Project Summary Metastasis is responsible for 90% of cancer deaths, yet there is a severe lack of effective anti-metastatic targeted therapeutics. The major mode of metastasis for many cancers is collective invasion, where heterogeneous packs of cells travel together. Our lab has previously shown that these collective packs include specialized “leader” and “follower” cells, and we created the first purified leader and follower cell cultures utilizing the SaGA technique (Spatiotemporal Genomic and Cellular Analysis), which leverages a combination of live-cell confocal microscopy and fluorescence-activated cell sorting. Using these novel cell lines, we showed that cooperativity between leaders and followers is crucial for successful collective invasion, with leaders promoting the invasion of followers, and followers promoting the survival and proliferation of leaders. Importantly, we now report the first known panel of leader- and follower-specific gene mutations, identified via RNA-seq analysis. Notably, leader cells exclusively harbor a specific missense mutation in the ARP3 protein, a crucial component of the Arp2/3 complex that drives cell motility by promoting actin polymerization. Furthermore, leader cells display increased lamellipodia and more highly organized actin filaments compared to follower cells, suggesting increased Arp2/3 activity in leaders. Understanding how this ARP3 mutation drives the emergence and invasive biology of leader cells could be a crucial step toward identifying therapeutic targets to inhibit metastasis in cancer patients. We will therefore test the overall hypothesis that ARP3 mutations lead to increased Arp2/3 activity in rare cancer cells, thereby driving the unique leader cell phenotype and promoting collective invasion. To probe this hypothesis, we will determine 1) the molecular mechanism by which this ARP3 mutation drives the emergence and invasive biology of leader cells in vitro, and 2) if this ARP3 mutation drives lung cancer collective invasion and metastasis in vivo. In Aim 1 we will create stable cell lines expressing mutant ARP3, and subsequently perform 3-D spheroid invasion assays and co-immunoprecipitation experiments to determine how this mutation affects protein ubiquitination, Arp2/3 activation, and overall emergence of the leader cell phenotype. In Aim 2, we will use a mouse orthotopic lung cancer xenograft model and subsequent pathological analysis to determine the effects of this ARP3 point mutation on collective invasion and metastasis in vivo. These studies will provide critical insight into the biological events that drive collective invasion, which could ultimately set a new paradigm for the treatment and prevention of metastasis in patients.