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Mammalian conception involves a multitude of reciprocal interactions via a molecular dialogue between mother and conceptus. Extracellular vesicles (EVs) are secreted membrane-encapsulated particles that mediate cell-to-cell communication in various contexts. EVs, which are present in seminal, follicular, oviductal, and endometrial fluids, as well as in embryo secretions, carry molecular constituents that impact gamete maturation, fertilization, early embryo development, and embryo-maternal communication. The distribution, concentration, and molecular cargo of EVs are regulated by steroid hormones and the health status of the tissue of origin, and thus are influenced by menstrual phase, stage of conception, and the presence of infertility-associated diseases. EVs have been recognized as a novel source of biomarkers and potential reproductive medicine therapeutics, particularly for assisted reproductive technology (ART). There are still many technological and scientific hindrances to be overcome before EVs can be used in clinical diagnostic and therapeutic ART applications. Issues to be resolved include the lack of standardized measurement protocols and an absence of absolute EV quantification technologies. Additionally, clinically suitable and robust EV isolation methods have yet to be developed. In this review, we provide an overview of EV-mediated interactions during the early stages of reproduction from gamete maturation to embryo implantation and then outline the technological progress that must be made for EV applications to be translated to clinical settings.
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Vesículas Extracelulares , Animales , Implantación del Embrión , Embrión de Mamíferos , Femenino , Fertilización , Mamíferos , ReproducciónRESUMEN
Extracellular vesicles (EVs) have attracted considerable interest due to their role in cell-cell communication, disease diagnosis, and drug delivery. Despite their potential in the medical field, there is no consensus on the best method for separating micro- and nanovesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation of a single class of EVs, such as exosomes, is complex because blood and cell culture media contain many nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high-purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles usually requires harsh conditions that hinder their use in certain types of downstream analysis. A novel capture and release approach for small extracellular vesicles (sEVs) is presented based on DNA-directed immobilization of antiCD63 antibody. The flexible DNA linker increases the capture efficiency and allows for releasing EVs by exploiting the endonuclease activity of DNAse I. This separation protocol works under mild conditions, enabling the release of vesicles suitable for analysis by imaging techniques. In this study, sEVs recovered from plasma were characterized by established techniques for EV analysis, including nanoparticle tracking and transmission electron microscopy.
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Exosomas , Vesículas Extracelulares , Nanopartículas , Sistemas de Liberación de Medicamentos , Fenómenos MagnéticosRESUMEN
The relevance of extracellular vesicles (EVs) has grown exponentially, together with innovative basic research branches that feed medical and bioengineering applications. Such attraction has been fostered by the biological roles of EVs, as they carry biomolecules from any cell type to trigger systemic paracrine signaling or to dispose metabolism products. To fulfill their roles, EVs are transported through circulating biofluids, which can be exploited for the administration of therapeutic nanostructures or collected to intercept relevant EV-contained biomarkers. Despite their potential, EVs are ubiquitous and considerably heterogeneous. Therefore, it is fundamental to profile and identify subpopulations of interest. In this study, we optimized EV-labeling protocols on two different high-resolution single-particle platforms, the NanoFCM NanoAnalyzer (nFCM) and Particle Metrix ZetaView Fluorescence Nanoparticle Tracking Analyzer (F-NTA). In addition to the information obtained by particles' scattered light, purified and non-purified EVs from different cell sources were fluorescently stained with combinations of specific dyes and antibodies to facilitate their identification and characterization. Despite the validity and compatibility of EV-labeling strategies, they should be optimized for each platform. Since EVs can be easily confounded with similar-sized nanoparticles, it is imperative to control instrument settings and the specificity of staining protocols in order to conduct a rigorous and informative analysis.
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Vesículas Extracelulares/metabolismo , Fluorescencia , Colorantes Fluorescentes/química , Nanopartículas/química , Nanotecnología/métodos , Coloración y Etiquetado/métodos , Vesículas Extracelulares/química , Citometría de Flujo/métodos , Células HEK293 , Células HT29 , HumanosRESUMEN
The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role.
