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Background Understanding intricate cellular interactions within the tumor microenvironment (TME) is essential for understanding disease progression and advancing cancer treatments, such as immunotherapy. The cancer ecosystem is complex, composed of cells with dysregulated metabolism and signaling pathways, contributing to tumor heterogeneity, growth and differential treatment response. Targeting metabolic and signaling pathways represents a growing strategy to enhance immunotherapy treatments, often in combination with standard of care treatments. Imaging Mass Cytometry™ (IMC™) is a spatial biology imaging technique utilizing CyTOF™ technology, which enables deep characterization of 40-plus markers in TME simultaneously. IMC offers scalable and high-throughput acquisition while generating high-quality data with true dynamic range of signal without amplification or fluorescence-based limitations such as spectral overlap and autofluorescence.Methods IMC was used to explore the TME and interrogate key pathways in metabolic reprogramming and signaling by utilizing antibody panels that integrate markers from the Human Immuno-Oncology IMC Panel (201509) combined with the Human Metabolism IMC Panel (201521) or Human Cell Signaling IMC Panels (201522). This enabled investigation of energy production, cellular homeostasis and mitogenic signaling pathways in human breast cancer samples. To phenotype immune and tumor cells and assess the activation status of immune cells, we used Preview Mode to acquire the whole tissue, followed by higher-resolution imaging of regions of interest using Cell Mode or whole tissue sections using Tissue Mode ( figure 1).Results IMC analysis elucidated the spatial organization and metabolic profile of cells in breast cancer ( figure 2A). Heterogeneity within tumor is highlighted by differential utilization of energy sources across the tumor. Immune cells primarily infiltrated tumor areas utilizing fatty acid oxidation, while tumor cells using anaerobic metabolism or aerobic respiration were classified as immune deserts. Differences in signaling pathways were also observed in these tumor cell populations (figure 2B). Elevated glycolysis and mTOR pathway activation suggested adaptations to hypoxia and anabolic growth. Wnt signaling and PTEN expression were mainly localized in tumor cells, whereas MAP kinase signaling was localized in stroma. Unsupervised pixel clustering and hierarchical clustering using MCD™ SmartViewer highlighted metabolic activity and activation of signaling pathways within tumor regions.Conclusions Comprehensive spatial profiling using IMC technology illuminates the heterogeneity of metabolism and signaling pathways in tumors. IMC allows us to detect many clinically relevant targets simultaneously with intact spatial resolution. This is crucial for developing future prognostic assessments and guiding more effective, personalized cancer therapies.For Research Use Only. Not for use in diagnostic procedures.Abstract 1219 Figure 1Whole tissue evaluation of metabolic activity in human breast cancer. Whole slide Tissue Mode image, human breast cancer stained with IMC panels. A) Multi-color IMC image, metabolic activity in breast cancer. B) Shows differential activation of signaling cascades in breast cancer cellsAbstract 1219 Figure 2High-resolution evaluation of metabolic and cell signaling activity in human breast cancer. Cell Mode IMC images from 3 separate regions of interest from human breast cancer stained with A) Human Metabolism IMC Panel or B) Human Cell Signaling IMC Panels