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INDEX: development of an integrated platform to provide EV-based liquid biopsy for lung cancer

D. Brambilla1, M. Araya Farias2, S. Descroix2, E. Chiodi3, M. S. Ünlü3, R. Baek4, M. Jorgensen4, N.

Zarovni5, M. Chiari1

1 Dipartimento di Scienze e Tecnologie Chimiche “Giulio Natta”, Consiglio Nazionale delle Ricerche (CNR – SCITEC), 20131 Milan, Italy

2 Laboratoire Physico‐Chimie Curie CNRS UMR 168, Institut Curie, PSL Research University, 75005 Paris, France

3 Department of Electrical Engineering, Boston University, 02215 Boston, United States 4 Department of Clinical Immunology, Aalborg University Hospital, 9000 Aalborg, Denmark

5 HansaBioMed Life Sciences Ltd, 12618 Tallinn, Estonia INDEX project (Integrated Nanoparticle isolation and Detection system for complete on chip analysis of EXosomes) aimed to develop an integrated platform, able to provide in line separation of extracellular vesicles (EVs) from complex biological fluids and their phenotyping on the same instrument. The platform is divided into two main modules: the first one is the separation device where magnetic beads, functionalized using antibodies against CD63 through DNA-directed immobilization (DDI), constantly recirculate into a microfluidic fluidized bed chamber which allows a better surface contact between the fluid and beads. The presence of a dsDNA linker between antibodies and beads on one side increase the exposure of the antibody and thus its binding efficiency, but also allows the release of intact EVs upon treatment with DNAse I, an endonuclease that catalyzes the cleavage of dsDNA. Released EVs are then transferred into the second module for their detection, sizing and phenotyping. EVs are immobilized on the surface of a microarray chip using specific antibodies (in particular antibodies directed against CD9, EpCAM, TSPAN8, EGFR and CD151) and immobilized using Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS), a label free technique that analyze the shift of reflectance along a surface and elaborate pictures where single EVs are depicted as areas of increased contrast. The combination of the multiplexed microarray surface and interferometric imaging is able to provide information regarding number, size and surface composition of EVs within a single analysis. For the validation of the INDEX instrument, a cohort of 26 people was enrolled; 14 were healthy individuals, while the remaining 12 were affected by lung cancer. Plasma samples were collected from the 26 individuals and EVs were separated using magnetic beads functionalized with DNA-directed antiCD63. Purified sample were then sent to detection chips and analyzed. The diagnostic potential of the INDEX system to distinguish samples from healthy controls from samples from lung cancer patients was tested. Single markers, ratio between markers and multivariate data analysis were evaluated to distinguish between the two groups of individuals. Using ratios in the diagnostic calculation revealed a greater potential than using the markers individually. Best results were obtained with the ratio between the counts of EpCAM and EGFR spots. Using a cut-off value of 0.763 the test will have a specificity of 0.82 (CI 0.54-1.0) and sensitivity of 0.80 (CI 0.6-0.93). These data and evidences strongly confirm the great potential of the platform developed in the frame of INDEX project to bring EV-based liquid biopsy into clinical use. Supported by the European Union Horizon 2020 under Grant 766466.

Identification of viral particles copurifying with tomato-derived nanovesicles by shotgun proteomics

Ramila Mammadova 1,2, Immacolata Fiume 1, Ramesh Bokka 1, Gabriella Gellén 3, Gitta Schlosser 3

and Gabriella Pocsfalvi 1

1 Extracellular Vesicles and Mass Spectrometry Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy, Naples, Italy

2 University of Campania Luigi Vanvitelli, Department of Mental and Physical Health and Preventive Medicine, Naples, Italy

3 MTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, ELTE Eötvös Loránd University, Institute of Chemistry, Budapest, Hungary

Cell-derived submicron particles isolated from plant resources are gaining increasing attention as complex biomaterials with health promoting effects and delivery vectors for exogenous substances. Nanometer-sized vesicles (NVs) have been isolated from many different plants, such as ginger, carrots, citrus, grapes, tomato, blueberry, coconut, broccoli, wheat, etc., and even dried plant material. Several of these NV isolates were shown to have anticancer, anti-inflammatory, or anti-senescence activities in vitro or in vivo. Tomato plants are continuously confronted by various viral, bacterial, fungal and pest pathogens. Over recent years, several viral diseases have emerged affecting the quality of the fruit and contributing to the pre- and post-harvest losses both in field and greenhouse tomato. Emerging viruses spread very fast and require prompt intervention for their control and prevention. Most of the viral diseases in tomato is attributed to single-stranded RNA (ssRNA) viruses. Tomato fruit was homogenized, crude NVs were isolated by differential ultracentrifugation. NVs were further separated using sucrose or iodixanol gradient density ultracentrifugation. The fractions obtained were analysed by SDS-PAGE and LC-ESI-MS/MS-based shotgun proteomics. Identified proteins were analysed by bioinformatics tools using the omicsbox. Some of the crude tomato NV samples (dUC isolates) showed an unusual SDS-PAGE profile with prominent bands at the low molecular region. The combination of SDS-PAGE profiling with proteomics analysis revealed that these samples contained tomato viruses that co-purified with the nanovesicles in dUC. Tomato NVs are spherical structures with a diameter 110±10 nm and characterized by a low buoyant density of 1.070-1.120 g/mL. On the other hand, most of the known plant viruses are rod-shaped with coat proteins forming a tube surrounding the viral genome and have high density ranging from 1.35–1.4 g/cm3. Due to these differences, we showed the buoyant density-based separation is able to separate plant NVs from viruses. Here we showed that both sucrose and iodixanol gUC were indeed effective to enrich NVs in the low-density fractions and viral particles in the high-density fractions. In our hands, iodixanol gUC provided a better separation. The methods used in this work turned out to straightforward in the detection and identification of plant viruses that genome sequences were available. However, further studies are needed to ascertain if the method is capable to distinguish spherical virions from the membrane vesicles. Viral particles were identified based on their coat proteins on other viral proteome components by mass-spectrometry-based proteomics. In the two isolates studied, we identified both commonly occurring viruses, like ToMV, TSWV, PVY and STV and emerging ones, like ToBRFV and ToMMV. Most of them were rod-shaped, unless TSWV which is roughly spherical shape. We cannot exclude the presence of other viruses, which genome information is unavailable.

Extracellular vesicles from microalgae: anti-oxidant bioactivity and cellular uptake studies

Giorgia Adamo1,2, Sabrina Picciotto1,2, Daniele Romancino1,2, Angela Paterna1,3, Estella Rao1,3,

Samuele Raccosta1,3, Antonella Cusimano1,2, Rosina Noto1,3, Rita Carrotta1,3, Nicolas Touzet5, Mauro Manno1,3 and Antonella Bongiovanni1,2

1 Cell-Tech HUB, National Research Council of Italy (CNR), Palermo 90146, Italy 2 Institute for Research and Biomedical Innovation, National Research Council of Italy (CNR),

Palermo 90146, Italy 3 Institute of Biophysics, National Research Council of Italy (CNR), Palermo 90146, Italy.

4 Institute of Biosciences and BioResources (IBBR), National Research Council (CNR), Napoli, Italy

5 Centre for Environmental Research Innovation and Sustainability, Institute of Technology Sligo, Sligo, Ireland

Extracellular vesicles (EVs) are lipid bilayer nano-sized vesicles released by various cell types for intercellular communication. EVs also constitute cross-species communication means and have been found in all kingdoms of life. The exploitation of the biotechnological potential of EVs as nanocarriers of bioactive compounds, such as nucleic acids, proteins, peptides, lipids, synthetic drugs or other cargo, for different theranostic applications is of increasing interest. Here, we describe the newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes [1,2]. Specifically, we focus on cellular uptake of the nanoalgosomes, staining them with different specific dyes. We confirm that they are able to bypass mammalian cell membrane, localizing in specific intra-cellular compartments. In particular, nanoalgosomes were separated from a suspension of Tetraselmis chuii cells using differential ultracentrifugation (dUC) or tangential flow filtration (TFF). After the biophysical characterization (according to MISEV-2018 guidelines) [3], they were labelled with different lipophilic fluorescent dyes to follow their intra-cellular localization in mammalian cells, over time. Moreover, in vitro studies on normal and tumour cells underlined the ability of nanoalgosomes to scavenge free radicals and protect mammalian cells in vitro against hydrogen peroxide damage. The studies performed demonstrated that nanoalgosomes can be efficiently taken up by mammalian cells where they, thanks to high value microalgae substances, protect from oxidant stress, confirming the cross-kingdom communication potential of EVs. Nanoalgosomes will be further explored as novel and natural delivery systems, or up-stream loaded bioactive molecules and synthetic drugs. (This work has been developed under the projects VES4US and BOW, which have received funding from the European Union’s Horizon 2020 research and innovation programme, under grant agreements No 801338 and 952183, respectively). [1] Adamo, et al. J. Extracellular Vesicles 10, e12081, 2021. doi:10.1002/jev2.12081 [2] Picciotto, et al. Biomaterials Science 9, 2917-2930, 2021. doi:10.1039/d0bm01696a [3] Théry, et al. J. Extracellular Vesicles. 7, 1, 2018. doi:10.1080/20013078.2018.1535750

We present our investigation on the association of antithrombin (AT) isoforms in human plasma extracellular vesicles (EVs). AT is a glycoprotein produced by the liver and acts as the most important antagonist of clotting factors. A deficit in AT production or function leads to coagulation disorders. Two kinds of AT deficiencies are reported, named quantitative (or type I) and qualitative (or type II) defects. The first is characterized by low levels of AT in the bloodstream, the latter by impaired AT activity related to dysfunctional domains of AT and it is challenging to diagnose. Although being a soluble protein, evidence of AT transported by plasma EVs has been found [1] but the physicochemical features of the association of AT to EVs are missing. We separated and characterized EVs from the plasma of healthy subjects, focusing on AT association. We found AT is localized on the external leaflet of the EV membrane. Furthermore, 2D-electrophoresis conducted on plasma and EVs of healthy subjects highlighted that specific AT glycoforms are selectively enriched onto the EVs with respect to whole plasma, suggesting that glycosylation plays a role in the partitioning of AT between the EV surface and liquid plasma phase, and ultimately on the EV exofacial topology. Finally, we separated EVs from the plasma of 8 patients affected by type II AT defect. The comparison of the AT 2D-electrophoretic pattern of patients and healthy subjects highlighted a difference in AT adsorption onto EV surface, supporting the role of EVs in coagulation and suggesting a promising approach to improve diagnosis and management of type II AT deficiencies. References

[1] Levin, G. Y., and E. Sukhareva, 2015. Blood Transfus 13(4):688-9, doi:10.2450/2015.0016-15

Specific antithrombin glycoforms adsorb onto plasma extracellular vesicles

A. Zendrini1,2, S. Alacqua1, V. Previcini1, F. Todaro1, G. Martini3, D. Ricotta1,6, P. Bergese1,2,4,5, and A. Radeghieri1,2

1 Dept. of Molecular and Translational Medicine, University of Brescia, Italy 2 Center for Colloid and Surface Science (CSGI), Florence, Italy

3 Clinical Chemistry Laboratory, Spedali Civili Hospital, Brescia, Italy 4 Institute for Research and Biomedical Innovation (IRIB), National Research Council, Palermo, Italy 5 National Inter-university Consortium of Materials Science and Technology (INSTM), Firenze, Italy 6 GLG Klinikum Barnim GmbH, Werner Forßmann Klinikum Eberswalde, Rudolf-Breitscheid-Straße

100, Eberswalde, Germany

Circulating extracellular vesicles as biomarkers of response for immune-checkpoint inhibitors in advanced NSCLC

Orazio Fortunato1, Diego Signorelli2, Claudia Proto2, Valentina Capizzuto3, Francesca Pontis1, Ilaria Petraroia1, Gabriella Sozzi1, Mario Paolo Colombo3, Marina Chiara Garassino2 and Elena Jachetti3

1 Tumor Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori

2 Thoracic Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori 3 Molecular Immunology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori

INTRODUCTION: Programmed death ligand-1 (PD-L1) expression is the only predictive biomarker in clinical practice for immune-checkpoints inhibitors (ICI) in NSCLC. Even if ICI efficacy is higher in PD-L1 strong positive tumours (Tumour Proportion Score, TPS>50%), some patients (pts) with low (TPS 1-49%) or absent (TPS<1%) PD-L1 can achieve a durable benefit. Extracellular vesicles (EVs) were used as biomarkers for cancer progression and could express PD-L1 on their surface (EVs-PD-L1). The main goal of this study was to evaluate EVs and EVs-PD-L1 in advanced NSCLC pts with low or absent PD-L1 expression in order to find biomarkers for ICI in this pts subgroup. MATERIAL AND METHODS: EVs were isolated using ultracentrifuge from plasma of advanced NSCLC pts treated with ICI at our Institute. Pts were classified in responders (R) if they achieved a complete or partial response by RECIST 1.1, non-responders (NR) otherwise. EVs-PD-L1 expression was assessed by flow cytometry (FC). EVs characterization was performed following the MISEV guidelines. T cells of healthy donors were isolated and stimulated with CD3/CD28 Beads in presence of patients derived EVs, and analyzed by FC. RESULTS: Plasma samples were prospectively collected from 61 pts: 23 (37.7%) TPS low, 38 (62.3%) TPS absent, treated with ICI as first (n=23) or further (n=38) line; 19.7% were R, 80.3% NR. EVs-PD-L1 did not correlate with tumour PD-L1 expression. Stratifying baseline circulating EVs-PD-L1 levels according to median value, higher values were related to worse progression free survival (median: 2.2 vs 3.8 months, HR 1.78, 95%CI 1.03-3.08, p=0.036). In 20 patients (10 R, 10 NR), evaluated during ICI therapy (range: 3-13 weeks), EVs-PD-L1 levels increased in R compared to NR (PD-L1 fold change: R= 1.89 ± 0.24 vs NR= 0.91 ± 0.15; p<0.01). No differences were observed in size, total particles count and expression of EVs-markers. Epcam surface expression was increased on R-EVs surface (median fluorescence: R=2114 vs NR=540, p<0.05), suggesting an epithelial origin of these EVs. We cultured in vitro CD8 T cells and CD4 T cells with EVs isolated from NR (n=12) or R (n=8) pts. EVs from both cohorts increased T cell proliferation compared to control T cells stimulated alone. Compared to NR-EVs, R-EVs induced higher T cell activation, evaluated as IFNg and Granzyme B production, and reduced the frequency of CD4 Treg within cultures. We did not find any significant difference in checkpoint receptors expression on T cell surface (PD1, Tim3, Vista, Lag3). CONCLUSION: Plasma EVs from NSCLC pts treated with ICI showed different features in terms of size, surface markers and activation of T cells. Increased T cell activation induced by R-EVs might reflect the presence of an activated immune-status in these pts. PD-L1 on EVs could represent a promising biomarker for ICI in NSCLC.

Decoding distinctive features of plasma extracellular vesicles in amyotrophic lateral sclerosis

Laura Pasetto1, Stefano Callegaro2, Alessandro Corbelli1, Fabio Fiordaliso1, Deborah Ferrara3, Laura Brunelli1, Giovanna Sestito1, Roberta Pastorelli1, Elisa Bianchi1, Marina Cretich4, Marcella Chiari4, Cristina Potrich5, Cristina Moglia6, Massimo Corbo7, Gianni

Sorarù8, Christian Lunetta9, Andrea Calvo6, Adriano Chiò6, Gabriele Mora10, Maria Pennuto11,12, Alessandro Quattrone3, Francesco Rinaldi2, Vito Giuseppe D’Agostino3,

Manuela Basso1,3*, and Valentina Bonetto1*

1 Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milano (Italy) 2 Department of Mathematics “Tullio Levi-Civita”, University of Padova

3 Department of Cellular, Computational and Integrative Biology – CIBIO, University of Trento, Trento, Italy

4 Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC-CNR), Milano, Italy

5 Centre for Materials and Microsystems, Fondazione Bruno Kessler, Trento, Italy & Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Trento, Italy

6 ‘Rita Levi Montalcini’ Department of Neuroscience, Università degli Studi di Torino, Torino, Italy 7 Department of Neurorehabilitation Sciences, Casa Cura Policlinico (CCP), Milano, Italy

8 Department of Neuroscience, University of Padova, 35122 Padova, Italy 9 NEuroMuscular Omnicentre (NEMO), Serena Onlus Foundation, Milano

10 Department of Neurorehabilitation, ICS Maugeri IRCCS, Milano, Italy 11 Department of Biomedical Sciences (DBS), University of Padova, 35131 Padova, Italy.

12 Veneto Institute of Molecular Medicine (VIMM), 35129 Padova, Italy. *These are co-corresponding and co-last authors.

Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease's complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell's physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification. We analyzed plasma-derived EVs of ALS patients (n=106) and controls (n=96), and SOD1G93A and TDP-43Q331K mouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis. Our procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. There were more particles in the plasma of ALS patients and the two mouse models of ALS while their average diameter was smaller. HSP90 was differentially represented in ALS patients and mice compared to the controls. In terms of disease progression, the levels of cyclophilin A, with the EV size distribution, distinguished fast and slow disease progressors, suggesting a new means for patient stratification. We also measured the levels of phosphorylated TDP-43 and showed that is not an intravesicular cargo of plasma-derived EVs. Our analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis. Supported by the Italian Ministry of Health (GR-2016-02361552)

Human amniotic fluid stem cell-extracellular vesicles support cardiomyocyte renewal

A. Costa1, A. De Palma2, D. Ceresa3,C. Balbi4, S. Santamaria1, K. Cortese1, D. Paladini5, D. Coviello5, L. Barile6, P. Malatesta1,3, P. Mauri2 and S. Bollini1

1 Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy; 2 Proteomics and Metabolomics Unit, Institute for Biomedical Technologies, Milan, Italy;

3 IRCCS Ospedale Policlinico San Martino, Genova, Italy; 4 Cellular and Molecular Cardiology, Cardiocentro Ticino Foundation, Lugano, Switzerland;

5 IRCCS Istituto Giannina Gaslini, Genova, Italy; 6 Cardiovascular Theranostic, Cardiocentro Ticino Foundation, Lugano, Switzerland

Functional replacement of cardiomyocytes following injury represents a milestone in cardiac regeneration. We reported that human amniotic fluid-derived stem cell (hAFS) secretome exerts cardioprotective and proliferative effects on murine ischemic myocardium [1]. Here, we analysed whether the paracrine potential of extracellular vesicles (EVs) from II trimester hAFS of leftover fluid samples of amniocentesis (fetal hAFS-EVs) over III trimester cells from C-section delivery clinical waste (perinatal hAFS-EVs) can be influenced by gestational stage; moreover, we validated the hAFS-EVs cardio-active effect in stimulating cardiomyocyte renewal. Fetal and perinatal hAFS-EVs (f-hAFS-EVs and p-hAFS-EVs) were concentrated by serial ultracentifugation [1] from hAFS conditioned medium after 1%O2 cell preconditioning. f-hAFS-EVs and p-hAFS-EVs were profiled by transmission electron microscopy and nanoparticle tracking analysis, proteomics and miRNA sequencing (miRNAseq). Neonatal mouse ventricular cardiomyocytes (mNVCM) were enzymatically isolated from R26pFUCCI2 transgenic pups endowed with fluorescent tagging of cell cycle stages by mutual nuclear signal (G1-phase by mCherry; S-G2-M-phase by mVenus [2]). mNVCM were stimulated by f- hAFS-EVs versus p-hAFS-EVs for 48h to evaluate the most cardio-active formulation for further in vivo investigation in a R26pFUCCI2 4-day-old mouse pup model of myocardial infarction (MI). While f-hAFS-EVs and p-hAFS-EVs presented similar size distribution, ranging between 70-220 nm, and cytokine and chemokine profiling and miRNAseq revealed a stable miRNA core of hAFS-EVs cargo, proteomics reported significant enrichment of Agrin in f-hAFS-EVs (****p<0.0001), which is an important mediator for heart regeneration in mice [3]. f-hAFS-EVs sustained in vitro cardiomyocyte transition from S-M-phase by 2.3-fold (*p<0.05) and increased cytokinesis by 4.5-fold (*p<0.05), as detected by Aurora B kinase at midbodies (a marker of the authentic cell division) compared to untreated cells, while their perinatal counterpart was not effective. f-hAFS-EVs also decreased gene expression of Cofilin-2 (a sarcomeric disassembly marker [4]) by 0.6-fold (***p<0.001), suggesting cardiomyocyte de-differentation as required for cell cycle re-entry. f-hAFS-EVs confirmed in vivo cardiomyocyte S-M phase transition by 2.0-fold (*p<0.05) in the remote zone of myocardium after 3 days from MI. Our preliminary findings suggest that f-hAFS-EVs may trigger cardiomyocyte renewal for regenerative purposes. Further analyses are ongoing to define f-hAFS-EVs cardio-active mechanism of action focusing on Agrin as molecular candidate. References [1]C. Balbi et al., Int. J. Cardiol., Jul. 2019. [2]T. Abe et al., Dev., Jan. 2013. [3]E. Bassat et al., Nature, Jul. 2017. [4]C. Torrini et al., Cell Rep., May 2019.

COMT × DRD2 epistasis modulates a putative emotional connectomic intermediate phenotype for schizophrenia

H. Cao1, A. Schäfer1, O. Grimm1, L. Haddad1, H. Walter2, A. Heinz2, S. Witt1, M.

Rietschel1, S. Cichon3, H. Tost1, A. Meyer-Lindenberg1

1Central Institute of Mental Health, University of Heidelberg, Mannheim, Germany 2Charité Universitaetsmedizin Berlin, Department of Psychiatry and Psychotherapy, Berlin, Germany

3University of Bonn, Department of Genomics, Bonn, Germany Our prior work using graph theory based method, presented at the 43rd Annual Meeting of Society for Neuroscience, has identified a disrupted visual-limbic subnetwork in unaffected first-degree relatives of schizophrenia during emotion processing, and validated this connectomic finding as a robust and task-specific intermediate phenotype (presented at the 20th Annual Meeting of Organization for Human Brain Mapping) . Here, to further investigate the utility of this potential phenotype in imaging genetics, we examined the main effects and interaction of two dopaminergic risk variants on this identified subnetwork: a candidate variant for emotion dysregulation (COMT val158met) [1] and a genome-wide supported schizophrenia risk variant in DRD2 (rs2514218) [2]. Our sample consisted of 289 healthy individuals of European ancestry without a first-degree relative with mental illness (mean age 33.73±9.78 years, 155 females). The subjects were recruited from the communities in Mannheim, Bonn and Berlin. For each individual, the COMT val158met polymorphism was directly genotyped by the DNA arrays while DRD2 rs2514218 genotype was imputed with Impute2 using reference haplotypes derived from the 1000 Genomes Project [3]. Following the procedures of the previous studies, the carriers of the presumed protective alleles were combined into one group (COMT: val/val + val/met; DRD2: risk C allele number <1.5). The observed genotype distributions did not deviate from Hardy-Weinberg equilibrium (COMT: 207 Val-carriers, 82 Met/Met, P=0.79; DRD2: 174 T-carriers, 115 CC, P=0.36). The four genotype groups for both variants did not show significant differences in demographic, psychological and fMRI performance data (all P values >0.10). All the subjects underwent a well-established emotional face-matching task. The image preprocessing followed the standard procedures implemented in SPM8. The mean time series were extracted from each of the 90 anatomical regions defined by AAL template and corrected for noises. Whole-brain connectivity matrices were subsequently computed by the pairwise correlations between the corrected time series of each of the 90 nodes. Here, the averaged connectivity measures of the same subnetwork reported in our previous studies were extracted and entered as the dependent variable into an ANCOVA model with genotypes (COMT and DRD2) as variables of interest and age, sex and site as covariates of non-interest. Significance was measured at P<0.05. No significant main effects for both COMT and DRD2 genes on this phenotype measures were found (COMT: P=0.95, DRD2: P=0.15). However, the epistatic study demonstrated a strong COMT × DRD2 interaction (p=0.01). Specifically, in the group of COMT met/met homozygotes, there was a significant decrease of the phenotype in DRD2 CC homozygotes compared to T carriers (p=0.01). In contrast, no significant difference was found between DRD2 genotypes in the group of COMT val carriers (p=0.36), indicating that COMT val158met is epistatic to DRD2 rs2514218 on the identified phenotype. This study showed that genetic epistasis between COMT val158met and DRD2 rs2514218 could modulate the identified emotional connectomic phenotype for schizophrenia and highlighted the utility of this potential phenotype in imaging genetics. Supported by the National Science Centre grants 2012/05/E/NZ3/00487

References [1] Mier, D., Kirsch, P., Meyer-Lindenberg, A., 2010. Neural substrates of pleiotropic action of genetic variation in COMT: a meta-analysis. Molecular Psychiatry 15(9), 918-927. [2] Schizophrenia Working Group of the Psychiatric Genomics Consortium, 2014. Biological insights from 108 schizophrenia-associated genetic loci. Nature 511(7510), 421-427.

Human induced pluripotent stem cells as inexhaustible source of extracellular vesicles for acellular therapy

M. Barilani1, V. Peli1, P. Manzini1, F. Pischiutta2, E. Caruso2, A. Cherubini1, F. Rusconi1, V. Dolo3, V. Bollati4, G. Damia2, E. Zanier2, L. Lazzari1

1Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy 2Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy

3University of L’Aquila, L’Aquila, Italy 4University of Milan, Milan, Italy

Extracellular vesicles (EV) are under investigation as cell-free therapeutics to overcome limitations of their parental cell counterparts. Mesenchymal stromal cells (MSC) are the most studied cell type as source of EV for tissue protection and regeneration, mainly due to previous clinical experience with MSC-based therapies. Yet, EV production for clinical application entails large-scale manufacturing via cGMP-compliant processes that must meet strict quality requirements. In this framework, primary MSC have two main drawbacks: limited life-span caused by replicative senescence; potentially relevant intra- and inter-donor heterogeneity. Therefore, we investigated whether human induced pluripotent stem cells (hiPSC), generated from clinical-grade HLA-typed cord blood MSC, may act as an inexhaustible source of massively produced therapeutic EV. hiPSC-EV were isolated by serial centrifugation and purified by size-exclusion chromatography and sucrose density gradient. MISEV2018-compliant characterization was performed by nanoparticle tracking analysis, western analysis, flow cytometry and electron microscopy. Physical features were compatible with small EV, while biological features showed presence of EV and pluripotency markers and absence of contaminating proteins. Kinetics of EV release by hiPSC and internalization by neural cells were tested in vitro, showing robust EV production and dose-dependent uptake. Proof-of-concept therapeutic potential was observed in an in vitro model of brain ischemia on cortical organotypic slices by oxygen and glucose deprivation. hiPSC-EV induced significant brain protection and downregulation of TNFa and IFNg, two key drivers of the inflammatory response, supporting the use of hiPSC as source of EV with inherent immunomodulatory properties. Further efforts are needed to determine hiPSC-EV mechanism of action and to set up a translatable manufacturing process, which is currently focused on cGMP culture of hiPSC in bioreactors. Supported by Fondazione Regionale per la Ricerca Biomedica, Regione Lombardia (Regione Lombardia), Project nr. CP_10/2018

Extracellular vesicle-model membranes interaction: a non-total fusion mechanism

Loredana Casalis1, Fabio Perissinotto1,2, Valeria Rondelli3, Paola Brocca3, Pietro Parisse4

1 Elettra Sincrotrone Trieste, Trieste, Italy

2 Center for Infection and Immunity of Lille, INSERM U1019, Institut Pasteur de Lille, Lille, France 3 Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano,

Italy 4 CNR-IOM, Trieste, Italy

Extracellular vesicles (EVs) are renown as a potent intercellular communication system [1]. Such small vesicles transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. Due to their specific biological functions they have been proposed as biomarkers for various diseases and as optimal candidates for therapeutic applications [2]. Despite their extreme biological and therapeutical relevance, the precise mechanisms of interaction with recipient cells through their membranes are still hotly debated [3]. Here, we performed a multiscale investigation based on atomic force microscopy, small angle X-ray and neutron scattering and neutron reflectometry to reveal structure–function correlations of purified EVs from umbilical cord multipotent Mesenchymal Stem (Stromal) Cells in interaction with model membrane systems of variable compositions featuring raft-like domains. With our investigation platform we were able to spot the role of different membrane phases on the vesicle internalization routes. Our analysis highlighted the role of the different membrane phases on vesicle internalization revealing a preferential interaction with the borders of the raft-like phase domains. Moreover, we found that vesicles broke up on the model membrane surface and that the biomolecules carried by/on the surface of EVs diffuse with different kinetics rates, in a process distinct from simple fusion [4]. The biophysical platform proposed here has clear implications on the modulation of EVs cellular uptake by targeting specific domains at the plasma cell membrane and, as a consequence, on EV-based therapy. References [1] M. Mathieu, et al., Nat. Cell Biol., 21 (2019): 9. [2] S. Fais, et al., ACS Nano, 10, (2016): 3886–3899. [3] L. Margolis et al., PLos Biol., 17, (2019): e3000363. [4] F Perissinotto, et al., Nanoscale, 13 (2021); 5224–5233

Circulating extracellular vesicles are endowed with enhanced procoagulant activity in SARS-CoV-2 infection

Carolina Balbi 1,2*, Jacopo Burrello 3*, Sara Bolis 1,3*, Edoardo Lazzarini 3, Vanessa Biemmi 3, Enea Pianezzi 4, Alessio Burrello 5, Elena Caporali 6, Lorenzo Gauthier Grazioli 7, Gladys Martinetti 4, Tanja Fusi-

Schmidhauserg 7, Giuseppe Vassalli 1,2,8, Giorgia Melli 8,9+, Lucio Barile 3,8,10+.

1 Laboratory of Cellular and Molecular Cardiology, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland

2Center for Molecular Cardiology, Zurich, Switzerland 3Laboratory for Cardiovascular Theranostics, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale

Lugano, Switzerland 4 Laboratory of Microbiology, Ente Ospedaliero Cantonale, Bellinzona, Switzerland

5 Department of Electrical, Electronic and Information Engineering (DEI), University of Bologna, Bologna, Italy

6 Cardiology Department, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland 7 Internal Medicine Department, Ospedale Regionale di Lugano, Ente Ospedaliero Cantonale, Lugano,

Switzerland 8 Faculty of Biomedical Sciences, Universita della Svizzera italiana, Lugano, Switzerland

9 Laboratory for Biomedical Neurosciences, Neurocenter of Southern Switzerland, Lugano, Switzerland 10 Institute of Life Science, Scuola Superiore Sant'Anna, Pisa, Italy

Background: Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents. Methods: We analyzed serum-derived EV from 67 participants who underwent nasopharyngeal swabs molecular test for suspected SARS-CoV-2 infection (34 positives and 33 negatives) and from 16 healthy controls (HC), as referral. A sub-analysis was performed on subjects who developed pneumonia (n = 28). Serum-derived EV were characterized for their surface antigen profile and tested for their procoagulant activity. A validation experiment was performed pre-treating EV with anti-CD142 antibody or with recombinant FVIIa. Serum TNF-α levels were measured by ELISA. Findings: Profiling of EV antigens revealed a surface marker signature that defines circulating EV in COVID-19. A combination of seven surface molecules (CD49e, CD209, CD86, CD133/1, CD69, CD142, and CD20) clustered COVID (+) versus COVID (-) patients and HC. CD142 showed the highest discriminating performance at both multivariate models and ROC curve analysis. Noteworthy, we found that CD142 exposed onto surface of EV was biologically active. CD142 activity was higher in COVID (+) patients and correlated with TNF-α serum levels. Interpretation: In SARS-CoV-2 infection the systemic inflammatory response results in cell-release of substantial amounts of procoagulant EV that may act as clotting initiation agents, contributing to disease severity.

Generation of spike-extracellular vesicles (S-EVs) as a tool to mimic SARS-CoV-2 interaction with host cells

Roberta Verta1, Cristina Grange2, Renata Skovronova1 and Benedetta Bussolati1

1Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 2Department of Medical Science, University of Torino, Torino, Italy

Extracellular vesicles (EVs) and viruses share common features in size, structure, biogenesis and uptake while being different entities1,2. The aim of our work was to generate and characterize EVs, which express the spike protein of SARS-CoV-2 on their surface (S-EVs). S-EVs can be used as a tool to study spike protein binding, internalization in human cells and its modulation. To identify possible molecular therapeutic targets, we studied the effect of colchicine, which is already used in clinical trials, for COVID-19 treatment3 and anti-ACE2 blocking antibody that may impact SARS-2-S/angiotensin-converting enzyme2 (ACE2) binding. We generated SARS-CoV-2 spike expressing HEK-293T cells by stable transfection with a spike protein plasmid coding for the S1 and S2 subunits. Subsequently, we collected the EVs by ultracentrifugation. S-EVs were characterized using Nanosight, ExoView and super-resolution microscopy (Nanoimager). Finally, we used HUVEC (ACE2 positive cells) for assessing the internalization of labelled S-EVs by using cytofluorometric analysis. We obtained virus-like vesicles ranging in size between 50 to 200 nm (evaluated by Nanosight tracking analysis). Using a chip-based analysis (ExoView), we showed that S-EVs represented around 50% of the released-EVs population, while simultaneously expressing CD9, CD63, and CD81. Single vesicle analysis was performed using super-resolution microscopy, used to validate the co-expression of S-EVs with tetraspanins. Analysis using super resolution microscopy confirmed the co-expression of spike and tetraspanins on EVs surface. We demonstrated that S-EVs bind to endothelial cells through an ACE2 dependent-interaction, in the same manner as the SARS-CoV-2 virus. S-EVs uptake was statistically (P<0.01) reduced by ACE2 blocking antibody. Furthermore, colchicine treatment significantly (P<0.01) reduced the S-EVs entry into endothelial cells, due to its possible indirect effect on ACE2 expression4. S-EVs represent a safe and valuable tool for the study of host-virus interactions facilitating the development of novel therapeutics by minimizing the gap between preclinical and clinical studies.

1. Urbanelli, L. et al. The role of extracellular vesicles in viral infection and transmission. Vaccines 7, 1–20

(2019). 2. Hassanpour, M. Since January 2020 Elsevier has created a COVID-19 resource centre with free

information in English and Mandarin on the novel coronavirus COVID- 19 . The COVID-19 resource centre is hosted on Elsevier Connect , the company ’ s public news and information . (2020).

3. Deftereos, S. G. et al. Effect of Colchicine vs Standard Care on Cardiac and Inflammatory Biomarkers and Clinical Outcomes in Patients Hospitalized with Coronavirus Disease 2019: The GRECCO-19 Randomized Clinical Trial. JAMA Netw. Open 3, 1–14 (2020).

4. Weber, A., Iturri, J., Benitez, R., Zemljic-Jokhadar, S. & Toca-Herrera, J. L. Microtubule disruption changes endothelial cell mechanics and adhesion. Sci. Rep. 9, 1–12 (2019).