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Vesículas Extracelulares/química , Ensayos Analíticos de Alto Rendimiento , Microscopía de Fuerza Atómica , Nanotecnología , Animales , Ascaris suum/química , Bovinos , Células HCT116 , Humanos , Liposomas/química , Leche/químicaRESUMEN
STUDY QUESTION: Are there differences in the proteomic profile of exosomes isolated from seminal plasma of normozoospermic (NSP) and severe asthenozoospermic (SA) men, potentially contributing to sperm features? SUMMARY ANSWER: A relevant group of proteins known to positively regulate sperm functions were over-represented in seminal exosomes of NSP men, i.e. cysteine-rich secretory protein-1 (CRISP1), while the inhibitory protein glycodelin was enriched in exosomes of SA subjects. WHAT IS KNOWN ALREADY: Exosomes are secreted along the male reproductive tract and are thought to be involved in spermatozoa maturation and function. Ejaculated spermatozoa are still able to capture exosomes; exosomes of NSP individuals improve sperm motility and prompt capacitation, while exosomes of SA men fail to exert similar features. STUDY DESIGN, SIZE, DURATION: Semen samples from NSP and SA men, aged 18 to 55 and registered at a single IVF center, were considered for this study project. Subjects were subdivided into three groups: a discovery cohort (five NSP men and six SA patients), a validation cohort (seven NSP and seven SA men) and the 'glycodelin analysis' cohort (20 NSP and 37 SA men). Exosomes were purified from semen of every participant. PARTICIPANTS/MATERIALS, SETTING, METHODS: Exosomes were characterized by nanoparticle tracking analysis, transmission electron microscopy and western blot. Comprehensive proteomics analysis of the exosomal proteome was performed by nanoscale liquid chromatographic tandem mass spectrometry analysis. Funrich software was used to determine statistical enrichment of pathways, networks and Gene Ontology terms of the identified proteins. Validation of differentially expressed proteins was performed through ELISA and western blot analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The comprehensive proteomic analysis identified a total of 2138 proteins for both groups. There were 89 proteins found to be differentially expressed in exosomes of NSP versus SA subjects, of which 37 were increased in the NSP group and 52 were increased in the SA group. One-third of the exosomes-associated proteins highly expressed in NSP samples were involved in the reproductive process; conversely, the over-expressed proteins in exosomes of SA samples were not functionally specific. Quantitative data were confirmed on seminal exosomes from different cohorts of subjects. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Transfer of the proteins from exosomes to spermatozoa has been only partially demonstrated and up-take mechanisms are still poorly defined. WIDER IMPLICATIONS OF THE FINDINGS: Seminal exosomes carry proteins that are potentially able to either favour or inhibit the reproductive process in humans. A better understanding of these phenomena might pave the way for novel intervention measures in terms of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Italian Ministry of Health through an Institution Seed Grant. None of the authors has any competing interests.
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Astenozoospermia/metabolismo , Exosomas/metabolismo , Glicodelina/metabolismo , Semen/metabolismo , Espermatozoides/metabolismo , Adolescente , Adulto , Humanos , Masculino , Persona de Mediana Edad , Proteómica , Análisis de Semen , Motilidad Espermática/fisiología , Adulto JovenRESUMEN
Exosomes are naturally secreted nanovesicles that have recently aroused a great interest in the scientific and clinical community for their roles in intercellular communication in almost all physiological and pathological processes. These 30-100nm sized vesicles are released from the cells into the extracellular space and ultimately into biofluids in a tightly regulated way. Their molecular composition reflects their cells of origin, may confer specific cell or tissue tropism and underlines their biological activity. Exosomes and other extracellular vesicles (EVs) carry specific sets of proteins, nucleic acids (DNA, mRNA and regulatory RNAs), lipids and metabolites that represent an appealing source of novel noninvasive markers through biofluid biopsies. Exosome-shuttled molecules maintain their biological activity and are capable of modulating and reprogramming recipient cells. This multi-faceted nature of exosomes hold great promise for improving cancer treatment featuring them as novel diagnostic sensors as well as therapeutic effectors and drug delivery vectors. Natural biological activity including the therapeutic payload and targeting behavior of EVs can be tuned via genetic and chemical engineering. In this review we describe the properties that EVs share with conventional synthetic nanoparticles, including size, liposome-like membrane bilayer with customizable surface, and multifunctional capacity. We also highlight unique characteristics of EVs, which possibly allow them to circumvent some limitations of synthetic nanoparticle systems and facilitate clinical translation. The latter are in particular correlated with their innate stability, ability to cross biological barriers, efficiently deliver bioactive cargos or evade immune recognition. Furthermore, we discuss the potential roles for EVs in diagnostics and theranostics, and highlight the challenges that still need to be overcome before EVs can be applied to routine clinical practice.
