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

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Molecular & Cellular Analysis Technologies
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PROJECT SUMMARYMicrobes are increasingly recognized as a critical component of the tumor microenvironment of cancers thatarise at epithelial barrier surfaces, such as human colorectal cancer (CRC). Spatial interactions betweenmicrobes and between microbes and host tissues, are fundamental to the mechanisms by which microbiota drivecarcinogenesis in CRC, yet these interactions remain poorly studied. This lack of knowledge is in large part dueto fundamental limitations of the tools available to study microbes and microbiomes. Microbiome studies primarilyrely on shotgun DNA sequencing, which destroys all information about the spatial context of microbes and theirfunctional interactions, or imaging methodologies that are limited to identifying a small number of organismsusing general species marker tags.In this project, we will invent and apply spatially resolved metagenomics (SRM), a revolutionarymolecular analysis technology that enables to create micro-scale maps of the locations of thousands ofdifferent bacterial species in dense microbial communities. SRM takes advantage of optical barcoding andspectral imaging-based barcode decoding, enabling the identification of bacterial species by their unique 16Sribosomal RNAs, and even quantification of host gene expression. SRM is a flexible and inexpensive technologythat increases the number of unique microbial species that can be identified over existing methods by at leasttwo orders of magnitude and is well supported by pilot data. We will investigate three aims. First, we will refinea host of innovative technologies that together lay the foundation for SRM, including but not limited to softwarefor the automated design of hybridization probes, spectral imaging procedures and software for the automatedannotation 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 willimprove the range of possible applications of SRM. Third, we will perform rigorous validation of SRM inexperiments that address highly timely questions in human CRC. The functional roles of cancer-promotingmicrobes in CRC, the role for biofilm formation as a consequential, early event in CRC development, and thepresence of a persistent microbiome in CRC tumors, are all very recent landmark discoveries, that we will beable 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 thereforeprovide a powerful, novel means to study the functional role of microbiota in the initiation and progression ofCRC, a disease that accounts for more than 50,000 deaths annually in the US.