Caenorhabditis elegans as model system to study EV bioactivity and biodistribution in vivo

Pamela Santonicola1, Giorgia Adamo2, Sabrina Picciotto2, Daniele P. Romancino2, Angela Paterna3, Estella Rao3, Samuele Raccosta3, Rosina Noto3, Nicolas Touzet4, Mauro Manno3,

Antonella Bongiovanni2 and Elia Di Schiavi1

(1) Institute of Biosciences and BioResources (IBBR), National Research Council (CNR), Napoli, Italy

(2) Cell-Tech HUB and Institute for Research and Biomedical Innovation, National Research Council of Italy (CNR), Palermo, Italy

(3) Cell-Tech HUB and Institute of Biophysics, National Research Council of Italy (CNR), Palermo, Italy

(4) Centre for Environmental Research Innovation and Sustainability, Institute of Technology Sligo, Sligo, Ireland

Caenorhabditis elegans is a unique model to study EV bioactivity and biodistribution in vivo, thanks to its small size, powerful genetics, short life, body transparency, and low costs. Moreover, being an invertebrate raises less ethical issues, fulfills the 3Rs principles and is highly supported by EU (Resolution 5/05/2009) and Italian Law (D.L. 4 marzo 2014/26). Indeed, C.elegans is largely used for drug and toxicology screening and has been adopted by the National Toxicology Program and the FDA in US. In the context of the VES4US European project (https://ves4us.eu), we used C. elegans to test in vivo the bioactivity and biodistribution of nanoalgosomes, a new subtype of small extracellular vesicles derived from microalgae [1, 2]. The chronic treatment with tangential flow filtration (TFF)-separated nanoalgosomes from the chlorophyceaen photosynthetic marine microalgae strain Tetraselmis chuii, did not cause any toxic effect on C. elegans survival, development and fertility at different concentrations and time of exposure. Interestingly, nanoalgosomes are attractant for worms and improve their motility in a dose dependent manner, suggesting cross kingdoms communication. The enhancement in motility has been previously ascribed to antioxidant activity, and indeed we observed a rescue in old animals of the age-related impairment in motility. Thanks to the unique possibility of visualizing fluorescent signal in living animals, while a biological process takes place, we tested the uptake of fluorescently labelled nanoalgosomes in vivo. We thus demonstrated that nanoalgosomes are uptaken by intestinal cells through an active mechanism, and they persist up to three days after treatment. We will present, the subcellular localization, the molecular mechanisms underlying uptake and bioactivity, and the potential neuroprotective effects on animal models of neurodegenerative pathologies, such as Spinal Muscular Atrophy, Parkinson and Alzheimer diseases. Supported by the European Union’s Horizon 2020 research and innovation programme under grant agreements No 801338 and 952183.

References [1] Adamo, G. et al., 2021. Nanoalgosomes: Introducing extracellular vesicles produced by microalgae. J Extracell Vesicles. 10(6):e12081. [2] Picciotto, S. et al., 2021. Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds. Biomater Sci 9(8):2917-2930.

Extracellular vesicles carrying human endogenous retroviruses as modulators of immune response after environmental stimuli: functional

studies in the zebrafish model

Luca Ferrari1, Marco Cafora1,2, Federica Rota1, Paola Monti3, Mirjam Hoxha1, Giulia Solazzo1, Serena Del Bue4, Anna Pistocchi2, Valentina Bollati1

1 EPIGET LAB, Department of Clinical Sciences and Community Health, Università degli Studi di Milano,

Milan, Italy

2 Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano,

Milan, Italy

3 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Unit of Occupational Medicine, Milan,

Italy

4 Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Milan, Italy

Extracellular vesicles (EVs) have a crucial role in the modulation of immune response after environmental

stimuli. In this context, among the EV-membrane-bound determinants, a growing interest has been turned

to human endogenous retroviruses (HERVs). The expression of HERVs is strongly modulated by the

environment, since HERV promoters are normally forced into a repressed state by DNA methylation, but

can be switched on in response to environmental triggers. HERVs are involved in a plethora of

physiological processes, such as trophoblast fusion during placental development and modulation of the immune response, which may turn to pathogenic, for example by promoting cell invasion and immune

escape during tumorigenesis. As mechanisms underlying this evidence are still unclear, we hypothesize

that HERV-positive EVs might modulate inflammation and immune suppression after environmental

stimuli. To investigate this hypothesis, we considered cell lines of different origins (i.e. epithelial and

monocytes) and treated them with different compounds, such as cortisol, to mimic the effects of stress

as a paradigmatic environmental trigger that is known to directly affect both immunity and inflammation.

EVs were quantified by nanoparticle tracking analysis, and HERV-positive EV concentrations were

measured by flow cytometry. The effect of EVs isolated from untreated and cortisol-treated cell lines on

the innate immune response was functionally evaluated by injecting them in zebrafish embryos and

assessing the expression levels of pro-inflammatory cytokines interleukin 1β (il1-β), interleukin 6 (il-6),

interleukin 8 (il-8) and the neutrophil marker myeloperoxidase (mpx), and the anti-inflammatory markers

Interleukin 10 (il-10), Interleukin-13 (il-13) by real-time qPCR. Interestingly, both HERV-W and HERV-K

positive EVs injected in zebrafish embryo circulation were significantly associated with a reduced expression of proinflammatory markers supporting our hypothesis that HERV-positive EVs may act as

immunomodulators. The obtained results open new perspectives about the modulation mechanisms of

immunity after environmental stimuli.

Astrocyte-derived extracellular vesicles from specific brain regions rescue apoptosis and preserve mitochondrial function in a cellular model of

Parkinson’s disease

L. Leggio1, F. L’Episcopo2, A. Magrì3, M. J. Ulloa-Navas4, G. Paternò1, S. Vivarelli1, C. A. P. Bastos5, C. Tirolo2, N. Testa2, S. Caniglia2, P. Risiglione3, F. Pappalardo1, N. Faria5, L. Peruzzotti-

Jametti6, S. Pluchino6, J. M.García-Verdugo4, A. Messina3, B. Marchetti1,2, N. Iraci1.

1Dept. of Biomedical and Biotechnological Sciences, Univ. of Catania, Catania, Italy. 2Oasi Research Institute-IRCCS, Troina, Italy.

3Dept. of Biological, Geological and Environmental Sciences, Univ. of Catania, Catania, Italy. 4Laboratory of Compared Neurobiology, Univ. of Valencia-CIBERNED, Paterna, Spain.

5Dept. of Veterinary Medicine, Univ. of Cambridge, Cambridge, United Kingdom. 6Dept. of Clinical Neurosciences, Univ. of Cambridge, Cambridge, United Kingdom.

Parkinson’s disease (PD) is a chronic neurodegenerative disease characterized by the progressive loss of dopaminergic (DAergic) neurons in the ventral midbrain (VMB) and their terminals in the striatum (STR). In this context, astrocytes (AS) are key players in the homeostatic regulation of DAergic neurons, with either destructive or beneficial functions. Interestingly, when activated by the chemokine CCL3, AS exert a robust DAergic neuroprotection both in cellular and pre-clinical models of PD [1]. However, the complex intercellular signaling between AS and neurons in PD has not been fully elucidated, yet. Recently, extracellular vesicles (EVs) emerged as a novel mechanism used by cells to transfer information to adjacent or distant regions. EVs are nanometric structures containing cell derived material (e.g., RNAs, proteins, lipids), released by almost all cells into the microenvironment. To evaluate the possible involvement of EVs in AS-neuron crosstalk, we isolated EVs derived from VMB- and STR-AS under basal and CCL3-treated conditions. EVs were purified by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, Immunogold-Transmission Electron Microscopy and Western Blotting. We found that AS primarily secrete small EVs of ⁓100 nm, positive for tetraspanin markers (CD63/CD9) and Alix. Under basal conditions, VMB-AS release more EVs per cell than STR-AS, and only VMB-AS respond to CCL3 by producing more EVs, demonstrating that the AS-EV secretion rate is specified by the brain region of origin [2]. Next, AS-EVs were labeled with the lipophilic dye PKH26 and their internalization was investigated in differentiated neuronal SH-SY5Y target cells. Via confocal microscopy, we found that AS-EVs co-localize with tyrosine hydroxylase, thus demonstrating their uptake by target cells. To investigate the functional impact of AS-EVs from the nigrostriatal system in degenerative conditions, we exposed SH-SY5Y cells to two distinct sources of toxicity – hydrogen peroxide (H2O2) and the neurotoxin MPP+ – mimicking oxidative stress and mitochondrial dysfunction found in PD. Neurons were pre-treated with AS-EVs, and then exposed to neurodegenerative stimuli for 24h. By immunofluorescence, we found that CCL3-AS-EVs from both VMB and STR fully counteract H2O2-induced caspase-3 activation. Furthermore, by High Resolution Respirometry, we found that the loss of neuronal mitochondrial complex I function induced by MPP+ is completely rescued by all AS-EVs. Notably, only VMB-AS-EVs fully restore ATP production in MPP+-injured neurons, further supporting the regional diversity of astrocytes. Overall, these results show the specificity of AS-EV secretion in the brain, with possible functional implications in the context of PD. Supported by Fondazione con il Sud, “Brain to South” grant 2015- PDR-0219. References [1] L’Episcopo F, et al. (2011). Reactive astrocytes and Wnt/β-catenin signaling link nigrostriatal injury to repair

in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s disease. Neurobiol Dis, 41:508–527. [2] Leggio L, et al. (2021). Small Extracellular Vesicles Secreted by Region-specific Astrocytes Ameliorate the

Mitochondrial Function in a Cellular Model of Parkinson’s Disease. bioRxiv. doi: 10.1101/2021.04.23.441135.

Extracellular vesicles (EVs) are known to be implicated in the spread of pathogenic proteins. For this

reason, inhibitors of enzymes critical in EV biogenesis, such as Neutral Sphingomyelinase 2 (nSMase2),

have been studied as therapeutic agents for several diseases. We recently discovered a new potent,

selective, orally available, and brain penetrable nSMase2 inhibitor, called PDDC1,2. We have reported

that PDDC reduces EV release in vitro and in vivo, but the specific cell populations affected by nSMase2

inhibition have not been explored. In this study, we took advantage of our biosensor based on Surface

Plasmon Resonance imaging (SPRi)3 to evaluate which brain-derived EVs were affected by PDDC in

response to a neuroinflammatory insult.

Specifically, mice were fed vehicle or PDDC-containing chow and after 6 days they were administered

an intra-striatal IL-1β injection. Then, striatum and plasma were collected in order to test the nSMAse2

activity and to isolate plasma EVs. In particular, EVs released by neurons, oligodendrocytes, astrocytes

and microglia were detected on a SPRi biochip by probing specific EV membrane molecules.

We found that IL-1β-induced injury selectively increased the levels of plasma CD171+ EVs (neurons)

and PLP1+ EVs (oligodendroglia) that were normalized by PDDC, while GLAST+ EVs (astrocytes) were

unchanged. Moreover, IL-1β injection increased the amount of EVs released from activated microglia,

compared to EVs released from non-activated microglia in plasma. The increase in activated microglial

EVs was normalized with PDDC.

Our results demonstrated that PDDC is able to normalize the increased release of neuronal-,

oligodendrocyte- and activated microglial-derived EVs into plasma following an acute brain injury and

can have a potential immune modulatory role. These data support the use of nSMase2 inhibitors as

therapeutic agents for acute brain injury, the use of circulating brain-derived EVs as neuroinflammation

indicators and the use of SPRi to evaluate the efficacy of therapeutics. Supported by the Italian Ministry of Health, NIH grants P30MH075673 and R01AG059799 and a Tau Pipeline

Enabling grant T-PEP-18-579974C jointly funded by the Alzheimer's Association and Rainwater Charitable

Foundation.

References 1. M. Sala et al. Journal of Medicinal Chemistry. Doi: 10.1021/acs.jmedchem.0c00278 2. C. Rojas et al. Br J Pharmacol. Doi: 10.1111/bph.14789 3. S. Picciolini et al. Journal of Pharmaceutical and Biomedical Analysis. Doi: 10.1016/j.jpba.2020.113649.

Evaluation of neural extracellular vesicles release after the inhibition of

neutral sphingomyelinase in acute brain injury

1IRCCS Fondazione Don Carlo Gnocchi ONLUS, Laboratory of Nanomedicine and Clinical Biophotonics (LABION), Milan, Italy

Johns Hopkins Drug Discovery2, Neurology3, Oncology4, Medicine5, Pharmacology and Molecular

Sciences6, Psychiatry and Behavioral Science7, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

Silvia Picciolini1, Carolyn Tallon2,3, Alice Gualerzi1, Francesca Rodà1, Seung‐Wan Yoo3, Ajit G. Thomas2, Arindom Pal2,3, Jesse Alt2, Norman J. Haughey3, Rana Rais2,3, Barbara S. Slusher2-7, Marzia Bedoni1

Pro-inflammatory activity of extracellular vesicles

released by senescent-like muscle cells

Serena Maggio1, Andrea Cioccoloni1, Paola Ceccaroli1, Emanuela Polidori1, Michela Battistelli1,

Giovanni Piccoli1, Gianluca Storci2, Massimiliano Bonafè2, Vilberto Stocchi3, Michele Guescini1*

1Department of Biomolecular Sciences (DISB), University of Urbino Carlo Bo, Via I Maggetti, 26, 61029 Urbino, Italy.

2Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Italy. 3Department of Human Sciences for the Promotion of Quality of Life, University San Raffaele Roma, Italy.

* Correspondence: michele.guescini@uniurb.it; Tel.: +39-0722-303424; Fax: +39-0722-303401

Sarcopenia is a geriatric syndrome involving progressive and generalized loss of skeletal muscle mass and

strength. Aged muscles are characterized by an altered mitochondrial function, an increased protein

degradation, inflammation, and a reduction of satellite cell number and function. It has been suggested that

an altered secretory pattern involving inflammatory, growth-promoting, and remodelling factors, called

senescence-associated secretory phenotype (SASP), could induce sarcopenia. Although the SASP has long

been considered composed of soluble factors, growing evidence has highlighted the role of extracellular

vesicles (EVs) as key players in the senescent cell secretome of sarcopenic muscles.

This study aims to investigate whether the EVs released by senescent-like myocytes could affect the behavior

of surrounding myoblasts and macrophages, critical players in the muscle niche.

Two models of senescent-like myocytes were set up: C2C12 cells, an in vitro model of myogenic differentiation,

were cultured for more than 40 passages (high passages, HP) or subjected to oxidative stress (OX). HP and OX

EVs were obtained using serial ultracentrifugation and density gradient, quantified through Nanoparticles

Tracking Assay (NTA), characterized by Western Blot (WB), and used to treat the recipient cells. Expression

analyses were performed by RT-qPCR and WB.

HP and OX myocytes displayed senescence-associated characteristics such as defective myogenic

differentiation process and a significantly inflamed status compared to C2C12 cells cultured in standard

condition at low passages (control, LP). The EV quantification showed that HP myocytes release more EVs

compared to LP cells, enriched in nucleic acids, especially DNA, compared to control. Moreover, senescent-

like EVs induced the expression levels of IL-6 in LP C2C12, and IL-1β and IFN-1β in RAW264.7 without

affecting STING, the cytosolic DNA sensor. Of note, the induction of IFN-1β was significantly reduced when

RAW264.7 cells were pre-exposed to chloroquine, an inhibitor of endosomal toll-like receptors (TLRs).

In summary, the reported data suggest that EVs released by senescent-like myocytes induce inflammation of

the cells typical of the muscle niche. These results led us to hypothesize a functional relationship between the

derangement of the myogenic process found in HP differentiating myocytes and the inflammatory response

induced by stress-derived EVs. Of note, EV-associated DNA seems to play a key role in triggering the

inflammation process as suggested by IFN-1β expression; moreover, this mechanism likely takes place in the

endosomal compartments by TLR stimulation.

The role of oxygen tension on 3D Neuroblastoma spheroids and their shredded exosomes: could hypoxic exosomes modulate

Mesenchymal Stem Cells behavior? N. Torriero1,2, M.R. Esposito1,2, P. Fusco1,2, L. Zanella1, E. Cimetta1,2

1 Department of Industrial Engineering (DII) University of Padua, Padua, Italy 2 Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy

Neuroblastoma (NB) is an embryonal malignancy with more than half cases classified as High Risk, showing 5-year overall survival less than 40% from the diagnosis.1 Hypoxia, the condition of low oxygen tension, is a known hallmark of solid tumors, leading to altered metabolism in the cellular microenvironment (µENV) and consequent increase in tumor aggressiveness.2 The aim of the study is to understand the role of exosomes (EXOs) released under different oxygen concentrations in driving NB aggressiveness. We based our studies on a 3D model capable of recapitulating in vivo tumor features in a simple yet more realistic way. Uniformly sized NB spheroids were formed using the SK-N-AS cell line through AggreWell™ plates and grown under 3 different oxygen concentrations (normoxia: 20% O2, hypoxia: 1.5%O2 and reoxygenation: hypoxia followed by normoxia). We also focused on the separation and analysis of the inner hypoxic core and the proliferating periphery of these in vivo-like 3D NB spheroids since we hypothesize that each compartment has a specific role in determining the aggressive phenotype in recipient target cells. We first purified and characterized EXOs released by SK-N-AS spheroids. The ExoView platform confirmed the successful isolation of a population of extracellular vesicles with mean diameters of 50-60 nm and expressing the most common exosomal markers CD63, CD81 and CD9. Transmission electron microscope (TEM) acquisition further confirmed the correct morphology of EXOs. In order to highlight differences between the core and periphery, we performed a stereomicroscope-guided mechanical separation on the spheroids. We then performed single cell-RNA Seq analysis (BD-Rhapsody Single Cell analysis system) on the two populations for the following 6 samples: core-shell normoxia, core-shell hypoxia, core-shell reoxygenation. This platform enables generating a whole transcriptome analysis and the consequent identification of heterogeneity in the differentially expressed genes between the two cell populations and µENV oxygenation conditions.3 For a finer separation of the core and periphery regions, we recently developed a new method. Briefly, after 14 days of culture, spheroids were placed on gelatin coated wells for additional 3 days: differently from the core cells, peripheric cells migrate through the gelatin, allowing a distinct and clear separation of the two populations. Released EXOs were isolated from both the hypoxic core and the periphery of the spheroids. Based on our existing data on the effects of hypoxic exosomes from 2D NB cell cultures on Bone Marrow-Mesenchymal Stem Cells (BM-MSCs) differentiation, we are now investigating the role of EXOs derived from the two distinct compartments of our 3D model in inducing changes on the morphology and behavior of recipient BM-MSCs, pushing cells towards a more aggressive phenotype. Our study will provide a detailed molecular comprehension of tumor complexity as well as highlight the peculiar key signals delivered by exosomes from different component in determining NB aggressiveness. Blocking these signals could hold the potential to unlock novel treatment strategies to prevent NB dissemination. Supported by ERC Starting Grant – MICRONEX UERI17_01 References 1. Luksch R, Castellani MR, Collini P, et al. Neuroblastoma (Peripheral neuroblastic tumours). Crit Rev Oncol Hematol. 2016;107:163-181. 2. Kim Y, Lin Q, Glazer PM, Yun Z. Hypoxic tumor microenvironment and cancer cell differentiation. Curr Mol Med. 2009;9(4):425-434. 3. Shum EY, Walczak EM, Chang C, Christina Fan H. Quantitation of mRNA Transcripts and Proteins Using the BD Rhapsody™ Single-Cell Analysis System. Adv Exp Med Biol. 2019;1129:63-79.

nEVs increase tumor metastasis in a 4T1 mouse triple-negative breast cancer (TNBC) model

Malvina Koni1, Cristina Grange1, Tatiana Lopatina1, Massimo Cedrino2, Emilio Venturelli1, Giovanni Camussi1, and Maria Felice Brizzi1

1Department of Medical Sciences, University of Turin, Turin, Italy

22i3T Scarl University of Turin, Turin, Italy

Breast cancer (BC) is the most diagnosed cancer in women worldwide and the second cause of cancer death in women, with expensive health care costs [1]. Triple-negative breast cancer (TNBC) is an aggressive phenotype of BC and accounts higher risks of recurrence and distant metastasis rates than other BC subtypes. Thus, understanding the machinery supporting metastatic colonization has an enormous implication in improving the development of new therapies and patient’s clinical outcomes. We have shown for the first time that TNBC samples expressed the receptor for the proinflammatory cytokine interleukin-3 (IL-3Rα) [2]. IL-3Rα expression was also evaluated in 7 TNBC cell lines. Then we showed that IL-3 stimulation or IL-3Rα blocking did not influence tumor cell viability, cell cycle or autocrine IL-3 secretion. Whereas changed extracellular vesicles (EVs) properties. In particular, EVs released by TEC after IL-3Rα blockade (anti-IL-3R-EVs) decreased the metastasis and tumor angiogenesis in vivo [3]. Since IL-3 is a well described myeloid trophic factor, we hypothesized that IL-3 stimulation in the tumor microenvironment could be relevant for the immune regulation by tumor EVs. The aim of this study was to assess the metastatic development in immune competent mice BALB/C using a syngenic TNBC model. We stimulated the established 4T1 metastasis with native TEC-EVs (nEVs) or anti-IL-3R-EVs for 3 weeks. After this period, we showed that treatment with nEVs increased the number of metastases and tumor angiogenesis while anti-IL-3R-EVs was able to reduce both of them possibly by acting on tumor immune regulation. Based on these findings, we suggested that nEVs support tumor growth and metastasis also by regulating the immune response and that IL-3 signalling could be the ideal target to break this pro-tumorigenic action.

References

1) Mohammad Fahad Ullah. Breast Cancer: Current Perspectives on the Disease. 2019;1152:51-64. doi: 10.1007/978-3-030-20301-6_4.

2) Lopatin et al. Targeting IL-3Rα on tumor-derived endothelial cells blunts metastatic spread of triple-negative breast cancer via extracellular vesicle reprogramming. Oncogenesis. 2020 Oct 10;9(10):90. doi: 10.1038/s41389-020-00274-y.

3) Lombardo et al. IL-3R-alpha blockade inhibits tumor endothelial cell-derived extracellular vesicle (EV)-mediated vessel formation by targeting the β-catenin pathway 2018 Mar;37(9):1175-1191. doi: 10.1038/s41388-017-0034-x.

POSTER PRESENTATIONS

Plants as a “green” source of biologically active compounds: isolation and characterization of biologically active compounds and cellular particles from European

spruce (Picea Abies) needles

M. Jeran1,2, D. Božič1,2, U. Novak3, M. Hočevar4, R. Barrios-Francisco5, A. Romolo1, A. Iglič2,6, V. Kralj-Iglič1

1University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana,

Slovenia 2University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana,

Slovenia 3National Institute of Chemistry, Laboratory for Biomolecular Structure, Ljubljana, Slovenia

4Institute of Metals and Technology, Physics and Chemistry of Materials, Ljubljana, Slovenia 5División Ingeniería Química, Tecnológico Nacional de México/TES de San Felipe del Progreso, San Felipe

del Progreso, Mexico 6University of Ljubljana, Faculty of Medicine, Laboratory of Clinical Biophysics, Ljubljana, Slovenia

Plants have an incredible ability to synthesize many active compounds on which today's drugs and therapies are based. This study focused on the European spruce (Picea abies), limonene and borneol in spruce needles macerates, and cellular particles in the spruce needles homogenates. Macerates of spruce needles were prepared in ethanol or n-hexane and analyzed by Fourier-transform infrared spectroscopy (FTIR). Microparticles were isolated by differential centrifugation from three different spruce needles homogenates prepared in 0.9 % saline (homogenized in mixer grinder only, additionally treated with ultrasound, or supplemented with lecithin). Samples throughout the centrifugation processing were analysed by flow cytometry (FCM), scanning electron microscopy (SEM) and FTIR. FTIR spectroscopy revealed high yield of limonene and borneol in macerates in both solvents. Limonene was better macerated in n-hexane while borneol was better macerated in ethanol. It was concluded that the solvent (and its chemical and physical properties) plays a key role in the maceration process and that the yield depends on the structure of the compound. In analysis of samples taken throughout the differential centrifugation of spruce needles homogenates, numerous submicron particles were observed in the supernatant after centrifugation at 2.000 × g, some having typical shapes of membrane-enclosed vesicles without internal structure. Visualization also revealed remnants of cells and amorphous material. Addition of lecithin in the homogenization step resulted in abundancy of particles with smooth contours. The morphology of particles seemed similar in supernatants and pellets throughout the differential centrifugation processing, however, with varying sizes and concentrations. In FTIR spectra, two main peaks were recognized in the spectrum – one characteristic for carbohydrates (1180–960 cm-1, corresponding to C-O and C-C stretching vibrations in combination with C-O-H, C-O-C), and one characteristic for lipids (3000–2850 cm-1 corresponding to CH2 and CH3 stretching vibrations of fatty acids). Higher lipid to carbohydrate ratio was detected in the isolate prepared at 50.000 × g for 60 min than in the one prepared at 17.500 × g for 60 min, suggesting that particles with higher lipid content were pelleted at higher forces. Our results indicate that cellular particles found in the isolates from spruce needles homogenates were formed mainly during processing of the samples by mechanical forces and that they reflect the properties of the sample as well as the parameters of the processing. Supported by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 801338 (VES4US project) & Slovenian Research Agency, ARRS grands: P2-0232, P3-0388, L3-2621, J1-1705 and J2-2486.

Biophysical analysis of small extracellular vesicles (sEVs) and their interaction with model plasma membrane

C. Paba 1, B. Senigagliesi 2, V. Rondelli 3, Mario Gimona 4, P.Parisse 5, L.Casalis 6

1 University of Trieste, Trieste, Italy, 2 SISSA, Trieste, Italy, 3 University of Milan, Milan,Italy,4

Paracelsus Medical University, Salzburg, Austria, 5 IOM-CNR, Trieste, Italy, 6 Elettra Sincrotrone Trieste, Trieste, Italy

Small extracellular vesicles (sEVs) are nanometer-sized vesicles (30-200 nm) that are released from almost all types of cells both in pathological or physiological conditions. These biological nanoparticles have shown potential for cancer diagnostics/therapeutics as by travelling in body fluids (blood, saliva) they carry “biological information” between near and far cells. However, the complexity of EVs origin, their composition, and nanometric size, make them a challenge to many available bioanalytical methods. Indeed, EVs are heterogenous nano-carriers with complex cargoes (proteins, lipids, and nucleic acids, etc.), whose population mostly changes in accordance with the type of the parental cell and the specific physiological or pathological conditions at the moment of their packaging and secretion. Cell type specificity of EVs biogenesis, release, and uptake is still debated, as well as the involved targeting cell receptors and internalization pathways (e.g. cell membrane fusion and endocytosis).

Here we propose to use an assay based on Atomic Force Microscopy (AFM), in order to investigate structure-function correlations of EVs from two different breast cancer cell lines with different aggressiveness, with lipid model membrane systems. The AFM imaging was and will be performed by using lipid bilayers of variably composition, in different environmental conditions. Preliminary vesicle-membrane interaction analyses were carried out with sEVs isolated from human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) as a reference sample. Results obtained revealed different interaction mechanisms of EVs with lipid model membranes depending on the salt concentration and preferentially with liquid-ordered raft-like lipid domains. These first results open to a better understanding of the EV uptake mechanisms by recipient cells and the most relevant parameters involved in the EV selective targeting in tumor microenvironments.

References:

- URIBE, Johana, et al. A supported membrane platform to assess surface interactions between extracellular vesicles and stromal cells. ACS Biomaterials Science & Engineering, 2020. - E Van Der Pol, AG Hoekstra, A Sturk, Cornelis Otto, TG Van Leeuwen, and R Nieuwland. Optical and non-optical methods for detection and characterization of microparticles and exosomes. Journal of Thrombosis and Haemostasis, 8(12):25962607, 2010. - Shivani Sharma, Haider I Rasool, Viswanathan Palanisamy, CliFF Mathisen, Michael Schmidt, David T Wong, and James K Gimzewski. Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative afm, fesem, and force spectroscopy. ACS nano, 4(4):19211926, 2010. - Clotilde Théry, Kenneth W Witwer, Elena Aikawa, Maria Jose Alcaraz, Johnathon D Anderson, Ramaroson Andriantsitohaina, Anna Antoniou, Tanina Arab, Fabienne Archer, Georgia K, Atkin-Smith, et al. 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. Journal of extracellular vesicles, 7(1):1535750, 2018. - Edwin Van der Pol, Anita N B•oing, Paul Harrison, Augueste Sturk, and Rienk Nieuwland. Classication, functions, and clinical relevance of extracellular vesicles. Pharmacological reviews, 64(3):676705, 2012 - YÁÑEZ-MÓ, María, et al. Biological properties of extracellular vesicles and their physiological functions. Journal of extracellular vesicles, 2015, 4.1: 27066. - AKUMA, Precious; OKAGU, Ogadimma D.; UDENIGWE, Chibuike C. Naturally occurring exosome vesicles as potential delivery vehicle for bioactive compounds. Frontiers in Sustainable Food Systems, 2019, 3: 23. - MULCAHY, Laura Ann; PINK, Ryan Charles; CARTER, David Raul Francisco. Routes and mechanisms of extracellular vesicle uptake. Journal of extracellular vesicles, 2014, 3.1: 24641.

Role of Extracellular Vesicles from adipose tissue- and bone marrow-mesenchymal stromal

cells in endothelial proliferation and chondrogenesis

Maria Elisabetta Federica Palamà1, Cansu Gorgun1,2, Daniele Reverberi3, Maria Cristina Gagliani1, Katia Cortese1, Roberta Tasso1, Chiara Gentili1

1 Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy

2 U.O. Cellular Oncology, IRCCS Ospedale Policlinico San Martino, Genova, Italy. 3 U.O. Molecular Pathology, IRCCS Ospedale Policlinico San Martino, Genoa, Italy.

The secretome of mesenchymal stromal cells (MSCs) derived from different tissue sources is considered an innovative therapeutic tool for regenerative medicine. Although adipose tissue-and bone marrow-derived MSCs (ADSCs and BMSCs, respectively) share many biological features, the different tissue origins can be mirrored by variations in their secretory profile, and in particular in the secreted extracellular vesicles (EVs). Since EVs trigger specific cellular responses, reflecting the status and composition of the parental cell, in our study we aimed to identify morphological and functional differences between BMSC- and ADSC-derived EV subpopulations. EVs were isolated from the cell conditioned media by serial differential centrifugations, to collect both medium-sized (mEVs) and small-sized vesicles (sEVs). Their involvement in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics. E17.5 (for vessel sprouting assay) and E15.5 (for bone growth) fetuses were removed from pregnant C57BL/6 wild-type mice and metatarsals were dissected under stereomicroscope. For vessel sprouting assay, isolated metatarsal bones were cultured in 24 well plates for 96h in complete medium. After 96h, attached bones covered by fibroblasts were selected and treated with ADSC- and BMSC-derived EVs. After 7 days, bones were analyzed for the expression of CD31 by immunohistochemistry, to visualize the vascular network around the bone. For metatarsal bone growth, isolated metatarsal bones were cultured in 24 well plate in suspension, in presence of ADSC- or BMSC-derived EVs. Bone growth was monitored daily and, after 10 days, bones were fixed, and paraffin embedded. Histological and immunohistochemical stainings were performed to analyze the morphology of the growth plate. Although EVs separated from both cell sources presented similar characteristics in terms of size, concentration and marker expression, they exhibited different characteristics in terms of protein content and functional effects. We showed that the EV-encapsulated components of the ADSC secretome over-expressed pro-angiogenic factors in comparison to the BMSC-counterpart, showing also greater angiogenic capabilities compared to BMSC-counterparts. This confirmed that ADSCs could be preferred over other MSC populations in therapeutic approaches dependent upon angiogenesis. On the other hand, BMSC-EVs contained a higher amount of pro-differentiation and chemotactic proteins, and they were able to prompt growth plate organization. Contrary to ADSC, BMSC-sEVs release signals that guide the spatial organization of chondrocytes in the growth plate that will dictate bone growth. The present study highlights the importance of selecting the appropriate cell source of EVs for targeted therapeutic applications. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671 to C.Ge. and M.E.F.P and the European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska- Curie grant agreement No. 721432 CarBon to C.G. and R.T. The materials presented and views expressed here are the responsibility of the authors(s) only.

Drugs inducing lysosomal impairment drives the release of Extracellular Vesicles carrying autophagy-associated markers

L. Urbanelli1, K. Sagini2, S. Buratta1, F. Delo1, R.M. Pellegrino1, C. Emiliani1,3

1 Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy

2 Department of Surgery, Division of Cancer Biology and Therapeutics, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA

3 CEMIN-Center of Excellence for Innovative Nanostructured Material, University of Perugia, Perugia, Italy

Extracellular vesicles (EVs) are now considered an additional manner to transmit intercellular

signals, but they were initially identified as a system to dispose extracellularly unnecessary cell material, instead of digesting it into lysosomes. Because of the important role played by the endolysosomal system also in the biogenesis and secretion of EVs, we investigated their release upon treatment with drugs impairing lysosomal function. First, we developed a cell model engineered to produce fluorescently labelled vesicles, by expressing mCherry-CD63 fusion protein in HEK-293 cells, then we treated cells with 1) amiodarone (AM), a cationic amphiphilic drug known to induce phospholipidosis (PLD), i.e. the accumulation of phospholipids into multilamellar structures within organelles of the endosomal-lysosomal system; 2) chloroquine (CQ), a drug commonly used to impair autophagy by blocking autophagosome-lysosome fusion; 3) bafilomycin A1 (BafA1), known to disrupt autophagic flux by independently inhibiting V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion. EVs were isolated from cell medium by differential ultracentrifugation. Both medium/large (10K fraction) and small (100K fraction) EVs were collected, and their fluorescence measured. We observed that all 3 drugs induced the release of a higher number of EVs, mostly of medium/large size, although in the case of BafA1, also of small size. EVs released upon treatment did not display significant morphological changes or altered size distribution but both fractions showed a dose dependent increase of autophagy associated markers. Interestingly, proteolytic digestion shows that one of these markers, LC3B, was localized within EVs, indicating a higher release of EVs with an autophagosome-like phenotype. Analysis of EV phospholipid content by LC/MS provided evidence that medium/large EVs released upon AM treatment contain more phospholipids than those released by untreated cells, whereas those release upon CQ and AM treatment did not, indicating that AM-treated cells release EVs enriched in phospholipids to possibly alleviate their intracellular accumulation. This result confirms that the lysosomal degradative capability is a key factor in determining undigested material fate (intracellularly degraded or extracellularly released) and suggests that this could be a feasible target to attenuate abnormalities induced by undigested substrates deposition in pathologies characterized by lysosomal dysfunction, such as Lysosomal Storage Disorders and neurodegenerative disease associated with accumulation of misfolded proteins.

NSC-34-derived EVs promote AChR clustering through

canonical and non-canonical Wnt signaling

Serena Maggio1, Paola Ceccaroli1, Emanuela Polidori1, Manuela Ferracin2, Andrea Cioccoloni1,

Michela Battistelli1, Massimiliano Bonafè2, Fabiola Olivieri3, 4, Vilberto Stocchi5 and Michele Guescini1 *

1Department of Biomolecular Sciences (DISB), University of Urbino Carlo Bo, Via I Maggetti, 26, 61029 Urbino, Italy.

2Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Italy. 3Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy.

4Center of Clinical Pathology and Innovative Therapy, IRCCS INRCA, National Institute, Ancona, Italy. 5Department of Human Sciences for the Promotion of Quality of Life, University San Raffaele Roma, Italy.

* Correspondence: michele.guescini@uniurb.it; Tel.: +39-0722-303424; Fax: +39-0722-303401

The neuromuscular junction (NMJ) is a chemical synapse formed between motor neuron and skeletal muscle.

Before the NMJ takes place, aneural acetylcholine receptors (AChRs) are widespread on the muscle membrane,

but during the NMJ development, they clusterize in regions exactly opposite to motor neuron contact sites, a

process called muscle pre-patterning. A correct establishment of the NMJ requires an appropriate

communication between motor neurons and muscle fibers; however, the molecular mechanisms leading to

NMJ formation have not yet been fully elucidated. The aim of this study is to investigate the potential role of

EVs in motor neuron-muscle communication during the NMJ establishment.

NSC-34, a motor neuron-like cell in vitro model, and C2C12, an in vitro model of myogenic differentiation, were

used. NSC-34-derived EVs have been isolated from differentiation media during motor neuron development

using a serial ultracentrifugation protocol specifically adjusted to isolate high and low density EVs (named

HD-EVs and LD-EVs), quantified through Nanoparticles Tracking Assay (NTA), characterized by Western

Blot (WB) and Next-Generation Sequencing (NGS), and used to treat cultured myotubes.

The obtained data showed that LD-EVs have a diameter of about 110 nm while HD-EVs exhibit a much more

variable size with an average diameter of 240 nm. Furthermore, WB analysis revealed that LD-EVs are negative

for calnexin, an endoplasmic reticulum marker, positive for Alix, a well-established exosome marker, and LD-

EVs and HD-EVs are both positive for Hsp70 and Tsg101, two well-defined EV markers. NGS analysis of the

motor neuron-derived EVs identified a total of 353 miRNAs, among these, by using several predictive analytic

tools, we found many miRNAs potentially targeting genes involved in Axon guidance, Wnt- and TGF-β

signaling pathways.

In addition, WB analyses showed that some Wnts are encapsulated into both HD-EVs and LD-EVs: of note

Wnt3a is secreted at the early time of differentiation while Wnt11 is preferably found in EVs released by fully

differentiated motor neurons.

Then, to investigate the role of NSC-34-derived EVs in muscle pre-patterning, cultured myotubes were treated

with EVs secreted by differentiated NSC-34. Firstly, immunofluorescence staining with α-bungarotoxin

highlighted an increase of AChR clusters in response to EV-treatments. Subsequently, the activity of WNTs in

promoting AChR clustering both through canonical and non-canonical Wnt signalling pathways was

investigated. WB and immunofluorescence analyses showed an increase in GSK3β phosphorylation and β-

catenin nuclear translocation, clearly demonstrating a positive stimulation of canonical Wnt signalling.

Moreover, WB analysis evidenced the increase of JNK phosphorylation, supporting also an involvement of the

non-canonical Wnt pathway.

Altogether these data suggest that EVs released from differentiated motor neurons carry multimodal signals,

miRNAs and Wnts, which can stimulate AChR clustering in myotubes, a fundamental preparatory stage for

NMJ formation.

Hydrophobic effects in membrane sensing peptides for small

Extracellular Vesicle isolation and analysis

A. Strada1,2, R. Frigerio1, A. Musicò1, G. Bergamaschi 1, R. Vago 3, M. Cretich1 and A. Gori1

1 Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC) - Consiglio Nazionale delle Ricerche, Milano, Italy

2 Politecnico di Milano - Department of Chemistry, Materials, and Chemical Engineering ‘‘Giulio Natta’’

3 IRCCS San Raffaele Scientific Institute, Milano, Italy

Small extracellular vesicles (EVs) present fairly distinctive lipid membrane features in the

extracellular environment. EV membrane could be then considered as a ‘universal’ marker,

alternative or complementary to traditional characteristic surface-associated proteins. Our recent

work has identified a class of membrane-sensing peptides derived from Bradykinin protein as a novel

class of molecular ligands for integrated small EV isolation and analysis [1]. The membrane

recognition and binding mechanisms are based on complementary electrostatic interactions

between the peptide and the phospholipids on the outer membrane leaflet, that subsequently can

lead to the insertion of hydrophobic residues into the membrane defects [2].

In the present work, we investigate the role of such hydrophobic aminoacids on the membrane

recognition efficiency. To this aim, we synthetized a small library of membrane-sensing peptides

derived from Bradykinin protein by replacing phenylalanine with residues with different degree of

hydrophobicity. To assess their binding capacity, the peptides were immobilized on microarrays chip

and tested with both exosome-mimicking liposomes [3] and fluorescent EVs from HEK293 cell

culture.

These substitutions showed how the hydrophobic component of peptide sequence play an important

role in membrane sensing. In addition, the same profile of binding for both types of particles

demonstrated the possibility to use synthetic liposomes as model for biological extracellular vesicles.

Finally, a preliminary computational approach was used to determine the free-energy profile of these

peptides in order to provide a more in-depth structural and energetics insight into the peptide

interaction with the phospholipid bilayer.

These results represent a step forward in the development of a new generation of membrane-

sensing peptides for EV isolation and analysis.

Supported by European Union’s Horizon 2020 research and innovation program under grant agreements No.

951768 (project MARVEL) and Regione Lombardia&Fondazione Cariplo, grant n° 2018-1720 (project

HYDROGEX).

References:

1. Gori, A. et al. Membrane-binding peptides for extracellular vesicles on-chip analysis. J. Extracell. Vesicles 9, (2020).

2. Antonny, B. Mechanisms of membrane curvature sensing. Annu. Rev. Biochem. 80, 101–123 (2011).

3. Lu, M. et al. Comparison of exosome-mimicking liposomes with conventional liposomes for intracellular delivery of siRNA. Int. J. Pharm. 550, 100–113 (2018).

Extracellular Vesicles (EVs) as targetable drug delivery vehicles

Maria Chiara Ciferri1, Daniele Reverberi2, Cansu Gorgun1, Chiara Gentili1, Rodolfo Quarto1,2, Roberta Tasso1

1Department of Experimental Medicine (DIMES), University of Genova, Italy

2IRCCS Ospedale Policlinico San Martino, Genova, Italy

Thanks to their activity as short and long-distance mediators of intercellular communication, EVs have been recently considered as a new class of nanocarriers. Their ability to transport functional cargoes to target cells makes them a valid delivery vehicle (1). EV surface can be modified to incorporate ligands for targeted therapy making natural nanoparticles valuable tools for the target-therapy of various diseases (2). Besides the EV delivering properties, the possibility of drug loading and the substantially successful preclinical results have encouraged researchers to study EVs as drug delivery vehicles. The aim of this work is to engineer the EV surface to create a membrane anchoring tool to be used as platform to bind molecules and obtain a tissue-specific delivery system. Plasma and serum represent the selected EV source. EVs have been isolated by size exclusion chromatography (SEC). Two different exogenous methods of membrane functionalization have been examined: i) Click chemistry (a highly efficient reaction between an alkyne and an azide that forms a triazole linkage), and ii) phospholipidic insertion. A fluorescent labelling based on copper free Click chemistry (3) and a membrane insertion of a lipid anchor DMPE-PEG-STREPTAVIDIN/BIOTIN (4) have been performed to detect and quantify the EV surface modification. The first method is based on the reaction of a Dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS ester) tied to the EV membrane with a fluorescent Azide. The second one concerns the presence of the biotin-streptavidin recognition system. In vitro studies on cancer cells have been performed to evaluate the uptake of the engineered vesicles. Flow cytometry and fluorimetry have been chosen to detect the fluorescent signal associated to an accomplished EV functionalization in both experimental methods. Click chemistry turned out to be a more efficient targeting system in comparison to the lipid anchoring approach, and this result was confirmed by in vitro experiments showing a significant uptake of the click-modified EVs by responding cells. Refrerences 1- Natalia L. Klyachko, Camryn J. Arzt, Samuel M. Li, Olesia A. Gololobova and Elena V. Batrakova, 2020. Extracellular Vesicle-Based Therapeutics: Preclinical and Clinical Investigations. Pharmaceutics, 12, 1171. doi: 10.3390/pharmaceutics12121171 2- Seunglee Kwon, Sol Shin, Minjae Do, Byeong Hoon Oh, Yeari, Song, Van Dat Bui, Eun Sook Lee, Dong-Gyu Jo, YongWoo Cho, Deok-Ho Kim, Jae Hyung Park, 2020. Engineering approaches for effective therapeutic applications based on extracellular vesicles. Journal of Controlled Release. https://doi.org/10.1016/j.jconrel.2020.11.062 3-Lizhou Xua, Farid N. Faruqua, Revadee Liam-ora, Omar Abu Abeda,b, Danyang Lia, Kerrie Vennerc, Rachel J Erringtond, Huw Summerse, Julie Tzu-Wen Wanga and Khuloud T. Al-Jamala, 2020. Design of experiment (DoE)-driven in vitro and in vivo uptake studies of exosomes for pancreatic cancer delivery enabled by copper-free click chemistry-based labelling. Journal of extracellular vesicles, Vol. 9, 1779458 https://doi.org/10.1080/20013078.2020.1779458 4-Travis J. Antes, Ryan C. Middleton, Kristin M. Luther, Takeshi Ijichi, Kiel A. Peck, Weixin Jane Liu, Jackie Valle, Antonio K. Echavez and Eduardo Marbán, 2018. Targeting extracellular vesicles to injured tissue using membrane cloaking and surface display. Journal of Nanobiotechnology, 16:61 https://doi.org/10.1186/s12951-018-0388-4

Fourier-transform infrared spectroscopy (FT-IR) to characterize EV

subpopulations from plasma of subjects with Parkinson’s disease and

healthy controls

L. Paolini1,2, S. Federici3,4, A. Radeghieri1,2, P. Bergese1,5

1Department of Molecular and Translational Medicine, Università degli Studi di Brescia, Brescia.

2Consorzio Sistemi a Grande Interfase (CSGI), Department of Chemistry, University of Florence,

Sesto Fiorentino (FI)

3Department of Mechanical and Industrial Engineering, Università degli Studi di Brescia, Brescia.

4National Interuniversity Consortium of Materials Science and Technology (INSTM), Florence.

5Institute for Research and Biomedical Innovation (IRIB) - National Research Council of Italy

(CNR), Palermo.

Extracellular Vesicle (EV) field is evolving to systematically separate and compare all secreted EV

subpopulations. EV subpopulation characterization is an open challenge since distinct

subpopulations bear different biochemical and biophysical properties. Common strategies are

based on searching molecular components, analyzing physical and biological functions to

univocally define the target subpopulation. These approaches present major risks like opting for an

unsuitable marker set, the need to use different techniques and equipment making them

inadequate to routinely deal with an intrinsic high level of heterogeneity. Our previous work

demonstrated that Fourier-transform Infrared (FT-IR) spectroscopy combined with multivariate

tools such as Principal Component Analysis (PCA) is an alternative method to collectively

fingerprint large (LEV), medium (MEV) and small (SEV) EVs from different cell lines [1]. In this

contribution we probed FT-IR to characterize EV subpopulations from plasma (1 ml) of subjects

with Parkinson’s disease (PD) and healthy controls (H) separated by serial (ultra) centrifugation.

LEV, MEV and SEV were fully characterized according to MISEV2018 [2] for their morphology,

size distribution, purity assessment and presence of biochemical markers. Total plasma, LEV,

MEV and SEV FT-IR spectra were acquired and regions between 3100-2800 cm-1 and 1880-900

cm-1, corresponding to spectral common features as lipids and proteins, were processed by PCA.

Results showed that multivariate analysis of plasma FT-IR spectra cannot discriminate among PD

and controls, while LEV, MEV and SEV show different fingerprints among PD and H subjects, even

if with different degree of accuracy: MEV from PD and H can be strongly discriminated, LEV/SEV

from PD and H are slightly separated. We can speculate that differences from pathological and

healthy subjects mirror differences (or similarities) in EV lipid and protein composition. Our

preliminary data confirmed the versatility of FT-IR combined with PCA analysis to characterize

different EV subpopulations.

References

[1] Paolini L, Federici S, Consoli G, Arceri D, Radeghieri A, Alessandri I, Bergese P. 2020. Fourier-transform

Infrared (FT-IR) spectroscopy fingerprints subpopulations of extracellular vesicles of different sizes and

cellular origin. J Extracell Vesicles. 30;9(1):1741174. doi: 10.1080/20013078.2020.1741174.

[2] Thery C et al. 2018. 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. 23;7(1):1535750. doi: 10.1080/20013078.2018.1535750

Funding

This work was funded by BIOMANE grant (Università degli Studi di Brescia), CSGI through the evFOUNDRY

project, Horizon 2020-Future and emerging technologies (H2020-FETOPEN), ID: 801367.

Characterisation of surface markers in mesenchymal stromal cell-derived extracellular vesicles in normal or apoptotic condition

Renata Skovronova1, Veronica Dimuccio1, Cristina Grange2, Maria Chiara Deregibus3,

Giovanni Camussi2 and Benedetta Bussolati1

1Department of Molecular Biotechnology and Health Sciences, University of Turin, Italy.

2Department of Medical Sciences, University of Turin, Italy.

32i3T Business Incubator and Technology Transfer, University of Turin, Italy

Extracellular vesicles released by mesenchymal stromal cells (MSC-EVs) are a rising resource for regenerative medicine. Current EVs research focuses on the small MSC-EVs enriched fraction for therapeutic applications or amelioration of tissue injury. In this study, sequential ultracentrifugation at different speed was used to separate and thoroughly investigate different fractions of MSC-EVs. Following the minimal criteria (MISEV2018) of the International Society for Extracellular Vesicles a bulk analysis was applied to characterize MSC-EV identity and purity, coupled with assessing single EV morphology, size and integrity. We used different orthogonal methods to provide a quantitative analysis of size and surface marker expression in small and large MSC-EV fractions. Three sources of MSC-EVs (bone marrow, adipose tissue, and umbilical cord) were compared extracted from the standard or apoptotic condition. The apoptosis was induced using a directed anti-Fas antibody. A small EV size population <100 nm, as detected by electron microscopy, was confirmed by super-resolution microscopy and ExoView analysis, at variance with nanoparticle tracking analysis provided a biger EV size. Quantitative single‐vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanin expressions, being MSC-EV sub-fractions single, double and triple positive in variable tetraspanins proportion. The ExoView analysis provided single small MSC-EV analysis of tetraspanin and selected mesenchymal markers. Finally, semiquantitative beads based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the larger EV fraction. These results suggest that quantitative EV analysis methods are necessary methodologies to be applied for the characterization of MSC-EVs to generate insights for the safe and practical application of MSC-EVs in regenerative medicine.

Tunning density gradients for the separation of lipoproteins from plasma-derived extracellular vesicles

Ana Paulina Ramos Juarez1,2, Ramila Mammadova1,2, Giovambattista Capasso 2,3, Francesco Trepiccione2,3 and Gabriella Pocsfalvi1

1Extracellular Vesicles and Mass Spectrometry Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy, Naples, Italy.

2Department of Translational Medical Science University of Campania Luigi Vanvitelli, Naples, Italy. 3Biogem Research Institute, Ariano Irpino, Italy

INTRODUCTION. Extracellular vesicles are membrane-enclosed particles present in living organisms and secreted ubiquitously by all cells, hence they are present in body fluids. EVs are implicated in intercellular communication, they carry specific sets of molecules including proteins, lipids, metabolites and nucleic acids that can be a snapshot of the cell of origin. EVs isolated from biofluids have been proposed as a resource of biomarkers for different diseases. Especially, plasma-derived EVs are under development for next-generation liquid biopsies. However, this has been hindered by the complexity of this biofluid, especially by the presence of lipoproteins which not only outnumber the quantity of EVs but also are similar in size and density. Different methods have been developed to reduce the number of lipoproteins from plasma-derived EV samples. Here we used a combination of size exclusion chromatography (SEC), molecular filtration and density gradient ultracentrifugation (DGUC) methods to separate lipoproteins and EVs. The rationale behind is that SEC help in the removal of high-density lipoproteins (HDLs) and low-molecular-weight proteins similar in size to EVs, then the pooled fractions are concentrated by filtration and layered on top of a high-performance density gradient to separate the low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL) from the EVs. Here we studied the effect of the slope of the gradient curve on the separation of lipoproteins from EVs aiming to obtain samples more specifically enriched in EVs. MATERIALS AND METHODS. Blood samples were collected into 3.2% (w/v) sodium citrate tubes from fasting donors. Platelet-depleted plasma (PFP) was obtained by centrifugation of the blood at 2,500g for 15 min at room temperature two times. Then PFP was aliquoted into 2 mL tubes snap freeze with liquid nitrogen and stored at −80 °C until use. An in house-made SEC column was prepared using 10 mL of sepharose CL-2B (GE Healthcare) packed in a 15 mL Chromabond column with a polyethylene (PE) frit. 2 ml of plasma were loaded on top of the column, and 1 ml fractions were collected, fraction 3 and 4 were selected as the EV enriched fractions with less contaminants. The sample was concentrated using a 100 kDa molecular weight cut-off (MWCO) filter to a final volume of 200 µL. Then sample was loaded on the top of a density gradient prepared using three different sets of iodixanol cushions in a 12 mL tube to compare the separation of the lipoproteins from the EVs. Samples were centrifuged in SW41 Ti rotor for 18 h at 100,000g, at 4 C. A total of 12 fractions of 1 mL were collected. RESULTS. Protein concentration was assessed using Qubit 2.0 fluorometer, the protein total quantity recovered from the pool samples of SEC fractions was on average of 1.88 mg, after the concentration step using the 100 kDa MWCO the protein quantity obtained was on average .40 mg. The protein quantity of the 12 fractions collected using the iodixanol cushions with a percentage ranging from 5% to 40% showed a peak in protein quantity in fractions 2 and 3 while fractions 5 to 10 showed similar protein distribution and less protein was observed in fractions 11 and 12. Density determination was assessed using a spectrophotometer at 244 nm in samples diluted 5,000 fold, fractions with the buoyant density similar to lipoproteins were from fractions 1 to 5 while the fractions with similar buoyant to EVs appeared in fractions ranging from 6 to 9 using iodixanol cushions ranging from 5% to 40%. Western blot showed a positive signal to Alix from fractions 5 to 10 with a higher signal on fractions 9 and 10, CD81 showed a positive signal from fractions 4 to 10, Flotillin-1 and CD63 showed positive signal on fractions 3 to 5, the markers to assess the presence of lipoproteins ApoB gave signal from fractions 2 to 5 with higher signal in fractions 2 and 3 while ApoA showed signal in fractions 2 and 3; and albumin showed signal from fraction 1 to 5 with a higher signal on fraction 2 and 3. Results show that subpopulation of Alix-positive EVs has different densities than CD81 and CD63-positive EVs.

Characterization of mesenchymal stromal cell-derived extracellular vesicles isolated by two scalable methods

Ricardo Malvicini1,2,3, Giada De Lazzari1,2,4, Anna Maria Tolomeo1,2,4, Michela

Pozzobon4,5, Natalia Pacienza3, Maurizio Muraca1,2,4, Gustavo Yannarelli3.

1 Laboratory of Extracellular Vesicles as Therapeutic Tools, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy

2 L.i.f.e.L.a.b. Program, Consorzio per la Ricerca Sanitaria (CORIS), Veneto Region, Padua, Italy.

3 Laboratorio de Regulación Génica y Células Madre, Instituto de medicina traslacional, trasplante y bioingenieria (IMeTTyB-CONICET), Buenos Aires, Argentina.

4 Department of Women’s and Children’s Health, University of Padova, Padua, Italy 5 Laboratory of Stem Cells and Regenerative Medicine, Fondazione Istituto di Ricerca

Pediatrica Città della Speranza, Padua, Italy

Mesenchymal stromal cells (MSCs) are a well-characterized population of multipotent progenitor cells that has shown to exert important immunomodulatory, anti-oxidant and anti-inflammatory functions in numerous in vivo studies and clinical trials. Nowadays, it is well known that several beneficial effects of MSCs are mediated by paracrine factors, mainly by extracellular vesicles (EV), which can be isolated and purified from conditioned culture media. Even though there are numerous methods to isolate these vesicles, few of them are compatible with clinical applications, as they are not scalable. Moreover, as each method isolates EV based on different physicochemical properties, it results in the isolation of different EV subpopulations with different characteristics and a different degree of contaminants. In the present work, we compare two isolation methods that are feasibly scalable and suitable for clinical production of EV, i.e. ion exchange chromatography (IEX) and ultrafiltration (UF) and evaluate their impact on the composition and the activity of the EV. We assessed both nanoparticle size and distribution by NTA and tRPS and morphology by TEM following isolation from conditioned cultured medium by IEX and UF. We also measured protein concentration (BCA assay), lipid concentration (sulfo-phospho-vainillin assay) and RNA concentration (absorbance at 260nm) and immunophenotyped both populations by flow cytometry (Macsplex assay). Moreover, immunomodulatory activity was tested with a macrophage polarization assay and with a T cell stimulation assay. We found that IEX and UF yielded a comparable amount of total particles (7.43E+10 vs 7.04E+10) with similar size and distribution, (mode 96nm vs 97nm and 94nm vs 101nm by tRPS and NTA, respectively). In addition, a similar amount of protein (559±126µg vs 479±83µg) and lipids (16±0.21µg vs 18±0.29ug) was obtained. However, IEX yielded a 10-fold higher RNA quantity (13.4 vs 1.54 µg) with respect to UF. MSC-EVs isolated from IEX and UF were positive for the exosomal markers CD9, CD63 and CD81, and showed a very similar expression pattern of surface markers. Finally, both populations showed a similar anti-inflammatory activity in vitro as they prevented the acquisition of the M1 phenotype in LPS stimulated macrophages an inhibited the acquisition of the activation markers CD69 and CD25 on T cells. In conclusion, MSC-EV isolated by the two above-mentioned methods displayed similar physicochemical, phenotypic and functional characteristics. Further studies are ongoing to characterize the higher RNA content in IEX isolates. Funding: Supported by Exo Biologics, Niel, Belgium.

Stability of haemoglobin-filled erythrocyte vesicles as assessed by dynamic and static light scattering

D. Božič1,2, M. Jeran1,2, M. Pajnič1, L. Pađen1, K. Kogej3, A. Iglič2,4, V. Kralj-Iglič1

1University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia

2University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana, Slovenia 3University of Ljubljana, Faculty of Chemistry and Chemical Technology, Chair of Physical

Chemistry, Ljubljana, Slovenia 4University of Ljubljana, Faculty of Medicine, Laboratory of Clinical Biophysics, Ljubljana, Slovenia

Natural and synthetic nanovesicles present an interesting form of molecular assembly with many already established applications (drug delivery systems) as also many still developing uses (diagnostics, theranostics). To assure validity of analysis conducted on vesicular systems, as also safety and effectivity when used as medicine formulations, evaluation of vesicle integrity prior to their use may be of a fundamental value. We aimed to evaluate the potential of erythrocyte vesicles to be used as an in-house reference, and suitability of light scattering techniques to be used for integrity-check-up as they are non-destructive to the sample, do not require any chemical changes of the sample and are relatively less time consuming compared to other methods or their combinations that might be employed for this purpose. In the present study we analysed haemoglobin-filled vesicles (hbEVs) obtained from in vitro aged erythrocyte suspension. Vesicles were isolated by differential centrifugation, and subjected to pH and osmolarity changes (pH 4-10, and osmolarity between 0.05-1.00 Osm/L), heating (15-60 °C) and gradual solubilization by detergent Triton X-100 (added at increasing concentration until complete solubilization). Samples were analysed by dynamic and static light scattering at multiple angles (30-150 °) using a red laser (λ = 630 nm). We observed only minimal changes in size distribution of particles in treated samples, while intensity of scattered light dropped substantially in samples with pH below 6 and above 8, in hyperosmolar media above 0.45 Osm/L, and at temperatures above 45 °C. hbEVs in 0.30 and 0.05 osmolar media showed the same characteristics of static light scattering, corresponding to spherical particles with Gaussian distribution of mass. We conclude that hbEVs are fairly stable in hypotonic media (assessed down to 0.05 Osm/L), and in 0.30 Osm/L phosphate-buffer-saline-citrate media in the non-buffered range of pH 6-8, and that they start to degrade at 45 °C. Short-term changes in topology of vesicles detected in diluted samples used in this study were minor and events such as leakage of contents or fusion of single vesicles into larger ones were not observed. It would need to be tested if subtler changes could be detected with a shorter wave-length light source. Our results demonstrate that the mean hydrodynamic radius itself may be preserved even at severe loss of sample integrity, it is therefore suggested that in order to assess sample preservation by light scattering techniques, size distribution should always be considered together with scattering intensity, and compared to the initial sample. Supported by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 801338 (VES4US project) and Slovenian Research Agency (ARRS. Research core foundlings No. P2-0232, P3-0388, P1-0201, and projects No. L3-2621)

Formation of colloidal small particles during processing of samples

V. Kralj-Iglič1, D. Božič1,2, M. Jeran1,2, A. Iglič 2,3

1University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia

2University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana, Slovenia 3University of Ljubljana, Faculty of Medicine, Laboratory of Clinical Biophysics, Ljubljana, Slovenia

The acknowledged view on the submicron-sized particles in biological samples is that there are three main types of them: apoptotic bodies, microvesicles and exosomes. However, once in the sample that is being processed and analyzed, the origin of a particular particle is not decisive. This is the consequence of the limitations of the existing methods for particle harvesting. The understanding of the formation mechanisms is still inadequate. We have studied budding and vesiculation of biological membranes theoretically and experimentally. In our experimental studies we isolated submicron-sized particles from different biological samples, including blood and suspensions of giant phospholipid vesicles. We found that besides the above mechanisms, a large pool of submicron-sized particles in the isolates from plasma is formed during the processing of samples. While erythrocytes shed vesicles in the final stage of the membrane budding, the platelets undergo fragmentation in the shear stress. Such particles have transient identity and shapes of minimal membrane free energy pertaining to colloidal systems. Formation of colloidal particles can be manipulated by changing the parameters of processing which is of advantage for production of therapeutic preparations from biological samples. We present the review of the studies leading to these conclusions. Supported by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 801338 (VES4US project) and Slovenian Research Agency (ARRS. Research core foundlings No. P2-0232, P3-0388, P1-0201, and projects No. L3-2621)

Evolving reversible immunocapturing by membrane sensing peptides: versatile platforms for scalable extracellular vesicles

isolation and analysis

M. Cretich1, R. Frigerio1, A. Musicò1, A. Strada1, G. Bergamaschi1, A. Mussida1, M. Chiari1 and A. Gori1

1Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”

(SCITEC), Milano, Italy

The use of Extracellular Vesicles (EVs) as either therapeutic agents or as a source of diagnostic biomarkers strictly relies on the high yield isolation and recovery of pure, homogeneous EV sub-populations from complex biological matrices. Recently, a novel immune-capture and release approach was presented based on DNA-directed immobilization of antiCD63 and EV releasing by exploiting the endonuclease activity of DNAse I [1]. In this scenario, a particularly arduous task is to design robust, scalable and economically viable affinity capturing processes to achieve such goal beyond the analytical scale. Versatile, and cost-effective methodologies to enable scalable EV isolation from bio-samples in high purity and from laboratory analysis (μL to mL) to the manufacturing (>1L) scale, are still necessarily demanded to open new perspectives in EV-based therapeutics and diagnostics. Our solution to these limitations is a paradigm shift from antibodies to peptides as an alternative class of affinity ligands characterized by high efficiency of EV capturing. Key figures of merit for peptide ligands in contrast to antibodies are the ease of chemical manipulation, offering virtually unlimited possibilities for system integration, batch to batch reproducibility, stability, and the low costs of preparation with well-known possibilities for scale-up of production. Here we introduce the use of membrane-sensing peptides (MSP) [2] as novel ligands for the size-selective capturing and release of small EVs, unbiased by differential surface protein expression and using membrane as universal EV marker. Thanks to its versatile and modular nature, system integration of our technology result in a diverse range of tools for EV analysis and recovery, ranging from microchips to magnetic beads and from lateral flow assays to tangential flow filtration membranes. Such platform is used for EV isolation on different scales, opening new scenarios for both diagnostics and therapeutics. Supported by European Union’s Horizon 2020 research and innovation programme under grant agreement No. 951768 – Project MARVEL References [1] Brambilla, D., Sola, L., Ferretti, A.M., Chiodi, E., Zarovni, N., Fortunato, D., Criscuoli, M., Dolo, V., Giusti, I., Murdica, V., Kluszczyńska, K., Czernek, L., Düchler, M., Vago, R., Chiari, M., 2021. EV Separation: Release of Intact Extracellular Vesicles Immunocaptured on Magnetic Particles. Anal. Chem. 93, 5476–5483. [2] Gori, A., Romanato, A., Bergamaschi, G., Strada, A., Gagni, P., Frigerio, R., Brambilla, D., Vago, R., Galbiati, S., Picciolini, S., Bedoni, M., Daaboul, G.G., Chiari, M., Cretich, M., 2020. Membrane‐binding peptides for extracellular vesicles on‐chip analysis. J. Extracell. Vesicles 9, 1751428.

A novel cost-effective biosensing platform based on silicon nanowires for liquid biopsy in ovarian cancer

Leonardi AA, Battaglia R, Morganti D, Lo Faro MJ, Fazio B, Caponnetto A, Ferrara C, De Pascali C, Francioso L, Palazzo G, Mallardi A, Purrello M, Priolo F, Musumeci P, Irrera A, Di Pietro C

The contribution of small Extracellular Vesicles (sEVs) in human pathology has been widely demonstrated, and sEVs are recently emerging as strategic biomarkers of cancer, neurodegenerative, cardiovascular diseases, and as potential and important targets for therapeutic intervention. However, their use for real medical application is still limited due to the selectivity and sensitivity limits of the commonly applied approaches.

Novel sensing solutions based on nanomaterials are arising as strategic tools able to surpass the nowadays traditional sensor limits. Among them, silicon nanowires (Si NWs) realized with a cost-effective industrially compatible approach arise as a perfect candidate for sEVs detection [1]. Our group recently demonstrated an industrially compatible approach for the realization of room temperature (RT) luminescent Si NWs that can be used as transducer for high-sensitive and high-selective protein and pathogen genome biosensors [2,3].

In this talk, we report the realization of a selective sensor able to isolate, concentrate quantify and analyze sEVs CD81+, from minimal volumes of biofluid and its promising application in early diagnosis, monitoring disease progression and treatment efficacy in ovarian cancer [4,5].

