Maja Kosanović

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

Isolation of urinary extracellular vesicles from Tamm- Horsfall protein–depleted urine and their application in the development of a lectin-exosome-binding assay

Urine is a readily available source of relatively large quantities of extracellular vesicles (EVs). However, the isolation of urinary EVs (uEVs) is complicated by the presence of Tamm-Horsfall protein (THP), which polymerizes and co-precipitates as a contaminant. This may make glycan analysis of uEVs difficult since THP is heavily glycosylated. To facilitate glycosylation analysis and address the need for elimination of non-uEV glycans, we present a modification of the uEV isolation procedure and use the isolated uEVs in the development of a lectin-exosome binding assay. Salt precipitation was employed to remove THP under conditions originally described for its separation from urine, followed by differential centrifugation. The quality of the isolated uEVs was examined by electron microscopy, SDS-PAGE, and immunoblotting. The uEVs were subsequently immobilized on solid phase and probed with labeled plant lectins using the lectin-exosome binding assay. Our results indicate that the isolated uEVs had preserved structural integrity and reacted with labeled plant lectins in a selective, carbohydrate-dependent manner. The basic lectin binding pattern of uEVs obtained by our method can be used as a reference for assessing the composition of their surface glycans in different physiological and pathological conditions.

Fibronectin Pattern in Benign Hyperplasia and Cancer of the Prostate

Fibronectin (FN) is a multifunctional glycoprotein involved in cell-matrix interactions. It exhibits a complex pattern of forms differing in respect to aminoacid and oligosaccharide composition. In this study we examined glycobiochemical and functional properties of the FN in benign prostatic hyperplasia (BPH) and prostatic cancer (PCa), attempting to resolve disease-related differences. Two BPH sera pools and three PCa sera pools were used as the FN source. The affinity-purified molecule was characterized by SDS-PAGE, immuno- and lectin blot, lectin-affinity chromatography and adhesion assay. BPH FN existed as intact molecule, giving the main immunoreactive band at 220 kDa. In contrast, PCa FN comprised three main immunoreactive fragments of 140, 110 and 90 kDa. As for glycosylation the ratio of altogether lectin-reactive PCa FN was different from that of BPH FN manifested as a decrease of Con A- and an increase of LCA-reactive moieties. Fibroblasts adhered to both FN preparations in a concentration dependent manner, but with a significantly lower efficiency to PCa FN. The results obtained showing distinct structural characteristics of PCa FN compared to BPH FN could be important for modulation of its ligand and recognition properties expressed as gain or loss of functions or as specific markers of its origin.

GLYCANS AS A TARGET IN THE DETECTION OF REPRODUCTIVE TRACT CANCERS

Glycans as a Target in the Detection of Reproductive Tract Cancers The significance of changes in glycosylation for the beginning, progress and outcome of different human diseases is highly recognized. In this review we summarized literature data on the alteration of glycans in cancer, especially glycoforms of tumor markers of reproductive tract cancers: prostate-specific antigen (PSA) and cancer antigen 125 (CA125). We aimed to highlight the diagnostic potential and relevance of glycan microheterogeneity and to present some novel methods for cancer detection. A computerized search of articles published up to 2007 was performed through the PubMed database. Search terms utilized included prostate/ovarian cancer glycosylation, prostate/ovarian cancer detection, PSA/CA125 glycosylation. Additional sources were identified through cross-referencing and researching in available biomedical books. The comparative studies of sugar chain structures of the PSA and CA125 indicated specific structural alterations associated with malignant transformation, in relation to glycan branching, sialylation and fucosylation. These glycan modifications should be better in distinguishing between benign and malignant conditions than the measurement of marker concentrations alone, which is widely used in practice. Cancer-associated changes in the glycosylation could yield more sensitive and discriminative diagnostic tests for reproductive tract cancer detection, i.e. for improvement of the clinical utility of known tumor markers or the discovery of new ones. Glikani Kao Mete U Detekciji Kancera Reproduktivnog Trakta Poznat je značaj promena glikozilacije za nastanak, razvoj i ishod različitih bolesti. U ovom radu sumirani su podaci iz literature o promenama glikana kod kancera, posebno glikoformi tumorskih markera kancera reproduktivnog trakta: specifičnog antigena prostate (PSA) i kancerskog antigena 125 (CA125). Cilj ovog rada je da se naglase dijagnostički potencijal i značaj mikroheterogenosti glikana kao i da se prezentuju neke nove metode za detekciju kancera. Izvršena je pretraga radova objavljenih do 2007. godine u bazi podataka PubMed, pomoću termina > prostate/ovarian cancer glycosylation<, >prostate/ovarian cancer detection<, >PSA/CA125 glycosylation<. Kao dodatni izvor podataka korišćene su i dostupne biomedicinske knjige. Uporedna studija strukture šećernih lanaca PSA i CA125 ukazala je na specifične promene povezane sa malignom transformacijom, koje se tiču grananja, sijalinizacije i fukozilacije glikana. Ovakve glikanske modifikacije tumorskih markera mogle bi imati veći potencijal u smislu diferencijalne dijagnostike benignih i malignih stanja nego samo merenje njihove koncentracije, koje se sada široko primenjuje u praksi. Promene glikozilacije, vezane za kancer, mogu biti osnov za razvoj senzitivnijih i specifičnijih dijagnostičkih testova za detekciju kancera reproduktivnog trakta, tj. poboljšati kliničku upotrebu poznatih ili doprineti pronalaženju novih tumorskih markera.

