ABSTRACT BOOK

ORAL COMMUNICATIONS

Mesenchymal stromal cell-derived extracellular vesicles (EVs) as

mediators of anti-inflammatory effects: endorsement of

macrophage polarization

C. Lo Sicco1, D. Reverberi1, L. Pascucci2, C. Franzin3, M. Pozzobon3,4, F. Villa1, C. Gorgun1,5, R. Tasso1,5

1 IRCCS Ospedale Policlinico San Martino, Genova, 2 Department of Veterinary Medicine, University of Perugia, 3 Stem Cells and Regenerative Medicine Lab, Fondazione Istituto di

Ricerca Pediatrica Città della Speranza, Padova, 4 Department of Women and Children Health, University of Padova, 5 Department of Experimental Medicine, University of

Genova

Mesenchymal Stromal Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues [1]. Preliminary results indicate that the paracrine activity of MSC promotes a functional switch of macrophages from a pro- (M1) to an anti-inflammatory (M2) state [2]. Since extracellular vesicles (EVs) are relevant components of the MSC secretome, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived-MSCs to investigate their involvement as modulators of MSC anti-inflammatory effects. The EV-isolation method was based on differential centrifugations of the medium conditioned by MSC exposed to either normoxic or hypoxic conditions (EVNormo and EVHypo). Both types of EVs were efficiently internalized by responding bone marrow-derived macrophages, eliciting their switch from a M1 to a M2 phenotype. Observations that different macrophage subsets are associated with different stages of muscle regeneration led us to investigate whether EV treatment could influence macrophage polarization from M1 to M2 phenotype in vivo. Taking advantage of a cardiotoxin (CTX) injury mouse model, we observed a down-regulation of IL6 and Nos2, concurrent to a significant up-regulation of Arg1 and Ym1, late markers of alternative activation in injured and EV-treated muscles. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EVHypo administration. Collectively, these data indicate that MSC-EVs possess effective anti-inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs.

References

[1] Kode, J., Mukherjee, S., Joglekar, M., Hardikar, A., 2009. Mesenchymal stem cells: immunobiology and role in immunomodulation and tissue regeneration. Cytotherapy 1(4), 377-391.

[2] Ulivi, V., Tasso, R., Cancedda, R., Descalzi, F., 2014. Mesenchymal stem cell paracrine activity is modulated by platelet lysate: induction of an inflammatory response and secretion of factors maintaining macrophages in a proinflammatory phenotype. Stem Cells and Development 23(16),1858-1869.

MSC extracellular vesicles for treatment of alpha-1-antitrypsin deficiency

pulmonary diseases

Bari Elia1, Ferrarotti Ilaria2,3, Perteghella Sara1,4, Di Silvestre Dario5, Rossi Rossana5, Mauri Pierluigi5, Grisoli Pietro1, and Corsico Angelo Guido2,3,4, Torre Maria Luisa1,4

1 University of Pavia, Department of Drug Sciences, Pavia, Italy 2 Center for Diagnosis of Inherited Alpha1-antitrypsin Deficiency, Pavia, Italy

3 Pneumology Unit, IRCCS San Matteo Hospital Foundation, Pavia, Italy 4 PharmaExceed s.r.l., Pavia, Italy

5 Institute for Biomedical Technologies, Segrate, Milan, Italy Recently, we defined a scalable GMP-compliant production process for Mesenchymal Stem/Stromal Freeze-Dried secretome (lyo-secretome): proteomic investigation revealed that this product contains alpha-1-anti-trypsin (AAT), the main inhibitor of pulmonary neutrophil elastase [1]. Here, we propose lyo-secretome for the treatment of AAT deficiency-associated lung diseases. MSC-secretome release was obtained by platelet lysate starvation (ST) or by ST and dexamethasone (DEX) and/or IL-1β stimulation. Supernatants were purified by ultrafiltration, added with mannitol and freeze-dried. Expression of SERPINA1 transcripts in MSCs was evaluated by RT-PCR. MSC-secretome was fractioned to separate the extracellular vesicles (EV)-enriched fraction (EV, >300 kDa), the low molecular weight protein fraction (LMW, 5-100 kDa) and the high molecular weight protein fraction (HMW, 100-300 kDa). Protein content was determined by BCA Protein Assay Kit, AAT was measured by a rate immune nephelometric method and proteomic analysis was performed by LC-MS/MS. Elastase inhibition activity was assessed spectrophotometrically and calculated as Abscontrol-Abssample/Abscontrol x 100. Logarithmic lowering of the bacterial population was calculated against Gram-positive (S.aureus, S. aureus MRSA) and Gram-negative (K. pneumoniae, Ps. aeruginosa) bacteria. Results are reported as mean ± standard deviation, n=3. Proteomic investigation revealed that MSC-secretome contains AAT, 73 other proteins involved in protease/antiprotease balance and 48 proteins involved in the response to bacteria. Treatment with DEX and/or IL-1β increased MSC-secretome total protein content, but not AAT. The expression of AAT transcripts increased after ST and after IL-1β+DEX treatment. Secretome fraction separation revealed that the AAT content, expressed as µg/mg of freeze-dried sample, was 1.37 ± 0.25 for EV fraction, 1.38 ± 0.64 for LMW fraction and 0.16 ± 0.05 for HMW fraction. The EV fraction showed the highest anti-elastase activity (29.80 % ± 4.336, at 100 mg/ml) with respect to protein fractions. All the batches exhibited a good dose-dependent anti-elastase activity: at the highest dose (100 mg/ml), the inhibition rates were 46.17% ± 13.546, 39.97% ± 9.609, 41.77% ± 10.446 and 32.74% ± 15.196, for ST, DEX, IL-1β and IL-1β+DEX stimulated secretome, respectively. Finally, lyo-secretome showed antimicrobial activity on Gram-negative bacteria, especially for K. pneumoniae. We proved that MSC-secretome contains AAT with high anti-elastase in vitro activity. AAT is present both in the soluble fraction and associated with EVs, that can act as a natural carrier, promoting AAT in vivo stability and activity. Finally, MSC-secretome was active on Gram-negative bacteria. This paves the way for the use of MSC-secretome in the treatment of AAT-deficiency lung diseases.

References

[1] E. Bari et al. Pilot Production of Mesenchymal Stem/Stromal Freeze-Dried Secretome for Cell-Free Regenerative Nanomedicine: A Validated GMP-Compliant Process, Cells 7(190) (2018).

Urinary extracellular vesicles carrying klotho improve the recovery of renal function in acute tubular injury model

E. Papadimitriou1, C. Grange2, V. Dimuccio1, C. Pastorino1, J. Molina1, R. O’Kelly3, LJ

Niedernhofer3, PD Robbins3, G. Camussi3, B. Bussolati1.

1Department of Molecular Biotechnology and Health Sciences, 2Department of Medical Sciences; University of Torino, Turin, Italy, 3Department of Medical Sciences; University of Torino, Turin, Italy. 3Institute on the Biology of Aging and Metabolism, University of Minnesota, Minneapolis, MN.

Acute kidney injury (AKI), defined by a rapid deterioration of renal function, is a common complication in hospitalized patients [1]. Recent therapeutic strategies, exploiting the properties of extracellular vesicles (EVs), have been gaining increasing interest as the demand for new approaches targeting AKI is considerably high [2]. In physiological conditions, renal cells have been described to release bioactive EVs along the nephron and target downstream cells, facilitating an intra-nephron communication. In the present study we investigate the possible regenerative effect of renal-derived EVs, isolated from normal human urine (uEVs), in a murine model of acute kidney injury generated by glycerol injection. Our data showed the unique presence of reno-protective Klotho within uEVs and demonstrated that intravenously injected uEVs in AKI mice resulted in accelerated renal recovery. Tubular cell proliferation was stimulated, while functional and histological abnormalities were alleviated. uEV administration reduced the expression of renal injury and inflammation markers and induced the expression of endogenous Klotho, lost upon damage. Labelled uEVs were found preferentially localized within injured kidneys, transferring their miRNA cargo, a mechanism previously described involved in tissue repair. Similar results in tissue repair were observed, when EVs isolated from mouse urine were administered in AKI mice. However, the use of uEVs from Klotho null mice showed no regenerative effect. The repairing capacity of the latter was further achieved when engineered with human recombinant Klotho, prior to their administration. On the same line, the engineering with recombinant Klotho of ineffective fibroblast-derived EVs resulted in the acquisition of regenerative capacity. Overall this study suggests a new therapeutic strategy for acute kidney injury using uEVs, highlighting the importance of renoprotective Klotho. [1] Morigi M, Rota C, Remuzzi G. Mesenchymal Stem Cells in Kidney Repair. Methods Mol Biol. 2016. [2] Grange C, Iampietro C, Bussolati B. Stem cell extracellular vesicles and kidney injury. Stem Cell Investig. 2017.

VES4US, a Horizon 2020-Future and Emerging Technology project: Selection and characterization of natural source derived extracellular

vesicles (EVs)

Giorgia Adamo(1), Antonella Cusimano(1), Daniele Romancino(1), Sabrina Picciotto(1), Samuele Raccosta(2), Vincenzo Martorana(2), Rosina Noto(2), Rita Carrotta(2), Elia Di Schiavi(3), Giovanna L.

Liguori(4), Christopher Stanly(3), Rachel Parkes(5), David Fierli(5), Anita Aranyos(5), Maria Elena Barone(5), Annamaria Kisslinger(6), Katharina Landfester(7), Blanca Rodriguez(8), Svenja

Morsbach(7), Darja Božič(9), Ales Iglic(9), Veronika Iglic(9), Laura Corcuera(8), Paolo Arosio(10), Mauro Manno(2), Gabriella Pocsfalvi(3), Nicolas Touzet(5) and Antonella Bongiovanni(1).

(1) Institute for Biomedical Research and Innovation (IRIB), National Research Council of Italy

(CNR), Palermo, Italy; (2) CNR, Institute of Biophysics, Palermo, Italy;

(3) CNR, Institute of Biosciences and BioResources (IBBR), Napoli, Italy; (4) CNR, Institute of Genetics and Biophysics (IGB), Napoli, Italy;

(5) Institute of Technology Sligo (ITSligo), Sligo, Ireland; (6) CNR, Institute of Experimental Endocrinology and Oncology (IEOS), Napoli, Italy;

(7) Max-Planck Institute for Polymer Research, Mainz, Germany; (8) Zabala Innovation Consulting, Navarra, Spain;

(9) Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia; (10) ETH Zurich Institute for Chemical and Bioengineering, Zurich, Switzerland.

VES4US is a European project funded under the Horizon 2020 Future and Emerging Technology (FET) Open programme. Its aim is to develop an innovative platform for the efficient production of extracellular vesicles (EVs) from a renewable biosource (https://ves4us.eu). Several recent studies and few applications have focused on cell-free therapies based on the use of EVs owing to their properties such as low immunogenicity, long circulating half-life, ability to bypass biological barriers and mediation of inter-species cell communication. One objective of VES4US is to create a biocompatible and cost-effective EV-based delivery system, which could be applied in a variety of sectors such as target therapies to cosmetics. In this view, we have already selected an innovative natural source and the first phase of the project has focused on identifying the best species of this EV-producing natural source. Next, we have optimized its cultivation and the isolation of EVs using differential ultracentrifugation and tangential flow filtration. The physiochemical characterization of EVs was carried out according to MISEV-2018 guidelines1 using: 1) biochemical methods (BCA analyses and immunoblotting) to verify the presence of specific EV-biomarkers, 2) dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) to assess the particles number and distribution, 3) electronic scanning microscopy (SEM) and atomic force microscopy (AFM) for imaging analyses and 4) bilayer-specific fluorescence staining to assess the purity of the EV preparations. We generated ‘EV Identity Cards’ containing the EV features from the sources they were isolated from in order to facilitate the selection of the most promising one to up-scale. The next steps will be dedicated to the scalable production, functionalization and engineering of EVs. Their bio-activity will also be explored using both in vitro and in vivo models. This research will allow the development of novel natural nanocarriers with unprecedented features, which fit as biocompounds in the context of future therapeutics, cosmetics or nutraceutical formulations. Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 801338.

1Théry 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;7(1):1535750

Extracellular vesicles from adipose mesenchymal stromal cells promote

cardio-renal protection in DOCA-salt hypertensive model

Rafael Soares Lindoso 1, Jarlene Alécia Lopes 1, Renata Binato 2, Eliana Abdelhay 2, Christina Maeda Takiya 1, Benedetta Bussolati 3, Adalberto Vieyra 1, Federica Collino 1,4.

1 Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro,

Brazil, 2 National Institute of Cancer, Rio de Janeiro, Brazil, 3 Department of Molecular Biotechnology and Health Sciences, University of Torino, Turin, Italy, 4 Department of Biomedical

Sciences, University of Padova, Padua, Italy.

Extracellular vesicles (EVs). derived from mesenchymal stromal cells have been demonstrated to play an important role in the protection from the transition of acute kidney injury (AKI) to chronic kidney disease (CKD) and its progression.

Hypertension (HTN) is. a multifactorial disease that acts as comorbidity factor for the development of cardiovascular disease and CKD. Here, we investigated the potential of EVs derived from adipose-mesenchymal stromal cells (ASC-EVs) to be used as therapeutic tool for the treatment of cardio-renal syndrome (CRS) associated with CKD, using the uninephrectomized DOCA-salt hypertensive rat model.

We demonstrated that multiple administration of ASC-EVs induced a protective effect on renal tissue, by reducing tubular (9.3 ± 0.21 vs 7.5 ± 0.5, p <0.05 compared to DOCA) and vascular damage (3.1 ± 0.0 vs 2.2 ± 0.04, p <0.05 compared to DOCA) thus preserving kidney function as demonstrated by the maintenance of normal glomerular filtration rate and the absence of proteinuria in respect to DOCA-salt untreated rats. Also, the glomerular region was significantly affected in DOCA-salt rats, as demonstrated by the increased in the glomerular damage marker, Desmin and the presence of glomerular lesions, which were significantly reduced in both the UNX and DOCA-EV groups (p <0.05).

EV administration significantly reduced inflammatory response in the kidney by downregulating the pro-inflammatory molecules monocyte chemoattracting protein-1 (MCP-1) and plasminogen activating inhibitor-1 (PAI1) (p< 0.05) and reducing the recruitment of macrophages in the kidney (ED-1 positive cells). Consequence of the attenuation of the inflammatory response by ASC-EVs infusion, was the significant reduction of fibrosis, as detected by the decrease in collagens (Col1A1, Col4A1) and fibronectin (FN) expression in respect to pathological DOCA-salt animals. Moreover, the kidney of DOCA-salt rats showed a strong the interstitial and periglomerular accumulation of a-SMA, that was almost absent in ASC-EV animals. ASC-EVs were able to act in multiple organs, preventing fibrosis and inflammation development also in the heart of DOCA-salt rats, therefore maintaining blood pressure within normal level during the 8-weeks of treatment.

Kidney miRNA microarray profile of DOCA-salt rats identified a distinctive miRNA signature associated with the damage. The most representative pathway in the kidney was associated with the epithelial-mesenchymal transition. Moreover, we demonstrated that the miR-200 family was altered by DOCA-salt treatment and EV administration reverted their reduction, thereby affecting the TGF-β signaling and preventing renal damage. In conclusion, our results indicate that EVs can be an effective therapeutic strategy to prevent and rescue hypertension-induced CKD by acting on the kidney-heart crosstalk.

Supported by the Brazilian National Research Council (CNPq) and the Carlos Filho Rio de Janeiro State Research

Foundation (FAPERJ).

Extracellular vesicles surface protein profile as biomarkers to characterize allograft rejection in heart transplanted patients

C. Castellani1, J. Burrello2, M. Fedrigo1, S. Bolis2, A. Di Francesco1, A. Burrello3, F.

Tona4, T. Bottio4, G. Toscano4, A. Gambino4, G. Gerosa4, G. Vassalli2,5, A. Angelini1, L. Barile2,5.

1 – Cardiovascular Pathology and Pathological Anatomy Unit, Dept. of Cardiac-Thoracic and Vascular Sciences and Public Health, University of Padua, Italy

2 - Laboratory of Cellular and Molecular Cardiology and Laboratory for Cardiovascular Theranostics Cardiocentro Ticino Foundation, Lugano, Switzerland.

3 - Department of Electrical, Electronic and Information Engineering "Guglielmo Marconi" (DEI), University of Bologna, Italy

4 – Division of Cardiac Surgery, Dept. of Cardiac-Thoracic and Vascular Sciences and Public Health, University of Padua, Italy.

