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Cancer Research Frontiers. 2017; 3(1): 83-111. doi: 10.17980/2017.83

Prostate Cancer under the Light of Tumor Cells-derived Extracellular Vesicles

Irène Tatischeff*

Honorary CNRS (Centre National de la Recherche Scientifique, Paris, France) and UPMC (Université Pierre et Marie Curie, Paris, France) Research Director; Founder of RevInterCell (www.revintercell.com), a Scientific Consulting Service, Orsay, 91400, France.

 

*Corresponding author: Dr. I. Tatischeff. E-mail:

Citation: Irène Tatischeff. Prostate Cancer under the Light of Tumor Cells-derived Extracellular Vesicles. Cancer Research Frontiers. 2017; 3(1): 83-111. doi: 10.17980/2017.83

Copyright: @ 2017 Irène Tatischeff. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Competing Interests: The author declares no competing financial interests.

Received Feb 17, 2017; Revised Apr 17, 2017; Accepted July 20, 2017. Published Oct 30, 2017

 

 

Abstract

Introduction: The aim of this study is to gather the main results obtained about « Prostate Cancer and EVs », in the six yearly Meetings of the International Society for Extracellular Vesicles (ISEV) from 2012 to 2017, in order to get a leading thread for future research.

Methods: Altogether, 167 abstracts (72 Oral Communications and 95 Posters) dealing with « Prostate » were analyzed and classified as a function of their main respective goal : characterization of the various prostate cancer-derived EVs, search for biomarkers at the proteomics or RNAs levels, influence of prostate cancer-derived EVs from various cell lines upon normal cells, EVs clinical measurements in different human biofluids and in vivo models. New set-ups for sensitive exosome measurements, having a clinical potential for diagnostics, were also reported.

Summary/Conclusion: The main results of this scientific monitoring will be presented. Many interesting observations have already been collected about the characterization and functional properties of prostate cancer cells-derived EVs. However, a better research coordination about EVs potentials for diagnosis, prognosis and therapy of prostate cancer would be worth seeking, with a more defined leading thread for research. The final aim of the paper is to try to interest practicians, deeply involved with prostate cancer, but not yet familiar with the future potentialities of EVs for theranostics of this cancer.

Keywords: Prostate cancer (PCa), extracellular vesicles (EVs), intercellular communication, PCa diagnosis, Castration-Resistant Prostate cancer (CRPC) prognosis

 

 

Introduction

Prostate cancer (PCa) (1) represents the second most frequent cancer in men, but with a rather low relative mortality. As for other cancers, an early diagnosis greatly enhances the efficiency of subsequent therapy. For a long time, detection of prostate cancer relied on prostate palpation and histological examination of micro-biopsies, using the Gleason Score, which remains one of the most powerful prognostic factors for prostate cancer (2). Since 1986, the Prostate Specific Antigen (PSA) blood test (3) brought a great help to the medical diagnosis. However, it is now widely recognized that PSA has a low specificity and diagnoses many false positive patients, who may be submitted to inappropriate therapy with subsidiary great inconvenience. Although the PCA3 urinary test (4) has improved the blood PSA test, PCa diagnosis is still not recommended by the American Cancer Society for PCa prevention at a large scale (5). The need for other more reliable diagnostic tools of prostate cancer is therefore a current challenge.

 

 

1715 FIG1

Figure 1. Yearly search, using Web of Science from 2000 to 2016, for « Extracellular vesicles (EVs) & Cancer » and « Extracellular vesicles (EVs) & Prostate Cancer », showing their simultaneous huge increase with time in the last five years.

 

Depending on the classification grade of a given prostate cancer, the therapy might involve a simple medical monitoring (active surveillance), chemotherapy, prostate surgical removal and radiotherapy and, lastly, androgen deprivation therapy after castration. The outcome of non-aggressive prostate cancer is rather good, with long survival expectation as the cancer evolution remains slow with advancing age. However, after castration and an efficient androgen deprivation therapy, well controlled by the PSA test, some patients become resistant with time and the so-called castration-resistant prostate cancer (CPRC) becomes irredeemably out of control and of very bad prognosis (6). Therefore, the two major problems of prostate cancer are, first, elaborating a secure early diagnostic tool and, secondly, providing an efficient means of preventing acquired chemo-resistance after castration.

With the support of many 3 to 4 decades old observations, a new field has recently emerged in biology, i.e. the one of extracellular vesicles (EVs), extending the cell well beyond the cell membrane, as active vectors of intercellular communication (7). Prostasomes are nanosized microvesicles secreted by the acinar epithelial cells of the prostate gland. They were one of the earliest exosome-like EVs, evidenced about four decades ago and thoroughly studied by Ronquist. G. et al. (8). Research was first centered about the characterization of prostasomes and the study of their biological effects promoting the fertilizing ability of spermatozoa. Later, the ability to synthesize and export prostasomes to the extracellular space was also observed in malignant prostate cells, suggesting their role in malignant cell growth and proliferation.

In 2012, the international EV disseminated works, both in fundamental biology and in different medical fields, have been gathered into an International Society for the study of Extracellular Vesicles (ISEV), with annual international meetings and a dedicated Journal for EV publications (JEV). The ISEV participants are greatly increasing with time, from about 500 in 2012 to about twice as many in 2016. Almost all medical fields are increasingly impacted by this new EV approach, especially oncology, as shown by a yearly search, using Web of Science, for “Extracellular vesicles and Cancer” / “Extracellular vesicles and Prostate Cancer” (Fig.1). Besides many specialized papers published in the field (9), I published in 2015 a review to stress the interest of EVs in Cancer Research (10).

The aim of the present paper is to focus on PCa and to analyze the different works dealing with prostate and EVs as reported in the abstracts of the six annual ISEV meetings (in 2012 (11); 2013 (12); 2014, (13); 2015, (14), 2016, (15) and 2017 (16)). A first short report about the four previous ISEV meetings (2012-2015) has been presented as a poster at the 2016 ISEV meeting ((15), p64)). The current paper will give an overview of this study and will more specifically address the more recent works reported on the matter, with my own comments and suggestions for discussion.