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Vesículas Extracelulares , Neoplasias/terapia , Animales , Humanos , Nanomedicina Teranóstica , TropismoRESUMEN
Clinical implementation of exosome based diagnostic and therapeutic applications is still limited by the lack of standardized technologies that integrate efficient isolation of exosomes with comprehensive detection of relevant biomarkers. Conventional methods for exosome isolation based on their physical properties such as size and density (filtration, ultracentrifugation or density gradient), or relying on their differential solubility (chemical precipitation) are established primarily for processing of cell supernatants and later adjusted to complex biological samples such as plasma. Though still representing gold standard in the field, these methods are clearly suboptimal for processing of routine clinical samples and have intrinsic limits that impair their use in biomarker discovery and development of novel diagnostics. Immunoisolation (IA) offers unique advantages for the recovery of exosomes from complex and viscous fluids, in terms of increased efficiency and specificity of exosome capture, integrity and selective origin of isolated vesicles. We have evaluated several commercially available solutions for immunoplate- and immunobead-based affinity isolation and have further optimized protocols to decrease non-specific binding due to exosomes complexity and matrix contaminants. In order to identify best molecular targets for total exosome capture from diverse biological sources, as well as for selective enrichment in populations of interest (e.g. tumor derived exosomes) several exosome displayed proteins and respective antibodies have been evaluated for plate and bead functionalisation. Moreover, we have optimized and directly implemented downstream steps allowing on-line quantification and characterization of bound exosome markers, namely proteins and RNAs. Thus assembled assays enabled rapid overall quantification and validation of specific exosome associated targets in/on plasma exosomes, with multifold increased yield and enrichment ratio over benchmarking technologies. Assays directly coupling selective immobilization of exosomes to a solid phase and their immune- and or molecular profiling through conventional ELISA and PCR analysis, resulted in easy-to-elaborate, quantitative readouts, with high low-end sensitivity and dynamic range, low costs and hands-on time, minimal sample handling and downscaling of a working plasma volumes to as few as 100 µl.
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Biomarcadores de Tumor/aislamiento & purificación , Neoplasias del Colon/sangre , Exosomas/química , Proteínas de Neoplasias/aislamiento & purificación , Neoplasias de la Próstata/sangre , ARN Neoplásico/aislamiento & purificación , Transporte Biológico , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Fraccionamiento Celular/métodos , Línea Celular Tumoral , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Masculino , Proteínas de Neoplasias/genética , Células Neoplásicas Circulantes , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Neoplásico/sangre , ARN Neoplásico/genética , UltracentrifugaciónRESUMEN
Extracellular vesicles (EVs) have mostly been investigated as carriers of biological therapeutics such as proteins and RNA. Nevertheless, small-molecule drugs of natural or synthetic origin have also been loaded into EVs, resulting in an improvement of their therapeutic properties. A few methods have been employed for EV cargo loading, but poor yield and drastic modifications of vesicles remain unsolved challenges. We tested a different strategy based on temporary pH alteration through incubation of EVs with alkaline sodium carbonate, which resulted in conspicuous exogenous molecule incorporation. In-depth characterization showed that vesicle size, morphology, composition, and uptake were not affected. Our method was more efficient than gold-standard electroporation, particularly for a potential therapeutic toxin: the plant Ribosome Inactivating Protein saporin. The encapsulated saporin resulted protected from degradation, and was efficiently conveyed to receiving cancer cells and triggered cell death. EV-delivered saporin was more cytotoxic compared to the free toxin. This approach allows both the structural preservation of vesicle properties and the transfer of protected cargo in the context of drug delivery.
