It is unclear how a melanocyte transitions from a premalignant nevus to an invasive tumor cell; even less studied is the direct examination of this transition to motility in vivo. Zebrafish allows for high resolution imaging of tumor cell motility and zebrafish have melanocyte distribution that is similar to humans. Additionally, the progression of premalignant nevi to an early melanoma and later to an aggressive tumor is conserved between fish and humans. Thus, I will use human-in-fish xenotransplantation approaches as well as transgenic zebrafish strains to directly visualize melanoma cell motility. I will focus on how tumor associated macrophages facilitate specific steps of the metastatic cascade, specifically identifying the cell biological mechanisms regulating invadopodium formation by taking advantage of the amenability to high resolution imaging of zebrafish. I will then validate our key findings in mouse models with imaging window and fate mapping with photoconversion techniques. Importantly, I will discover how inflammatory response pathways regulate melanoma metastasis to better understand how immune cells and melanoma cells communicate in the microenvironment. The candidate for this career development award is a postdoctoral fellow, and the research proposed in this grant application will be conducted under the primary mentorship of Dr. Cecilia Moens, and under the co- mentorship of Dr. John Condeelis (Albert Einstein College of Medicine, NY) during the K99 phase of the award. The Moens lab at the Fred Hutchinson Cancer Research Center is an ideal environment for these studies due to the expertise of reverse genetics approaches, cell signaling, and live cell imaging strategies in zebrafish embryos. Dr. Condeelis is a world leader in the tumor microenvironment, and has expertise in both in vitro cytoskeletal regulation of tumor cell invasion, and intravital imaging of tumor cells in murine models. I will benefit immensely from the mentorship of Drs. Moens and Condeelis, and will bridge the expertise of both mentors toward understanding the dynamic cell-cell interactions regulating melanoma progression in fish and mouse models. The Fred Hutchinson Cancer Research Center is a world renowned cancer institute, and provides a wealth of cancer expertise: I have established collaborations with melanoma experts and physician- scientists within the Fred Hutchinson Cancer Research Center, at nearby University of Washington campuses, and with a melanoma clinical expert at MD Andersen. I am committed to a career as an independent investigator at an academic institution studying cancer cell biology, specifically studying the dynamic cytoskeletal mechanisms regulating tumor cell movements during metastasis. My current postdoctoral fellowship ends in August 2014. As I only been in Dr. Moens' lab for less than a year, the K99 award will give me enough time in her lab to develop this model further for me to set up the foundation for ongoing projects in my independent lab. My immediate goal is to define the tumor stromal signaling mechanisms regulating melanoma cell motility during metastasis. My long-term goal is to discover new markers and therapeutic targets for melanoma treatment.