5- Faculty of Biomedical Sciences, Università Svizzera Italiana, Lugano, Switzerland Background - Exosomes, secreted extracellular vesicles (EV), may represent a non-invasive diagnostic tool as they are easily detectable in biological fluids and contain specific set of nucleic acids, proteins and lipids based on their cell origin and environment. In the context of heart transplantation they might support endomyocardial biopsies (EMBs) for the diagnosis of cardiac allograft rejection and for monitoring post-transplant status with considerable benefits for patient. Purpose - We aimed at investigating differences in EV surface protein expression profile as reproducible and objective measures to characterized acute cellular rejection (ACR) and antibody-mediated rejection (pAMR), and to monitor the immunological profile and evolution of allograft rejection in heart transplanted patients. Methods - Plasma was collected from 53 patients at the time of EMBs monitoring for allograft heart rejection: at EMBs 12 displayed ACR, 8 pAMR, and 33 no rejection, which served as controls. After serial centrifugations, EVs were quantified by nanoparticle tracking analysis (NTA) and 37 EV-surface markers were characterized by an antibody-coated bead-based multiplex flow cytometric assay. According to differential EV-markers expression two diagnostic model were built and validated through supervised learning algorithms (linear discriminant analysis – LDA – and random forest – RF – model). Results – The number of circulating plasma-EV was significantly increased and particle diameter significantly decreased in patients with either pAMR or ACR (P < 0.001). Noteworthy, the FACS analysis on CD9, CD63 and CD81 confirmed the increased concentration of EVs (1.99-3.56 fold-increase). Among EV-surface markers, CD3, CD2, ROR1, SSEA-4, HLA-ABC, and CD41b were identified as discriminants between controls and ACR, whereas CD19, HLA-DRDPDQ, CD2, CD25, ROR1, SSEA-4, HLA-ABC, CD41b, CD326, and CD20 discriminated controls from patients with pAMR (P < 0.05 for all comparisons). Univariate analysis and ROC curves confirmed a reliable diagnostic performance for each single marker (AUC range 0.785-0.944 for ACR, 0.761- 0.871 for pAMR). Finally, the combination of all differentially expressed potential biomarkers, through an LDA model or a RF analysis respectively, resulted in the accurate detection of 90.6% and 100.0% of patients with either ACR or pAMR. Conclusion - By profiling EV surface proteins we were able to discriminate different types of allograft rejections. Circulating plasma-derived EV may have a strong potential as a new tool to characterize, monitor and predict cardiac allograft rejection.

Exosomal HSP60 levels and related miRNAs in brain tumors

C. Caruso Bavisotto 1,2,3, F. Graziano 4, F. Rappa 1,2, A. Marino Gammazza 1,2, A. Pitruzzella 1,2, S. David 1, E. Conway de Macario 5, A.J.L. Macario 2,5, P.L. San Biagio 3, D. Bulone 3, F.

Bucchieri 1, F. Cappello 1,2, D.G. Iacopino 4, C. Campanella 1,2

1 Department of Biomedicine, Neurosciences and Advanced Diagnostic (BiND), Institute of Human Anatomy and Histology, University of Palermo, Palermo, Italy.

2 Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy. 3 Institute of Biophysics, National Research Council, Palermo, Italy

4 Department of Biomedicine, Neurosciences and Advanced Diagnostic (BiND), Section of Neurosurgery, University of Palermo, Palermo, Italy.

5 Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore-Institute of Marine and Environmental Technology (IMET), Baltimore, Maryland.

In cancer, Extracellular Vesicles (EVs), such as exosomes, contribute to tumor progression by regulating local and systemic parameters [1,2]. Since exosomes are released into body fluids, they may be used in nanomedicine as a valuable source of diagnostic biomarkers [3]. The prognosis of brain tumors is poor even after surgical resection followed by post-operatory chemo- and radio-therapies and it is cogent to find innovative treatments. The discovery that molecular chaperones can be determinant factors in tumorigenesis and the increasing understanding of exosomes, particularly in what refers to their release by tumor cells and contents, including chaperones and miRNA, provide elements to develop novel treatment strategies and means [4]. We measured the chaperone HSP60 and related miRNAs in primary brain tumors and peritumoral cells in vivo to determine levels and distribution. In addition, the presence and levels of HSP60 and miRNAs involved in its regulation were investigated in blood exosomes isolated from tumor patients before and after ablative surgery. Blood and pathological tissue samples were taken on the surgery day, blood was collected at one week, one month, and three months after surgery, and exosomes were isolated. The results revealed distinctive changes in the levels of miRNAs involved in HSP60 regulation in brain tumors, pointing to their potential as biomarkers for diagnosis and patient monitoring during treatment.

Keywords: molecular chaperones, HSP60, exosomes, brain tumor, new therapeutic tools. Supported by the Euro Mediterranean Institute of Science and Technology, and funded with the Italian National Operational Programme for Research and Competitiveness 2007-2013 grant awarded to the project titled "Cyber Brain – Polo di innovazione" (Project code: PONa3_00210, European Regional Development Fund). CCB was partially supported by a STSM bursary from the EU COST Action BM1202. References [1] Santiago-Dieppa, D. R.; Steinberg, J.; Gonda, D.; et al. Extracellular vesicles as a

platform for ‘liquid biopsy’ in glioblastoma patients. Expert Rev. Mol. Diagn. 2014, 14, 819–825.

[2] Roma-Rodrigues, C.; Fernandes, A. R.; Baptista, P. V. Exosome in tumour microenvironment: overview of the crosstalk between normal and cancer cells. Biomed Res. Int. 2014, 2014, 179486.

[3] Cappello, F.; Logozzi, M.; Campanella, C.; et al. Exosome levels in human body fluids: tumor marker by themselves? Eur. J. Pharm. Sci. 2017, 96.

[4] Caruso Bavisotto, C.; Graziano, F.; Rappa, F.; et al. Exosomal Chaperones and miRNAs in Gliomagenesis: State-of-Art and Theranostics Perspectives. Int. J. Mol. Sci. 2018, 19, 2626.

Biophotonics-based biosensor for the detection of circulating

extracellular vesicles as biomarkers in personalized neurorehabilitation

Silvia Picciolini1, Alice Gualerzi1, Cristiano Carlomagno1, Marzia Bedoni1

1Laboratory of Nanomedicine and Clinical Biophotonics (LABION), Fondazione Don Carlo Gnocchi IRCCS S. Maria Nascente, Milan, Italy

One of the main hurdle in the rehabilitation and treatment of neurological diseases is the lack of easily accessible and sensitive biomarkers for the prediction of the progression rate and the

evaluation of therapy efficacy. Extracellular vesicles (EVs) represent promising biomarkers of neurological diseases, being involved in the onset and progression of Alzheimer’s disease (AD) [1], and in the regenerative and repair processes occurring after ischemic stroke [2], that are two of the most common causes of disability in our aging society. In the strive for a reliable and sensitive method to analyze EVs, we propose a biophotonics-based biosensor that takes advantage of Surface Plasmon Resonance imaging (SPRi) technique. We applied our recently optimized SPRi biosensor [3] for the detection and characterization of EVs isolated from the blood of stroke and AD patients. The SPRi-array was designed to separate simultaneously EVs released by neurons, astrocytes, microglia, oligodendrocytes, endothelial cells and apoptotic bodies, and to evaluate the presence and the relative amount of specific surface molecules related to pathological or recovery processes. Our results showed differences in the relative amount of specific cell-derived EV populations, and also in their cargo during the disease progression or resolution. In particular, variations in the amount of specific receptors related to neuroinflammation and neuroregeneration were observed in the circulating EVs of stroke patients before and after rehabilitation. Similarly, the characterization of EVs from AD patients demonstrated the presence of EVs loaded with altered cargoes compared to healthy subjects: differences in the lipid moieties present on neuronal EVs and variations in the activation phenotype of microglia EVs were observed. Our results provide support for using the SPRi-based biosensor for the detection and characterization of circulating EVs to evaluate their potential as peripheral biomarkers for the prediction of the recovery rate after stroke, the monitoring of AD patients and the evaluation of the rehabilitation outcomes in a personalized way.

References [1] Joshi P, et al. International Journal of Molecular Sciences. 2015;16(3):4800-4813. [2] Zhang ZG, Chopp M. Journal of Clinical Investigation. 2016;126(4):1190-1197. [3] Picciolini S, et al. Analytical Chemistry. 2018;90(15):8873-8880.

MicroRNA signatures loaded on small vesicles spread some features of senescent phenotype from senescent

to younger endothelial cells

Emanuela Mensà1, Michele Guescini

2, Angelica Giuliani

1, Manuela Ferracin

3, Gianluca Fulgenzi

1, Laura

Graciotti1, Maria Giulia Bacalini

3, Paolo Garagnani

3, Rina Recchioni

4, Fiorella Marcheselli

4, Gianluca Storci

3,

Anna Rita Bonfigli4, Maria Rita Rippo

1, Jacopo Sabbatinelli

1, Massimiliano Bonafè

3 and Fabiola Olivieri

1,4

1Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy.

2 Department

of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, Italy; 3 Department of Experimental, Diagnostic

and Specialty Medicine (DIMES), University of Bologna, 4IRCCS INRCA, National Institute, Ancona, Italy.

Abstract

Epigenetic mechanisms play a pivotal role in modulating inflammaging, currently recognized as one of the main

risk factors for clinical morbidity/mortality in the elderly (1). Inflammagig is fostered by the accrual of senescent

cells during aging and endothelial senescent cells could contribute to such phenomenon. Yet, the relative

contribution of extracellular vesicles (EVs)-carried microRNAs in vascular ageing is still poorly explored.

To verify if endothelial cells (HUVECs) and cognate EVs share the same miRNAs repertoire and if replicative

senescence affect the release of EVs and their miRNA cargoes, here we analysed EVs, both small (SV) and large

(LV) vesicles, released by non senescent (CON) and senescent (SEN) HUVECs and compared miRNAs expressed

in cells and their cognate EVs.

We observed that SEN released a three-fold greater number of SVs/cell than CON. Interestingly, 33% of miRNAs

detectable in cells was also loaded in EVs. Surprisingly, SVs and LVs shared only 24% of miRNAs, supporting

the hypothesis of a selective miRNAs loading in different EVs. Notably, miR-21-5p and miR-217 were

significantly hyper-expressed in SEN and their SVs. Notably, miR-21 and miR-217 can target DNMT1 and

SIRT1, enzymes hypo-expressed in SEN and involved in methylation pattern maintenance. To disentangle the

biological effects of senescent SVs on younger cells, CON cells were treated with SVs released by SEN. A

significant reduction of DNMT1 and SIRT1 proteins was observed, as well as a reduction of replicative rate, in

association with reduced cyclin-D expression. These effects appear miR-21-5p and miR-217 dependent.

Furthermore, we previously described an inverse U-shaped age-related trend for miR-21-5p in plasma levels

during normal aging and significantly increased miR-21-5p plasma levels in patients affected by cardiovascular

diseases and type 2 diabetes (2; 3). Here, we analysed miR-21-5p loaded on SVs circulating in plasma and

demonstrate for the first time that miR-21-5p shuttled by circulating SVs shows the same inverse U-shaped trend

that we observed in plasma, and that this trend is highlighted when centenarians are included in the analysis. This

finding strongly supports the hypothesis that circulating miR-21-5p loaded on SVs could be an archetypal

biomarker of cellular senescence and inflammaging.

Overall, our data suggest that miR-21 and miR-217 loaded on senescent SVs spread pro-senescence signals

interfering with DNA methylation and cell proliferation.

References

1) Fulop T, Witkowski JM, Olivieri F, Larbi A. The integration of inflammaging in age-related diseases. Semin Immunol.

2018 Dec;40:17-35. doi: 10.1016/j.smim.2018.09.003.

2) Olivieri, F., Spazzafumo, L., Santini, G., Lazzarini, R., et al., 2012. Age-related differences in the expression of

circulating microRNAs: miR-21 as a new circulating marker of inflammaging. Mech Ageing Dev 133, 675-685.

3) Prattichizzo, F., Micolucci, L., Cricca, M., De Carolis, S., Mensa, E., Ceriello, A., Procopio, A.D., Bonafe, M., Olivieri,

F., 2017. Exosome-based immunomodulation during aging: A nano-perspective on inflamm-aging. Mech Ageing Dev 168,

44-53.

Quantitative assessment of RNA targets on NBI-isolated extracellular

vesicles from the blood of metastatic colorectal cancer patients.

Vito G. D’Agostino1, Luigi Pasini2, Paola Ulivi2, Luca Frassineti2, Manuela Gariboldi3, Filippo

Pietrantonio3.

1Department CIBIO, University of Trento, Italy

2Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola, Italy

3Istituto Nazionale dei Tumori (INT), Milano, Italy

Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their

potential cargo of diagnostic markers. An efficient and high-throughput study of EVs is needed to

understand the clinical utility of somatic alterations potentially found at RNA level. We recently

designed the nickel-based isolation (NBI) procedure to rapidly isolate EVs preserving their integrity

and dispersity in solution, while minimizing particle aggregation. Therefore, we combined this

approach with a new protocol of droplet digital PCR (ddPCR), allowing for direct encapsulation of

EVs into generated oil droplets, to ultrasensitively detect cancer biomarkers from liquid biopsy of

oncological patients. From a retrospective cohort of 27 metastatic colorectal cancer (mCRC)

patients, we identified somatic BRAF and KRAS transcript mutations matching 100% of

concordance with tissue diagnostics and higher sensitivity and specificity compared with

immunoenrichment of tumor-derived EVs. We also had a chance to validate the obtained liquid

biopsy data in cases where further DNA analyses on additional matching FFPE tissues were

available, proposing these strategies as a valuable approach to probe the tumor heterogeneity in

advanced states of disease. Here we also show that a quantitative approach of RNA analysis on

circulating EVs can be exploited to detect EGFR expression levels in mCRC patients subjected to

anti-EGFR therapy.

Maternal-placental messaging through extracellular vesicles is influenced

by particulate air pollution exposure

Cantone L.1, Iodice S. 1, Mariani J.1, Ferrari L. 1, Pergoli L. 1, Hoxha M. 1, Dolo V. 3, Persico N. 2, Bollati

V. 1

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

Milano, Milan, Italy.

2 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. 3 Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

BACKGROUND: Growing evidences have shown that maternal exposure to particulate matter (PM) might be associated with an impaired fetal development and adverse birth outcomes but to date the biological mechanisms are still unknown. Extracellular vesicles (EVs) might be the ideal candidate to mediate the effects of PM exposure on pregnancy: potentially they could be responsible for the complex signaling occurring between the maternal tissues representing the primary target of PM exposure, such as the lungs, and the developing fetus. The main objective of the present study was thus to determine the effects of short term (day before blood drawing) and long-term (90 days) exposure to PM on EV production in a cross-sectional sample of 199 healthy pregnant women recruited at the 11th week of pregnancy. METHODS: Ambient concentrations PM2.5 were obtained from the regional air quality monitoring network. Size and cellular origin of plasma EVs were characterized by nanoparticle-tracking and flow-cytometry analysis. Association between PM exposure and EVs was evaluated by multivariable regression model adjusted for age, BMI, smoking habits, season, apparent temperature, warm/cold months. RESULTS:

Short-term exposure to PM2.5 (10 µg/m3 increase) was associated with increased release of CD14+ EVs (Δ% = +6.5%, p=0.008) derived from macrophages/ monocytes, as well as hERV-w+ EVs (Δ% = +6.6%, p=0.008). Long-term exposure to PM2.5 was in turn associated with lower concentration of CD14+ EVs (Δ% = -62.5%, p<0.001) and hERV-w+ EVs (Δ% = -62.7%, p<0.001). In addition, CD66+ EVs produced from neutrophils were reduced (Δ% = -53.6%, p<0.001). CONCLUSION:

Our study sheds light on the potential mechanisms underlying the adverse effects of air pollution exposure during pregnancy. The positive effect of short term exposure to PM is suggestive of a pro-inflammatory reaction. This reactive ability might be desensitized once the high exposure is maintained for a long period (i.e.90 days).

Circulating extracellular vesicles as biomarkers in patients after ST-segment elevation myocardial infarction.

J. Burrello1,2, S. Bolis1, E. Provasi1, E. Caporali Elena1, L. Grazioli Gauthier1, A.

Burrello3, L. Barile1,4, G. Vassalli1,4.

1 - Laboratory of Cellular and Molecular Cardiology and Laboratory for Cardiovascular Theranostics Cardiocentro Ticino Foundation, Lugano, Switzerland.

2 - Division of Internal Medicine and Hypertension Unit, Department of Medical Sciences, University of Turin, Turin, Italy

3 - Department of Electrical, Electronic and Information Engineering "Guglielmo Marconi" (DEI), University of Bologna, Italy.