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Nanomedicine aims to exploit the improved and often novel physical, chemical, and biological properties of materials at the nanometric scale, possibly with the highest level of biomimetism, an approach that simulates what occurs in nature. Although extracellularly released vesicles include both microvesicles (MVs) and exosomes, only exosomes have the size that may be considered suitable for potential use in nanomedicine. In fact, recent reports have shown that exosomes are able to interact with target cells within an organ or at a distance using different mechanisms. Much is yet to be understood about exosomes, and currently, we are looking at the visible top of an iceberg, with most of what we have to understand on these nanovesicles still under the sea. In fact, we know that exosomes released by normal cells always trigger positive effects, whereas those released by cells in pathological condition, such as tumor or infected cells, may induce undesired, dangerous, and mostly unknown effects, but we cannot exclude the possibility that exosomes may also be detrimental for the body in normal conditions. However, whether we consider extracellular vesicles as a whole, thus including MVs, it appears that even in normal conditions, extracellular vesicles may lead to unwanted effects, depending on gender and age. This review aims to critically emphasize existing data in the literature that support the possible roles of exosomes in both diagnostic and therapeutic scopes.
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Sistemas de Liberación de Medicamentos , Exosomas/química , Exosomas/metabolismo , Nanopartículas/química , Neoplasias/diagnóstico , Neoplasias/terapia , Animales , Biomarcadores de Tumor/análisis , Humanos , Lípidos/química , MicroARNs/química , MicroARNs/metabolismo , Proteínas/química , Proteínas/metabolismoRESUMEN
Extracellular vesicles (EVs) are lipid membrane-bound particles involved in cell-to-cell communication through a delivery of regulatory molecules essential for physiological processes. Since EVs efficiently vectorize specific cargo molecules, they have been proposed as suitable vehicles for therapeutic agents. Drug loading into EVs can be achieved by active, exogenous strategies or by genetic modifications of vesicle-producing cells. With the aim to produce EVs conveying therapeutic proteins, we genetically engineered and compared HEK293 to tumor cells. Tetraspanin-based RFP fusions were found to be more stable and preferentially sorted into EVs in HEK293. EVs isolated from genetically modified HEK293 cells media were captured by cancer cells, efficiently delivering their cargo. Cathepsin B cleavage site introduced between CD9/CD81 and RFP was recognized by tumor specific proteases allowing the release of the reporter protein. Our results indicate HEK293 cells as a preferential system for the production of EVs and pave the way to the development of nano-platforms for the efficient delivery of therapeutic proteins and prodrugs to tumor cells.
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Vesículas Extracelulares , Neoplasias , Humanos , Células HEK293 , Vesículas Extracelulares/metabolismo , Proteínas/metabolismo , Transporte de Proteínas , Neoplasias/metabolismo , Comunicación Celular , Tetraspanina 28/metabolismo , Tetraspanina 29/metabolismoRESUMEN
BACKGROUND: Blood plasma is available with minimal invasive sampling, it has significant diagnostic utility, and it is a valuable source of extracellular vesicles (EVs). Nevertheless, rich protein content, the presence of lipoproteins (LPs) that share similar biophysical properties, and relatively low abundance of EVs, especially those of rare subpopulations, make any downstream application a very challenging task. The growing evidence of the intricate surface interactome of EVs, and the association of EVs with LPs, impose further challenges during EV purification, detection, and biomarker analyses. OBJECTIVES: In this study, we tackled the fundamental issues of plasma EV yield and LP co-isolation and their implications in the subsequent marker analyses. METHODS: Moderate acidification of plasma was combined with size exclusion chromatography (SEC) and/or differential centrifugation (DC) to disrupt LPs and improve recovery of EVs and their subsequent detection by immunoassays and single-particle analysis methods. RESULTS: Our results demonstrate a surprisingly efficient enrichment of EVs (up to 3.3-fold higher than at pH 7) and partial depletion of LPs (up to 61.2%). Acidification of blood plasma samples enabled a quick single-step isoelectric precipitation of up to 20.4% of EVs directly from plasma, upon short low-speed centrifugation. CONCLUSION: Thus, acidification holds potential as a simple and inexpensive methodological step, which improves the efficacy of plasma EV enrichment and may have implications in future biomarker discoveries.
