Solunulkoiset vesikkelit : tulevaisuudennäkymiä biomarkkerien löytämiseen ja lääkekuljetukseen

Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies are a heterogeneous population of membrane particles released by cells to the extracellular space and into biofluids during normal physiological and pathological processes. EVs have been recognized as powerful vehicles for intercellular communication due to their capacity to transfer lipids, proteins, and nucleic acids, thereby influencing the properties and functions of recipient cells. Cells generate EVs with a unique composition based on their characteristics, which has a special relevance in the study of diseases such as cancer. Since specific molecular signatures can be passed on to tumor EVs, they are prime candidates for implementation as cancer biomarkers and in the delivery of therapeutics. Thus, exhaustive research is currently targeted towards elucidating the role of EVs in cell-to-cell communication and their therapeutic and diagnostic use. This thesis aims at broadening our understanding of the applicability and functional relevance of the use of EVs as prostate cancer biomarkers and therapeutic delivery vehicles. First, the practical use of EVs as a source of nucleic acid biomarkers in prostate cancer was assessed by exploring the DNA and RNA content of vesicles. Genomic DNA analysis of apoptotic bodies, microvesicles, and exosomes were performed to detect mutations within the EV cargo. The results were validated in plasma EVs of prostate cancer patients, from which the presence of prostate cancer-relevant genes was identified. Next, the prostate cancer-specific messenger RNA signatures of microvesicles and exosomes were analyzed. Unique nucleic acid signatures distinctive for the cell origin were found in the form of differential levels of mRNA transcripts from EV subpopulations. Overall, the nucleic acid content of EVs provided a new source of diagnostic information that could contribute to early prediction and diagnosis of prostate cancer, especially if combined. The role of EV-mediated intercellular communication was shown by comparing the uptake efficiencies and functional effects of EVs from prostate cancer cells of different metastatic status with non-cancer EVs. Additionally, the ability of EVs to carry and deliver a chemotherapeutic drug, together with their cytotoxic effects on prostate cancer cells were also analyzed. While EV uptake, in general, was an active and continuous process, the internalization rate and the subsequent functional effects of EVs on recipient cells differed based on the vesicle origin. EVs derived from cells of a metastatic source were more efficiently internalized than primary prostate cancer or benign prostate epithelial cell-derived EVs. Similarly, those EVs also induced a more proliferative and migratory phenotype in the recipient cells. Applying prostate cancer EVs in the in vitro delivery of paclitaxel to prostate cancer cells, resulted in an enhanced cytotoxic effect of paclitaxel mediated by EV delivery compared to the free drug. In summary, the results presented in this thesis support the concept that EVs can be utilized in both biomarker discovery and drug delivery fields as multifunctional tools for diagnosis and treatment of diseases such as prostate cancer. The studies presented here will also contribute to set the bases for further functional analysis of the roles of EVs in cell-to-cell communication. This new era of research could lead to faster, non-invasive, and more individualized diagnosis and improved treatments tailored to the specific needs of the patients.Solunulkoiset vesikkelit (engl. extracellular vesicles, EV), mukaan lukien mm. eksosomit, mikrovesikkelit ja apoptoottiset vesikkelit, ovat heterogeeninen joukko lipidikalvollisia partikkeleita, joita solut vapauttavat ympärilleen kehon eri nesteisiin ja eritteisiin sekä fysiologisissa että patologisissa olosuhteissa. EV:t on todettu tehokkaiksi solujenvälisen kommunikaation välittäjiksi, koska ne voivat kuljettaa lipidejä, proteiineja ja nukleiinihappoja ja siten vaikuttaa vastaanottavan solun ominaisuuksiin ja toimintakykyyn. Solut tuottavat ominaisuuksistaan riippuen yksilöllisiä EV:itä, mikä on merkittävää sairauksien, kuten syövän tutkimisessa. Koska solujen spesifit tunnuspiirteet voivat siirtyä syöpäperäisiin EV:ihin, ne ovat ensiluokkaisia ehdokkaita sovellettaviksi syöpäbiomarkkereina ja terapeuttisten aineiden kuljettimina. Siksi EV:iden tehtäviä solujen välisessä vuorovaikutuksessa ja käyttöä hoidoissa ja diagnostiikassa pyritäänkin nyt selvittämään perinpohjaisesti. Tämän väitöskirjan tavoitteena oli laajentaa käsitystämme EV:iden käyttökelpoisuudesta ja merkityksestä prostatasyövän biomarkkereina ja terapeuttisina kuljettimina. Ensimmäiseksi EV:iden käyttöä nukleiinihappobiomarkkerien lähteenä arvioitiin tutkimalla niiden DNA- ja RNA-sisältöä. Genomista DNA:ta voitiin eristää apoptoottisten vesikkelien, mikrovesikkelien ja eksosomien kuljettamasta (molekyyli)lastista ja sitä analysoitiin mutaatioiden löytämiseksi. Tulokset validoitiin prostatasyöpäpotilaisen plasmaperäisistä EV-näytteistä osoittamalla, että niistä voitiin tunnistaa prostatasyöpään liittyviä geenejä. Seuraavaksi analysoitiin mikrovesikkeleille ja eksosomeille tunnuspiirteisiä, syöpäspesifejä lähetti-RNA:ita prostatasyöpäsolulinjojen näytteistä. Yksilöllisiä ja solualkuperälle ominaisia nukleiinihappotunnuspiirteitä löydettiin EV-alaryhmistä eri määrinä tiettyjä lähetti-RNA:ita. Erityisesti EV:iden RNA- ja DNA-nukleiinihapposisällön yhteisanalyysi voisi tarjota uuden diagnostisen tiedonlähteen, joka voi auttaa prostatasyövän varhaisen ennusteen tekemisessä ja diagnosoimisessa. EV:ien vaikutus solujen väliseen kommunikaatioon osoitettiin vertailemalla niiden sisäänottoa ja niiden toiminnallisia vaikutuksia vastaanottajasoluissa riippuen siitä, oliko tuottava prostatasolulinja metastaattinen vai ei-syöpäsolu. Lisäksi EV:iden kykyä kuljettaa ja siirtää kemoterapeuttista lääkettä analysoitiin arvioimalla EV:iden kuljettaman lääkkeen syöpäsoluihin kohdistamaa sytotoksista vaikutusta. EV:iden sisäänotto soluihin oli aktiviinen ja jatkuva prosessi, kun taas solujen vastaanottamien EV:iden määrä ja sitä seuranneet toiminnalliset muutokset vastaanottajasoluissa olivat riippuvaisia vesikkelien lähtösolujen syöpäasteesta. Metastaattisista soluista peräisin olevat EV:t otettiin vastaanottajasoluihin tehokkaammin kuin primäärisyövästä tai hyvälaatuisesta kasvaimesta peräisin olevat EV:t. Nämä EV:t myös aiheuttivat tehokkaammin jakautuvan ja liikkuvan solufenotyypin vastaanottajasolussa. In vitro kokeissa prostatasyöpäperäisten EV:iden käyttäminen paclitaxelin kuljettamiseen tehosti lääkkeen sytotoksista vaikutusta prostatasyöpäsoluissa verrattuna suoraan annettuun lääkkeeseen. Yhteenvetona voidaan sanoa, että tämän väitöskirjan tulokset tukevat ajatusta, että EV:itä voidaan hyödyntää monipuolisina välineinä biomarkkerien löytämiseen ja lääkekuljetukseen sairauksien, kuten prostatasyövän, diagnosoinnissa ja hoidossa. Esitetyt tutkimustulokset luovat myös osaltaan perusteita EV-välitteisen solukommunikaation lisätutkimuksille. Tämä uusi tutkimusalue voi johtaa nopeampaan, ei-kajoavaan ja yksilöllisempään diagnostiikkaan sekä edistyneempiin hoitokeinoihin, jotka voidaan räätälöidä potilaiden tarpeiden mukaisesti

