Logistics and distribution of small extracellular vesicles from the subcutaneous space to the lymphatic system

Small extracellular vesicles (sEVs) are small, cell-derived particles with sizes of approximately 100 nm. Since these particles include cargos such as host cell-derived proteins, messenger RNAs, and micro RNAs, they serve as mediators of cell-cell communication. While the analysis of the pharmacokinetic of sEVs after the intravenous injection have been reported, the lymphatic transport of sEVs remains unclear. The objective of this study was to provide insights into the intra-lymphatic trafficking and distribution of sEVs when they are injected into an interstitial space both in normal skin tissue and in cancerous tissue. When sEVs were Subcutaneously administered into the tail base and the tumor tissue, they preferably accumulated in the lymph nodes (LNs), rather than in the liver and the spleen. The findings reported herein show that the lymphatic transport of sEVs was drastically changed in model mice, in which a surgical treatment was used to modify to allow the dominant lymphatic flow from the footpad directly to the axillary LN via the inguinal LN. Based on the results, we conclude that when sEVs are injected into the subcutis space, they are preferably delivered to the LN via the lymphatic system. Further, the extent of accumulation of sEVs in the LN after subcutaneous injection was reduced when they were preliminarily incubated with Proteinase K. These results suggest that the lymphatic drainage of sEVs in normal skin tissue is regulated by membrane proteins on their surface. This reduction, however, was not observed in the case of cancer tissue. This discrepancy can be attributed to the presence of highly permeable lymphatic vessels in the tumor tissue. Further, the major cell subtypes that captured sEVs in the LN were LN-resident medullary sinus macrophages. These collective findings indicate that the lymphatic drainage of sEVs are mediated by proteins and, that they may appear to contribute to the control of the function of immune-responsive cells in the LNs.