Proteomic analysis of exosomes from lymphatic affluents reveals their implications in developing premetastatic niche in melanoma

Melanoma is an aggressive form of skin cancer that often spreads via lymphatic pathways to regional and distant sites. Melanoma-derived lymphatic exosomes play a crucial role in forming a tumor-supportive environment for metastasis, or premetastatic niche, within the first tumor draining lymph node, also known as the sentinel lymph node (SLN). Therefore, analyzing the proteomic content of tumor-draining lymphatic exosomes that deliver oncogenic molecules to the SLN is important in understanding the premetastatic niche. To reveal the proteomic landscape of lymphatic exosomes, we performed multidimension liquid chromatography-tandem mass spectrometry with multiplexing (18-samples) using tandem mass tag (TMT) labeling to profile the lymphatic exosomal proteomes obtained from afferent lymphatic channels leading to the SLN of patients with melanoma (n=6), control afferent lymphatic channels from prophylactic mastectomy (n=3) and non-cancer post-operative lymphatic fluid (n=9). Lymphatic fluid from postoperative lymphadenectomy drains served as another control to filter out non-melanoma alteration in lymph that may be related to the procedure of surgical resection and wound healing process. Our proteomic analysis identified 3929 proteins in the lymphatic exosomes, of which 968 were unique proteins absent from the current exosomal database. Interestingly, melanoma lymphatic exosomes possess distinctive proteomic cargo, which is significantly associated with cancer-associated and cellular structural remodeling pathways (FDR <0.05). Moreover, proinflammatory wound healing pathways are predominantly present in melanoma and postoperative lymph fluid compared to normal control afferent lymphatic channels. We identified a total of 17 uniquely modulated proteins in melanoma compared to control and postoperative lymph, which are critically involved in the process of melanoma tumorigenesis. At least ten upregulated proteins strongly correlate with each other at the gene expression levels in melanoma tumors compared to controls and may serve as a signature panel for melanoma (p = 2.13 x 10 ^-58 ). In summary, this study represents the first comparative analysis of the lymphatic exosomal proteome and highlights distinct exosomal proteins that may support premetastatic niche formation in the SLN.