Lymph-Borne Melanoma-Derived Proteins as Determinants of Lymph Node Metastasis

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PROJECT SUMMARY Tissues are primed for metastasis prior to the arrival of tumor cells. Primary tumors drive this transformation by shedding tumor-derived factors (TDFs), including extracellular vesicles and secreted/shed proteins (TSPs) into tumor-associated blood and lymphatic vasculature. Despite being the most common site of metastasis across solid tumor types, we know very little about how the lymph node (LN) is primed for metastasis by TDFs. My preliminary data demonstrates that primary melanomas initiate remodeling in their draining LNs that enhances metastatic outgrowth relative to contralateral, non-draining and naïve LN controls. Furthermore, I have demonstrated that TDFs uniquely activate this pro-metastatic LN phenotype rather than factors derived from benign dermis or mouse albumin. The focus of this field has been on the role tumor-derived extracellular vesicles play in establishing this pre-metastatic niche, however my data further demonstrates TSPs are also sufficient to activate this niche. Using a novel cell-specific labeling strategy termed BONCAT (biorthogonal non-canonical amino acid tagging), I have identified several candidate TSPs transported to the pre-metastatic LN. In this proposal, I will test the hypothesis that TSPs initiate reprogramming events within resident cell populations of the draining LN that support metastatic growth through two specific aims. The first aim will employ a scRNAseq approach to identify cellular networks, TSP-induced crosstalk between the resident LN stroma and seeded tumor cells, that provide trophic support to metastatic cells and the extent to which LN lymphangiogenesis is required for LN metastasis. The second aim will investigate a specific TSP I have identified, CSPG4 (Melanoma- Associated Chondroitin Sulfate Proteoglycan). This protein is shed by melanoma cells, has known melanoma cell-intrinsic roles of invasion and is elevated in human melanoma patients, yet, it is unknown whether this TSP contributes to pre-metastatic niche formation in distant sites. The completion of this work will provide foundational information on TSP-dependent pre-metastatic niche development in the tdLN, and may uncover CSPG4 as a novel driver of niche development. The proposed work will provide me with comprehensive training opportunities integrating new bench techniques with high dimensional data analysis, ultimately preparing me for a modern independent career as a researcher. Other training opportunities proposed will strengthen my professional skills in writing, presenting, teaching/mentoring, and networking, laying the foundation for my long-term goals of leading a research lab at an academic institution. My training objectives are supported by my sponsor (Dr. Amanda Lund; Departments of Dermatology and Pathology) and co-sponsor (Dr. Itai Yanai; Departments of Biochemistry and Molecular Pharmacology and Director of the Institute for Computational Medicine) who bring expertise in cancer biology and immunology, and sequencing data analysis and integration, respectively.