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Vesículas Extracelulares , Lipopolisacáridos , Humanos , Lipopolisacáridos/metabolismo , Vesículas Extracelulares/metabolismo , Lipoproteínas/metabolismo , Cromatografía en Gel , Plasma/metabolismo , Concentración de Iones de Hidrógeno , BiomarcadoresRESUMEN
Urine features an ideal source of non-invasive diagnostic markers. Some intrinsic and methodological issues still pose barriers to its full potential as liquid biopsy substrate. Unlike blood, urine concentration varies with nutrition, hydration and environmental factors. Urine is enriched with EVs from urinary-genital tract, while its conservation, purification and normalization can introduce bias in analysis of EV subsets in inter-and intra-individual comparisons. The present study evaluated the methods that decrease such biases such as appropriate and feasible urine storage, optimal single-step EV purification method for recovery of proteins and RNAs from small urine volumes and a normalization method for quantitative analysis of urine EV RNAs. Ultracentrifugation, chemical precipitation and immuno-affinity were used to isolate EVs from healthy donors' urine that was stored frozen or at room temperature for up to 6 months. Multiple urine biochemical and EV parameters, including particle count and protein content, were compared across urine samples. To this purpose nanoparticle tracking analysis (NTA) and protein assessment by BCA, ELISA and WB assays were performed. These measurements were correlated with relative abundances of selected EV mRNAs and miRNAs assessed by RT-PCR and ranked for the ability to reflect and correct for EV content variations in longitudinal urine samples. All purification methods enabled recovery and downstream analysis of EVs from as few as 1 ml of urine. Our findings highlight long term stability of EV RNAs upon urine storage at RT as well as excellent correlation of EV content in urine with some routinely measured biochemical features, such as total urine protein and albumin, but not creatinine most conventionally used for urine normalization. Comparative evaluation of mRNA and miRNAs in EV isolates revealed specific RNAs, in particular RNY4 and small miRNA panel, levels of which well reflected the inter-sample EV variation and therefore useful as possible post-analytical normalizers of EV RNA content. We describe some realistic urine processing and normalization solutions for unbiased readout of EV biomarker studies and routine clinical sampling and diagnostics providing the input for design of larger validation studies employing urine EVs as biomarkers for particular conditions and diseases.
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Vesículas Extracelulares , MicroARNs , Vesículas Extracelulares/metabolismo , MicroARNs/genética , Biomarcadores/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Albúminas/metabolismoRESUMEN
BACKGROUND: Intercellular communication is mediated by extracellular vesicles (EVs), as they enclose selectively packaged biomolecules that can be horizontally transferred from donor to recipient cells. Because all cells constantly generate and recycle EVs, they provide accurate timed snapshots of individual pathophysiological status. Since blood plasma circulates through the whole body, it is often the biofluid of choice for biomarker detection in EVs. Blood collection is easy and minimally invasive, yet reproducible procedures to obtain pure EV samples from circulating biofluids are still lacking. Here, we addressed central aspects of EV immunoaffinity isolation from simple and complex matrices, such as plasma. METHODS: Cell-generated EV spike-in models were isolated and purified by size-exclusion chromatography, stained with cellular dyes and characterized by nano flow cytometry. Fluorescently-labelled spike-in EVs emerged as reliable, high-throughput and easily measurable readouts, which were employed to optimize our EV immunoprecipitation strategy and evaluate its performance. Plasma-derived EVs were captured and detected using this straightforward protocol, sequentially combining isolation and staining of specific surface markers, such as CD9 or CD41. Multiplexed digital transcript detection data was generated using the Nanostring nCounter platform and evaluated through a dedicated bioinformatics pipeline. RESULTS: Beads with covalently-conjugated antibodies on their surface outperformed streptavidin-conjugated beads, coated with biotinylated antibodies, in EV immunoprecipitation. Fluorescent EV spike recovery evidenced that target EV subpopulations can be efficiently retrieved from plasma, and that their enrichment is dependent not only on complex matrix composition, but also on the EV surface phenotype. Finally, mRNA profiling experiments proved that distinct EV subpopulations can be captured by directly targeting different surface markers. Furthermore, EVs isolated with anti-CD61 beads enclosed mRNA expression patterns that might be associated to early-stage lung cancer, in contrast with EVs captured through CD9, CD63 or CD81. The differential clinical value carried within each distinct EV subset highlights the advantages of selective isolation. CONCLUSIONS: This EV isolation protocol facilitated the extraction of clinically useful information from plasma. Compatible with common downstream analytics, it is a readily implementable research tool, tailored to provide a truly translational solution in routine clinical workflows, fostering the inclusion of EVs in novel liquid biopsy settings.