4Faculty of Biomedical Sciences, Università Svizzera Italiana, Lugano, Switzerland Circulating extracellular vesicles (EV) are released from a variety of cells (e.g., platelets, endothelial, and inflammatory cells), reflecting their activation in response to physiological or pathological stimuli. Thus, EV have been defined as a form of “fluid biopsy” and a systematic screening for specific EV-associated antigens might be useful to define new disease-related biomarkers. After acute myocardial injury, changes in number and composition of EV are expected to occur even earlier to troponin increase and may have diagnostic relevance. The aims of the study are: (1) to investigate early changes in EV profile in patients after ST-segment elevation myocardial infarction (STEMI); (2) to identify candidate biomarkers among surface membrane-proteins carried by EV; (3) to develop a reliable prediction model for the diagnosis of STEMI, based on surface-markers expressed on circulating EVs. Blood was collected from 26 patients after STEMI and 26 controls with documented absence of coronary disease. STEMI patients were evaluated before coronary angioplasty (pre-PTCA), 12-36h, and 48-72h after reperfusion. After serial centrifugations, EVs were quantified by nanoparticle tracking analysis (NTA) and 37 EV-surface markers were characterized by an antibody-coated bead-based multiplex flow cytometric assay. A diagnostic model was built and validated through a linear discriminant analysis (LDA) on the base of differentially expressed surface-EV markers. At NTA, EV concentration was increased in STEMI patients vs. controls (3.5-fold increase at pre-PTCA evaluation); EV diameter was significantly higher in samples before reperfusion. Noteworthy, the EV higher concentration was detected earlier than the peak of troponin, thus supporting the hypothesis that changes in EV profile anticipated the death of cardiomyocytes. Among EV-surface markers, SSEA-4, CD40, CD62P, CD41b, CD42a, and CD31 were identified as potential discriminants between groups. Their expression was significantly associated with the diagnosis of STEMI at univariate analysis and was correlated to troponin levels and cardiac ejection fraction, thus being associated not only with the occurrence of myocardial infarction, but also with its severity. At ROC curve evaluation, the diagnostic performance of each single marker was not inferior to troponine, whereas the combination of all surface markers in the LDA model correctly detected 100% of patients with STEMI at pre-PTCA evaluation. In conclusion, we proposed an easy-reproducible prediction model based on EV-surface marker signature, which allows the accurate discrimination of patients with STEMI. Our data confirm that profiling of circulating EV can be considered a valuable diagnostic tool after cardiac injury.

Microproteomics workflow for exosome biomarker discovery:

enabling single mouse analysis on longitudinal models

Federica Anastasi1,2, Marialaura Dilillo1, Francesco Greco1,3, Eleonora Vannini4, Valentina Cappello5, Matteo Caleo4, Liam McDonnell1

1 Fondazione Pisana per la Scienza ONLUS, 56107 San Giuliano Terme, Pisa, Italy 2NEST, Scuola Normale Superiore, 56127 Pisa, Italy

3 Institute of Life Sciences, Sant’Anna School of Advanced Studies, 56127 Pisa, Italy 4CNR Neuroscience Institute, 56124 Pisa, Italy

5Istituto Italiano di Tecnologia, Center for Nanotechnology Innovation @NEST, 56127 Pisa, Italy

Proteomics analysis of exosomes by bottom-up-LC-MS/MS are overwhelmingly performed on exosomes purified by ultracentrifugation, which requires large sample volume and thus cannot be used to analyze the extracellular vesicles (EVs) isolated from the serum of individual mice. While xenograft mouse models are among the most frequently studied cancer models, longitudinal molecular analysis of body fluids during tumor development has been undermined by the lack of EVs purification methods compatible with the low sample volumes that may be obtained from a single mouse at each time point (maximum serum volume for such longitudinal studies is about 75 µl every 14 days). Here a micro proteomics work-flow for proteome analysis of exosomes purified from 50 µl of mouse serum is reported. This procedure enables the parallel study of the serum proteome and serum exosome proteome from the longitudinal serum samples of individual mice.

We compared two EVs purification procedures: precipitation (PPT) and size exclusion chromatography (SEC), and analyzed the vesicles using a modified SP3 protocol1. This led to the identification of 274±21 and 334±38 protein groups for PPT and SEC respectively. We were able to identify more EVs markers for SEC (25) than PPT (10)2. Transmission Electron Microscopy (TEM) reported vesicles diameters consistent with exosomes for both procedures (15nm to 135 nm), but also revealed the presence of aggregates in the PPT-exosome samples.

The methodological comparison revealed that SEC led to purer exosomes. We then scaled down this protocol to 50 µl of serum and further optimized the proteolytic digestion. This led to the identification of 277±2 protein groups from the exosomes of 50 µl of mouse serum. This method was then used to compare the exosome proteome from the serum of naive mice and glioblastoma (GBM) bearing mice (induced using GL261 cells). Statistical analysis revealed 6 and 6 down regulated proteins in the GBM samples, and which included proteins involved in cancer progression (Reelin, Myosin9, Talin). The procedure was then used for a longitudinal study of GBM progression, using individual GBM mice, each of which was sampled at three time points. The study revealed an increasing trend in the expression of 7 proteins, including extracellular activators of the PI3K-AKT pathway (Angiopoietin-1, Laminin subunit β1, Vitronectin).

References

(1) de Graaf, E. L.; Pelligrini, D.; McDonnell, L. A. A Set of Novel Automated Quantitative Microproteomics Protocols for Small Sample Amounts and Its Application to Kidney Tissue Substructures. J. Proteome Res. 2016, acs.jproteome.6b00889.

(2) Théry, C. et al. Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles . J. Extracell. Vesicles 2019, 8 (1), 1535750.

Characterization of the whole profile of extracellular vesicles isolated

from synovial fluid from different load-bearing joints.

D. D'Arrigo1,2, C. Arrigoni1, S. Bolis3, M. Cantù4, C. Candrian1, M. Moretti1,5 1 Regenerative Medicine Technologies Laboratory, Ospedale Regionale di Lugano, Unità di

Traumatologia e Ortopedia, Lugano, Switzerland 2 Department of Biotechnology and Bioscience, Università degli studi di Milano-Bicocca, Milano,

Italy 3 Laboratory of Cellular and Molecular Cardiology, Cardiocentro Ticino Foundation, Lugano,

Switzerland. 4 Laboratory of Clinical Biochemistry and Pharmacology, Department of Laboratory Medicine

EOLAB, Ente Ospedaliero Cantonale, Bellinzona, Switzerland.

5 Cell and Tissue Engineering Laboratory, IRCCS Istituto Ortopedico Galeazzi, Milano, Italia

Osteoarthritis (OA) is a multifactorial degenerative disease with an increasing incidence on the ageing population. The major issue related to this pathology consists in the lack of both early biomarkers and effective therapeutic treatments [1]. In this scenario, extracellular vesicles (EVs) represent one of the most promising approaches not only in the research of early and stage-specific OA markers but also in the development of new and effective regenerative clinical treatments [2]. We collected synovial fluid (SF) from different human joints of arthritic and non-arthritic patients subjected to joint surgeries. For the isolation of extracellular vesicles, we firstly digested the hyaluronic acid of the SF samples with hyaluronidase type -S (1000 U/mL) to make the SF less viscous. After that, we centrifuged samples at 1000g to eliminate cells and debris and subsequently all the EVs were isolated and separated based on their size. We then characterized the EVs belonging to each of the sub-populations by different parameters, among which the number, the size and the expression of 39 different superficial markers. Once the characterization of the EVs was completed, we obtained the EVs profile of the specific joint and we compared the profiles of different joints. Moreover, we also performed a preliminary assessment of the physiopathologic role of the EVs on the different articular cell populations in a microfluidic model of an OA joint previously developed in our laboratory. Briefly, in the device we seeded in a 3D matrix primary articular cells (human chondrocytes and synovial fibroblasts obtained from biopsies) separated by a channel in which human SF was injected. In the same channel we then added EVs and evaluated their effects on articular cells. We successfully isolated, separated and compared different subpopulations of SF-derived EVs based on their size collected from different joints. The EVs profile of different joints resulted slightly different. The analysis of the 39 EVs superficial markers revealed a positive expression of CD9, CD63 and CD81 the surface of the small size EVs (ranging from 50 to 200 nm, classically named exosomes), confirming the effectiveness of the assay. These preliminary results demonstrated the potential of our approach in the research of new biomarkers for the OA. In addition, thanks to our OA microfluidic model, we were able to evaluate and compare the physiopatologic role of the different subpopulations of SF-derived EVs. This could give the opportunity to also assess if in the OA progression different EVs subpopulations have a dissimilar biological effect, as demonstrated for other tissues [3].

References

[1] Cosenza, S., et al., 2017. Mesenchymal stem cells derived exosomes and microparticles protect cartilage and bone from degradation in osteoarthritis. Sci Rep. 24;7(1):16214. [2] Malda, J., et al., 2016. Extracellular vesicles — new tool for joint repair and regeneration. Nat Rev Rheumatol. 12(4):243-9. [3] Lasser, C., et al., 2018. Subpopulations of extracellular vesicles and their therapeutic potential. Mol Aspects Med. 60:1-14

Size-dependent Extracellular Vesicles lipid composition. In vitro studies

from a lymph-node metastatic melanoma cell line.

Accattatis F.M.1, Granata A. 1, Mazza S. 1, Baroni, S.1, Merlo L.1, Huber V.3, Vergani E.3, Corsini A.1,2, Arnaboldi L1.

1Dipartimento di Scienze Farmacologiche e Biomolecolari DiSFeB, Università Degli Studi di Milano 2IRCCS Multimedica, Milano

3IRCCS Istituto Nazionale Tumori; Laboratorio di Immunoterapia dei Tumori Umani, MIlano

Extracellular vesicles (EVs) are structures physiologically secreted by cells. Their main feature, the transport of protein, lipid and nucleic acids transfer to distant cells, suggests an involvement in physiological and pathological processes. Unfortunately, lack of characterization and separation methods impairs the comprehension of their functions. Following latest works, EVs are classified, based on their size, in small- (50-80nm), large- (80-120nm) exosomes and microvesicles (120-1000nm). Nevertheless, a new distinct population of non-membranous nanoparticles (exomeres; <50 nm) emerged1.

We are strongly convinced that separation and characterization of these particle subsets is of paramount importance not only to understand their different roles and behavior, but also to promote new targeted pharmacological approaches aimed at controlling EVs-dependent pathologies.

Therefore, we aimed to find a valid and reproducible method to separate different EVs subpopulations, using a lymph node melanoma metastatic cell line (LM-16), by differential ultracentrifugation (DC)2 and to characterize their lipid/protein content.

We cultivate cells in 150mm dishes with RPMI-10%FCS for three days. Cells are washed twice with PBS, supplemented with serum-free medium for further three days and then EVs isolated by DC, to obtain populations of the aforementioned theoretical size, further analyzed by TEM, GLC and colorimetric assays. We are currently setting up western-blot, dot-blot analysis and conditions to assess number, distribution and purity of recovered pellets by atomic force microscopy and by COlorimetric NANoplasmonic assay (CONAN)3.

Our preliminary data (n=2) obtained with these new separation conditions show a relative and continuous increase in saturated fatty acids (SFA) ranging from microvesicles (36.9%) to exomeres (65.8%), counterbalanced by a decrease in unsaturated fatty acids (UFA) from 49.4% (microvesicles) to 30.3% (exomeres) and an increase in fatty alchools (from 3.8% to 13.6% respectively). These results support our previous analysis where we isolated five different ranges of nanoparticles (< 50nm, 50-100 nm, 100-150 nm, 150-200 nm, > 200 nm) and where SFA gradually increased from 36.8±2.2 (>200 nm) to 59.0±3.3 (<50 nm).

Moreover, also the differences found in Phospholipid/Free Cholesterol ratio among the different populations (0.44 <50nm: 0.58 50-80: 0.56 80-120: 0.65 120-200: 0.78 200 nm) are in accordance with our previous data obtained with the former separation method, showing a relative decrease in PL/FC ratio ranging from smaller to bigger nanoparticles and reflecting an increased content in phospholipids (up to 2.5 folds) and free cholesterol (up to 8 folds) vs parental cells. The increased content in SFA concomitant with EVs size decrease, possibly due to higher presence of palmitic and stearic acid in phosphatidylcholine and phosphatidylethanolamine is currently being examined.

Altogether, these data show that a specific lipid signature not only characterizes EVs subset function (e.g. rigidity and biological fluids resistance) but may represent a useful marker for the effect of the pharmacological modulations. Since EVs are mainly constituted by cholesterol and phospholipids, modulation of the pathways involved in their synthesis is now under investigation by supplementing parental cells with statins, cholesterol, the neutral sphingomyelinase inhibitor GW4869 and siRNAs.

We will utilize this separation method to administer different nanoparticles subsets harvested from pharmacologically-treated and control cells to test their effects in functional tests (e.g. proliferation, apoptosis, migration, invasion), hoping that EVs alterations (number, distribution, composition and secretion) will shed light on their pathophysiological roles.

1) Zhang H. et al., 2018 Nature Cell Biology; 332. 2) Livshits M.A. et al., 2015. Sci Rep. 5; 17319 3) Maiolo D. et al., 2015. Anal. Chem.87; 4168

Potential roles of extracellular vesicles in brain cell-to-cell communication

F. Carfì Pavia1, M. A. Di Bella2, V. Brucato1, V. Blanda1, F. Zummo3, I. Vitrano1, C. M. Di Liegro3,

G. Ghersi3, G. Schiera3, I. Di Liegro2.

1Department of Engineering, University of Palermo, I-90128 Palermo; 2Department of Biomedicine, Neurosciences and Advanced Diagnostics, University of Palermo,

I-90127 Palermo; 3Department of Biological Chemical and Pharmaceutical Sciences and Technologies, University of

Palermo, I-90128 Palermo, Italy

Extracellular vesicles (EVs) are released into the extracellular space from both cancer and normal brain cells, and are probably able to modify the phenotypic properties of receiving cells1. EVs released from astrocytes and neurons contain FGF2 and VEGF2-3 and induce a ‘blood-brain barrier’ (BBB) phenotype in cultured brain capillary endothelial cells (BCECs, unpublished results), On the other hand, EVs from G26/24 oligodendroglioma induce apoptosis in neurons and astrocytes4-5. These effects are probably due to Fas Ligand and TRAIL, present in G26/24 vesicles4-5. Moreover, G26/24 EVs contain extracellular matrix remodeling proteases (such as ADAMTS)6, H1.0 histone protein, and H1.0 mRNA7. In particular, we previously hypothesized that G26/24 cells, and tumor cells in general, can escape differentiation cues, and continue to proliferate by eliminating proteins, such as the H1° linker histone (and its mRNA) 7, which could otherwise block proliferation. To study vesicle release in a system that can better resemble in vivo conditions, astrocytes and BCECs were cultured on poly-L-lactic acid (PLLA) scaffolds and tested for their ability to grow and survive on this three-dimensional structures. We analyzed in parallel the cell growth in 2D and 3D culture systems and observed the differences in cell morphology by fluorescence analysis: three-dimensional scaffolds have the ability to guide cell growth, provide support, encourage cell adhesion and proliferation. Astrocytes8 and BCECs (unpublished results) adapted well to these porous matrices, not only remaining on the surface, but also penetrating inside the scaffolds. EVs released by astrocytes in these scaffolds are probably exosomes, as suggested by transmission electron microscopy pictures, and by the presence of intracellular structures resembling multivesicular bodies. This 3D cell culture system could be further enriched to host different brain cell types, in order to set , for example, an in vitro model of BBB, that may be useful for drug delivery studies, and for the formulation of new therapeutic strategies for the treatment of neurological diseases. References [1] Schiera, G., Di Liegro, C.M., Di Liegro I. Int J Mol Sci. 2017, 18(12). pii: E2774. [2] Schiera, G., Proia, P., Alberti, C., Mineo, M., Savettieri, G., Di Liegro, I., 2007. J Cell Mol Med. 2007, 111(6), 1384-94. [3] Proia, P., Schiera, G., Mineo, M., Ingrassia, A.M. Santoro, G., Savettieri, G., Di Liegro, I. Int J Mol Med. 2008, 21(1), 63-7. [4] D'Agostino, S., Salamone, M., Di Liegro, I., Vittorelli, ML., Int J Oncol. 2006, 29(5),1075-85. [5] Lo Cicero, A., Schiera, G., Proia, P., Saladino, P., Savettieri, G., Di Liegro, C.M., Di Liegro, I. Int J Oncol. 2011, 39(6):1353-7. [6] Lo Cicero, A., Majkowska, I., Nagase, H., Di Liegro, I., Troeberg, L., Matrix Biol. 2012, 31(4), 229-33. [7] Schiera, G., Di Liegro, C.M., Saladino, P., Pitti, R., Savettieri, G., Proia, P., Di Liegro, I. Int J Oncol. 2013, 43(6), 1771-6. [8] Carfì Pavia, F., Di Bella, M.A., Brucato, V., Blanda, V., Zummo, F., Vitrano, I., Di Liegro, C.M., Ghersi, G., Di Liegro, I., Schiera, G. Mol Med Rep. 2019 [Epub ahead of print]. [9] Di Bella M.A., Zummo F., Carfì Pavia F., Brucato V., Di Liegro I., Schiera G. 2017, In: Microscopy and Imaging Science: practical approaches to applied research and education, pp 260-264. Ed: A. Méndez-Vilas Publisher, Formatex Research Center (Spain), ISBN-13, 978-84-942134-9-6.