 

Main results reported about “Prostate” at the (2012-2015) ISEV Meetings

At ISEV 2012 (11), most of the works were performed in vitro. Exosomes were prepared, using human non-PCa (PNT2C2, RWPE-1) and PCa (PC3, P346C, VCAP, RWPE-2, DU145, LNCaP, 22Rv1) cell lines. Three works were aimed to develop new methods for sensitive detection of exosomes or microvesicles for further diagnosis of cancers in the clinics, in particular for PCa. Like all other EVs, Prostate-derived EVs were not well defined: they could be characterized as prostasomes, oncosomes (up to 10 µm in diameter), apoptotic bodies, microvesicles (ectosomes) or exosomes, as a function of their cell origin and preparation method. Characterization of PCa (exosomes / oncosomes/ microvesicles) through proteomics and miRNAs profiling for further search of PCa biomarkers was well represented. PCa-derived EVs were also characterized from biofluids, such as blood and urine. The influence of tumor EVs on normal recipient cells was actively investigated, stressing the important role of EVs as mediators of intercellular communication. With regard to PCa, recipient cells might involve normal prostate cells, stromal cells of the tumor environment or human natural killer (NK) T cells. It was shown that tumor cells can use oncosome formation to actively export pro-oncogenic miRNAs. Some specific miRNAs (miR-1228, miR-150, miR-373, miR-135a, miR-34b and miR-491-3p) were involved in cancer proliferation and migration, whereas miR-135a and miR-373 promoted a mesenchymal to amoeboid transition, stimulated cell invasion, and were up- regulated in metastatic prostate cancer. Table 1 summarizes the abstracts related to “Prostate” at the first ISEV International Meeting (11).

At ISEV 2013 (12), most of the works about “Prostate” dealt with PCa cell lines and PCa patient body fluids. Only Corrigan et al. (p94) questioned the physiological role of prostate exosomes in reproduction, using an in vivo prostate Drosophila model. Five works, using PCa cell lines, human blood, plasma or urine samples, focused on different methods for preparing EVs beside differential centrifugation. Yuana et al. (p31) proposed a standardization of EVs collection and handling and compared the different methods for their characterization. Puhka et al. (p31) elaborated a biobank from patient urine samples amenable for MVs extraction, aimed to prostate cancer research. Most of the works (14/29) were concentrated on characterization of prostate derived EVs and search for PCa biomarkers, either at the proteomic or at the miRNA level. Liorente et al. (p43) questioned the less studied EV lipidomic composition. Seven works addressed the influence of tumor PCa EVs upon normal prostate cells, mesenchymal stem cell differentiation, or upon the stromal environment of PCa tumors. Their active role in reversal of chemosensitivity, induction of angiogenesis and propagation of cell malignancy was demonstrated, whereas some signalling pathways of these biological functions were studied. Table 2 classifies all the reported works about “Prostate” and their main results at the second ISEV International Meeting (12).

At ISEV 2014 (13), many works were still concerned with improving the EV isolation methods and solving some EV-related technological problems, such as their fluorescent labelling. However, most of the works were still concerned with EV characterization, principally aimed at improving clinical PCa diagnosis and prognosis. Interestingly, Lazaro-Ibanez et al. (p80) showed that the three main EV subpopulations (apoptotic bodies, microvesicles and exosomes) from three PCa cell lines carry, not only proteins and RNAs but different gDNA contents. Some works were concerned with the mechanisms of EV internalization and influence on recipient cells, with regard to PCa progression. The only in vivo work by Goberdhan et al. (p103), using the Drosophila prostate model, evidenced a critical step for exosome secretion after formation of vesicles in multivesicular bodies (MVBs). Table 3 depicts the different works about “Prostate” reported at the third ISEV International Meeting (13).

At ISEV 2015 (14), less works dealt with technological improvements for EV preparation. The search for EV-mediated PCa biomarkers remained an increasing goal and more search for prognosis of Castration-Resistant Prostate Cancer (CRPC) through EVs also appeared. For example, Ito et al. (p58) asserted that “detection of exosomal P-gp and ITGβ4 in blood would allow to select treatment and predict prognosis, as well as to diagnose drug-resistance and the stage of CRPC progression”. Some works were still concerned with tumoral EVs-induced modifications of the tumor environment. The same team, working as before with Drosophila in vivo, found analogies of EV trafficking with the one of mamalian exosome secretion, but suggested that EV trafficking to the cell surface may not just involve late endosomal compartments. Table 4 summarizes the main results of all the reported works about “Prostate” at the fourth ISEV International Meeting (14).

The subjective choice of monitoring all the works dealing with “Prostate” through the first four international ISEV meetings gives an extended overview of all matters of concern, when introducing EVs in the field of biological research and medical applications of health and disease involving prostate, especially PCa. For the ISEV 2016 meeting, I chose to differently classify and detail the abstracts involved with “Prostate”, as a function of their main field of interest, with the hope of, thus, better contributing to a current general clarification of the field.

 

Main results reported about “Prostate” at the ISEV 2016 Meeting (15)

