Diederick Duijvesz

Exosomes as biomarker treasure chests for prostate cancer.

CONTEXT Although progress has been made with regard to types of markers (protein, DNA, RNA, and metabolites) and implementation of improved technologies (mass spectrometry, arrays, and deep sequencing), the discovery of novel biomarkers for prostate cancer (PCa) in complex fluids, such as serum and urine, remains a challenge. Meanwhile, recent studies have reported that many cancer-derived proteins and RNAs are secreted through small vesicles known as exosomes. OBJECTIVE This narrative review describes recent progress in exosome research, focusing on the potential role of exosomes as novel biomarkers for PCa. The purpose of this review is to acquaint clinicians and researchers in the field of urology with the potential role of exosomes as biomarker treasure chests and with their clinical value. EVIDENCE ACQUISITION Medline and Embase entries between 1966 and September 2010 were searched using the keywords exosomes, microvesicles, prostasomes, biomarkers, prostate cancer, and urology. Leading publications and articles constructively contributing to exosome research were selected for this review. EVIDENCE SYNTHESIS Exosomes are small vesicles (50-100 nm) secreted by almost all tissues; they represent their tissue origin. Purification of prostate- and PCa-derived exosomes will allow us to profile exosomes, providing a promising source of protein and RNA biomarkers for PCa. This profiling will contribute to the discovery of novel markers for the early diagnosis and reliable prognosis of PCa. CONCLUSIONS Although the initial results are promising, further investigations are required to assess the clinical value of these exosomes in PCa.

Proteomic Profiling of Exosomes Leads to the Identification of Novel Biomarkers for Prostate Cancer

Background Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, the complexity of body fluids often hampers biomarker discovery. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ∼100 nm, which contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific biomarker discovery. Materials and Methods Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Candidate biomarkers were validated by Western blotting and Immunohistochemistry. Results Proteomic characterization resulted in the identification of 248, 233, 169, and 216 proteins by at least 2 peptides in exosomes from PNT2C2, RWPE-1, PC346C, and VCaP, respectively. Statistical analyses revealed 52 proteins differently abundant between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. Conclusions Identification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer.

α‐Blockers to assist stone clearance after extracorporeal shock wave lithotripsy: a meta‐analysis

Study Type – Therapy (meta‐analysis)
Level of Evidence 1a

Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer.

Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa‐derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time‐resolved fluorescence immunoassay (TR‐FIA) for capture/detection of PCa‐derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin‐coated wells. After application of exosomes, Europium‐labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR‐FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR‐FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR‐FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR‐FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease‐specific detection assays.

EVIDENCE-BASED UROLOGY IN PRACTICE: PUBLICATION BIAS

B J U I N T E R N A T I O N A L

28 Detectie van exosomen uit urine van mannen met prostaatkanker door middel van een gevoelige ELISA

SamenvattingExosomen zijn kleine blaasjes die uitgescheiden worden door vrijwel alle cellen.