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4. Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics

Author

Dhondt, B. (Bert), Pinheiro, C. (Cláudio), Geeurickx, E. (Edward), Tulkens, J. (Joeri), Vergauwen, G. (Glenn), Van Der Pol, E. (Edwin), Nieuwland, R. (Rienk), Decock, A. (Anneleen), Miinalainen, I. (Ilkka), Rappu, P. (Pekka), Schroth, G. (Gary), Kuersten, S. (Scott), Vandesompele, J. (Jo), Mestdagh, P. (Pieter), Lumen, N. (Nicolaas), De Wever, O. (Olivier), Hendrix, A. (An), Dhondt, B. (Bert), Pinheiro, C. (Cláudio), Geeurickx, E. (Edward), Tulkens, J. (Joeri), Vergauwen, G. (Glenn), Van Der Pol, E. (Edwin), Nieuwland, R. (Rienk), Decock, A. (Anneleen), Miinalainen, I. (Ilkka), Rappu, P. (Pekka), Schroth, G. (Gary), Kuersten, S. (Scott), Vandesompele, J. (Jo), Mestdagh, P. (Pieter), Lumen, N. (Nicolaas), De Wever, O. (Olivier), and Hendrix, A. (An)

Abstract

The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT; six preservation and five non-preservation) and three blood processing intervals (BPI; 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies.

Published
2023

5. Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles

Author

Roux, Q. (Quentin), Boiy, R. (Robin), De Vuyst, F. (Felix), Tkach, M. (Mercedes), Pinheiro, C. (Claudio), de Geyter, S. (Sofie), Miinalainen, I. (Ilkka), Théry, C. (Clotilde), De Wever, O. (Olivier), Hendrix, A. (An), Roux, Q. (Quentin), Boiy, R. (Robin), De Vuyst, F. (Felix), Tkach, M. (Mercedes), Pinheiro, C. (Claudio), de Geyter, S. (Sofie), Miinalainen, I. (Ilkka), Théry, C. (Clotilde), De Wever, O. (Olivier), and Hendrix, A. (An)

Abstract

Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration.

Published
2023

6. Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility

Author

Van Dorpe, S. (Sofie), Lippens, L. (Lien), Boiy, R. (Robin), Pinheiro, C. (Cláudio), Vergauwen, G. (Glenn), Rappu, P. (Pekka), Miinalainen, I. (Ilkka), Tummers, P. (Philippe), Denys, H. (Hannelore), De Wever, O. (Olivier), Hendrix, A. (An), Van Dorpe, S. (Sofie), Lippens, L. (Lien), Boiy, R. (Robin), Pinheiro, C. (Cláudio), Vergauwen, G. (Glenn), Rappu, P. (Pekka), Miinalainen, I. (Ilkka), Tummers, P. (Philippe), Denys, H. (Hannelore), De Wever, O. (Olivier), and Hendrix, A. (An)

Abstract

Background: Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. Results: Automated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine. Conclusions: In conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.

Published
2023

8. Performance evaluation of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

Author

Bert Dhondt, Gary P. Schroth, Celine Everaert, Avila Cobos F, Jasper Anckaert, Nurten Yigit, Jill Deleu, Eva Hulstaert, Van Maerken T, Vanden Eynde E, Piofczyk T, Scott Kuersten, Annelien Morlion, De Wever O, Anneleen Decock, De Wilde J, Kathleen Schoofs, Pieter Mestdagh, Kimberly Verniers, Thibaut D’huyvetter, An Hendrix, Justine Nuytens, Philippron A, Hetty Helsmoortel, Jo Vandesompele, Van Paemel R, Nele Nijs, and Carolina Fierro

Subjects
Transcriptome, Messenger RNA, RNA, RNA extraction, Computational biology, Sample collection, Biology, Blood Collection Tube, Gene, Extracellular RNA
Abstract

The use of blood-based extracellular RNA (cell-free RNA; exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing, and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated ten blood collection tubes, three time intervals between blood draw and downstream processing, and eight RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics on deep transcriptome profiling of both small and messenger RNA from healthy donors’ plasma or serum was assessed for each pre-analytical variable separately and for interactions between a selected set of pre-analytics, resulting in 456 extracellular transcriptomes. Making use of 189 synthetic spike-in RNAs, the processing and analysis workflow was controlled. When comparing blood collection tubes, so-called preservation tubes do not stabilize exRNA well, and result in variable RNA concentration and sensitivity (i.e., the number of detected RNAs) over time, as well as compromised reproducibility. We also document large differences in RNA purification kit performance in terms of sensitivity, reproducibility, and observed transcriptome complexity, and demonstrate interactions between specific blood collection tubes, purification kits and time intervals. Our results are summarized in 11 performance metrics that enable an informed selection of the most optimal sample processing workflow for a given experiment. In conclusion, we put forward robust quality control metrics for exRNA quantification methods with validated standard operating procedures (SOPs), representing paramount groundwork for future exRNA-based precision medicine applications.

Published
2021
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9. Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions

Author

Vergauwen, G. (Glenn), Tulkens, J. (Joeri), Pinheiro, C. (Cláudio), Cobos, F. A. (Francisco Avila), Dedeyne, S. (Sándor), De Scheerder, M.-A. (Marie-Angélique), Vandekerckhove, L. (Linos), Impens, F. (Francis), Miinalainen, I. (Ilkka), Gevaert, K. (Kris), Mestdagh, P. (Pieter), Vandesompele, J. (Jo), Denys, H. (Hannelore), De Wever, O. (Olivier), and Hendrix, A. (An)

Subjects
separation, transcriptomics, proteomics, blood, biomarkers, exosomes, extracellular vesicles, corona, isolation, lipoprotein particles
Abstract

Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV.

Published
2021

15. Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction

Author

Tulkens, J. (Joeri), Vergauwen, G. (Glenn), Van Deun, J. (Jan), Geeurickx, E. (Edward), Dhondt, B. (Bert), Lippens, L. (Lien), De Scheerder, A. (Angélique), Miinalainen, I. (Ilkka), Rappu, P. (Pekka), De Geest, B. G. (Bruno G.), Vandecasteele, K. (Katrien), Laukens, D. (Debby), Vandekerckhove, L. (Linos), Denys, H. (Hannelore), Vandesompele, J. (Jo), De Wever, O. (Olivier), Hendrix, A. (An), Tulkens, J. (Joeri), Vergauwen, G. (Glenn), Van Deun, J. (Jan), Geeurickx, E. (Edward), Dhondt, B. (Bert), Lippens, L. (Lien), De Scheerder, A. (Angélique), Miinalainen, I. (Ilkka), Rappu, P. (Pekka), De Geest, B. G. (Bruno G.), Vandecasteele, K. (Katrien), Laukens, D. (Debby), Vandekerckhove, L. (Linos), Denys, H. (Hannelore), Vandesompele, J. (Jo), De Wever, O. (Olivier), and Hendrix, A. (An)

Published
2020

16. Feasibility of mechanical extrusion to coat nanoparticles with extracellular vesicle membranes

Author

Van Deun, J. (Jan), Roux, Q. (Quentin), Deville, S. (Sarah), Van Acker, T. (Thibaut), Rappu, P. (Pekka), Miinalainen, I. (Ilkka), Heino, J. (Jyrki), Vanhaecke, F. (Frank), De Geest, B. G. (Bruno G.), De Wever, O. (Olivier), Hendrix, A. (An), Van Deun, J. (Jan), Roux, Q. (Quentin), Deville, S. (Sarah), Van Acker, T. (Thibaut), Rappu, P. (Pekka), Miinalainen, I. (Ilkka), Heino, J. (Jyrki), Vanhaecke, F. (Frank), De Geest, B. G. (Bruno G.), De Wever, O. (Olivier), and Hendrix, A. (An)

Abstract

Biomimetic functionalization to confer stealth and targeting properties to nanoparticles is a field of intense study. Extracellular vesicles (EV), sub-micron delivery vehicles for intercellular communication, have unique characteristics for drug delivery. We investigated the top-down functionalization of gold nanoparticles with extracellular vesicle membranes, including both lipids and associated membrane proteins, through mechanical extrusion. EV surface-exposed membrane proteins were confirmed to help avoid unwanted elimination by macrophages, while improving autologous uptake. EV membrane morphology, protein composition and orientation were found to be unaffected by mechanical extrusion. We implemented complementary EV characterization methods, including transmission- and immune-electron microscopy, and nanoparticle tracking analysis, to verify membrane coating, size and zeta potential of the EV membrane-cloaked nanoparticles. While successful EV membrane coating of the gold nanoparticles resulted in lower macrophage uptake, low yield was found to be a significant downside of the extrusion approach. Our data incentivize more research to leverage EV membrane biomimicking as a unique drug delivery approach in the near future.