  1. Characterization of prostate cancer EVs

Due to the heterogeneity of cancer, it is unlikely to find a single specific cancer marker, so many different approaches tried to evidence an outstanding characterization of PCa EVs. Miglarese et al. (Phoenix, AZ, USA, p20) developed a novel multiplexed platform (ADAPT) for profiling exosomes from cancer cells, identifying cancer-associated proteins expressed on exosomes, and able to be deployed against multiple cancer types. Exosomes from two prostate cancer cell lines, VCaP and LNCaP, were first used. Binding partners of ODNs bound to VCaP exosomes were identified, including ESCRT endosomal sorting proteins (CHMP1b/2a/4b, VPS28, Syntenin-1). Components of ESCRT participate in exosomes biogenesis and are overexpressed in human cancers. In addition, the chemokine ITAC, which is overexpressed in blood and tissue of men with advanced prostate adenocarcinoma was found. A cancer associated splicing factor, hnRNP-1, and the cold shock proteins RNPL and A18 hnRNP were also identified. Knockdown of these cold shock proteins has been shown to enhance chemotherapeutic killing of prostate cells. Reis-Sobreiro et al. (Los Angeles, CA, USA, p24) questioned whether alterations in the protein assemblies, that link the cytoskeleton with the nucleoskeleton (LINC) complex, were associated with the amoeboid phenotype, having propensity to metastasize and shedding EVs. They showed that loss of the cytoskeletal regulator DIAPH3 drives the transition of cancer cells to an amoeboid phenotype, promoting nuclear envelope blebbing and shedding of non-apoptotic EVs with nuclear content. LINC complex disruption, through the silencing of emerin and lamin A/C, promotes increased migration, formation of nuclear envelope blebs, increased shedding of non-apoptotic EVs and ROCK pathway activation. Lamin A/C is downregulated in metastatic PCa patients. Circulating tumour cells (CTCs), isolated from PCa patients, express low emerin levels, sugesting new markers of lethal disease in PCa, and showing that LINC complex disruption is a critical step for metastatic spread. It also identifies a mechanism that promotes inclusion of nuclear content into EVs. Kosgodage et al. (London, UK, p29), stating that microvesicle (MV) release from tumor cells plays an important role in cancer drug resistance, studied a range of potential inhibitors of microvesicle release with the potential to enhance effectivity of cancer chemotherapy. The maximum MV inhibition was with 500 µM ethylene glycol tetra-acetic acid (EGTA) and 10 µM bis-indolymaleimide I, resulting in 48 and 34%, inhibition, respectively. Roberts-Dalton et al. (Cardiff, Wales, UK, p30) searched to understand how exosomes, capable of delivering macromolecular signal mediators, deliver their cargo to recipient cells. A novel thiol-based conjugation method was used for the attachment of Alexa-488 to well characterized (Du145) PCa exosomes. Uptake was analyzed in fibroblasts and HeLa cells. Alexa 488-exosomes retained their function to differentiate fibroblasts into myofibroblasts and were effectively internalized into fibroblasts and HeLa cells as motile punctate cytoplasmic structures. SiRNA and inhibitor studies in HeLa cells highlighted actin-dependent macropinocytosis, as a major pathway mediating cell uptake, suggesting that exosomes may be activating their own internalization. Brzozowski et al. (New South Wales, Australia, p52) compared lipid profiling of normal and tumorigenic cell-derived extracellular vesicles. EVs from the RWPE1 cell line, an immortalized normal prostate cell line, and the WPE1-NB26 cell line, a chemically modified and tumorigenic derivative of the RWPE1 cell line, were collected using an ultrafiltration procedure, and lipids were extracted and detected by LCMS. Quantification of 265 lipid metabolites, including phospholipids (PC, PE, PI, PS), ceramides, sphingolipids (SM), cholesterol esters (CE) and di- and tri-acyl-glycerides (DG, TG), was conducted in a lipid-targeted approach to identify differences in EVs from normal and tumorigenic prostate cells. All 265 lipid metabolites targeted were detected in EVs. Of the 265 metabolites detected, 71 WPE1-NB26 EV metabolites including various CE, PC, PE and SM species were significantly different from RWPE1 metabolites. There is a selective and identifiable difference in lipid metabolites between EVs derived from normal and tumorigenic cells. Saari et al. (Helsinki, Finland, p58) improved large-scale EV production by using a two-compartment bioreactor. PC-3 prostate cancer cells (ATCC, USA) were grown both in T-175 cell culture flasks and in a Celline AD 1000 bioreactor (Integra). The cell-conditioned medium (CM) was harvested three times per week from the 2D cultures and once per week from the bioreactor cell compartment. The EVs were collected by differential centrifugation into 20k x g and 110k x g pellets and analyzed by electron microscopy, western blotting, nanoparticle tracking analysis (NTA) and Apogee flow cytometry. The PC-3 cells in the bioreactor produced approximately 1-2 x 1012 EVs collected in the 20k pellet and 2-4 x 1012 EVs in the 110k pellet from 15 ml of CM, while a single 2D cell culture flask produced approximately 1-2 x 1010 and 8-11 x 109 EVs, respectively, from 25 mL of CM over 7 days. Bioreactors have the potential to improve EV production, but the produced EVs need careful comparison with the EVs produced in standard 2D cultures. Puhka et al. (Helsinki, Finland, p109) explored the metabolomics of urinary EVs, i.e. the main pathways, normalization and potential for biomarker studies. EVs purified from urine samples of three PCa patients before and after radical prostatectomy and of three controls, were subjected together with their source urine samples to UPLC-MS-MS analysis of 102 metabolites. For normalization, EV-derived (CD9, particle number and volume) or urine-derived parameters (volume and creatinine) and calculation of metabolite proportions were tested. 32-55% of the screened metabolites were quantified in EV samples, containing as little as 6 x 109 EVs, or in EVs from 10 mL of urine. The EV metabolome differed clearly from the urine. While most of the metabolites were more abundant in urine, some were better detected in the EVs. All EV samples contained nucleotides, amino acids, vitamin B, carnitine, amines or related metabolites representing mainly the urea cycle, purine nucleotide, glutathione and carnitine shuttle pathways. Glucuronate, ribose-5-phosphate and isobutyrylcarnitine were reduced in cancer samples, compared to control or post-prostatectomy samples. A relatively small number of EVs is sufficient for detection of EV metabolites. The observed changes in the cancer samples relate to cancer metabolism and suggest that EV metabolites could act as PCa biomarkers. Panaretakis et al. (Stockholm, Sweden, p119) compared energy-requiring uptake of normal seminal prostasomes and a PCa cell line, PC3, cell-derived exosomes into non-malignant and malignant cells. Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles, called prostasomes, belonging to the exosome family, and having an ATP forming capacity. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and both of them were able of generating ATP, when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes, due to a high ATPase activity, contrary to an elevated net ATP level for PC3 exosomes, because of their low ATPase activity. The uptake of the two types of EVs by normal prostate epithelial cells (CRL2221) and PCa cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. The internalization of EVs into recipient cells is an energy-requiring process, also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process. Rikkert et al. (Amsterdam, The Netherlands, p171) reported that, in the Dutch Cancer-ID program, 8 universities and 23 companies worked together to develop a platform, capable of reliable identification and characterization of tumor vesicles in human body fluids. Methods involved were atomic force microscopy, Coherent anti-Stokes Raman spectroscopy, flow cytometry, scanning electron microscopy, Raman microspectroscopy, RNA sequencing, smart microsieves, and surface plasmon resonance imaging. To compare results, EV-containing samples from prostate cancer cell lines (PC3 and LNCaP) and blood products (platelets, red blood cells, and plasma) were distributed to obtain a first assessment of specificity and sensitivity of all methods. Both flow cytometry and Raman microspectroscopy are operational, whereas other methods are currently being optimized for EV detection. Flow cytometry was able to identify tumor EVs (CD63+, EpCAM+) in PC3-EVs and LNCaP-EVs spiked plasma samples at a ratio of 1: 80,000. The heterogeneity of EVs in morphology, size and refractive index comprises a major challenge to develop a single platform, capable of reliable identification and characterization of tumor EVs in liquid biopsies. Due to a detection limit of 1 tumor EV among 80,000 EVs, the sample needs to be enriched prior to analysis of clinical samples. Vergauwen et al. (Ghent, Belgium, p173) described a two-step protocol for high-purity isolation of EVs in plasma: size-exclusion chromatography (SEC), followed by iodixanol density gradient ultracentrifugation (ODG). SEC separates EVs from the bulk of protein contaminants such as albumin, and Ago2. The EV-rich SEC fractions contain 31% of total particles of plasma, while only less than 1% of proteins. Combinations of SEC fractions show a x250-fold enrichment in EVs compared to proteins, but still show the presence of lipoprotein particles. Addition of density gradient ultracentrifugation further purifies the CD9 and flotilin-1 positive EVs from lipoproteins and Ago2 protein complexes. This protocol was further validated on plasma from breast, ovarian and prostate cancer patients, and showed the ability to detect EV-enriched markers (CD9, flotillin-1) from 6 ml plasma of cancer patients on western blots, without contaminating Ago2 and ApoA-1.

