Abstract 1503: Melanoma-derived lymphatic extracellular vesicles suppress dendritic cell differentiation and maturation in the sentinel lymph node
Authors: Suman Shankar , Nevala Wendy K. , Jakub James W. , Guo Ray , Leontovich Alexey A. , Moore Raymond M. , Atherton Chathu L. , Geng Liyi , Johnson Jeffrey E. , McLaughlin Sarah A. , Markovic Svetomir N. 
Published: March 2024

Spread to regional lymph nodes is the most common site of metastasis from melanoma and almost all solid tumors. Lymphatic metastases begin in the first lymph nodes draining from the primary tumor, i.e., the sentinel lymph nodes (SLN), preceded by the development of the premetastatic niche (PMN) initiated by primary cutaneous melanoma. Melanoma mediators are known to play a critical role in developing the PMN through the immune subversion in SLN. The DCs are a vital component of LN immunity that directly relates to eliciting T-cell functions. Our multiplex immunofluorescence (MxIF) analysis of SLNs indicates significantly decreased levels of DCs and cytotoxic-T cells in both core and interface areas of tumors in recurred patients treated with immunotherapy compared to non-recurred melanoma patients. The goal of this study is to determine whether the lymphatic extracellular vesicles (L-EVs) of melanoma patients regulate DC tolerization in SLN in order to stimulate the development of PMN. To identify whether L-EVs stimulate DC suppression, we extracted L-EVs from the lymphatic fluid of patients with melanoma downstream of primary cutaneous melanomas, compared it with controls (non-malignant post-operative fluid collected from the lymph node dissection field, and evaluated their effect on DC maturation by challenging these L-EVs in monocytic (CD14+) and hematopoietic stem cells (CD34+) cells. We found that L-EVs derived from melanoma patients suppress differentiation of CD11c+HLA-DR+ DCs and their maturation (defined by CD83 and CD86) compared to control L-EVs in CD14+ based DC maturation assay. We also found a reduction of conventional type 1 DC (cDC1) in CD34+-based DC maturation assay those challenged with patients’ L-EVs compared to control. To understand the mechanism of DC suppression in patients' L-EVs, we also conducted a total RNA sequencing analysis of subsets of DCs from maturation experiments to evaluate differently regulated transcriptomic profiles. The gene ontology analysis indicates that patient-derived L-EVs interfere with tRNA processing, oncostatin M & IL-10 signaling, and other immunoregulatory pathways to alter the hematopoietic cell lineage pathways in the DC maturation. Furthermore, the proteomic analysis of L-EVs also indicated several differentially regulated cargo-identified proteins in patient L-EVs, which are associated with immunomodulation, including regulation of DC function compared to control L-EVs. In conclusion, our study revealed that the patient's L-EVs carry differential proteomic cargoes secreted by cutaneous melanoma, which may be involved in regulating DC differentiation and maturation to diminish their functions and support the developing PMN in SLN.

Citation Format: Shankar Suman, Wendy K. Nevala, James W. Jakub, Ray Guo, Alexey A. Leontovich, Raymond M. Moore, Chathu L. Atherton, Liyi Geng, Jeffrey E. Johnson, Sarah A. McLaughlin, Svetomir N. Markovic. Melanoma-derived lymphatic extracellular vesicles suppress dendritic cell differentiation and maturation in the sentinel lymph node [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1503.