Published
2020

17. Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine

Author

Dhondt, B. (Bert), Geeurickx, E. (Edward), Tulkens, J. (Joeri), Van Deun, J. (Jan), Vergauwen, G. (Glenn), Lippens, L. (Lien), Miinalainen, I. (Ilkka), Rappu, P. (Pekka), Heino, J. (Jyrki), Ost, P. (Piet), Lumen, N. (Nicolaas), De Wever, O. (Olivier), Hendrix, A. (An), Dhondt, B. (Bert), Geeurickx, E. (Edward), Tulkens, J. (Joeri), Van Deun, J. (Jan), Vergauwen, G. (Glenn), Lippens, L. (Lien), Miinalainen, I. (Ilkka), Rappu, P. (Pekka), Heino, J. (Jyrki), Ost, P. (Piet), Lumen, N. (Nicolaas), De Wever, O. (Olivier), and Hendrix, A. (An)

Abstract

Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.

Published
2020

18. 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

Author

Théry, C, Witwer, KW, Aikawa, E, Alcaraz, MJ, Anderson, JD, Andriantsitohaina, R, Antoniou, A, Arab, T, Archer, F, Atkin-Smith, GK, Ayre, DC, Bach, JM, Bachurski, D, Baharvand, H, Balaj, L, Baldacchino, S, Bauer, NN, Baxter, AA, Bebawy, M, Beckham, C, Bedina Zavec, A, Benmoussa, A, Berardi, AC, Bergese, P, Bielska, E, Blenkiron, C, Bobis-Wozowicz, S, Boilard, E, Boireau, W, Bongiovanni, A, Borràs, FE, Bosch, S, Boulanger, CM, Breakefield, X, Breglio, AM, Brennan, M, Brigstock, DR, Brisson, A, Broekman, MLD, Bromberg, JF, Bryl-Górecka, P, Buch, S, Buck, AH, Burger, D, Busatto, S, Buschmann, D, Bussolati, B, Buzás, EI, Byrd, JB, Camussi, G, Carter, DRF, Caruso, S, Chamley, LW, Chang, YT, Chaudhuri, AD, Chen, C, Chen, S, Cheng, L, Chin, AR, Clayton, A, Clerici, SP, Cocks, A, Cocucci, E, Coffey, RJ, Cordeiro-da-Silva, A, Couch, Y, Coumans, FAW, Coyle, B, Crescitelli, R, Criado, MF, D’Souza-Schorey, C, Das, S, de Candia, P, De Santana, EF, De Wever, O, del Portillo, HA, Demaret, T, Deville, S, Devitt, A, Dhondt, B, Di Vizio, D, Dieterich, LC, Dolo, V, Dominguez Rubio, AP, Dominici, M, Dourado, MR, Driedonks, TAP, Duarte, FV, Duncan, HM, Eichenberger, RM, Ekström, K, EL Andaloussi, S, Elie-Caille, C, Erdbrügger, U, Falcón-Pérez, JM, Fatima, F, Fish, JE, Flores-Bellver, M, Försönits, A, Frelet-Barrand, A, and HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.

Subjects
ectosomes, microparticles, standardization, minimal information requirements, exosomes, guidelines, Biochemistry and Cell Biology, extracellular vesicles, microvesicles, reproducibility, rigor
Abstract

© 2018, © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles. 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.

Published
2019

19. Splenic metabolic activity as biomarker in cervical cancer

Author

De Jaeghere, E.A., primary, Laloo, F., additional, Lippens, L., additional, De Man, K., additional, Van Bockstal, M., additional, Van de Vijver, K., additional, Tummers, P., additional, Makar, A., additional, De Visschere, P., additional, De Wever, O., additional, Amant, F., additional, Denys, H., additional, and Vandecasteele, K., additional

Published
2019
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20. Stromal integrin alpha 11 regulates PDGFRβ signaling and promotes breast cancer progression

Author

Primac, I. (Irina), Maquoi, E. (Erik), Blacher, S. (Silvia), Heljasvaara, R. (Ritva), Van Deun, J. (Jan), Smeland, H. Y. (Hilde Y.H.), Canale, A. (Annalisa), Louis, T. (Thomas), Stuhr, L. (Linda), Sounni, N. E. (Nor Eddine), Cataldo, D. (Didier), Pihlajaniemi, T. (Taina), Pequeux, C. (Christel), De Wever, O. (Olivier), Gullberg, D. (Donald), Noel, A. (Agnès), Primac, I. (Irina), Maquoi, E. (Erik), Blacher, S. (Silvia), Heljasvaara, R. (Ritva), Van Deun, J. (Jan), Smeland, H. Y. (Hilde Y.H.), Canale, A. (Annalisa), Louis, T. (Thomas), Stuhr, L. (Linda), Sounni, N. E. (Nor Eddine), Cataldo, D. (Didier), Pihlajaniemi, T. (Taina), Pequeux, C. (Christel), De Wever, O. (Olivier), Gullberg, D. (Donald), and Noel, A. (Agnès)

Abstract

Cancer-associated fibroblasts (CAFs) are key actors in modulating the progression of many solid tumors, such as breast cancer (BC). Herein, we identify an integrin α11/PDGFRβ–positive CAF subset displaying tumor-promoting features in BC. In the preclinical MMTV-PyMT mouse model, integrin α11 deficiency led to a drastic reduction of tumor progression and metastasis. A clear association between integrin α11 and PDGFRβ was found at both transcriptional and histological levels in BC specimens. High stromal integrin α11/PDGFRβ expression was associated with high grades and poorer clinical outcome in human BC patients. Functional assays using 5 CAF subpopulations (1 murine, 4 human) revealed that integrin α11 promotes CAF invasion and CAF-induced tumor cell invasion upon PDGF-BB stimulation. Mechanistically, the proinvasive activity of integrin α11 relies on its ability to interact with PDGFRβ in a ligand-dependent manner and to promote its downstream JNK activation, leading to the production of tenascin C, a proinvasive matricellular protein. Pharmacological inhibition of PDGFRβ and JNK impaired tumor cell invasion induced by integrin α11+ CAFs. Collectively, our study uncovers an integrin α11+ subset of protumoral CAFs that exploits the PDGFRβ/JNK signaling axis to promote tumor invasiveness in BC.