 

  1. PCa diagnosis by means of EVs

Beside a better characterization of prostate tumor EVs, a search for EV-mediated diagnosis of PCa was also investigated. Shephard et al. (Cardiff, UK, p64) asked whether exosome-associated heparan sulphate proteoglycans (HSPGs), present on the surface of exosomes, might serve as potential new markers for the detection of PCa. Exosomes were isolated from cell-conditioned media and from PCa cells cultured in bioreactor flasks. Exosome concentration was determined by nanoparticle tracking analysis, and total protein content measured by BCA-assay. The particle/protein ratio was used as a measure of exosome purity. Size-exclusion chromatography proved as a successful method for isolation of exosomes from serum, enabling detection of several HSPGs present on exosomes from multiple PCa cell lines, and with increased levels on serum-derived exosomes from PCa patients, compared to healthy individuals.

 

2a. PCa diagnosis by EV-specific proteins

Shin et al. (Postech, Pohang, Korea, p18) suggested a PCa diagnosis using EVs isolated from urine by aqueous two-phase systems (ATPS), with an efficiency of 100% and total processing time ~35 min. Twenty benign prostate hyperplasia (BPH) patients were recruited. Ten millilitres of each patient’s urine was collected, and EVs were isolated, using the ATPS for measuring the expression level of PCA3 (PCa mRNA marker) and actin for normalization. The optimized ATPS isolation method recovered ~100% of EVs from the urine, whereas ultracentrifuged-pellet methods recovered only ~30% of total EVs. Significant differences in PCA3/ACTIN demonstrated that PCa might be successfully distinguishable from BPH. Olivan et al. (Barcelona, Spain, p61) identified novel biomarkers by comprehensive proteomics in urinary exosomes for early non-invasive detection of PCa. Protein biomarker candidates for PCa were initially identified in urinary exosomes obtained after digital rectal examination. Label-free liquid chromatography coupled to mass spectrometry (LC-MS/MS) protein quantitation was performed on 24 samples: 8 benign samples, 8 low-risk PCa samples and 8 high-risk PCa samples (Gleason >7). Proteins significantly changing in abundance were selected for further selected reaction monitoring (SRM) validation in 53 urinary exosomes samples from PCa patients and 54 from benign counterparts. 1673 proteins including PSA, PSMA and ACPP were identified and a panel of 64 candidates was selected for validation. A profile of two novel urinary exosome-associated protein biomarkers was identified after the comparison between benign and PCa patients and also a promising profile of 5 proteins able to significantly distinguish between high (Gleason =7 (4+3)) and low (Gleason =7 (3+4)) risk patients. Bijnsdorp et al. (Amsterdam, The Netherlands, p61) identified PCa protein biomarkers by proteomics of urinary extracellular vesicles, isolated either by ultracentrifugation or by a novel EV isolation method that captures EVs, using heat shock protein (HSP)-binding peptides, aggregating HSP-decorated EVs. Proteins related to PCa were determined using label-free proteomics in urine EVs (isolated by ultracentrifugation) from 3 controls, 3 indolent and 3 aggressive PCa patients. The protein profile in HSP-isolated EVs was compared by proteomics in urine samples of 2 controls and 2 PCa patients. In urine EVs, >3000 proteins were identified. Hierarchical cluster analysis separated aggressive from indolent and control patients. Selected candidate proteins (259) were functionally related to translation and cell migration. Known PCa-markers were differentially expressed, including PSA, PSMA, integrins, and CDH1. Several non-reported candidates were identified. The overlap between the identified proteins isolated by HSP-kit and ultracentrifugation across all 4 patients was large (86%). Urinary EV isolation by HSP-binding peptides offers new opportunities in the identification and application of PCa biomarkers. Llorente et al. (Oslo, Norway, p62) analyzed the proteome of urinary exosomes by mass spectrometry, to identify proteins differentially expressed in 16 PCa patients compared to 15 healthy male controls, and validated potential biomarkers by methods more often used in the clinic, such as antibody-based methods. 246 proteins were significantly changed in urinary exosomes from healthy controls, compared to PCa patients. At 100% specificity, 17 of these proteins displayed individual sensitivities above 60%. The highest sensitivity, 94%, was observed for transmembrane protein 256. Western blot and ELISA were used to validate the results of the mass spectrometry analysis. The validation of some proteins was challenging, due to their low amounts and/or lack of good antibodies, but the potential of urinary exosomal proteins as PCa biomarkers was clearly demonstrated. Khomyakova et al. (Moscow, Russia, p118) performed a surface markers profiling of PCa exosomes, through expression of CD9, CD81, CD147, CD117, CD29 and EGF on the exosomes isolated from the urine of patients with histologically confirmed PCa and age matched patients without cancer, collected after digital rectal exam (DRE). Exosomes were isolated either by differential centrifugation or by using Exosome Precipitation solution (Machery-Nagel). The purity of exosome population was analyzed using TEM. Pre-purified exosomes (5 ml of urine per sample) were incubated with CD9 exosomes dynabeads for 36 hrs at 4°C followed by antibody staining and flow analysis. The highest CD81 fluorescence signal was observed for exosomes purified by differential centrifugation. The initial data showed essential differences between the patients in expression of CD9/CD81 markers and markers associated with PCa progression, but they should be further confirmed at a larger scale.

 

2b. PCa diagnosis by EV-specific miRNAs             

Brennan et al. (Sydney, Australia, p61) investigated the potential of urinary exosomal miRNAs as biomarkers for PCa. Human urine was collected from healthy, age-matched volunteers and PCa patients, both before and after radical prostatectomy. Exosomes were isolated using ultrafiltration, followed by ultracentrifugation. RNA was extracted from the exosomes and analyzed. All exosome samples, isolated from urine, contained particles of the expected size with minimal contamination, and the markers CD9 and TSG101 were abundant, while AGO2 could not be detected. Exosome samples contained mostly small RNAs, with 24 highly upregulated exosomal miRNAs, and 4 of these marker candidates became massively reduced in patient urine post radical prostatectomy, showing great promise as a source of diagnosis/prognosis markers for PCa. Richard et al. (Moncton, Canada, p62) tested a novel urinary EV-based PCa assay, correctly predicting cancer grade as identified in the biopsy sample. Patients scheduled for prostate biopsy provided post-DRE urine. Urinary EVs were isolated either by ultracentrifugation (UC) or by using the fast and easy Vn96 peptide (Vn) protocol. EV protein markers and prostate-specific protein markers were assessed by western blot (WB). Total and small RNAs were extracted from EVs and urine sediment and analyzed for prostate-specific markers. WB revealed the presence of EVs and prostate-specific protein markers and often showed a greater abundance of these biomarkers with Vn-EVs compared to UC-EVs, and improved test specificity for PCA3, when used with the Vn-EVs compared to urine sediment. Vn-based isolation increases PCA3/PSA ratio specificity by 14%. Urinary EV marker investigation led to the discovery of a 5-gene panel (5-bio) and a 10-biomarker panel (10-bio) with predictive PCa diagnosis, based on biopsy outcome. 5-bio used with Vn isolation showed better accuracy than with the UC isolation. 10-bio increased the specificity to 90% and the sensitivity to 75%. Where high-grade PCa (Gleason=7) was identified on the biopsy sample, the PCa assay, using the Vn-EVs, was able to predict the outcome with a sensitivity of 100%. Pink et al. (Edmonton, Canada, p63) developed a biomarker-based extracellular vesicle assay for PCa prognosis, in biofluids (plasma, serum, urine and semen) from healthy men and PCa patients. Five prostate-specific membrane antigen (PSMA) antibodies were tested, and the J591 antibody, which detects an extracellular domain of PSMA, was shown to be optimal. A cohort of 500 samples of plasma and urine were prospectively collected from men prior to prostate biopsy. Detection of PSMA+ EVs was shown to be linear with concentration and assay volume. Preliminary analysis of EVs from platelet-depleted plasma collected pre-biopsy revealed that men testing positive for PCa (+Gleason) had significantly more EVs, than men testing negative (About 1.6-fold increase). PSA levels in these samples were significantly (p=0.0021) but weakly (r2=0.06) correlated with EVs staining positive for PSMA.