References

[1] Fazio B, Artoni P, Iatì MA, D'andrea C, Lo Faro MJ, Del Sorbo S, Pirotta S, Gucciardi PG, Musumeci P, Vasi CS, Saija R, Galli M, Priolo F, Irrera A. Strongly enhanced light trapping in a two-dimensional silicon nanowire random fractal array. Light Sci Appl 2016, 5(4):e16062-e16062. doi:10.1038/lsa.2016.62

[2] Irrera A, Leonardi AA, Di Franco C, Lo Faro MJ, Palazzo G, D’andrea C, Manoli K, Franzò G, Musumeci P, Fazio B, Torsi L, Priolo F. New Generation of Ultrasensitive Label-Free Optical Si Nanowire-Based Biosensors. ACS Photonics 2018, 5(2):471-479. doi:10.1021/acsphotonics.7b00983

[3] Leonardi AA, Lo Faro MJ, Petralia S, Fazio B, Musumeci P, Conoci S, Irrera A, Priolo F. Ultrasensitive Label- and PCR-Free Genome Detection Based on Cooperative Hybridization of Silicon Nanowires Optical Biosensors. ACS Sensors 2018, 3(9):1690-1697. doi:10.1021/acssensors.8b00422

[4] Leonardi AA, Battaglia R, Morganti D, Lo Faro MJ, Fazio B, De Pascali C, Francioso L, Palazzo G, Mallardi A, Purrello M, Priolo F, Musumeci P, Di Pietro C, Irrera A. A novel silicon platform for selective isolation, quantification, and molecular analysis of small extracellular vesicles. Int J. Nanomed. 2021, just accepted

[5] Chang, L; Ni, J; Zhu, Y; Pang, B; Graham, P; Zhang, H; Li, Y Liquid biopsy in ovarian cancer: Recent advances in circulating extracellular vesicle detection for early diagnosis and monitoring progression. Theranostics 2019, Vol. 9, Issue 14. DOI:10.7150/thno.34692

Extracellular vesicles (EVs) participate to physiological and pathological processes, thanks to the ability to transfer their cargo and signaling molecules from cell to cell. Nowadays, OMICs are powerful and helpful tool to dissect EV functionality and to identify new biomarkers of various diseases. Therefore, new insights into size, protein and lipid composition may help in characterizing functionality of EVs and may also infer with their fast development as delivery tools. Unfortunately, despite latest size-based classifications, which divide EVs in small (50-80nm) or large (80-120nm) exosomes, microvesicles (<1000nm) and the new smallest (<50nm) population (exomeres), overlapping of different EV populations and unproper separation methods impair the comprehension of their biological roles. To overcome this problem, we set up a reproducible sequential ultracentrifugation method for a size-based separation of different EV populations derived from a metastatic melanoma cell line (LM-16), taking into account physical and dynamic parameters (EVs, medium, centrifuge rotor and dimensions), as described by an algorithm developed by Livshits et al1. We characterized each population by size with transmission electron microscopy (TEM) and dynamic light scattering (Zetasizer), fatty acid profile by gas chromatography (GLC) and protein content by mass spectrometry. Ingenuity Pathway Analysis (IPA) was then utilized to describe signaling pathways as related to EV biological functions. TEM analysis documented the existence of five size-based different EV populations. Importantly, when analyzed by Zetasizer, the fractions not only confirmed the differences in size but also reflected the theoretical sizes calculated by the algorithm. GLC analysis performed on parental cells and on the same fractions revealed a gradual and continuous percentage increase in saturated fatty acids from cells to smaller EVs (33.61%±0.21 to 64.79%±9.47), suggesting different origin and membrane rigidity among fractions. Descriptive proteomics identified a total of 2003 proteins differentially distributed among the five EV populations (n=1654, 1621, 1079, 819 and 697, ranging from bigger to smaller ones). Interestingly, some proteins were unique for each fraction (252, 141, 14, 17, 97 respectively) and may be considered as biomarkers. IPA analysis demonstrated that some signaling pathways were common to all or at least to part of the fractions, but also that others were enriched in specific populations, suggesting possible typical roles of these fractions, at least in melanoma cell functions. In conclusion, melanoma-released EVs include vesicles of different size, fatty acid and protein composition. These differences may translate into distinct behaviors and functions in biological fluids and may help to define the role of specific EV populations in physiological and pathological processes. Finally, these results pave the road to new pharmacological treatments to modulate EV functions or to use EVs as pharmaceutical tools. Supported by EXTRALIPO “New lipid-oriented pharmacological and chemical approaches to discriminate and unravel extracellular vesicles biological functions” Bando SEED – PSR 2019. References [1] Livshits et al. 2015 “Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol,” Sci. Rep., 5 (14); 1–14

Size, lipid and protein characterization of different populations of extracellular vesicles obtained by sequential centrifugation from a

metastatic melanoma cell line

F. Accattatis1, S. Mazza1, A. Granata1, E. Vergani2, M. Rodolfo2, S. Bellosta1, A. Corsini1, L. Arnaboldi1

1Dipartimento di Scienze Farmacologiche e Biomolecolari DISFeB, Università degli Studi di Milano, Milan, Italy

2Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy

Extracellular vesicles from microalgae: biophysical/biomechanical characterisation

Samuele Raccosta1,2 , Angela Paterna1,2, Estella Rao1,2, Giorgia Adamo1,3, Sabrina Picciotto1,3,

Daniele P. Romancino1,3, Antonella Cusimano1,3, Rosina Noto1,2, Rita Carrotta1,2, Vincenzo Martorana2, Fabio Librizzi1,2, Nicolas Touzet4, Antonella Bongiovanni1,3 and Mauro Manno1,2

1 Cell-Tech HUB, National Research Council of Italy (CNR), Palermo 90146, Italy.

2 Institute of Biophysics, National Research Council of Italy (CNR), Palermo 90146, Italy. 3 Institute for Research and Biomedical Innovation, National Research Council of Italy (CNR),

Palermo 90146, Italy. 4 Centre for Environmental Research Innovation and Sustainability, Institute of Technology Sligo,

Sligo, Ireland. Extracellular vesicles (EVs) are nanometer-sized particles “made by cells for cells” to shuttle lipids, proteins and nucleic acids, thus acting as potent signal transducers and intercellular and inter-organismal communicators. We recently identified nanoalgosomes, that are EVs secreted by microalgae, a sustainable natural source [1,2]. Here, we use an integrated biochemical-biophysical approach for an extensive characterisation of their structural properties. Specifically, combined experiments of immunoblotting analysis for commonly used EV-biomarkers (IB), Atomic Force Microscopy (AFM), Small-Angle X-ray Scattering (SAXS) and Small-Angle Neutron Scattering (SANS), Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA), along with a dedicated analysis, are used to cope with the heterogeneity of such complex systems. Our effort is also dedicated to the development of Standard Operating Procedures which may guide the characterisation of size distributions in EV sample and meet the minimal requirements for studies of EVs (MISEV) [3]. In this frame, we have used AFM to assess the biomechanical properties of EVs, along with their size distribution. Our experimental strategy consists in deposit EVs on functionalised substrates with different affinity for specific EVs components (proteins, sugars, lipids), so to perform AFM imaging on selected EVs subpopulations. Our study provides the means for a comprehensive structural characterization of EVs, which is expected to be crucial in the design of engineered vesicles to be employed in different fields, such as nanomedicine, nutraceutics and cosmetics. (This work has been developed under the projects VES4US and BOW, which have received funding from the European Union’s Horizon 2020 research and innovation programme, under grant agreements No 801338 and 952183, respectively). [1] Adamo, et al. Journal of Extracellular Vesicles 10, e12081, 2021. doi: 10.1002/jev2.12081 [2] Picciotto, et al. Biomaterials Science 9, 2917-2930, 2021. doi: 10.1039/d0bm01696a [3] Théry, et al. J. Extracellular Vesicles. 7, 1, 2018. doi: 10.1080/20013078.2018.1535750

Characterization of TF positive small extracellular vesicles by microarrays and flow cytometry approach

Marta Brambilla1, Roberto Frigerio2, Paola Canzano1, Marina Cretich2, Alessia Becchetti1, Daniela

Trabattoni1, Marina Camera1,3

1Centro Cardiologico Monzino, IRCCS, 2National Research Council of Italy (SCITEC-CNR), 3Dept. of Pharmaceutical Sciences, Università degli Studi di Milano

The plasma procoagulant potential is primarily driven by circulating microvesicles (MVs). Their thrombin generation capacity rely on the exposure of anionic phospholipids on their membrane. The presence of tissue factor (TF), the main activator of the coagulation cascade, on subsets of MVs also significantly contributes to their thrombin generation capacity. This procoagulant potential increases in different pathologic states, including cancer and cardiovascular diseases. However, whether it is hold by large MVs only or rather it is also supported by small vesicles (sEV) is still unknown mostly due to methodological issues. Indeed, while MVs are quite easily analyzed using straight forward flow cytometry approaches even in whole blood, the characterization of sEV is hampered from one side by instrument resolution limits and, from the other side, by the complex composition of blood leading to the co-isolation of lipoproteins and protein aggregates in the size range or density of sEV. Thus, our goal was to characterize TF expression in sEV by using two complementary immunological-based approaches, i.e. flow cytometry and a multiparametric microarray platform that digitally count, size and phenotype sEV by fluorescence immuno-staining. This combined strategy was set up analyzing plasma from healthy subjects and then applied to characterize sEVs isolated from plasma of patients with patent foramen ovale (PFO; n=8), a cardiac defect often associated with increased platelet activation and MV release, before (T0) and 6 months after (T1) PFO closure. In order to overcome the resolution limit of flow cytometry we took advantage from the use of the MACSPlex Exosome kit based on the use of antibody-conjugated beads to capture exosomes. Results indicated that TFpos sEV were present in plasma from HS. PFO patients had a 1.5 fold higher number of TFpos sEV compared to HS with a trend toward reduction to physiological levels 6 months after PFO closure. By contrast, no difference among these groups were observed when the mean intensity fluorescence relative to sEV-associated TF expression was analyzed. Next, to complement this analysis and to exclude aspecific binding, a more sensitive and selective microarray based approach was applied. To this aim, we used chips spotted with a membrane sensing peptide able to capture sEV based on membrane curvature, thus unbiased, unlike flow cytometry analysis, by non EV-structures. Chips were immune-stained with anti-CD9 and anti-TF antibodies, labelled with Cy3 and Cy5 respectively. Analysis was focused on particles sized 50-130 nm with a positive co-localization of CD9 and TF. Results showed in all tested patients a statistically significant (p=0.0013) decrease from T0 to T1 in the number of CD9/TFpos sEV, becoming similar to those measured in HS. Overall, by using two different approaches, i.e. analysis by flow cytometry and by a multiparametric microarray platform, we provided evidence that in human plasma TF is associated to even the smallest subpopulation of EVs. Further studies will be required to define sEV cell origin and whether TFpos sEV are functionally active and thus able to support thrombin generation.

Comparison between Bronchoalveolar Lavage (BAL) and blood-derived microvesicles in lung diseases

M. Tinè1, E. Bazzan1, C. M. Radu2, D. Biondini1, M. Damin1, U. Semenzato1, A. Casara1, P. Simioni3, M. G. Cosio4, M. Saetta1

1Department of Cardiac, Thoracic, Vascular Sciences and Public Health, University of Padova, Padova, Italy 2Department of Women’s and Children’s Health, University of Padova, Padova, Italy 3Department of Medicine, University of Padova, Padova, Italy 4Meakins-Christie Laboratories, Respiratory Division, McGill University, Montreal, Canada Background Cell derived extracellular vesicles (EVs) found in the circulation and body fluids contain biomolecules that could be used as biomarkers for lung and other diseases. EVs from BAL might be more informative of lung abnormalities than EVs from blood, where information might be diluted. Aim To compare EVs characteristics in BAL and blood in smokers with and without COPD. Methods Same day BAL and blood samples were obtained in 5 nonsmokers, 11 smokers w/o COPD and 9 with COPD (FEV1: 63±3% pred). After differential centrifugation, microvesicles (MV) (200-500 nm diameter) were identified by flow cytometry and labelled with cell-type specific antigens: CD14 for macrophages-derived MV (AMCD14), EPCAM for epithelial-derived MV (EpiEPCAM), CD146 for endothelial-derived MV (EndCD146) and CD62E for activated-endothelial-derived MV (ActivendCD62E). Results In BAL, AMCD14-MV were increased in smokers compared to nonsmokers [383(56-566) vs 172(115-582) events/μl; p=0.04] and further increased in COPD patients [618(223-887)] compared to both smokers (p=0.04) and nonsmokers (p=0.019). EpiEPCAM-MV were increased in smokers [759(47-2855) events/μl, p=0.01] and in COPD [1054(194- 11491), p=0.001] when compared to nonsmokers [15(0-68)]. EndCD146 and ActivendCD62E-MV were similar in the three groups. In plasma, the volumes of all MVs (events/μl) where markedly increased when compared to BAL, regardless of the cell origin, but were similar in the nonsmokers, smokers and COPD groups. Conclusions In BAL significant differences in epithelial and macrophage-derived MVs can be clearly detected between nonsmokers, smokers and COPD, while these differences were not found in plasma. This suggests that BAL is a better media than blood to study MVs in lung diseases.

Proteins associated with cancer risk are delivered into plasma exosomes early after extracorporeal circulation in cardiac surgery patients without a

history of malignancies V. Casieri1, A. Carrozzo1,2, D. Di Silvestre3, A. Dushpanova1, P. Mauri3, V. Lionetti1,4

1Institute of Life Sciences, Scuola Superiore Sant’Anna, Pisa, Italy; 2Department of Cardiac Surgery, ICLAS, GVM Care & Research, Rapallo, Italy;

3 Proteomics and Metabolomic Lab, ITB-CNR, Segrate, Italy; 4 UOS Anesthesiology and Intensive Care, Fondazione Toscana G. Monasterio, Pisa/Massa, Italy.

Background: Emerging evidence indicates that some patients without a history of malignancies undergoing to on-pump cardiac surgery may develop cancer. Although biological mechanisms remain unclear, it is conceivable that postoperative cancer susceptibility is related to soluble mediators released during extracorporeal circulation (ECC). Plasma exosomes (pEXOs) are the smallest extracellular nanovesicles that emerge as critical mediators of cell-to-cell communication in cancer. We analyzed the protein cargo of pEXOs released early after ECC in order to detect established cancer-related factors. Methods: We have performed mass spectrometry-based proteomics analysis of the cargo of pEXOs isolated from adult cardiac patients (NYHA II-III) who underwent minimally invasive coronary artery bypass (CABG; n=15) or heart valve surgery (mitral valve repair, n=15; aortic valve replacement, HVS; n=15). Exosomes were isolated at induction of anesthesia (T0) and at 3 hours (T1) after aortic unclamping. Results: pEXOs levels increased at T1 (P < 0.01) in all groups. A total of 340 exosomal proteins were identified and the analysis of pEXOs at T1 vs ones at T0 of each type of on-pump cardiac surgery demonstrated changes in protein cargo. In HVS-derived T1-pEXOs, in particular, we detected runt-related transcription factor 3 (RUNX3) and keretin 6B (KRT6B) which are proteins involved in the development of a variety of cancers. In CABG-derived T1-pEXO, moreover, we found jagged-2 (JAG2), synaptonemal complex protein 3 (SYCP3) and thousand and one kinase 3 (TAOK3) that are proteins involved in tumorigenesis. Conclusion We demonstrated that pEXOs circulating at 3 hours after aortic unclamping deliver specific proteins involved in tumorigenesis, which are known to confer a higher risk of cancer regardless of the type of minimally invasive on-pump cardiac surgery.

Emerging role of urinary and serum extracellular vesicles to identify biomarkers in children with immune-related nephrotic

syndrome. G. Cricri’1,2, L. Bellucci1, S. Bruno3, C. Tamburello4, W. Morello4, G. Montini 1,2,4 and F.

Collino1,2

1 Laboratory of Translational Research in Paediatric Nephro-urology, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy.

2 Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy. 3Department of Medical Sciences, University of Turin, Turin, Italy.

4 Pediatric Nephrology, Dialysis and Transplant Unit, Fondazione Ca’ Granda IRCCS, Policlinico di Milano, 20122 Milan, Italy.

IDIOPATHIC NEPHROTIC SYNDROME (NS) IS A GLOMERULAR DISEASE AFFECTING PAEDIATRIC PATIENTS THAT USUALLY PRESENT OEDEMA, PROTEINURIA, HYPOALBUMINEMIA [1]. PATIENTS NOT RESPONSIVE TO STANDARD CORTICOSTEROID TREATMENTS MAY PROGRESS TO END-STAGE RENAL DISEASE, REQUIRING DIALYSIS OR KIDNEY TRANSPLANT. PROGRESSIVE IMPROVEMENTS IN THE EFFICACY OF IMMUNOSUPPRESSIVE AGENTS IN COMBINATION WITH STEROIDS INCREASE THE RESPONSE TO THE TREATMENT IN OVER NINETY PERCENT OF PATIENTS (STEROID-SENSITIVE), WHILE TEN PERCENT ARE STILL RESISTANT (STEROID-RESISTANT). STEROID-RESPONSIVE PATIENTS MAY EXPERIENCE MULTIPLE RELAPSES AFTER THE INITIAL THERAPY (STEROID-DEPENDENT) [1]. THE MAIN CAUSE OF CHILDHOOD NS IS STILL NOT FULLY UNDERSTOOD. IT HAS BEEN PROPOSED THAT CHILDREN ARE MORE LIKELY TO DEVELOP NS IF THEY PRESENT AN OVERACTIVATION OF THE IMMUNE SYSTEM [2]. UNDERSTANDING THE IMMUNOLOGICAL CONTRIBUTION IN NS APPEARANCE MAY PROVIDE NEW THERAPEUTIC TOOLS TO POTENTIATE CONVENTIONAL DRUG EFFECTS OR TO CREATE PERSONALIZED THERAPY. BIOFLUID-DERIVED EVS CAN FUNCTION AS CARRIERS OF PREDICTIVE BIOMARKERS FOR THE MONITORING OF KIDNEY DAMAGE AND IMMUNE ACTIVATION IN NS CHILDREN.

TO INVESTIGATE THE MOLECULAR PROFILE OF URINARY AND SERUM EVS (UEVS AND SEVS), QUANTITATIVE/QUALITATIVE ANALYSES OF EVS USING NTA AND MACSPLEX WERE PERFORMED. THE NTA ANALYSIS DEMONSTRATED THAT UEVS AND SEVS WERE HETEROGENEOUS IN SIZE, WITH AN ENRICHMENT OF 100 NM PARTICLES. IN ALL THE NS SUBGROUPS, PHENOTYPING OF UEVS CONFIRMED THEIR IDENTITY AS EXOSOMES (CD63, CD81, CD9). INTERESTINGLY, AT THE DISEASE ONSET, THEY SHOWED A STRONGER EXPRESSION OF EPITHELIAL (CD326, CD133, CD24) AND LYMPHOCYTIC (B CELLS AND T CELLS DERIVATION) MARKERS. SEVS WERE ENRICHED IN SURFACE EXPRESSION OF THE EXTRACELLULAR MATRIX REGULATOR CD29 AND ALSO OF CD41B AND CD40, SUPPORTING THE ONGOING PLATELET AND B-CELL ACTIVATION IN NS [3]. NEXT, WE DEEPLY CHARACTERIZED THE IMMUNE CELL PROFILE OF NS PATIENTS IN RELAPSE OR REMISSION, FOCUSING ON THE FOLLOWING B-CELL (NAÏVE/SWITCHED MEMORY AND TRANSITIONAL B CELLS) AND T-CELL COMPARTMENTS (TH1 AND TH17 T-HELPER, T-REGULATORY, CD8-CYTOTOXIC AND T MEMORY CELLS).

OVERALL, DATA ON EV CONTENT, IMMUNE CELL PHENOTYPE AND CLINICAL DIAGNOSIS WILL PROVIDE A SIGNATURE PREDICTING KIDNEY DAMAGE AND ITS CORRELATION WITH B- AND T- DYSFUNCTIONS IN IMMUNE-RELATED NS CHILDREN.

References

[1] Damien G Noone, Kazumoto Iijima, Rulan Parekh. Idiopathic nephrotic syndrome in children. Lancet 2018 Jul (London, England); 392(10141): 61–74.

[2] Wagner de Fátima Pereira, Gustavo Eustáquio Alvim Brito-Melo, Fábio Tadeu Lourenço Guimarães, Thiago Guimarães Rosa Carvalho, Elvis Cueva Mateo, Ana Cristina Simões e Silva. The role of the immune system in idiopathic nephrotic syndrome: a review of clinical and experimental studies. Inflamm Res 2014 Jan; 63(1):1-12.

[3] Ula Štok, Elizabeta Blokar, Metka Lenassi, Marija Holcar, Mojca Frank-Bertoncelj, Andreja Erman, Nataša Resnik, Snežna Sodin-Šemrl, Saša Čučnik, Katja Perdan Pirkmajer, Aleš Ambrožič, and Polona Žigon. Characterization of Plasma-Derived Small Extracellular Vesicles Indicates Ongoing Endothelial and Platelet Activation in Patients with Thrombotic Antiphospholipid Syndrome. Cells. 2020 May; 9(5): 1211.

Extracellular vesicle miRNAs track cognitive symptoms in multiple sclerosis

patients.

Maria Teresa Golia1, Federica Scaroni1, Visconte Caterina2, Martina Gabrielli1, Maria Serpente2, Marijn

Huiskamp3, Elio Scarpini2, Daniela Galimberti2, Brigit A. de Jong3, Chiara Fenoglio2, Claudia Verderio1

1CNR Institute of Neuroscience, via Vanvitelli 32, 20129 Milano, Italy. 2Fondazione IRCCS Ca' Granda

Ospedale Maggiore Policlinico, Neurodegenerative Diseases Unit, Milan, Italy. 3Department of Neurology,

Amsterdam UMC, location VUmc, the Netherlands.

ALTHOUGH MULTIPLE SCLEROSIS (MS) HAS BEEN LONG CONSIDERED A WHITE MATTER

DISORDER WITH A SECONDARY NEURODEGENERATIVE COMPONENT, CURRENT KNOWLEDGE

INDICATES THAT SYNAPTIC LOSS AND AXONAL DEGENERATION ARE EARLY EVENTS IN THE

DISEASE THAT COULD OCCUR INDEPENDENTLY OF DEMYELINATION. BETWEEN 40% AND 70%

OF MS PATIENTS SUFFER SYMPTOMS ARISING FROM NEURONAL DERANGEMENT, INCLUDING

COGNITIVE DEFICITS THAT STRONGLY AFFECT THEIR QUALITY OF LIFE. HOWEVER, NO

COGNITIVE MS BIOMARKERS ARE CURRENTLY AVAILABLE TO MONITOR PATIENT FOLLOW-UP

AND THERAPEUTIC TREATMENT EFFICACY. EXTRACELLULAR VESICLES (EVs) CONTAIN

MARKERS OF PARENTAL CELLS AND ARE ABLE TO PASS FROM THE BRAIN INTO PERIPHERAL

BLOOD, REPRESENTING A VALUABLE SOURCE OF DISEASE BIOMARKERS. AMONG EV

CARGOES, microRNAs (miRNAs) IN MICROGLIAL EVs HAVE BEEN REPORTED TO INDUCE

SYNAPTIC ALTERATIONS IN THE MOUSE BRAIN AND MAY BE RESPONSIBLE FOR THE DEFECTS

IN SYNAPTIC FUNCTION AND COGNITION IN MS PATIENTS, THUS REPRESENTING GOOD

CANDIDATES AS COGNITIVE BIOMARKERS IN MS.

IN THIS STUDY, WE ISOLATED TOTAL EVs AND MYELOID IB4+ EVs (RELEASED FROM

MICROGLIA/PERIPHERAL MACROPHAGES) FROM THE PLASMA OF COGNITIVELY PRESERVED

(CP) AND COGNITIVE IMPAIRED (CI) MS PATIENTS (10 PER GROUPS), AND PROBED THEM FOR A

SET OF 13 miRNAs WITH PREDICTED SYNAPTIC TARGETS IN HUMANS. COGNITIVE FUNCTIONS

WERE ASSESSED BY A BATTERY OF 10 NEUROPSYCHOLOGICAL TESTS (STROOP, SDMT, PAST,

BVMT-R, COWAT, LLT, AKTG, D-KEFS, and BADS). PRELIMINARY ANALYSIS BY REAL-TIME PCR

REVEALED NO ALTERATIONS IN TOTAL PLASMA EVs FROM CI vs CP PATIENTS, WHEREAS

HIGHER LEVELS OF miR-150-5p AND LOWER LEVELS OF let-7b WERE DETECTED IN IB4+ EVs

FROM CI PATIENTS COMPARED TO CP PATIENTS. THIS IS THE FIRST STUDY THAT PROVIDES

POSSIBLE BIOMARKERS FOR COGNITIVE DISABILITIES IN MS.

Cigarette smoke extract induces in airway epithelial cells the release of EVs conveying oxidatively modified proteins.

S. Buratta1, E. Chiaradia2, A. Gambelunghe3, L. Urbanelli1, R.M. Pellegrino1, A. Tognoloni2, G.

Cerrotti1, R. Latella1, B. Tancini1, D. Manfredelli2, M. Dell’Omo3, C. Emiliani1

1Department of Chemistry, Biology and Biotechnology, University of Perugia (Italy) 2Department of Veterinary Medicine, University of Perugia (Italy)

3Department of Medicine, University of Perugia (Italy) The aim of the present study is to examine the biochemical and biophysical properties of Extracellular Vesicles (EVs) released by human lung epithelial BEAS-2B cells exposed to cigarette smoke extract (CSE). Many studies demonstrated that CSE induces the release of EVs by different cell types, including airway epithelial cells [1,2], which are the first line of contact with inhaled cigarette smoke. As CSE causes oxidative stress, we evaluated if EVs released under CSE-induced oxidative conditions carry oxidatively modified proteins. In a first series of experiments, we evaluated viability and intracellular levels of ROS and carbonylated proteins in BEAS-2B cells exposed for 24h to increasing concentration of CSE (0.5-10%). Cell viability and ROS levels were assayed by MTT and DCFH-DA, respectively. The levels of protein carbonyl groups, which are most harmful irreversible oxidative modification induced by the oxidative stress, were evaluated by Oxyblot. CSE treatment reduced cell viability and elevated intracellular levels of ROS and carbonylated proteins, in a dose-depend manner. Then, we isolated EVs from culture media of cells incubated in the absence or presence of 1% CSE, a concentration that did not affect cell viability but increased the intracellular levels of ROS and carbonylated proteins. The quality of EVs, isolated by a differential centrifugation protocol [3], was checked by NTA, SEM and Immunoblotting. Biophysical analysis showed a rounded morphology and a diameter of the released EVs generally < 100 nm, characteristic of small vesicles of endosomal origin. Immunoblotting showed the presence in vesicle preparations of EV marker (CD81 and flotillin-1), whereas calnexin (marker of endoplasmic reticulum) and actin were not detected in our EV preparations. Notably, 1 % CSE increased levels of carbonylated proteins both in cells and in released EVs. These results suggest that CSE-treated cells can remove oxidized toxic proteins via EVs in order to reduce their harmful effects and maintain cellular homeostasis. Considering that CSE-treated cells release a higher number of EVs compared to controls [1, 2], these findings indicate that BEAS-2B exposed to CSE release a high amount of oxidatively modified proteins via EVs. The further identification of specific oxidized protein species in CSE-treated cells and EVs will be useful to predict the effects exerted by CSE-EVs on neighboring or distant target cells. The in vitro results of our study could be a starting point for the utilization of circulating EVs as biomarkers for the diagnosis of CSE-induced lung damage and/or cigarette smoke-related diseases, after the confirmation that specific oxidized proteins are present in EVs derived from bronchoalveolar lavage fluid or plasma. References [1] Benedikter, B.J., Volgers, C., van Eijck, P.H., Wouters, E.F.M., Savelkoul, P.H.M., Reynaert, N.L., Haenen, G.R.M.M., Rohde, G.G.U., Weseler, A.R., Stassen, F.R.M., 2017. Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants. Free Radic Biol Med 108, 334-344. [2] Benedikter, B.J., Bouwman, F.G., Heinzmann, A.C.A., Vajen, T., Mariman, E.C., Wouters, E.F.M., Savelkoul, P.H.M., Koenen, R.R., Rohde, G.G.U., van Oerle. R., Spronk. H.M., Stassen, F.R.M., 2019. Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles. J Ext Vesicles 8, 1585163. [3] Kowal, J., Arras, G., Colombo, M., Jouve, M., Morath, J.P., Primdal-Bengtson, B., Dingli, F., Loew, D., Tkach, M., Théry, C., 2016. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Natl Acad Sci USA 113, E968-E977.

Evaluation of small extracellular vesicles microRNA cargo in FTD patients and their interactome

Garofalo M.1,2, Sproviero D.1, Dragoni F.1,2, Corridori E.1,3, Costa A.4,5, Perini G.5, Cotta Ramusino M.5, Crippa V.3, Pansarasa O1, Poletti A.3, Cereda C.1, Gagliardi S.1

1 Genomic and post-Genomic Unit, IRCCS Mondino Foundation, Pavia, Italy 2 Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Pavia, Italy

3Dipartimento di Scienze Farmacologiche e Biomolecolari, Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milano, Italy

4Department of Brain and Behavioral Sciences, University of Pavia, 27100 Pavia, Italy 5Unit of Behavioral Neurology, IRCCS Mondino Foundation, 27100 Pavia, Italy

Role of extracellular vesicles (EVs) in neurodegenerative disorders has been investigated. In particular, EVs content in terms of small RNAs cargo has been evaluated. Fronto-Temporal Dementia (FTD) is characterized by aggregation of proteins (TDP-43 and Tau) in the frontal and temporal lobes with microvacuolation and relevant deregulation of RNA-binding proteins (RBPs). We investigated miRNA cargo of small EVs (SEVs) derived from plasma of FTD patients and healthy controls. The purpose was to evaluate deregulated microRNAs in patients to identify new peripheral biomarkers. Moreover, we aimed to identify mRNA targets involved in FTD pathogenesis. SEVs were isolated from plasma of 9 FTD patients and 9 healthy volunteers by differential centrifugation and characterized by Nanosight. MicroRNA libraries were generated using Small RNA-Seq Library Prep Kit (Lexogen) and sequenced on a NextSeq 500/550 (Illumina). Interaction prediction was carried out on TarBase v.8 database. We found a total of 197 Differentially Expressed (log Fold Change (FC) >1 and <-1) microRNAs, 99 up-regulated and 98 down-regulated. Then, we looked for directly validated mRNA targets of the most deregulate microRNAs (5 up and 5 down-regulated) in our analysis. Interestingly, hsa-miR-522-5p, down-regulated in our profiling, targets RTN3 that in turn interacts with and modulates BACE1, and the up-regulation of hsa-miR-203a-3p may have an impact on TNF and IL-12 levels, reduced in CSF of FTD patients. Moreover, hsa-miR-181c-5p was up-regulated and its role was already linked to a negative feedback network of TDP43. We also evaluated the deregulation of microRNA already associated to other dementia types and we found a down-regulated microRNA in common with Alzheimer’s disease, hsa-miR-1260b, involved in Wnt pathway. In conclusion, our data highlight the importance of microRNAs cargo examination in EVs of FTD patients. In fact, their potential is exploitable both for biomarkers discovery and for study of gene expression alteration in FTD pathogenesis. Funding: Fondazione Cariplo, Italy n. 2017-0747; GR-2016-02361552 Italian Ministry of Health; RC2017-2019 Italian Ministry of Health.

Molecular characterization and angiogenic properties of extracellular vesicles in uncomplicated and preeclamptic amniotic fluid

Natalia Gebara1, Julia Scheel3, Renata Skovronova1, Luca Marozio2, Shailendra Gupta3, Veronica

Giorgione4, Chiara Benedetto2 , Asma Khalil4,5 , Benedetta Bussolati1

1.Department of Molecular Biotechnology and Health Sciences; University of Turin, Italy 2. Department of Surgical Sciences, Obstetrics and Gynecology, University of Turin, Italy 3.Department of Systems Biology and Bioinformatics, University of Rostock 4.Vascular Biology Research Centre, Molecular and Clinical Sciences Research Institute, St George's University of London, UK 5.Foetal Medicine Unit, St George's University Hospitals NHS Foundation Trust, UK Amniotic fluid surrounding the developing fetus is a complex biological fluid, rich in metabolically active bio-factors. It has been previously reported that the majority of EVs within the amniotic fluid are of urinary origin (Keller et al., 2007). We characterized term amniotic fluid EVs with regards to surface marker expression and their molecular content using different orthogonal techniques. EVs expressed a variety of markers of renal, placental, epithelial and stem cell origin and appeared as a heterogeneous population. In addition, we compared amniotic fluid EVs from uncomplicated pregnancies and pregnancies affected by preeclampsia, a hypertensive pregnancy disorder affecting up to 8% of pregnancies worldwide (Brown et al., 2018). We set up a method for EV isolation from amniotic fluid using differential ultracentrifugation. The size and concentration were checked with nanoparticle tracking analysis. Super resolution microscopy was used to visualize single vesicles and their surface markers. EVs surface receptor analysis was performed by bead-based cytofluorimetric analysis (MACSPlex) and confirmed using a chip-based analysis, ExoView. MiRNA array was performed to check the molecular content of amniotic fluid EVs. Angiogenic function of NP- and PE-EVs was assessed using a tube formation assay. Super-resolution microscopy showed a heterogenous EV population with 25% of the EVs co-expressing all three tetraspanins markers. We found no significant differences between the concentration of EVs from preeclamptic amniotic fluid in contrast to normal pregnancy. ExoView and MACSPlex uncovered a plethora of surface markers expressed on the EVs, where few, including CD105 (endoglin) were differentially expressed on preeclampsia-EVs. At a functional level, preeclampsia derived EVs but not normal pregnancy EVs showed an anti-angiogenic effect, possibly due to decoy effect of endoglin. In addition, several miRNAs were differentially expressed in EVs derived from PE-EVs and directly related to angiogenesis modulation. Our results provide a characterization of term amniotic fluid EVs, supporting their origin from foetal and placental cells. Moreover, the anti-angiogenic characteristic of EVs in preeclamptic amniotic fluid support the pathogenic role of EVs described in preeclampsia.

The study was supported by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant iPlacenta, agreement No.765274 Biliography: Brown, M. A. et al. (2018) ‘Hypertensive disorders of pregnancy: ISSHP classification, diagnosis, and management recommendations for international practice’, Hypertension, 72(1), pp. 24–43. doi: 10.1161/HYPERTENSIONAHA.117.10803. Keller, S. et al. (2007) ‘CD24 is a marker of exosomes secreted into urine and amniotic fluid’, Kidney International. Elsevier Masson SAS, 72(9), pp. 1095–1102. doi: 10.1038/sj.ki.5002486.

Mass spectrometry-based characterization of extracellular vesicles from

sporadic Amyotrophic Lateral Sclerosis: lipidomic

Silvia De Siervi1, Maria Chiara Mimmi1, Daisy Sproviero1, Orietta Pansarasa1, Cristina Cereda1

1Genomic and Post Genomic Unit, IRCCS Mondino Foundation, Pavia, Italy

There is no accurate blood-based biomarker for sporadic Amyotrophic Lateral Sclerosis (sALS). Extracellular Vesicles (EVs) have the potential to solve this unmet diagnostic need thanks to their involvement in the development and progression of neurodegeneration. Although current knowledge of their distribution and function is limited, lipids are significant molecular components of EVs and are involved in crucial pathways for ALS pathogenesis, including autophagy, energy metabolism, and neuroinflammation. This study aimed to identify potential biomarker by investigating the lipidic composition of plasma-derived EVs with High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS). We isolated small and large vesicles (sEVs and lEVs) from plasma of 20 sALS patients and matched healthy controls (CTRLs), by differential centrifugation/ultracentrifugation. Following that, we characterized sEVs, lEVs, and plasma through Nanoparticle Tracking Analysis (NTA), Western Blotting, and subsequently by HPLC-MS, targeting a panel of 200 lipids. Statistical analysis included univariate and multivariate approaches, such as PCA (Principal Component Analysis) and PLS-DA (Partial Least Squares-Determinant Analysis). NTA and WB analysis confirmed the correct isolation of lEVs and sEVs. HPLC-MS revealed some lipid classes discriminating between sALS subjects and CTRLs, including glycerophospholipids, as lysophosphatdylcholines (LPC), phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylinositols (PI), and sphingolipids, as ceramides (Cer), mono/di-hexosylceramides (M/DHC) and sphingomyelines (SM). In particular, it was proven the increase of PC(34:1), MHC(24:1), and Cer(24:1) in plasma, lEVs, and sEVs from sALS and the up-regulation of PI(36:3), PE(36:2) and SM(36:2) only in ALS EVs. It was also observed a decrease of LPC(18:2) in plasma and lEVs from sALS. In conclusion, using HPLC-MS approach, we identified reliable lipid markers of sALS that overlap with those discovered in previous studies on cerebrospinal fluid (CSF), supporting the idea of plasma-derived EVs as an easily available source of robust biomarkers.

A miRNA fingerprint in Plasma-derived extracellular vesicles of hSOD1G93A transgenic swine

E Berrone1, M Garofalo2,3, S Gagliardi2, L Messa2,4, S Carelli4,5, G Cagnotti6, F Sammartano6, P Coppo6, V Benedetti1, M Gallo1, C Galli7, A Perota7, R Duchi7, L Bergamaschi7, C Cereda2, C Casalone1, C Corona1

1SC Neuroscienze, Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta; Turin, Italy 2 Genomic and post-Genomic Unit, IRCCS Mondino Foundation, Pavia, Italy

3 Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Pavia, Italy 4 Department of Biomedical and Clinical Sciences "L. Sacco", University of Milan, Milan, Italy

5 Pediatric Clinical Research Center Fondazione "Romeo ed Enrica Invernizzi", University of Milan, Milan, Italy

6 Department of Veterinary Science, University of Turin, Grugliasco (TO), Italy 7Avantea, Cremona, Italy

Two goals of Amyotrophic Lateral Sclerosis (ALS) research are: a) validation of new experimental models b) identification of diagnostic biomarkers, in order to speed up the diagnosis, to monitor its progression and to assess whether a new therapy may be effective. Extracellular vesicles (EVs) and their content may be reliable clinical biomarkers for ALS, as they have yet been used for the diagnosis and prognosis of various diseases. In this context, we developed a hSOD1G93A transgenic swine characterized by a reproducible preclinical and clinical phase in order to clarify certain ALS etiopathogenetic aspects. In particular, EVs characterization could elucidate their role in relation to main key pathogenetic mechanisms involved in ALS. Therefore, this study aimed at evaluating of miRNA into EVs isolated from hSOD1G93A transgenic swine plasma, in preclinical and clinical phase. EVs were isolated from plasma of hSOD1G93A and wild type (WT) pigs by a modified precipitation method. EVs were characterized by Nanosight, flow cytometry and Western blotting (WB). miRNAs were sequenced on a NextSeq 500/550. Phenotype characterization confirmed that the majority of EVs were exosomes expressing the typical exosome markers (CD63, TSG101, Flotillin 1, Alix). As regard miRNA analysis, few miRNAs were deregulated in transgenic swine, representing a specific expression pattern in the model. Also, an evident up-regulation of miR-206 was observed. This miRNA is involved in the proper formation and regeneration of the mature neuromuscular junction. Interestingly, its increased exosomal expression was also detected comparing hSOD1G93A samples at clinical phase with their corresponding preclinical samples. Nonetheless, miR-206 was up-regulated in homozygous transgenic swine compared to heterozygous ones. In conclusion, these data show that in the swine model, biomarkers already associated to ALS are expressed and that miRNAs may be used as monitoring tools for disease severity. Supported by POR FESR 2014-2020 and Italian Ministry of Health

The predictive role of extracellular vesicles in ischemic stroke

Angelica Carandina1, Chiara Favero2,3, Roberto M. Sacco1,3, Nicola Montano1,3, Eleonora Tobaldini1,3, Valentina Bollati2,3

1 Department of Internal Medicine, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico,

Milan, Italy

2 EPIGET - Epidemiology, Epigenetics and Toxicology Lab, Milan, Italy 3 Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy

Since the burden of ischemic stroke (IS) in terms of mortality-to-incidence and disability remains challenging and given the lack of objective non-invasive tools for the prognostic assessment of patients, the aim of our study is to verify the role of Extracellular vesicles (EVs) as a relevant prognostic factor for acute and mid-term outcomes in IS.