VES4US, a Horizon 2020 Future and Emerging Technology project: Biophysical experimental tools for EVs structural characterisation

Samuele Raccosta,a Rita Carrotta,a Fabio Librizzi,a Vincenzo Martorana,a Rosina Noto,a

Giorgia Adamo,b Antonella Cusimano,b Sabrina Picciotto,b Daniele Romancino,b

Christopher Stanly,c Elia Di Schiavi,c Giovanna L. Liguori,d Annamaria Kisslinger,e

Katharina Landfester,f Svenja Morsbach,f Ales Iglic,g Veronika Iglic,g

Laura Corcuera,h Paolo Arosio,i Gabriella Pocsfalvi,c Nicolas Touzet, j Antonella Bongiovanni,b

Mauro Manno a

a. National Research Council of Italy (CNR), Institute of Biophysics (IBF), Palermo, Italy; b. CNR, Institute for Biomedical Research and Innovation (IRIB), Palermo, Italy;

c. CNR, Institute of Biosciences and BioResources (IBBR), Napoli, Italy; d. CNR, Institute of Genetics and Biophysics (IGB), Napoli, Italy;

e. CNR, Institute of Experimental Endocrinology and Oncology (IEOS), Napoli, Italy; f. Max-Planck Institute for Polymer Research, Mainz, Germany;

g. Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia; h. Zabala Innovation Consulting, Navarra, Spain;

i. ETH Zurich Institute for Chemical and Bioengineering, Zurich, Switzerland; j. Institute of Technology Sligo (ITSligo), Sligo, Ireland

VES4US is a European project which aims to develop an innovative platform for the efficient production of extracellular vesicles (EVs) from a renewable biosource, and then use them as safe and efficient tailor-made nano-delivery systems (https://ves4us.eu).

We identified a natural source to constitute a cost-effective and sustainable source of EVs and then we are optimizing the experimental activities for harvesting, manipulating, storing, characterizing and treating EVs.

Here, we focus on the first basic step of our novel bioprocessing approach, that is an extensive characterisation of their structural properties. Specifically, combined experiments of Atomic Force Microscopy (AFM), 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 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).

Also, we show how the structural organization of the lipid bilayer of the nano-sized extracellular vesicle membrane may be determined by Small-Angle X-ray Scattering (SAXS) and Small-Angle Neutron Scattering (SANS). The combination of SAXS and SANS measurements on the same sample becomes crucial to clearly identify the structural features of the lipid belayer in such a complex and heterogeneous system.

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 field, such as nanomedicine, nutraceutics and cosmetics.

(This project has received funding from the European Union’s Horizon 2020 research and innovation programme, under grant agreement No 801338).

Projecting extracellular vesicles in two dimensions

Paolo Bergese1,6, Debora Berti2,6, Marco Brucale3,6, Sara Busatto4,6, Stefania Federici5, Costanza Montis2,6, Lucia Paolini1,6, Annalisa Radeghieri1,6, Andrea Ridolfi3,6, Annalisa

Salvatore2,6, Francesco Valle3,6, Andrea Zendrini1

1Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, Brescia, Italia

2Dipartimento di Chimica, Università di Firenze, Firenze, Italia. 3ISMN-CNR, Bologna, Italia.

4Department of Transplantation Medicine; Department of Physiology and Biomedical Engineering, Mayo Clinic, Jacksonville, FL, USA

5Dipartimento di Ingegneria Meccanica e Industriale, Università degli Studi di Brescia, Brescia, Italia.

6CSGI - Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Firenze, Italia

We report on solid supported lipid bilayers made from nanosized EVs (EVSLBs) [1,2] and their interaction with gold nanoparticles. EVSLBs are the first attempt ever to harness EVs in two dimensions. It was prompted by the guess that EVSLBs would be innately endowed with intermediate complexity between synthetic bilayer mimics and natural cellular membranes, while also linked to phenotype and functions of the originating cells. EVSLBs are of fundamental interest per se, may advance understanding of EV-surface interaction (to date mostly unexplored) and foster new experiments, such as the study by surface-based techniques of so far inaccessible properties of natural and synthetic nanosized vesicles (e.g. EV membrane proteins in native conformation). From a translational perspective, EVSLBs can provide streamlined fabrication of hybrid surfaces that mimic structure and function of biological membranes, bypassing the full synthetic approach, which still poses severe challenges. This can contribute to momentous progress of breakthrough “surface biotechnologies”, including realization of assays and biosensors which are simpler, more specific and possibly personalized, new biogenic interfaces for cell and organoid-on-a-chip support/interaction and alternative routes for bio-camouflage of inorganic nanomaterials.

[1] Federici, S., Ridolfi, A., Zendrini, A., Radeghieri, A., Bontempi, E., Depero, L., et al. (2018). Interaction of Extracellular Vesicles with Si Surface Studied by Nanomechanical Microcantilever Sensors. Applied Sciences 8, 404–11. doi:10.3390/app8030404. [2] Montis, C., Busatto, S., Valle, F., Zendrini, A., Salvatore, A., Gerelli, Y., et al. (2018). Biogenic Supported Lipid Bilayers from Nanosized Extracellular Vesicles. Adv. Biosys. 2, 1700200–7. doi:10.1002/adbi.201700200.

Membrane sensing peptide microarrays for Extracellular Vesicles

M. Cretich1, M. Chiari1, M.T. Odinolfi1, A. Romanato1, R. Vago2, S. Picciolini3 and A. Gori1

1Istituto di Chimica del Riconoscimento Molecolare (ICRM), CNR, Milano, Italy 2Università Vita e Salute San Raffaele, Milano, Italy

3LABION, IRCCS Fondazione Don Carlo Gnocchi, Milano, Italy The use of protein markers for extracellular vesicles (EVs) capturing and detection is biased by individual differences in surface protein expression and by possible contamination by soluble antigens. Yet, EVs membranes show unique traits in the extracellular space such as the high curvature, lipid packing defects and the presence of rare lipids such as phosphatidylserine and ceramide. These features can be targeted by membrane binding proteins and their derived peptides. Here we present the first example of EVs capturing on microarrays by membrane sensing peptides. These are a unique class of ligands that can sense the physical state of membrane, likely being a more universal molecular tool for EVs than those based on protein markers and being applicable to sensing for diagnostic purposes as well as for targeting in therapy. The peptides were designed based on the structural analysis of known membrane binding protein domains. A chemoselective binding method based on a clickable polymer allowed us to immobilize different peptides, in a oriented, linear and multivalent presentation [1-2], on silicon slides for Single Particle Interferometric Detection (SP-IRIS) [3] and on gold chips for Surface Plasmon Resonance Imaging (SPRi). Capturing of vesicles from various types of samples, including EVs isolated by ultracentrifugation and by size exclusion chromatography, was demonstrated by label free counting of particles and colocalization of tetraspanins CD9, CD63 and CD81 by immune-staining. Peptides were able to selectively capture EVs with a higher binding capacity compared to anti-tetraspanins antibodies, even from pure serum samples without the need of pre-isolation. A discussion on the importance of chemoselective peptide immobilization on microarray surfaces, on the effect of ligand multivalency on EVs capturing yield and on the possible nano-particle binding mechanism is presented. Acknowledgements: - INDEX: European Union’s Horizon 2020 research and innovation programme under grant agreement No 766466 - HYDROGEX: Avviso congiunto per la concessione di contributi a sostegno del trasferimento della conoscenza nel settore dei Materiali avanzati; Fondazione Cariplo & Regione Lombardia; rif. 2018-1720 References [1] A. Gori, L. Sola, P. Gagni, G. Bruni, M. Liprino, C. Peri, G. Colombo, M. Cretich*, M. Chiari Screening Complex Biological Samples with Peptide Microarrays: the Favorable Impact of Probe Orientation via Chemoselective Immobilization Strategies on Clickable Polymeric Coatings Bioconjugate Chemistry (2016), 27, 2669-2677 [2] A. Gori, M. Cretich, R. Vanna, L. Sola, P. Gagni, M. Liprino, G. Bruni, F. Gramatica, S. Burastero, M. Chiari. Multiple epitope presentation and surface density control enabled by chemoselective immobilization leads to enhanced performance in IgE-binding fingerprinting on peptide microarrays Analytica Chimica Acta (2017) 983: 189-197 [3] G. Daaboul, P. Gagni, L. Benussi, P.Bettotti, M. Ciani, M. Cretich, D. Freedman, R. Ghidoni, A. Yalcin Ozkumur, C.Piotto, D. Prosperi, B.Santini, M. Selim Ünlü and M. Chiari, Digital detection of exosomes by interferometric imaging Scientific Reports (2016) 6: 3746

The Possible role of Extracellular Vesicles in

Neuromuscular Junction pre-patterning

Serena Maggio1, Paola Ceccaroli1, Emanuela Polidori1, Manuela Ferracin3, Andrea Cioccoloni1, Gianluca Storci3, Michela Battistelli1, Massimiliano Bonafè3, Fabiola Olivieri2, 4, Vilberto Stocchi1

and Michele Guescini 1* 1Department of Biomolecular Sciences, 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 Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Italy. 4Center of Clinical Pathology and Innovative Therapy, IRCCS INRCA, National Institute, Ancona, Italy.

* Correspondence: michele.guescini@uniurb.it; Tel.: +39-0722-303424; Fax: +39-0722-303401 The neuromuscular junction (NMJ) is a chemical synapse formed between motoneuron and skeletal muscle. NMJ translates the electrical impulses delivered by the motoneuron into action potentials in the juxtaposed muscle fiber. Before the neuromuscular junction takes place, aneural acetylcholine receptors (AChRs) are clustered in a central region of the muscle, a process called muscle pre-patterning. The aim of this study is to investigate the potential role of EVs in modulating motoneuron-muscle communication during the NMJ establishment. Under differentiation condition, NSC-34, a motoneuron-like cell model, showed significant neurite growth with long branching processes. These morphological observations are in agreement with the upregulation of some NSC-34 differentiation markers, such as MAP2 (microtubule-associated protein), GAP-43 (growth associated protein 43) and ChAT (choline acetyltransferase) at 4-6 days of differentiation. EVs have been isolated during motoneuron development using a serial ultracentrifugation protocol specifically adjusted to isolate large and small EVs (named LEVs and SEVs) based on their differential sedimentation properties. The nanoparticle tracking and transmission electron microscopy analyses of purified EVs showed that, in our conditions, SEVs have a diameter of about 110nm while LEVs exhibit a much more variable size with an average diameter of 240 nm. Furthermore, western blot analysis showed that SEVs are negative for calnexin, a marker of the endoplasmic reticulum and positive for Alix, a well-established exosome marker, and SEVs and LEVs are both positive for Hsp70 and Tsg101, two well-defined EV markers. NGS analysis allowed us to examine miRNA expression profile of EVs released during motoneuron development. A total number of 353 miRNAs was detected in at least one replicate of the four types of the analyzed samples, such as LEVs and SEVs released respectively at early and late NSC-34 differentiation time. Different predictive analytic tools were used to have a global picture of the signal pathways finely tuned by the EV-miRNAs released by differentiating NSC-34; this analysis highlighted that Axon guidance, Wnt- and TGF beta signaling pathways show the highest scores. To investigate the role of NSC-34-derived EVs on the modulation of muscle membrane pre-pattering, cultured myotubes were treated with EVs purified from differentiated NSC-34. Immunofluorescence staining with α-bungarotoxin allowed us to highlight a slight but significant increase of AChR clusters in response to EV-treatment. These data corroborate with the significant stimulation of RSpo2 mRNA found in treated myotubes, which is known to promote AChR clustering at the neuromuscular junction and to be involved in Wnt signaling. Altogether this data suggest that differentiated motoneuron release EVs which could stimulate AChR aggregation on myotube membrane, a fundamental preparatory stage for NMJ formation.

Differential contribution of microvesicles derived from platelets and monocytes of coronary artery

disease patients to endothelial dysfunction

Marta Brambilla1, Maria Talmon2, Paola Canzano1, Luigia Fresu2, Sandra Brunelleschi2, Elena Tremoli1,

Marina Camera1,2 1Centro Cardiologico Monzino I.R.C.C.S., Milan, Italy 2Dept of Health Sciences, University of Piemonte

Orientale, Novara; 3Dept of Pharmacological and Biomolecular Sciences,Università degli Studi di Milano,

Milan, Italy

BACKGROUND. Microvesicles (MVs) are biomarkers of vascular injury and inflammation in

atherothrombosis and coronary artery disease (CAD). Moreover, plasma MVs have been demonstrated to

induce endothelial dysfunction in many pathological conditions, including cardiovascular disorders.

However, the specific contribution of platelet (PLT) and monocyte (Mo)–derived MVs, the two MVs

subtypes most frequently implicated in the prothrombotic disorders, are poorly defined.

AIM. The aim of this study is to assess how two of the main processes involved in the vessel damage, such

as oxidative stress and inflammation, are affected by MVs obtained in vitro from PLT and Mo of healthy

subjects and coronary artery disease patients.

METHODS. PLT and Mo, isolated from healthy subjects (HS, n=15) and CAD patients (n=15), were treated

with TRAP-6 (20 µM, for 30 min at 37°C) or LPS (10µg/ml, for 16h at 37°C), respectively, or vehicle in order

to produce MVs which were isolated from the supernatant and characterized by flow cytometry. MVs were

then added to the culture medium of human vascular endothelial cells (hECV). Superoxide anion

production, an index of oxidative stress, and the mRNA expression of inflammatory markers (such as IL6,

TNFalpha, NF-κB) were analyzed.

RESULTS. Platelets, isolated from CAD patients, released 1336 [991-1548] MV/µl, an amount which is 4-

times higher compared to that obtained from resting HS platelets (345 [126-695] MV/µl) but comparable to

that produced by TRAP-stimulated HS platelets (1962 [1028-4700] MV/µl). By contrast, Mo isolated from

HS and CAD subjects released a similar amount of MVs (206 [156-266] and 261 [199-323] MV/µl,

respectively). Overnight incubation with LPS determined a comparable mean increase of 1.60.5

(p<0.0001) in the MV production by both HS and CAD Mo (285 [252-425] and 433 [312-553] MVs/l, for HS

and CAD Mo, respectively). Incubation of hECV with HS MVs produced by unstimulated cells did not result

in an increase of superoxide anion production. By contrast, the stimuli-induced MVs, both from PLT and

Mo, were able to trigger the oxidative burst (3-fold increase) in a concentration dependent manner,

suggesting that a prothrombotic/proinflammatory microenvironment could affect PLT and Mo behaviour

inducing the production of MVs with an oxidizing phenotype. Interestingly, in pathological condition, MVs

spontaneously released by CAD-PLT were able to induce an oxidative stress comparable to that produced

by the same number of MVs produced by stimulated healthy PLT. Conversely, MV produced by

unstimulated CAD-Mo were not able to increase the superoxide anion production, which was doubled only

after incubation with MVs from LPS-stimulated Mo. Finally, we evaluated the effect of PLT- and Mo-derived

MVs on endothelial inflammatory marker expression. Similarly to what observed for oxidative stress, in

healthy condition, both PLT and Mo derived MVs were able to upregulate the IL6, TNFalpha and NF-κB

mRNA only if produced by stimulated cells. In pathological condition, MVs spontaneously released by PLT,

and not those released by Mo, were able to affect the endothelial expression of these inflammatory

markers.

CONCLUSION. Our data indicate that, in healthy status, MVs physiologically released by PLT and Mo do not

induce endothelial oxidative stress and inflammation which can be affected by MVs produced by stimulated

cells. Interestingly, in pathological condition such as CAD, MV spontaneously released from unstimulated

PLT, and not those produced by Mo, are able to produce a potentially damaging oxidative stress and

proinflammatory phenotype in endothelial cells. This suggests a pathological role for PLT-derived MVs in

contributing to the atherotrombotic process and identifies a potential target for a pharmacological

antiplatelet intervention.

RNA binding proteins (RBPs) control sequence-specific miRNAs sorting in extracellular vesicles (EVs)

Giovanna Sabarese1, Cecilia Battistelli1, Laura Santangelo2, Veronica Riccioni1, Sabrina Garbo1,

Raffaele Strippoli1, 2, Michela Terri2, Claudia Montaldo2, Andres Ramos3, Carla Cicchini1* and Marco Tripodi1, 2*

1Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy

2National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy. 3Department of Structural and Molecular Biology, University College of London, London, UK.

*Co-contribution In spite of the evidence that Extracellular Vesicles (EVs, e.g. exosomes and micro-vesicles)

embed a defined cellular state-specific repertoire of microRNAs (miRNAs), the

molecular mechanisms controlling the specific sorting of these functional ncRNA molecules are still

poorly understood.

We previously provided evidence on a mechanism allowing the hepatocyte to selectively load

exosomes with miRNAs, this by identifying and determining the functional role of both a specific

miRNAs motif and a specific interactor of it, the RNA Binding Protein (RBP) SYNCRIP

(Synaptotagmin-binding Cytoplasmic RNA-Interacting Protein also known as hnRNPQ) (1).

SYNCRIP knock-down impairs sorting of miRNAs in exosomes. Furthermore, SYNCRIP directly

binds to specific miRNAs enriched in exosomes sharing a common extra-seed sequence (hEXO

motif). We also characterized SYNCRIP amino-terminal domain, which was previously thought to

mediate protein-protein interactions, showing that it is a cryptic, conserved and sequence-specific

RNA-binding domain, designated NURR (N-terminal unit for RNA recognition). The NURR domain

mediates the specific recognition of the hEXO sequence defining SYNCRIP exosomal miRNA

targets (2). Furthermore, the hEXO motif was proven to have a role in the exosome miRNAs

loading, since its embedment into a poorly-exported miRNA enhances the sorting of this molecule

into exosomes (1).