Published
2019

21. 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

Author

Thery, C., Witwer, K. (Kenneth), Aikawa, E. (Elena), Alcaraz, M.J. (Maria Jose), Anderson, J.D. (Johnathon D), Andriantsitohaina, R. (Ramaroson), Antoniou, A. (Anna), Arab, T. (Tanina), Archer, F. (Fabienne), Atkin-Smith, G.K. (Georgia K), Ayre, D.C. (D Craig), Bach, J.-M. (Jean-Marie), Bachurski, D. (Daniel), Baharvand, H. (Hossein), Balaj, L. (Leonora), Baldacchino, S. (Shawn), Bauer, N.N. (Natalie N), Baxter, A.A. (Amy A), Bebawy, M. (Mary), Beckham, C. (Carla), Bedina Zavec, A. (Apolonija), Benmoussa, A. (Abderrahim), Berardi, A.C. (Anna C), Bergese, P. (Paolo), Bielska, E. (Ewa), Blenkiron, C. (Cherie), Bobis-Wozowicz, S. (Sylwia), Boilard, E. (Eric), Boireau, W. (Wilfrid), Bongiovanni, A. (Antonella), Borràs, F.E. (Francesc), Bosch, S. (Steffi), Boulanger, C.M. (Chantal), Breakefield, X. (Xandra), Breglio, A.M. (Andrew M), Brennan, M.Á. (Meadhbh Á), Brigstock, D.R. (David R), Brisson, A. (Alain), Broekman, M.L.D. (Marike), Bromberg, J.F. (Jacqueline F), Bryl-Górecka, P. (Paulina), Buch, S. (Shilpa), Buck, A.H. (Amy H), Burger, D. (Dylan), Busatto, S. (Sara), Buschmann, D. (Dominik), Bussolati, B. (Benedetta), Buzas, E. (Edit), Byrd, J.B. (James Bryan), Camussi, G. (Giovanni), Carter, D.R.F. (David RF), Caruso, S. (Sarah), Chamley, L.W. (Lawrence W), Chang, Y.-T. (Yu-Ting), Chaudhuri, A.D. (Amrita Datta), Chen, C. (Chihchen), Chen, S. (Shuai), Cheng, L. (Lesley), Chin, A.R. (Andrew R), Clayton, A. (Aled), Clerici, S.P. (Stefano P), Cocks, A. (Alex), Cocucci, E. (Emanuele), Coffey, R.J. (Robert J), Cordeiro-da-Silva, A. (Anabela), Couch, Y. (Yvonne), Coumans, F.A.W. (Frank AW), Coyle, B. (Beth), Crescitelli, R. (Rossella), Criado, M.F. (Miria Ferreira), D’Souza-Schorey, C. (Crislyn), Das, S. (Saumya), de Candia, P. (Paola), De Santana, E.F. (Eliezer F), De Wever, O. (Olivier), Del Portillo, H. (Hernando), Demaret, T. (Tanguy), Deville, S. (Sarah), Devitt, A. (Andrew), Dhondt, B. (Bert), Di Vizio, D. (Dolores), Dieterich, L.C. (Lothar C), Dolo, V. (Vincenza), Dominguez Rubio, A.P. (Ana Paula), Dominici, M. (Massimo), Dourado, M.R. (Mauricio R), Driedonks, T.A.P. (Tom AP), Duarte, F.V. (Filipe V), Duncan, H.M. (Heather M), Eichenberger, R.M. (Ramon M), Ekström, K. (Karin), EL Andaloussi, S. (Samir), Elie-Caille, C. (Celine), Erdbrügger, U. (Uta), Falcon-Perez, J.M. (Juan), Fatima, F. (Farah), Fish, J.E. (Jason E), Flores-Bellver, M. (Miguel), Försönits, A. (András), Frelet-Barrand, A. (Annie), Fricke, F. (Fabia), Fuhrmann, G. (Gregor), Gabrielsson, S. (Susanne), Gámez-Valero, A. (Ana), Gardiner, C. (Chris), Gärtner, K. (Kathrin), Gaudin, R. (Raphael), Gho, Y.S. (Yong Song), Giebel, B. (B.), Gilbert, C. (Caroline), Gimona, M. (Mario), Giusti, I. (Ilaria), Goberdhan, D.C.I. (Deborah CI), Görgens, A. (André), Gorski, S.M. (Sharon M), Greening, D.W. (David W.), Gross, J.C. (Julia Christina), Gualerzi, A. (Alice), Gupta, G.N. (Gopal N), Gustafson, D. (Dakota), Handberg, A. (Aase), Haraszti, R.A. (Reka A), Harrison, P. (Paul), Hegyesi, H. (Hargita), Hendrix, A. (An), Hill, A.F. (Andrew F), Hochberg, F.H. (Fred H), Hoffmann, K.F. (Karl F), Holder, B. (Beth), Holthofer, H. (Harry), Hosseinkhani, B. (Baharak), Hu, G. (Guoku), Huang, Y. (Yiyao), Huber, V. (Veronica), Hunt, S. (Stuart), Ibrahim, A.G.-E. (Ahmed Gamal-Eldin), Ikezu, T. (Tsuneya), Inal, J.M. (Jameel), Isin, M. (Mustafa), Ivanova, A. (Alena), Jackson, H.K. (Hannah K), Jacobsen, S. (Soren), Jay, S.M. (Steven M), Jayachandran, M. (Muthuvel), Jenster, G.W. (Guido), Jiang, L. (Lanzhou), Johnson, S.M. (Suzanne M), Jones, J.C. (Jennifer C), Jong, A. (Ambrose), Jovanovic-Talisman, T. (Tijana), Jung, S. (Stephanie), Kalluri, R. (Raghu), Kano, S.-I. (Shin-ichi), Kaur, S. (Sukhbir), Kawamura, Y. (Yumi), Keller, E.T. (Evan T), Khamari, D. (Delaram), Khomyakova, E. (Elena), Khvorova, A. (Anastasia), Kierulf, P. (Peter), Kim, K.P. (Kwang Pyo), Kislinger, T. (Thomas), Klingeborn, M. (Mikael), Klinke, D.J. (David J), Kornek, M. (Miroslaw), Kosanović, M.M. (Maja M), Kovács, Á.F. (Árpád Ferenc), Krämer-Albers, E.-M. (Eva-Maria), Krasemann, S. (Susanne), Krause, M. (Mirja), Kurochkin, I.V. (Igor V), Kusuma, G.D. (Gina D), Kuypers, S. (Sören), Laitinen, S. (Saara), Langevin, S.M. (Scott M), Languino, L.R. (Lucia R), Lannigan, J. (Joanne), Lässer, C. (Cecilia), Laurent, L.C. (Louise C), Lavieu, G. (Gregory), Lázaro-Ibáñez, E. (Elisa), Le Lay, S. (Soazig), Lee, M.-S. (Myung-Shin), Lee, Y.X.F. (Yi Xin Fiona), Lemos, D.S. (Debora S), Lenassi, M. (Metka), Leszczynska, A. (Aleksandra), Li, I.T.S. (Isaac TS), Liao, K. (Ke), Libregts, S.F. (Sten), Ligeti, E. (Erzsebet), Lim, R. (Rebecca), Lim, S.K. (Sai Kiang), Linē, A. (Aija), Linnemannstöns, K. (Karen), Llorente, A. (Alicia), Lombard, C.A. (Catherine A), Lorenowicz, M.J. (Magdalena J), Lörincz, Á.M. (Ákos M), Lötvall, J. (Jan), Lovett, J. (Jason), Lowry, M.C. (Michelle C), Loyer, X. (Xavier), Lu, Q. (Quan), Lukomska, B. (Barbara), Lunavat, T.R. (Taral R), Maas, S.L.N. (Sybren), Malhi, H. (Harmeet), Marcilla, A. (Antonio), Mariani, J. (Jacopo), Mariscal, J. (Javier), Martens-Uzunova, E.S. (Elena), Martin-Jaular, L. (Lorena), Martinez, M.C. (M Carmen), Martins, V.R. (Vilma Regina), Mathieu, M. (Mathilde), Mathivanan, S. (Suresh), Maugeri, M. (Marco), McGinnis, L.K. (Lynda K), McVey, M.J. (Mark J), Meckes, D.G. (David G), Meehan, K.L. (Katie L), Mertens, I. (Inge), Minciacchi, V.R. (Valentina R), Möller, A. (Andreas), Møller Jørgensen, M. (Malene), Morales-Kastresana, A. (Aizea), Morhayim, J. (Jess), Mullier, F. (Francois), Muraca, M. (Maurizio), Musante, L. (Luca), Mussack, V. (Veronika), Muth, D.C. (Dillon C), Myburgh, K.H. (Kathryn H), Najrana, T. (Tanbir), Nawaz, M. (Muhammad), Nazarenko, I. (Irina), Nejsum, P. (Peter), Neri, C. (Christian), Neri, T. (Tommaso), Nieuwland, C.C.M. (Carolien) van, Nimrichter, L. (Leonardo), Nolan, J.P. (John P), Nolte-’t Hoen, E.N.M. (Esther NM), Hooten, N.N. (Nicole Noren), O’Driscoll, L. (Lorraine), O’Grady, T. (Tina), O’Loghlen, A. (Ana), Ochiya, T. (Takahiro), Olivier, M. (Martin), Ortiz, A. (Alberto), Ortiz, L.A. (Luis