In this perspective study conducted at the Policlinico Hospital (Milan), we enrolled 47 patients with a diagnosis of IS within 4.5h from the onset of the event. Stroke severity was evaluated at the admission and after one week using the National Institutes of Health Stroke Scale (NIHSS) and after 3 months by means the Modified Rankin Scale (mRS). To assess circulating EV levels, blood samples were collected at the admission (T0), immediately after recanalization treatment or 2 hours after the first blood sample (T1), and after one week (Tw). Total count of EVs was obtained by Nanosight analysis and analysis on EV subtypes was obtained by flow cytometry.

No association was found between total EV count and early and long-term outcomes. However, the amount of platelet-derived EVs from the blood sample at admission was positively associated with both the severity of the ultra-early outcome and the severity of the early and mid-term outcome. Endothelial and Treg-derived EV levels at the admission were positively related to the early IS outcome. Finally, monocyte/macrophage-derived EVs at T1 and Tw were positively related to the early IS outcome.

The present study suggests that specific EV subtypes are associated with stroke severity and both short- and long-term outcomes, contributing to the pathophysiological processes of AIS with a specific temporal profile. The evidence of our study could contribute to risk stratification in patients with ischemic stroke, to the choice of the best medical therapy and to post-recanalization treatment monitoring.

Evaluation of extracellular vesicles as potential myositis biomarkers

Chiara Franco1, Anna Ghirardello1, Mariele Gatto1, Andrea Doria1

1 Unit of Rheumatology, Department of Medicine (DIMED), University of Padova, Padova, Italy.

IDIOPATHIC INFLAMMATORY MYOPATHIES (IIM) ARE RARE CHRONIC AUTOIMMUNE

DISEASES, CLINICALLY SUB-CLASSIFIED, CHARACTERIZED BY MUSCULAR WEAKNESS,

INFLAMMATION, AND EXTRA-MUSCULAR INVOLVEMENT [1]. IIM PATHOGENESIS

INVOLVES INFLAMMATORY AND NON-INFLAMMATORY MECHANISMS, IMMUNE [2] AND

NONIMMUNE ABNORMALITIES [3], GENETIC AND ENVIRONMENTAL PREDISPOSING

FACTORS [2]. THE HETEROGENEOUS GROUP OF EXTRACELLULAR VESICLES (EVs) [4, 5]

HAVE BEEN PROPOSED AS IMMUNOLOGICAL EFFECTORS AND/OR DISEASES

BIOMARKERS [7, 8]. THE AIM OF OUR STUDY IS TO CHARACTERIZE AND INVESTIGATE EVs

AS POTENTIAL IIM BIOMARKERS.

EVS WERE ISOLATED FROM PLATELET-FREE PLASMA OF IIM PATIENTS AND AGE/SEX-

MATCHED HEALTHY DONORS (HDs) THROUGH SIZE EXCLUSION CHROMATOGRAPHY

AND ENRICHED BY ULTRAFILTRATION STEP. EVs SAMPLES WERE OBSERVED BY

TRANSMISSION ELECTRON MICROSCOPY (TEM) AND QUANTIFY BY NANOPARTICLE

TRACKING ANALYSIS (NTA).

BY TEM, EVs WERE SEEN MORE CONCENTRATED IN IIM PATIENTS (N=4) THAN IN HDs

(N=5) SAMPLES. EVs APPEARED INTACT, ROUNDISH, AND HETEROGENEOUS IN SIZE,

WITH A PREVALENCE OF SMALLER EVs IN BOTH GROUPS. NTA MEASUREMENTS

SHOWED AN EVs CONCENTRATION MEAN OF 1.74E+10 (+/- 4.2E+9 STANDARD DEVIATION

(SD)) [EVs/mL] AND A MEAN MODE DIAMETER OF 133.2 (+/- 10.8 SD) nm IN THE PATIENTS’

SAMPLES (N=7). AMONG THE ANALYZED PATIENTS’ SAMPLES, EVs CONCENTRATION OF

THE THERAPEUTICALLY UNTREATED ONE WAS 2.47E+10 [EVs/mL], INCREASED

COMPARED TO TREATED PATIENTS. THE MEAN EVs CONCENTRATION OF HDs SAMPLES

(N=7) WAS 1.56E+10 (+/- 2.5E+9 SD) [EVs/mL] WITH A MEAN MODE DIAMETER OF 153.1 (+/-

9.5 SD) nm. P-VALUE IN STATISTICAL UNPAIRED T TEST OF EVs MODE SIZE WAS 0.0033,

SIGNIFICANTLY DIFFERENT (P<0.05) BETWEEN THE PATIENTS AND HDs GROUPS.

PRELIMINARY DATA BASED ON TEM OBSERVATIONS SHOWED THE PREDOMINANCE OF

SMALL EVs IN PATIENTS AND HDs SAMPLES. NTA MEASUREMENTS SHOWED

SIGNIFICANTLY SMALLER EVs SIZE IN PATIENTS SAMPLES COMPARED TO HDs

SAMPLES. THE SIMILAR EVS CONCENTRATION IN THE TWO GROUPS REQUIRES THE

INTRODUCTION OF SAMPLES FROM UNTREATED PATIENTS. FURTHER EVs

CHARACTERIZATION EMPLOYING IMAGING FLOW CYTOMETRY IS ONGOING.

REFERENCES

1. Cerezo LA, Vencovský J, Šenolt L. Cytokines and inflammatory mediators as promising markers

of polymyositis/dermatomyositis. Curr Opin Rheumatol. 2020 Nov;32(6):534-541. doi:

10.1097/BOR.0000000000000744. PMID: 32941247.

2. Schmidt J. Current Classification and Management of Inflammatory Myopathies. J Neuromuscul

Dis. 2018;5(2):109–29.

3. Loredo Martinez M, Zampieri S, Franco C, et al. Nonimmune mechanisms in idiopathic

inflammatory myopathies. Curr Opin Rheumatol. 2020;32(6):515–22.

4. Yáñez-Mó M, Siljander PR, Andreu Z, et al. Biological properties of extracellular vesicles and their

physiological functions. J Extracell Vesicles. 2015 May 14;4:27066. doi: 10.3402/jev.v4.27066.

PMID: 25979354; PMCID: PMC4433489.

5. Théry C, Witwer KW, Aikawa E, et al. 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 Nov 23;7(1):1535750. doi:

10.1080/20013078.2018.1535750. PMID: 30637094; PMCID: PMC6322352.

6. Wu WC, Song SJ, Zhang Y, et al. Role of Extracellular Vesicles in Autoimmune Pathogenesis.

Front Immunol. 2020 Sep 23;11:579043. doi: 10.3389/fimmu.2020.579043. PMID: 33072123;

PMCID: PMC7538611.

7. Lu M, DiBernardo E, Parks E, et al. The Role of Extracellular Vesicles in the Pathogenesis and

Treatment of Autoimmune Disorders. Front Immunol. 2021 Feb 24;12:566299. doi:

10.3389/fimmu.2021.566299. PMID: 33732229; PMCID: PMC7959789.

Comparative characterization of prostate cancer biomarkers in EVs isolated from patients’ plasma through different methods

Vera Mugoni1, Yari Ciani1, Federico Vannuccini1, Orsetta Quaini1,

Simone Tomasini1, Antonello Veccia2, Vito D’Agostino1, Orazio Caffo2, Caterina Nardella1, Francesca Demichelis1

1. Department of Cellular, Computational and Integrative Biology; University of Trento;

Trento, Italy 2. Department of Medical Oncology, Santa Chiara Hospital, Trento, Italy

In the last few years, extracellular vesicles (EVs) and their associated RNAs (EV-

RNAs) have been investigated as a source of biomarkers in prostate cancer (PC)

patients liquid biopsies. Specifically, EVs from patients’ urines have already been

proven suitable for prostate cancer detection. Furthermore, EVs circulating in the

blood are being utilized for the development of multi-analytes liquid biopsies based on

the analysis of EV-RNAs and other cancer-released circulating materials, such as cell

free DNA or proteins.

In order to maximize the informative power of EV-RNAs from healthy donors’ (HD) and

from PC patients’ (from the PRIME consortium study (1)) plasma samples, we

evaluated the detection of prostate–specific biomarkers in EVs samples isolated

through diverse methodological approaches, including ultracentrifugation, size

exclusion chromatography and a recently developed charge–based isolation method

(2).

Here we characterized the differently isolated EVs populations obtained from aliquots

of the same blood drawn by imaging technologies, such as nanoparticle tracking

analysis technology and/or imaging flowcytometry (ImageStream). Accordingly, we

analyzed exosome-specific biomarkers across the differently isolated EVs samples.

We then assessed the similarity of EV-RNA cargo obtained through the reported

isolation methods. RNA-seq studies demonstrated that the distinct methods allowed

the recovery of diverse proportions of analogous RNA species, including protein-

coding RNA (e.g. mRNAs) and a variety of non-coding RNA (such as miRNAs, RNAs

associated to intergenic regions and miscellaneous small RNA). To further validate

the diverse EVs isolation methods for their potential application in liquid biopsies, we

tested the relevant EV-RNA cargo for the detection of PC-specific biomarkers

(including AR, AR-v7, PSMA and PCA3) by ultra-sensitive ddPCR analysis and/or

immune-based assays.

Altogether, our results support the use of plasma, in alternative to urine, as a source

of EVs for PC patients’ liquid biopsies studies and suggest that EV-RNAs obtained

through diverse EV isolation methods provide useful information for PC biomarkers

detection.

REFERENCES

1. UK CR. <https://news.cancerresearchuk.org/2018/09/14/prime-time-how-team-science-is-bringing-liquid-biopsies-into-the-clinic-for-prostate-cancer>.

2. Notarangelo M, Ferrara D, Potrich C, Lunelli L, Vanzetti L, Provenzani A, et al. Rapid Nickel-based Isolation of Extracellular Vesicles from Different Biological Fluids. Bio-protocol 2020;10(3):e3512 doi 10.21769/BioProtoc.3512.

Profiling circulating extracellular vesicle surface antigens in primary aldosteronism

Jacopo Burrello1,2, Martina Tetti1*, Vittorio Forestiero1*, Vanessa Biemmi2,3, Sara Bolis2, Margherita

Alba Carlotta Pomatto4, Martina Amongero5, Dario Di Silvestre6, Pierluigi Mauri6, Giuseppe Vassalli2, Giovanni Camussi4, Tracy Ann Williams1,7, Paolo Mulatero1, Lucio Barile2,3#, and Silvia Monticone1#.

1 Division of Internal Medicine 4 and Hypertension Unit, Department of Medical Sciences, University of Turin, Italy. 2 Laboratory for Cardiovascular Theranostics and Laboratory of Cellular and Molecular Cardiology, Cardiocentro Ticino Institution, Lugano, Switzerland. 3 Faculty of Biomedical Sciences,

Università della Svizzera Italiana, Lugano, Switzerland. 4 Molecular Biotechnology Center, Department of Medical Sciences, University of Torino, Italy. 5 Department of Mathematical Sciences

G. L. Lagrange, Polytechnic University of Torino, Italy. 6 Proteomic and Metabolomic Laboratory, Institute for Biomedical Technologies – National Research Council (ITB-CNR), Segrate (Milan), Italy.

7 Medizinische Klinik und Poliklinik IV, Klinikum der Universität, Ludwig-Maximilians-Universität München, Munich, Germany. * Contributed equally and should be considered as joint second authors.

# Contributed equally and should be considered as joint last authors. Primary aldosteronism (PA) is a frequent cause of endocrine hypertension, with a prevalence of 6% among the general hypertensive population. PA is characterized by an inappropriate secretion of aldosterone by the adrenal glands and may result from either aldosterone producing adenoma (APA) or bilateral hyperaldosteronism (BiPA). Chronic aldosterone excess has detrimental effects on cardiovascular system, including endothelial dysfunction and vascular inflammation. Circulating extracellular vesicles (EVs) are central players in the crosstalk between endothelium, vascular structures, and inflammatory cells, and they may be involved in the development of organ damage in patients with PA.

The aim of the present study was to assess the potential role of EVs in aldosterone-related vascular injury by evaluating a comprehensive panel of 37 EV surface antigens through an innovative flow cytometry multiplex bead-based platform. Serum EVs were isolated by immuno-capture from 32 patients with PA (20 with an APA and 12 with BiPA), 29 patients with essential hypertension (EH) and from 22 normotensive (NT) controls. Patients with an APA were re-evaluated after curative adrenalectomy, whereas patients with BiPA after 6 months from the beginning of specific medical treatment with mineralocorticoid receptor antagonists. EVs were characterized by western blotting, nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry. EV concentration was higher and diameter lower in patients with PA compared to both patients with EH and NT controls and the number of particles decreased after unilateral adrenalectomy. Nineteen EV surface antigens were differentially expressed in patients with PA compared to patients with EH or NT controls, including markers from activated platelets, endothelial and immune/inflammatory cells. Particle concentration and several EV antigens were directly correlated to blood pressure and aldosterone, and inversely correlated to potassium levels. Supervised learning was used to combine the expression levels of EV surface markers in a specific molecular signature which discriminated patients with PA from controls (EH and NT), whereas after specific treatment the profile became similar to EH. Stimulation of human endothelial cells with PA patient-derived EVs, but not with EVs from EH, resulted in the overexpression of 5 selected genes (AKT1, CALR, CSNK2A1, FN1, and PIK3R1), previously identified by bioinformatics analysis as potential targets of differentially expressed EV antigens. Notably, the effect was specifically dependent on EV surface antigens, since it was abolished by removal of EV membrane associated proteins. The overexpression of CALR and FN1 was confirmed also at protein level. EVs may interact with target cells by their surface antigens, thus influencing biomolecular pathway involved in inflammatory response, platelets activation, and endothelial cell function. Our data suggest that EVs represent biomarkers of vascular inflammation and endothelial dysfunction in patients with PA, and also potential biovectors contributing to endothelial dysfunction, vascular inflammation, and accelerated organ damage by multiple signalling processes.

The immune-profiling of plasma- and CSF- derived extracellular vesicles allows the discrimination between Parkinson’s Disease and atypical

parkinsonisms

Elena Vacchi1,2, Jacopo Burrello3, Alessio Burrello4, Sara Bolis3, Lucio Barile2,3, Alain Kaelin-Lang1,2,5,6, Giorgia Melli1,2,5

1. Laboratory for Biomedical Neurosciences, Neurocenter of Southern Switzerland, Ente Ospedaliero Cantonale, Lugano, Switzerland

2. Faculty of Biomedical Sciences, Università della Svizzera Italiana, Lugano, Switzerland 3. Cellular and Molecular Cardiology Laboratory and Laboratory for Cardiovascular Theranostics,

Cardiocentro Ticino Foundation, Lugano, Switzerland 4. Department of Electrical, Electronic and Information Engineering, University of Bologna,

Bologna, Italy 5. Neurology Department, Neurocenter of Southern Switzerland, Ente Ospedaliero Cantonale,

Lugano, Switzerland 6. Department of Neurology, Inselspital, Bern University Hospital, University of Bern, Bern,

Switzerland

The clinical discrimination between Parkinson’s Disease (PD) and atypical parkinsonisms (AP) is still challenging. With the aim to build a diagnostic model via artificial intelligence able to discriminate these neurodegenerative proteinopathies disorders, we characterized and compared distinctive extracellular vesicles (EVs) subpopulations from plasma and CSF. Plasmatic EVs were collected from 27 PD, 19 matched healthy controls (HC), 9 AP with multiple system atrophy (MSA) and 9 AP with tauopathies (AP-Tau). CSF-derived EVs were collected from 4 PD, 4 MSA, 4 AP-Tau. The expression of 37 EV-surface markers, related to inflammatory and immune cells, were measured by MACSPlex in plasm and CSF. Random forest (RF) diagnostic models based on EV markers expression were built via supervised machine learning algorithms. In plasma, 17 EV-surface markers resulted statistically different between groups. Among these, 8 were observed also in CSF of PD, 10 in MSA and 6 in AP-Tau. A diagnostic model based on the these 17 differentially expressed markers in plasma discriminates patients from HC with high sensitivity (100%) and specificity (83.3%). Integrating CSF-derived data a potentiated model was able to differentiate PD from not-PD patients with 96.6% sensitivity and 88.0% specificity. The immune profiling of EVs in plasma and CSF allows the stratification of patients with different neurodegenerative disorders, leading the way to potential, non-invasive, low-cost blood test for the diagnosis of PD and AP patients.

Klotho inside uEVs and its potential role as biomarker for kidney physiopathology

Cristina Grange1, Dario Tomanin2, Luca Deorsola3, Carlo Pace Napoleone3 and Benedetta

Bussolati2 1Department of Medical Sciences, University of Turin, 2Department of Molecular Biotechnology and

Health Sciences, University of Turin; 3Regina Margherita Pediatric Hospital, Turin, Italy.

Entering an era in which biomarkers are gaining considerable importance in the clinical world, we decided to investigate Klotho levels within urinary extracellular vesicles (uEVs) as an efficient indicator of the overall state of the kidneys. We recently described that uEV also carry Klotho, an anti-aging hormone released by the kidney with protective activity on the cardiovascular and neurological systems. We here aimed to characterize the uEVs of a population of pediatric patients affected by single ventricle congenital heart disease. In particular, we aimed to evaluate Klotho levels as a potential biomarker of renal damage.

In this study, we isolated uEVs from first morning urine of pediatric patients affect by single ventricle defects undergone to Fontan procedure (n=15), and from aged match young healthy subjects. We subsequently characterized uEVs, combining the cytofluorimetric analysis of uEV surface markers, obtained with MACSplex Exosome Kit, with the quantification of uEVs-Klotho measured by ELISA. Finally, Klotho and classical exosomal and renal markers were confirmed by single-molecule super resolution microscopy. Renal function was evaluated by creatinine and NGAL levels.

We isolated a comparable number of uEVs from pediatric patients and healthy subjects. We observed the presence of renal progenitor markers (SSEA4, CD133, CD24) and of the epithelial marker CD326 in both experimental conditions. However, the expression of renal uEVs markers was decreased in single ventricle patients. Of interest, the quantification of Klotho levels within uEVs revealed a significantly higher expression of Klotho in uEVs from healthy subjects compared with uEVs from single ventricle patients. On the contrary, no differences in renal functional parameters were observed.

The results presented here suggest that in patients affected by single ventricle congenital heart disease, uEV characterization and Klotho expression might predict pre-clinical damage to the renal tissue. Klotho loss could be involved in the systemic alterations observed in these patients.

References

1) Grange C, Papadimitriou E, Dimuccio V, Pastorino C, Molina J, O'Kelly R, Niedernhofer LJ, Robbins PD, Camussi G, Bussolati B. Urinary Extracellular Vesicles Carrying Improve the Recovery of Renal Function in an Acute Tubular Injury Model. Mol Ther. 2020 Feb 5;28(2):490-502. doi: 10.1016/j.ymthe.2019.11.013.

2) Erdbrügger U, Blijdorp CJ, Bijnsdorp IV, Borràs FE, Burger D, Bussolati B, Byrd JB, Clayton A, Dear JW, Falcón-Pérez JM, Grange C, et al. Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles. J Extracell Vesicles. 2021 May;10(7):e12093. doi: 10.1002/jev2.12093.

Fast quantification of extracellular vesicles levels in early breast cancer patients by Single Molecule Detection Assay (SiMoA)

Alessandra Ricciardi1#, Carlo Morasso1#, Daisy Sproviero2, Marta Truffi1, Sara Albasini3, Francesca Piccotti1, Federico Sottotetti4, Ludovica Mollica4, Cristina Cereda2, Luca Sorrentino5, Fabio Corsi3,6*

1 Laboratory of Nanomedicine, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy.

2Genomic and post-Genomic Center, IRCCS Mondino Foundation, Pavia, Italy. 3Breast Unit, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy.

4Medical Oncology Unit, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy. 5Colorectal Surgery Unit, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan,

Italy. 6Dipartimento di Scienze Biomediche e Cliniche “L. Sacco”, Università di Milano, 20157

Milano, Italy.

Breast cancer (BC) is the most commonly diagnosed cancer and the main cause of cancer deaths in women worldwide. The identification of new biomarkers of the disease thus remains a priority for biomedical research. In this regard, extracellular vesicles (EVs), lipid bilayer vesicles, nanometer-sized secreted from many cell types, have been recently considered as a potential biomarker in BC [1]. The predictive role of EVs in BC is supported by the higher concentration of tetraspanin CD9, CD63, and CD24 receptors in EVs originated by cancer cells than those derived from normal epithelial mammary cells [2]. Unfortunately, there is a lack of clinical data that supporting the diagnostic and prognostic value of extracellular vesicles in BC due to the difficulty associated with the EVs quantification through the traditional analytical approaches. To solve this problem, we have developed an innovative ultrasensitive assay based on Single Molecule Array (SiMoA) Technology, that can quantify very low concentrations of EVs present in the biological fluids. In the assay, we used a couple of antibodies targeting CD63 and CD9, two tetraspanins well know for their expression on EVs surface. Thanks to this innovative technique, it is possible to analyze EVs without any previous purification process using only 4 µl of plasma for each analysis. This is made possible by the fact that the SiMoA assay intrinsically includes an isolation step for EVs based on a magnetic extraction. The magnetic beads, conjugated with an anti CD63 antibody, are mixed with the samples and let to form an immunocomplex with the target EVs and with the anti CD9 detection antibody. Afterwards, each immunocomplex is individually distributed by the SiMoA system in a microwell. Since the microwells have a volume of about 40 femtoliters, the local concentration of the target molecule resulted amplified, and the system thus allows measuring much lower target concentrations (LOQ: 1.03 ng/ml) than those detectable through a traditional ELISA. Using this assay, we determined the EVs concentration in plasma from 70 early BC patients (stage I-III) and 45 healthy controls (HC). The obtained results showed that EVs levels resulted significantly higher in BC (p < 0.001) than in HC. To check the accuracy of an hypothetical diagnostic test based on our SiMoA assay, a ROC curve was calculated, obtaining an AUC of 0.84 with a sensitivity of 76% and specificity of 84% (PPV:85.1% and NPV: 77%). Our results thus support the idea that EVs may be a promising biomarker for BC disease. Besides, considering the low amount of plasma used for the analysis, this new technology offers the possibility to study rare subpopulations of EVs more specifically related to BC or other pathological conditions. References [1] Riches, A., Campbell, E., Borger, E. & Powis, S. Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway. Eur. J. Cancer 50, 1025–1034 (2014). [2] Rupp, A.-K. et al. Loss of EpCAM expression in breast cancer derived serum exosomes: role of proteolytic cleavage. Gynecol. Oncol. 122, 437–446 (2011).

Neuronal Extracellular Vesicles content during Alzheimer’s disease progression

Silvia M. Masciandaroa, Roberta Giannellia, P. Beffyad, Renata Del Carratorea*, Paolo Bongioannibc,

a Institute of Clinical Physiology, National Research Council, Pisa.

b Azienda Ospedaliero-Universitaria Pisana, Pisa. c NeuroCare onlus, Pisa.

d ICCOM National Research Council, Pisa.

Cells from the central nervous system, (neurons, astrocytes, oligodendrocytes) are able to secrete small extracellular vesicles (EV) into the bloodstream, in both physiological and pathological conditions. EV through their miRNA, nucleic acids and proteins content might thus reflect the disorders of nerve cells, and contribute to the diagnosis of neurodegenerative diseases. The specific investigation could be difficult, because in blood are present the total exosomes from the whole body, where the neuronal ones are less than 5%. In order to enrich peripheral blood samples in neuronal-derived EV (NDEV), an immunochemical method based on specific antibody against neural L1 cell adhesion molecule (L1CAM), was developed in our laboratory. The aim of our work was to evaluate the content of NDEV during the onset and/or progression of Alzheimer’s Disease (AD). Increasing epidemiological evidence shows that AD is characterized by a long presymptomatic preclinical period before the definitive diagnosis that could take several years before leading to cognitive dysfunction. This makes necessary to look for diagnostic biomarkers for early intervention. At present, diagnostic research criteria for AD include emission tomography with amyloid tracer, which is a complex and very expensive tool, and the cerebrospinal fluid pathogenetic protein detection, that implies the invasive procedure of sample collection by lumbar puncture. In this scenario, circulating biomarkers would be of help for differential diagnosis and possibly for monitoring cognitive decline. In our work a group of 20 patients and as many controls were enrolled for the molecular studies and periodically underwent medical examination at our hospital. MiRNA plays an important role in regulating AD-related neuropathological hallmarks include the deposition of senile plaques resulting from the excessive accumulation of β-amyloid protein (Aβ) and the formation of neurofibrillary tangles derived from hyperphosphorylated tau proteins leading globally to neural dysfunction. In summary, our study can give important information for the prevention, progression and for future therapeutic strategies. Nevertheless, although there are several studies describing miRNAs as biomarkers of neurodegenerative diseases, more research into miRNAs that are secreted by the brain under pathophysiological conditions is required. Mir-132 is the most common downregulated miRNA in the postmortem AD brain, and is involved in the progression of Aβ and tau pathology. Aβ and tau content, and a specific AD miRNA signature in NDEV could be used as potential diagnostic biomarkers. MiR-124 has been used to check the quality of the enrichment process and miR-132, and miR-9, some of the principal miRNA involved in ALZ progression and present in EV, are being tested in the first disease onset phases and during the disease progression. A large number of blood samples diachronically collected and chemically analysed, from more than 50 AD patients, supports our study. The NDEV biomarkers related to the disease severity overtime gave significant association.

Supported by the GAE P2444-NEWDEM Region Health Grant 2018

References

Meng Zhang, et al. 2021. Alzheimer's Disease and microRNA-132: A Widespread Pathological Factor and Potential Therapeutic Target Front. Neurosci., 24 https://doi.org/10.3389/fnins.2021.687973.

Anastasi F, et al. 2021. Proteomics Profiling of Neuron-Derived Small Extracellular Vesicles from Human Plasma: Enabling Single-Subject Analysis. P.Int J Mol Sci. 22: 2951. doi: 10.3390/ijms22062951

New targets and new approaches in multiple myeloma: extracellular

vesicles as functional liquid biomarkers

Antonia Reale1*, Tiffany Khong1, Irena Carmichael2, Rong Xu3,4, Haoyun Fang5, Sridurga Mithraprabhu1, Malarmathy Ramachandran1, Maoshan Chen1, David W. Greening4,5,6*,

Andrew Spencer1,6,7*

1. Myeloma Research Group, Australian Centre for Blood Diseases, Central Clinical School,

Monash University/Alfred Health, Melbourne, VIC, Australia 2. Monash Micro Imaging-AMREP, Monash University, Melbourne, VIC, Australia

3. Nanobiotechnology Laboratory, Australian Centre for Blood Diseases, Central Clinical School, Monash University, Melbourne, Victoria, Australia

4. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia

5. Molecular Proteomics, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia. 6. Central Clinical School, Monash University/Alfred Health, Melbourne, VIC, Australia

7. Malignant Haematology and Stem Cell Transplantation, The Alfred Hospital, and Department of Clinical Haematology, Monash University, Melbourne, VIC, Australia

Introduction: Circulating small extracellular vesicles (sEV) represent promising non-invasive biomarkers that may aid in the diagnosis and risk-stratification of multiple myeloma (MM), an incurable blood cancer. We have previously demonstrated that: 1) peripheral blood plasma (PBPL)-sEV can be isolated from 1 mL of both fresh or frozen PBPL using a commercial kit. 2) MM-derived PBPL-sEV regulate stromal cell (HS5) proliferation and migration. In turn, pre-educated stromal cells favour human MM cell lines (HMCL) adhesion. Interestingly, PBPL-sEV isolated from patients with both pre-malignant plasma cell disorders (monoclonal gammopathy of undetermined significance, MGUS; smouldering MM, SMM) and newly diagnosed MM (NDMM) have a similar ability to promote cell migration and adhesion, suggesting a role for both malignant and pre-malignant PBPL-sEV in disease progression. 3) Preliminary proteomic profiling of (x2) NDMM-sEV revealed enrichment of factors implicated in cell migration and adhesion, in comparison to (x2) healthy donors (HD)-sEV. Methods: sEV were isolated from 1 mL of fresh PBPL using a commercial kit. Proteomic profiling (nLC and high-resolution mass spectrometry, Orbitrap HF-X) of PBPL-sEV derived from 10 HD, 8 NDMM, 4 SMM,10 MGUS patients, and functional studies using a co-culture system (HS5:HMCL) were performed. Results: NDMM-sEV favoured HMCL proliferation and drug resistance to the proteasome inhibitor bortezomib when HMCL were co-cultured for 24h with HS5 cells pre-treated with NDMM-sEV compared to untreated HS5 cells or pre-treated with HD-sEV or MGUS-sEV or SMM-sEV. A total of 412 proteins were detected and quantified by proteomic profiling of PBPL-sEV with 13 reported as highly enriched in EV marker databases (ExoCarta top 100) and 8/13 corresponding to universal cancer markers as proposed by Hoshino et al (Cell, 2020). Comparative analysis of sEV between the 4 cohorts revealed 241/412 co-identified proteins. Gene ontology analysis of co-identified proteins (G:Profiler; p<0.05) revealed enrichment for cellular component terms such as “extracellular vesicles”, “exosomes”, and for several biological processes including “cell communication”, “endocytosis”, “cell migration”, “cellular response to stimulus”, “immune response”. A specific protein signature identified in NDMM-sEV was found in >30% of NDMM-sEV but <30% HD-sEV which warrants further validation. Specific protein signatures were also identified in MGUS-sEV (>30% of MGUS-sEV but <30% HD-sEV or NDMM-sEV or SMM-sEV) and SMM-sEV (>30% of SMM-sEV but <30% HD-sEV or NDMM-sEV or MGUS-sEV). Conclusions: The characterization and proteomic profiling of disease-specific circulating sEV as a biomarker discovery strategy may provide translational applications in MM. Importantly, MM-sEV may play an important role in disease progression by re-programming the tumour microenvironment.

Extracellular vesicles from acute coronary syndrome patients and myocardial ischemia-reperfusion injury

Saveria Femminò1, Fabrizio D’Ascenzo2, Francesco Ravera1, Filippo Angelini2, Andrea

Caccioppo2, Luca Franchin2, Alberto Grosso2, Stefano Comità4, Claudia Cavallari3, Claudia Penna4, Gaetano Maria De Ferrari2, Giovanni Camussi1, Pasquale Pagliaro4, and Maria Felice

Brizzi1

1 Department of Medical Sciences, University of Turin, Turin, Italy 2 Division of Cardiology, Department of Medical Sciences, University of Turin, Turin, Italy

3 2i3T Scarl, University of Turin, Turin, Italy 4 Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy

The Remote Ischemic PreConditioning (RIPC) procedure fails to reduce Ischemia/Reperfusion (I/R) injury in Acute Coronary Syndrome (ACS) patients that have undergone Percutaneous Coronary Intervention (PCI). Extracellular vesicles (EV) have attracted interest as mediators of tissue damage and/or recovery upon I/R injury. In the present study, we investigated the potential effect of serum-derived EV recovered from ACS patients (Non-ST Elevation Myocardial Ischemia and Unstable Angina), subjected to the PCI and the RIPC procedure or otherwise, in preventing I/R injury. A double-blind, randomized, sham-controlled study (NCT02195726) has been extended, and EV recovered from 30 ACS patients who were randomly assigned (1:1) to undergo the RIPC- (EV-RIPC) or sham-procedures (EV-naive) before PCI. Of note, we found that troponin (TnT) was enriched in EV, compared to healthy subjects, regardless of diagnosis. Moreover, we demonstrated that EV-naive, unlike EV-RIPC, induced protection against I/R, both in-vitro and in the whole heart with STAT-3 as a crucial mediator of EV-naive protection. Interestingly, Erk-1/2 targeting rescued STAT-3 activation and protection upon EV-RIPC treatment. EV were screened for 84 Human Cardiovascular Disease mRNAs and DUSP6 mRNA was significantly enriched in EV-naive. Therefore, DUSP6 silencing in EV-naive prevented STAT-3 phosphorylation and their cardio-protective properties. This study first demonstrates the cardio-protective activity and mechanism of action of EV-naive and validates a potency test to predict biological activity ex-vivo. Moreover, it provides insight on the unsuccessfulness of the RIPC-procedure in humans. Finally, we propose EV-TnT content as a sensitive marker to estimate I/R damage in clinics.

Extracellular vesicles derived from human liver stem cells attenuate chronic kidney disease development in an in vivo experimental model of

renal ischemia and reperfusion injury.

S. Bruno1,2, M. Cedrino2, G. Chiabotto1, E. Ceccotti1, C. Pasquino1,2, S. Tritta2, G. Camussi1,2.

1Department of Medical Sciences, University of Torino, Italy 2Molecular Biotechnology Center, University of Torino, Italy.

In order to test the potential therapeutic effect of extracellular vesicles (EVs) derived from human liver stem cells (HLSCs) in acute kidney injury (AKI), with subsequent development of chronic kidney disease (CKD), we set up a model of renal ischemia reperfusion injury (IRI). EVs were obtained by ultracentrifugation and characterized in accordance with ISEV guidelines. Mice were subjected to 30 minutes IRI and EVs (1x109) were iv administered immediately after the surgery and three days after. To evaluate AKI, mice were sacrificed 2 days after the surgery. Renal function (creatinine and BUN plasma levels), histology and molecular analyses of specific markers of AKI (lipocalin-2/NGAL, kidney injury molecule 1 KIM1) have been evaluated using specific kits, immunohistochemical staining and real time PCR analyses. To evaluate development of CKD, mice were sacrificed 2 months after the injury. Renal function, histology and molecular analyses of specific markers of fibrosis development (alpha-SMA, collagen I, transforming growth factor beta 1) and of inflammation (TNF-alpha, IL1-beta and IL-6) have been quantified using the same methods described above. In AKI experiments, we found that EV-treatment induced an amelioration of renal histology (tubular necrosis), an increase in tubular cell proliferation and a slight reduction of lipocalin-2/NGAL expression, considered as a biomarker of renal proximal tubular injury. In CKD experiments, we found that treatment of IRI-mice with HLSC-EVs, limited the development of interstitial fibrosis at histological and molecular levels. Furthermore, the expression levels of pro-inflammatory genes TNF-alpha, IL1-beta and IL-6 were significantly reduced by EV-treatment. Treatment of mice with renal IRI with HLSC-EVs, induces AKI amelioration and interferes with the development of subsequent interstitial fibrosis in CKD.