 

  1. Castration-Resistant Prostate Cancer (CRPC) & EVs

Foroni et al. (Brescia, Italy, p16) introduced exosome DNA, as a new promising liquid biopsy-based diagnostic tool for personalized management of aggressive (PCa) patients. They compared advantages of liquid biopsy methods based on extracellular vesicle-associated DNA (EV-DNA) and cell-free DNA (cfDNA) to improve the power of detecting the androgen receptor (AR) gene status in biofluid samples. They stated that tumor EV-DNA is more appropriate than total cfDNA for specific and sensitive liquid biopsy approaches for the clinical implementation of novel personalized strategies for PCa patients. Nanou et al. (Enschede, The Netherlands, p26) studied circulating tumor cells (CTC), tumor extracellular vesicles (tdEV) and serum cytokeratins (CK) in the blood of 87 CRPC patients and 16 healthy donors, and investigated their relation with clinical outcome. The levels of circulating cytokeratin 18 (M65) and cleaved cytokeratin 18 (M30) were evaluated with ELISA in the corresponding plasma samples. CTC, tdEV, M30 and M65 values of patients were all significantly higher compared to the values in healthy donors. The presence of ‰¥ 700 U/L of M65, ‰¥ 5 CTC and ‰¥ 500 tdEV in 7.5 mL blood of CRPC patients predicted significantly shorter overall survival in the patient cohort, whereas M30 could not predict survival. Soekmadji et al. (Brisbane, Australia, p62) identified EV-derived miRNAs in advanced PCa. The proliferation and survival of PCa cells are regulated by the presence of steroid hormone androgens, which are also influencing the secretion of EVs in advanced PCa. The LNCaP prostate cancer cells were grown in the presence of fetal bovine serum or in androgen-depleted charcoal-stripped serum (CSS). Cells grown in CSS were treated with 10 nM physiological androgen dihydrotestosterone (DHT) or 10 µM anti-androgen enzalutamide. EVs were isolated from conditioned medium by differential ultracentrifugation and confirmed by EM; miRNAs were extracted and analyzed. Growing cells in the presence and absence of androgens do not significantly alter the yield of total RNA found in isolated EVs. A total of 288 miRNAs were identified across EV samples, with 141 miRNAs (49%) commonly found in all samples, and compared with miRNAs found in the corresponding parental cells (401 miRNAs), where 53.1% (213 miRNAs) were found in all cell samples. 34 candidates miRNAs, which secretion in EVs was increased by >1.5-fold upon DHT treatment, were discovered in comparison with cells grown in androgen-deprived condition. Among these, miR-454 was previously reported to regulate PCa proliferation. In PCa, androgens such as DHT can influence the secretion through EVs of small RNAs, including miRNAs. Fonseca et al. (Solna, Sweden, p62), stressing the need for biomarkers, that can predict disease progression and response to therapy, suggested PCa exosomes as molecular predictors of response to abiraterone acetate. Cancer cell lines, that show increased resistance to abiraterone acetate, were used to isolate exosomes through differential ultracentrifugation. Characterization of these exosomes was followed by their molecular profiling based on a proteomics approach, whereas the protein profile of these exosomes was compared with the one of exosomes from the parental, drug-sensitive cell lines, in order to find a predictive signature of response to therapy. This signature was then validated in exosomes isolated from human plasma samples derived from patients non-responsive to abiraterone acetate. Cells that are resistant to abiraterone acetate secrete more exosomes than their sensitive counterparts. A number of putative markers, that may constitute a predictive signature of response to therapy, were identified. El Sayed et al. (Paris, France, p64) showed that CRIPTO 1 (CR-1), the founding member of EGF-CFC protein superfamily, is associated with tumor aggressiveness in PCa. Prostate normal and cancerous tissue specimens were examined by immunohistochemistry and PCa cell lines were engaged in experimental studies. Significant CR-1 expression was observed in 37.9% of PCa, while being absent or marginally detected in benign conditions. CR-1 overexpression plays a functional role in PCa cells by promoting an epithelial-mesenchymal transition (EMT), associated with enhanced migration capacity and survival under stress conditions, due to propensity to stimulate PI3K/AKT and FGFR1/ERK signaling pathways. Cells overexpressing CR-1 excessively secreted vesicles and CR-1-rich vesicles, a new exosomal form of this protein, stimulated the aggressiveness in prostate cancer.

 

