Publications by authors named "Hector Peinado"

77 Publications

Endoglin in the Spotlight to Treat Cancer.

Int J Mol Sci 2021 Mar 20;22(6). Epub 2021 Mar 20.

Molecular Pathology of Sarcomas, Institute of Biomedicine of Sevilla (IBiS), Virgen del Rocio University Hospital, CSIC, University of Sevilla, CIBERONC, 41013 Seville, Spain.

A spotlight has been shone on endoglin in recent years due to that fact of its potential to serve as both a reliable disease biomarker and a therapeutic target. Indeed, endoglin has now been assigned many roles in both physiological and pathological processes. From a molecular point of view, endoglin mainly acts as a co-receptor in the canonical TGFβ pathway, but also it may be shed and released from the membrane, giving rise to the soluble form, which also plays important roles in cell signaling. In cancer, in particular, endoglin may contribute to either an oncogenic or a non-oncogenic phenotype depending on the cell context. The fact that endoglin is expressed by neoplastic and non-neoplastic cells within the tumor microenvironment suggests new possibilities for targeted therapies. Here, we aimed to review and discuss the many roles played by endoglin in different tumor types, as well as the strong evidence provided by pre-clinical and clinical studies that supports the therapeutic targeting of endoglin as a novel clinical strategy.
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http://dx.doi.org/10.3390/ijms22063186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003971PMC
March 2021

Could Extracellular Vesicles Contribute to Generation or Awakening of "Sleepy" Metastatic Niches?

Front Cell Dev Biol 2021 2;9:625221. Epub 2021 Mar 2.

Microenvironment and Metastasis Laboratory, Molecular Oncology Programme, Spanish National Cancer Research Center (CNIO), Madrid, Spain.

Pre-metastatic niches provide favorable conditions for tumor cells to disseminate, home to and grow in otherwise unfamiliar and distal microenvironments. Tumor-derived extracellular vesicles are now recognized as carriers of key messengers secreted by primary tumors, signals that induce the formation of pre-metastatic niches. Recent evidence suggests that tumor cells can disseminate from the very earliest stages of primary tumor development. However, once they reach distal sites, tumor cells can persist in a dormant state for long periods of time until their growth is reactivated and they produce metastatic lesions. In this new scenario, the question arises as to whether extracellular vesicles could influence the formation of these metastatic niches with dormant tumor cells? (here defined as "sleepy niches"). If so, what are the molecular mechanisms involved? In this perspective-review article, we discuss the possible influence of extracellular vesicles in early metastatic dissemination and whether they might play a role in tumor cell dormancy. In addition, we comment whether extracellular vesicle-mediated signals may be involved in tumor cell awakening, considering the possibility that extracellular vesicles might serve as biomarkers to detect early metastasis and/or minimal residual disease (MRD) monitoring.
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http://dx.doi.org/10.3389/fcell.2021.625221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960773PMC
March 2021

Postlymphadenectomy Analysis of Exosomes from Lymphatic Exudate/Exudative Seroma of Melanoma Patients.

Methods Mol Biol 2021 ;2265:345-359

Microenvironment and Metastasis Laboratory, Molecular Oncology Programme, Spanish National Cancer Research Center (CNIO), Madrid, Spain.

Circulating extracellular vesicles in biofluids have become an interesting approach to analyse disease biomarkers. There are multiple methods for isolation of extracellular vesicles, though differential ultracentrifugation is still considered as the gold-standard isolation technique for exosomes. Furthermore, exosomes purified by this method have been demonstrated to display functional activity in vitro and in vivo and exhibit great versatility for subsequent analysis including proteomics, electron microscopy, mass spectrometry, or nucleic acid analysis. Here, we describe the method for isolation of exosomes from lymphatic exudate (seroma) obtained postlymphadenectomy for liquid biopsy approaches.
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http://dx.doi.org/10.1007/978-1-0716-1205-7_25DOI Listing
April 2021

DNA-Loaded Extracellular Vesicles in Liquid Biopsy: Tiny Players With Big Potential?

Front Cell Dev Biol 2020 21;8:622579. Epub 2021 Jan 21.

Microenvironment and Metastasis Laboratory, Molecular Oncology Programme, Spanish National Cancer Research Center (CNIO), Madrid, Spain.