It is conceivable that the specific miRNAs sorting process might involve several molecular players,

each governing specific classes of miRNAs. By analyzing by MALDI-TOF/TOF MS the protein

interactors able to specifically bind to exosome-enriched or intracellular-retained miRNAs, we also

identified other interactors. Current studies are now focused on the characterization of the role of

these RBPs as further molecular regulators of specific loading in exosomes.

These new insights into the mechanisms of miRNAs exosome sorting process open the way for the

possible selective modification of the miRNAs exosome cargo in biotechnological approaches.

References (1) Santangelo et al., Cell Reports 2016; (2) Hobor et al., Nature Communication 2018.

Preparative and Analytical ultracentrifugation of EVs

C. Stanly1, R. Bokka1, M. Moubarak1,14, A. P. Ramos Juarez1,13, G. Antonucci1,12, I. Fiume1, A. Cusimano2, G. Adamo2, D. Romancino2, S. Picciotto2, S. Raccosta2, V. Martorana3, R. Noto3, R.

Carrotta3, P. Santonicola1, E. Di Schiavi1, G. L. Liguori4, R. Parkes5, D. Fierli5, A. Aranyos5, M. E. Barone5, A. Kisslinger6, K. Landfester7, B. Rodriguez8, S. Morsbach7, D. Božič9, A. Iglic9, V. Iglic9,

L. Corcuera8, P. Arosio10, M. Manno3, N. Touzet5, and A. Bongiovanni2, A. Bhattacharya11, L. Ehrdardt11 and G. Pocsfalvi1.

1 EVs-MS Research Group, Institute of Biosciences and BioResources (IBBR), National Research

Council, (CNR), Napoli, Italy; 2Institute for Biomedical Research and Innovation (IRIB), National Research Council, (CNR),

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

4Institute of Genetics and Biophysics (IGB), National Research Council, (CNR), Napoli, Italy; 5Institute of Technology Sligo (ITSligo), Sligo, Ireland,

6Institute of Experimental Endocrinology and Oncology (IEOS), National Research Council, (CNR) Napoli, Italy;

7Max-Planck Institute for Polymer Research, Mainz, Germany; 8Zabala Innovation Consulting, Navarra, Spain;

9Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia; 10ETH Zurich Institute for Chemical and Bioengineering, Zurich, Switzerland

11Beckman Coulter GmbH 12University of Federico II, Department of Biology, Napoli, Italy

13University of Campania Luigi Vanvitelli, Department of clinical and experimental medicine, Caserta, Italy

14University of Damanhour, Faculty of Agriculture, Egypt There has been a vast increase in the number of new methods that the extracellular vesicles (EVs) scientific community can use for the enrichment of EVs. All these methods are based on a few physical principles like filtration, centrifugation, precipitation, liquid-liquid extraction and solid-liquid partitioning (chromatographic methods). Even though the number of commercial kits available for EV isolation are increasing every day, ultracentrifugation (UC) is still considered as the gold standard in laboratory setups[1]. The vast majority of studies on extracellular vesicles published in the past two decades employed UC for the isolation of EV. However, UC itself may prove insufficient means to separate vesicles based on size and it also carries the risk of contamination of other non-vesicle substances. These include aggregates, polymeric substances, lipoproteins, etc. that are also pelleted by a high speed spin. Especially, biological fluids are affected from the shortcomings of differential centrifugation (DC) (i.e. co-purification of uromodulin in urine and lipoproteins in plasma). In fact, MISEV 2018 guidelines emphasize the use of gradient UC for the enrichment of EV for these reasons[2]. Gradient UC has a number of advantages respect to DC: 1). Allows for a better separation and enrichment of vesicles when extra purity is needed, 2). Allows separation of vesicles based on size UC offers more than just a preparative tool for EV research. Analytical ultracentrifugation (AUC), for example is an exceptional technique. AUC is very promising for the investigation of EVs due to the fact that the vesicles are analyzed in their native state in a liquid environment. There by, just one experiment can provide us the information about the shape, diameter, mass, purity, heterogeneity, and aggregation. The talk will highlight our experience gained during the isolation of extracellular vesicles of non-mammalian origin within the VES4US European project funded by the Horizon 2020-Future and Emerging Technology (FET) Open program. It will also emphasize the need of new characterization techniques, like AUC in the field of EV research. Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 801338.

References

1. Stanly, C., Fiume, I., Capasso, G. & Pocsfalvi, G. Isolation of exosome-like vesicles from plants by ultracentrifugation on sucrose/deuterium oxide (D2O) density cushions. in Methods in Molecular Biology (2016). doi:10.1007/978-1-4939-3804-9_18

2. Théry, C. 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). doi:10.1080/20013078.2018.1535750

Nanomechanics of individual extracellular vesicles

Andrea Ridolfi1,4, Marco Brucale1,4, Lucia Paolini2,4, Costanza Montis3,4, Debora Berti3,4, Paolo Bergese2,4, Francesco Valle1,4

1 Istituto per lo studio dei materiali nanostrutturati (ISMN), Consiglio nazionale delle ricerche (CNR),

Bologna, Italia; 2 Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, Brescia, Italia;

3 Dipartimento di Chimica, Università di Firenze, Firenze, Italia; 4 Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGI), Firenze,Italia

The quantitative measurement of selected physical properties of individual extracellular vesicles (EVs) and/or their subcomponents is still a largely open issue in EV research. In particular, even though EV nanomechanical characteristics are considered to have an impact on their biological function, their measurement remains challenging [1-4]. We will herein present our latest results regarding the nanomechanical characterization of individual EVs via atomic force microscopy (AFM)-based force spectroscopy (FS), discuss their significance, and outline possible perspectives. Our experimental approach entails depositing EVs on substrates with controlled surface properties. The mechanical characteristics of individual EVs are then analyzed through a series of specifically designed AFM experiments to obtain a nanomechanical fingerprint of the EV population. We show how this type of characterization can be exploited to differentiate between EVs and other organic envelopes of similar size and/or composition. We will conclude discussing how a nanomechanics-based taxonomy could prove to be an important tool for a deeper understanding of EV types. This research has received funding from the Horizon 2020 Framework Programme under the grant FETOPEN-801367 evFOUNDRY. [1] Calò A, Reguera D et al. Nanoscale 6, 2275–2285 (2014).

[2] Sorkin R, Huisjes R et al. Small 14, 1–8 (2018).

[3] Vorselen D, Mackintosh F. et al. ACS Nano 11, 2628–2636 (2017).

[4] Vorselen D, van Dommelen SM et al. Nat. Commun. 9, 4960 (2018).

Improved loading of plasma-derived extracellular vesicles to

encapsulate antitumour miRNAs

M. A. C. Pomatto1, B. Bussolati2, S. D’Antico3, S. Ghiotto3, C. Tetta4, M. F. Brizzi1, G. Camussi1

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

3Blood Bank, A.O.U. Città della Salute e della Scienza, Turin, Italy 4Unicyte srl, Turin, Italy

Introduction: Extracellular vesicles (EVs) are particles released by cells that carry a complex cargo

of molecules and mediate intercellular communication. Recently, they have raised great interest as

drug delivery systems and several engineering methods are currently under investigation.

Numerous factors, however, influence the transfection yield, including protocol variability and EV

damage.

Methods: The electroporation was investigated as method to directly load miRNAs in plasma-

derived EVs. Different parameters (voltage and number of pulses) were compared for their effect

on EV morphology and loading capacity of a synthetic miRNA, cel-39, including miRNA enrichment

in EVs and its transfer to target cells. Next, analyses were performed to evaluated the transfection

effect on EV endogenous cargo and the exogenous miRNA protection from RNAse degradation.

Then, EVs were loaded with antitumour miRNAs and their proapoptotic effect was evaluated on a

cell line of hepatocellular carcinoma, HepG2 cells.

Results: The comparison of different electroporation settings demonstrated the importance of

choosing the more appropriate protocol parameters to obtain an efficient EV transfection yield,

understood as both molecules loading and EV damage. In particular, we observed the superiority

of one electroporation protocol (using 750 Volt and 10 pulses) that allowed the most efficient

miRNA packaging and transfer to target cells, without structurally damaging EVs. The most

efficient electroporation protocol was also proven to allow a more efficient miRNA loading in

respect to incubation, better protecting miRNA from enzymatic digestion. In addition, our findings

suggested that electroporation preserved the naïve EV cargo, including RNAs and proteins, and

did not alter their uptake in cells. EVs engineered with antitumor miRNAs (miR-31 and miR-451a)

successfully promoted the apoptosis of HepG2 cells, downregulating their target genes related to

apoptotic pathways.

Summary/Conclusion: In conclusion, our findings indicate an efficient and functional miRNA

encapsulation in plasma-derived EVs following an electroporation protocol that preserves EV

integrity.

Supported by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.) and Unicyte AG

(Switzerland)

CD73+ extracellular vesicles inhibit angiogenesis through adenosine A2B

receptor signalling

Roberta Angioni1-2 PhD; Stephanie Herkenne3 PhD; Cristina Liboni1-2 MS; Ricardo Sánchez-Rodríguez1-2 PhD; Giulia Borile2 PhD; Maurizio Muraca2-4 Prof.; Bianca Calì1-2 PhD; Antonella

Viola1-2 Prof.

1Department of Biomedical Sciences, University of Padua, Padua, Italy; 2Istituto di Ricerca Pediatrica, Fondazione Città della Speranza, Padua, Italy; 3Department of Biology, University of Padua, Padua, Italy; and 4Department of Women's and Children's Health, University of Padua,

Italy.

Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce extracellular vesicles that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A2B adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic extracellular vesicles to inhibit tumor progression in vivo. Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis.

Physiactisome: a new drug from engineered muscle cells that

mimics physical exercise

R. Barone1,2, F. Macaluso3, E.Trovato1, A. Marino Gammazza1, C. Campanella1, F. Cappello1,2, F. Farina1, D. Coletti4,5,7, S. Adamo4, Paolo Gasco6, Valentina Di Felice1,7

1 Dipartimento di Biomedicina, Neuroscienze e Diagnostica Avanzata, Via del vespro

129, 90127 Palermo, Italy. 2 Istituto Euromediterraneo di Scienza e della Tecnica, Palermo.

3 SMART Engineering Solutions & Technologies Research Center, eCampus University, Novedrate, Italy.

4 Section of Histology & Medical Embryology, Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Interuniversity Institute of Myology, Sapienza

University of Rome, Rome, Italy. 5 Sorbonne Université, CNRS UMR 8256-INSERM ERL U1164, Biological Adaptation

and Aging B2A, Paris, France. 6 Nanovector srl, Via Livorno, 60, 10144, Torino.

7 iBioTHex, c/o Dipartimento di Biomedicina, Neuroscienze e Diagnostica Avanzata, Via del vespro 129, 90127 Palermo, Italy.

Physiactisome® is a new anti-cachectic drug, based in the secretion of vesicles (exosomes)

produced by genetically modified muscle cells. The final product seems to mimic what occurs

naturally in an organism following physical exercise. These vesicles contain substances with

beneficial properties for muscle tissue and can be used to counteract severe forms of muscular

atrophy, such as those observed in conditions of chronic diseases such as cancer, Alzheimer's

and kidney or heart dysfunction. Physiactisome®, currently validated in animal testing, aims

to be the first specific drug against muscular atrophy, with the particularity of being

customizable. The method used to obtain Physiactisome® may be applied to muscle stem

cells of the patient himself, obtainable by simple biopsy. Since muscle atrophy, in its most

severe form (cachexia), is often the cause of death because it interferes with therapy,

Physiactisome®, by contrasting muscle atrophy, can not only increase patients' survival and

quality of life , but also to improve the effectiveness of other therapies.

Pluripotent EV as carriers of regenerative prompts in aged cells

M. Barilani1, V. Peli1, R. Piras1, A. Cherubini1, L. Dioni2, V. Bollati2, V. Dolo3, L. Lazzari1 1Laboratory of Regenerative Medicine – Cell Factory, Department of Transfusion Medicine and Hematology, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy 2EPIGET – Epidemiology, Epigenetics and Toxicology Lab, Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy 3Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy In the frame of regenerative medicine, our previous work focused on the anti-apoptotic and anti-inflammatory properties of human mesenchymal stem cells (hMSC) isolated from cord blood (CB) and bone marrow (BM) [1]. In an in vitro model of acute kidney injury, we observed a protective effect of extracellular vesicles mediated in part by the horizontal transfer of anti-inflammatory interleukin 10 full-length mRNA [2]. Herein, hCB-MSC were reprogrammed to pluripotency to overcome primary cell limited lifespan, preserving the advantages of cord blood as cell source. Peculiar features in comparison to conventional induced pluripotent stem cells generated from skin fibroblasts were addressed. Next, the generation of extracellular vesicles from pluripotent stem cells was studied by transmission and scanning electron microscopy, nanoparticle tracking analysis (NTA), flow cytometry and western analysis. Protein markers for vesicle identity (CD63, CD81, CD9, flotillin-2, hsp70), purity (UCP2 for mitochondria, H3 for nucleus, calreticulin for endoplasmic reticulum, GAPDH and β-actin for cytoplasm) and parental cell type (embryonic SSEA-4, stem ROR1, epithelial CD326) were evaluated. Kinetics of extracellular vesicle production of pluripotent stem cells in exponential growth were determined. Thorough characterization of stemness-related transcriptome, miRNome and circRNome was performed. In particular, the incorporation from parental cells to extracellular vesicles of full-length mRNA of pluripotency factors Oct4A, Sox2, Klf4, c-Myc, Lin28A, Nanog and miRNA-302b cluster was studied. To demonstrate a potential therapeutic role in regenerative medicine approaches, pluripotent extracellular vesicles were challenged to revert senescence- and disease-related phenotypes of cells isolated by young (CB) and old (BM) tissues, previously studied by our group [3,4]. The modulation of proliferation rate by XCelligence automated system, of senescence by qPCR (p16, p21, p53) and β-galactosidase assay, and of metabolic parameters (ROS production, ATP content) was evaluated. Finally, a role as crucial player in this context was proposed for Lin28A. Supported by Ricerca corrente 2016-2018, Minister of Health, Italy. References [1] Montemurro, T., Viganò, M., Ragni, E., Barilani, M., Parazzi, V., Boldrin, V., Lavazza, C., Montelatici, E., Banfi, F., Lauri, E., Giovanelli, S., Baccarin, M., Guerneri, S., Giordano, R., Lazzari, L., 2016. Angiogenic and anti-inflammatory properties of mesenchymal stem cells from cord blood: soluble factors and extracellular vesicles for cell regeneration. European Journal of Cell Biology 95(6-7):228-38. [2] Ragni, E., Banfi, F., Barilani, M., Cherubini, A., Parazzi, V., Larghi, P., Dolo, V., Bollati, V., Lazzari, L., 2017. Extracellular Vesicle-Shuttled mRNA in Mesenchymal Stem Cell Communication. Stem Cells 1093-1105. [3] Barilani, M., Lavazza, C., Viganò, M., Montemurro, T., Boldrin, V., Parazzi, V., Montelatici, E., Crosti, M., Moro, M., Giordano, R., Lazzari, L., 2015. Dissection of the cord blood stromal component reveals predictive parameters for culture outcome. Stem Cells and Development 24(1):104-14. [4] Angelova, P.R., Barilani, M., Lovejoy, C., Dossena, M., Viganò, M., Seresini, A., Piga, D., Gandhi, S., Pezzoli, G., Abramov, A.Y., Lazzari, L., 2018. Mitochondrial dysfunction in Parkinsonian mesenchymal stem cells impairs differentiation. Redox Biology 14:474-484.

Cardiac progenitor cell exosome-associated periostin triggers reentry of cardiomyocytes into cell cycle

Balbi C.1, Bolis S.1, Barile L.2,3, Vassalli G.1,2

1Molecular and Cell Cardiology Lab., CardioCentro Ticino, Lugano, Switzerland, 2Università Svizzera Italiana, Lugano, Switzerland,

3Cardiovascular Theranostics Lab., CardioCentro Ticino, Lugano, Switzerland.

Introduction: Nanovesicles known as exosomes (Exo) from cardiac-derived progenitor cells (CPCs) are cardioprotective and improve cardiac function after myocardial infarction; however the mechanisms of benefit are incompletely understood, especially with respect to endogenous cardiomyocytes (CM) renewal. Periostin (POSTN), a secreted extracellular matrix protein, is emerging as a matricellular factor that can trigger CM proliferation. We have identified POSTN as a protein secreted by CPC and enriched in their exosomal fraction.

Purpose: We sought to determine whether Exo-CPC can induce proliferation of CM and to explore the role of exosomal POSTN in inducing reentry of CM into the cell cycle.