  1. Functions of extracellular vesicles in PCa & CRPC

Chatterjee et al. (Providence, Rhode Island, USA, p3) evidenced an extracellular vesicle-mediated reversal of CRPC. EVs isolated from human mesenchymal stem cells (hMSC) were co-cultured with enzalutamide (Enz) sensitive (C4-2B) or resistant (C4-2BR) PCa cells. hMSC-EV treatment of C4-2BR cells restored sensitivity to Enz via the transfer of Raf kinase inhibitor protein (RKIP). RKIP inhibited STAT3 activation in C4-2BR cells, induced apoptosis, inhibited anchorage independent growth, cell invasion and tumor xenograft formation. hMSC-EVs can reverse the malignant “education” of recipient cells and provide the promising basis to investigate the therapeutic utility of hMSC- EVs for the treatment of CRPC. Lee et al. (Los Angeles, USA, p23) investigated the immunomodulatory properties of large oncosomes from PCa cells. Large oncosomes (LO) contain tumor-derived macromolecules, such as microRNA1227 (miR1227), enriched in EVs from tumorigenic, but not from benign cells. miR1227 was predicted to target the ring finger protein 125 (RNF125), an E3 ubiquitin protein ligase and a known inhibitor of the retinoic acid inducible gene 1 (RIG-I). Macrophages and dendritic cells (DC) take up LO avidly. RNA125 mRNA levels were significantly reduced in DC treated with LO from parental or miR1227 overexpressing-PC3 cells. In the same conditions, DC exhibited altered mRNA levels of select cytokines downstream of the RIG-I pathway (CCL5, IL6, IFNβ1 and TNF). LO might inhibit RNF125 that, in turn, can activate the RIG-I pathway, thus reprogramming the immune system in favour of tumor progression, a new molecular mechanism underlying the modulation of the immune response to PCa. Yeung et al. (Cardiff, UK, p63) identified components that regulate the secretion of stroma-activating exosomes in PCa. With the assumption of coexisting parallel pathways for generating and trafficking distinct MVs, hence providing exosome heterogeneity, they investigated the roles of 6 proteins, CD9, Rab5a, Rab11b, Rab35, VAMP7 and VPS25, putatively implicated in exosome biogenesis/secretion, and their subsequent functional impact on stromal cells. A lentiviral-based delivery of shRNAs was used to selectively knockdown targets of interest in DU145 cells. Successful stable knockdown of all targets, by at least 80%, was demonstrated at both mRNA and protein levels. Knockdown of Rab35 or VPS25 resulted in the greatest attenuation of exosome secretion, with a corresponding failure to induce stromal activation, compared to functionally competent control DU145 cells. Both Rab35 and VPS25 are relevant factors for modulating exosome secretion by DU145 prostate cancer cells. Targeting these proteins may be sufficient to attenuate exosome-mediated stromal cell activation and slow tumor growth. Salimu et al. (Cardiff, UK, p63) studied the immunosuppressive effects of tumor exosomes on dendritic cells cross-presenting tumor antigens. DU145 cells were transduced with either shRNA lentiviral particles to knockdown Rab27a (DU145KD) or an irrelevant control (DU145C). Rab27a deficiency was characterized for attenuation of exosome secretion. Cross-presentation of the tumor-associated antigen 5T4 from DU145KD cells generated significantly stronger anti-5T4 T-cell responses compared to that from DU145C cells. This enhanced T-cell response was reduced, when purified exogenous DU145 exosomes were added back to the cross-presentation model incorporating DU145KD cells. Knockdown of exosome secretion alleviates tumor-associated immunosuppression. While exosomes did not directly inhibit CD8+T-cell function, they exerted a negative effect on DC function. DC exposed to exosomes had an elevated expression of CD73, an ecto-5-nucleotidase responsible for AMP to adenosine hydrolysis. DC constitutively express CD39 (ectonucleotidase responsible for ATP to AMP hydrolysis) which, when co-expressed with CD73, resulted in ATP-dependent inhibition of TNFα and IL-12 release. Inhibition of PGE2 receptors inhibited exosome-dependent CD73 expression on DC. The results revealed a hitherto unknown effect of tumor exosomes on the suppression of DC function via exosomal PGE2, adding a new element to tumor-immune cell crosstalk. Minciacchi et al. (Los Angeles, CA, USA, p79) observed that large oncosomes reprogram prostate fibroblasts (NAF) towards an angiogenic phenotype. Active AKT1 is significantly more expressed and functional in LO than in exosomes (Exo). Patients with metastatic disease express abundant active AKT1 in plasma LO. Uptake of LO harbouring active AKT1 by NAF results in AKT1 and c-MYC activation. Conditioned media from LO-treated NAF, but not from Exo-treated NAF, promoted endothelial morphogenesis. The dynamin (DNM) inhibitor dynasore (Dyn) inhibited LO uptake, as well as MYC activation and tube formation. LO uptake occurs by phagocytosis. MYC activation is an early event in cancer development. Tumor-derived LO induced a novel, c-MYC mediated, pro-tumorigenic reprogramming of fibroblasts that can be reverted by selectively inhibiting LO uptake. Van Driel et al. (Rotterdam, The Netherlands, p126) questioned the positive effects of vitamin D (1a, 25-OH2D3) in bone metastasis and the role for EVs. The aim was to investigate the potential of vitamin D, as a novel bone-targeted therapy for bone metastatic cancer and study underlying mechanisms involving EVs, with a human co-culture model of differentiating osteoblasts (SV-HFO) and bone metastatic PCa cells (PC-3). EVs released by PC-3 cells were isolated via ultracentrifugation (20,000 x g and 100,000 x g) and quantified on a NanoSight NS300. The effect of 1a, 25-OH2D3 (10-7 M) was studied on (1) osteoblast differentiation (alkaline phosphatase (ALP) activity), mineralization (calcium) and PC-3 growth (FACS), (2) EV production by PC-3 cells (NanoSight) and (3) PC-3 EV interaction with osteoblasts (ALP activity). 1a, 25-OH2D3 treatment recovered the negative effects of PC-3 cells on osteoblast differentiation and mineralization and significantly reduced cancer cell growth by 80%. Interestingly, the number of EVs produced by PC-3 cells is increased by 1a, 25-OH2D3 (20,000 x g fraction 10-fold, 100,000 x g fraction 2-fold). EVs from bone metastatic PCa cells negatively affect osteoblast differentiation. This is the first observation, that vitamin D may act as a therapeutic agent in bone metastasis, with an involvement of EVs.

 

  1. Therapeutical use of extracellular vesicles

R.Gupta et al. (Louisville, Kentucky, USA, p2) proposed milk-derived exosomes, as a platform nanocarrier to enhance anti-proliferative, anti-inflammatory and anti-cancer activities of small drug molecules against multiple human cancers, including PCa. All tested agents as Exo formulation showed 2- to 20-fold higher antiproliferative activity against the various cancer cell lines versus free agents.

 

Main results reported about “Prostate” at the ISEV 2017 Meeting (16)

The recent ISEV 2017 Meeting brought new promising insights into the field. Indeed, the 30 abstracts dealing with “Prostate” were still disseminated in 20 different sessions, with only six posters gathered in the session entitlled ” Body-fluid Biomarkers of Cancer”, showing that there is still no precise research guiding method for the best utilization of EVs for PCa theranostics. Nevertheless, some outstanding observations were reported, concerning the propagation of the EV-mediated metastatic process in PCa. First, oncosomes with a diameter > 900 nm, were shown to be specifically released by actively extravasating cancer cells in vivo (F. Deng et al. p.81), stressing the importance of considering the whole EV panel for tracking the cancer process and not only exosomes. The oncosomes, previously identified in tumor tissue and in plasma of prostate cancer patients, were now detected in urine samples of prostate cancer patients, providing an alternative, non-invasive access to circulating diagnosis/prognosis molecular signatures of cancer cells. (T. Vagner et al. p. 169). Secondly, for the first time, it was clearly demonstrated that among the already recognised heterogeneity of exosomes, exhibiting different protein profiles, only about 20% were actively involved for triggering a cancer associated fibroblast phenotype and mediate pro-invasive behaviour within tumor stroma spheroids (V. Yeung et al. p.30). Lastly, normal epithelial cells (RWPE-1) were incubated for 96h with exosomes isolated from plasma from individuals without cancer (n=10), PCa patients with Gleason score 6 (n=10), > or=7 (n=10), and castration-resistant disease (CRPC) (n=5). It was shown that the content of circulating exosomes is modified according to malignancy of PCa and triggers phenotypical changes that may promote cancer progression and metastasis, with a greater efficiency for CRPC patient-derived exosomes (E. Andahur et al. p. 112). The importance of these observations will be further discussed in the conclusion.