A), Osteikoetxea, X. (Xabier), Ostegaard, O. (Ole), Ostrowski, M. (Matias), Park, J. (Jaesung), Pegtel, D.M. (D. Michiel), Peinado, H. (Hector), Perut, F. (Francesca), Pfaffl, M.W. (Michael W), Phinney, D.G. (Donald G), Pieters, B.C.H. (Bartijn CH), Pink, R.C. (Ryan C), Pisetsky, D.S. (David S), Pogge von Strandmann, E. (Elke), Polakovicova, I. (Iva), Poon, I.K.H. (Ivan KH), Powell, B.H. (Bonita H), Prada, I. (Ilaria), Pulliam, L. (Lynn), Quesenberry, P. (Peter), Radeghieri, A. (Annalisa), Raffai, R.L. (Robert L), Raimondo, S. (Stefania), Rak, J. (Janusz), Ramirez, M.I. (Marcel I.), Raposo, L. (Luís), Rayyan, M.S. (Morsi S), Regev-Rudzki, N. (Neta), Ricklefs, F.L. (Franz L), Robbins, P.D. (Paul D), Roberts, D.D. (David D), Rodrigues, S.C. (Silvia C), Rohde, E. (Eva), Rome, S. (Sophie), Rouschop, K.M.A. (Kasper MA), Rughetti, A. (Aurelia), Russell, A.E. (Ashley E), Saá, P. (Paula), Sahoo, S. (Susmita), Salas-Huenuleo, E. (Edison), Sánchez, C. (Catherine), Saugstad, J.A. (Julie A), Saul, M.J. (Meike J), Schiffelers, R.M. (Raymond), Schneider, R. (Raphael), Schøyen, T.H. (Tine Hiorth), Scott, A. (Aaron), Shahaj, E. (Eriomina), Sharma, S. (Shivani), Shatnyeva, O. (Olga), Shekari, F. (Faezeh), Shelke, G.V. (Ganesh Vilas), Shetty, A.K. (Ashok K), Shiba, K. (Kiyotaka), Siljander, P. (Pia), Silva, A.M. (Andreia M), Skowronek, A. (Agata), Snyder, O.L. (Orman L), Soares, R.P. (Rodrigo Pedro), Sódar, B.W. (Barbara W), Soekmadji, C. (Carolina), Sotillo, J. (Javier), Stahl, P.D. (Philip D), Stoorvogel, W. (Willem), Stott, S.L. (Shannon L), Strasser, E.F. (Erwin F), Swift, S. (Simon), Tahara, H. (Hidetoshi), Tewari, M. (Muneesh), Timms, K. (Kate), Tiwari, S. (Swasti), Tixeira, R. (Rochelle), Tkach, M. (Mercedes), Toh, W.S. (Wei Seong), Tomasini, R. (Richard), Torrecilhas, A.C. (Ana Claudia), Tosar, J.P. (Juan Pablo), Toxavidis, V. (Vasilis), Urbanelli, L. (Lorena), Vader, P. (Pieter), Balkom, B.W.M. (Bas) van, van der Grein, S.G. (Susanne G), Van Deun, J. (Jan), van Herwijnen, M.J.C. (Martijn JC), Van Keuren-Jensen, K. (Kendall), van Niel, G. (Guillaume), Royen, M.E. (Martin), van Wijnen, A.J. (Andre J), Vasconcelos, M.H. (M Helena), Vechetti, I.J. (Ivan J), Veit, T.D. (Tiago D), Vella, L.J. (Laura J.), Velot, É. (Émilie), Verweij, F.J. (Frederik J), Vestad, B. (Beate), Viñas, J.L. (Jose L), Visnovitz, T. (Tamás), Vukman, K.V. (Krisztina V), Wahlgren, J. (Jessica), Watson, D.C. (Dionysios C), Wauben, M.H.M. (Marca), Weaver, A. (Alissa), Webber, J.P. (Jason P), Weber, V. (Viktoria), Wehman, A.M. (Ann M), Weiss, D.J. (Daniel J), Welsh, J.A. (Joshua A), Wendt, S. (Sebastian), Wheelock, A.M. (Asa M), Wiener, Z. (Zoltán), Witte, L. (Leonie), Wolfram, J. (Joy), Xagorari, A. (Angeliki), Xander, P. (Patricia), Xu, J. (Jing), Yan, X. (Xiaomei), Yáñez-Mó, M. (María), Yin, H. (Hang), Yuana, Y., Zappulli, V. (Valentina), Zarubova, J. (Jana), Žėkas, V. (Vytautas), Zhang, J.-Y. (Jian-ye), Zhao, Z. (Zezhou), Zheng, L. (Lei), Zheutlin, A.R. (Alexander R), Zickler, A.M. (Antje M), Zimmermann, P. (Pascale), Zivkovic, A.M. (Angela M), Zocco, D. (Davide), Zuba-Surma, E.K. (Ewa K), Thery, C., Witwer, K. (Kenneth), Aikawa, E. (Elena), Alcaraz, M.J. (Maria Jose), Anderson, J.D. (Johnathon D), Andriantsitohaina, R. (Ramaroson), Antoniou, A. (Anna), Arab, T. (Tanina), Archer, F. (Fabienne), Atkin-Smith, G.K. (Georgia K), Ayre, D.C. (D Craig), Bach, J.-M. (Jean-Marie), Bachurski, D. (Daniel), Baharvand, H. (Hossein), Balaj, L. (Leonora), Baldacchino, S. (Shawn), Bauer, N.N. (Natalie N), Baxter, A.A. (Amy A), Bebawy, M. (Mary), Beckham, C. (Carla), Bedina Zavec, A. (Apolonija), Benmoussa, A. (Abderrahim), Berardi, A.C. (Anna C), Bergese, P. (Paolo), Bielska, E. (Ewa), Blenkiron, C. (Cherie), Bobis-Wozowicz, S. (Sylwia), Boilard, E. (Eric), Boireau, W. (Wilfrid), Bongiovanni, A. (Antonella), Borràs, F.E. (Francesc), Bosch, S. (Steffi), Boulanger, C.M. (Chantal), Breakefield, X. (Xandra), Breglio, A.M. (Andrew M), Brennan, M.Á. (Meadhbh Á), Brigstock, D.R. (David R), Brisson, A. (Alain), Broekman, M.L.D. (Marike), Bromberg, J.F. (Jacqueline F), Bryl-Górecka, P. (Paulina), Buch, S. (Shilpa), Buck, A.H. (Amy H), Burger, D. (Dylan), Busatto, S. (Sara), Buschmann, D. (Dominik), Bussolati, B. (Benedetta), Buzas, E. (Edit), Byrd, J.B. (James Bryan), Camussi, G. (Giovanni), Carter, D.R.F. (David RF), Caruso, S. (Sarah), Chamley, L.W. (Lawrence W), Chang, Y.-T. (Yu-Ting), Chaudhuri, A.D. (Amrita Datta), Chen, C. (Chihchen), Chen, S. (Shuai), Cheng, L. (Lesley), Chin, A.R. (Andrew R), Clayton, A. (Aled), Clerici, S.P. (Stefano P), Cocks, A. (Alex), Cocucci, E. (Emanuele), Coffey, R.J. (Robert J), Cordeiro-da-Silva, A. (Anabela), Couch, Y. (Yvonne), Coumans, F.A.W. (Frank AW), Coyle, B. (Beth), Crescitelli, R. (Rossella), Criado, M.F. (Miria Ferreira), D’Souza-Schorey, C. (Crislyn), Das, S. (Saumya), de Candia, P. (Paola), De Santana, E.F. (Eliezer F), De Wever, O. (Olivier), Del Portillo, H. (Hernando), Demaret, T. (Tanguy), Deville, S. (Sarah), Devitt, A. (Andrew), Dhondt, B. (Bert), Di Vizio, D. (Dolores), Dieterich, L.C. (Lothar C), Dolo, V. (Vincenza), Dominguez Rubio, A.P. (Ana Paula), Dominici, M. (Massimo), Dourado, M.R. (Mauricio R), Driedonks, T.A.P. (Tom AP), Duarte, F.V. (Filipe V), Duncan, H.M. (Heather M), Eichenberger, R.M. (Ramon M), Ekström, K. (Karin), EL Andaloussi, S. (Samir), Elie-Caille, C. (Celine), Erdbrügger, U. (Uta), Falcon-Perez, J.M. (Juan), Fatima, F. (Farah), Fish, J.E. (Jason E), Flores-Bellver, M. (Miguel), Försönits, A. (András), Frelet-Barrand, A. (Annie), Fricke, F. (Fabia), Fuhrmann, G. (Gregor), Gabrielsson, S. (Susanne), Gámez-Valero, A. (Ana), Gardiner, C. (Chris), Gärtner, K. (Kathrin), Gaudin, R. (Raphael), Gho, Y.S. (Yong Song), Giebel, B. (B.), Gilbert, C. (Caroline), Gimona, M. (Mario), Giusti, I. (Ilaria), Goberdhan, D.C.I. (Deborah CI), Görgens, A. (André), Gorski, S.M. (Sharon M), Greening, D.W. (David W.), Gross, J.C. (Julia Christina), Gualerzi, A. (Alice), Gupta, G.N. (Gopal N), Gustafson, D. (Dakota), Handberg, A. (Aase), Haraszti, R.A. (Reka A), Harrison, P. (Paul), Hegyesi, H. (Hargita), Hendrix, A. (An), Hill, A.F. (Andrew F), Hochberg, F.H. (Fred H), Hoffmann, K.F. (Karl F), Holder, B. (Beth), Holthofer, H. (Harry), Hosseinkhani, B. (Baharak), Hu, G. (Guoku), Huang, Y. (Yiyao), Huber, V. (Veronica), Hunt, S. (Stuart), Ibrahim, A.G.