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http://dx.doi.org/10.3389/fcell.2020.622579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872099PMC
January 2021

Extracellular Vesicles From Liver Progenitor Cells Downregulates Fibroblast Metabolic Activity and Increase the Expression of Immune-Response Related Molecules.

Front Cell Dev Biol 2020 12;8:613583. Epub 2021 Jan 12.

Center for Cooperative Research in Biosciences, Bizkaia Technology Park, Bizkaia, Spain.

Extracellular vesicles (EVs) mediate cell-to-cell crosstalk whose content can induce changes in acceptor cells and their microenvironment. MLP29 cells are mouse liver progenitor cells that release EVs loaded with signaling cues that could affect cell fate. In the current work, we incubated 3T3-L1 mouse fibroblasts with MLP29-derived EVs, and then analyzed changes by proteomics and transcriptomics. Results showed a general downregulation of protein and transcript expression related to proliferative and metabolic routes dependent on TGF-beta. We also observed an increase in the ERBB2 interacting protein (ERBIN) and Cxcl2, together with an induction of ribosome biogenesis and interferon-related response molecules, suggesting the activation of immune system signaling.
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http://dx.doi.org/10.3389/fcell.2020.613583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835421PMC
January 2021

Covalently Labeled Fluorescent Exosomes for In Vitro and In Vivo Applications.

Biomedicines 2021 Jan 16;9(1). Epub 2021 Jan 16.

Unidad de Medicina y Cirugía Experimental, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), 28007 Madrid, Spain.

The vertiginous increase in the use of extracellular vesicles and especially exosomes for therapeutic applications highlights the necessity of advanced techniques for gaining a deeper knowledge of their pharmacological properties. Herein, we report a novel chemical approach for the robust attachment of commercial fluorescent dyes to the exosome surface with covalent binding. The applicability of the methodology was tested on milk and cancer cell-derived exosomes (from U87 and B16F10 cancer cells). We demonstrated that fluorescent labeling did not modify the original physicochemical properties of exosomes. We tested this nanoprobe in cell cultures and healthy mice to validate its use for in vitro and in vivo applications. We confirmed that these fluorescently labeled exosomes could be successfully visualized with optical imaging.
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http://dx.doi.org/10.3390/biomedicines9010081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829962PMC
January 2021

The influence of secreted factors and extracellular vesicles in ovarian cancer metastasis.

EJC Suppl 2020 Aug 22;15:38-48. Epub 2020 Aug 22.

Microenvironment and Metastasis Group, Department of Molecular Oncology, Spanish National Cancer Research Center (CNIO), Madrid 28029, Spain.

Ovarian cancer cells mainly metastasise within the peritoneal cavity, the lethal consequence of tumour progression in this cancer type. Classically, changes in tumour cells, such as epithelial to mesenchymal transition, involve the down-regulatinon of E-cadherin, activation of extracellular proteases and integrin-mediated adhesion. However, our current understanding of ovarian tumour progression suggests the implication of both intrinsic and extrinsic factors. It has been proposed that ovarian cancer metastases are a consequence of the crosstalk between cancer cells and the tumour microenvironment by soluble factors and extracellular vesicles. Characterisation of the alterations in both the tumour cells and the surrounding microenvironment has emerged as a new research field to understand ovarian cancer metastasis. In this mini review, we will summarise the most recent findings, focusing our attention on the role of secreted factors and extracellular vesicles in ovarian cancer metastasis.
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http://dx.doi.org/10.1016/j.ejcsup.2019.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573474PMC
August 2020

Metabolomic profile of cancer stem cell-derived exosomes from patients with malignant melanoma.

Mol Oncol 2021 02 25;15(2):407-428. Epub 2020 Nov 25.

Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Spain.

