Metastases are responsible for ~90% of human cancer-related deaths, yet our understanding of the stages of metastasis and the regulating features that drive secondary, tertiary etc. tumors is sorely lacking. In particular, the early niche surrounding disseminated cells appears critical for survival, dormancy, and/or successful development of progressing micrometastases. Indeed, not infrequently, breast cancer patients succumb to recurrent or metastatic disease years to decades after treatment that had rendered the disease undetectable. In fact, greater than 67% of breast cancer deaths occur beyond the 5-year survival window and some patients present with recurrence after more than a decade of being “disease-free”. Yet, our understanding of the intrinsic and environmental factors that initiate and maintain programs of dormancy versus metastatic progression remains extremely limited. Here, we seek to elucidate fundamental physical and molecular mechanisms that govern cell fate in ectopic sites. To date, numerous technical hurdles have impeded our ability to study the genetic and microenvironmental drivers of dormancy and recurrence, particularly in vivo where these events are rare and not easily controlled. Indeed, in vitro platforms that permit control of the cell microenvironment and permit cell isolation based on cell state (i.e. dormant vs. progressing) are required to identify and characterize molecular mechanisms governing these behaviors that can be validated and targeted in vivo. To address these significant challenges, this proposal leverages expertise in cancer biology and cancer bioengineering through numerous innovative technologies (e.g. microfluidic generation of metastatic niches, advanced optical imaging, cutting edge cell engineering with CRISPR technologies etc.) that uniquely enable us to drastically improve our understanding of how dormancy is regulated in vivo. Here, we hypothesize that dormancy or colony proliferation in metastatic niches is dictated by lock-and-key behavior between cancer cells with specific genetic and epigenetic signaling and the initial and evolving properties of the ectopic microenvironment. Our hypotheses will be tested in the following Specific Aims: (1) Define specific extracellular matrix compositions that drive survival, dormancy, or colonization using high-throughput micro- engineering metastatic environments (MEME) technology; (2) Dissect the molecular mechanisms governing survival, dormancy, or colonization in defined metastatic niche microenvironments; (3) Define the specific influence of bone marrow-derived and tissue-specific resident macrophages in carcinoma cell survival, dormancy, or colonization. Through these efforts we will dissect the mechanistic drivers of disseminated tumor cell dormancy or proliferation, which will elucidate therapeutic targets to prevent dormant tumor cells from evading therapy. Additionally, these studies will reveal therapeutic targets to kill dormant cells directl...