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.
Keywords: biomarkers; clear cell renal cell carcinoma; extracellular vesicles; liquid biopsies; sucrose gradient; tumor exosomes; ultracentrifugation.