-E. (Ahmed Gamal-Eldin), Ikezu, T. (Tsuneya), Inal, J.M. (Jameel), Isin, M. (Mustafa), Ivanova, A. (Alena), Jackson, H.K. (Hannah K), Jacobsen, S. (Soren), Jay, S.M. (Steven M), Jayachandran, M. (Muthuvel), Jenster, G.W. (Guido), Jiang, L. (Lanzhou), Johnson, S.M. (Suzanne M), Jones, J.C. (Jennifer C), Jong, A. (Ambrose), Jovanovic-Talisman, T. (Tijana), Jung, S. (Stephanie), Kalluri, R. (Raghu), Kano, S.-I. (Shin-ichi), Kaur, S. (Sukhbir), Kawamura, Y. (Yumi), Keller, E.T. (Evan T), Khamari, D. (Delaram), Khomyakova, E. (Elena), Khvorova, A. (Anastasia), Kierulf, P. (Peter), Kim, K.P. (Kwang Pyo), Kislinger, T. (Thomas), Klingeborn, M. (Mikael), Klinke, D.J. (David J), Kornek, M. (Miroslaw), Kosanović, M.M. (Maja M), Kovács, Á.F. (Árpád Ferenc), Krämer-Albers, E.-M. (Eva-Maria), Krasemann, S. (Susanne), Krause, M. (Mirja), Kurochkin, I.V. (Igor V), Kusuma, G.D. (Gina D), Kuypers, S. (Sören), Laitinen, S. (Saara), Langevin, S.M. (Scott M), Languino, L.R. (Lucia R), Lannigan, J. (Joanne), Lässer, C. (Cecilia), Laurent, L.C. (Louise C), Lavieu, G. (Gregory), Lázaro-Ibáñez, E. (Elisa), Le Lay, S. (Soazig), Lee, M.-S. (Myung-Shin), Lee, Y.X.F. (Yi Xin Fiona), Lemos, D.S. (Debora S), Lenassi, M. (Metka), Leszczynska, A. (Aleksandra), Li, I.T.S. (Isaac TS), Liao, K. (Ke), Libregts, S.F. (Sten), Ligeti, E. (Erzsebet), Lim, R. (Rebecca), Lim, S.K. (Sai Kiang), Linē, A. (Aija), Linnemannstöns, K. (Karen), Llorente, A. (Alicia), Lombard, C.A. (Catherine A), Lorenowicz, M.J. (Magdalena J), Lörincz, Á.M. (Ákos M), Lötvall, J. (Jan), Lovett, J. (Jason), Lowry, M.C. (Michelle C), Loyer, X. (Xavier), Lu, Q. (Quan), Lukomska, B. (Barbara), Lunavat, T.R. (Taral R), Maas, S.L.N. (Sybren), Malhi, H. (Harmeet), Marcilla, A. (Antonio), Mariani, J. (Jacopo), Mariscal, J. (Javier), Martens-Uzunova, E.S. (Elena), Martin-Jaular, L. (Lorena), Martinez, M.C. (M Carmen), Martins, V.R. (Vilma Regina), Mathieu, M. (Mathilde), Mathivanan, S. (Suresh), Maugeri, M. (Marco), McGinnis, L.K. (Lynda K), McVey, M.J. (Mark J), Meckes, D.G. (David G), Meehan, K.L. (Katie L), Mertens, I. (Inge), Minciacchi, V.R. (Valentina R), Möller, A. (Andreas), Møller Jørgensen, M. (Malene), Morales-Kastresana, A. (Aizea), Morhayim, J. (Jess), Mullier, F. (Francois), Muraca, M. (Maurizio), Musante, L. (Luca), Mussack, V. (Veronika), Muth, D.C. (Dillon C), Myburgh, K.H. (Kathryn H), Najrana, T. (Tanbir), Nawaz, M. (Muhammad), Nazarenko, I. (Irina), Nejsum, P. (Peter), Neri, C. (Christian), Neri, T. (Tommaso), Nieuwland, C.C.M. (Carolien) van, Nimrichter, L. (Leonardo), Nolan, J.P. (John P), Nolte-’t Hoen, E.N.M. (Esther NM), Hooten, N.N. (Nicole Noren), O’Driscoll, L. (Lorraine), O’Grady, T. (Tina), O’Loghlen, A. (Ana), Ochiya, T. (Takahiro), Olivier, M. (Martin), Ortiz, A. (Alberto), Ortiz, L.A. (Luis A), Osteikoetxea, X. (Xabier), Ostegaard, O. (Ole), Ostrowski, M. (Matias), Park, J. (Jaesung), Pegtel, D.M. (D. Michiel), Peinado, H. (Hector), Perut, F. (Francesca), Pfaffl, M.W. (Michael W), Phinney, D.G. (Donald G), Pieters, B.C.H. (Bartijn CH), Pink, R.C. (Ryan C), Pisetsky, D.S. (David S), Pogge von Strandmann, E. (Elke), Polakovicova, I. (Iva), Poon, I.K.H. (Ivan KH), Powell, B.H. (Bonita H), Prada, I. (Ilaria), Pulliam, L. (Lynn), Quesenberry, P. (Peter), Radeghieri, A. (Annalisa), Raffai, R.L. (Robert L), Raimondo, S. (Stefania), Rak, J. (Janusz), Ramirez, M.I. (Marcel I.), Raposo, L. (Luís), Rayyan, M.S. (Morsi S), Regev-Rudzki, N. (Neta), Ricklefs, F.L. (Franz L), Robbins, P.D. (Paul D), Roberts, D.D. (David D), Rodrigues, S.C. (Silvia C), Rohde, E. (Eva), Rome, S. (Sophie), Rouschop, K.M.A. (Kasper MA), Rughetti, A. (Aurelia), Russell, A.E. (Ashley E), Saá, P. (Paula), Sahoo, S. (Susmita), Salas-Huenuleo, E. (Edison), Sánchez, C. (Catherine), Saugstad, J.A. (Julie A), Saul, M.J. (Meike J), Schiffelers, R.M. (Raymond), Schneider, R. (Raphael), Schøyen, T.H. (Tine Hiorth), Scott, A. (Aaron), Shahaj, E. (Eriomina), Sharma, S. (Shivani), Shatnyeva, O. (Olga), Shekari, F. (Faezeh), Shelke, G.V. (Ganesh Vilas), Shetty, A.K. (Ashok K), Shiba, K. (Kiyotaka), Siljander, P. (Pia), Silva, A.M. (Andreia M), Skowronek, A. (Agata), Snyder, O.L. (Orman L), Soares, R.P. (Rodrigo Pedro), Sódar, B.W. (Barbara W), Soekmadji, C. (Carolina), Sotillo, J. (Javier), Stahl, P.D. (Philip D), Stoorvogel, W. (Willem), Stott, S.L. (Shannon L), Strasser, E.F. (Erwin F), Swift, S. (Simon), Tahara, H. (Hidetoshi), Tewari, M. (Muneesh), Timms, K. (Kate), Tiwari, S. (Swasti), Tixeira, R. (Rochelle), Tkach, M. (Mercedes), Toh, W.S. (Wei Seong), Tomasini, R. (Richard), Torrecilhas, A.C. (Ana Claudia), Tosar, J.P. (Juan Pablo), Toxavidis, V. (Vasilis), Urbanelli, L. (Lorena), Vader, P. (Pieter), Balkom, B.W.M. (Bas) van, van der Grein, S.G. (Susanne G), Van Deun, J. (Jan), van Herwijnen, M.J.C. (Martijn JC), Van Keuren-Jensen, K. (Kendall), van Niel, G. (Guillaume), Royen, M.E. (Martin), van Wijnen, A.J. (Andre J), Vasconcelos, M.H. (M Helena), Vechetti, I.J. (Ivan J), Veit, T.D. (Tiago D), Vella, L.J. (Laura J.), Velot, É. (Émilie), Verweij, F.J. (Frederik J), Vestad, B. (Beate), Viñas, J.L. (Jose L), Visnovitz, T. (Tamás), Vukman, K.V. (Krisztina V), Wahlgren, J. (Jessica), Watson, D.C. (Dionysios C), Wauben, M.H.M. (Marca), Weaver, A. (Alissa), Webber, J.P. (Jason P), Weber, V. (Viktoria), Wehman, A.M. (Ann M), Weiss, D.J. (Daniel J), Welsh, J.A. (Joshua A), Wendt, S. (Sebastian), Wheelock, A.M. (Asa M), Wiener, Z. (Zoltán), Witte, L. (Leonie), Wolfram, J. (Joy), Xagorari, A. (Angeliki), Xander, P. (Patricia), Xu, J. (Jing), Yan, X. (Xiaomei), Yáñez-Mó, M. (María), Yin, H. (Hang), Yuana, Y., Zappulli, V. (Valentina), Zarubova, J. (Jana), Žėkas, V. (Vytautas), Zhang, J.-Y. (Jian-ye), Zhao, Z. (Zezhou), Zheng, L. (Lei), Zheutlin, A.R. (Alexander R), Zickler, A.M. (Antje M), Zimmermann, P. (Pascale), Zivkovic, A.M. (Angela M), Zocco, D. (Davide), and Zuba-Surma, E.K. (Ewa K)

