PROJECT SUMMARY Metastasis is a defining feature of advanced cancer, often representing a transition from curable to incurable disease. It is largely driven by stochastic processes, and remains challenging to predict when it will occur. We undertake an investigation into the mechanisms driving the increased metastatic potential of circulating tumor cell (CTC) clusters through a combination of systems biology, in vivo experiments in zebrafish, and theoretical ecology. Melanoma, the most lethal of skin cancers, shows a particularly stark difference between the outcomes of patients with local versus metastatic disease. CTC clusters have been isolated from the blood of patients with melanoma, among other cancer types, and portend a poor clinical prognosis. CTC clusters are important in metastases, but despite their importance many key mechanisms underlying their formation, increased metastatic capacity, and potential for therapeutic targeting remain largely unexplored, particularly in melanoma. Our study takes advantage of the zebrafish model of metastatic melanoma, including zebrafish melanoma cell lines capable of transplantation into transparent Casper zebrafish, providing a powerful tool for investigating the cellular processes driving the increased metastatic potential of CTC clusters. For this fellowship, we will address two properties of CTC clusters and how they relate to metastatic fitness. (Aim 1) We hypothesize that the trade-off between group size and number?integral to ecological dispersal?is key in metastasis formation by CTC clusters. We will test this hypothesis by applying quantitative statistical analysis to melanoma clusters of varying size transplanted into zebrafish, characterizing the metastatic fitness landscape of melanoma CTC clusters. We will then introduce genetic perturbations specifically targeting hypothesized mechanisms of cluster cooperation in order to elucidate the mechanisms underlying CTC cluster fitness. (Aim 2) We hypothesize that high intra-cluster diversity promotes overall metastatic fitness despite the presence of some cells with lower individual fitness. We will test this hypothesis by engineering clusters with melanoma-specific forms of genetic heterogeneity. We will apply quantitative statistical analysis comparing high- and low-diversity clusters transplanted into zebrafish, evaluating the role of compositional heterogeneity in CTC cluster metastatic fitness. These two approaches, combined with validation in mammalian models, will generate new insights into the size and compositional trade-offs underlying CTC cluster fitness that can inform the development of new diagnostic, prognostic and therapeutic strategies in melanoma and more broadly.