HLSC-EVs alleviate kidney fibrosis by partially interfering with the β-catenin pathway through miR29b.

Sharad Kholia1,3, Maria Beatriz Herrera Sanchez2,3, Maria Chiara Deregibus2,3, Marco Sassoè-

Pognetto4, Giovanni Camussi1, Maria Felice Brizzi1*.

1Department of Medical Sciences, University of Torino, Torino, Italy. 22i3T Società per la gestione dell'incubatore di imprese e per il trasferimento tecnologico Scarl,

University of Torino, Torino, Italy. 3Molecular Biotechnology Centre, University of Torino, Torino, Italy.

4Department of Neurosciences, University of Torino, Torino, Italy. Abstract Inadequate therapeutic strategies for the prevention, treatment and progression of chronic kidney disease (CKD) remain a major burden on global health care systems 1. Alternative therapies therefore need to be developed and implemented to alleviate this burden. Aristolochic acid (AA) nephropathy (AAN) is a rapidly progressive form of nephrotoxic CKD characterised by inflammation, tubular damage, and interstitial fibrosis that leads to end stage renal disease 2. Stem cell derived extracellular vesicles (EVs) in particular human liver stem cell derived EVs (HLSC-EVs) have been reported to exhibit therapeutic properties in various pre-clinical disease models including kidney injury 3,4. Previously, in a mouse model of AAN, we have shown that administration of AA caused deterioration in kidney function, tubular damage, infiltration of inflammatory cells, and interstitial fibrosis, which was significantly ameliorated following treatment with HLSC-EVs 5. In addition, the Wnt/β-catenin pathway was found to be one of the pathways to be regulated by HLSC-EVs following transcriptomic analysis. The current study confirms the expression of the active form of β-catenin to be elevated in AA kidneys as well as in activated fibroblasts in vitro. Furthermore, this upregulation was significantly reduced following treatment with HLSC-EVs both in vitro and in vivo at a molecular and protein level. Silencing of the β-catenin gene in fibroblasts prevented the upregulation of the pro-fibrotic genes α-Sma and collagen type 1 post activation with AA injured mTECs in-vitro, therefore confirming the role of Wnt/β-catenin pathway in fibroblast activation. Interestingly, AA mice kidneys and fibroblasts treated with HLSC-EVs also showed elevated levels of the anti-fibrotic miRNA miR29b. However, upon treatment of fibroblasts with the transcription inhibitor α-amanitin, or antimir29b In-vitro, this upregulation of miR29b together with the downregulation of β-catenin and other pro-fibrotic genes by HLSC-EVs was significantly reduced. Taken together these data suggest a novel regulatory mechanism of action by HLSC-EV whereby β-catenin is downregulated by miR29b induced directly by HLSC-EVs in fibroblasts in vitro. Supported by funding from: University of Torino and Unicyte srl.

References

1 Coresh, J. et al. Prevalence of chronic kidney disease in the United States. Jama 298, 2038-2047, doi:10.1001/jama.298.17.2038 (2007).

2 Debelle, F. D., Vanherweghem, J. L. & Nortier, J. L. Aristolochic acid nephropathy: a worldwide problem. Kidney international 74, 158-169, doi:10.1038/ki.2008.129 (2008).

3 Correa, R. R., Juncosa, E. M., Masereeuw, R. & Lindoso, R. S. Extracellular Vesicles as a Therapeutic Tool for Kidney Disease: Current Advances and Perspectives. International journal of molecular sciences 22, doi:10.3390/ijms22115787 (2021).

4 Bruno, S. et al. Human Liver Stem Cells: A Liver-Derived Mesenchymal Stromal Cell-Like Population With Pro-regenerative Properties. Frontiers in cell and developmental biology 9, 644088, doi:10.3389/fcell.2021.644088 (2021).

5 Kholia, S. et al. Human Liver Stem Cell-Derived Extracellular Vesicles Prevent Aristolochic Acid-Induced Kidney Fibrosis. Frontiers in immunology 9, 1639, doi:10.3389/fimmu.2018.01639 (2018).

Human liver stem cell-derived extracellular vesicles target hepatic stellate cells and attenuate their pro-fibrotic phenotype

G. Chiabotto1, E. Ceccotti1, M. Tapparo1,2, G. Camussi1,2, S. Bruno1,2

1Department of Medical Sciences, University of Torino, Italy. 2Molecular Biotechnology Center, University of Torino, Italy.

Liver fibrosis occurs in response to chronic liver injury and is characterized by an excessive deposition of extracellular matrix. Activated hepatic stellate cells are primarily responsible for this process. A possible strategy to counteract the development of hepatic fibrosis could be the reversion of the activated phenotype of hepatic stellate cells. Extracellular vesicles (EVs) are nanoscale lipid membranes involved in intercellular communication. Our previous study have demonstrated that EVs derived from human liver stem cells (HLSCs), a multipotent population of adult stem cells with mesenchymal-like phenotype, exert in vivo anti-fibrotic activity in a murine model oh chronic liver disease. However, the mechanism of action of these EVs remains to be determined. We set up an in vitro model of hepatic fibrosis using a human hepatic stellate cell line (LX-2) activated by transforming growth factor-beta 1 (TGF-β1). Then, we investigated the effect of EVs obtained from HLSCs and from human bone marrow-derived mesenchymal stromal cells (MSCs) on activated LX-2. TGF-β1 increased the expression of pro-fibrotic markers alpha-smooth muscle actin (α-SMA) and collagen I alpha-1 (COL1A1) in LX-2. The release of pro-COL1A1 in LX-2 supernatant was also up-regulated by TGF-β1. The incubation of activated LX-2 with HLSC-EVs reduced the expression level of α-SMA. On the other hand, MSC-EVs induced an increase in the expression of pro-fibrotic markers in activated LX-2. The analysis of the RNA cargo of HLSC-EVs revealed the presence of several miRNAs involved in the regulation of fibrosis and inflammation. Predictive target analysis indicated that several microRNAs (miRNAs) contained into HLSC-EVs could possibly target α-SMA. In particular, we demonstrated that HLSC-EVs shuttled miR-146a-5p and treatment with HLSC-EVs increased miR-146a-5p expression levels into LX-2. In conclusion, this study demonstrates in vitro that MSC-EVs support the activation of LX-2, whereas HLSC-EVs can attenuate the activated phenotype of LX-2 by delivering miRNAs, such as miR-146a-5p, that reduce the expression of pro-fibrotic marker α-SMA.

Renal progenitor-derived extracellular vesicles ameliorate podocyte function in a human in vitro model of glomerular permeability

Lola Buono1, Corinne Iampietro1, Maddalena Arigoni1, Raffaele Calogero1, Benedetta Bussolati1

1Department of Biotechnology and Health Sciences, University of Turin, 10126 Turin, Italy

Idiopathic Nephrotic Syndrome is the most common glomerular disease in children. The large majority of patients are steroid sensitive while 10-20% of children appear resistant, and frequently progress to end-stage renal disease. We here recovered podocytes from the urines of children affected by steroid resistant glomerular diseases and evaluated the effects of renal progenitor-derived extracellular vesicles (EVs) on their filtration ability, and the mechanisms involved.

Freshly collected urine derived from three nephropathologic patients were centrifuged and pelleted podocytes were kept in culture. The patients were all resistant to classic therapies and presented, respectively, a mutation in podocin gene, a mutation in ift172/kif14 with a defect in cilia formation and, finally, a diabetic nephropathy. Urine-derived podocytes were seeded on culture inserts and a filtration assay was performed by measuring the transit of FITC-albumin from the lower compartment to the upper one. EVs, used for the treatment of podocytes, were isolated from immortalized renal progenitor cells isolated from a preterm neonate of 25 weeks. The cells were expanded and starved overnight and supernatants were ultracentrifuged to obtain EVs. EVs were characterized by MACSPlex flow cytometry for the expression of CD63, CD81, and CD9, and the main mesenchymal, platelet, and endothelial surface markers. Podocytes were treated with EVs Total RNA sequencing was performed on podocytes untreated and treated with EVs.

The treatment with the renal progenitor-derived EVs was able to significantly reduce FITC-albumin permeability in all the different patient-derived podocytes tested. Total RNA sequencing unraveled several EV-regulated genes with a role in kidney physiology.

In conclusion, we established a human in vitro model that functionally reproduces the podocyte filtration barrier of the nephron, as a model for personalized medicine. We identified renal progenitor-derived EVs as regulators of podocyte permeability, showing that their administration can significantly ameliorate podocyte function in genetic-driven steroid resistant glomerulonephritis.

Extracellular vesicles derived from mesenchymal stromal cells cultured in a clinical-grade medium promote human cartilage recovery.

M.E.F. Palamà1, G. Shaw2, D. Reverberi3, D. Pisignano4,5, K. Cortese6, F. Barry2, M. Murphy2, C. Gentili1

1Department of Experimental Medicine (DIMES), University of Genoa, Italy, 2Regenerative Medicine Institute (REMEDI), National University of Ireland Galway (NUI Galway), Galway, Ireland,

3U.O. Molecular Pathology, IRCCS Policlinico San Martino, Genoa, Italy, 4Nanoscience Institute CNR-NANO (NEST), Pisa, Italy, 5Department of Physics, University of Pisa, Pisa, Italy, 6DIMES,

Department of Experimental Medicine, Human Anatomy, University of Genoa, Italy.

Osteoarthritis (OA) is a disabling joint disorder causing articular cartilage degeneration. Currently, treatments are mainly pain- and symptom-modifying, rather than disease-modifying. Human bone marrow stromal cells (hBMSCs) have emerged as a promising paracrine mechanism-based approach for the treatment of OA. Many studies demonstrate that MSCs attend to tissue repair through secretion of trophic factors or extracellular vesicles [1.2]. We developed a “donor-to-patient” closed, scalable and automated system for aseptic therapeutic cell manufacturing using a xeno-free medium. We validated the potential therapeutic benefits of secreted extracellular vesicles (EVs) isolated from MSC culture in this innovative culture system, for cartilage repair.

We characterized extracellular vesicles (EVs) derived from hBMSCs, grown in a xeno-free culture system (XFS) compared to a conventional fetal bovine serum (FBS) culture system, in normoxic and hypoxic culture setting. We investigated also the therapeutic potential of EVs in an in vitro model of OA. We characterized the miRNA content of EVs in different culture setting to select putative miRNA that could be involved in a biological function.

The biological effects of XFS- and FBS-cultured hBMSC-derived EVs was tested on IL-1α treated hACs in an experiment designed to mimic the OA environment. We observed that under inflammatory conditions hACs are able to recruit and internalize more MSC-derived EVs, especially those derived from cells cultured in our XFS system and in hypoxic condition. Analysis of miRNA content showed upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as miR-17, miR-140, miR-145, miR-30a, miR-29a, miR-130a, miR-199a. Interestingly, most of the miRNA found in our samples seem to be involved in cartilage homeostasis and influence TGF-beta signalling.

In conclusion, the XFS medium was found to be suitable for isolation and expansion of hBMSCs with increased production of EVs. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

References

[1] Mancuso, P., Raman, S., Glynn, A., Barry, F., and Murphy, J. M. (2019). Mesenchymal stem cell therapy for osteoarthritis: the critical role of the cell secretome. Front. Bioeng. Biotechnol. 7:9. doi: 10.3389/fbioe.2019.00009.

[2] Cosenza, S., Ruiz, M., Toupet, K., Jorgensen, C., and Noël, D. (2017). Mesenchymal stem cells derived exosomes and microparticles protect cartilage and bone from degradation in osteoarthritis. Sci. Rep. 7:16214. doi: 10.1038/s41598-017- 15376- 8

The role of extracellular vesicles in TDP-43 proteinopathies

E. Casarotto1, D. Sproviero2, E. Corridori2, M.C. Gagliani3, B. Tedesco1, R. Cristofani1, V. Ferrari1, M. Chierichetti1, M. Cozzi1, F. Mina1, P. Rusmini1, M. Galbiati1, S. Gagliardi2, K.

Cortese3, C. Cereda2, A. Poletti1, V. Crippa1

1Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018-

2022, University of Milan, Milano (Milano), Italy. 2 Genomic and post-Genomic Center, IRCCS - Mondino Foundation, Pavia (Pavia), Italy

3 Section of Anatomy, Department of Experimental Medicine, University of Genoa, Genova (Genova), Italy

TDP-43 proteinopathies are a group of diseases characterized by an abnormal accumulation of pathological inclusions of the TAR DNA-binding protein of 43 KDa (TDP-43), in the cytosol of affected cells. Among these, there are Amyotrophic lateral scerosis (ALS), Frontotemporal lobar degeneration (FTLD), Alzheimer’s disease (AD) and Parkinson’s disease (PD). Disease-related inclusions contain insoluble forms of TDP-43 and of its C-terminal fragments of 35 (TDP-35) and 25 KDa (TDP-25), that are neurotoxic for cells. Interestingly, TDP-43 and its C-terminal fragments have been also found extracellularly, both secreted as free proteins or into extracellular vesicles (EVs), in particular into exosomes. These extracellular species are able to spread from one cell to another, therefore they could be toxic for the recipient cells and contribute to the disease. However, they could also represent a protective mechanism for the secreting cell, working together with the intracellular protein quality control (PQC) system, in the clearance of TDP species. In order to better understand the role of extracellular TDP-43 in health and disease, we investigated the secretion of TDP-43 by taking into account both large and small vesicles (LVs and SVs, respectively). In particular, we carefully analysed LVs and SVs protein cargo in terms of TDP-43 species (FL vs fragments) and solubility (soluble vs insoluble), by comparing it with the intracellular TDP-43. Moreover, we analysed how and if PQC blockage might affect TDP-43 secretion. For these purposes, we isolated EVs produced by an immortalized neuronal cell line using the differential ultracentrifugation method, and we analysed them for size and count through the Nanoparticle Tracking Analysis, for morphology through the transmission electron microscopy and for their protein content through western blot analysis. We found that both TDP-43 and its C-terminal fragments (especially TDP-35) were physiologically secreted in EVs, in particular into LVs; interestingly, secreted TDP species were mainly insoluble, while intracellular TDPs were in a soluble form. Moreover, we discovered that LVs were immunoreactive for the lipidated form of the Microtubule-associated protein 1B-light chain 3 (MAP1BLC3-II), that has been recently proven to be a crucial component of a new secretory autophagy pathway, the LC3-dependent extracellular vesicle loading and secretion (LDELS), and they contained important other PQC components, known to be involved in TDPs clearance. Finally, we observed that a complete PQC blockage boosted EVs secretion, in particular that of MAP1BLC3-II positive LVs, further increasing the secretion of TDP C-terminal fragments and PQC components. In conclusion, we demonstrated that, in physiological condition, EVs, in particular LVs, possibly secreted via LDELS, could represent an important mechanism for the clearance of insoluble TDPs species. This mechanism is specifically boosted when PQC is impaired. This aspect is particularly relevant, since PQC impairment is a common condition observed in TDP-43 proteinophaties. Funding Fondazione Cariplo, Italy n. 2017-0747; Italian Ministry of University and Research (MIUR), PRIN n. 2017F2A2C5; Bando SEED 2019 - Bando Straordinario per Progetti Interdipartimentali dell'Università Statale di Milano; PSR2020 - Piano di sostegno alla ricerca dotazione 2020 LINEA 2 AZIONE A dell'Università Statale di Milano.

Tau seeds in extracellular vesicles induce tau accumulation indegradative organelles of cells

Giona Pedrioli1,2, Ester Piovesana1,3, Claudia Magrin1,3, Martina Sola1,3,Giorgia Senesi1, Stéphanie Papin1, Paolo Paganetti1,3

1. Neurodegeneration Research Group, Ente Ospedaliero Cantonale, Lugano, Switzerland2. PhD Program of the Biozentrum, University of Basel, Switzerland

3. Università della Svizzera italiana, Faculty of Biomedical Sciences, Lugano, Switzerland

Clinical progression of tauopathies may result from transcellular propagation of pathogenic

Tau seeds with the possible involvement of extracellular vesicles as transport vectors. The

mechanism regulating extracellular vesicle cargo delivery to recipient cells is poorly

understood. We established a cell model for investigating the transport of proteins by

extracellular vesicle. In this model, extracellular vesicles are readily internalized and

accumulate in degradative organelles. For the first time, we show that in this acidic

compartment, profibrillogenic Tau delivered by extracellular vesicles directly interacts with

Tau expressed by the recipient cells and cause its accumulation by a process that involves

the participation of autophagy. Thus, the degradative compartment of cells may represent

the subcellular site initiating a cascade of events resulting in early hallmarks of tauopathies.

These are characterized by seeded Tau accumulation, pathology-associated epitopes,

degradative organelle stress and cytotoxicity. The involvement of autophagy to this process

and the relative accessibility of the degradative pathway for extracellular agents, support

possible modes of intervention to slow-down the progression of neurodegeneration

Extracellular Vesicles analysis in cerebrospinal fluid of transgenic pigs: a model for biomarker discovery in Amyotrophic Lateral Sclerosis

M. T. Golia1, S. Pucci1, S. Colombo1, M. Cretich2, R. Frigerio2, M. Chiari2, C. Corona3,

C. Palmitessa3, C. Testori3 , E. Berrone 3 , G. Cagnotti 4, A. Perota 5, R. Duchi 5, L. Bergamaschi 5, C. Galli 5 and C. Verderio1

1Consiglio Nazionale delle Ricerche, Istituto di Neuroscienze (IN), Milano, Italy

2 Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Milano, Italy

3 Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta; Torino, Italy 4 Università degli Studi di Torino, Dipartimento di Scienze Veterinarie; Torino, Italy

5 Avantea, Cremona, Italy.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease gradually leading to paralysis of the whole body. Recently, a transgenic pig model expressing the human pathogenic superoxide dismutase SOD1 gene (hSOD1G93A) has been generated that displays behavioral, neurodegenerative and molecular features of ALS and is characterized by a quite long pre-symptomatic phase, offering the unique opportunity to investigate early ALS biomarkers. Extracellular vesicles (EVs) contain markers of parental cells and represent a valuable source of biomarkers for neurodegenerative diseases. In this study we measured the concentration and size distribution of EVs present in the cerebrospinal fluid (CSF) of heterozygotes hSOD1G93A pigs [1] at different disease stages (early pre-symptomatic, late pre-symptomatic and symptomatic) and we characterized their cell origin. TRPS and NTA analysis both showed that the concentration of EVs increases with age in wild type but not in transgenic pigs, with a statistically significant difference at symptomatic stages (32-40 months). In parallel experiments, EVs were isolated by differential centrifugation from the CSF (2ml) and analyzed by western blot for the neuronal marker CD171, the astrocyte marker ADHL1, the microglial marker TME119 and for the EV markers AnnexinA1 e Alix. We found a larger proportion of neuron-derived EVs at late pre-symptomatic stages and of astrocytic EVs compared to wild type animals. Microglia-derived EVs tended to be more abundant at both late pre-symptomatic and symptomatic stages in hSOD1G93Apigs, but the difference was not statistically significant. Western blot analysis of cervical and lumbar tracts of the spinal cords revealed increased expression of the astrocytic markers GFAP and pentraxin3 in symptomatic pigs, indicating a positive correlation between the level of astrocyte activation and EV production from these cells. Furthermore, in agreement with defective EV production in transgenic pigs, we found a decreased expression of Alix, a protein involved in EV biogenesis, in symptomatic pigs. Supported by Regione Lombardia (project INTERSLA ID 1157625) References [1] Crociara, P., Chieppa, M.N., Vallino Costassa, E., Berrone, E., Gallo, M., Lo Faro, M., Pintore, M.D., Iulini, B., D’Angelo, A., Perona, G., Botter, A., Formicola, D., Rainoldi, A., Paulis, M., Vezzoni, P., Meli, F., Peverali, F.A., Bendotti, C., Trolese, M.C., Pasetto, L., Bonetto, V., Lazzari, G., Duchi, R., Perota, A., Lagutina, I., Quadalti, C., Gennero, M.S., Dezzutto, D., Desiato, R., Boido, M., Ghibaudi, M., Valentini, M.C., Caramelli, M., Galli, C., Casalone, C., Corona, C., 2019. Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS. Neurobiol. Dis. 124, 263–275.

Role of IL-5 in the release of eosinophilic extracellular vesiclesTommaso Neri 1, Erica Bazzan 2, Valentina Scalise 1, Chiara Sanguinetti 1, Dario Nieri 1,3, Stefania Lombardi 4,

Roberto Pedrinelli 1, Pierluigi Paggiaro 1, Alessandro Celi 1,3

1 Centro Dipartimentale di Biologia Cellulare CardioRespiratoria, Dipartimento di PatologiaChirurgica, Medica, Molecolare e dell’Area Critica, Università di Pisa, Pisa, Italy

2 Dipartimento di Scienze Cardio-Toraco Vascolari e Sanità Pubblica, Università di Padova, Padova, Italy3 Sezione a Valenza Dipartimentale di Fisiopatologia Respiratoria e Riabilitazione

Respiratoria Universitaria, Dipartimento Cardio-Toraco Vascolare, AziendaOspedaliero Universitaria Pisana, Pisa, Italy

4 SSD Analisi ChimicoCliniche ed ImmunoAllergologia, USL Toscana Nordovest,Massa Carrara, Italy

Over the last few years, several studies have demonstrated an involvement of extracellular vesicles(EV) in the pathogenesis of asthma. Eosinophil-derived EV are increased in asthmatic patients [1];these EV also act on structural lung cells to produce epithelial damage, increase smooth muscleproliferation, and modify the expression of several proinflammatory cytokines and signaling factors [2].Moreover, EV from asthmatic patients differ also in composition and function compared with those fromhealthy subjects, again lending support to the hypothesis that EV are involved in the regulation ofasthma pathology [3]. Taken together, these observations are consistent with the hypothesis that EVderived from eosinophils, not detected with standard laboratory techniques, exert biological activitiessimilar to those of the parental cell.Based on these observations, we investigated the role of IL-5, one of the key cytokines responsible forthe development of asthma, in the release of EV from eosinophils. In an in vitro model, we used ahuman eosinophil cell line (EOL-1); cells were treated with different concentrations of IL-5, and therelease of EV was assessed by a functional assay and by flow cytometry. Benralizumab, an anti-IL-5receptor monoclonal antibody, developed for the treatment of asthma, was used to confirm thespecificity of this observation. IL-5 upregulates the release of EV by EOL-1 in a dose-dependentmanner (Fig 1) and benralizumab inhibits this effect (Fig 2a, b).

These preliminary data show an involvement of IL-5 in the release of EV by cells of the eosinophiliclineage, EOL-1. Additionally, an IL5 receptor blocking antibody, benralizumab, currently used to treatsevere asthma, inhibits this release, confirming the specificity of IL-5 involvement. These observationslay the groundwork for further investigating a currently unexplored role for IL-5 in the pathogenesis ofasthma.

References: [1] Mazzeo, C., et al. Exosome secretion by eosinophils: A possible role in asthma pathogenesis. JAllergy Clin Immunol. (2015).[2] Cañas, J.A., et al. Exosomes from eosinophils autoregulate and promote eosinophil functions. J LeukocBiol. (2017).[3] Torregrosa Paredes, P., et al. Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotrieneproduction in allergic asthma. Allergy. (2012)

Macrophages, Extracellular Vesicles and Immune Function: a new

crosstalk in metabolic diseases and related alterations

S. Tacconi2, C. Sbarigia1, S. Longo2, M. Fidaleo 1,3, A.M. Giudetti2, L. Dini1,3,4

1 Department of Biology and Biotechnology “C. Darwin”, University of Rome Sapienza, Rome, Italy;

2 Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento, Lecce, Italy;

3 Research Center for Nanotechnology for Engineering of Sapienza (CNIS), University of Rome Sapienza, Rome, Italy;

4 CNR Nanotec, Lecce, Italy

Growing evidence describes how the immune system is actively involved in the etiopathogenesis of several metabolic diseases, such as obesity, type 2 diabetes (T2D), insulin resistance, and dyslipidemia, which are often associated with a status of systemic inflammation. Among the innate immunity cells, macrophages are important modulators of inflammation, due to their capacity to differentiate towards the M1 pro-inflammatory or M2 anti-inflammatory phenotype. Next to soluble factors, such as cytokines and chemokines, macrophages can modulate the activity of other target cells by releasing extracellular vesicles (EVs). Recently, a new macrophage phenotype, called “metabolically activated macrophages” (MMe), associated with obesity and induced by several metabolic stimuli, such as free-fatty acids (FFAs), high insulin and glucose has been described. The functions of EVs-derived macrophages in metabolic disorders, such as T2D, which are characterized not only by insulin signaling impairments, but also by systemic inflammation, have not been extensively studied. Therefore, a comprehensive understanding of EVs-derived macrophages and their role in disease progression and treatment is needed. For this reason, the aim of this work was to determine the effects on macrophage polarization and EVs release after exposure to a condition of hyperlipidemia, alone or associated with hyperglycemia. In our in vitro system, THP1-derived macrophages (M0) were treated with a mix of fatty acids (palmitate and oleate), in absence (M0/FFAs) or presence of 15 mM glucose (MG15/FFAs) for 24 hours. After the analysis of the polarization phenotype, using specific inflammatory markers and cytokines, metabolic adaptations and EVs fractions (microvesicles fraction, MVs; and exosome fraction, EXOs) of macrophages were analyzed. Specifically, EVs-enriched fractions were characterized both morphologically and quantitatively by electron microscopy, Nanotracking analysis and EVs-related markers expression. The obtained data showed that exposure to lipids and/or glucose overload determines the polarization of macrophages towards the pro-inflammatory MMe phenotype and diverse metabolic adaptations, in terms of insulin response, lipid profile and metabolism, and metabolites composition. In order to identify possible modifications in lipid and metabolite contents of vesicles, a qualitative lipidomic and metabolomic analysis was performed on EVs-enriched fractions (including MVs and EXOs) from MMe-activated macrophages. This revealed a different lipid and metabolic profile of EVs in response to FFAs or glucose challenge. Taken together, our results showed that nutrient excess, a hallmark of metabolic distresses, modulates the inflammatory and metabolic response of macrophages leading to clear changes in lipids and metabolites composition of released EVs.

Role of plasma extracellular vesicles in lung cancer progression

Ilaria Petraroia1, Francesca Pontis1, Paola Suatoni2, Ugo Pastorino2, Gabriella Sozzi1 and Orazio Fortunato1

1 Tumor Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori 2 Thoracic Surgery Unit, Fondazione IRCCS Istituto Nazionale dei Tumori

Introduction: Today, lung cancer is one of the major causes of cancer related-deaths worldwide. Despite of improvements in early diagnosis and treatments with the development of immunotherapeutic drugs the survival is still very low. Recently, it has emerged that extracellular vesicles (EVs) are critical players in tumor progression, due to their ability to delivery functional biomolecules such as miRNAs or proteins, to recipient cells. The aim of this project is to evaluate EVs as biomarker of prognosis in early stage lung cancer patients and to evaluate the role of EVs in lung carcinogenesis. Materials and methods: Plasma EVs were isolated by ultracentrifugation from a group of 72 lung cancer patients with different prognosis (19 dead within two years (EV-D), and 53 of these patients alive after five years (EV-A) from the diagnosis). EV’s characterization was performed accordingly to the MISEV guidelines and their origin and functional properties were investigated using in vitro assays. EVs characterization was performed using FC (Flow cytometry), NTA (Nano Tracking Analysis) and Western Blot. The functional role of EVs was evaluated using Endothelial Cells (HUVEC) by FC, Real Time and Matrigel assay. Results: The results showed no differences in terms of size and particles number, between EV-A and EV-D. In addition we noted the presence of Tetraspanins, CD9+, CD81+ and CD63+ on EVs surface without any differences between the two groups. To investigate the EVs-organotropism in vitro we treated stromal cells (endothelial, fibroblast, macrophages and epithelial cells) for 24h with 1ug of PKH26-labelled-EVs. EVs are incorporated mostly by endothelial cells, macrophages and fibroblast without any difference between the two groups. Following these results, we treated endothelial cells with 15ug of plasma EV’s and we evaluated the modulation of pro-angiogenic features such as expression of adhesion molecules and growth factors by Real time PCR and FC. The results showed that the mRNA levels of cytokines (CXCL1, CXCL2, CXCL11) and surface receptors (VCAM-1, VEGR and CXCR4) are up-regulated in EV-D-treated HUVEC compared to EV-A. The increase of CXCR4 and VCAM on the surface of EV-D treated cells was confirmed by FC. No differences were observed in the formation of capillary-like structures comparing the two groups. Conclusion: Our preliminary findings demonstrated that EVs from lung cancer patients with a worse prognosis are able to activate the endothelium towards a pro-angiogenic phenotype. Further studies are needed to better elucidate the role of EVs in lung tumorigenesis.

Surface marker analysis of extracellular vesicles from tumor extracellular matrix

S. Tassinari1, E. D’Angelo2,3, F. Caicci4, M. Agostini2,3, F. Collino5,6, B. Bussolati1

1 Department of Molecular Biotechnology and Health Sciences, University of Torino, Turin, Italy.

2 First Surgical Clinic, Department of Surgery, Oncology and Gastroenterology, University of Padova, Padua, Italy.

3 Institute of Pediatric Research, Fondazione Città della Speranza, Padua, Italy. 4 Department of Biomedical Sciences, University of Padova, Padua, Italy.

5 Laboratory of Translational Research in Paediatric Nephro-urology, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy.

6 Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy.

The tumor microenvironment is a complex entity composed of cellular and non-cellular elements that surround the tumor and promote its growth. The non-cellular components include the extracellular matrix (ECM), that represents the most abundant element, and the secreted metabolites, including soluble factors and extracellular vesicles (EV). It is of interest to better characterize EV within ECM considering their crucial role in tumor progression and diffusion. Several reports showed that EV cargo influences the stroma by activating a molecular pathway that differs, in part, from the ones modulated by soluble factors. Since EV cargo contains information on cell sub-proteome, it can either be used as a source of disease biomarkers and cancer staging. This project aims to identify and characterize EV from decellularized tumor biopsies, at different stages. A model of ECM has been used, obtained by decellularization of human colon-rectal cancer (CRC) biopsy and normal colon biopsy (HT) as control. We developed a protocol of mild enzymatic digestion, which was necessary to detach EV trapped within the ECM, followed by sequential centrifugations and ultra-centrifugations. Using Nanoparticle Tracking Analysis, we observed EV average yield of 1.70*108

particles/mg tissue. Transmission electron microscopy was conducted to confirm the presence of intact vesicles. Moreover, cytofluorimetric analysis has been performed to characterize EV surface markers using MACSPlex exosome kit. Exosomal markers including, CD9, CD63, CD81 were detected in our preparations, showing that the exosome enriched EVs fractions were present in decellularized ECM from both tumor and normal colon tissue. We detected that CD81 had the lowest expression among the samples. A change in the expression of immune and adhesion EV surface markers between the normal and cancer decellularized ECM highlighted the presence of different pattern. In conclusion, ECM-EV can be a powerful tool for the identification of new biomarkers for cancer diagnosis and staging. Furthermore, the surface markers characterization of EV may provide useful information to understand how vesicles migrate between tissues under both physiological and pathological conditions.

The relationships between cancer cells and the surrounding non-neoplastic cells are of

crucial importance in several aspects of tumor development. It has been known for a while

that cancer cells are metabolically distinct from other non-transformed cells [1]. This

metabolic phenotype is not peculiar to cancer cells but reflects the characteristics of the

tumor microenvironment. Recently, it has been shown that extracellular vesicles (EVs) are

involved in the metabolic switch occurring in cancer and tumor-stroma cells and sustain the

growth of tumor cells [1]. Previously, we showed that differentiation process affects the

content of EVs released by Colorectal Cancer-Cancer Stem Cells (RC-CSCs) [2,3]. This

study aims to analyze the effects of differentiation on cancer-associated fibroblasts (CAFs)

metabolism reprogramming. We found that CRC-CSCs-released EVs carry significant levels

of aminoacids and metabolites. By proteomic analysis, we demonstrated that EVs released

during CRC-CSCs differentiation are enriched in glycolytic enzymes related to cancer

metabolism compared to EVs released by undifferentiated counterparts. Moreover, we

showed, by metabolomic analysis, that CAFs treated with EVs released by differentiated

CRC-CSCs switch from oxidative to glycolytic metabolism. Evaluation of glycolytic-related

genes and proteins, lactate production and glucose uptake by RT-PCR, western blot, lactate

and glucose assays, respectively, confirmed metabolomic data. Our findings reveal a role of

EVs in metabolic reprogramming of CAFs during the differentiation process of CRC-CSCs.

[1] Lucchetti D, Ricciardi Tenore C, Colella F, Sgambato A. Extracellular Vesicles and Cancer: A Focus on Metabolism, Cytokines, and Immunity. Cancers (Basel). 2020;12(1):171. PMID: 32015297 [2] Lucchetti D, Calapà F, Palmieri V, Fanali C, Carbone F, Papa A, De Maria R, De Spirito M, Sgambato A. Differentiation Affects the Release of Exosomes from Colon Cancer Cells and Their Ability to Modulate the Behavior of Recipient Cells. Am J Pathol. 2017;187(7):1633-1647. PMID: 28619275. [3] Lucchetti D, Colella F, Perelli L, Ricciardi-Tenore C, Calapà F, Fiori ME, Carbone F, De Maria R, Sgambato A. CD147 Promotes Cell Small Extracellular Vesicles Release during Colon Cancer Stem Cells Differentiation and Triggers Cellular Changes in Recipient Cells. Cancers (Basel). 2020;12(2):260. PMID: 31973205.

