Chemokines and their G-protein coupled receptors (GPCRs) are key mediators of intercellular signaling between cancer cells and multiple stromal cell types. Cancer cell proliferation, local invasion, systemic metastasis, recruitment of immune cells and angiogenesis all are regulated by spatial and temporal dynamics of chemokines. The central role of chemokines in tumor biology has made them an attractive target for therapies designed to inhibit or enhance chemokine signaling. However, much of the complex biochemistry of chemokines remains to be discovered, due to the technical challenges of tracing their presence and function in living systems. Chemokines are effective at signaling at very low levels, and their spatial distribution is complex due to their ability to bind to cell surfaces and extracellular matrix. Furthermore, internalization of chemokines by GPCRs and scavenger receptors shape local chemokine gradients by sequestering and degrading chemokines. Experimental evidence and computational modeling by our lab show that short-range, steep local gradients of chemokine CXCL12 over distances of a single cell most effectively drive signaling and chemotaxis through receptor CXCR4. To enable transformative studies of chemokines and ultimately advance applications of drugs targeting chemokines in cancer, we propose to develop a new suite of quantitative molecular imaging tools to measure the spatial and temporal dynamics of chemokine distribution, association, and signaling in hundreds of single cells. These tools will allow us to measure cell signaling patterns during chemotaxis and identify potential mechanisms underlying intercellular heterogeneity in responses to chemokines. We will 1) Generate an integrated, multiplexed bioluminescence and fluorescence microscopy toolbox for single-cell imaging of extracellular and intracellular steps in chemokine signaling; and 2) Test our chemokine imaging technology as a generalizable, quantitative approach applicable to patient-derived cells. We will uniquely combine dynamic, single-cell bioluminescence and fluorescence microscopies technologies to measure numerous molecular events cells use to convert chemokine inputs into signaling outputs and chemotaxis. We will couple our multiplexed imaging readouts with advanced image processing methods to generate high-dimensional quantitative data sets needed for basic studies of chemokines and integration with other large-scale data. Our innovative imaging tools will allow an unprecedented view of chemokine gradients and heterogeneity of cellular responses in complex environments. This technology will transform investigations of chemokines in cancer and advance ongoing efforts to target chemokines for therapy.