Malignant melanoma (MM) is the most aggressive and life-threatening form of skin cancer. It is characterized by an extraordinary metastasis capacity and chemotherapy resistance, mainly due to melanoma cancer stem cells (CSCs). To date, there are no suitable clinical diagnostic, prognostic or predictive biomarkers for this neoplasia. Therefore, there is an urgent need for new MM biomarkers that enable early diagnosis and effective disease monitoring. Exosomes represent a novel source of biomarkers since they can be easily isolated from different body fluids. In this work, a primary patient-derived MM cell line enriched in CSCs was characterized by assessing the expression of specific markers and their stem-like properties. Exosomes derived from CSCs and serums from patients with MM were characterized, and their metabolomic profile was analysed by high-resolution mass spectrometry (HRMS) following an untargeted approach and applying univariate and multivariate statistical analyses. The aim of this study was to search potential biomarkers for the diagnosis of this disease. Our results showed significant metabolomic differences in exosomes derived from MM CSCs compared with those from differentiated tumour cells and also in serum-derived exosomes from patients with MM compared to those from healthy controls. Interestingly, we identified similarities between structural lipids differentially expressed in CSC-derived exosomes and those derived from patients with MM such as the glycerophosphocholine PC 16:0/0:0. To our knowledge, this is the first metabolomic-based study aimed at characterizing exosomes derived from melanoma CSCs and patients' serum in order to identify potential biomarkers for MM diagnosis. We conclude that metabolomic characterization of CSC-derived exosomes sets an open door to the discovery of clinically useful biomarkers in this neoplasia.
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http://dx.doi.org/10.1002/1878-0261.12823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858120PMC
February 2021

Publisher Correction: ITGB3-mediated uptake of small extracellular vesicles facilitates intercellular communication in breast cancer cells.

Nat Commun 2020 09 15;11(1):4730. Epub 2020 Sep 15.

Translational Molecular Pathology, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-18674-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493978PMC
September 2020

Neuroblastoma-secreted exosomes carrying miR-375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells.

J Extracell Vesicles 2020 Jun 3;9(1):1774144. Epub 2020 Jun 3.

Department of Pediatric Onco-Hematology and Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement.
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http://dx.doi.org/10.1080/20013078.2020.1774144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448845PMC
June 2020

ITGB3-mediated uptake of small extracellular vesicles facilitates intercellular communication in breast cancer cells.

Nat Commun 2020 08 26;11(1):4261. Epub 2020 Aug 26.

Translational Molecular Pathology, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.

Metastasis, the spread of malignant cells from a primary tumour to distant sites, causes 90% of cancer-related deaths. The integrin ITGB3 has been previously described to play an essential role in breast cancer metastasis, but the precise mechanisms remain undefined. We have now uncovered essential and thus far unknown roles of ITGB3 in vesicle uptake. The functional requirement for ITGB3 derives from its interactions with heparan sulfate proteoglycans (HSPGs) and the process of integrin endocytosis, allowing the capture of extracellular vesicles and their endocytosis-mediated internalization. Key for the function of ITGB3 is the interaction and activation of focal adhesion kinase (FAK), which is required for endocytosis of these vesicles. Thus, ITGB3 has a central role in intracellular communication via extracellular vesicles, proposed to be critical for cancer metastasis.
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http://dx.doi.org/10.1038/s41467-020-18081-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450082PMC
August 2020

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

Cell 2020 08 13;182(4):1044-1061.e18. Epub 2020 Aug 13.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
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http://dx.doi.org/10.1016/j.cell.2020.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522766PMC
August 2020

Proteomic Profiling of Retinoblastoma-Derived Exosomes Reveals Potential Biomarkers of Vitreous Seeding.

Cancers (Basel) 2020 06 12;12(6). Epub 2020 Jun 12.

Department of Pediatric Hematology/Oncology and Cell and Gene Therapy, IRCCS, Ospedale Pediatrico Bambino Gesù, Piazza Sant'Onofrio 4, 00165 Rome, Italy.