Abstract

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 the

Published
2019
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22. The generation and use of recombinant extracellular vesicles as biological reference material

Author

Geeurickx, E. (Edward), Tulkens, J. (Joeri), Dhondt, B. (Bert), Van Deun, J. (Jan), Lippens, L. (Lien), Vergauwen, G. (Glenn), Heyrman, E. (Elisa), De Sutter, D. (Delphine), Gevaert, K. (Kris), Impens, F. (Francis), Miinalainen, I. (Ilkka), Van Bockstal, P.-J. (Pieter-Jan), De Beer, T. (Thomas), Wauben, M. H. (Marca H. M.), Nolte-‘t-Hoen, E. N. (Esther N. M.), Bloch, K. (Katarzyna), Swinnen, J. V. (Johannes V.), van der Pol, E. (Edwin), Nieuwland, R. (Rienk), Braems, G. (Geert), Callewaert, N. (Nico), Mestdagh, P. (Pieter), Vandesompele, J. (Jo), Denys, H. (Hannelore), Eyckerman, S. (Sven), De Wever, O. (Olivier), Hendrix, A. (An), Geeurickx, E. (Edward), Tulkens, J. (Joeri), Dhondt, B. (Bert), Van Deun, J. (Jan), Lippens, L. (Lien), Vergauwen, G. (Glenn), Heyrman, E. (Elisa), De Sutter, D. (Delphine), Gevaert, K. (Kris), Impens, F. (Francis), Miinalainen, I. (Ilkka), Van Bockstal, P.-J. (Pieter-Jan), De Beer, T. (Thomas), Wauben, M. H. (Marca H. M.), Nolte-‘t-Hoen, E. N. (Esther N. M.), Bloch, K. (Katarzyna), Swinnen, J. V. (Johannes V.), van der Pol, E. (Edwin), Nieuwland, R. (Rienk), Braems, G. (Geert), Callewaert, N. (Nico), Mestdagh, P. (Pieter), Vandesompele, J. (Jo), Denys, H. (Hannelore), Eyckerman, S. (Sven), De Wever, O. (Olivier), and Hendrix, A. (An)

Abstract

Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications.

Published
2019

23. Reproducible isolation of high-purity urinary extracellular vesicles for uro-oncological biomarker studies

Author

Dhondt, B., primary, Vergauwen, G., additional, Van Deun, J., additional, Geeurickx, E., additional, Tulkens, J., additional, Lippens, L., additional, Buelens, S., additional, De Bleser, E., additional, Poelaert, F., additional, Claeys, T., additional, Miinalainen, I., additional, Rappu, P., additional, Heino, J., additional, Hendrix, A., additional, De Wever, O., additional, and Lumen, N., additional

Published
2018
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24. 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

