SUMMARY/ABSTRACT: PROJECT 1 Lymph node (LN) metastasis precedes further dissemination for most solid malignancies and confers a stage III diagnosis. Nonetheless, the mechanistic role of LN metastasis in further disease progression is poorly understood. We discovered in mice that in colonizing LNs, tumor cells activate transcriptional programs that enable their induction of tumor-specific immune tolerance through interactions with LN leukocytes. These leukocytes subsequently recirculate throughout the host, resulting in systemic immune tolerance facilitating metastatic seeding of distant sites. Importantly, we found the same conserved transcriptional signature in LN metastases in humans, suggesting that the mechanisms driving LN metastasis and immune tolerance in our mouse model are pertinent to human cancers. We hypothesize that local interactions between tumor cells, leukocytes, and stroma within LNs before, during, and following LN colonization alter distant tissues to promote disease progression. We expect these interactions to involve homotypic and heterotypic cell-cell interactions, orchestration of architectural changes within LNs, tumors, and distant sites, and trafficking of various populations throughout the host. We will use syngeneic mouse models to identify the mechanisms by which these interactions facilitate disease progression through the following approaches. 1: Determine the kinetics by which tumors generate systemic immune tolerance. Using a high-dimensional imaging platform (CODEX), we will uncover longitudinal changes in local microenvironments across the host. 2: Interrogate the nature of heterotypic cell-cell interactions within LNs and the mechanisms by which these interactions facilitate immune evasion and induction of immune tolerance. We will use scRNA-seq to identify transcriptional changes in immune and stromal populations interacting with malignant cells within LNs. Using spatial transcriptomics, we will determine how the gene expression patterns of cells interacting with malignant cells differ from those at a distance. To confirm the functional significance of these targets identified and those identified in Project 2, we will combine CRISPR- mediated gene editing of LN metastatic tumors, along with cell depletion studies and knockout mice or inducible mouse models. 3: Determine how trafficking of immune cells from tumor-involved LNs establish a pro-metastatic microenvironment at distant sites. We will employ a novel cell labeling platform to evaluate trafficking of leukocytes and stromal cells from tumor-involved LNs to distant sites and back to the primary tumor. These studies will reveal the mechanisms whereby LN metastases promote tumor spread to distant sites and identify novel targets for the prevention and treatment of metastatic disease.