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BACKGROUND: The analysis of liquid biopsies brings new opportunities in the precision oncology field. Under this context, extracellular vesicle circular RNAs (EV-circRNAs) have gained interest as biomarkers for lung cancer (LC) detection. However, standardized and robust protocols need to be developed to boost their potential in the clinical setting. Although nCounter has been used for the analysis of other liquid biopsy substrates and biomarkers, it has never been employed for EV-circRNA analysis of LC patients. METHODS: EVs were isolated from early-stage LC patients (n = 36) and controls (n = 30). Different volumes of plasma, together with different number of pre-amplification cycles, were tested to reach the best nCounter outcome. Differential expression analysis of circRNAs was performed, along with the testing of different machine learning (ML) methods for the development of a prognostic signature for LC. RESULTS: A combination of 500 µL of plasma input with 10 cycles of pre-amplification was selected for the rest of the study. Eight circRNAs were found upregulated in LC. Further ML analysis selected a 10-circRNA signature able to discriminate LC from controls with AUC ROC of 0.86. CONCLUSIONS: This study validates the use of the nCounter platform for multiplexed EV-circRNA expression studies in LC patient samples, allowing the development of prognostic signatures.
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BACKGROUND: The identification of novel biomarkers for the early detection and monitoring of gastric (GC) and colorectal cancer (CRC) is of paramount importance. TM9SF4 is a newly described V-ATPase interacting protein involved in the malignant progression of cancer cells. While TM9SF4 expression pattern and cellular localization have been described in in vitro in tumor cell lines of different histotypes, its expression in gastrointestinal tumor tissues has never been investigated. METHODS: In this study, we detected by immunohistochemistry (IHC) in tumor and surrounding healthy tissues TM9SF4, in comparison with clinically adopted biomarkers CEA and CA 19-9 to evaluate TM9SF4 potential as a novel tissue marker for early detection and monitoring of GC and CRC cancers. RESULTS: The expression of TM9SF4, CEA and CA 19-9 was evaluated in samples from 108 cancer patients (68 with GC and 40 CRC) and in healthy tissues from 20 non-cancer patients. Our results clearly suggest that TM9SF4 expression was significantly increased in GC and CRC samples and significantly correlated to disease stage in both cancer types. CONCLUSIONS: We propose TM9SF4 as highly specific cancer biomarker, exploitable for disease detection and staging of gastrointestinal cancers patients, with tumor tissue levels of expression outperforming those of clinically adopted markers such as CEA and CA 19-9.
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We evaluated the advantages and the reliability of novel protocols for the enrichment of tumor extracellular vesicles (EVs), enabling a blood-based test for the noninvasive parallel profiling of multiple androgen receptor (AR) gene alterations. Three clinically relevant AR variants related to response/resistance to standard-of-care treatments (AR-V7 transcript, AR T878A point mutation and AR gene amplification) were evaluated by digital PCR in 15 samples from patients affected by Castration-Resistant Prostate Cancer (CRPC). Plasma was processed to obtain circulating RNA and DNA using protocols based on tumor EVs enrichment through immuno-affinity and peptide-affinity compared to generic extraction kits. Our results showed that immuno-affinity enrichment prior to RNA extraction clearly outperforms the generic isolation method in the detection of AR-V7, also allowing for a distinction between responder (R) and non-responder (NR) patients. The T878A mutation was detected, overall, in nine out of 15 samples and no approach alone was able to reveal mutations in all harboring samples, showing that the employed methods complement each other. AR amplification was detected in the majority of CRPC samples analysed using either cell-free DNA (cfDNA) or exosome isolation kits (80%). We demonstrated that selective isolation of a subset of circulating exosomes enriched for tumor origin, rather than analysis of total plasma exosomes, or total plasma nucleic acids, increases sensitivity and specificity for the detection of specific alterations.
