Proteomic analysis of small extracellular vesicles from lymphatic affluents in developing premetastatic niche in melanoma

Melanoma is an aggressive form of skin cancer that often metastasizes through lymph nodes (LNs). Lymphatic small extracellular vesicles (sEVs) derived from melanoma play a crucial role in establishing a premetastatic niche (PMN) within the sentinel-LN (SLN). Therefore, analyzing the proteomic content of tumor-draining lymphatic sEVs that deliver oncogenic signals to the SLN is vital in understanding the PMN. To investigate this, we performed multiplexing (18-samples) using tandem mass tag labeling to profile the lymphatic sEVs proteomes obtained from afferent lymphatic channels leading to the SLN of melanoma patients (n=6), non-cancer-associated afferent lymphatic channels (n=3), and postoperative lymphatic fluid after LN dissection (n=9). We identified 595 new proteomic cargoes compared to those reported in ExoCarta, and 1003 new cargoes relative to three previously reported lymphatic EVs datasets. The analysis revealed 145 differentially expressed proteins (DEPs) of melanoma sEVs that link to increased cellular stress and injury pathways and a decrease in extracellular matrix organization [-log(p-value)>7.0]. Analysis of the top 50 DEPs included expressions of normal, primary, and metastatic samples across multiple omics datasets. Hierarchical clustering with postoperative samples demonstrated 9 upregulated and 2 downregulated proteins specific to melanoma sEVs, which are associated with melanoma progression (p < 0.05). Notably, several common proteins associated with melanoma and postoperative samples were related to the wound healing mechanism. The multiplex immunofluorescence analysis of selected proteins reveals significantly increased expression levels of CD38, galectin 9 (LGALS9), and tenascin C (TNC) in the lymphatic sinuses of SLN (-) compared to the control LN sinuses. Moreover, higher levels of LGALS9 in LN tissue are associated with poor overall survival of melanoma patients (p= 0.0018). In summary, this study reveals an altered landscape of sEV proteome in the afferent lymphatic fluid of melanoma, highlighting distinct sEV proteins that are uniquely present in the SLN during PMN development.