Ion-exchange chromatography purification of extracellular vesicles.

Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.

On Chip Immuno-Affinity Profiling of Cancer- and Benign Hyperplasia-Associated Free Prostate Specific Antigen

Prostate specific antigen (PSA) exhibits pronounced heterogeneity in both primary structure and glycan composition, resulting in the existence of different molecular forms. Investigation of PSA structure is a demanding task facing limitations due to inadequate sensitivity of analytical techniques and low concentrations of the different forms. This study aimed to profile free PSA (fPSA), especially lower molecular mass species lacking detailed classification, in normal seminal plasma and in sera from subjects with benign hyperplasia (BPH) or cancer of the prostate (PCa) as samples of known clinical relevance. fPSA forms were separated from complex proteomes on chips with immobilized anti-fPSA antibody followed by detection using surface-enhanced laser desorption/ionization time of flight mass spectrometry. At least 39 fPSA-immunoreactive species, ranging from 3–29 kDa were detected in seminal plasma. General fPSA profiles in seminal plasma and sera were similar, but differed in the abundance and presence of particular peaks/clusters of the lower molecular mass species. No striking difference in fPSA forms was observed between BPH and PCa samples, but some distinct peaks varied in intensity and frequency within or between groups. Obtained data verify fPSA heterogeneity that might be important for better exploration of all their molecular and marker potentials.

Evaluation of the Pattern of Human Serum Glycoproteins in Prostate Cancer

Evaluation of the Pattern of Human Serum Glycoproteins in Prostate Cancer Glycoprotein profiling at the level of cells, tissues and biological fluids is aimed at discovering new cancer biomarkers and also at finding specific cancer-related structural alterations of known tumor markers. In this study we comparatively evaluated the glycoprotein patterns of human prostate cancer (PCa)- and normal human sera regarding sialylation and fucosylation as structural characteristics relevant for cancer progression. Glycoproteins were isolated using affinity chromatography on Sambucus nigra agglutinin- and Lens culinaris agglutinin-columns and subsequently characterized by SDS-PAGE and on-chip normal phase-surface capture combined with surface-enhanced laser/desorption ionization time of flight mass spectrometry. Comparative analysis of the glycoproteins purified from healthy and PCa sera indicated differences and redundancy of the isolated molecules in terms of the microheterogeneity of counterpart glycans, the relative abundance and the presence/absence of particular molecular species. In PCa there was a general increase in sialylation and decrease in fucosylation of human serum glycans compared to normal sera. Taken together, the results obtained indicated that an affinity-approach based on the use of lectins of narrow specificity reduced the complexity of the examined samples and at this discovery-phase of our study pointed to specific glyco-changes that may be relevant for improving the monitoring of PCa progression. Procena Profila Humanih Serumskih Glikoproteina Kod Kancera Prostate Glikoproteomska istraživanja na nivou ćelija, tkiva i bioloških tečnosti imaju za cilj otkrivanje novih biomarkera kao i strukturnih promena već poznatih biomarkera, koje su specifične za kancer. U ovom radu je izvršena komparativna evaluacija glikoproteinskog profila normalnog humanog seruma i seruma pacijenata sa kancerom prostate (PCa), u smislu sijalinizacije i fukozilacije, kao strukturnih promena relevantnih za progresiju kancera. Odgovarajući glikoproteini su izolovani lektinskom afini tetnom hromatografijom na kolonama sa imobilisanim Sambucus nigra aglutininom i Lens culinaris aglutininom i okarakterisani tehnikama SDS-PAGE i vezivanja za proteinski čip sa normalnom fazom u kombinaciji sa SELDI-TOF masenom spektrometrijom. Komparativna analiza humanih serumskih glikoproteina izolovanih iz pulova normalnih seruma i PCa-seruma je ukazala na brojnost izolovanih molekula i na razliku u smislu njihovog pojedinačnog prisustva, mikroheterogenosti i relativne zastupljenosti. Generalno, uočeno je povećanje sijalinizacije i smanjenje fukozilacije kod PCa. Rezultati dobijeni u ovom radu ukazuju na to da pristup u kome se u inicijalnoj fazi ispitivanja koriste lektini uske specifičnosti smanjuje kompleksnost uzorka, kao i na specifične promene glikoproteinskog profila, koje mogu biti relevantne za poboljšanje praćenja progresije PCa.

