Extracellular vesicles (EVs) are secreted by healthy and tumor cells and are involved in cell–cell communication. Tumor-released EVs could represent a new class of biomarkers from liquid biopsies. The aim of this study was to identify tumor-specific EV markers in clear cell renal carcinoma (ccRCC) using cell lines and patient-derived tissue samples. EVs from ccRCC cell lines (786-O, RCC53, Caki1, and Caki2) and patient tissues were isolated via ultracentrifugation. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting using exosome and putative tumor markers (epithelial cell adhesion molecule (EpCAM), carbonic anhydrase 9 (CA9), CD70, CD147). The tumor markers were verified using immunohistochemistry. CA9 was expressed in Caki2 cells and EVs, and CD147 was found in the cells and EVs of all tested ccRCC cell lines. In tumor tissues, we found an increased expression of CA9, CD70, and CD147 were increased in cell lysates and EV fractions compared to normal tissues. In contrast, EpCAM was heterogeneously expressed in tumor samples and positive in normal tissue. To conclude, we developed an effective technique to isolate EVs directly from human tissue samples with high purity and high concentration. In contrast to EpCAM, CA9, CD70, and CD147 could represent promising markers to identify tumor-specific EVs in ccRCC.

Extracellular vesicles (EVs) secreted by cancer cells have been shown to take a pivotal part in the process of local and systemic tumor progression by promoting the formation of a supportive local tumor microenvironment and preparing premetastatic niches in distant organ systems. In this study, we analyzed the organ-specific uptake of EVs secreted by urological cancer cells using an innovative in-vivo approach. EVs from benign and malignant prostate, kidney, and bladder cells were isolated using ultracentrifugation, fluorescence-labeled and injected intravenously in immunodeficient mice. After 12 or 24 h, the animals were sacrificed, their organs were harvested and analyzed for the presence of EVs by high-resolution fluorescence microscopy. Across all entities, EVs were taken up fast (12 h > 24 h), and EVs from malignant cells were taken up more efficiently than EVs from benign cells. Though not entirely organ-specific, EVs were incorporated in different amounts, depending on the entity (prostate: lung > liver > brain; kidney: brain > lung > liver; bladder: lung > liver > brain). EV uptake in other organs than lung, liver, brain, and spleen was not observed. Our results suggest a role of EVs in the formation of premetastatic niches and an organotropism in EV uptake, which have to be examined in more detail in further studies.