Author

Thery, C, Witwer, KW, Aikawa, E, Jose Alcaraz, M, Anderson, JD, Andriantsitohaina, R, Antoniou, A, Arab, T, Archer, F, Atkin-Smith, GK, Ayre, DC, Bach, J-M, Bachurski, D, Baharvand, H, Balaj, L, Baldacchino, S, Bauer, NN, Baxter, AA, Bebawy, M, Beckham, C, Zavec, AB, Benmoussa, A, Berardi, AC, Bergese, P, Bielska, E, Blenkiron, C, Bobis-Wozowicz, S, Boilard, E, Boireau, W, Bongiovanni, A, Borras, FE, Bosch, S, Boulanger, CM, Breakefield, X, Breglio, AM, Brennan, MA, Brigstock, DR, Brisson, A, Broekman, MLD, Bromberg, JF, Bryl-Gorecka, P, Buch, S, Buck, AH, Burger, D, Busatto, S, Buschmann, D, Bussolati, B, Buzas, E, Byrd, JB, Camussi, G, Carter, DRF, Caruso, S, Chamley, LW, Chang, Y-T, Chen, C, Chen, S, Cheng, L, Chin, AR, Clayton, A, Clerici, SP, Cocks, A, Cocucci, E, Coffey, RJ, Cordeiro-da-Silva, A, Couch, Y, Coumans, FAW, Coyle, B, Crescitelli, R, Criado, MF, D'Souza-Schorey, C, Das, S, Chaudhuri, AD, de Candia, P, De Santana Junior, EF, De Wever, O, del Portillo, HA, Demaret, T, Deville, S, Devitt, A, Dhondt, B, Di Vizio, D, Dieterich, LC, Dolo, V, Dominguez Rubio, AP, Dominici, M, Dourado, MR, Driedonks, TAP, Duarte, F, Duncan, HM, Eichenberger, RM, Ekstrom, K, Andaloussi, SEL, Elie-Caille, C, Erdbrugger, U, Falcon-Perez, JM, Fatima, F, Fish, JE, Flores-Bellver, M, Forsonits, A, Frelet-Barrand, A, Fricke, F, Fuhrmann, G, Gabrielsson, S, Gamez-Valero, A, Gardiner, C, Gaertner, K, Gaudin, R, Gho, YS, Giebel, B, Gilbert, C, Gimona, M, Giusti, I, Goberdhan, DC, Goergens, A, Gorski, SM, Greening, DW, Gross, JC, Gualerzi, A, Gupta, GN, Gustafson, D, Handberg, A, Haraszti, RA, Harrison, P, Hegyesi, H, Hendrix, A, Hill, AF, Hochberg, FH, Hoffmann, KF, Holder, B, Holthofer, H, Hosseinkhani, B, Hu, G, Huang, Y, Huber, V, Hunt, S, Ibrahim, AG-E, Ikezu, T, Inal, JM, Isin, M, Ivanova, A, Jackson, HK, Jacobsen, S, Jay, SM, Jayachandran, M, Jenster, G, Jiang, L, Johnson, SM, Jones, JC, Jong, A, Jovanovic-Talisman, T, Jung, S, Kalluri, R, Kano, S-I, Kaur, S, Kawamura, Y, Keller, ET, Khamari, D, Khomyakova, E, Khvorova, A, Kierulf, P, Kim, KP, Kislinger, T, Klingeborn, M, Klinke, DJ, Kornek, M, Kosanovic, MM, Kovacs, AF, Kraemer-Albers, E-M, Krasemann, S, Krause, M, Kurochkin, I, Kusuma, GD, Kuypers, S, Laitinen, S, Langevin, SM, Languino, LR, Lannigan, J, Lasser, C, Laurent, LC, Lavieu, G, Lazaro-Ibanez, E, Le Lay, S, Lee, M-S, Lee, YXF, Lemos, DS, Lenassi, M, Leszczynska, A, Li, ITS, Liao, K, Libregts, SF, Ligeti, E, Lim, R, Lim, SK, Line, A, Linnemannstoens, K, Llorente, A, Lombard, CA, Lorenowicz, MJ, Lorincz, AM, Lotvall, J, Lovett, J, Lowry, MC, Loyer, X, Lu, Q, Lukomska, B, Lunavat, TR, Maas, SLN, Malhi, H, Marcilla, A, Mariani, J, Mariscal, J, Martens-Uzunova, ES, Martin-Jaular, L, Martinez, MC, Martins, VR, Mathieu, M, Mathivanan, S, Maugeri, M, McGinnis, LK, McVey, MJ, Meckes, DG, Meehan, KL, Mertens, I, Minciacchi, VR, Moller, A, Jorgensen, MM, Morales-Kastresana, A, Morhayim, J, Mullier, F, Muraca, M, Musante, L, Mussack, V, Muth, DC, Myburgh, KH, Najrana, T, Nawaz, M, Nazarenko, I, Nejsum, P, Neri, C, Neri, T, Nieuwland, R, Nimrichter, L, Nolan, JP, Nolte-'t Hoen, ENM, Noren Hooten, N, O'Driscoll, L, O'Grady, T, O'Loghlen, A, Ochiya, T, Olivier, M, Ortiz, A, Ortiz, LA, Osteikoetxea, X, Ostegaard, O, Ostrowski, M, Park, J, Pegtel, DM, Peinado, H, Perut, F, Pfaffl, MW, Phinney, DG, Pieters, BCH, Pink, RC, Pisetsky, DS, von Strandmann, EP, Polakovicova, I, Poon, IKH, Powell, BH, Prada, I, Pulliam, L, Quesenberry, P, Radeghieri, A, Raffai, RL, Raimondo, S, Rak, J, Ramirez, M, Raposo, G, Rayyan, MS, Regev-Rudzki, N, Ricklefs, FL, Robbins, PD, Roberts, DD, Rodrigues, SC, Rohde, E, Rome, S, Rouschop, KMA, Rughetti, A, Russell, AE, Saa, P, Sahoo, S, Salas-Huenuleo, E, Sanchez, C, Saugstad, JA, Saul, MJ, Schiffelers, RM, Schneider, R, Schoyen, TH, Scott, A, Shahaj, E, Sharma, S, Shatnyeva, O, Shekari, F, Shelke, GV, Shetty, AK, Shiba, K, Siljander, PR-M, Silva, AM, Skowronek, A, Snyder, OL, Soares, RP, Sodar, BW, Soekmadji, C, Sotillo, J, Stahl, PD, Stoorvogel, W, Stott, SL, Strasser, EF, Swift, S, Tahara, H, Tewari, M, Timms, K, Tiwari, S, Tixeira, R, Tkach, M, Toh, WS, Tomasini, R, Torrecilhas, AC, Pablo Tosar, J, Toxavidis, V, Urbanelli, L, Vader, P, van Balkom, BWM, van der Grein, SG, Van Deun, J, van Herwijnen, MJC, Van Keuren-Jensen, K, van Niel, G, van Royen, ME, van Wijnen, AJ, Helena Vasconcelos, M, Vechetti, IJ, Veit, TD, Vella, LJ, Velot, E, Verweij, FJ, Vestad, B, Vinas, JL, Visnovitz, T, Vukman, KV, Wahlgren, J, Watson, DC, Wauben, MHM, Weaver, A, Webber, JP, Weber, V, Wehman, AM, Weiss, DJ, Welsh, JA, Wendt, S, Wheelock, AM, Wiener, Z, Witte, L, Wolfram, J, Xagorari, A, Xander, P, Xu, J, Yan, X, Yanez-Mo, M, Yin, H, Yuana, Y, Zappulli, V, Zarubova, J, Zekas, V, Zhang, J-Y, Zhao, Z, Zheng, L, Zheutlin, AR, Zickler, AM, Zimmermann, P, Zivkovic, AM, Zocco, D, Zuba-Surma, EK, Thery, C, Witwer, KW, Aikawa, E, Jose Alcaraz, M, Anderson, JD, Andriantsitohaina, R, Antoniou, A, Arab, T, Archer, F, Atkin-Smith, GK, Ayre, DC, Bach, J-M, Bachurski, D, Baharvand, H, Balaj, L, Baldacchino, S, Bauer, NN, Baxter, AA, Bebawy, M, Beckham, C, Zavec, AB, Benmoussa, A, Berardi, AC, Bergese, P, Bielska, E, Blenkiron, C, Bobis-Wozowicz, S, Boilard, E, Boireau, W, Bongiovanni, A, Borras, FE, Bosch, S, Boulanger, CM, Breakefield, X, Breglio, AM, Brennan, MA, Brigstock, DR, Brisson, A, Broekman, MLD, Bromberg, JF, Bryl-Gorecka, P, Buch, S, Buck, AH, Burger, D, Busatto, S, Buschmann, D, Bussolati, B, Buzas, E, Byrd, JB, Camussi, G, Carter, DRF, Caruso, S, Chamley, LW, Chang, Y-T, Chen, C, Chen, S, Cheng, L, Chin, AR, Clayton, A, Clerici, SP, Cocks, A, Cocucci, E, Coffey, RJ, Cordeiro-da-Silva, A, Couch, Y, Coumans, FAW, Coyle, B, Crescitelli, R, Criado, MF, D'Souza-Schorey, C, Das, S, Chaudhuri, AD, de Candia, P, De Santana Junior, EF, De Wever, O, del Portillo, HA, Demaret, T, Deville, S, Devitt, A, Dhondt, B, Di Vizio, D, Dieterich, LC, Dolo, V, Dominguez Rubio, AP, Dominici, M, Dourado, MR, Driedonks, TAP, Duarte, F, Duncan, HM, Eichenberger, RM, Ekstrom, K, Andaloussi, SEL, Elie-Caille, C, Erdbrugger, U, Falcon-Perez, JM, Fatima, F, Fish, JE, Flores-Bellver, M, Forsonits, A, Frelet-Barrand, A, Fricke, F, Fuhrmann, G, Gabrielsson, S, Gamez-Valero, A, Gardiner, C, Gaertner, K, Gaudin, R, Gho, YS, Giebel, B, Gilbert, C, Gimona, M, Giusti, I, Goberdhan, DC, Goergens, A, Gorski, SM, Greening, DW, Gross, JC, Gualerzi, A, Gupta, GN, Gustafson, D, Handberg, A, Haraszti, RA, Harrison, P, Hegyesi, H, Hendrix, A, Hill, AF, Hochberg, FH, Hoffmann, KF, Holder, B, Holthofer, H, Hosseinkhani, B, Hu, G, Huang, Y, Huber, V, Hunt, S, Ibrahim, AG-E, Ikezu, T, Inal, JM, Isin, M, Ivanova, A, Jackson, HK, Jacobsen, S, Jay, SM, Jayachandran, M, Jenster, G, Jiang, L, Johnson, SM, Jones, JC, Jong, A, Jovanovic-Talisman, T, Jung, S, Kalluri, R, Kano, S-I, Kaur, S, Kawamura, Y, Keller, ET, Khamari, D, Khomyakova, E, Khvorova, A, Kierulf, P, Kim, KP, Kislinger, T, Klingeborn, M, Klinke, DJ, Kornek, M, Kosanovic, MM, Kovacs, AF, Kraemer-Albers, E-M, Krasemann, S, Krause, M, Kurochkin, I, Kusuma, GD, Kuypers, S, Laitinen, S, Langevin, SM, Languino, LR, Lannigan, J, Lasser, C, Laurent, LC, Lavieu, G, Lazaro-Ibanez, E, Le Lay, S, Lee, M-S, Lee, YXF, Lemos, DS, Lenassi, M, Leszczynska, A, Li, ITS, Liao, K, Libregts, SF, Ligeti, E, Lim, R, Lim, SK, Line, A, Linnemannstoens, K, Llorente, A, Lombard, CA, Lorenowicz, MJ, Lorincz, AM, Lotvall, J, Lovett, J, Lowry, MC, Loyer, X, Lu, Q, Lukomska, B, Lunavat, TR, Maas, SLN, Malhi, H, Marcilla, A, Mariani, J, Mariscal, J, Martens-Uzunova, ES, Martin-Jaular, L, Martinez, MC, Martins, VR, Mathieu, M, Mathivanan, S, Maugeri, M, McGinnis, LK, McVey, MJ, Meckes, DG, Meehan, KL, Mertens, I, Minciacchi, VR, Moller, A, Jorgensen, MM, Morales-Kastresana, A, Morhayim, J, Mullier, F, Muraca, M, Musante, L, Mussack, V, Muth, DC, Myburgh, KH, Najrana, T, Nawaz, M, Nazarenko, I, Nejsum, P, Neri, C, Neri, T, Nieuwland, R, Nimrichter, L, Nolan, JP, Nolte-'t Hoen, ENM, Noren Hooten, N, O'Driscoll, L, O'Grady, T, O'Loghlen, A, Ochiya, T, Olivier, M, Ortiz, A, Ortiz, LA, Osteikoetxea, X, Ostegaard, O, Ostrowski, M, Park, J, Pegtel, DM, Peinado, H, Perut, F, Pfaffl, MW, Phinney, DG, Pieters, BCH, Pink, RC, Pisetsky, DS, von Strandmann, EP, Polakovicova, I, Poon, IKH, Powell, BH, Prada, I, Pulliam, L, Quesenberry, P, Radeghieri, A, Raffai, RL, Raimondo, S, Rak, J, Ramirez, M, Raposo, G, Rayyan, MS, Regev-Rudzki, N, Ricklefs, FL, Robbins, PD, Roberts, DD, Rodrigues, SC, Rohde, E, Rome, S, Rouschop, KMA, Rughetti, A, Russell, AE, Saa, P, Sahoo, S, Salas-Huenuleo, E, Sanchez, C, Saugstad, JA, Saul, MJ, Schiffelers, RM, Schneider, R, Schoyen, TH, Scott, A, Shahaj, E, Sharma, S, Shatnyeva, O, Shekari, F, Shelke, GV, Shetty, AK, Shiba, K, Siljander, PR-M, Silva, AM, Skowronek, A, Snyder, OL, Soares, RP, Sodar, BW, Soekmadji, C, Sotillo, J, Stahl, PD, Stoorvogel, W, Stott, SL, Strasser, EF, Swift, S, Tahara, H, Tewari, M, Timms, K, Tiwari, S, Tixeira, R, Tkach, M, Toh, WS, Tomasini, R, Torrecilhas, AC, Pablo Tosar, J, Toxavidis, V, Urbanelli, L, Vader, P, van Balkom, BWM, van der Grein, SG, Van Deun, J, van Herwijnen, MJC, Van Keuren-Jensen, K, van Niel, G, van Royen, ME, van Wijnen, AJ, Helena Vasconcelos, M, Vechetti, IJ, Veit, TD, Vella, LJ, Velot, E, Verweij, FJ, Vestad, B, Vinas, JL, Visnovitz, T, Vukman, KV, Wahlgren, J, Watson, DC, Wauben, MHM, Weaver, A, Webber, JP, Weber, V, Wehman, AM, Weiss, DJ, Welsh, JA, Wendt, S, Wheelock, AM, Wiener, Z, Witte, L, Wolfram, J, Xagorari, A, Xander, P, Xu, J, Yan, X, Yanez-Mo, M, Yin, H, Yuana, Y, Zappulli, V, Zarubova, J, Zekas, V, Zhang, J-Y, Zhao, Z, Zheng, L, Zheutlin, AR, Zickler, AM, Zimmermann, P, Zivkovic, AM, Zocco, D, and Zuba-Surma, EK

Abstract

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.

