PROJECT 3 SUMMARY Cancers commonly spread from their primary site and colonize regional lymph nodes (LNs), representing a hallmark of progression and a key parameter for staging. The prognostic impact of LN metastasis and utility of complete LN dissection in patients with micrometastatic LN disease, however, varies between solid tumor types and patient subsets. The clinical observation that removal of micrometastatic LN disease does not by definition improve survival has highlighted a gap in our understanding of regional LN metastasis and calls into question the simple model of sequential metastasis. Given that LNs are both an early site of metastasis and immunological organs, the interplay between dissemination and immunity may be critical to understanding how LNs impact outcome. Here we will investigate the biology of tumor-draining LNs to ask whether regional draining LNs act as an educational site that ultimately shifts the systemic macroenvironment to favor distant tumor outgrowth. In this proposal we test the hypothesis that sentinel LNs undergo a cascade of cellular and molecular events that prime the host to be receptive to tumor progression. To test this hypothesis, we will build an unbiased, temporally, and spatially resolved map of regional draining LNs. This will enable us to identify clinically relevant mechanisms that determine outcome by integrating a deep, mechanistic understanding of lymphatic and LN biology together with high-dimensional imaging and novel, computational tools in mice and humans. To build this map, we will leverage fresh frozen LNs from surgical cases, archived, FFPE matched primary and metastasis pairs, and a clinically relevant mouse model, and determine the early changes that support tumor cell seeding and the melanoma cell states that first arrive in LNs (Aim 1). We will further test causal programs, including type I interferon signaling, that may functionally initiate this tumor permissive state and install local mechanisms of adaptive immune suppression that limit systemic immune surveillance and thereby distant metastasis (Aim 2). Finally, we will leverage the extensive resource and expertise of Core B to investigate the utility of immune markers to stratify patient risk from archived sentinel LN tissue and thereby predict recurrence in Stage II and III melanoma patients (Aim 3). The work we propose here promises to significantly reimagine the role of the sentinel LN in systemic melanoma progression. This conceptual shift may inform personalized strategies to manage local disease and thereby mobilize anti-tumor immunity and systemic tumor control across solid tumor types and identify immune-based LN features to stratify risk for recurrence in melanoma and guide clinical care. The resources and knowledge generated through this proposal will be made publicly accessible through NYULH MetNet Center Cores B and C, which will enable extension of these observations to other solid tumor types (e.g. breast cancer) ...