Retinoblastoma (RB) is the most common tumor of the eye in early childhood. Although recent advances in conservative treatment have greatly improved the visual outcome, local tumor control remains difficult in the presence of massive vitreous seeding. Traditional biopsy has long been considered unsafe in RB, due to the risk of extraocular spread. Thus, the identification of new biomarkers is crucial to design safer diagnostic and more effective therapeutic approaches. Exosomes, membrane-derived nanovesicles that are secreted abundantly by aggressive tumor cells and that can be isolated from several biological fluids, represent an interesting alternative for the detection of tumor-associated biomarkers. In this study, we defined the protein signature of exosomes released by RB tumors (RBT) and vitreous seeding (RBVS) primary cell lines by high resolution mass spectrometry. A total of 5666 proteins were identified. Among these, 5223 and 3637 were expressed in exosomes RBT and one RBVS group, respectively. Gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified in RBVS exosomes upregulated proteins specifically related to invasion and metastasis, such as proteins involved in extracellular matrix (ECM) remodeling and interaction, resistance to anoikis and the metabolism/catabolism of glucose and amino acids.
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http://dx.doi.org/10.3390/cancers12061555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352325PMC
June 2020

Platelet factor 4 is a biomarker for lymphatic-promoted disorders.

JCI Insight 2020 07 9;5(13). Epub 2020 Jul 9.

Center for Vascular and Developmental Biology, Feinberg Cardiovascular and Renal Research Institute, Northwestern University, Chicago, Illinois, USA.

Genetic or acquired defects of the lymphatic vasculature often result in disfiguring, disabling, and, occasionally, life-threatening clinical consequences. Advanced forms of lymphedema are readily diagnosed clinically, but more subtle presentations often require invasive imaging or other technologies for a conclusive diagnosis. On the other hand, lipedema, a chronic lymphatic microvascular disease with pathological accumulation of subcutaneous adipose tissue, is often misdiagnosed as obesity or lymphedema; currently there are no biomarkers or imaging criteria available for a conclusive diagnosis. Recent evidence suggests that otherwise-asymptomatic defective lymphatic vasculature likely contributes to an array of other pathologies, including obesity, inflammatory bowel disease, and neurological disorders. Accordingly, identification of biomarkers of lymphatic malfunction will provide a valuable resource for the diagnosis and clinical differentiation of lymphedema, lipedema, obesity, and other potential lymphatic pathologies. In this paper, we profiled and compared blood plasma exosomes isolated from mouse models and from human subjects with and without symptomatic lymphatic pathologies. We identified platelet factor 4 (PF4/CXCL4) as a biomarker that could be used to diagnose lymphatic vasculature dysfunction. Furthermore, we determined that PF4 levels in circulating blood plasma exosomes were also elevated in patients with lipedema, supporting current claims arguing that at least some of the underlying attributes of this disease are also the consequence of lymphatic defects.
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http://dx.doi.org/10.1172/jci.insight.135109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406300PMC
July 2020

Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence.

Nat Commun 2020 06 2;11(1):2761. Epub 2020 Jun 2.

ISGlobal, Hospital Clínic - Universitat de Barcelona, Barcelona, 08036, Spain.

Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the uptake of EVs from healthy individuals. Moreover, this uptake induces specific upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After this uptake, P. vivax-infected reticulocytes obtained from patients show specific adhesion properties to hSFs, reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen.
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http://dx.doi.org/10.1038/s41467-020-16337-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265481PMC
June 2020

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

Nat Cell Biol 2019 11 4;21(11):1403-1412. Epub 2019 Nov 4.

Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP exosomes. Moreover, uptake of CEMIP exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.
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http://dx.doi.org/10.1038/s41556-019-0404-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354005PMC
November 2019

Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3.

Cell Rep 2019 06;27(13):3956-3971.e6

Epigenetics & Cellular Senescence Group, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1 2AT, UK; Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT, UK. Electronic address:

Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.
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http://dx.doi.org/10.1016/j.celrep.2019.05.095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613042PMC
June 2019

Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and mutation.

J Exp Med 2019 05 11;216(5):1061-1070. Epub 2019 Apr 11.

Microenvironment and Metastasis Laboratory, Molecular Oncology Program, Spanish National Cancer Research Center, Madrid, Spain

Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse.
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http://dx.doi.org/10.1084/jem.20181522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504207PMC
May 2019

Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo.

Dev Cell 2019 02 7;48(4):554-572.e7. Epub 2019 Feb 7.

INSERM UMR_S1109, Strasbourg 67200, France; Université de Strasbourg, Strasbourg 67200, France; Fédération de Médecine Translationnelle de Strasbourg, Strasbourg 67200, France. Electronic address:

Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs' hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo.
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http://dx.doi.org/10.1016/j.devcel.2019.01.014DOI Listing
February 2019

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.