Determination of Prostate-Specific Antigen in Serum and a Reference Material by On-Chip Immunoaffinity Chromatography

Glycoprotein tumor markers are striking examples of heterogeneous analytes. The complexity of their structural forms in biological fluids is generally not reflected in reference materials. Therefore, they are not specified to consist of a distinct form, but rather to contain a mixture of molecular species. In this study, the question of the heterogeneity of free prostate-specific antigen (free PSA) is addressed in reference materials to define the immunoreactive molecular species and compare them to those in clinical serum. The reference material for free PSA and serum samples of subjects with benign prostatic hyperplasia and prostate cancer was examined for immunoreactivity to epitope I-specific anti-free PSA antibody using on-chip immunoaffinity chromatography in combination with mass spectrometry for the determination of bound forms. The mass spectra of the reference material for free PSA and clinical serum, obtained by on-chip immunoaffinity chromatography, were similar. The cluster of major free PSA-immunoreactive peaks at 28–29 kDa corresponding to the mature glycosylated PSA molecule overlapped in both analytes. However, the reference material displayed a more restricted pattern of low molecular mass species corresponding to nicked PSA fragments or PSA degradation products. The PSA concentration in clinical serum seems to consist of more species than equivalent concentrations of reference material. Regarding analysis of heterogeneous proteins, immunoaffinity capture combined with mass-specific detection represents a rapid means for selective detection of distinct molecular species, exceeding the analytical performance of current formats of immunoassays.

Nano-sized CA125 antigen glycocamouflage: Mucin - Extracellular vesicles alliance to watch?

Mucin 16 (MUC16) is a transmembrane type mucin and its released extracellular portion is designated as CA125 antigen. It is considered to be part of a supramolecular glycoprotein complex having a complicated epitope map and extreme structural heterogeneity. Starting from the initial transmembrane localization of MUC16/CA125 antigen and its alternative routes of release by shedding or putative secretion, CA125 antigen from human amniotic fluid soluble and extracellular vesicles (EVs)-containing fractions were characterized aiming at the possible glycosylation-associated mode of distribution as a factor contributing to the reported conflicting structural data. Ultracentrifugation, sucrose density gradient centrifugation, ion-exchange chromatography and TEM were used for analysis. The results indicated that the smeared abundantly glycosylated high molecular mass CA125-immunoreactive species, which follow the wheat germ agglutinin-binding pattern, were shared across amniotic fluid soluble and particulate fractions. A lower molecular mass glycoprotein-like CA125-immunoreactive species which follows the peanut agglutinin-binding pattern and was specifically associated with the EVs-enriched fraction was observed. CA125 presentation in the particulate amniotic fluid fraction was found to be shaped by a complex interactome partially involving lactose-sensitive galectin-3 binding. The MUC16 - EVs alliance as well as heterogeneous mucin/macromolecular complexes, at membranes or extracellularly, may represent cryptic pools of distinct CA125 species.