CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To understand the role of CD36 in melanoma, we first analyzed the SKCM dataset for clinical prognosis, evaluated the percentage of CD36 in lymphatic fluid-derived EVs (LEVs), and tested whether melanoma-derived EVs increase CD36 expression and induce M2-macrophage-like characteristics. Furthermore, we performed a multiplex immunofluorescence (MxIF) imaging analysis to evaluate the CD36 expression and its colocalization with various other cells in the lymph node (LN) of patients and control subjects. Our findings show that cutaneous melanoma patients have a worse clinical prognosis with high CD36 levels, and a higher percentage of CD36 in total LEVs were found at baseline in melanoma patients compared to control. We also found that monocytic and endothelial cells treated with melanoma EVs expressed more CD36 than untreated cells. Furthermore, melanoma-derived EVs can regulate immunosuppressive macrophage-like characteristics by upregulating CD36. The spatial imaging data show that cells in tumor-involved sentinel LNs exhibit a higher probability of CD36 expression than cells from control LNs, but this was not statistically significant. Conclusively, our findings demonstrated that CD36 plays a vital role in controlling the immunosuppressive microenvironment in the LN, which can promote the formation of a protumorigenic niche.

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 lymph node (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 sEV 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 with those reported in ExoCarta and 1003 new cargo proteins relative to three previously reported lymphatic EV datasets. The analysis revealed 145 differentially expressed proteins 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 differentially expressed proteins included expressions of normal, primary, and metastatic samples across multiple omics datasets. Hierarchical clustering with postoperative samples demonstrated nine upregulated and two 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 with the control LN sinuses. Moreover, higher levels of LGALS9 protein 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.