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.