# Proteomic Analysis of Small Extracellular Vesicles From Lymphatic Affluents in Developing Premetastatic Niche in Melanoma

## TL;DR

This study identifies key proteins in small extracellular vesicles from melanoma patients that contribute to the formation of a premetastatic niche in lymph nodes.

## Contribution

The study reveals 145 differentially expressed proteins in melanoma-derived sEVs linked to premetastatic niche development and identifies CD38, LGALS9, and TNC as potential biomarkers.

## Key findings

- Melanoma sEVs contain 145 differentially expressed proteins compared to control lymphatic fluid, linked to cellular stress and decreased extracellular matrix organization.
- CD38, LGALS9, and TNC are significantly upregulated in lymphatic sinuses of sentinel lymph nodes in melanoma patients.
- Higher LGALS9 expression in lymph node tissue is associated with poor overall survival in melanoma patients.

## Abstract

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.

•Proteomic analysis of small extracellular vesicles (sEVs) from lymphatic fluid in melanoma patients and control subjects using TMTpro labeling identified key proteins related to premetastatic niche development.•Melanoma sEV cargoes revealed 145 differentially expressed proteins compared with control lymphatic fluid obtained from the lymphatic afferent channels.•Multiple omics dataset comparison reveals that melanoma-associated lymphatic sEV cargoes can be involved in metastatic progression.•Melanoma-negative sentinel lymph node carries more contents of the sEV cargoes, CD38, LGALS9 (galectin-9), and TNC (tenascin-C), in the lymphatic sinuses compared with control lymph nodes.•LGALS9 was upregulated in melanoma-derived samples, and higher expression is also linked with melanoma prognosis.

Proteomic analysis of small extracellular vesicles (sEVs) from lymphatic fluid in melanoma patients and control subjects using TMTpro labeling identified key proteins related to premetastatic niche development.

Melanoma sEV cargoes revealed 145 differentially expressed proteins compared with control lymphatic fluid obtained from the lymphatic afferent channels.

Multiple omics dataset comparison reveals that melanoma-associated lymphatic sEV cargoes can be involved in metastatic progression.

Melanoma-negative sentinel lymph node carries more contents of the sEV cargoes, CD38, LGALS9 (galectin-9), and TNC (tenascin-C), in the lymphatic sinuses compared with control lymph nodes.

LGALS9 was upregulated in melanoma-derived samples, and higher expression is also linked with melanoma prognosis.

Metastatic melanoma commonly spreads to distant organs via the lymphatic system. Melanoma-derived small extracellular vesicles (sEVs) play a crucial role in the development of the premetastatic niche (PMN) in regional lymph nodes. Quantitative proteomic analysis of sEVs from lymphatic fluid in melanoma patients and control subjects identified 145 proteins that may be associated with PMN development through immunological and extracellular matrix organization pathways. High levels of CD38, LGALS9, and TNC were found in the sinuses of the lymph node, supporting potential immunological targets for the PMN in melanoma.