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.

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INTRODUCTION AND OBJECTIVE:Multiple studies have demonstrated recurrent genomic alterations in advanced prostate cancer in recent years. However, genomic characterization of metastatic hormone-naïve prostate cancer (mHNPC) has been limited. Recently, we identified clinical parameters associated with prognosis in mHNPC, and it would be helpful to characterize genomic alterations in mHNPC for developing a more accurate prognostic model. In this study, we performed whole-exome sequencing (WES) of mHNPC and aimed to detect somatic genomic alterations and discover the relevance between the findings and clinical course.METHODS: Prostate biopsies were performed in 19 mHNPC patients and the specimens were halved. One section from each was used for pathological analysis, while the other was stored for DNA extraction. We extracted DNA from the stored section when the paired one had cancer cells, and matched germline DNA was isolated from blood. We then performed WES of DNA from tumor tissue and lymphocytes, and the following criteria were applied for variant calling: 1) variant allele frequency (VAF) of ! 5% after removing base calls with base quality or mapping quality of < 20; 2) the P-value of Fisher's exact test comparing VAF in tumor to that in matched germline of < 0.001; 3) single nucleotide polymorphisms that were registered in databases were excluded. The relevance between genomic alterations and clinicopathological features were assessed.RESULTS: Of the 19 samples, five samples exhibited an exceptionally large number of somatic mutations, and the average tumor mutational burden (TMB) was 14.9 per megabase. The average TMB of the rest of 14 samples was 2.5 per megabase, which was about half that of castration-resistant prostate cancer. Of the five samples with high TMB, four had aberrations in DNA repair genes such as CDK12, ATM and ERCC2. Recurrently mutated genes were SPOP, FOXA1, KMT2C and ZMYM3, and all were mutated in five samples each. There was no relationship between TMB and clinicopathological features such as PSA progression-free survival, overall survival, PSA value and Gleason score. No specific mutation correlated with survival in this cohort.CONCLUSIONS: SPOP and FOXA1, which are key genes in AR signaling, were most frequently mutated in mHNPC. Patients with DNA repair genes mutations had higher TMB, which was not associated with worse survival in this study.