 

Conclusion

This six years survey of the abstracts devoted to Prostate” in the successive ISEV yearly meetings from 2012 to 2017 depicts the important international involvement of the teams convinced upon the potential interest of EVs for the future theranostics of Prostate Cancer. Many interesting observations have already been collected about the characterization and functional properties of PCa cells-derived EVs, and some of the most noteworthy ones are reported below for following the evolution of research about EVs and PCa.

During the whole 2012-2017 period, very few reports were concerned with the physiologic function of normal prostate cell-derived EVs or in vivo studies. From the beginning, the preponderant aims were the search for PCa biomarkers and the disclosure of the mechanisms involved in the biological functions of tumor cell-derived EVs.

At ISEV 2012, three new methods for detection of exosomes / MVs were described, but the major goal was to characterize PCa cell-derived EVs. The searches were first mainly focused on the proteomic content of exosomes or MVs released by different cell lines, related either to normal prostate or to PCa at various stages of disease. One team was also interested in the lipidomic content of PCa-derived exosomes, when searching for novel biomarkers and therapeutic targets for CRPC. One team analyzed for the first time MVs in blood and urine, correlated with health and disease, including PCa. Only one study questioned the microRNA profiling of PCa cell-derived oncosomes, with identification of a signature for PCa progression and metastasis. The abundance of tumor cell-released MVs correlated positively with the advanced grade of cancer progression. The mode of tumor cell-derived EVs by normal recipient cells was rapidly questioned, and the uptake of exosomes was shown to occur rather by endocytosis than by fusion with the cell plasma membrane. The biological functions of PCa EVs as mediators of intercellular communication with the tumoral environment was also early investigated. Cancer cell exosomes can trigger normal-prostate stromal cell to acquire a phenotype and function of disease-derived stroma. This exosome effect was TGF-β1 dependent and could not be reproduced with soluble TGF-β1. PCa-derived exosomes were also shown to mediate immune evasion and to transfer docetaxel resistance, partly by exosomal MDR1/P-gp transfer.

These first observations were quite promising, and the number of prostate-related reports increased in 2013 from 18 to 29, but remained about the same yearly thereafter. At ISEV 2013 many new methods for isolation of exosomes/MVs were described and tested with prostate cell lines EVs. Biobanking of microvesicles of patient samples was elaborated in Helsinki urological biobank for users specifically working on PCa. PCa-derived MVs were enumerated for follow-up after prostatectomy or analyzed by multicolour flow cytometry. Moreover, PCa-derived EVs were further characterized by proteomics, lipidomics and microRNA profiling, either from cell lines or, more often, from patient’s body fluids. Exosomes were found to be highly enriched in glycosphingolipids, sphingomyelin and cholesterol. Different integrins proteins were identified in exosomes of PCa cell lines and expression of these integrins was significantly higher in urine exosomes of metastatic PCa patients. Transcription factors were identified in circulating MVs from PCa patients. The influence of normal / tumor EVs on recipient cells was actively continued, specifically targeting reversal of chemosensitivity, induction of cell malignancy, inhibition of mesenchymal stem cell differentiation and suppression of immune cells proliferation.

At ISEV 2014, characterization of PCa-derived EVs was mostly focused on patients and search for RNA biomarkers. However, a study compared, for the first time, gDNA fragments present in different EV subpopulations (exosomes, microvesicles, apoptotic bodies). The uptake mechanisms of exosomes and large oncosomes were more precisely assigned to active endocytic processes. EV-mediated differentiation of mesenchymal stem cells into myofibroblasts was further described. An exosomes-mediated reduction of proliferation of androgen-sensitive PCa cells was also reported, as well as the EV-mediated reversal of the malignant phenotype in PCa.

At ISEV 2015, comparison of isolation methods of exosomes from biofluids showed that plasma proteins were removed (> 95%), using size exclusion chromatography (SEC), but not with ultracentrifugation (UC) or filtration and gradient UC. The huge diversity of EVs in normal human ejaculates was pointed out, whereas the properties of large oncosomes were compared with those of exosomes. The studies dealing with the characterization of EVs from patients at different stages of PCa, compared with healthy controls, increased. Exosomal markers for taxane resistance and progression of PCa were identified. A urine exosome gene signature predictive of aggressive PCa was announced. PCa biomarkers were also found in platelets. Exosomes were shown to confer pro-survival signals to alter the phenotype of PCa cells in their surrounding environment. Exosome-mediated transfer of alphaV-integrins was evidenced as a novel pathway leading to increased PCa metastasis in distinct distant sites. Androgens were shown to regulate the secretion of EV subpopulations. The influence of hypoxia on EV biogenesis was questioned and targeted exosome delivery began to be considered.

At ISEV 2016, beside the three main topics about PCa-derived EVs studied as previously and, more and more in depth, other approaches appeared. Large-scale EV production was improved by using a two-compartment Celline AD 1000 bioreactor (Integra) for growth of PC-3 prostate cancer cells (ATCC, USA) and T-175 cell culture flasks for comparison. The cell-conditioned medium (CM) was harvested three times per week from the 2D cultures and once per week from the bioreactor cell compartment. The PC-3 cells in the bioreactor produced approximately 1-2 x 1012 MVs and 2-4 x 1012 exosomes from 15 ml of CM, while a single 2D cell culture flask produced approximately 1-2 x 1010 and 8-11 x 109 of those EVs, respectively, from 25 mL of CM over 7 days, showing that bioreactors have the potential to improve EV production, but this needs a further careful comparison of EVs produced in both conditions. The metabolomics of urinary EVs began to be explored: EVs purified from urine samples of three PCa patients before and after radical prostatectomy and of three controls, were subjected together with their source urine samples to UPLC-MS-MS analysis of 102 metabolites. 32-55% of the screened metabolites were quantified in EV samples, containing as little as 6 x 109 EVs, or in EVs from 10 mL of urine. The EV metabolome differed clearly from the one of urine. While most of the metabolites were more abundant in urine, some were better detected in EVs. All EV samples contained nucleotides, amino acids, vitamin B, carnitine, amines or related metabolites representing mainly the urea cycle, purine nucleotide, glutathione and carnitine shuttle pathways. A relatively small number of EVs is sufficient for detection of EV metabolites. The observed changes in the cancer samples suggest that EV metabolites could also act as PCa biomarkers.