Methods: Exo were isolated from CPC condioned medium by density gradient ultracentrifugation. Fractions were analyzed by Western blotting for the presence of POSTN as well as specific Exo markers (TSG101, CD9). POSTN-depleted Exo (ExoCPC_SiPOSTN) were obtained by transfecting CPC with specific siRNA. Active DNA synthesis was assessed on primary cell culture of rat neonatal CM by EdU incorporation and pH3 staining. H9C2 cardiomyoblast cells were used to assess by real-time RT-PCR the expression of downstream genes Hippo/Yes-associated protein (YAP) signaling pathway. H9C2 cardiomyocyte cells were also used to investigate the signaling pathway by Western Blot analysis.

Results: Western blotting analysis allowed to specifically determining the presence of Exo markers and POSTN in the different fractions of secreted vesicles. Smaller fractions (f1-f3) have the highest amount of TSG101 and CD9 as well as POSTN, thus suggesting that CPC secrete POSTN associated with Exo. The silencing of POSTN in cells resulted in a 60% reduction of Exo-associated POSTN compared to naïve ExoCPC. ExoCPC but not ExoCPC_SiPOSTN, were able to increase phosphorylation of FAK, AKT and ERK in H9C2 cells. YAP phosphorylation and its degradation was decreased resulting in the activation of his downstream genes. By real-time PCR, AurkB Ccn2, Birc5 and Ccnd1 expression resulted increase with ExoCPC and less with ExoCPC_SiPOSTN, compared to cells not exposed to Exo. ExoCPC were also able to increase EdU incorporation and pH3 positivity in cardiac troponin-positive primary rat CM at p0 and p7. ExoCPC_SiPOSTN did not affect proliferation.

Conclusion: These results suggest that POSTN may promote cardiomyocyte proliferation through the binding with INTEGRIN in the target cells inducing FAK phosphorylation and consequent activation of the AKT/ERK/Hippo-Yap pathway. Exosomes released by CPC are an important source of POSTN and may have a potential for promoting cardiac regeneration.

Mesenchymal stromal cell secretory activity: the role of

extracellular vesicles in angiogenesis

C. Gorgun1,2, D. Reverberi3, C. Balbi4, L. Barile5,6, R. Quarto1,2, R. Tasso1,2

1Department of Experimental Medicine, University of Genova, Genova, Italy 2U.O. Cellular Oncology, IRCCS Ospedale Policlinico San Martino, Genova, Italy

3U.O. Molecular Pathology, IRCCS Ospedale Policlinico San Martino, Genova, Italy 4Molecular and Cell Cardiology Laboratory, CardioCentro Ticino, Lugano, Switzerland

5Università Svizzera Italiana, Lugano, Switzerland 6Cardiovascular Theranostic Lab, Lugano, Switzerland

For many years, mesenchymal stromal cells (MSC) have been considered a remarkable cell source for regenerative medicine applications. More recently, a paradigm shift has emerged suggesting that their beneficial effects is often due to their paracrine activity [1]. Since MSC have a regulatory phenotype and respond actively to the environmental signals, their secrotome activity can be modulated by cues from the microenvironment, mimicking the milieu established during an injury event. Although several cell-secreted factors have been identified, the exact role of extracellular vesicles (EV) in some aspects of the healing process, i.e. in angiogenesis, is currently unknown. In this context, EVs are recognized as important components of the MSC secretome and the term defines a heterogeneous population of exosomes (EXO), microvesicles (MV) and apoptotic bodies. During the traditional EV isolation methods, discarding the pellet after 10000 xg (10K) centrifugation or filtering the supernatant with 0.22 μm filters, could in part exclude larger vesicles, such as shedding MV. However, recent papers highlighted the importance of considering the different subtypes that could hold different functional properties [2]. Within this reason, we aim to determine the role of the injury microenvironment in modulating the angiogenic potential of the MSC secretome fractions. Following the incubation of human MSCs for 24 hours with the inflammatory factors TNF- (50 ng/ml) and IL1- (50 ng/ml), either under normoxic or hypoxic conditions; total conditioned media (CM), corresponding MV and EXO and EV-free depleted media (DM) were collected. EVs were analyzed by western blot to identify proteins specifically expressed by either MV or EXO, by flow-cytometry to evaluate the expression of tetraspanin markers, and by nanoparticle tracking analysis to check their size and amount. To evaluate their angiogenic potential, all secretome fractions were investigated in vitro by analyzing their effect on Human Umbilical Vein Endothelial Cells (HUVEC) migration, proliferation and differentiation and an additional ex vivo metatarsal assay was performed to confirm in vitro results. Our data confirm the importance of a proper pre-conditioning to positively influence the cell paracrine activity. Addition of inflammatory factors manipulates MSCs to release secretomes with different functional properties. Under these environmental cues, EV fractions, and in particular exosomes, stimulate the different aspects of the angiogenic process, highlighting their potential role for the generation of successful cell-free therapies. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 721432. [1] Caplan, A. I. ,2009. Why are MSCs therapeutic? New data: new insight. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland, 217(2), 318-324. [2] Tkach, M., Kowal, J., & Théry, C., 2017. Why the need and how to approach the functional diversity of extracellular vesicles. Philosophical Transactions of the Royal Society B: Biological Sciences, 373(1737), 20160479.

Cancer EVs convey metastatic traits through horizontal gene transfer Mohamed Abdouh1, Matteo Floris2, Zu-Hua Gao3, Vincenzo Arena4, Manuel Arena5,

Goffredo Orazio Arena1,6 1McGill University Health Centre-Research Institute (MUHC-RI), Montreal, Canada

2Department of Biomedical Sciences, Sassari University, Sassari, Italy 3Department of Pathology, MUHC-RI, Montreal, Canada

4Department of Obstetrics and Gynecology, Santo Bambino Hospital, Catania, Italy 5Department of Surgical Sciences, University of Catania, Catania, Italy

6Department of Surgery, McGill University, Montreal, Canada

Metastasis is the leading cause of cancer related mortality. It is considered to be a multistep biological process, controlled by distinct genes and signaling pathways, initiated by the detachment of cancer cells that invade the neighboring tissues, penetrate the basement membrane, disseminate via the blood flow and engraft in distant niche. Recently, our group and others have brought forward the hypothesis that the metastatic process might not be solely due to primary tumor cells spreading but might occur via transfer of biologically active cancer circulating factors to susceptible target cells located in distant organs (1-4). We demonstrated that circulating cancer EVs induce malignant transformation of target cells through horizontal transfer of malignant traits (3). Notably, cancer EVs carry in their lumen genetic materials and proteins that favor tumor growth, alter the tumor niche microenvironment, and guide the homing of cancer cells (3-6). Interestingly, our experiments confirmed that different types of oncosuppressor mutated cell lines are able to turn malignant when exposed to either EVs collected from cancer cells’ conditioned media or from cancer patients’ sera. Notably the exposed cells displayed features resembling those of the primary tumors with gain of immunohistochemical features similar or identical to the cancer cells that release the EVs (3). In the present study, we shed light on the molecular mechanisms behind this phenomenon. We profiled DNA, mRNA and miRNA expression in colorectal cancer-derived EVs and target cells prior and following exposure to these EVs. We confirmed that the phenotypical transformation of the exposed cells is associated with cancer EVs DNA transfer and we showed data suggesting that EVs DNA is actively transcribed in these cells. We further demonstrated that a definite set of miRNA families, transferred from the cancer EVs to the target cells, activate cell cycle progression and cell survival pathways. Remarkably, we noted that the uptake of cancer miRNAs was concomitant to downregulation of transcription factors that prompted a mesenchymal to epithelial transition of target cells that acquired epithelial features and immunohistochemistry markers, suggestive of complete colorectal cancer differentiation. These discoveries shed new light into the molecular mechanisms behind the horizontal transfer of malignant traits and confirm the notion that metastatic disease might be reproduced through transfer of circulating genetic material. We believe that these data may guide the development of new and more efficient therapeutic strategies targeting cancer EVs, with the ultimate goal of inhibiting the metastatic process in its earliest stages. Financial Support: This study was financially supported by Giuseppe Monticciolo. References: 1. Garcia-Olmo DC, et al. 2010. Cell-free nucleic acids circulating in the plasma of colorectal cancer

patients induce the oncogenic transformation of susceptible cultured cells. Cancer Res 70:560–7. 2. Trejo-Becerril C, et al. 2012. Cancer progression mediated by horizontal gene transfer in an in vivo

model. PLoS One 7:e52754. 3. Arena G, et al. 2017. Exosomes isolated from cancer patients’ sera transfer malignant traits and

confer the same phenotype of primary tumors to onsuppressor- mutated cells. J. Exp. Clin. Cancer Res 36:113.

4. Arena G, et al. 2014. Transfer of malignant trait to immortalized human cells following exposure to human cancer serum. J. Exp. Clin. Cancer Res 33:86.

5. Lee TH, et al. 2011. Microvesicles as mediators of intercellular communication in cancer–the emerging science of cellular ‘debris’. Semin Immunopathol 33:455–67.

6. Hoshino A, et al. 2015. Tumour exosome integrins determine organotropic metastasis. Nature 527:329-35.

Cardiac dysfunction after myocardial infarction: role of pro-

inflammatory extracellular vesicles Vanessa Biemmi1, Giuseppina Milano1, Jacopo Burrello1, Alessandra Ciullo1, Michele Dei Cas2, Rita Paroni3, Tiziano Tallone1, Sarah Longnus3, Giuseppe Vassalli1,4, Lucio Barile1,4.

1 - Cardiocentro Ticino Foundation, Lugano, Switzerland. 2-Department of Health Sciences of the University of Milan

3 - Department of Cardiovascular Surgery, Inselspital, Bern University Hospital, Bern, Switzerland.

4 - Faculty of Biomedical Sciences, Università Svizzera Italiana, Lugano, Switzerland Background- After myocardial infarction, necrotic cardiomyocytes release damage-associated proteins that stimulate innate immune pathways and macrophage tissue infiltration, which drives inflammation and myocardial remodeling. Circulating inflammatory extracellular vesicles play a crucial role in the acute and chronic phases of ischemia, in terms of inflammatory progression. Purpose-In this study, we hypothesize that the paracrine effect mediated by these vesicles induces direct cytotoxicity in cardiomyocytes. Thus, we examined whether reducing the generation of inflammatory vesicles within the first few hours after the ischemic event ameliorates cardiac outcome at short and long time-points. Methods-To test this hypothesis, myocardial infarction was induced in rats that were previously injected intraperitoneally with a chemical inhibitor of extracellular-vesicle biogenesis. Cytotoxic effects of circulating vesicles were also evaluated in vitro and ex vivo heart perfusion system. Results-The inflammatory reaction after myocardial infarction dramatically increased the number of circulating extracellular vesicles carrying alarmins such as IL-1, IL-1 and Rantes. Reducing the burst in inflammatory vesicles during the acute phase of ischemia resulted in preserved left ventricular ejection fraction in vivo. Hemodynamic analysis confirmed functional recovery by displaying higher velocity of left ventricular relaxation and improved contractility. When added to the perfusate of isolated hearts, post-infarction circulating vesicles induced significantly more cell death in adult cardiomyocytes, as assessed by cTnI release, compared to circulating vesicles isolated from healthy (non-infarcted) rats. In vitro inflammatory extracellular vesicles induce cell death by driving nuclear translocation of NF-κB into nuclei of cardiomyocytes. Conclusion-Our data suggest that targeting circulating extracellular vesicles during the acute phase of myocardial infarction would offer an effective therapeutic approach to preserve function of ischemic heart.

Intercellular miscommunication in the brain and periphery: characterization of extracellular vesicle in Amyotrophic Lateral

Sclerosis

Ferrara D.1, Brunelli L.2, Pasetto L.2, Ferrari S.1, Corsi J.1, Corbelli A.2, Fiordaliso F.2, Calvo A.3, Chiò A.3, Corbo M.4, Lunetta C.5, Mora G.6, Piazza S.1, Beltrame L.2, Cretich M.7, Pastorelli R.2,

D’Agostino VG.1, Bonetto V.2, Basso M.1,2

1 Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento,

Trento, Italy 2 IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy

3 CRESLA, department of neuroscience "Rita Levi Montalcini", Università degli Studi di Torino, Torino, Italy

4 Department of Neurorehabilitation Sciences, Casa Cura Policlinico (CCP), Milan, Italy 5 NeuroMuscular Ominicentre, Fondazione Serena Onlus, Milano, Italy

6 ICS Maugeri-IRCCS, Milano, Italy 7 National Research Council of Italy, Istituto di Chimica del Riconoscimento Molecolare, Italy

Amyotrophic Lateral Sclerosis (ALS) is a progressive and devastating motor neuron disease that affects upper and lower motor neurons. Among several factors, the progression of the disease is mediated by altered intercellular communication in the spinal cord between neurons and glial cells. One of the possible ways in which the cell-to-cell communication occurs is through extracellular vesicles (EVs) that transport proteins, lipids and nucleotides from one cell to the other [1,2]. To characterize EVs in ALS, we are using a novel bead-based methodology, called NBI [3], allowing rapid and efficient isolation of EVs. By this approach, it is possible recover not only a higher and purer amount of astrocyte-derived EVs, but also to preserve the integrity and the stability of vesicles and analyze their cargo content. Our preliminary results suggest that EVs derived from astrocytes of a transgenic mouse model of ALS, overexpressing mutant TDP-43 (Q331K), transmit toxicity to wild type neurons and are different in number and size compared with control mice, suggesting that in ALS, EV biogenesis is altered. We further focused on which component of the EVs would be responsible for propagation of toxicity. We set up novel methods to generate i) EVs unloaded of the majority of the protein cargos and ii) EVs containing ‘inactive’ RNA. At the same time, we performed an unbiased characterization of the protein and RNA cargos through a proteomic and small RNA sequencing analysis. Surprisingly, we failed to detect mutant TDP-43 as an EV protein cargo. Accordingly, small RNA sequencing did not reveal notable differences between samples, suggesting that in ALS, RNA and RNA binding proteins do not propagate toxicity through astrocyte EVs. Interestingly, the protein cargos differ significantly between control and disease condition. Finally, to test whether the defect is systemic or CNS-specific, we investigated the similarities between astrocyte-derived EVs and plasma EVs obtained from two different ALS mouse models and human patients. To detect whether the neuron and glia-derived EVs are present in the plasma, we set up a new methodology to define the relative percentage of CNS-derived EVs in periphery. Concluding, ALS EVs present different biochemical properties compared to controls, suggesting that their characterization could be of use for biomarker discovery and mechanistic investigations. Supported by the MSCA-IF ExItALS (752470) and the Italian Ministry of Health (GR-2016-02361552). References

[1] Basso M, Pozzi S, Tortarolo M, Fiordaliso F, Bisighini C, Pasetto L, Spaltro G, Lidonnici D, Gensano F, Battaglia E, Bendotti C, Bonetto V. Mutant copper-zinc superoxide dismutase (SOD1) induces protein secretion pathway alterations and exosome release in astrocytes: implications for disease spreading and motor neuron pathology in amyotrophic lateral sclerosis. J Biol Chem. 2013 May 31;288(22):15699-711. doi: 10.1074/jbc.M112.425066. Epub 2013 Apr 16. [2] Ferrara D, Pasetto L, Bonetto V, Basso M. Role of Extracellular Vesicles in Amyotrophic Lateral Sclerosis. Front Neurosci. 2018 Aug 17;12:574. doi: 10.3389/fnins.2018.00574. eCollection 2018. Review. [3] Notarangelo M, Zucal C, Modelska A, Pesce I, Scarduelli G, Potrich C, Lunelli L, Pederzolli C, Pavan P, la Marca G, Pasini L, Ulivi P, Beltran H, Demichelis F, Provenzani A, Quattrone A, D'Agostino VG. Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood. EBioMedicine. 2019 May;43:114-126. doi: 10.1016/j.ebiom.2019.04.039. Epub 2019 Apr 29.

Role of multiple myeloma exosomal miRNAs in the inhibition of osteoblast

differentiation

S. Raimondo1, A. Conigliaro1, O. Urzì1, S. Parisi1, G. Lo Bosco2, M. Carlisi3, S. Siragusa1,R. Alessandro1

1Dipartimento di Biomedicina, Neuroscienze e Diagnostica Avanzata, University of Palermo, Palermo. 2Dipartimento di Matematica e Informatica, University of Palermo, Palermo.

3Dipartimento di Promozione della Salute, Materno-Infantile, di Medicina Interna e Specialistica di Eccellenza “G. D’Alessandro”, University of Palermo, Palermo.

