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

项目总结 微生物日益被认为是癌症微环境的关键组成部分， 发生在上皮屏障表面，如人类结直肠癌(CRC)。空间上的相互作用 微生物之间以及微生物和宿主组织之间的关系，是微生物区系驱动机制的基础 在结直肠癌的致癌作用，然而这些相互作用的研究仍然很少。这种知识的缺乏在很大程度上是由于 可用于研究微生物和微生物群的工具的根本局限性。微生物组研究主要 依赖于鸟枪式DNA测序，它摧毁了关于微生物和他们的空间背景的所有信息 功能相互作用，或仅限于识别少数生物体的成像方法 使用通用物种标记标签。 在这个项目中，我们将发明并应用空间分辨元基因组学(SRM)，这是一项革命性的技术 分子分析技术，能够创建数以千计的 在密集的微生物群落中有不同的细菌种类。SRM利用光学条形码和 基于光谱成像的条形码解码，能够通过细菌独特的16S识别细菌物种 核糖体RNA，甚至宿主基因表达的量化。SRM是一种灵活且廉价的技术 这增加了与现有方法相比可以识别的独特微生物种类的数量至少 两个数量级，并得到了试点数据的很好支持。我们将调查三个目标。首先，我们将提炼 一系列共同奠定SRM基础的创新技术，包括但不限于软件 用于自动化设计杂交探针、光谱成像程序和软件的自动化 图像的注释。第二，我们将设计和建造一个定制的宽波长共焦显微镜， 这将使SRM的多路复用度和速度提高一个数量级，进而 扩大开关磁阻电机的可能应用范围。第三，我们将在以下方面执行严格的SRM验证 解决人类CRC中非常及时的问题的实验。促癌物质的功能作用 结直肠癌中的微生物，作为结直肠癌发展早期事件的生物膜形成的作用，以及 在结直肠癌肿瘤中存在持久的微生物群，都是最近的里程碑式的发现，我们将 能够利用SRM的特点，以前所未有的空间和系统发育分辨率进行研究。 SRM不仅能够调查谁在那里，而且还可以调查谁在谁旁边，谁在什么旁边，因此 为研究微生物区系在疾病发生和发展中的功能作用提供了一种强有力的新手段 CRC是一种疾病，每年在美国造成5万多人死亡。

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.