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Extracellular vesicles (EVs) derived from different parts of the male reproductive tract can be internalized by human spermatozoa affecting their maturation and regulating their functions. Here we demonstrate that EVs derived from the female tract can be uptaken by sperm and affect their competence. Primary endometrial cells release EVs with a diameter between 50 and 350 nm and bear the standard vesicle and exosome marker proteins CD63, CD9, TSG101 and ALIX. The uptake of dye-labelled endometrial cell-derived EVs by spermatozoa, quantified as fluorescence intensity, was significantly higher when EVs were derived from cells in the proliferative phase. Vital, motile fluorescent sperm could be appreciated after a 48-hour co-incubation with endometrial cells previously labelled with the Vybrant™ DiO dye. EV internalization by sperm was blocked at 4 °C and by incubation with filipin, suggesting an energy-dependent process probably attributable to the lipid-raft domain mediated-endocytosis. Sperm ability to undergo capacitation and acrosome reaction was stimulated by endometrial cell-derived EVs as manifested by the increased protein tyrosine phosphorylation and evident reactivity when stimulated with a calcium ionophore. Based on these findings, EVs exchange may be suggested as an emerging way through which female reproductive tract cells can interact with the passing spermatozoa.
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Vesículas Extracelulares/metabolismo , Espermatozoides/metabolismo , Ionóforos de Calcio/farmacología , Células Cultivadas , Frío , Proteínas de Unión al ADN/metabolismo , Endocitosis/efectos de los fármacos , Endometrio/citología , Endometrio/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Femenino , Filipina/farmacología , Humanos , Masculino , Microdominios de Membrana/metabolismo , Tamaño de la Partícula , Fosforilación/efectos de los fármacos , Tetraspanina 30/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Embryo implantation has been defined as the "black box" of human reproduction. Most of the knowledge on mechanisms underlining this process derives from animal models, but they cannot always be translated to humans. Therefore, the development of an in vitro/ex vivo model recapitulating as closely and precisely as possible the fundamental functional features of the human endometrial tissue is very much desirable. Here, we have validated endometrial organoids as a suitable 3D-model to studying epithelial endometrial interface for embryo implantation. Transmission and scanning electron microscopy analyses showed that organoids preserve the glandular organization and cell ultrastructural characteristics. They also retain the responsiveness to hormonal treatment specific to the corresponding phase of the menstrual cycle, mimicking the in vivo glandular-like aspect and functions. Noteworthy, organoids mirroring the early secretive phase show the development of pinopodes, large cytoplasmic apical protrusions of the epithelial cells, traditionally considered as reliable key features of the implantation window. Moreover, organoids express glycodelin A (GdA), a cycle-dependent marker of the endometrial receptivity, with its quantitative and qualitative features accounting well for the profile detected in the endometrium in vivo. Accordingly, organoids deriving from the eutopic endometrium of women with endometriosis show a GdA glycosylation pattern significantly different from healthy organoids, confirming our prior data on endometrial tissues. The present results strongly support the idea that organoids may closely recapitulate the molecular and functional characteristics of their cells/tissue of origin.
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Implantación del Embrión , Embrión de Mamíferos/fisiología , Endometrio/fisiología , Modelos Biológicos , Organoides/fisiología , Adulto , Forma de la Célula/efectos de los fármacos , Implantación del Embrión/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Endometriosis/genética , Endometriosis/patología , Endometrio/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Glicodelina/metabolismo , Hormonas/farmacología , Humanos , Ciclo Menstrual/efectos de los fármacos , Organoides/efectos de los fármacos , Organoides/ultraestructura , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Adulto JovenRESUMEN
Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients' stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner. Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E. To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide. When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation. Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.