Multiple myeloma (MM) is a hematologic malignancy characterized by abnormal proliferation of plasma cells within the bone marrow which leads to paraprotein release in the serum, renal failure, anemia, and osteolytic bone disease. In contrast to normal bone remodeling, during multiple myeloma progression, the functional balance between osteoclasts (OCs) and osteoblasts (OBs) is definitively perturbed. Recently, extracellular vesicles (EVs) have emerged as novel vehicles of tumor-derived molecules and thus players in the crosstalk between cancer cells and the tumor microenvironment. We and others have recently reported that MM cell-derived exosomes play a relevant functional role in activating osteoclast differentiation while they inhibit the differentiation of human mesenchymal stromal cells (hMSCs) in osteoblasts [1][2]. Important questions that remain to be addressed are to understand how MM cell-derived exosomes may act on osteoblast differentiation Our study aims to correlate the exosome-mediated osteogenic inhibition of hMSCs with vesicle content, focusing on miRNAs. Exosomes were isolated from MM cell lines and bone marrow aspirates from MM patients by ultracentrifugation. TaqMan Array Human MicroRNA Cards were used to identify the microRNA profile of MM1.S exosomes and of the parental cells. We found that, among the most enriched exosome-miRNAs, 25% of them have been previously correlated with osteogenesis regulation. Among those, hsa-miR-30c, hsa-miR-127-5p, hsa-miR-129-5p, and hsa-miR-146a have already been identified as correlated with OB differentiation since their validated and predicted targets are OB differentiation markers (TargetScan, miRTarBase, miRTargetLink Human and miRSystem bioinformatic tools were used to identify miRNA targets). Therefore, we firstly focused on these miRNAs. Starting from Exosomes isolated from the BM aspirates of MM patients (n=6) we found enrichment in the selected miRNAs compared with the parental plasma cells. Moreover, treatment of hMSCs with MM patient exosomes induced an increase of miR-127-5p, miR-129-5p and miR-146a-5p, thus suggesting an exosome-mediated transport of these miRNAs from MM cells to hMSCs. To investigate a possible role of the selected miRNAs on the exosome-mediated osteogenic inhibition of hMSCs, we transfected hMSCs with the miRNA mimics and subsequently analyzed the putative miRNA’ targets involved in osteoblast differentiation. Among the investigated osteoblast markers, all the selected miRNAs down-regulated the expression of the early differentiation marker Alkaline Phosphatase (ALP) both at the mRNA and protein level. Interestingly, ALP mRNA is a predicted target only for the miR-129-5p. Therefore, it is conceivable to hypothesize that miR-127-5p and miR-146a-5p act upstream of ALP transcription. Further study is required to better clarify this point. Supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) grant n°18783 References [1] Raimondo S, Saieva L, Vicario E, Pucci M, Toscani D, Manno M, Raccosta S, Giuliani N, Alessandro R. Multiple myeloma-derived exosomes are enriched of amphiregulin (AREG) and activate the epidermal growth factor pathway in the bone microenvironment leading to osteoclastogenesis. J Hematol Oncol. 2019 Jan 8;12(1):2. [2] Faict S, Muller J, De Veirman K, De Bruyne E, Maes K, Vrancken L, Heusschen R, De Raeve H, Schots R, Vanderkerken K, Caers J, Menu E. Exosomes play a role in multiple myeloma bone disease and tumor development by targeting osteoclasts and osteoblasts. Blood Cancer J. 2018 Nov 8;8(11):105.

Extracellular vesicles from liver stem cells inhibit renal cancer stem cell-derived tumor growth in vitro and in vivo

A. Brossa1, V. Fonsato2, C. Grange3, S. Tritta1, M. Tapparo3, R. Calvetti1, S. Fallo1, G. Camussi3, B.

Bussolati1.

1Department of Molecular Biotechnology and Health Sciences, 22i3T Scarl,, 3Department of Medical Sciences; University of Torino, Turin, Italy.

Renal cell carcinoma (RCC) is a common solid tumor characterized by metastatic spread, high recurrence and resistance to common therapies [1]. Many studies demonstrated that cancer stem cells (CSCs) are the responsible of initiation, maintenance and the recurrence of solid tumors, thus representing the key for tumor eradication [2]. We previously demonstrated the anti-tumor activity of extracellular vesicles (EVs) derived from a resident population of human liver mesenchymal stem cells (HLSCs) on different solid tumors [3]. In this study, we tested the effect of HLSC-EVs on renal CSCs, both in vitro and in vivo. In vitro, HLSC-EVs displayed a pro-apoptotic, anti-proliferative and anti-invasive effect of on renal CSCs. In addition, pre-treatment of renal CSCs with HLSC-EVs before subcutaneous injection in SCID mice delayed tumor onset in vivo. We subsequently investigated the in vivo effect of HLSC-EVs systemic administration on renal tumor progression. In particular, HLSC-EVs significantly impaired subcutaneous tumor growth by reducing tumor vascularization and inducing tumor cell apoptosis. Moreover, HLSC-EVs improved the metastasis-free survival of i.v treated mice. In order to investigate HLSC-EVs mechanism of action, we analyzed the expression of a panel of anti-tumor miRNAs, known to be downregulated in RCC, in HLSC-EVs treated tumors. Interestingly, we observed both the transfer and the induction of anti tumor miRNAs, such as miR-145 and some members of miR-200 family. In this study, we demonstrated that the systemic treatment with HLSC-EVs reduced development, growth and improved metastasis-free survival of a renal cell carcinoma obtained by injection of renal CSCs. HLSC-EVs targeted the tumor and induced transfer and upregulation of selected anti-tumor microRNAs, able to affect CSC growth, invasion and survival. This study provides further evidence on the clinical use of HLSC-EVs in renal tumor treatment.

References

1. Makhov P, Joshi S, Ghatalia P, Kutikov A, Uzzo RG, Kolenko VM. Resistance to Systemic Therapies in Clear Cell Renal Cell Carcinoma: Mechanisms and Management Strategies. Mol Cancer Ther. 2018. 2. Bussolati B, Bruno S, Grange C, Ferrando U, Camussi G. Identification of a tumor-initiating stem cell population in human renal carcinomas. FASEB J. 2008. 3. Fonsato V, Collino F, Herrera MB, Cavallari C, Deregibus MC, Cisterna B, Bruno S, Romagnoli R, Salizzoni M, Tetta C, Camussi G. Human liver stem cell-derived microvesicles inhibit hepatoma growth in SCID mice by delivering antitumormicroRNAs. Stem Cells. 2012.

Microscale technologies to decode the role of hypoxia-derived exosomes in determining Neuroblastoma dissemination and

aggressiveness

P Fusco1,2, S. Micheli1,2, MR Esposito1,2, M. Sorgato1, L. Zanella1, L. Bova1,2, M Manfredi3, E Marengo3, E Cimetta1,2

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

3 University of Eastern Piedmont, Department of Sciences and Technological Innovation

Over the past decades, miniaturization has become a dominant trend in biomedical research with

engineers playing a crucial role in binding technology and biology. In our bodies cells reside in a

complex milieu, the microenvironment (µEnv), regulating their fate and function. Most of this complexity is lacking in standard laboratory models, leading to readouts poorly predicting the in

vivo situation. This is particularly felt in cancer research, as tumors are extremely heterogeneous

and capable of conditioning both the local µEnv and distant organs. Neuroblastoma (NB) is a heterogeneous pediatric malignancy of the sympathetic nervous system accounting for up to 10%

of childhood cancers with a strong tendency to metastasize. Secreted exosomes (EXO) are means

by which NBs reshape their µEnv and induce local and long-range changes in cells, regulating

progression and prognosis. Hypoxia is a key feature of solid tumors, and is specifically known to i. favor NB metastasis and dedifferentiation towards immature stem cell-like phenotypes and to ii.

stimulate release of exosomes (EXO), facilitating intercellular communication at distant sites.

We are developing microfluidic bioreactors (µBRs) and testing their edge over classical approaches in decoding the role of EXO and of the µEnv in NB. Our µBRs generate time and space-resolved concentration gradients, support fast dynamic changes and reconstruct complex

interactions between cells and tissues while performing multifactorial and parallelized experiments.

In this study, we also characterized the proteomic and miRNAs cargo of EXO isolated from NB cell

lines cultured at different oxygen concentrations to identify an exosomal signature associated with NB metastatic dissemination.

SKNAS and SKNDZ NB cell lines were cultured for 48h in standard (20% O2) and hypoxic (1.5% O2) conditions. EXO were purified and characterized by scanning electron microscopy (SEM) and

qNANO giving populations of round particles within the expected diameters (50-120nm). Proteome and miRNA cargo profiles were analyzed by quantitative mass spectrometry and FirePlex

Discovery Panel (on 405 miRNAs), respectively. Surface markers analysis (MACSplex technology)

revealed that NB hypoxia-derived EXO express an increase of proteins associated with

angiogenesis, adhesion, stemness and immune function such as CD105, CD29, CD49e, SSEA4, HLA-DR and HLA-ABC. A list of miRNAs and proteins that are differentially expressed in i. each

cell line and ii. each oxygenation level have been identified by Principal Components Analysis

(PCA) and Projection to Latent Structures by Partial Least Squares for Discriminant Analysis (PLS-DA). We also confirmed that treatment with hypoxic EXOs stimulated tube forming ability and

increased motility of Human Umbilical Vein Endothelial Cells (HUVECs), and increased the

expression of stemness-related markers in Human Mesenchymal Stem Cells (MCS). We hypothesize that the expression of specific markers guides the EXOs towards the target tissues

and organs where they condition the so-called pre-metastatic niche.

Several microbioreactor prototypes were designed, produced and validated, proving successful in generating stable concentration gradients in shear-protective regimes over monolayers of cultured

cells. Target cells (ie. MSCs and HUVECs) were exposed to concentration gradients of EXOs isolated from NB cultures at different oxygenation conditions.

These promising results are the starting point for the identification of predictive biomarkers to be used to detect and monitor metastatic spread in NB.

Large Oncosomes detection in diverse solid tumors showed a priming effect on tumor engraftment in vivo

Chiara Ciardiello1, Rossella Migliorino1, Maria S. Roca1, Rita Lombardi1, Paola Lanuti2, Michele Minopoli3, Vincenzo Gigantino4, Carlo Vitagliano1, Maria R. Milone1, Biagio Pucci1,

Tania Moccia1, Elena Di Gennaro1, Francesca Bruzzese1, Maria V. Carriero3, Valentina Minciacchi5, Dolores Di Vizio6, Marco Marchisio2, Alessandra Leone1, Alfredo Budillon1.

1Experimental Pharmacology Unit, 3Neoplastic Progression Unit, 4Pathology Unit, Istituto Nazionale Tumori – IRCCS-Fondazione G. Pascale, Napoli, Italy. 2Department of Medicine and Aging Sciences, University "G. d'Annunzio", Chieti-Pescara, Italy. 5Georg-Speyer-Haus Institute for tumor biology and experimental therapy, Frankfurt, Germany. 6Departments of Surgery, Pathology & Lab Medicine, and Biochemical Science, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

Extracellular vesicles (EVs) are considered as one of the most effective vehicle of information among cells and recent findings demonstrated them playing an important role in cancer development, progression [1] and drug resistance [2]. Untypically large EVs, which are plasma membrane derived and called “Large Oncosomes” (LO), have been observed to be released by prostate [3] and other types of cancer cells, i.e. glioblastoma [4]. Recent findings unveiled that LO derived from prostate cancer (PCa) models carried activated AKT [5] and were enriched in chromosomal DNA compared to smaller EVs [6]. However, their function and content is far to be elucidated, especially in consideration of their diverse content, reflecting the source cells. Several isogenic models developed by our group were used in order to study LO content and functions. 1) prostate cancer DU145 cells and their derived subline DU145R80 selected as resistant to mevalonate-pathway inhibitors, showing different proteomic profile compared to parental cells and unveiling a more aggressive phenotype [7]; 2) the colorectal cancer CO147 primary cell line, cultured either as differentiated cells or as cancer stem cells enriched spheres; 3) the ovarian cancer model TOV_112D parental cell line and TOV_112D miRes cisplatin resistant counterpart [8] the latter showing a different proteomic profile compared to parental cells (R. Lombardi et al., manuscript in preparation). Both DU145R80 cells and CO147 spheres showed enriched stem like features and higher large EVs shedding. DU145R80-derived LO carried increased amounts of key-molecules that we previously found overexpressed in DU145R80 cells. In particular, we showed that integrin alpha V (αV-integrin) on LO surface was functionally responsible of both increased adhesion and invasion of DU145 recipient cells, via AKT activation. Indeed either the pre-incubation of LO with a αV-integrin blocking antibody, or a specific AKT inhibition in recipient cells are able to revert the LO-induced functional effects. Furthermore, in order to explore an hypothetic systemic role of LO, we run an “instigator/responder” experiment [9] where DU145R80 cells (instigator tumors), injected in the flank of nude mice, were able to enhance the engraftment of DU145 cells, which have been injected on the opposite flank of the same mouse (responder tumor). Intriguingly, both TOV_112D parental cell line and TOV_112D miRes resistant counterpart were able to spontaneously bleb, but a different proteome profile was unveiled analyzing LO derived from both cell lines. In conclusion, our results demonstrate that LO can be detected in diverse solid tumors as colon, ovarian and prostate cancers. Their altered composition drives tumor cells aggressive feature, being associated with the development of drug resistance. In prostate cancer, we also demonstrated that LO might act as priming agent to help tumor cells to engraft, by either a paracrine or systemic level, with a potential key role of αV-integrin. 1.Ciardiello C, Cavallini L, Spinelli C et al. Focus on Extracellular Vesicles: New Frontiers of Cell-to-Cell Communication in Cancer. Int J Mol Sci 2016; 17: 175. 2.Li I, Nabet BY. Exosomes in the tumor microenvironment as mediators of cancer therapy resistance. Mol Cancer 2019; 18: 32. 3.Di Vizio D, Kim J, Hager MH et al. Oncosome formation in prostate cancer: association with a region of frequent chromosomal deletion in metastatic disease. Cancer Res 2009; 69: 5601-5609. 4.Garnier D. Reprogramming of GBM microenvironment by large oncosomes: 'Traveling' V-ATPases are doing more than acidification. EBioMedicine 2019; 41: 15-16. 5.Minciacchi VR, Spinelli C, Reis-Sobreiro M et al. MYC Mediates Large Oncosome-Induced Fibroblast Reprogramming in Prostate Cancer. Cancer Res 2017; 77: 2306-2317. 6.Vagner T, Spinelli C, Minciacchi VR et al. Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma. J Extracell Vesicles 2018; 7:1505403. 7.Milone MR, Pucci B, Bifulco K et al. Proteomic analysis of zoledronic-acid resistant prostate cancer cells unveils novel pathways characterizing an invasive phenotype. Oncotarget 2015; 6: 5324-5341. 8.Sonego M, Pellizzari I, Dall'Acqua A et al. Common biological phenotypes characterize the acquisition of platinum-resistance in epithelial ovarian cancer cells. Sci Rep 2017; 7: 7104.9.Bruzzese F, Hagglof C, Leone A et al. Local and systemic protumorigenic effects of cancer-associated fibroblast-derived GDF15. Cancer Res 2014; 74: 3408-3417.

Glioma-derived miRNA-containing extracellular vesicles induce angiogenesis by reprogramming brain endothelial cells

Rocco Lucero1,*, Valentina Zappulli2,3,4,*, Alessandro Sammarco2,3,4, Oscar D. Murillo1, Pike See

Cheah3,4,5, Srimeenakshi Srinivasan6, Eric Tai7,8, David T. Ting7,8, Zhiyun Wei9, Matthew E. Roth1, Louise C. Laurent6, Anna M. Krichevsky9, Xandra O. Breakefield3,4, Aleksandar Milosavljevic1

1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA 2Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy 3Departments of Neurology and Radiology, Massachusetts General Hospital, Boston, MA 02114, USA 4Neuroscience Program, Harvard Medical School, Boston, MA 02115, USA 5Department of Human Anatomy, Faculty of Medicine and Health Sciences, University Putra Malaysia, Seri Kembangan, Selangor, Malaysia 6Department of Obstetrics, Gynecology, and Reproductive Sciences and Sanford Consortium for Regenerative Medicine, University of California, San Diego, La Jolla, CA 92037, USA 7Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA 8Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA 9Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA * These authors contributed equally

Glioblastoma multiforme (GBM) is an invasive, highly polymorphic and universally lethal brain malignancy. The close relationship of glioma stem cells (GSC) with aberrant brain microvasculature facilitates diffuse growth and post-intervention tumor recurrence which invariably thwart treatment. The general failure of anti-angiogenic therapies implicates unrecognized mechanisms of neovascularization in GBM progression. GSC-derived extracellular vesicles (GSC-EVs) and their RNA cargoes are proangiogenic in vitro, and their contribution to tumor vascularization through effects on target cell gene-expression has not been extensively explored. However, prior work has established a predominant role of EV-derived miRNAs in conveying growth promoting and angiogenic signaling in GBM. In this study we focused on the potential transfer of genetic information from GSCs derived from primary human GBM to primary human brain microvascular endothelial cells (HBMVECs) via extracellular RNAs (exRNAs) carried in GSC-derived EVs. To elucidate gene regulatory mechanisms of neovascularization both in vitro and in vivo, we performed RNA-seq and DNA methylation profiling of the response of HBMVECs to GSC-EVs or to growth factors as well as histoepigenetic analysis of GBM molecular profiles in the TCGA collection by applying the Epigenomic Deconvolution (EDec) method. HBMVECs treatment by growth factors or GSC-derived EVs resulted in visually similar vascularization patterns in a tube formation matrigel-based assay. However remarkably divergent transcriptional and epigenomic changes were observed revealing that GSC-EVs and normal growth factors stimulate highly distinct gene regulatory responses converging on angiogenesis. Two hundred thirty-one (231) genes were transcriptionally up-regulated (>2-fold) by either EV (229) or GF (2) treatment. Twenty-six (26) genes were down-regulated by either EV (18) or GF (8) treatment with only one in common downregulated by both treatments. Epigenomic changes were also divergent with GF treatment increasing and EV treatment decreasing genome-wide methylation. Methylation changes over gene bodies and promoters were also divergent. Transcriptional and epigenomic perturbations induced by growth factors in vitro in HBMVECs largely resembled those within human glioblastoma tumors as explored by EDec. Additionally, a subset of transcriptional perturbations observed in endothelial cells within human glioblastomas correlated with GBM EV-induced changes and were associated with post-transcriptional silencing by specific miRNAs. Silenced genes were partially associated with VEGF-independent angiogenic signaling. These results may provide an explanation for the failure of current anti-angiogenic therapy for GBM that exclusively targets growth factor signaling. We identified specific miRNAs that may mediate this EV-induced angiogenesis, thus potentially serving as liquid biopsy biomarkers and as targets for anti-angiogenic combination therapies that may extend lifespan in GBM patients.

