Proteomic analysis of urinary extracellular vesicles from high Gleason score prostate cancer.
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ABSTRACT:
SUBMITTER: Fujita K
PROVIDER: S-EPMC5314323 | BioStudies | 2017-01-01
REPOSITORIES: biostudies
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Project description:Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches-such as selected reaction monitoring (SRM)-as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients.
1000-01-01 | S-EPMC5354884 | BioStudies
Project description:Urine contains extracellular vesicles (EVs) that concentrate molecules and protect them from degradation. Thus, isolation and characterisation of urinary EVs could increase the efficiency of biomarker discovery. We have previously identified proteins and RNAs with differential abundance in urinary EVs from prostate cancer (PCa) patients compared to benign prostate hyperplasia (BPH). Here, we focused on the analysis of the metabolites contained in urinary EVs collected from patients with PCa and BPH. Targeted metabolomics analysis of EVs was performed by ultra-high-performance liquid chromatography-mass spectrometry. The correlation between metabolites and clinical parameters was studied, and metabolites with differential abundance in PCa urinary EVs were detected and mapped into cellular pathways. We detected 248 metabolites belonging to different chemical families including amino acids and various lipid species. Among these metabolites, 76 exhibited significant differential abundance between PCa and BPH. Interestingly, urine EVs recapitulated many of the metabolic alterations reported in PCa, including phosphathidylcholines, acyl carnitines, citrate and kynurenine. Importantly, we found elevated levels of the steroid hormone, 3beta-hydroxyandros-5-en-17-one-3-sulphate (dehydroepiandrosterone sulphate) in PCa urinary EVs, in line with the potential elevation of androgen synthesis in this type of cancer. This work supports urinary EVs as a non-invasive source to infer metabolic changes in PCa.
2018-01-01 | S-EPMC5944373 | BioStudies
Project description:Extracellular vesicles (EVs) secreted by prostate cancer (PCa) cells contain specific biomarkers and can be isolated from urine. Collection of urine is not invasive, and therefore urinary EVs represent a liquid biopsy for diagnostic and prognostic testing for PCa. In this study, we optimised urinary EV isolation using a method based on heat shock proteins and compared it to gold-standard ultracentrifugation. The urinary EV isolation protocol using the Vn96-peptide is easier, time convenient (?1.5 h) and no special equipment is needed, in contrast to ultracentrifugation protocol (>3.5 h), making this protocol clinically feasible. We compared the isolated vesicles of both ultracentrifugation and Vn96-peptide by proteome profiling using mass spectrometry-based proteomics (n = 4 per method). We reached a depth of >3000 proteins, with 2400 proteins that were commonly detected in urinary EVs from different donors. We show a large overlap (>85%) between proteins identified in EVs isolated by ultracentrifugation and Vn96-peptide. Addition of the detergent NP40 to Vn96-peptide EV isolations reduced levels of background proteins and highly increased the levels of the EV-markers TSG101 and PDCD6IP, indicative of an increased EV yield. Thus, the Vn96-peptide-based EV isolation procedure is clinically feasibly and allows large-scale protein profiling of urinary EV biomarkers.
2017-01-01 | S-EPMC5505003 | BioStudies
Project description:Prostate cancer (PCa) is defined by dysregulated lipid signaling and is characterized by upregulation of lipid metabolism-related genes including fatty acid binding protein 5 (FABP5), fatty acid synthase (FASN), and monoacylglycerol lipase (MAGL). FASN and MAGL are enzymes that generate cellular fatty acid pools while FABP5 is an intracellular chaperone that delivers fatty acids to nuclear receptors to enhance PCa metastasis. Since FABP5, FASN, and MAGL have been independently implicated in PCa progression, we hypothesized that FABP5 represents a central mechanism linking cytosolic lipid metabolism to pro-metastatic nuclear receptor signaling. Here, we show that the abilities of FASN and MAGL to promote nuclear receptor activation and PCa metastasis are critically dependent upon co-expression of FABP5 in vitro and in vivo. Our findings position FABP5 as a key driver of lipid-mediated metastasis and suggest that disruption of lipid signaling via FABP5 inhibition may constitute a new avenue to treat metastatic PCa.
