Quantitative proteomics of small extracellular vesicles from lymphatic fluid for analyzing the 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 small extracellular vesicles (sEVs) 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 sEVs that deliver oncogenic molecules to the SLN is important in understanding the premetastatic niche. To reveal the proteomic landscape of lymphatic sEVs we performed multidimension liquid chromatography-tandem mass spectrometry with multiplexing (18-samples) using tandem mass tag (TMT) pro labeling to profile the lymphatic sEVs 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.