PROJECT SUMMARY Lymph node (LN) metastasis is a key negative prognostic factor in melanoma and other solid tumors. Recent clinical trials in regionally advanced melanoma have suggested that complete LN dissection does not improve survival outcomes, and that neoadjuvant therapy is efficacious. If the standard of care of regional disease evolves to encompass these results, we will soon be treating many more patients with intact tumor-draining LN (tdLN), both metastatic and non-metastatic. Consequently, it is essential we understand the therapeutic potential – or liability – of the tdLN. While the LN has historically been considered a passive conduit for metastasis, recent studies have illuminated the role of LN metastasis in promoting a state of systemic immunosuppression permissive of distant metastasis. However, the specific mechanisms activated by LN metastasis that suppress systemic immune surveillance are poorly understood. We have generated a comprehensive spatiotemporal map of lymph node remodeling during metastasis using paired single cell and spatial transcriptomic data in an inducible BrafV600E; Pten-/- cutaneous melanoma model that spontaneously metastasizes to the tdLN. Integrated preliminary analysis of this multimodal transcriptional data suggests that effective anti-tumor immune surveillance is dependent on production of the alarmin IL-33 by lymphatic endothelial cells (LECs), which activates mast cells to produce tumor necrosis factor (TNF). TNF can support many inflammatory processes; in our data, appears to be essential for maintaining natural killer (NK) cell activation and proliferation. Here, we test the hypothesis that LEC-derived IL-33 is essential for maintaining local immune surveillance against LN metastasis. In Aim 1, we validate our transcriptional data and investigate the proposed niche through imaging colocalization and flow cytometry analysis of metastatic tdLN and tdLN that lack TNF or IL-33 production. In Aim 2, we interrogate the relationship between IL-33 production and local and systemic anti-tumor immune surveillance through assays that quantify lung and LN metastatic burden. In Aim 3, we correlate the expression of IL-33 with human LN metastasis. Additionally, we analyze human metastatic LN from patients with cutaneous melanoma to corroborate that this signaling network may be relevant to human metastatic progression. Collectively, this work will provide a comprehensive understanding of the immunological changes occurring in the tdLN and outline the role of IL-33 in local and systemic immune surveillance. Completion of this proposal will provide me an extensive training in translational tumor immunology and specific opportunities to improve my skills in computational analysis, scientific communication, and mentorship. This training will directly prepare me for a career as an independent investigator and clinical oncologist at a major academic cancer center. My training objectives are supported by my sponsor (Dr...