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR) of Italy, Palermo, Italy.

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.
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http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018

Expression profiles of exosomal miRNAs isolated from plasma of patients with desmoplastic small round cell tumor.

Epigenomics 2019 04 20;11(5):489-500. Epub 2018 Dec 20.

Department of Pediatric Hematology/Oncology, Bambino Gesù Children's Hospital, IRCCS, Piazza di Sant' Onofrio, 4, 00165 Rome, Italy.

Aim: Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive mesenchymal tumor, lacking biomarkers for diagnosis, treatment stratification and prognosis. We investigated the exosomal miRNA profile in plasma samples collected from DSRCT patients, evaluating their potential as circulating biomarkers for this tumor.

Patients & Methods: We isolated exosomes from plasma of three DSRCT adolescents and four age-matched healthy controls; expression of circulating miRNAs was quantified by qPCR.

Results: We identified 55 miRNAs significantly modulated compared with healthy controls. Among these miRNAs, 14 were highly dysregulated in at least one patient and 5 were expressed in all patients.

Conclusion: To our knowledge, this is the first report describing exosomal miRNAs as promising biomarkers to characterize disease status in DSRCT patients.
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http://dx.doi.org/10.2217/epi-2018-0179DOI Listing
April 2019

Lipid-induced lysosomal damage after demyelination corrupts microglia protective function in lysosomal storage disorders.

EMBO J 2019 01 7;38(2). Epub 2018 Dec 7.

Department of Molecular Neuropathology, Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Madrid, Spain

Neuropathic lysosomal storage disorders (LSDs) present with activated pro-inflammatory microglia. However, anti-inflammatory treatment failed to improve disease pathology. We characterise the mechanisms underlying microglia activation in Niemann-Pick disease type A (NPA). We establish that an NPA patient and the acid sphingomyelinase knockout (ASMko) mouse model show amoeboid microglia in neurodegeneration-prone areas. microglia ablation worsens disease progression in ASMko mice. We demonstrate the coexistence of different microglia phenotypes in ASMko brains that produce cytokines or counteract neuronal death by clearing myelin debris. Overloading microglial lysosomes through myelin debris accumulation and sphingomyelin build-up induces lysosomal damage and cathepsin B extracellular release by lysosomal exocytosis. Inhibition of cathepsin B prevents neuronal death and behavioural anomalies in ASMko mice. Similar microglia phenotypes occur in a Niemann-Pick disease type C mouse model and patient. Our results show a protective function for microglia in LSDs and how this is corrupted by lipid lysosomal overload. Data indicate cathepsin B as a key molecule mediating neurodegeneration, opening research pathways for therapeutic targeting of LSDs and other demyelinating diseases.
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http://dx.doi.org/10.15252/embj.201899553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331723PMC
January 2019

Neutrophils instruct homeostatic and pathological states in naive tissues.

J Exp Med 2018 11 3;215(11):2778-2795. Epub 2018 Oct 3.

Area of Developmental and Cell Biology, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain

Immune protection relies on the capacity of neutrophils to infiltrate challenged tissues. Naive tissues, in contrast, are believed to remain free of these cells and protected from their toxic cargo. Here, we show that neutrophils are endowed with the capacity to infiltrate multiple tissues in the steady-state, a process that follows tissue-specific dynamics. By focusing in two particular tissues, the intestine and the lungs, we find that neutrophils infiltrating the intestine are engulfed by resident macrophages, resulting in repression of transcription, reduced G-CSF in plasma, and reinforced activity of distant bone marrow niches. In contrast, diurnal accumulation of neutrophils within the pulmonary vasculature influenced circadian transcription in the lungs. Neutrophil-influenced transcripts in this organ were associated with carcinogenesis and migration. Consistently, we found that neutrophils dictated the diurnal patterns of lung invasion by melanoma cells. Homeostatic infiltration of tissues unveils a facet of neutrophil biology that supports organ function, but can also instigate pathological states.
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http://dx.doi.org/10.1084/jem.20181468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219739PMC
November 2018

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation.

Nat Cell Biol 2018 03 19;20(3):332-343. Epub 2018 Feb 19.

