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. 2009 Jan 12;7:4.
doi: 10.1186/1479-5876-7-4.

Can Urinary Exosomes Act as Treatment Response Markers in Prostate Cancer?

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Free PMC article

Can Urinary Exosomes Act as Treatment Response Markers in Prostate Cancer?

Paul J Mitchell et al. J Transl Med. .
Free PMC article

Abstract

Background: Recently, nanometer sized vesicles (termed exosomes) have been described as a component of urine. Such vesicles may be a useful non-invasive source of markers in renal disease. Their utility as a source of markers in urological cancer remains unstudied. Our aim in this study was to investigate the feasibility and value of analysing urinary exosomes in prostate cancer patients undergoing standard therapy.

Methods: Ten patients (with locally advanced PCa) provided spot urine specimens at three time points during standard therapy. Patients received 3-6 months neoadjuvant androgen deprivation therapy prior to radical radiotherapy, comprising a single phase delivering 55 Gy in 20 fractions to the prostate and 44 Gy in 20 fractions to the pelvic nodes. Patients were continued on adjuvant ADT according to clinical need. Exosomes were purified, and the phenotype compared to exosomes isolated from the prostate cancer cell line LNcaP. A control group of 10 healthy donors was included. Serum PSA was used as a surrogate treatment response marker. Exosomes present in urine were quantified, and expression of prostate markers (PSA and PSMA) and tumour-associated marker 5T4 was examined.

Results: The quantity and quality of exosomes present in urine was highly variable, even though we handled all materials freshly and used methods optimized for obtaining highly pure exosomes. There was approx 2-fold decrease in urinary exosome content following 12 weeks ADT, but this was not sustained during radiotherapy. Nevertheless, PSA and PSMA were present in 20 of 24 PCa specimens, and not detected in healthy donor specimens. There was a clear treatment-related decrease in exosomal prostate markers in 1 (of 8) patient.

Conclusion: Evaluating urinary-exosomes remains difficult, given the variability of exosomes in urine specimens. Nevertheless, this approach holds promise as a non-invasive source of multiple markers of malignancy that could provide clinically useful information.

Figures

Figure 1
Figure 1
Purification of urine-derived exosomes. Healthy donor urine was subjected to exosome purification, and at each step, 10 μl of sample was kept for electrophoretic analysis (4–20% gradient polyacrylamide gel, silver stained) (A), demonstrating effective removal of the principal non-exosomal protein bands such as that at ~80 Kd, and significant enrichment of diverse protein species in the final exosome product (A). Parallel gels were run for immuno-blot analyses, using antibodies against typical exosome proteins as indicated (B). Comparing the sucrose cushion method, with a simpler method of Pisitkun et al, where cell culture media (C) or fresh urine (D) were subject to centrifugation at 17,000 g followed by pelletting at 200,000 g. Exosomes (from sucrose method) and the 200,000 g pellet were normalised for protein differences, and 2.5 μg/well analysed by western blot for markers as indicated.
Figure 2
Figure 2
Quantification of urine-derived exosomes, in healthy donors, and Prostate Cancer patients. The quantity of exosomes present in each preparation was measured using the BCA protein assay. Values were corrected for urine-specimen volume, and are represented as ng Exosomes per ml of urine. Preparations from 10 healthy donors and 10 PCa patients undergoing standard therapy, at ADT4 (after 4 weeks ADT), ADT12 (after 3 months of ADT), and at RT20 (and after 20-fractions of radiotherapy) are compared. Bars represent mean+SE. *p < 0.5 using the Wilcoxon matched pairs test are shown.
Figure 3
Figure 3
Characterising exosomes produced by LNCaP-prostate cancer cell line. Prostate cancer cell lines (LNCaP and DU145), as indicated, were maintained in culture as a source of positive-control prostate cancer exosomes (for subsequent analyses). Whole cell lysates (CL) or exosomes (Exo) were analysed by SDS-PAGE (5 μg/well), with a panel of antibodies as indicated.
Figure 4
Figure 4
Characterising exosomes from healthy donor urine. Six healthy donors (detailed in Table 3), provided urine specimens and exosomes were purified. Western blots were performed with 5 μg urine-derived exosomes/well, or with 5 μg LNCaP-derived exosomes (Exo) or 5 μg LNCaP whole cell lysates (CL). Blots were probed with antibodies against PSA, TSG101, 5T4, CD9 and GAPDH, as indicated.
Figure 5
Figure 5
Characterising exosomes from PCa patients. Urinary exosomes (5 μg/well), isolated from 8 PCa patients (at ADT4, ADT12 or RT20), were subject to western blot analyses with a panel of antibodies as indicated. Whole cell lysates (CL) or exosomes (Exo) of LNCaP (5 μg/well) was included on each gel as positive controls.
Figure 6
Figure 6
Evaluating urine-mediated damage of exosomes. Exosomes coupled to microbeads were labelled with a luminal fluorescent dye (Calcein-AM), prior to incubation with various concentrations of NaCl (A) or with fresh urine specimens from four healthy donors (HD1-4) (B). In parallel, identical beads were set up, in the absence of Calcein-AM dye, stained instead with anti-MHC Class-I (RPE) conjugated antibody. After 1 h at room temperature, the fluorescence signal present in the FL-1 channel (Calcein) was compared to FL-2 fluorescence (Class-I-RPE). Graphs show ratio of Calcein to Class I fluorescence. To examine proteolytic damage of exosomes (C), western blot was performed for CD9, TSG101 and PSA on LNCaP-derived exosomes; which were incubated for 2 h or 18 h with fresh urine specimens (from three healthy donors), in the presence or absence of protease inhibitors. Trypsin was used as a positive control for proteolysis.

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