PROJECT SUMMARY Metastasis is the most dangerous stage of cancer, responsible for the vast majority of breast cancer-related deaths. Currently, we have no treatments that target metastasis; therefore, it is critical to understand metastatic progression at the molecular, cellular, and tissue levels to develop therapeutic strategies against this disease. This proposal investigates how the lymph node (LN) regulates tumor dormancy, a stage of quiescence responsible for delayed relapse which affects ~20% of metastatic breast cancer patients. Using this latency window as an opportunity for intervention requires an understanding of site-specific dormancy regulation. Although the LN is the most common site of metastasis, very little is known about how it sustains dormant DTCs and or what triggers reawakening. I hypothesize that stable LN endothelia deposit quiescence factors which are lost when the stable niche is disturbed during inflammation. This proposal combines animal studies, novel models of the LN microvascular niche (MVN), and intravital imaging to identify LN molecules that contribute to DTC quiescence and reawakening. ECM proteomics identified candidate quiescent factors including HSPG4, LOXL1, and TINAGL1 which will be validated in the LN MVNs. Candidates will be functionally tested in the LN MVNs and in vivo. Further, acute inflammation will be used to trigger dormant DTC reawakening in mouse models and by intravital imaging to see whether DTCs “wake up” when their niche is activated. These studies will provide the first mechanistic understanding of how the LN promotes tumor dormancy and demonstrate whether destabilizing the niche through inflammation is sufficient to wake dormant tumor cells up. In the K00 phase, I propose to investigate route of dissemination as a selective force that enriches metastasis-competent populations of tumor cells. Lymphatics are not only less hostile than blood circulation, but present an opportunity for immune escape as LN LEC induce peripheral tolerance in the T cell repertoire. LN LEC-mediated deletion of tumor-specific T cells27 may grant a survival advantage to lymph- exposed DTCs, priming them for immune escape. Metastatic advantage of lymphatic dissemination will be investigated using lymphatic-Cre transgenic mouse models to track dissemination route and metastatic outcome, T cell receptor sequencing to elucidate the systemic effect of local deletion on the peripheral repertoire, and molecular barcoding to track clonality and distribution of DTCs relative to their dissemination route. Understanding how dissemination influences metastasis will help identify which tumor cells are most likely to metastasize for elimination. The proposed aims will shed light on how the LN and lymphatic dissemination instruct metastasis, revealing actionable targets against tumor dormancy and metastatic fitness, provide invaluable experience studying the complex interactions between metastasis and immunity.