2019-01-01 | S-EPMC6908725 | BioStudies
Project description:Long noncoding (lnc)RNA modulate gene expression alongside presenting unexpected source of neoantigens. Despite their immense interest, little is known in their ability to be transferred and control adjacent cells. Extracellular Vesicles (EVs) offer a protective environment for nucleic acids, with pro and anti-tumorigenic functions by controlling the immune response. However, few studies have addressed the full RNA content of EVs in human fluids and none have compared them with their tissue of origin. Here, we performed Total RNA-Sequencing on 6 Formaldehyde-Fixed-Parafilm-Embedded (FFPE) prostate cancer (PCa) tumor tissues and their paired urinary (u)EVs to provide the first whole transcriptome comparison from the same patients. uEVs contain intron-free cytoplasmic transcripts, specific lnc/circular (circ)RNAs. Some uEV-circRNA were characterized as essential for prostate cell proliferation in vitro. 15 uEV-lncRNA were predicted encoding 750 unique high-affinity neoantigens. Our comparison of functional EVs-lnc/circRNAs in 3 PCa cell lines provides a fundamental resource to determine the role of uEV-lncRNA in PCa progression. Overall design: Here, we performed Total RNA-Sequencing on 6 Formaldehyde-Fixed-Parafilm-Embedded (FFPE) prostate cancer (PCa) tumor tissues and their paired urinary (u)EVs to provide the first whole transcriptome comparison from the same patients. Total RNA was extracted, checked for the quality and then converted into cDNA for total stranded RNA-sequencing.
2022-05-18 | GSE183070 | GEO
Project description:BACKGROUND:Prevention of unnecessary biopsies and overtreatment of indolent disease remains a challenge in the management of prostate cancer. Novel non-invasive tests that can identify clinically significant (intermediate-risk and high-risk) diseases are needed to improve risk stratification and monitoring of prostate cancer patients. Here, we investigated a panel of six DNA methylation biomarkers in urine samples collected post-digital rectal exam from patients undergoing prostate biopsy, for their utility to guide decision making for diagnostic biopsy and early detection of aggressive prostate cancer. RESULTS:We recruited 408 patients in risk categories ranging from benign to low-, intermediate-, and high-risk prostate cancer from three international cohorts. Patients were separated into 2/3 training and 1/3 validation cohorts. Methylation biomarkers were analyzed in post-digital rectal exam urinary sediment DNA by quantitative MethyLight assay and investigated for their association with any or aggressive prostate cancers. We developed a Prostate Cancer Urinary Epigenetic (ProCUrE) assay based on an optimal two-gene (HOXD3 and GSTP1) LASSO model, derived from methylation values in the training cohort, and assessed ProCUrE's diagnostic and prognostic ability for prostate cancer in both the training and validation cohorts. ProCUrE demonstrated improved prostate cancer diagnosis and identification of patients with clinically significant disease in both the training and validation cohorts. Using three different risk stratification criteria (Gleason score, D'Amico criteria, and CAPRA score), we found that the positive predictive value for ProCUrE was higher (59.4-78%) than prostate specific antigen (PSA) (38.2-72.1%) for all risk category comparisons. ProCUrE also demonstrated additive value to PSA in identifying GS ??7 PCa compared to PSA alone (DeLong's test p?= 0.039), as well as additive value to the PCPT risk calculator for identifying any PCa and GS ??7 PCa (DeLong's test p?=?0.011 and 0.022, respectively). CONCLUSIONS:ProCUrE is a promising non-invasive urinary methylation assay for the early detection and prognostication of prostate cancer. ProCUrE has the potential to supplement PSA testing to identify patients with clinically significant prostate cancer.
1000-01-01 | S-EPMC6260648 | BioStudies
Project description:BACKGROUND:Proteomic profiling of extracellular vesicles (EVs) from prostate cancer (PCa) and normal prostate cell lines, led to the identification of new candidate PCa markers. These proteins included the nuclear exportin proteins XPO1 (also known as CRM1), the EV-associated PDCD6IP (also known as ALIX), and the previously published fatty acid synthase FASN. In this study, we investigated differences in expression of XPO1 and PDCD6IP on well-characterized prostate cancer cohorts using mass spectrometry and tissue microarray (TMA) immunohistochemistry to determine their diagnostic and prognostic value. METHODS:Protein fractions from 67 tissue samples (n = 33 normal adjacent prostate [NAP] and n = 34 PCa) were analyzed by mass spectrometry (nano-LC-MS-MS). Label-free quantification of EVs was performed to identify differentially expressed proteins between PCa and NAP. Prognostic evaluation of the candidate markers was performed with a TMA, containing 481 radical prostatectomy samples. Samples were stained for the candidate markers and correlated with patient information and clinicopathological outcome. RESULTS:XPO1 was higher expressed in PCa compared to NAP in the MS data analysis (P > 0.0001). PDCD6IP was not significantly higher expressed (P = 0.0501). High cytoplasmic XPO1 staining in the TMA immunohistochemistry, correlated in a multivariable model with high Gleason scores (P = 0.002) and PCa-related death (P = 0.009). CONCLUSION:High expression of cytoplasmic XPO1 shows correlation with prostate cancer and has added clinical value in tissue samples. Furthermore, as an extracellular vesicles-associated protein, it might be a novel relevant liquid biomarker.
2019-01-01 | S-EPMC6594141 | BioStudies
Project description:The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu<sup>3+</sup>-chelate or Eu<sup>3+</sup>-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2-10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs.