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed 'exomeres' (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.
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http://dx.doi.org/10.1038/s41556-018-0040-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931706PMC
March 2018

Evaluation of Endoglin (CD105) expression in pediatric rhabdomyosarcoma.

BMC Cancer 2018 01 5;18(1):31. Epub 2018 Jan 5.

Department of Hematology/Oncology, Bambino Gesù Children's Hospital, IRCCS, Piazza di Sant'Onofrio, 4, 00165, Rome, Italy.

Background: The Intratumoral Microvessel Density (IMVD) is commonly used to quantify tumoral vascularization and is usually assessed by pan-endothelial markers, such as CD31. Endoglin (CD105) is a protein predominantly expressed in proliferating endothelium and the IMVD determined by this marker measures specifically the neovascularization. In this study, we investigated the CD105 expression in pediatric rhabdomyosarcoma and assessed the neovascularization by using the angiogenic ratio IMVD-CD105 to IMVD-CD31.

Methods: Paraffin-embedded archival tumor specimens were selected from 65 pediatric patients affected by rhabdomyosarcoma. The expression levels of CD105, CD31 and Vascular Endothelial Growth Factor (VEGF) were investigated in 30 cases (18 embryonal and 12 alveolar) available for this study. The IMVD-CD105 to IMVD-CD31 expression ratio was correlated with clinical and pathologic features of these patients.

Results: We found a specific expression of endoglin (CD105) in endothelial cells of all the rhabdomyosarcoma specimens analyzed. We observed a significant positive correlation between the IMVD individually measured by CD105 and CD31. The CD105/CD31 expression ratio was significantly higher in patients with lower survival and embryonal histology. Indeed, patients with a CD105/CD31 expression ratio < 1.3 had a significantly increased OS (88%, 95%CI, 60%-97%) compared to patients with higher values (40%, 95%CI, 12%-67%). We did not find any statistical correlation among VEGF and EFS, OS and CD105/CD31 expression ratio.

Conclusion: CD105 is expressed on endothelial cells of rhabdomyosarcoma and represent a useful tool to quantify neovascularization in this tumor. If confirmed by further studies, these results will indicate that CD105 is a potential target for combined therapies in rhabdomyosarcoma.
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http://dx.doi.org/10.1186/s12885-017-3947-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755407PMC
January 2018

Lysyl oxidase-like 3 is required for melanoma cell survival by maintaining genomic stability.

Cell Death Differ 2018 05 11;25(5):935-950. Epub 2017 Dec 11.

Departamento de Bioquímica, UAM, Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC-UAM, Arzobispo Morcillo 4, 28029, Madrid, Spain.

Lysyl oxidase-like 3 (LOXL3) is a member of the lysyl oxidase family comprising multifunctional enzymes with depicted roles in extracellular matrix maturation, tumorigenesis, and metastasis. In silico expression analyses followed by experimental validation in a comprehensive cohort of human cell lines revealed a significant upregulation of LOXL3 in human melanoma. We show that LOXL3 silencing impairs cell proliferation and triggers apoptosis in various melanoma cell lines. Further supporting a pro-oncogenic role in melanoma, LOXL3 favors tumor growth in vivo and cooperates with oncogenic BRAF in melanocyte transformation. Upon LOXL3 depletion, melanoma cells display a faulty DNA damage response (DDR), characterized by ATM checkpoint activation and inefficient ATR activation leading to the accumulation of double-strand breaks (DSBs) and aberrant mitosis. Consistent with these findings, LOXL3 binds to proteins involved in the maintenance of genome integrity, in particular BRCA2 and MSH2, whose levels dramatically decrease upon LOXL3 depletion. Moreover, LOXL3 is required for efficient DSB repair in melanoma cells. Our results reveal an unexpected role for LOXL3 in the control of genome stability and melanoma progression, exposing its potential as a novel therapeutic target in malignant melanoma, a very aggressive condition yet in need for more effective treatment options.
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http://dx.doi.org/10.1038/s41418-017-0030-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5907912PMC
May 2018

The influence of tumour-derived extracellular vesicles on local and distal metastatic dissemination.

Mol Aspects Med 2018 04 6;60:15-26. Epub 2017 Dec 6.

