Characterization of CD147, CA9, and CD70 as Tumor-Specific Markers on Extracellular Vesicles in Clear Cell Renal Cell Carcinoma.

Dirk Himbert,Kerstin Junker,Hiresh Ayoubian,Michael Stöckle,Joana Heinzelmann,Philip Zeuschner

doi:10.3390/diagnostics10121034

Dirk Himbert, Kerstin Junker + Show 4 more

Open Access

https://doi.org/10.3390/diagnostics10121034

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Abstract

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.

Highlights

Despite significant advances in diagnostics, there is a lack of adequate methods to detect cancers at early stages
In order to identify eligible tumor-specific exosomal markers in clear cell renal carcinoma (ccRCC), we examined the expression of these putative candidates in exosomes from renal cell carcinoma (RCC) cell lines and primary patient-derived tumor tissues
GM130 was detectable in all cells, but not in the corresponding exosome samples, underlining a high purification of exosomes without cellular contaminants (Figure 4A)

Summary

Introduction

Despite significant advances in diagnostics, there is a lack of adequate methods to detect cancers at early stages. Early detection of cancer is one of the most promising approaches to improve therapy outcomes combined with an individual prognostic evaluation to enhance therapy selection [1,2,3,4,5,6] In this regard, biomarkers are increasingly important in clinical management [7,8,9]. In contrast to the larger microvesicles, exosomes with a characteristic size of 30–150 nm are actively secreted by almost all cells and play an important role in cell–cell communication and cell homeostasis They are involved in physiological processes, and in tumor development and progression. Their molecular properties at least partly reflect their parental cells, rendering them a promising source of biomarkers from liquid biopsies [13,14,15,16,17,18,19,20,21,22]

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