2019-01-01 | S-EPMC6624270 | BioStudies
Project description:OBJECTIVES:To assess the ability of multiparametric MRI (mp-MRI) of the prostate to exclude prostate cancer (PCa) progression during monitoring patients on active surveillance (AS). METHODS:One hundred forty-seven consecutive patients on AS with mp-MRI (T2WI, DWI, DCE-MRI) at 3T were initially enrolled. Fifty-five received follow-up mp-MRI after a minimum interval of 12 months and subsequent targeted MR/US fusion-guided biopsy (FUS-GB) plus concurrent systematic transrectal ultrasound-guided (TRUS-GB) biopsy as reference standard. Primary endpoint was the negative predictive value (NPV) of the follow-up mp-MRI to exclude histopathologic tumor progression using PRECISE recommendations. Secondary endpoints were the positive predictive value (PPV), sensitivity, specificity, Gleason score (GS) upgrades, and comparison of biopsy method. RESULTS:Of 55 patients, 29 (53%) had a GS upgrade on re-biopsy. All 29 patients showed a tumor progression on follow-up mp-MRI. Fifteen of 55 patients (27%) displayed signs of tumor progression, but had stable GS on re-biopsy. None of the 11 patients (20%) without signs of progression on follow-up mp-MRI had a GS upgrade on re-biopsy. The NPV was 100%, PPV was 66%, sensitivity was 100%, and specificity 42%. FUS-GB resulted in GS upgrade significantly more often (n = 28; 51%) compared with TRUS-GB (n = 12; 22%; p < 0.001). CONCLUSIONS:(Follow-up) Mp-MRI can reliably exclude PCa progression in patients on AS. Standard serial re-biopsies might be waived if follow-up mp-MRIs are stable. Over 60% of patients with signs of tumor progression on mp-MRI during AS had a GS upgrade on re-biopsy. Targeted re-biopsies should be performed if cancer progression or higher-grade PCa is suspected on mp-MRI. KEY POINTS:• None of the patients with unsuspicious mp-MRI had a GS upgrade in re-biopsy and mp-MRI might replace serial biopsies in these cases • More than 60% of patients with mp-MRI signs of tumor progression had subsequent Gleason score (GS) upgrades • Targeted re-biopsies should be performed in case of higher GS cancer suspicion on mp-MRI.
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Project description:Background:In order to improve postoperative functional outcome, including urinary continence and erectile function, nerve sparing surgery is recommended for patients with clinically localized prostate cancer (PCa). However, due to poor diagnosis accuracy at the preoperative stage, upstaging occurs in a considerable proportion of patients. Multiparametric magnetic resonance imaging (mpMRI) and the Prostate Imaging Reporting and Data System version 2 (PI-RADS v2) have recently shown excellent performance in diagnosis and staging of PCa. The aim of this study was to develop a predictive model based on PI-RADS v2 for postoperative upstaging in patients with low-intermediate risk PCa. Methods:The medical records of 314 patients with low-intermediate risk PCa [prostate-specific antigen (PSA) level ?20 ng/mL, Gleason score (GS) <8, and clinical stage < T3] who underwent preoperative mpMRI and radical prostatectomy in the Department of Urology, Peking University First Hospital between January 2012 and July 2019 were reviewed retrospectively. Clinicopathological characteristics were collected. All MRI reports were done at our institution as part of routine clinical practice before prostate biopsy and there was no re-reporting occurred. Using PI-RADS v2, the mpMRI results were assigned to three groups: "negative", "suspicious", and "positive". Multivariate logistic regression analysis was used to assess factors associated with postoperative pathological upstaging, defined as the presence of pT3 at final pathology. A regression coef?cient based model for predicting postoperative upstaging was constructed and internally validated using 1,000 bootstrap resamples. The performance of the model was assessed using the area under the receiver operating characteristic curve (AUC). With the optimal cutoff point the performance of the model was assessed through analysis of sensitivity, specificity, positive predictive value, and negative predictive value. Results:Upstaging was observed in 119 (37.9%) patients. The univariate and multivariate analyses revealed that PSA density, biopsy Gleason grade group (GGG), and mpMRI findings were significantly independent predictors for postoperative upstaging (all P<0.05). A predictive model showing very favorable calibration characteristics and higher accuracy than the single variables was constructed (AUC =0.74; P<0.001). At the optimal cutoff point, the model demonstrated a sensitivity and negative predictive value of 87.4% and 87.0%, respectively. Conclusions:PI-RADS v2 assessment proved to be one of the most valuable predictors for postoperative upstaging in patients with low-intermediate risk PCa. The predictive model, based on PI-RADS v2 assessment, PSA density, and biopsy GGG, may help to select suitable candidates for nerve sparing radical prostatectomy among patients with low-intermediate risk PCa.
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