Project Summary Tumors reside within a complex multicellular ecosystem comprised of malignant and non- malignant cells, where interacting cells and molecules are organized in space and time. The diversity of these cells and their interactions affect cancer progression and drug response and resistance, and present opportunities for more precise diagnostics and therapeutics. In this proposal, we build upon our recent invention of Slide-tags, a single-cell spatial genomics technology, to enable facile, spatial profiling of human tumors. We will apply Slide-tags across a broad range of tumor specimens to enable future adoption with minimal training and equipment. We will extend Slide-tags to measure not only transcription, but also epigenetic states and genome variation within individual cells in the tumor microenvironment. Our novel computational pipelines will allow the seamless integration of molecular, cellular and histological understanding in tumors: they will enable the spatial localization of cell types within complex tumor environments, the identification of spatially varying gene expression patterns driven by pathology, as well as the organization of cellular niches. The innovations from this project will yield a robust, multiomic spatial platform for the routine profiling of human tumors at scale. Applying this platform will revolutionize our ability to discover changes in tumor spatial and molecular organization during disease progression and treatment, provide new biomarkers for diagnostics and prognostics, and highlight new therapeutic avenues.