ABSTRACT/SUMMARY - Project 1: Murine Modeling of Tumor-Mediated Immunosuppression Like many tumors, melanoma and head and neck squamous cell carcinoma (HNSCC) often metastasize first to regional lymph nodes, and patients with such metastases typically have poor prognoses. It remains unclear why lymph nodes, with their capacity to recognize distinctive antigens such as those produced by tumors, would be hospitable for metastatic outgrowth of the primary tumor. Previous studies of other cancers have identified mediators of tissue-specific metastases to sites such as the lungs, bone, and brain. However, most of these studies have utilized immunodeficient mice and thus have not queried the role of the adaptive immune response in inhibiting or facilitating spread to those tissues. We hypothesize that lymph node metastasis constitutes an essential first step in the metastatic cascade of melanomas and HNSCC in that such metastases act locally upon the adaptive immune system within the lymph nodes to induce tolerance to the tumor and that leukocytes recirculating from these nodes carry the tolerance to distant sites. Our objectives are to establish whether lymph node metastases induce perturbations in anti-tumor immunity and to identify the mechanisms of these perturbations. Starting with murine models, then validating in human tissue, we will a) characterize differences in local and systemic immune responses to metastatic tumors; b) identify differential regulators of tolerance induction by metastatic cells through the use of genomic profiling; and c) identify the molecular mediators of metastatic tolerance induction. Through the use of serial in vivo passaging in a murine model, we have already developed a panel of syngeneic melanoma cell lines that exhibit enhanced lymph node metastatic potential and will create similar lines in HNSCC. We will compare the activation states of these immune cells, cytokine profiles, T cell polarization, and cytolytic activity toward tumor cells using single cell proteomic methods, in partnership with Project 2. Using cytokine profiling and RNA sequencing on the lines, we will apply computational systems biology approaches, in partnership with Project 3, to identify the molecules relevant for induction of tolerance. Samples collected by the Biospecimen and Data Management Core will be used to corroborate our findings in human tissue. If our hypothesis is proven correct that lymph node metastasis is an obligate step in the generation of systemic disease due to tolerance induction, targeting the molecules responsible for lymph node metastasis induced tolerance could prevent and treat metastatic disease.