A minimally invasive methodology based on morphometric parameters for day 2 embryo quality assessment

[EN] The risk of multiple pregnancy to maternal fetal health can be minimized by reducing the number of embryos transferred. New tools for selecting embryos with the highest implantation potential should be developed. The aim of this study was to evaluate the ability of morphological and morphometric variables to predict implantation by analysing images of embryos. This was a retrospective study of 135 embryo photographs from 112 IVF ICSI cycles carried out between January and March 2011. The embryos were photographed immediately before transfer using Cronus 3 software. Their images were analysed using the public program ImageJ. Significant effects (P < 0.05), and higher discriminant power to predict implantation were observed for the morphometric embryo variables compared with morphological ones. The features for successfully implanted embryos were as follows: four cells on day 2 of development; all blastomeres with circular shape (roundness factor greater than 0.9), an average zona pellucida thickness of 13&#8201;µm and an average of 17695.1&#8201;µm2 for the embryo area. Embryo size, which is described by its area and the average roundness factor for each cell, provides two objective variables to consider when predicting implantation. This approach should be further investigated for its potential ability to improve embryo scoring.Molina Botella, MI.; Lázaro Ibáñez, E.; Pertusa, J.; Debón Aucejo, AM.; Martinez Sanchis, JV.; Pellicer Bofill, AJ. (2014). A minimally invasive methodology based on morphometric parameters for day 2 embryo quality assessment. Reproductive BioMedicine Online. 29(4):470-480. doi:10.1016/j.rbmo.2014.06.005S47048029

Multiomics analysis of naturally efficacious lipid nanoparticle coronas reveals high-density lipoprotein is necessary for their function

