Project Summary/Abstract “Novel approaches to study the intersection of cellular heterogeneity and tissue microanatomy” Positioning of cells in tissues is intimately linked to major cellular- and organ-level processes. Appropriate localization promotes exposure of cells to specific microenvironments and stimuli that define their differentiation states and functions. Cellular positioning also determines local tissue microanatomy, as well as the global macro-architecture, allowing for proper organ function and physiology. Thus, understanding the links among cellular positioning, heterogeneity and global tissue architecture are critical for biomedical research. However, current tools for understanding such relationships are limiting. Here, we will develop novel approaches to study cells using currently existing high-content tools, but importantly while also retaining information on the precise positioning of cells within tissues (Aim 1). This will allow us to interrogate how cellular heterogeneity and function is influenced by the exposure to distinct tissue microenvironments and localized stimuli. Further, we will generate novel computational approaches to understand how cellular positioning defines global tissue architecture (Aim 2). For this, we will develop a robust analytical methodology based on machine learning algorithms to study how complex patterns of cellular spatial positioning influences tissue organization and structure. These approaches will be broadly applicable across diverse disciplines of biology for both murine and human (or other species) studies, and will be highly pertinent in clinical settings, such as for cancer diagnostics. In summary, the proposed studies are significant, as they will provide a new toolbox for studying cellular and organ physiology based on cellular tissue positioning, as well as will lead to the development of new diagnostic tools for clinical medicine.