Project Summary Background: Malignant melanoma and pancreatic ductal adenocarcinoma (PDAC) typically spread to lymph nodes (LNs) prior to outgrowth in distant tissues. While metastasis to LNs is frequently attributed to passive drainage from tumor lymphatics, the mechanisms enabling LN metastasis and its functional role in tumor progression remain poorly understood. LNs are education hubs of the adaptive immune response and harbor the majority of potentially tumor-reactive lymphocytes. Recently, we discovered that in colonizing LNs, tumor cells induce tumor-specific immune tolerance through their interactions with leukocytes that subsequently circulate throughout the host, resulting in systemic tolerance that facilitates metastatic seeding of distant sites. Hypothesis and objective: We hypothesize that by targeting the induction of immune tolerance in LNs, we can both prevent distant metastasis and induce tumor regression. We will use multiple cutting-edge methods to identify the cellular and molecular mechanisms of LN tolerance induction and develop approaches for breaking it. These goals will be pursued in the following aims: Specific Aims: Aim 1: Identify the mechanisms by which lymph node metastases induce tumor-specific immune tolerance through their activation of immunosuppressive lymphocyte populations. Aim 2: Determine the role of epigenetic regulation in LN metastasis and tolerance induction. Aim 3: Investigate immunotherapeutic approaches targeting tumor immune tolerance in LNs. Study design and methods: Using high-content multiplexed microscopy, mass cytometry, photoconversion- based lineage tracing, TCR sequencing, and genetic mouse models of antigen presentation, we will dissect the cellular interactions in LNs that we hypothesize are responsible for tolerance induction and dissemination, and validate the findings in human tissues and datasets. LN metastatic tumor cells exhibit conserved and stable transcriptional profiles indicative of epigenetic reprogramming. We will assess the role of epigenetic alterations in conferring a pro-LN metastatic transcriptional signature using bisulfite sequencing and ATAC-seq, and employ T-ATAC-seq to elucidate changes in the epigenetic landscape within tolerized T cells that recognize tumor antigens. We will evaluate the ability of our novel immunostimulatory antibody conjugates, targeted specifically to LNs, to reprogram APCs in metastatic LNs, employ HDAC inhibitors to reprogram both malignant and lymphocyte populations away from tumor immune tolerance, and combine these strategies to elicit robust anti-tumor combination therapies in mouse models of melanoma and PDAC. Expected results and impact: Upon successful conclusion of this work, we will have identified the mechanisms by which LN metastasis induces systemic tolerance, and evaluated novel immunotherapeutic strategies to overcome these mechanisms.