Abstract Cancer is the second leading cause of death within the US with 90% of mortality due to metastases. The three major sites of breast cancer metastasis observed clinically are bone (78%), lung (35%) and liver (20%). Of these, the most difficult to access for in vivo, intravital optical imaging is the lung. This is because the lung is a vital organ enclosed within the thoracic cavity and undergoing perpetual motion. Until now, high resolution technologies have traditionally only been able to image fixed, sectioned tissues, or at best, ex vivo whole mount explants. In either case, the cellular dynamics and interactions are lost, and the perfusion of capillaries and oxygenation of cells is altered. By combining a previously published vacuum stabilized imaging window with a custom built two-laser multiphoton microscope specifically designed to optimize resolution and signal to noise, we developed a system capable of imaging the living breathing lung using high resolution multi-photon microscopy. Although these vacuum based window technologies have allowed imaging into the lung, these prior reported systems have focused either on pulmonary dynamics or on the surveillance of the lung by immune cells. The requirements for these studies are different than for imaging cancer growth and motility where subcellular resolution is required to image cell division, organelle content, protrusion and cellular dynamics rather than large fields of view used to capture cells with high velocities. We have attained complete tissue immobilization and exceptional subcellular resolution imaging of experimentally metastasized tumor cells. Using this biological system, we have been able to visualize not only tumor cell proliferation in vivo but also to define when and where tumor cells arrest themselves in the vasculature, and their motility while preparing for extravasation. We observe tumor cells arresting only in capillary vessels and switching from a locomotory – protrusion-high phenotype toward an invasive phenotype whose onset correlates with time of extravasation. We also are able to quantify interaction times between tumor cells and myeloid cells while arrested in the vasculature as well as once they reach the lung parenchyma. We observe that macrophage – tumor cell physical interaction at the extravascular space is extremely long lasting and without apparent effect on tumor cell viability. In vitro, this interaction time can be reduced when macrophages cannot respond to CCL3, molecule that could also control tumor cell – macrophage contact time in vivo. High-resolution imaging of early metastatic events is an extremely useful tool that will allow us not only to follow tumor cell behavior but also to study their interaction with other cell populations, including macrophages. The long-lasting interaction of tumor cells with macrophages, starting soon after tumor cell extravasation, suggests a main role for macrophages during tumor cell seeding in the lungs. Signaling molecules involved in this process can now be studied in details, including CCL3 and its downstream effectors. Citation Format: Carolina A. Rodriguez-Tirado, David Entenberg, Takanori Kitamura, John Condeelis, Jeffrey W. Pollard. In vivo subcellular resolution optical imaging in the lung reveals early metastastic proliferation, motility and tumor cell - macrophage interaction. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B62.