EVs released during the differentiation of Colorectal Cancer-Cancer Stem Cells induces a switch from oxidative to glycolytic metabolism in

cancer-associated fibroblasts

D. Lucchetti 1, F. Colella1, G. Artemi 1, C. Ricciardi-Tenore1, F. Calapà2, G. Lazzarino 3, M.E. Fiori2, F.

Vincenzoni 4, A. Urbani 4,5, B. Tavazzi 4,5, R. De Maria1,5, A. Sgambato 1,6 1 Department of Translational Medicine and Surgery, Faculty of Medicine, Catholic University of the

Sacred Heart, Rome, Italy; 2Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

3 UniCamillus-Saint Camillus International University of Health Sciences, 00131 Rome, Italy 4 Department of Basic Biotechnological Sciences, Intensive and Perioperative Clinics, Catholic

University of the Sacred Heart of Rome, 00168 Rome, Italy 5 Fondazione Policlinico Universitario "A. Gemelli"-IRCCS, 00168 Rome, Italy

6 IRCCS-Centro di Riferimento Oncologico della Basilicata (CROB), Rionero in Vulture (PZ), Italy

Notch pathway role in multiple myeloma progression via extracellular vesicles-mediated communication

Domenica Giannandrea1, Michela Colombo1, Natalia Platonova1, Valentina Citro1, Filippo Maltoni1,

Mara Mazzola2, Anna Pistocchi2, Raffaella Adami1, Vincenza Dolo3, Ilaria Giusti3, Laura Cantone4,

Valentina Bollati4, Mauro Turrini5, Raffaella Chiaramonte1

1 Department of Health Sciences, Università degli Studi di Milano, Italy 2 Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano,

Italy 3 Department of Life, Health and Environmental Sciences, Università degli Studi dell'Aquila, Italy 4 Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Italy 5 Division of Hematology, Valduce Hospital, Como, Italy

Multiple myeloma (MM) is a still incurable hematological disease of clonal plasma cells characterized by poor prognosis due to its localization in the bone marrow (BM). The homing of MM cells in the BM allows them to establish pathological interactions with the surrounding normal cells, inducing a pro-tumorigenic behavior. As a matter of fact, “educated” BM resident cells are reported to support neoplastic cell growth, inducing the formation of new vessels and osteolytic bone lesions, affecting MM patient’s quality of life and survival. Notch pathway plays a key role in the modulation of BM microenvironment. MM cells overexpressed Notch2 and its ligands, Jagged1 and 2, leading to Notch signaling activation in the same tumor cells and in the surrounding cells of the BM niche [1]. Myeloma-derived extracellular vesicles (MM-EVs) represent novel pro-tumorigenic players in the interplay between MM and BM by delivering molecular messengers [2]. Our previous preliminary in vitro study demonstrated a role for Notch pathway in MM-EV-mediated communication by showing that MM-EVs carry Notch2, Jagged1 and 2 and their presence was associated to MM-EVs ability to promote angiogenesis and osteoclastogenesis. Additionally, through a zebrafish embryo Notch reporter, we showed that MM-EVs were able to activate Notch pathway in recipient cells. Here, we have refined these promising preliminary results. Through a characterization of the morphology, size, and number of MM-EVs shed by human MM cell lines using transmission electronic microscopy and nanoparticle tracking analysis, we assessed that the amount of Notch members does not affect MM-EV size and number. A western blot analysis indicated that Jagged1 and 2, and Notch2 were present both in large and in small vesicles, separated by ultracentrifugation at 20 krpm or 110 krpm. Moreover, we confirmed that the observed functional effects of MM-EVs on endothelial cells and monocyte cells were associated with their uptake, using confocal microscopy and flow cytometric analysis. Importantly, we showed that EVs can transfer Notch pathway members to recipient cells, following the transfer of an HA-tagged Notch2 from engineered H293 sending cells to receiving cells and, finally, we confirmed that Notch signaling members expressed on MM-EV surface play a role in mediating the biological outcome since their neutralization through specific antibodies also inhibits the observed functional effect. Overall, our findings suggest that MM-EVs carry and transfer Notch pathway members to BM recipient cells affecting their pro-tumorigenic activity, leading to key processes involved in MM progression such as osteoclastogenesis and angiogenesis. The functional outcome is at least in part mediated by the activation of Notch signaling in target cells. These results strengthen our previous evidence that targeting the Notch pathway may be a valid therapeutic strategy to hamper the pro-tumorigenic role of EV in MM progression.

Supported by AIRC Investigator Grant n. 20614

References [1] M. Colombo, L. Mirandola, N. Platonova, L. Apicella, A. Basile, A.J. Figueroa, E. Cobos, M. Chiriva-Internati, and R.

Chiaramonte, Notch-directed microenvironment reprogramming in myeloma: a single path to multiple outcomes.

Leukemia 27 (2013) 1009-18.

[2] M. Colombo, D. Giannandrea, E. Lesma, A. Basile, and R. Chiaramonte, Extracellular Vesicles Enhance Multiple

Myeloma Metastatic Dissemination. International journal of molecular sciences 20 (2019).

Extrachromosomal FGFR2 gene is delivered in extracellular vesicles in two models of cancer of unknown primary

Irene Salamon1, Gianluca Storci1, Maria Naddeo1, Beatrice Fontana1, Elisa Porcellini1, Salvatore Serravalle2, Michele Guescini3, Massimiliano Bonafè1, Manuela Ferracin1

1 Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna,

Bologna, Italy. 2 Division of Pediatrics, IRCCS Azienda Ospedaliero-Universitaria di Bologna, 40138 Bologna,

Italy. 3 Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, Italy.

The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cells release EVs in the extracellular space, which can interact with recipient cells inducing regulation of gene expression, activation of specific pathways in pathological settings and stimulation of cancer progression. We focused our attention on EVs released by two cell line models of cancer of unknown primary site (CUP) we recently developed. This tumor type comprises 3-5% of new cancer cases and presents with metastases of unknown or uncertain origin and no apparent primary tumor. We developed two CUP cell line models that are both characterized by FGFR2 gene amplification. It has been recognized that tumors use circular extrachromosomal DNA (ecDNA) as a way to increase oncogenic amplification. Therefore, we hypothesized that FGFR2 amplification in our CUP cell lines could be linked to ecDNA generation and that this ecDNA could be loaded as cargo inside EVs. We identified FGFR2 amplification in ecDNA using FISH assay. Then, we isolated extracellular vesicles from culture medium using an ultracentrifugation-based protocol. We confirmed the loading of FGFR2 gene inside the vesicles, and its enrichment in the exosomes fraction. FGFR2 copy number was quantified using a probe-based droplet digital PCR assay. Then, we assessed the circular nature of the FGFR2 ecDNA using the Plasmid-Safe ATP-dependent DNAse to digest linear DNA. In conclusion, we identified a model of oncogene amplification and delivery in cancers of unknown primary that could explain the high metastatic potential of this cancer type.

Caveolin-1-overexpression induces extracellular vesicle

secretion in a model of rhabdomyosarcoma

Serena Maggio1, Emanuela Polidori1, Paola Ceccaroli1, Laura Graciotti2, Andrea Cioccoloni1,

Michela Battistelli1, Silvia Codenotti3, Gabriella Pocsfalvi4, Fabiola Olivieri2,5, Massimiliano

Bonafè6, Alessandro Fanzani3, Michele Guescini1*

1Department of Biomolecular Sciences (DISB), University of Urbino Carlo Bo, Via I Maggetti, 26, 61029 Urbino, Italy. 2Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy.

3Department of Molecular and Translational Medicine (DMMT), Università degli Studi di Brescia, Italy. 4 National Research Council of Italy Napoli, Campania, Italy.

5Center of Clinical Pathology and Innovative Therapy, IRCCS INRCA, National Institute, Ancona, Italy. 6Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Italy.

* Correspondence: michele.guescini@uniurb.it; Tel.: +39-0722-303424; Fax: +39-0722-303401

Caveolin-1 (CAV-1) is an integral membrane protein required to generate caveolae and cholesterol-enriched

lipid rafts of the plasma membrane. Moreover, CAV-1 binds to many proteins, controls cholesterol

homeostasis, and regulates various cell functions. CAV-1 has a controversial role in cancer, it is widely

accepted that loss of CAV-1 correlates with early-stage tumor progression, while its over-expression and

phosphorylation are associated with recurrence and metastatic disease. For example, it has been shown that

CAV-1 cooperates to tumor growth and metastatic potential in rhabdomyosarcoma (RD). A considerable body

of evidence suggests the possibility that extracellular CAV-1 may be relevant in cancer cell metastasis. The

present work aims to investigate if the increased aggressiveness of RD cells overexpressing CAV-1 correlates

with an altered extracellular vesicle (EV) release.

EVs were isolated by differential ultracentrifugation and density gradient separation methods from

conditioned media of control (RD-Ctrl) and metastatic RD cell lines overexpressing CAV1 (RD-CAV1).

Collected small (sEVs) and large (lEVs) extracellular vesicles were characterized by NanoparticleTracking

Analysis (NTA), transmission electron microscopy (TEM) and western blot analysis (WB).

The applied ultracentrifugation protocol allowed the isolation of two EV subpopulations, called sEVs and

lEVs, of about 93 nm and 170 nm in diameter, respectively. The obtained data showed that RD-CAV1 cells

release 3-4 fold more EVs compared to control cells; however, NTA revealed that CAV-1 overexpression seems

not to affect the sEV versus lEV production ratio with sEVs about 10 times more abundant than lEVs in both

RD cell models. The efficient separation between sEVs and lEVs was supported by western blot analysis: small

vesicles exhibit the exosomal markers Alix, Flot-1, Syntenin-1, and TSG101, whereas large EVs were positive

for Calnexin, an endoplasmic reticulum marker. Interestingly, RD-CAV1 EVs do not express the tetraspanins

CD63, CD81 and CD9, which instead were detected in RD-Ctrl EVs, suggesting that CAV-1 over-expression

induces an alteration of the EV biogenesis.

Proteomic analysis confirmed the absence of tetraspanins in the sEVs released from RD-CAV-1 and showed

that the lack of tetraspanin is associated with a significant decrease in EV protein loading; in fact, we identified

about 1000 different proteins in RD-Ctrl sEVs, whereas only 547 proteins were detectable in RD-CAV-1 sEVs.

The reported data suggest that, in addition to its well-established structural role, CAV-1 is a key regulatory

factor potentially involved in the remodelling of the tumor microenvironment by stimulating the release of

EVs deeply altered in protein composition.

Hypoxia-derived EVs deliver miR-210-3p to elicit a pro-metastatic phenotype in Neuroblastoma cells A. Fietta1, P. Fusco1,2, M.R. Esposito1,2, E. Cimetta1,2

1University of Padua, Department of Industrial Engineering (DII), Padua, Italy 2Fondazione Istituto di Ricerca Pediatrica Città della Speranza (IRP), Padua, Italy

Neuroblastoma (NB) is the most frequent extracranial solid tumor in children, causing 10% of all

pediatric oncology deaths. It arises in the embryonic neural crest due to an uncontrolled behavior of sympathetic nervous system progenitors, giving rise to heterogeneous tumors[1]. NB, like other solid cancers, is characterized by hypoxic areas leading to tissue necrosis and neovascularization. The role of hypoxia in NB has been correlated to a more aggressive behavior and to the increased presence of a stem cell-like sub population[2]. Besides undergoing broad intracellular molecular and metabolic adaptations, hypoxic tumor cells extensively communicate with their microenvironment (µENV) to concoct conditions favorable for their survival, growth and metastatic spread. This mode of communication is through diverse secretory factors including extracellular vesicles (EVs). EVs carry diverse molecular information including microRNA (miRNAs), a class of small non-coding RNAs that regulate cellular pathways, differentiation, apoptosis and stem cell maintenance[3]. EVs derived from tumor µENV may communicate with cancer cells in the vicinity by transferring miRNA to regulate the metastatic potential of recipient cells. miRNAs have generated great attention in oncology as they play a fundamental role in the regulation of gene expression and their aberrant expression is present in almost all types of tumors including pediatric ones[4].

We here analyzed the effects of the EVs isolated from SK-N-AS and SK-N-DZ NB cell lines cultured in normoxia (20% O2), hypoxia, (1.5% O2) and reoxygenation (1.5% followed by 20% O2) conditions on naïve NB cells. After validating the internalization of the EVs by immunofluorescence, we studied the effect of EVs derived from cells cultured in hypoxic condition on the migration and invasiveness of NB cells. We provided evidence that EVs derived from hypoxic conditions enhanced the migration and invasiveness of NB cells when compared to their normoxic counterpart.

Moreover, based on previous studies from our laboratory highlighting the relative abundance of a set of miRNAs, we here focused on miR-210-3p and proved that it was highly expressed in cells cultured in hypoxic condition and their derived EVs, compared with normoxic counterpart.

To clarify the role of miR-210-3p in NB, we investigated its effects when overexpressed and inhibited through transient transfection. We showed that EVs isolated from cells transfected with miR-210-3p-mimic and cultured in normoxic condition, increased the migration ability and invasiveness of NB cells. We also demonstrated that cells treated with EVs isolated from cells transfected with miR-210-3p-inhibitor and cultured in hypoxic condition, showed a reduced migration ability and invasiveness than cells treated with EVs isolated from cells transfected with miR-NC (negative control).

Overall, this study provides new evidence that hypoxic tumor cells have the potential to induce specific changes in normoxic tumor cells towards a malignant phenotype by transferring EVs carrying miR-210-3p. Our findings represent the starting point for further studies on the specific EVs-mediated biological processes favoring NB metastatic dissemination, with the ultimate goal of finding predictive biomarkers for better NB patients’ stratification and tailored therapeutic strategies. Supported by ERC Starting grant (ERC-StG) MICRONEX project (UERI17, PI E. Cimetta) References: 1. Huertas-Castano, C., et al., Hypoxia in the Initiation and Progression of Neuroblastoma Tumours. Int J Mol Sci, 2019. 21(1). 2. Kim, Y., et al., Hypoxic tumor microenvironment and cancer cell differentiation. Curr Mol Med, 2009. 9(4): p. 425-34. 3. Kumar, A. and G. Deep, Hypoxia in tumor microenvironment regulates exosome biogenesis: Molecular mechanisms and translational opportunities. Cancer Lett, 2020. 479: p. 23-30. 4. Galardi, A., et al., Exosomal MiRNAs in Pediatric Cancers. Int J Mol Sci, 2019. 20(18).

Adipocyte-derived extracellular vesicles drive prostate cancer progression

F. Fontana1, M. Marzagalli1, M. Raimondi1, E. Carollo2, P. Sartori1, P. Procacci1, D.

Carter2, P. Limonta1

1University of Milan, Department of Pharmacological and Biomolecular Sciences, Milan, Italy 2Oxford Brookes University, Department of Biological and Medical Sciences, Oxford, UK

It is known that an association exists between obesity and risk of prostate cancer (PCa). A crosstalk between adipose tissue and PCa has been demonstrated; however, the study of this dialog has been limited to metabolites and adipokines, although emerging evidence points to a key role of extracellular vesicles (EVs) in the control of tumor progression. Herein, we demonstrated that 3T3-L1 adipocyte conditioned media can affect PC3 and DU145 PCa cell features, inducing increased proliferation, associated with AKT phosphorylation, and invasion, correlated with MMP2 and 9 activation, E/N-cadherin switch and Snail upregulation. Moreover, after exposure to adipocyte conditioned media both PCa cell lines were found to accumulate lipid droplets and, more importantly, to undergo a neuroendocrine differentiation, accompanied by CD44 enhanced expression and docetaxel resistance. Notably, these results were confirmed in 3T3-L1 EV-treated PCa cells, where an increase in glucose consumption, mitochondrial activity, ATP production and ROS generation was also observed, suggesting that adipocyte EVs can reprogram PCa metabolism and drive its aggressiveness. Further studies will be performed to identify the adipocyte EV molecular cargo responsible for the modulation of this dialog.

M. Pucci1, O. Urzi’1, M. Moschetti1, A. Conigliaro1, M. Loria1, R. Gasparro1, R. Alessandro1, S.

Fontana1

1Department of Biomedicine Neuroscience and Advanced Diagnostics, University of Palermo, Sicily, Italy

Colorectal cancer-derived small extracellular vesicles induce epithelial to mesenchymal transition in normal hepatocytes: new insights on the hepatic

pre-metastatic niche formation in colorectal cancer liver metastases.

The liver is the main metastatic site for patients with colorectal cancer (CRC) and represents the most frequent cause of death for patients affected by this cancer. Numerous studies have shown that in the liver the dissemination of cancer cells is the result of complex mechanisms based on two-way interactions between metastatic cancer cells and resident cells. In the recent years, several studies have highlighted the crucial role played by small extracellular vesicles (sEVs) released by cancer cells in initiating pre-metastatic niche formation in the liver, specifically affecting the activities of non-parenchimal cells as Kupfer cells and hepatic stellate cells, while the involvement of the hepatocytes (Heps) remains still unknown. Even if this cell component is the most conspicuous in the liver and it is responsible for several physiological activities of this complex organ, its role in liver metastasis is partly described and related to the liver tumor cell colonization, while no data is available about its enrollment during the pre-metastatic niche formation. Our central hypothesis is that Heps may have a noticeable role in modulating liver metastatic cascade already during the pre-metastatic phase, before metastatic cells arriving. Our data show for the first time that sEVs derived from colon cancer cells (CRC-sEVs) modulate the properties of hepatocytes inducing an epithelial to mesenchymal transition (EMT) by sEVs_TGF-beta. To confirm our hypothesis, we tested the effects of Hesperetin in the EMT induction mediated by CRC-sEVs- showing that the co-treatment of normal hepatocytes with sEVs and Hesperetin reverted the effects induced by CRC-sEVs. To further confirm the central role of CRC-sEVs on EMT induction, we treated hepatocytes with sEVs isolated from plasma of CRC patients. Since it is known that EMT of hepatocytes led to the formation of a fibrotic environment, a well-known driver of metastasis, the obtained results indicate for the first time that hepatocytes can have an active role in regulating the pre-metastatic niche formation.

S. Mazza1, F. Accattatis1, A. Granata1, M. Ascagni2, E. Vergani3, M. Rodolfo3, S. Bellosta1,

A. Corsini1, L. Arnaboldi1 1Dipartimento di Scienze Farmacologiche e Biomolecolari DISFeB, 2UNITECH, Piattaforme

Tecnologiche di Ateneo, Università degli Studi di Milano, Milan, Italy 3Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Fondazione IRCCS Istituto

Nazionale dei Tumori di Milano, Milan, Italy

Pharmacological modulation of lipid metabolism in a human metastatic melanoma cell line alters protein expression and

signalling of secreted extracellular vesicles

Extracellular Vesicles (EVs) are becoming a very attractive pharmacological target due to their involvement in cell-cell communication, particularly in pathological conditions. EVs are composed by cholesterol and phospholipids; among these, bis-monoacylglycerophosphate (BMP), together with free cholesterol, plays a pivotal role in EV biogenesis, cargo recruiting (interaction with ALIX and Tsg101) and fate. In fact, the balance between these two lipids drives late endosomes towards secretion or lysosomal recycling. Based on these premises, we attempted to modulate cholesterol biosynthesis and BMP metabolism in the metastatic melanoma cell line LM-16 and we evaluated the effect of the pharmacological treatments on secreted EV protein content and on potential alterations in their biological functions. Simvastatin, an inhibitor of HMG-CoA reductase, the rate-limiting step of cholesterol biosynthesis, and/or KT182, an inhibitor of alfa-beta hydrolase containing domain 6 (ABHD6), the enzyme responsible for BMP degradation, have been administered to LM-16 cells for three days with medium plus 10% fetal calf serum, followed by subsequent three days with drugs in serum free conditions. EV fractions isolated following canonical ultracentrifugation protocols, substantially corresponding to microvesicles (10K) and exosomes (100K), have been characterized by Nanosight. Confocal microscopy analysis has also been conducted on control and treated cells. Cell free cholesterol mass and fatty acid composition of both cells and fractions have been analyzed in the presence or absence of the treatments by gas-chromatography. Proteomic analysis has been performed by mass spectrometry and results analyzed by Ingenuity Pathway Analysis (IPA). Simvastatin (0.1µM) and KT182 (50nM) alone or in combination, did not affect cell proliferation, EV size and number. Regarding cell lipid metabolism, simvastatin alone or in combination with KT182, decreased cholesterol biosynthesis, while KT182 increased it in a concentration-dependent manner. Neither cell cholesterol mass, nor fatty acid profile of cells or EVs have been significantly altered by the treatments. Intriguingly, confocal analysis showed that KT182 affects cell BMP and lysosome distribution, without affecting cell BMP mass. Among the 1294 (100K fraction) and 1192 (10K) proteins identified by proteomics, many demonstrated different expression patterns after treatments, with up- and down regulations. IPA analysis showed that treatments specifically decreased the expression of proteins involved in cellular movement, migration, proliferation, and cytoskeleton arrangement, both in 100K and 10K fractions, which may translate into altered signaling pathways and biological functions of EVs. In conclusion, cholesterol and BMP modulation of LM-16 cells significantly affects the protein content of released EVs, possibly leading to altered EV functionality, suggesting the potential of a lipid modulation in reshaping tumor cell-released EVs, in the light of the development of new therapeutic approaches. Supported by EXTRALIPO “New lipid-oriented pharmacological and chemical approach to discriminate and unravel extracellular vesicles biological functions” Bando SEED – PSR 2019.

Associations among PCSK9 levels, atherosclerotic-derived extracellular vesicles and miRNAs in adults with obesity

V. Bollati1, C. Macchi2, M. F. Greco2, C. Favero1, L. Dioni1, L. Vigna3, G. Solazzo1, A. Corsini2,4, A. C. Pesatori1, and M. Ruscica2

1 EPIGET Department of Clinical Sciences and Community Health, Università degli Studi di Milano

2 Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano 3 Occupational Medicine Unit, Fondazione Cà Granda, IRCCS Ospedale Maggiore Policlinico, Milan

4 IRCCS, Multimedica, Sesto San Giovanni, Milan

Atherosclerosis is a leading cause of morbidity and mortality worldwide. Obesity is one of the major risk factors for atherosclerosis, although the mechanisms explaining the association between an increased body mass index (BMI) and atherosclerosis are still poorly understood. Increasing evidence is emerging on the possible role of extracellular vesicles (EVs): Although it is generally thought that EV concentration is increased in patients with cardiovascular diseases [1], and a large amount of EVs has been observed within the atherosclerotic plaque, the protective role of EVs in atherosclerosis has been also reported [2]. To complement the possible role of EVs and their miRNA content in modulating these processes, a role can be also exerted by the proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the key regulators of low-density lipoprotein cholesterol (LDL-C). PCSK9 can directly increase atherosclerotic lesion inflammation and promote plaque monocyte infiltration and macrophage inflammation [3] Considering that EVs are vehicles of bioactive molecules (e.g., miRNA) which influence the intertwined relationship among players of the atherosclerotic process, the present study aims to investigate the interplay among PCSK9, EVs derived from cells relevant for the atherosclerotic process (i.e., platelets, endothelium, monocytes/macrophages, and neutrophils) and their miRNA content in a population of subjects with overweight or obesity, characterized by a high cardiovascular risk. EVs have been isolated from 936 individuals (body mass index= 33.6±5.6 Kg/m2 and HOMA-IR 3.1) and characterized by Nanoparticle Tracking and flow cytometry analyses. Circulating PCSK9 levels, measured by ELISA, were negatively associated either with the count of EV in the range 150-400 nm or with those derived from macrophages (CD14+), endothelium (CD105+), and neutrophils (CD66+). The association between PCSK9 and platelet-derived EVs (CD61+) was modified by platelet counts. 527 miRNAs (OpenArray) were expressed in at least one subject and, among them, PCSK9 was associated with five miRNAs (hsa-miRNA-362, -150, -1244, -520b-3p, -638) after Benjamini–Hochberg False Discovery Rate adjustment. Toll-like receptor 4 and Estrogen receptor 1 were targeted by all five miRNAs and Low-density lipoprotein receptor (LDLR) by four. The sum of these four miRNAs, as well as hsa-miRNA-150 alone, was negatively associated with LDLR expression measured in leukocytes. The present data show a potential impact of PCSK9 in regulating EV release and EV-derived miRNAs, especially those involved in inflammation and LDLR. Supported by the Fondazione Cariplo (2018-0511 to MR) and European Research Council (ERC-2011-StG 282,413 to VB). References [1] Boulanger CM, Loyer X, Rautou PE, Amabile N. Extracellular vesicles in coronary artery disease. Nat Rev Cardiol 2017;14(5):259-272.. [2] Hergenreider E, Heydt S, Treguer K, Boettger T, Horrevoets AJ, Zeiher AM, Scheffer MP, Frangakis AS, Yin X, Mayr M, Braun T, Urbich C, Boon RA, Dimmeler S. Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nat Cell Biol 2012;14(3):249-56. [3] Giunzioni I, Tavori H, Covarrubias R, Major AS, Ding L, Zhang Y, DeVay RM, Hong L, Fan D, Predazzi IM, Rashid S, Linton MF, Fazio S. Local effects of human PCSK9 on the atherosclerotic lesion. J Pathol 2016;238(1):52-62.

G. Solazzo1, J. Mariani1, S. Iodice1, L. Cantone1, P. Marraccini2, R. Ignar3, P. A. Bulsara3, M. S. Lombardi4, R.P. Howlin5, V. Bollati1, and L. Ferrari1

Exposure to particulate matter (PM) has been linked to the worsening of several respiratory conditions, including allergic rhinitis (AR), as it can trigger nasal and systemic inflammation. Although the underlying molecular

mechanisms remain largely unexplored, there is evidence that after environmental exposure bacterial nasal

microbiome (bNM) can interact with the host immune system through the Extracellular Vesicles and exacerbate

AR symptoms. To explore this hypothesis, we evaluated bNM composition and plasmatic EV release in 26 AR

patients and 26 matched healthy controls (HS) and assessed the role of PM10 and PM2.5 exposure in modulating

bNM and EV release. Daily PM10 and PM2.5 concentrations were estimated and attributed according to the

subject’s residence. bNM analysis was performed by 16S rRNA sequencing. Plasmatic EVs were counted by

nanoparticle tracking analysis and characterized by flow cytometry. bNM α-diversity indices were lower in the AR

group compared to HS and were associated with PM10 exposure. This evidence indicates that PM influences the

microbiome pattern. EV concentration was higher in AR compared to HS (p=0.009). AR showed a higher

concentration of all the investigated EV types, derived from platelets (CD61+), activated endothelium (CD62e+),

monocytes (CD14+), eosinophils (CD294+), neutrophils (CD177+), mastocytes (CD203c+), epithelial cells

(EPCAM+), Gram-positive (Lipoteichoic Acid+), and Gram-negative bacteria (LPS+). When we included PM effects, a significant association with EVs derived from both gram-positive and gram-negative bacteria in HS was

observed (β=0.038, p=0.01, β=-0.38, p=0.023, respectively). Interestingly, bNM composition modified these

associations. We conclude that PM exposure may exacerbate AR symptoms by modulating bNM diversity and

plasmatic EV release. These results further support the involvement of plasmatic EVs in the modulation of

environmental exposure effects. Moreover, they represent the first step for understanding the complex signaling

network linking external stimuli, bNM, and immunity.

Air pollution effects on nasal microbiome composition and plasmatic extracellular vesicle release in allergic rhinitis

1EPIGET LAB, Department of Clinical Sciences and Community Health, University of Milan 2Department of Preventive Medicine, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

3GlaxoSmithKline Consumer Healthcare, Warren, New Jersey, United States 4GlaxoSmithKline Consumer Healthcare, Nyon, Switzerland

5GlaxoSmithKline Consumer Healthcare, Weybridge, Surrey, United Kingdom

Circulating extracellular vesicles from individuals at high-risk of lung cancer induce pro-tumorigenic conversion of stromal cells through

transfer of miR-126 and miR-320

Francesca Pontis1, Luca Roz1, Mavis Mensah1, Miriam Segale1, Massimo Moro1, Giulia Bertolini1, Anna Maria Ferretti2, Paola Suatoni3, Ugo Pastorino3, Gabriella Sozzi1 and Orazio Fortunato1

1 Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133 Milan, Italy;

2 Istituto di Scienze e Tecnologie Chimiche-CNR, Via G. Fantoli 16/15 20138 Milan, Italy;

3 Thoracic Surgery Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy

Introduction: To date, lung cancer is still the leading cause of cancer-related deaths despite several improvements in early diagnosis and treatments using immunotherapeutic drugs. Extracellular vesicles (EVs) has been emerged as key players in tumor growth and cancer progression thanks to their ability to mediate cell to cell communication by delivering of bio functional molecules such as miRNAs. To date, many studies have been addressed to understand the function of tumor derived EV-miRNAs but only few studies analyzed the pro-tumorigenic potential of miRNAs inside plasma derived-EV from heavy-smokers cancer-free subjects. The analysis of the EVs and their miRNAs could be useful as valuable predictive biomarkers for lung cancer development and progression. Methods: Plasma-EVs were isolated by ultracentrifugation from 40 cancer-free heavy-smokers classified, accordingly to a validated 24-microRNA signature classifier (MSC), as high (MSCpos-EVs) or low (MSCneg-EVs) risk for lung cancer. EVs characterization was performed following the MISEV guidelines and their origin and functional properties were investigated both in vitro (2D, 3D cultures) and in vivo. The prognostic value of miRNAs inside EVs was assessed in training and in validation cohorts of 54 and 48 lung cancer patients, respectively. Results: We noted that MSCpos and MSCneg-EVs posses similar size and particle distribution but they differ in terms of membrane composition, biological cargo and pro-tumorigenic activity. Indeed, the analysis of the EV cargo revealed that MSCpos-EVs were enriched miR-126 and miR-320 and c-Myc content compared to MSCneg-EVs. Moreover, in vitro studies revealed that MSCpos-EVs were able to increase angiogenesis and that pre-treated endothelial cells are able to foster tumor growth in vivo. Moreover, MSCpos-EVs induced in vitro an immune suppressive phenotype of macrophages (M2 polarization) and prompted 3D proliferation of non-tumorigenic epithelial cells. In addition, we noted that hypoxic conditions increased the levels of c-Myc in fibroblasts-EVs, miR-126 in endothelial-EVs and miR-320 in granulocytes-EVs compared to normoxia conditions. Furthermore, high levels of miR-320 in lung cancer patients derived-EVs correlated with worse prognosis and short overall survival both in training [HR2.96] and validation [HR2.68] sets. Conclusion: Overall our data provide a new perspective on the pro-tumorigenic role of circulating EVs from high risk smokers and highlight the significance of miR-320-EVs as a new prognostic biomarker in lung cancer patients.

The phenotype of astrocytes from the spinal cord of late symptomatic SOD1G93A mice is modulated by mesenchymal stem cell-derived

extracellular vesicles

Roberta A Zerbo1, Francesca Provenzano1, Debora Giunti2,4, Carola Torazza1, Marco Milanese1, Benedetta Parodi2, Nicole Kerlero de Rosbo2, Cesare Usai3, Antonio Uccelli2,4, Giambattista

Bonanno1,4

1 Department of Pharmacy, Pharmacology and Toxicology Unit, University of Genoa, Genoa, Italy. 2Ophthalmology, Genetics, Rehabilitation and Child Health, Department of Neurosciences,

University of Genoa, Genoa, Italy. 3 Institute of Biophysics, National Research Council (CNR), Genoa, Italy.

4 IRCCS, San Martino Polyclinic Hospital, Genoa, Italy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects upper and lower motor neurons (MNs) leading to muscle atrophy and paralysis. ALS is a non-cell autonomous disease, in which astrocytes play a central role in clinical progression. In a previous work we investigated the effect of intravenous administration of mesenchymal stem cells (MSCs) in SOD1G93A mice and we demonstrated that a single intravenous injection of MSCs prolonged survival, improved motor skills and reduced reactive gliosis. These beneficial effects were determined by paracrine mechanisms, rather than by MSC differentiation. We postulated that MSC-derived extracellular vesicles (EVs) can be a mode to sustain the paracrine effects of MSCs. The expression of the astrocyte activation markers GFAP, S100β and vimentin was significantly increased in spinal cord astrocytes from 120 day-old SOD1G93A mice compared to age-matched WT astrocytes and exposure to EVs reversed these over-expressions. The expression of the inflammation complex NLRP3 was increased in SOD1G93A astrocytes and the increase was reversed after exposure to EVs. The expression and release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 significantly increased in astrocytes from SOD1G93A mice and reduced after EV treatment. Conversely, the expression of the anti-inflammatory cytokine IL-10 was decreased in SOD1G93A astrocytes and normalized after exposure to EVs. The viability of embryonic SOD1G93A mouse-derived MNs was significantly increased when seeded on EV-treated adult SOD1G93A astrocytes, compared to non-treated astrocytes. Our results suggest that the reactive phenotype and the inflammation state of SOD1G93A mouse astrocytes are ameliorated by EVs derived from IFNγ-primed-MSCs and this amelioration reflects into MN viability increase. These results open to the possibility to in-vivo preclinical trials with EVs in SOD1G93A mice.

Extracellular vesicles isolated from follicular fluid of antral and preovulatory follicles present a different miRNA profile related to follicular

development and breeding season in buffalo (bubalus bubalis)

E. Capra1, M. Andrzej Kosior2, B. Lazzari1, N. Cocchia2, C. Del Prete2, V. Longobardi2, F. Pizzi1, R. Frigerio3, M. Cretich3, A. Lange Consiglio4-5, B. Gasparrini2

1Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche IBBA CNR, Lodi. 2Università degli Studi di Napoli Federico II, Dipartimento di Medicina Veterinaria e Produzioni

Animali (DMVPA). 3Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”, Consiglio Nazionale delle Ricerche

SCITEC-CNR, Milano. 4Università degli Studi di Milano, Dipartimento di Medicina Veterinaria (DIMEVET).

5Centro Clinico-Veterinario e Zootecnico-Sperimentale di Ateneo, Università degli Studi di Milano.

Interest in buffalo (Bubalus bubalis) breeding has been steadily increasing worldwide, due to pivotal role played by this species as a protein producer in developing countries. In Italy, the economic importance of buffalo farming is linked to the production of mozzarella cheese, highly requested around the world. A major factor limiting the profitability of buffalo farming is reproductive seasonality. Although seasonal effects on oocyte competence are well known, the molecular characterization of the follicular environment has not yet been investigated. Extracellular vesicles (EVs) in the follicular fluid (FF) play an important role in modulating oocyte developmental competence and carry microRNAs (miRNAs) essential for in vitro fertilization (IVF) outcomes. This study characterizes the FF-EVs-miRNA cargo of antral (An) and preovulatory (pO) buffalo follicles collected in different breeding seasons. EVs were obtained from five biological replicates of FF isolated from An (n=20) and pO (n=1) follicles, collected in the in breeding (BS) and non-breeding (NBS) seasons, through Exoquick precipitation following previous reported method [1]. Size and concentration of EVs were evaluated by the NanoSight LM10 instrument. Total RNA was extracted from isolated EVs and libraries were obtained using a TruSeq Small RNA Library Preparation Kit and sequenced on NovaSeq instrument. An-FF present a higher content of EVs compared to pO-FF in both breeding (BS) and non-breeding (NBS) seasons (An: 2.17e+11 particles/ml in BS and 1.01e+11 particles/ml in NBS; pO: 2.51e+10 particles/ml in BS and 5.40e+10 particles/ml in NBS). Overall expression of 1335 miRNAs (538 known Bos taurus miRNAs, 324 homologous to known miRNAs from other species and 473 new candidate miRNAs) clearly separate An and pO FF-EVs. We identified 413 differentially expressed miRNAs (DE-miRNAs) (FDR<0.05) between An and oP groups. Comparison between BS and NBS showed 14 and 12 DE-miRNAs in An-FF-EVs and oP-FF-EVs, respectively. In conclusion, buffalo FF-EVs present a specific miRNA cargo in function of their developmental stage that is partially affected by the breeding season. These preliminary results suggest a strong paracrine regulation mediated by miRNAs during oocyte development and specific alteration of miRNA content related to breeding season. Further research is needed for a thorough characterization of EVs and to evaluate their potential employment in IVF experiments. References [1] Sohel MM, Hoelker M, Noferesti SS, Salilew-Wondim D, Tholen E, Looft C, Rings F, Uddin MJ, Spencer TE, Schellander K, Tesfaye D. 2013. Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence. PLoS One. 8(11):e78505.

