Project Summary Studying tumors in a spatial context will advance our understanding of intratumoral heterogeneity and how the complex interactions between cancer and surrounding non-cancer cells results in the growth of malignant subclones, which promises to address outstanding questions in cancer biology and improve the diagnosis and treatment of specific cancer subtypes. In both basic and translational cancer studies, the majority of biopsies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples, and a growing number of FFPE specimens are newly archived every year. Accordingly, spatial omics profiling in archived FFPE tissue can be invaluable for cancer research and potential novel biomarker or molecular regulators discovery. In this project, we propose to develop a first-of-its-kind technology for spatially resolved co-mapping of epigenome, transcriptome, and proteins in FFPE tissues at the cell level. Specifically, we will (Aim 1) develop spatial epigenome sequencing to measure not only gene expression but also epigenetic underpinning of cell type and state directly in FFPE tissues, (Aim 2) develop a novel deterministic barcoding strategy for joint profiling of accessible chromatin or histone modifications, mRNAs, and proteins in the same FFPE tissue section. This novel technology addresses the lack of capability for spatial multi-omics that can be integrated, scaled, and applied to FFPE tissue mapping. As FFPE samples are widely available and represent the most abundant format of archivable clinical tumor tissue samples, we envision that this work will open up new opportunities to revisit the huge resource of clinical tissue banks to study the mechanisms of pathophysiology and to discover new targets for diagnosis and treatment of human diseases.