Children's Cancer and Blood Foundation Laboratories, Department of Pediatrics, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medical College, New York, NY 10021, USA; Microenvironment and Metastasis Group, Department of Molecular Oncology, Spanish National Cancer Research Center (CNIO), Madrid 28029, Spain. Electronic address:

Extracellular vesicles (EVs) are key mediators of intercellular communication that have been ignored for decades. Tumour cells benefit from the secretion of vesicles as they can influence the behaviour of neighbouring tumour cells within the tumour microenvironment. Several studies have shown that extracellular vesicles play an active role in pre-metastatic niche formation and importantly, they are involved in the metastatic organotropism of different tumour types. Tumour-derived EVs carry and transfer molecules to recipient cells, modifying their behaviour through a process defined as "EV-driven education". EVs favour metastasis to sentinel lymph nodes and distal organs by reinforcing angiogenesis, inflammation and lymphangiogenesis. Hence, in this review we will summarize the main mechanisms by which tumour-derived EVs regulate lymph node and distal organ metastasis. Moreover, since some cancers metastasize through the lymphatic system, we will discuss recent discoveries about the presence and function of tumour EVs in the lymph. Finally, we will address the potential value of tumour EVs as prognostic biomarkers in liquid biopsies, specially blood and lymphatic fluid, and the use of these tools as early detectors of metastases.
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http://dx.doi.org/10.1016/j.mam.2017.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856602PMC
April 2018

Tumour-adipose tissue crosstalk: fuelling tumour metastasis by extracellular vesicles.

Philos Trans R Soc Lond B Biol Sci 2018 Jan;373(1737)

Microenvironment and Metastasis Group, Department of Molecular Oncology, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain

During metastasis, tumour cells must communicate with their microenvironment by secreted soluble factors and extracellular vesicles. Different stromal cell types (e.g. bone marrow-derived cells, endothelial cells and fibroblasts) influence the growth and progression of tumours. In recent years, interest has extended to other cell types in the tumour microenvironment such as adipocytes and adipose tissue-derived mesenchymal stem cells. Indeed, obesity is becoming pandemic in some developing countries and it is now considered to be a risk factor for cancer progression. However, the true impact of obesity on the metastatic behaviour of tumours is still not yet fully understood. In this 'Perspective' article, we will discuss the potential influence of obesity on tumour metastasis, mainly in melanoma, breast and ovarian cancer. We summarize the main mechanisms involved with special attention to the role of extracellular vesicles in this process. We envisage that besides having a direct impact on tumour cells, obesity systemically preconditions the tumour microenvironment for future metastasis by favouring the formation of pro-inflammatory niches.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.
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http://dx.doi.org/10.1098/rstb.2016.0485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717439PMC
January 2018

Proteomic Analysis of Neuroblastoma-Derived Exosomes: New Insights into a Metastatic Signature.

Proteomics 2017 Dec 12;17(23-24). Epub 2017 Sep 12.

Department of Hematology/Oncology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Around 70% of patients with metastatic disease at diagnosis present bone-marrow infiltration, which is considered a marker of poor outcome; however, the mechanism underlying this specific tropism has to be elucidated. Tumor-derived exosomes may support metastatic progression in several tumors by interacting with the microenvironment, and may serve as tumor biomarkers. The main objective of this study is to identify an exosomal signature associated with NB metastatic bone-marrow dissemination. Therefore, the proteomic cargo of exosomes isolated from NB cell lines derived from primary tumor and bone-marrow metastasis is characterized. The comparison among exosomal proteins show 15 proteins exclusively present in primary tumor-derived exosomes, mainly involved in neuronal development, and 6 proteins in metastasis-derived exosomes related to cancer progression. Significant proteins obtain with statistical analysis performed between the two groups, reveal that primary tumor exosomes contain a higher level of proteins involved in extra-cellular matrix (ECM) assembly and adhesion, as well as in neuronal development. Exosomes isolated from bone-marrow metastasis exhibit proteins involved in ameboidal cell migration and mitochondrial activity. This work suggests that proteomic profiling of NB-derived exosomes reflects the tumor stage and may be considered as potential tumor biomarker.
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http://dx.doi.org/10.1002/pmic.201600430DOI Listing
December 2017