Autocrine effect of extracellular vesicles in the canine mammary tumor

V. Moccia1, A. Sammarco1,2, S. Ferro1, V. Zappulli1

1 Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro,

Padova; 2 Department of Neurology and Radiology, Massachusetts General Hospital, Harvard

Medical School, Boston, MA, USA

Extracellular vesicles (EVs) are membrane bound vesicles which take part to cell communication through autocrine or paracrine interactions. This communication involves both normal and tumoral cells.[1] Since a few years ago ultracentrifugation (UC) was considered the gold standard for the isolation and concentration of EVs.[2] Although, more recently, also according to MISEV, several factors (e.g. required sensibility and sensitivity, type of downstream analyses) should be considered before choosing the isolation procedure.[2] Size exclusion chromatography (SEC) is considered an EV isolation method more able to separate EVs from contaminants and to keep EVs integrity for functional studies.[3] The aim of this study was to characterize EVs isolated with UC or SEC from a canine mammary tumour cell line (CIPp) and to compare these two isolation techniques evaluating EVs profile and autocrine effects on cell migration, invasiveness and proliferation in vitro. EVs were isolated from CIPp culture media using UC or SEC. The size distribution of particles was evaluated through Nanoparticle Tracking Analysis (NTA) and the presence of EVs markers was assessed through Western Blot (WB). Both analyses were performed on UC EVs and grouped SEC 7 to 18 fractions. EVs functionality on cell proliferation, migration and invasion was tested through in vitro assays at different time points. On NTA analysis, UC EVs and SEC EVS presented a similar particle size distribution, with particle diameter ranging from 60 to 600 nm. UC EVs particle concentration was approx. 1011/ml while in SEC fractions 7-8-9 particle concentration was approx.1010/ml, and decreased in the following fractions, reaching a concentration of approx.109/ml in fractions 10-11-12. Particles under detection ranges were obtained in controls, represented by unconditioned medium (UCM)-derived pellets (p)/fractions (f). The presence of EVs was confirmed by CD9 and Alix in UC pellet. In SEC fractions 7-8-9 and 10-11-12 the presence of Alix but not of CD9 was observed, while in the following fractions no EVs markers were detected. UC EVs and SEC EVs manifested a trend of slightly enhanced proliferation when compared to controls (cells treated with UCM p/f) and to un-treated cells. Considering transwell migration SEC EVs showed a higher effect than UC EVs, however both EVs effects were not higher than the relative control; no effects on migration were seen through the wound-healing assay. Transwell invasion assay allowed to see a minimal effect of EVs that was higher than controls, but indeed not different between SEC and UC EVs. The biological effects of EVs in these experiments was not remarkable as well as the difference between the two isolation procedures. The EVs concentration might represent a critical point and could be modified to define broader qualitative differences. Additionally, also the minimal difference of EVs vs controls (UCM p/f) might indicate a relevant role of dissolved/co-pelleted proteins as already discussed within the literature.[2] This was a preliminary study performed in canine oncology to open the field of EVs in veterinary pathology also for comparative analyses with humans. References

[1] Bebelman, M. P., Smit, M. J., Pegtel, D. M., Baglio, S. R., 2018. Biogenesis and function of extracellular vesicles in cancer. Pharmacol. Ther.188,1–11.

[2] Théry, C., et al., 2018. 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 7(1), 1535750.

[3] Coumans, F. A. W., et al., 2017. Methodological Guidelines to Study Extracellular Vesicles. Circ. Res. 120(10), 1632–1648.

“One health” extracellular vesicles: what messages from the veterinary medicine?

V. Moccia1, V. Zappulli1

1 Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro,

Padova

The heterogeneous world of extracellular vesicles (EVs) has been exponentially exploding in the last 20 years, mainly and reasonably in human-oriented research. However, in any field, a huge consideration is now given to the complex interconnections and interdependence of all living species and of environment - the concept of One health.[1] Why not looking at the most powerful interconnectors - EVs – also from within this big “one health” picture? To depict this picture, all EVs-related data coming from studies in veterinary medicine have been here explored and summarized. To date more than 200 papers dealing with EVs in animals (excluding lab animal models used for human research) have been published (exhaustive list of references available from first author) since1983.[2] A major growing field related to the one-health concept is comparative oncology, in which mainly pets share the same environment and life of their owners.[3] For this reason following the footprints of human medicine, a few studies (no. 7) focused on tumor-bearing dogs and on the possible use of EVs as cancer biomarkers. Moving to farm and wild animals a major area of one-health concern, as sadly demonstrated with the COVID-19 pandemia, is the management of zoonotic risks as well as pollution and contaminants impact.[4], [5] High productivity of farm animals is also very relevant both for economically established and developing societies.[6] These interests are reflected on a relative “high” number of publications (no.30) on productive animals investigating EVs content as biomarkers for pregnancy, infertility, and infections. Interestingly, researchers recently started looking at EVs also as possible biomarkers for wild/unconventional animals physiology/immunology (6 studies) and health (3 studies), the latter so closely related to the environment. Among animal-derived by products, milk received the highest attention with more than 100 EV-related publications produced since 2010; mainly bovine milk is a very attractive, natural, easy, cheap, and abundant source of EVs. Among major results, animal milk-derived EVs showed a proliferative and protective effects on many type of recipient cells (e.g. osteoblasts, enterocytes) and are now also strongly investigated as good drug carrier. This promising field of animal-derived EVs represents a relevant “small” world within the very complex and gigantic one-health planet and should and will certainly receive further attention. References

[1] Amuasi, J. H., Lucas, T., Horton, R., Winkler, A. S., 2020. Reconnecting for our future: The Lancet One Health Commission. The Lancet 395(10235), 1469–1471.

[2] Harding, C., Heuser, J., Stahl, P., 1983. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J. Cell Biol 97(2), 329–339.

[3] Howard, J., Wyse, C., Argyle, D., Quinn, C., Kelly, P., McCann, A., 2020. Exosomes as Biomarkers of Human and Feline Mammary Tumours; A Comparative Medicine Approach to Unravelling the Aggressiveness of TNBC. Biochim. Biophys. Acta - Rev. Cancer 1874(2), 188431.

[4] Tomley, F. M, Shirley, M. W., 2009. Livestock infectious diseases and zoonoses. Philos. Trans. R. Soc. B Biol. Sci. 364(1530), 2637–2642.

[5] Saegerman, C., Pussemier, L., Huyghebaert, A., Scippo, M. L., Berkvens, D., 2006. On-farm contamination of animals with chemical contaminants. OIE Rev. Sci. Tech. 25(2), 655–673.

[6] Kakkar, M., Chauhan, A. S., Bahl, T., Rahi, M., 2019. Opportunities for One Health policies to reduce poverty. Rev. Sci. Tech. 38(1), 135–144.

Quantitative and qualitative characterization of extracellular vesicle in three cetaceans cell lines

Cinzia Centelleghe1, Valentina Moccia1, Ilaria Giusti2, Vincenza Dolo2, Sandro Mazzariol1,

Valentina Zappulli1

1: Dept. of Comparative Biomedicine and Food Science, University of Padova 2: Dept. of Life, Health and Environmental Sciences, University of L'Aquila

During the last two decades, extracellular vesicles (EVs) have been increasingly recognized as potent intercellular communicators via transfer of a wide variety of molecular cargoes in humans and animals [1]. The study of EVs in the aquatic environment, despite still little, has underlined that EVs can modulate host-pathogen interaction and nutrient/toxins acclimation/scavenging [2]. In terrestrial mammals, EVs can be isolated from most body fluids, cell culture media, and tissues and many studies are focusing on their possible role as biomarkers for the assessment of health status. Preliminary studies have already been performed on serum-derived EVs and EV-related microRNAs and deaminated proteins in 5 cetacean species [3]. The aim of this preliminary study was to characterize EVs from cell culture media of immortalized fibroblasts cell lines isolated from skin of bottlenose dolphin (Tursiops truncatus), Risso’s dolphin (Grampus griseus) and Cuvier's beaked whale (Ziphius cavirostris). EVs were isolated by ultracentrifugation and the obtained EV-enriched pellet was analyzed to evaluate EVs size and distribution, using Nanoparticle Tracking Analysis (NTA), to asses EVs morphology via Scanning Transmission Electron Microscopy (STEM), and to assess the presence of EVs markers with Western Blot (WB) analysis. NTA showed in each pellet a concentration of 4.02*1010 particles in bottlenose dolphin, 2.24*1010 in Risso’s dolphins and 3.11*1010 in Cuvier's beaked whale. Particles had the typical size distribution within the range of EVs (50-700nm), with a mean diameter of 137,6±4,7nm, 142,1±2,8nm and 144,9±5,7nm, respectively for each cell line. STEM analysis revealed the presence of intact and rounded EVs. At WB analysis, EVs isolated from all the three species were CD9+ and Calnexin-, while only bottlenose dolphin EVs resulted Alix+. To the best of our knowledge, this is the first study that characterize EVs in three cetaceans cell lines. Further investigations will be addressed to differently isolate and better characterize EVs from these cell lines. This is a very preliminary study, however, it gives a very relevant system to evaluate how EVs are involved in the host/pathogen-toxic compounds/aquatic environment relation in marine mammals species to possibly develop relevant new biomarkers for assessing impact on health of waters pollution and pathogens. [1] Gill, S., Catchpole, R., Forterre P., 2019. Extracellular membrane vesicles in the three domains of life and beyond. FEMS Microbiology Reviews 43 (3), 273–303. [2] Schatz, D., Vardi, A., 2018. Extracellular vesicles - new players in cell-cell communication in aquatic environments. Current Opinion in Microbiology 43: 148-154 [3] Magnadóttir, B., Uysal-Onganer, P., Kraev, I., Svansson V., Hayes, P., LangeDeiminated, S., 2020. Deiminated proteins and extracellular vesicles - Novel serum biomarkers in whales and orca. Comparative Biochemistry & Physiology Part D Genomics Proteomics 34, 100676

PESTO: Preservation of Extracellular vesicles during STOrage

The impact of storage on extracellular vesicles: a systematic study

Authors: Stefano Gelibter1*, Giulia Marostica1*, Alessandra Mandelli1, Stella Siciliani3, Paola Podini2, Annamaria Finardi1, Roberto Furlan1

*Equally contributing first authors

Affiliations:

1Clinical Neuroimmunology Unit, Institute of Experimental Neurology, Division of Neuroscience, IRCCS Ospedale San Raffaele Milan, Italy.

2Neuropathology Unit, Institute of Experimental Neurology, Division of Neuroscience, IRCCS Ospedale San Raffaele Milan, Italy.

3Neuroimmunology Unit, Institute of Experimental Neurology, Division of Neuroscience, IRCCS Ospedale San Raffaele Milan, Italy.

Abstract

Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While -80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of -80°C storage and freeze-thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent-tagged EVs populations (GFP and mCherry), mixed before preservation. We found that -80°C storage reduces EVs concentration and sample purity in a time-dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size-charge plots. None of the tested conditions prevented the observed effects. Freeze-thaw cycles lead to an EVs reduction after the first cycle and to a cycle-dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double-positive EVs (GFP+-mCherry+). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non archival, samples in majority of cases.

Keywords: Extracellular vesicles, storage, fusion, tunable resistive pulse sensing, size exclusion chromatography, preservation, flow cytometry

A three-dimensional millifluidic glomerular model for the tracking of extracellular vesicles in kidney physiopathology.

F. Collino1,2, L. Bellucci 1,3, G. Montini 1,2, B. Bussolati 3

1 Laboratory of Translational Research in Paediatric Nephro-urology, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy.

2 Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy. 3 Department of Molecular Biotechnology and Health Sciences, University of Torino, Turin, Italy.

Dynamic three-dimensional (3D) cultures, characterized by continuous fluid reperfusion, replicate the organ physiology adequately. The fabrication of bioreactors and interconnected microchannels allows the simulation of bioactive molecules' absorption, distribution, and metabolism through precise manipulation of fluid flow that replicates blood circulation [1]. Extracellular vesicles (EVs) are small, rounded structures released by all active cells and involved in physiological and pathological process regulation. Their detrimental and repairing functions in kidney-related processes have been investigated in traditional two-dimensional (2D) and animal models. Moreover, numerous reports have supported the role of stem cell-derived EVs (SC-EVs) as a novel approach in the treatment of several renal diseases [2]. The purpose of this study was to develop a versatile 3D kidney model to 1. investigate the regenerative potential of therapeutic SC-EVs on the components of the glomerular filtration barrier (GFB) after an acute glomerular injury and 2. study the role of pathological EVs as biomarkers of the acute kidney injury (AKI) to chronic kidney disease (CKD) transition. For this purpose, we set up the 3D model by plating human glomerular endothelial cells (h-GECs) and human podocytes (h-PODs) inside a bioreactor on the opposite sides of type IV collagen-coated porous membrane. We were able to track the uptake of labelled cel-miR-39 EVs by kidney cells in the 3D glomerulus submitted to a continuous flow by immunofluorescence and molecular analysis. Moreover, we induced an acute glomerular injury to the 3D glomerulus through doxorubicin (DXR) treatment. DXR administration prompted intense damage to co-cultured h-PODs, by increasing the expression of apoptosis-related markers and alteration in the slit diaphragm proteins. The administration of therapeutic EVs protected the GFB by decreasing cell death and reverting the enhanced permeability induced by the DXR treatment. The AKI to CKD model was developed by direct administration of TGF-β1 in the 3D glomerular system. The co-cultured h-PODs responded to the treatment by undergoing epithelial-mesenchymal transition (EMT) within 24 hours. Moreover TGF-β1 administration enhanced the permeability of the GFB and induced a release of pathological EVs from both the endothelial and podocyte compartments. Pathological EVs were characterized for their phenotype, content and concentration. In conclusion, we developed a versatile 3D millifluidic model that can trace therapeutic and pathological EVs in time course studies and analyze the cellular and molecular responses that characterize the GFB in different kidney pathologies. References 1. Lee, J.B.; Sung, J.H. Organ-on-a-chip technology and microfluidic whole-body models for

pharmacokinetic drug toxicity screening. Biotechnol. J. 2013, 8, 1258–1266. 2. Tsuji, K.; Kitamura, S.; Wada, J. Immunomodulatory and regenerative effects of mesenchymal

stem cell-derived extracellular vesicles in renal diseases. Int. J. Mol. Sci. 2020, 21.

Role of Extracellular Vesicles in modulation of biomechanical properties of target cells

Beatrice Senigagliesi1,2*, Loredana Casalis2, Pietro Parisse2,3

1SISSA, Trieste, Italy

2Elettra-Sincrotrone S.C.p.A. Trieste, Trieste, Italy 3Istituto Officina dei Materiali, CNR-IOM, Trieste, Italy

Extracellular vesicles (EVs) mediated communication has been recently receiving a growing attention for its role in the development of cancer metastasis. EVs are small vesicles exchanged among cells that, owing to their biologically active content, can promote tumor–induced immune suppression, metastasis and angiogenesis, and constitute a potential target in cancer therapy. Yet, their role in priming the premetastatic niche and helping the spreading of cancer cells is still debated. It is recognized that the biomechanical properties play a crucial role during metastatic spreading. As a matter of fact, metastasis is thought to be easier for more deformable and, therefore, soft cancer cells, which can migrate through narrow pores of the matrix and vessels. Here, we focused on the effects of EVs on the biomechanical properties of target cells. To this purpose, we isolated and thoroughly characterized (through scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis, and western blot) metastatic breast cancer cell-derived EVs. We tested their ability to model biomechanical properties of recipient cells through AFM-based nanoindentation and immunofluorescence measurements. Our results indicate that triple negative breast cancer (TNBC)-derived small extracellular vesicles can actively induce cellular stiffness, cytoskeleton, nuclear and Yap activity rearrangements. In conclusion, we believe to demonstrate with this work that the analysis of the biomechanical response of target cells upon EV addition could represent a powerful resource in view of the application of EVs in theranostic field for metastatic TNBCs and also for other types of cancer. * E-mail: beatric.senigagliesi@gmail.com

Extracellular Vesicle analysis in the COVID-19 era: Insights on Serum Inactivation Protocols towards Downstream Isolation and

Analysis

A. Musicò1, R. Frigerio1, A. Strada1, M. Brucale 2, A, Ridolfi 2, R. Vago 3, S. Galbiati 3, F.

Valle 2, M. Chiari1, A. Gori1, M. Cretich1

1 Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC) - Consiglio Nazionale delle

Ricerche, Milano, Italy 2 Istituto per lo Studio dei Materiali Nanostrutturati (ISMN) - Consiglio Nazionale delle Ricerche,

Bologna, Italy

3 IRCCS San Raffaele Scientific Institute, Milano, Italy

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators’ safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis [1].

Supported by European Union’s Horizon 2020 research and innovation program under grant agreements No. 951768 (project MARVEL), No. 801367 (project EVfoundry), No. 952183 (project BOW), by Regione Lombardia (project INTERSLA ID 1157625) and Regione Lombardia&Fondazione Cariplo, grant n° 2018-1720 (project HYDROGEX). References

[1] Frigerio, R et al, Cells 2021, 10, 544

Immunological characterization of plasma-derived extracellular vesicles in patients with Amyotrophic Lateral Sclerosis.

D. Sproviero1, E. Corridori2, S. Gagliardi1, L. Diamanti3, M. Bordoni2, P. Bergese4, A. Radeghieri4,

O. Pansarasa1, C. Cereda1.

1 Genomic and Post-Genomic Unit, IRCCS Mondino Foundation, Pavia, Italy 2 Dipartimento di Scienze Farmacologiche e Biomolecolari, Centro di Eccellenza sulle Malattie

Neurodegenerative, Università degli Studi di Milano, Milano, Italy 3 Neuro-Oncology Unit, IRCCS Mondino Foundation, Pavia, Italy

4 Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy

Extracellular vesicles (EVs) play central role in inflammatory processes and they could be plausible targets in Amyotrophic Lateral Sclerosis (ALS), a disease marked by an immunological reaction to motor neuron loss [1]. We have previously demonstrated that leukocyte-derived EVs are upregulated in ALS patients and they can be considered markers of disease progression [2].

The aim of this study was to investigate specific immunological surface markers on Large and Small EVs (LEVs and SEVs) from plasma of sporadic ALS (sALS) patients and healthy donors (CTRLs).

EVs were isolated from plasma of 50 sALS and 28 CTRLs by differential centrifugation and characterized by Nanoparticle Tracking Analysis (NTA), Atomic Force Microscopy (AFM), and COlorimetric NANoplasmonic method (CONAN). For a simultaneous identification of 37 surface protein markers in each sample, we used a multiplex bead-based flow cytometric assay (MACSPlex Exosome Kit).

Endosome-specific tetraspanins (CD9 and CD63), endothelial marker (CD31), T-cell and Natural Killer markers (CD2, CD45, and CD69), MHC Class I (HLA-ABC), and T-cells homing regulator (CD44) were more expressed in SEVs derived from CTRLs than in sALS patients (P<0,05). Differently, the expression of the tetraspanin CD81, MHC class II (HLA-DRDPDQ), and the glycosphingolipid SSEA4 were higher in the SEVs derived from sALS patient compared to CTRLs. LEVs derived from CTRLs were enriched in endothelial cells and platelets adhesion molecule (CD31, CD42a, CD41b, and CD62P), CD63, CD9, and HLA-ABC (P<0,05) compared to sALS patients.

In conclusion, endosome-specific tetraspanins decrease in both LEVs and SEVs in accordance with the autophagy-endolysosomal system dysregulation described in ALS [3]. LEVs from sALS patients also had fewer platelet adhesion markers in line with the literature as suggested by the platelet variation in blood of ALS patients [4]. These data suggest that LEVs and SEVs carry different surface markers which might discriminate their role in ALS pathogenesis.

References 1. Cereda, C. et al. (2008). TNF and sTNFR1/2 plasma levels in ALS patients. J. Neuroimmunol. 194,

123–131. doi: 10.1016/j.jneuroim.2007.10.028 2. Sproviero, D. et al. (2019). Leukocyte derived microvesicles as disease progression biomarkers in slow

progressing Amyotrophic Lateral Sclerosis Patients. Front Neurosci. 13:344. doi: 10.3389/fnins.2019.00344.

3. Harris H, Rubinsztein DC. Control of autophagy as a therapy for neurodegenerative disease. Nat Rev Neurol. 2012; 8: 108–117.

4. Shrivastava M, Das TK, Behari M, Pati U, Vivekanandhan S. Ultrastructural variations in platelets and platelet mitochondria: a novel feature in amyotrophic lateral sclerosis. Ultrastruct Pathol. 2011; 35(2):52-9.

Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs)

R. Tasso1, C. Gorgun1,2, D. Ceresa2, R. Lesage3,4, F. Villa2, D. Reverberi2, C. Balbi5, S.

Santamaria1, K. Cortese1, P. Malatesta1,2, L. Geris3,4,6, R. Quarto1,2

1Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy 2IRCCS Ospedale Policlinico San Martino, Genova, Italy

3Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium 4Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven, Belgium

5Laboratory of Cellular and Molecular Cardiology, Cardiocentro Ticino Foundation, 6900, Lugano, Switzerland

6Biomechanics Research Unit, GIGA in Silico Medicine, University of Li`ege, Li`ege, Belgium

Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity [1]. Preconditioning represents an adaptive strategy to improve MSC therapeutic efficacy, preparing cells to survive in hostile conditions and enhancing their regulatory activities [2]. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. In particular, we compared the effect of the total conditioned medium (CM) with that exerted by the corresponding fractions composed by either small- or medium-sized extracellular vesicles (sEVs and mEVs), and with the vesicle-depleted medium, enriched only in soluble factors. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that a deep investigation into how preconditioning can specifically influence the different cell paracrine activities is fundamental to provide new insights for the therapeutic use of the MSC secretome. Supported by the the European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska- Curie grant agreement No. 721432 References [1] A.M. DiMarino, A.I. Caplan, T.L. Bonfield, 2013. Mesenchymal stem cells in tissue repair, Front. Immunol. 4, 201. [2] C. Hu, L. Li, 2018. Preconditioning influences mesenchymal stem cell properties in vitro and in vivo, J. Cell Mol. Med. 22, 1428–1442.

1. Arulselvan, P.; Fard, M.T.; Tan, W.S.; Gothai, S.; Fakurazi, S.; Norhaizan, M.E.; Kumar, S.S. Role of Antioxidants and Natural Products in Inflammation. Oxidative Med. Cell. Longev. 2016. 2. Urzì, O.; Raimondo, S.; Alessandro, R. Extracellular Vesicles from Plants: Current Knowledge and Open Questions. International Journal of Molecular Sciences. 2021. 3. Ju, S.; Mu, J.; Dokland, T.; Zhuang, X.; Wang, Q.; Jiang, H.; Xiang, X.; Deng, Z.B.; Wang, B.; Zhang, L.; et al. Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis. Mol. Ther. 2013. 4. Deng, Z.; Rong, Y.; Teng, Y.; Mu, J.; Zhuang, X.; Tseng, M.; Samykutty, A.; Zhang, L.; Yan, J.; Miller, D.; et al. Broccoli-Derived Nanoparticle Inhibits Mouse Colitis by Activating Dendritic Cell AMP-Activated Protein Kinase. Mol. Ther. 2017. 5. Rahimi Ghiasi, M.; Rahimi, E.; Amirkhani, Z.; Salehi, R. Leucine-rich Repeat-containing G-protein Coupled Receptor 5 Gene Overexpression of the Rat Small Intestinal Progenitor Cells in Response to Orally Administered Grape Exosome-like Nanovesicles. Adv. Biomed. Res. 2018. 6. Lawrence, T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol. 2009.

Chronic inflammation is associated with the occurrence of several diseases. Anti-inflammatory drugs have many side effects, therefore the discovery of new therapeutic strategies is necessary [1]. Plant vesicles are gaining increasing interest in the scientific community as they possess interesting biological properties [2, 3, 4, 5]. We isolated extracellular vesicles from Citrus limon juice (LEVs) through differential centrifugation followed by ultracentrifugation. We first characterized the size and morphology of LEVs through biophysical analyses. Further, we characterized flavonoids, limonoids, and lipids contained in the LEVs by means of HPLC-ESI-Q-ToF-MS. We found that LEVs were enriched in lipids, comparing vesicles content with the total juice and LEVs-deprived juice. We found that LEVs did not affect the cell viability of murine macrophages (RAW-264) and human primary PBMCs at all the doses and time points tested. To investigate the protective role of LEVs on the inflammatory process, murine and primary human macrophages were pre-treated with LEVs for 24h and then cells were stimulated with LPS. We found that the pre-treatment with LEVs decreased the gene and protein expression of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNFα. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) is a key pro-inflammatory pathway that can be activated by the ERK1-2 signaling cascade [6]. We found that the pre-treatment with LEVs reduced the nuclear translocation and phosphorylation of NF-kB in murine macrophages stimulated with LPS. The inhibition of the activation of NF-kB was associated with the reduction of ERK1-2 phosphorylation. Furthermore, LEVs showed the ability to decrease pro-inflammatory cytokines and increase anti-inflammatory molecules in human primary T lymphocytes, supporting the in vitro results. In conclusion, we demonstrated the anti-inflammatory properties of LEVs both in vitro and ex vivo; LEVs exerted these effects by inhibiting the ERK1-2/NF-kB signaling pathway.

The anti-inflammatory properties of lemon-derived extracellular vesicles are achieved through the inhibition of ERK/NF-kB signaling pathway

O. Urzì1, S. Raimondo1, M. Di Simone1,2, A. M. Corsale1,2, S. Meraviglia1,2, A. Conigliaro1, A. Palumbo Piccionello3, G. Polito3, F. Dieli1,2, R. Alessandro1

1 Dipartimento di Biomedicina, Neuroscienze e Diagnostica Avanzata, Università degli Studi di Palermo; 2 Central Laboratory of Advanced Diagnosis and Biomedical Research (CLADIBIOR), AOUP Paolo

Giaccone, Palermo, Italy; 3 Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Università degli Studi di Palermo

Cow, donkey, and goat milk extracellular vesicles OMIC characterization tells about their anti-inflammatory, immunomodulatory, and antioxidant

potential.

S. Mecocci1, S. Capomaccio1, D. Petrucci2, M. Milanesi2, L. Pascucci1, G. Chillemi2, K.Cappelli1

1 Università degli Studi di Perugia, Dipartimento di Scienze Veterinarie.

2 Dipartimento per la Innovazione nei sistemi biologici, agroalimentari e forestali.

Other than being a valuable nutrition source, milk represents a sophisticated signaling system that delivers maternal messages. This property seems to be mostly mediated by signaling molecules enclosed in micro/nano-sized membrane-bound structures called Extracellular Vesicles (EVs). EVs act as signal mediators between distant cells and/or tissues, exerting several biological effects [1,2]. Our aim was to characterize the molecular content of cow, donkey and goat milk EVs (mEVs) through RNA, metabolite and lipid omic analysis in view of prospective applications as immunomodulatory and anti-inflammatory compound, being milk a scalable and reliable source of EVs. Mass milk isolated EVs of the chosen species were subjected to transcriptomic and metabolomic/lipidomic analyses through massive parallel sequencing and mass spectroscopy (MS) coupled with Ultrahigh-performance liquid chromatography (UHPLC). RNA sequencing on both mRNA and smallRNA libraries was performed, targeting over 10,000 transcripts and 2,000 smallRNAs in each species. In order to characterize similarities and peculiarities between species, we compared the mEVs mRNA cargo selecting 10% of the most expressed one-to-one orthologous genes for each species (1223 in total). A total of 110 genes were present in the mEVs of the three species; donkey and goat being the most similar species with 335 shared entries. Also DGE analysis on all the orthologous genes highlighted cow distinct signature with 1055 and 875 up-regulated genes (FC>2, FDR<0.05) compared to goat and donkey, respectively. Concerning smallRNAs, donkey was found to be the most divergent species with 57% of the RPKM total amount referring to micro RNA (miRNA) and a conspicuous component of miscellaneous RNA (42%), mostly Y-RNA and Vault, while, for cow and goat, 99% of the RPKM referred to miRNA; the remaining part enclosed protein-coding genes, snoRNAs, snRNAs, lncRNAs and scaRNAs. Several miRNAs with known anti-inflammatory capabilities were found and target genes of the most expressed miRNAs (covered by 95% of RPKM) were also retrieved. Functional analysis on up-regulated species-specific genes (mRNA) and targets of miRNAs revealed similar results with enriched GO terms related to epigenetic regulation, macromolecules metabolic processes for cow; goat showed biological processes relative to mitochondrion, intracellular transport, protein formation and maturation and cellular component organization; donkey additionally showed a high number of terms related to peroxisomes. The metabolomic analysis, in accordance with the transcriptome results, revealed both common and specific mEV metabolites in all species comparisons. Enriched metabolic pathways were investigated and consistent signals were found for metabolites with immunomodulating effects such as arginine, asparagine, glutathione and lysine. The lipidomic analyses highlights a total of 3348 lipids, 2175 of these were shared among the three species and goat had nearly 1000 more. Flavones was the most represented lipid class for all the species but additional polyphenols were found with well-known anti-inflammatory and antioxidant properties confirming results of the other molecules. This in-depth molecular characterization highlights key molecules enclosed in mEVs of cow, donkey and goat particularly relevant for the immune and inflammatory response regulation and biological processes involved in oxidative stress and cell homeostasis which make these micro/nano-sized structures possible effectors potentially able to improve chronic inflammatory conditions. References [1] Robbins, P. D. & Morelli, A. E. Regulation of immune responses by extracellular vesicles. Nature Reviews Immunology 14, 195–208 (2014). [2] Ma, Z., Wang, Y. & Li, H. Applications of extracellular vesicles in tissue regeneration. Biomicrofluidics 14, 011501 (2020).

Extracellular vesicles released by tumor endothelial cells suppress immune defense by enhancing PDL1 expression on myeloid cells.

Tatiana Lopatina1, Malvina Koni1, Cristina Grange1, Massimo Cedrino2, Emilio

Venturelli1, Francesco Rivera1, Giovanni Camussi1, Maria Felice Brizzi1

1 Department of Medical Sciences, University of Turin. 2 2i3T Scarl University of Turin, Turin, Italy

Immune regulation by tumor cells is a key event for tumor establishment and

growth. We showed that tumor endothelial cells (TEC) release extracellular vesicles

(TEV) that favor tumor growth by multiple mechanisms, including immune regulation

(1). Previously we showed that TEC-EVs enhanced T regulatory cell formation (2).

Herein we demonstrate that TEV significantly enhance the number of myeloid cells in

vitro and these cells were significantly more positive for PDL1. Coculture of tumor cells

with PBMC stimulated with nTEV leads to significant increase in the number of tumor

cells with respect to the control. Since we previously showed that IL-3 signaling

pathway may influence pro-tumorigenic function of native TEV (nTEV) enhancing their

pro-metastatic properties (3), we analyze the immune effect of the TEV released after

blocking of IL-3R on TEC (αTEV). As expected, αTEV did not enhanced PDL1 expression

on monocytes and as consequence did not favor tumor cell viability in coculture with

PBMC. The same effects were seen in vivo using syngeneic model of triple negative

breast cancer. Balb/c mice were intravenously injected with 4T1 cells and treated or

not with nTEV/αTEV. Lung tissue of mice treated with nTEV contained significantly

more PDL1-positive myeloid cells and tumor cells with respect to control mice; vice

versa for lung tissue of mice treated with αTEV. We suggest that IL-3 signaling is crucial

for the regulation of tumor immune suppression through TEV.

References.

1. Marar C, Starich B, Wirtz D. Extracellular vesicles in immunomodulation and tumor progression. Nat Immunol. 2021 May;22(5):560–70.

2. Lopatina T, Favaro E, Danilova L, Fertig EJ, Favorov AV, Kagohara LT, et al. Extracellular Vesicles Released by Tumor Endothelial Cells Spread Immunosuppressive and Transforming Signals Through Various Recipient Cells. Front Cell Dev Biol [Internet]. 2020 [cited 2021 Feb 12];8. Available from: https://www.frontiersin.org/articles/10.3389/fcell.2020.00698/full

3. Lopatina T, Grange C, Cavallari C, Navarro-Tableros V, Lombardo G, Rosso A, et al. Targeting IL-3Rα on tumor-derived endothelial cells blunts metastatic spread of triple-negative breast cancer via extracellular vesicle reprogramming. Oncogenesis. 2020 Oct 10;9(10):1–14.

Zebrafish as a functional model to assess the effects of Extracellular Vesicles on innate immune response modulation

M. Cafora1,2, L. Ferrari1, M. Hoxha1, L. Cantone1, V. Bollati1, A. Pistocchi2

1 EPIGET LAB, Department of Clinical Sciences and Community Health, Università degli Studi di Milano,

Milan, Italy

2 Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano,

Milan, Italy

3 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Unit of Occupational Medicine, Milan,

Italy 4 Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Milan, Italy

The release of extracellular vesicles (EVs) is a common feature of all cell types, both prokaryotic and

eukaryotic, with a critical role in cell-to-cell communication. In particular, increasing evidence suggests

that bacteria belonging to the human microbiome are able to communicate not only with each other, but

also with the host immune cells through the release of bacterial EVs (bEVs). To functionally evaluate the

effects of bEVs on the host immune-modulation, we set-up a methodological approach by taking

advantage of the zebrafish model. In particular, we injected isolated EVs from different biological samples

into the zebrafish embryo circulation to follow in vivo the response of the innate immune system.

Compared to mammalian models that present limited accessibility of developmental stages, high

husbandry costs and the impracticality of genetic analyses, zebrafish is genetically tractable and, due to

its fecundity and small size, is suitable for large-scale studies. Furthermore, zebrafish embryos are

optically transparent, which facilitates the EVs injection and the development progression in real-time.

Moreover, it is possible to directly follow the immune response at a single cell level using transgenic

zebrafish lines where specific immune cell populations are fluorescent, such as the Tg(mpx:GFP) line with fluorescent neutrophils or the Tg(mpeg:mcherry) line with fluorescent macrophages. In addition, pro-

and anti-inflammatory cytokines are evolutionary conserved with human and the inflammation can be

measured by assessing cytokines expression by RT-qPCR or ELISA techniques. In this context, we are

isolating bEV from bacterial cultures of Moraxella catharralis, and injecting them into the circulation of

zebrafish embryos under different environmental conditions, such as under Particulate Matter (PM)

exposure, in order to investigate their ability to modulate the innate immune response. This approach

allows assessing the functional effects of specific EV sub-population by using zebrafish as a well-

characterized model system.