed of SRM by an additional order of magnitude, which in turn will improve the range of possible applications of SRM. Third, we will perform rigorous validation of SRM in experiments that address highly timely questions in human CRC. The functional roles of cancer-promoting microbes in CRC, the role for biofilm formation as a consequential, early event in CRC development, and the presence of a persistent microbiome in CRC tumors, are all very recent landmark discoveries, that we will be able to study with unprecedented spatial and phylogenetic resolution by taking advantage of the features SRM. SRM enables to survey not only who is there, but also who is next to who and who is next to what, and therefore provide a powerful, novel means to study the functional role of microbiota in the initiation and progression of CRC, a disease that accounts for more than 50,000 deaths annually in the US.