In terms of lipid nanoparticle (LNP) engineering, the relationship between particle composition, delivery efficacy, and the composition of the biocoronas that form around LNPs, is poorly understood. To explore this we analyze naturally efficacious biocorona compositions using an unbiased screening workflow. First, LNPs are complexed with plasma samples, from individual lean or obese male rats, and then functionally evaluated in vitro. Then, a fast, automated, and miniaturized method retrieves the LNPs with intact biocoronas, and multiomics analysis of the LNP-corona complexes reveals the particle corona content arising from each individual plasma sample. We find that the most efficacious LNP-corona complexes were enriched with high-density lipoprotein (HDL) and, compared to the commonly used corona-biomarker Apolipoprotein E, corona HDL content was a superior predictor of in-vivo activity. Using technically challenging and clinically relevant lipid nanoparticles, these methods reveal a previously unreported role for HDL as a source of ApoE and, form a framework for improving LNP therapeutic efficacy by controlling corona composition.</p

DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain barrier and be detected in peripheral blood of patients

Tumor-cell-secreted extracellular vesicles (EVs) can cross the disrupted bloodbrain barrier (BBB) into the bloodstream. However, in certain gliomas, the BBB remains intact, which might limit EVs release. To evaluate the ability of tumor-derived EVs to cross the BBB, we used an orthotopic xenotransplant mouse model of human glioma-cancer stem cells featuring an intact BBB. We demonstrated that all types of tumor cells-derived EVs−apoptotic bodies, shedding microvesicles and exosomes− cross the intact BBB and can be detected in the peripheral blood, which provides a minimally invasive method for their detection compared to liquid biopsies obtained from cerebrospinal fluid (CSF). Furthermore, these EVs can be readily distinguished from total murine EVs, since they carry human-specific DNA sequences relevant for GBM biology. In a small cohort of glioma patients, we finally demonstrated that peripheral blood EVs cargo can be successfully used to detect the presence of IDH1G395A, an essential biomarker in the current management of human gliomaWe are grateful for the financial support from the ‘Fondo de Investigaciones Sanitarias’ (FIS) (PI10/01069 and PI14/00077) and the ‘Miguel Servet Program’ (CP11/00147) from the ‘Instituto de Salud Carlos III’ (AAS), RTC-2015-3846-1 from Ministerio de Economía y Competitividad and FEDER fund

SOX2(+) Cell Population from Normal Human Brain White Matter Is Able to Generate Mature Oligodendrocytes

Peer reviewe

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Aislamiento y caracterización de microvesículas procedentes de Glioblastoma Multiforme y su futuro empleo como marcadores diagnóstico

Los gliomas malignos son los tumores cerebrales primarios más comunes, constituyendo además el tipo de tumor mayoritario en cerebro. Un tercio de estos tumores corresponde con Glioblastoma Multiforme (GBM), con una supervivencia media no superior a los 15 meses. En las células tumorales se ha observado un incremento en la formación y secreción de microvesículas exocíticas comparadas con células no tumorales. Estas microvesículas sintetizadas por las células del tumor están enriquecidas en proteínas, mRNA, miRNA, DNA y son capaces de atravesar la barrera hematoencefálica hacia el torrente sanguíneo. Trabajos recientes han relacionado las microvesículas procedentes de gliomas malignos con la trasferencia horizontal de proteínas y RNA a otros tipos celulares como las células endoteliales. En el presente trabajo se han asilado y caracterizado líneas enriquecidas con células iniciadoras de glioma a partir de muestras quirúrgicas. Estas células serán utilizadas como modelo in vitro para la optimización del protocolo de aislamiento y caracterización de microvesículas (MVs) así como para la caracterización del contenido de las mismas. Posteriormente, se empleará un modelo in vivo de ratón para distinguir entre DNA genómico humano y de ratón y con ello validar los biomarcadores candidatos. Por último, se validaran los genes candidatos utilizando muestras quirúrgicas de gliomas humanos y sangre periférica de pacientes tomada a lo largo del curso de la enfermedad. Por ello, el objetivo principal del presente trabajo es evaluar, aislar y caracterizar las MVs procedentes de los tres modelos y validar la utilización de secuencias de DNAs genómicos aisladas de los diferentes tipos de microvesículas como biomarcadores de tumores gliales malignos, con objeto de mejorar el diagnostico y pronóstico y de controlar la respuesta a los tratamientos convencionales y a los fármacos de nueva generación, de una forma no invasiva.Lázaro Ibáñez, E. (2011). Aislamiento y caracterización de microvesículas procedentes de Glioblastoma Multiforme y su futuro empleo como marcadores diagnóstico. http://hdl.handle.net/10251/15378Archivo delegad

Additional file 3: Table S2. of Distinct prostate cancer-related mRNA cargo in extracellular vesicle subsets from prostate cell lines

Top Regulated Genes: Distinctly different genes between PC-3 MVs vs PNT2 MVs. (XLSX 10 kb