PROJECT SUMMARY/ABSTRACT There is a fundamental gap in understanding the immunological paradox by which solid tumors first metastasize via lymphatic channels into the immune-rich lymph node. During this process, immune surveillance is disabled, ultimately allowing metastatic dissemination to the precise location where it should be first eliminated—the tumor-draining, sentinel lymph node (SLN). While notably prognostic, it is clear the metastatic status of the SLN alone is not sufficient in determining patient’s risk or relapse. Therefore, understanding the mechanisms underlining this chronic process of tumor mediated regional immunosuppression, commonly referred to as pre-metastatic niche (PMN) formation in the SLN will lend significant insights for developing improved prognostic and therapeutic tools to detect and reverse cancer dissemination early in the natural history of metastatic progression. Our long term goal is to develop therapeutic strategies capable of overcoming the immune compromise of the SLN PMN and thereby disrupt the first stage of cancer metastasis. Therefore, the objective of the current work is to mechanistically interrogate the process by which the subcellular component of the primary tumor lymphatic effluent directly mediates PMN formation. The central hypothesis proposes that in solid tumors, subcellular mediators derived from the primary tumor microenvironment actively traffic through the lymphatics and in a cargo-dependent manner create a PMN in the tumor-draining SLN. This hypothesis has been formulated on the basis of preliminary data produced in the applicant’s laboratory; namely the discovery and characterization of human lymphatic extracellular vesicles (L-EV) which have a demonstrated capacity to modulate immune function. The rationale asserts that in elucidating the factors and signatures that define PMN formation in the SLN, the knowledge gained will be significant as it will identify histopathologic biomarkers that could aid in patient risk stratification beyond the presence of melanoma cells in the SLN. Guided by strong preliminary data, this hypothesis will be tested in two specific aims: 1) identify mechanisms whereby lymphatic subcellular factors promote immune dysfunction in the pre-metastatic SLN beyond those already identified (i.e. S100A9); 2) evaluate the prognostic utility of these immune-modulating factors in predicting risk of recurrence by considering the comprehensive, interactive cellular landscape that defines the immunologically compromised SLN. The approach is innovative as it will use a mechanism-driven model to identify subcellular factors from a previously uncharacterized biological fluid, human lymph (Aim 1), complemented by a novel, multiplexed biomarker imaging approach in order to survey the SLN immunological landscape in a quantitative and spatially preserved manner to ultimately translate predictive features into clinically amenable platforms (Aim 2). Such findings will result in a ref...