Published
2018

27. Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research

Author

Vergauwen, G. (Glenn), Dhondt, B. (Bert), Van Deun, J. (Jan), De Smedt, E. (Eva), Berx, G. (Geert), Timmerman, E. (Evy), Gevaert, K. (Kris), Miinalainen, I. (Ilkka), Cocquyt, V. (Véronique), Braems, G. (Geert), Van den Broecke, R. (Rudy), Denys, H. (Hannelore), De Wever, O. (Olivier), Hendrix, A. (An), Vergauwen, G. (Glenn), Dhondt, B. (Bert), Van Deun, J. (Jan), De Smedt, E. (Eva), Berx, G. (Geert), Timmerman, E. (Evy), Gevaert, K. (Kris), Miinalainen, I. (Ilkka), Cocquyt, V. (Véronique), Braems, G. (Geert), Van den Broecke, R. (Rudy), Denys, H. (Hannelore), De Wever, O. (Olivier), and Hendrix, A. (An)

Abstract

Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.

Published
2017

28. The transcriptome of lung tumor-infiltrating dendritic cells reveals a tumor-supporting phenotype and a microRNA signature with negative impact on clinical outcome

Author

Pyfferoen, L, Brabants, E, Everaert, C, De Cabooter, N, Heyns, K, Deswarte, K, Vanheerswynghels, M, De Prijck, S, Waegemans, G, Dullaers, M, Hammad, H, De Wever, O, Mestdagh, P, Vandesompele, J, Lambrecht, Bart, Vermaelen, KY, Pyfferoen, L, Brabants, E, Everaert, C, De Cabooter, N, Heyns, K, Deswarte, K, Vanheerswynghels, M, De Prijck, S, Waegemans, G, Dullaers, M, Hammad, H, De Wever, O, Mestdagh, P, Vandesompele, J, Lambrecht, Bart, and Vermaelen, KY

Published
2017

31. Laminin α1 orchestrates VEGFA functions in the ecosystem of colorectal carcinoma

Author

Mammadova-Bach, E., primary, Rupp, T., additional, Spenlé, C., additional, Jivkov, I., additional, Shankaranarayanan, P., additional, Klein, A., additional, Pisarsky, L., additional, Méchine-Neuville, A., additional, Cremel, G., additional, Kedinger, M., additional, De Wever, O., additional, Ambartsumian, N., additional, Robine, S., additional, Pencreach, E., additional, Guenot, D., additional, Goetz, J.G., additional, Simon-Assmann, P., additional, Orend, G., additional, and Lefebvre, O., additional

Published
2017
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32. APOBEC3G expression correlates with T-cell infiltration and improved clinical outcomes in high-grade serous ovarian carcinoma

Author

Leonard, B. (Brandon), Starrett, G.J. (Gabriel J.), Maurer, M.J. (Matthew J.), Oberg, A.L. (Ann L.), Van Bockstal, M. (Mieke), Van Dorpe, J. (Jo), De Wever, O. (Olivier), Helleman, J. (Jozien), Sieuwerts, A.M. (Anieta), Berns, P.M.J.J. (Els), Martens, J.W.M. (John), Anderson, B.D. (Brett D.), Brown, W.L. (William L.), Kalli, K.R. (Kimberly R.), Kaufmann, S.H. (Scott H.), Harris, R.S. (Reuben), Leonard, B. (Brandon), Starrett, G.J. (Gabriel J.), Maurer, M.J. (Matthew J.), Oberg, A.L. (Ann L.), Van Bockstal, M. (Mieke), Van Dorpe, J. (Jo), De Wever, O. (Olivier), Helleman, J. (Jozien), Sieuwerts, A.M. (Anieta), Berns, P.M.J.J. (Els), Martens, J.W.M. (John), Anderson, B.D. (Brett D.), Brown, W.L. (William L.), Kalli, K.R. (Kimberly R.), Kaufmann, S.H. (Scott H.), and Harris, R.S. (Reuben)

Abstract

__Purpose:__ APOBEC3 DNA cytosine deaminase family members normally defend against viruses and transposons. However, deregulated APOBEC3 activity causes mutations in cancer. Because of broad expression profiles and varying mixtures of normal and cancer cells in tumors, including immune cell infiltration, it is difficult to determine where different APOBEC3s are expressed. Here, we ask whether correlations exist between APOBEC3 expression and T-cell infiltration in high-grade serous ovarian cancer (HGSOC), and assess whether these correlations have prognostic value. __Experimental Design:__ Transcripts for APOBEC3G, APOBEC3B, and the T-cell markers, CD3D, CD4, CD8A, GZMB, PRF1, and RNF128 were quantified by RT-qPCR for a cohort of 354 HGSOC patients. Expression values were correlated with each other and clinical parameters. Two additional cohorts were used to extend HGSOC clinical results. Immunoimaging was used to colocalize APOBEC3G and the T-cell marker CD3. TCGA data extended expression analyses to additional cancer types. __Results:__ A surprising positive correlation was found for expression of APOBEC3G and several T cell genes in HGSOC. Immunohistochemistry and immunofluorescent imaging showed protein colocalization in tumor-infiltrating T lymphocytes. High APOBEC3G expression correlated with improved outcomes in multiple HGSOC cohorts. TCGA data analyses revealed that expression of APOBEC3D and APOBEC3H also correlates with CD3D across multiple cancer types. __Conclusions:__ Our results identify APOBEC3G as a new candidate biomarker for tumor-infiltrating T lymphocytes and favorable prognoses for HGSOC. Our data also highlight the complexity of the tumor environment with respect to differential APOBEC family gene expression in both tumor and surrounding normal cell types. Clin Cancer Res; 22(18); 4746-55.

Published
2016
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37. Fibroblast activation protein-α, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations

Author

Koczorowska, M.M., primary, Tholen, S., additional, Bucher, F., additional, Lutz, L., additional, Kizhakkedathu, J.N., additional, De Wever, O., additional, Wellner, U.F., additional, Biniossek, M.L., additional, Stahl, A., additional, Lassmann, S., additional, and Schilling, O., additional

Published
2015
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38. Effect of the secretory small GTPases Rab27B on breast cancer growth, invasion, and metastasis

Author

Hendrix, A., Maynard, D., Pauwels, P., Braems, G., Denys, H., Van den Broecke, R., Lambert, J., Van Belle, S., Cocquyt, V., Gespach, C., Bracke, M., Seabra, MC., Gahl, WA., De Wever, O., and Westbroek, W.

Subjects
EXPRESSION, GRISCELLI-SYNDROME, MATRIX METALLOPROTEINASES, HEPATOCELLULAR-CARCINOMA, EXOCYTOSIS, GRANULES, SHOCK-PROTEIN 90, HSP90, MASS-SPECTROMETRY, CELL-MIGRATION
Abstract

Methods Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided.

Published
2010

42. The secretory small GTPase Rab27B as a marker for breast cancer progression

Author

Hendrix A, Braems G, Bracke M, Miguel Seabra, Gahl W, De Wever O, and Westbroek W

Subjects
EXPRESSION, MELANOCYTES, GRISCELLI-SYNDROME, PROTEINS, Breast Neoplasms, Prognosis, GENE, Gene Expression Regulation, Neoplastic, Oncology, Receptors, Estrogen, rab GTP-Binding Proteins, Lymphatic Metastasis, METASTASIS, CELLS, Biomarkers, Tumor, Disease Progression, Medicine and Health Sciences, Humans, GROWTH, Female, Neoplasm Invasiveness, Research Perspectives, Signal Transduction
Abstract

In contemporary oncology practice, an urgent need remains to refine the prognostic assessment of breast cancer. It is still difficult to identify patients with early breast cancer who are likely to benefit from adjuvant chemotherapy. Although invasion of cancer cells is the main prognostic denominator in tumor malignancy, our molecular understanding and diagnosis are often inadequate to cope with this activity. Therefore, deciphering molecular pathways of how tumors invade and metastasize may help in the identification of a useful prognostic marker. We recently discovered that the secretory small GTPase Rab27B, a regulator of vesicle exocytosis, delivers proinvasive signals for increased invasiveness, tumor size, and metastasis of various estrogen receptor (ER)-positive breast cancer cell lines, both in vitro and in vivo. In human breast cancer specimens, the presence of Rab27B protein proved to be associated with a low degree of differentiation and the presence of lymph node metastasis in ER-positive breast cancer.

Published
2010

48. JAK3 deregulation by activating mutations confers invasive growth advantage in extranodal nasal-type natural killer cell lymphoma

Author

Bouchekioua, A, primary, Scourzic, L, additional, de Wever, O, additional, Zhang, Y, additional, Cervera, P, additional, Aline-Fardin, A, additional, Mercher, T, additional, Gaulard, P, additional, Nyga, R, additional, Jeziorowska, D, additional, Douay, L, additional, Vainchenker, W, additional, Louache, F, additional, Gespach, C, additional, Solary, E, additional, and Coppo, P, additional

Published
2013
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