The High-Content Imaging & Analysis Core provides the three SASCO Center projects with experimental and computational resources to analyze different biological samples by multiplexed fluorescence microscopy and to extract quantitative data from microscopy-based images. The core will develop, validate, and apply highly multiplexed (iterative) imaging approaches, integrate subcellular imaging data collected across multiple rounds of iterative imaging, and build customized tools for image analysis, feature recognition, and extraction. The generated multi-parametric datasets will address specific needs of each project, enabling model-experiment iterations involving diverse cancer drivers, signaling pathways, and subcellular organelles in multiple biological contexts. Aim #1 supports the development, validation, and application of highly multiplexed immunofluorescence staining and imaging procedures to measure molecular features at cellular, subcellular, and organelle resolution. These procedures will enable the molecular analysis of mitotic signaling pathways and recruitment patterns in metaphase chromosomes derived from breast cancer organoids (Project 1), mitochondrial morphology, cell signaling and metabolic enzymes in colorectal cancer cells and tumors (Project 2), and post-translational modifications of key EGFR signaling molecules, protein-protein interactions, and their subcellular localization in glioblastoma cells (Project 3). Aim #2 customizes tools for quantitative image analysis, including image alignment and tracking, segmentation of cells and subcellular structures, as well as feature extraction and recognition. The High-Content Imaging & Analysis Core provides all three projects the necessary expertise and tools for quantifying information about cells, cultures, and tissues from image data to support hypothesis generation and model testing related to the overall hypotheses of the SASCO Center.