PCa diagnosis was mostly searched by means of EV-specific proteins from urine, seeking for new isolation methods, followed by comprehensive proteomics. For example, an optimized isolation of EVs from urine by aqueous two-phase systems (ATPS) was suggested, with an efficiency of 100% and total processing time ~35 min. This method recovered ~100% of EVs from the urine, whereas ultracentrifuged-pellet methods recovered only ~30% of total EVs. Novel biomarkers were identified in urinary exosomes for early non-invasive detection of PCa. Significant differences in the expression level of PCA3 (PCa mRNA marker) /ACTIN demonstrated that PCa might be successfully distinguishable from BPH. A profile of two novel urinary exosome-associated protein biomarkers was selected after the comparison between benign and PCa patients and a promising profile of 5 proteins was claimed to be able to significantly distinguish between high (Gleason =7 (4+3)) and low (Gleason =7 (3+4)) risk patients.

PCa diagnosis was also searched by means of EV-specific miRNas. Exosome samples from urine contained mostly small RNAs, with 24 highly upregulated exosomal miRNAs, and 4 of these marker candidates became massively reduced in patient urine post radical prostatectomy, showing great promise as a source of diagnosis/prognosis markers for PCa.

A biomarker-based extracellular vesicle assay in biofluids (plasma, serum, urine and semen) was developed from healthy men and PCa patients for PCa prognosis. Five prostate-specific membrane antigen (PSMA) antibodies were tested, and the J591 antibody, which detects an extracellular domain of PSMA, was shown to be optimal.

The interest for EVs entered also the field of CRPC. Cells overexpressing CRIPTO (CR-1), the founding member of EGF-CFC protein superfamily, excessively secreted vesicles and CR-1-rich vesicles, a new exosomal form of this protein, stimulated the aggressiveness in prostate cancer. Exosome DNA was introduced as a new promising liquid biopsy-based diagnostic tool, and found more appropriate than total cell-free DNA (cfDNA) to improve the power of detecting the androgen receptor (AR) gene status in biofluid samples, for personalized management of aggressive (PCa) patients. EV-derived miRNAs were also evidenced in advanced PCa. PCa exosomes were suggested as molecular predictors of response to abiraterone acetate. EVs isolated from human mesenchymal stem cells (hMSC) were able to reverse the malignant “education” of recipient cells and provide the promising basis to investigate the therapeutic utility of hMSC- EVs for the treatment of CRPC.

With regard to the functions of EVs in PCa, large oncosomes (LO) were found to mediate a new molecular mechanism, reprogramming the immune system in favor of tumor progression and underlying the modulation of the immune response to PCa. Large oncosomes were also observed to reprogram prostate fibroblasts (NAF) towards an angiogenic phenotype. Tumor-derived LO induced a novel, c-MYC mediated, pro-tumorigenic reprogramming of fibroblasts that could be reverted by selectively inhibiting LO uptake. On the other hand, the roles of 6 proteins, CD9, Rab5a, Rab11b, Rab35, VAMP7 and VPS25, putatively implicated in exosome biogenesis/secretion, were investigated. Knockdown of Rab35 or VPS25 resulted in the greatest attenuation of exosome secretion. Targeting these proteins may be sufficient to attenuate exosome-mediated stromal cell activation and slow tumor growth.

 For the first time, vitamin D was claimed to act as a therapeutic agent in bone metastasis, with an involvement of EVs, and milk-derived exosomes were proposed, as a platform nanocarrier to enhance anti-proliferative, anti-inflammatory and anti-cancer activities of small drug molecules against multiple human cancers, including PCa. All tested agents as Exo formulation showed 2- to 20-fold higher antiproliferative activity against the various cancer cell lines versus free agents.

While greatly increasing the knowledge about PCa- derived EVs, the diversity of observations at ISEV 2012-2016 gave the bad feeling that “too much informations kills the information”. It seemed then quite difficult to find the “right thread”, in order to efficiently unravel the most important processes involved in prostate cancerization under the light of tumor cells-derived EVs. Hence, it was not easy to have a clear view about how to use these EVs for PCa early diagnosis, CRPC prognosis and therapy. This disappointing conclusion fits with the ones of Junker et al. (17) who, using PubMed, performed a systematic literature search of articles published between 2005 and 2015 regarding EVs in different types of urologic tumor diseases. With regard to prostate cancer, they agree that “research has mainly focused on the content inventory of the PCa vesicles”.

However, the ISEV 2017 new findings stressed for the first time the importance of sorting the wide EV panel into well characterized subpopulations, in order to associate their specialized biological functions to the appropriate subpopulation(s). This will indeed be the next important challenge in the field. Beside the recent work of Park et al. (18), suggesting the mere amount of prostate-specific EVs, as a simple novel selective biomarker in human PCa, one might suggest that it is now time to search for (a) specific EV subpopulation (s) into prostate cells-derived EVs, as a function of the Gleason score. These PCa specific EVs might, f.ex., carry some known EV-transported proteins such as PSA/PMSA as prostate markers, and the tumor-suppressor PTEN (for Phosphatase TENsin homolog), known to be present in many tumors and in PCa as well, as cancer marker, together with a “house-keeping” compound such as GAPDH for normalisation. It would be worth checking if the analysis of such specifically designed EVs might positively help PCa diagnosis and prognosis of agressive CRPC.

 

Acknowledgments

I thank AM. & R. Stribley for their final English reading of the manuscript.

 

Abbreviations:

1a, 25-OH2D3, vitamin D;

ATPS, aqueous two-phase system;

BPH, begnin prostate hyperplasia;

CK, serum cytokeratin;

CM, conditioned medium;

CR1, CRIPTO-1 protein;

CRPC, castration-resistant prostate cancer;

DC, dendritic cell;

DHT, dihydrotestosterone;

DRE, digital rectal exam;

EVs, extracellular vesicles;

Exo, exosome;

hMSC, human mesenchymal stem cell;

HSP; heat shock protein;

ISEV, International Society for EVs;

LINC, complex linking cyto- and nucleo-skeleton

LO, large oncosome;

M30, cleaved CK18;

M65, circulating CK18;

miRNAs, micro-RNas;

MVs, microvesicles;

PCa, prostate cancer; 

PSA, prostate specific antigen;

PSMA, prostate specific membrane antigen;

SEC, size exclusion chromatography;

UC, ultracentrifugation;

Vn, Vn96 peptide protocol;

 

Human PCa cell lines: PC3, P346C, VCAP, RWPE-2, DU145, LNCaP, 22Rv1, WPE1-NB26

Human normal prostate cell lines: PNT2C2, RWPE-1, CRL2221

 

 

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