Imaging and Analysis of Melanoma-Secreted Extracellular Vesicles in Tumor-Draining Lymph Nodes

Small extracellular vesicles (sEVs) are key mediators of intercellular communication and play important roles in both physiological and pathological processes. In cancer, sEVs mediate communication between the tumor and its microenvironment but also contribute to metastatic organotropism and the formation of pre-metastatic niches in regional and distant organs. Lymph nodes (LNs) are central regulators of immune responses and primary sites of early metastasis, and, as such, they are critical targets for understanding cancer progression and improving immunotherapy. sEVs contribute to the conditioning of lymph nodes during pre-metastatic niche formation by altering lymphatic endothelial cell function and modulating immune cell populations, thereby promoting tumor progression. Because these interactions are complex and dynamic, their study requires in vivo models and specifically tailored sEVs-based techniques. In this chapter, we describe several approaches for studying sEVs in the LN context, including biodistribution analysis in the lymphatic system, imaging by immunofluorescence, and uptake measurement by flow cytometry.