Targeting extracellular vesicles in multiple myeloma: a new role for the Notch pathway

Domenica Giannandrea1, Michela Colombo1, Natalia Platonova1, Mara Mazzola2, Francesco

Baccianti1, Raffaella Adami1, Laura Cantone3, Elisa Milano1, Anna Pistocchi2, Valentina Bollati3,

Silvia Ancona1, Elena Lesma1, Mauro Turrini4, Raffaella Chiaramonte1

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

3 Department of Clinical Sciences and Community Health, Università degli Studi di Milano 4 Division of Hematology, Valduce Hospital, Como, Italy

Multiple myeloma (MM) is the second most frequently diagnosed hematological malignancy and

today is still incurable, mainly due to the interaction between the bone marrow (BM) niche. Indeed,

MM cells accumulate in the BM and establish complex interactions with the surrounding normal

cells, forcing them to assume a pro-tumor behavior. In this process, a key role is played by Notch2

and two Notch ligands Jagged1 and 2, whose deregulated expression causes an aberrant

activation of the Notch pathway both in MM cells and in the BM niche cells mediating a

pathological communication.

In this complex picture, new players have recently come to light: the extracellular vesicles (EVs).

EVs include a heterogeneous group of cell-derived membranous structures classified into

exosomes and microvesicles, which are key mediators in the communication between tumor and

stroma.

In this work, we aimed to elucidate the possible role of the Notch pathway in EV-mediated

communication between MM cells and non-tumor cells of the BM niche, by investigating the pro-

tumorigenic activity of EVs from the MM cell line RPMI8226 and the changes occurring in EVs from

RPMI8226 constitutively inhibited for Jagged1/2 (MMJ1/2KD) and Notch2 (MMN2KD).

The ability of MM-EVs to interact with normal cells and activate the Notch pathway in the recipient

cells was evaluated in vivo, by injecting MM-EVs in the duct of Cuvier of 2 days post fertilization

Notch-reporter Tg(T2KTp1bglob:hmgb1-mCherry)jh transgenic zebrafish embryos. Results showed

that MM-EVs are uptaken by embryo’s cells and induce Notch activation in the intersegmental vessels, caudal artery and in the area of the caudal hematopoietic tissue, the main hematopoietic

organ, analogous to the human BM. By contrast, this effect is reduced when EVs were produced

by MMJ1/2KD and MMN2KD cells.

We also demonstrated that MM-EVs promote three key processes involved in myeloma

progression: osteoclastogenesis, angiogenesis and drug resistance. Interestingly, Jagged1/2 or

Notch2 knock down reduces MM-EVs pro-tumor activity. Indeed, MMJ1/2KD-EVs and MMN2KD-EV lost

the pro-osteoclastogenic effect on the monocyte cell line Raw264.7 and the ability to induce human

endothelial cells to organize tubular structures, a key step of angiogenesis. Finally, the level of pro-

tumor cytokines released by the bone marrow stromal cells (BMSCs) HS5 was also reduced when

they were treated with MMJ1/2KD-EVs and MMN2KD-EVs along with their ability to promote MM cells

resistance to bortezomib and melphalan.

In this work, we provide the first evidence that targeting the Notch pathway may represent a

suitable strategy to hamper MM-EVs-mediated communication with the BM niche and the nasty

consequence on the progression of this disease.

Role of exosomes in acute coronary syndromes

Andrea Caccioppo1, Saveria Femminò1, Luca Franchin1, Filippo Angelini1, Alberto Grosso1,Francesco Ravera1, Claudia Cavallari1, Claudia Penna2, Giovanni Camussi1, Pasquale Pagliaro2,

Fabrizio D’Ascenzo1, Maria Felice Brizzi1

1Department of Medical Sciences, University of Turin, Turin, Italy2Department of Clinical and Biological Sciences, University of Turin, Torino. Italy

Background: Despite the improvement in the management of acute coronary syndromes,cardiovascular disease (CVD) still remains the leading cause of mortality worldwide. The challengeis to better characterize the mechanisms occurring in the ischemic tissue damage, particularlyduring the ischemia – reperfusion process. Exosomes (Exo) play an important role in the cross-talk between cells. Their effects mainly dependon their cargo which modulates pathways involved in cell survival or apoptosis and in thinning outthe inflammatory response driven by ischemia. The aim of the present study is to compare featuresand functional capabilities of circulating Exo in ischemic and in normal-perfused myocardium.

Method: 15 patients undergoing coronary angiography were enrolled. Among them 10 wereadmitted to the Cardiology department for unstable angina or Non-ST Elevation MyocardialInfarction (NSTEMI), while 5 patients, showing a low probability of coronary artery disease (CAD)and a normal perfused myocardium served as controls. For each patient, three arterial bloodsamples were collected before the beginning of the angiography. For each sample Exo wereisolated by precipitation methods and stored at -80° C for further analyses. GUAVA FACS analysis

was performed to identify their cell of origin-catenin, Cox-2, pSTAT3/STAT3 and GSK3 wereevaluated in Exo (mRNA and protein). Finally, their functional capability was investigated in vitro oncardiomyoblasts and microvascular endothelial cells subjected to hypoxia/reoxygenation condition.

Results: We demonstrated that Exo collected from ischemic patients mainly express markers of

inflammatory cells and contain a low level of STAT3 and GSK3mRNA. Furthermore, we found a

lower level of-catenin and STAT3 protein content in ischemic patients compared to controls. Exoderived from ischemic patients impair the viability of cells used for in vitro studies by interfering withthe survival signalings.

Conclusions: Our preliminary results suggest that Exo from ischemic patients may promotedamaging effects by transferring their cargo into recipient cells. Further in vivo studies are requiredto validate our observations.

Exosomes enriched for miR-199a-3p reverse tumor malignancy in Ewing sarcoma cells

Alessandra De Feo1*, Marika Sciandra1*, Alessandra Carè2 and Katia Scotlandi1

1Laboratory of Experimental Oncology, IRCCS Istituto Ortopedico Rizzoli, Via Di Barbiano 1/10, 40136 Bologna, Italy; 2Oncology Unit, Center for Gender Medicine, Istituto Superiore di Sanità,

Rome, Italy.

Ewing sarcoma (EWS) is a highly malignant mesenchymal tumor affecting children, adolescents

and young adults, whose aggressive cells are able to release exosomes (EXOs), a subset of

membrane-bound extracellular vesicles with diameters ranging from 50 to 150 nm.

Recently we demonstrated that EWS cells silenced for CD99, a cell membrane molecule

constantly overexpressed in EWS, deliver EXOs with oncosuppressive functions EXOs

onco(-), which significantly reduce tumor aggressiveness. miRNA characterization of the

cargo of these EXOs, identify a signature of 56 miRNAs (10 up-regulated; 46

downregulated). miR-199a-3p resulted as the most enriched miRNA in these vesicles.

Therefore, in this study we investigate the biological role of miR-199a-3p in EWS by

exposing cells to the mimic 199a-3p miRNA and or to its antagomir. Overexpression of

the miR-199a-3p resulted in inhibition of cell growth and migration, reduction of AP-1

activity and c-Fos levels together with induction of terminal neural cell differentiation,

thereby confirming the oncosuppresive functions of this miRNA in EWS. EXOs enriched

for the expression of this miRNA were able to induce similar oncosuppressive phenotypic

effects in tumor recipient cells. To verify the clinical potential of miR-199a-3p as circulating

biomarkers, EXOs isolated from blood of 39 patients were analysed for miR-199a-3p

expression and its levels will be correlated with patient clinical outcome. Overall, our data

provide evidence that endogenous miR-199a-3p and EXOs enriched for miR199a-3p expression

are able to reverse EWS malignancy in experimental models, supporting a potential novel strategy

for this tumor.

Grant:AIRC IG 18451

GLIAL MICROVESICLES IN MOTION AT THE NEURON SURFACE: IMPLICATION IN

ALZHEIMER’S DISEASE

Giulia D’Arrigo1-2, Martina Gabrielli2, Dan Cojoc3, Giuseppe Legname1, Nicola Origlia4, Ottavio Arancio5, Claudia Verderio2

1Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy.

2CNR Institute of Neuroscience, Milano, Italy. 3CNR – Institute of Materials, Area Science Park – Basovizza, Trieste, Italy.

4CNR Institute of Neuroscience, Pisa, Italy. 5Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University,

New York, USA.

Extracellular vesicles (EVs) released from astrocytes and microglia are key players in glia-neuron communication in the healthy and diseased brain. However, how EVs move across the extracellular space to reach target neurons and whether EVs interact with neurons at preferential sites remain elusive. Here, we show that gial EVs efficiently bind to both the cell body and neurites of primary hippocampal neurons. Surprisingly, after the neuron contact, a large fraction of EVs move along the surface of axons and dendrites in both retrograde and anterograde directions. Extracellular EV motion may be driven by binding to neuronal receptors, that ay drift on the plasma membrane following cytochalasin-sensitive and nocodazole-resistant cytoskeleton rearrangements. However, a fraction of MVs contain actin filaments and have an independent capacity to move along the gradient of neuronal receptors in an actin-dependent manner. Our results demonstrate, for the first time, that glial EVs exploit surface neuronal receptors to passively/actively reach their target sites. The implication of extracellulatr EV movement in the rise and propagation of synaptic dysfunction in the early stages of Alzheimer’s disease will be discussed.

Distinct microRNA signature in plasma derived exosomes from different

neurodegenerative diseases

Sproviero D. 1, Gagliardi S. 1, Zucca S. 1, Arigoni M. 2, Pandini C. 1,3, Olivero M. 2, Pansarasa O. 1, “Neurodegeneration Clinical Group”, Calogero R. 2, Cereda C.1.

“Neurodegeneration Clinical Group”: Ceroni M., Costa A., Diamanti L., Minafra B., Pacchetti C., Perini G., Sinforiani E., Zangaglia R.

1. Genomic and post-Genomic Center, IRCCS Mondino Foundation, Pavia, Italy.

2. Department of Molecular Biotechnology and Health Sciences, Bioinformatics and Genomics Unit, University of Turin, Italy.

3. Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy

Abstract

Exploring robust biomarkers is essential for early diagnosis of neurodegenerative diseases. Blood stream transports microvesicles (MVs) and exosomes (EXOs), which are extracellular vesicles of different sizes and biological functions (1,2). Aim of our study was to investigate mRNA/miRNA signatures in plasma derived MVs and EXOs of Amyotrophic Lateral Sclerosis (ALS), Parkinson’s disease (PD), Fronto-temporal Dementia (FTD) and Alzheimer’s Disease (AD) patients. MVs and EXOs were isolated from plasma of patients and healthy volunteers (CTR) by ultracentrifugation and RNA was extracted. Whole transcriptome and miRNA libraries were prepared by TruSeq Stranded Total RNA kit and TruSeq Small RNA Library kit (Illumina). Genes/miRNAs quantification and differential expression were performed using the available tools as part of the reproducible-bioinformatics.org project, to guarantee data analysis reproducibility. Our data suggest that EXOs and MVs RNA cargo varies among different diseases and in comparison to controls. miRNA analysis provides the most informative disease specific signatures in EXOs, as instead whole transcriptome analysis did not show any specific signature. ALS is characterized by a small but specific group of miRNAs. miRNAs profiling displayed that PD and FTD can be subgrouped in two classes, as instead AD appears to be a homogeneous population. Furthermore, miRNAs profiling show the presence of overlaps in the signatures between the analyzed diseases. miRNA profiling in MVs is similar to that observed in EXOs, although in MVs the overall differences between diseases are less marked. Our results show that miRNAs are the most interesting subpopulation of transcripts transported by plasma derived EXOs, for the detection of a miRNA signature for neurodegenerative diseases. In particular we have identified a small set of miRNAs only detectable in the plasma of ALS patients. Finally, MVs, do not represent the best choice for signature detections, because differences between disease signatures is less striking than in EXOs.

1. Cocucci E, Meldolesi J. Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends Cell Biol. 2015;25(6):364-72. doi: 10.1016/j.tcb.2015.01.004.

2. Tetta C, et al. Extracellular vesicles as an emerging mechanism of cell-to-cell communication. Endocrine. 2013;44(1):11-9. doi: 10.1007/s12020-012-9839-0.

CD147 promotes cell exosome release during colon cancer stem cells

differentiation and triggers cellular changes in recipient cells

D. Lucchetti1,2, F. Colella2, C. Ricciardi-Tenore2, F. Calapà2, M.E. Fiori3, F. Vincenzoni1,4, V. Corasolla1,4, A. Urbani1,4, R. De Maria1,2, A. Sgambato5

1 Fondazione Policlinico Universitario A. Gemelli IRRCS, Roma, Italy

2 Institute of General Pathology, Università Cattolica del Sacro Cuore, Roma, Italy

3Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

4Institute of Biochemistry and Clinical Biochemistry, Università Cattolica del Sacro Cuore, Rome, Italy

5 IRCCS-Referral Cancer Center of Basilicata (CROB), Rionero in Vulture (PZ), Italy

Cancer cells secrete exosomes that are involved in the remodeling of stroma microenvironment

and promoting tumor progression [1, 2]. The exosomal molecular key players involved in colon

cancer stem cells differentitation are poorly understood. This study aimed to analyze the role and

content of exosomes in the differentiation process of colorectal cancer stem cells. Here we show

that exosomes secretion during colon cancer stem cells (CSCs) differentiation is partially controlled

by CD147 which is involved in colon cancer tumorigenesis. CD147 expression increased on

exosomes surface during differentiation of both colon cancer cell lines and CSCs. CD147

knockdown as well as anti-CD147 antibodies impaired exosomes release and downstream effects

on recipient cells. Moreover, blocking multivesicular body maturation prevented the exosomes

release during the differentiation and reduced the membrane expression of CD147 on CSC.

Exosomal CD147 appeared to activate a signaling cascade in recipient cells by the induction of

metalloprotease and RhoGTPase expression as well as invasive features in colon cancer cells.

Moreover, exosomes treatment with anti-CD147 antibodies prevented the induction of alfa-sma

and vimentin expression in cancer associated fibroblasts. Our findings reveal a function of CD147

in promoting exosome release during the differentiation process of colon cancer stem cells and in

triggering cellular changes in recipient cells.

References

[1] 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.

[2] Han L, Lam EW, Sun Y. Extracellular vesicles in the tumor microenvironment: old stories, but new tales. Mol Cancer.

2019.

Ultrastructural and molecular characterization of astrocyte-derived extracellular vesicles from nigrostriatal brain regions: implications for

dopaminergic neuroprotection

L. Leggio1, F. L’Episcopo2, S. Vivarelli1, G. Paternò1, C. Tirolo2, N. Testa2, S. Caniglia2, C. Bastos3, N. Faria3, MJ. Ulloa-Navas4, JM. Garcia-Verdugo4, S. Pluchino5, B. Marchetti1,2, N. Iraci1.

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

3Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom. 4Institute Cavanilles,University of Valencia,Valencia, Spain.

5Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom. Parkinson’s disease (PD) is a long-term neurodegenerative disorder that affects cell bodies of dopamine-producing (DA) neurons in subtantia nigra pars compacta of the ventral midbrain (VM) and their terminals in the striatum (Str). An important role in maintaining DA neuron homeostasis in the context of PD is played by astrocytes (AS) that may influence positively or negatively the biology of DA neurons. Our previous work demonstrated that AS activated by chemokines, such as Ccl3, exert a robust DA neuroprotection against the neurotoxin MPTP, both in vitro and in vivo preclinical models of PD [1,2], but the mechanism(s) underlying the complex cross-talk between AS and neurons is still unknown. We hypothesize a possible involvement of AS-derived extracellular vesicles (AS-EVs) in this intercellular signaling. EVs are continuously released outside by cells both in health and disease, through specific pathways that define the vesicle type. Exosomes and shedding vesicles are the main classes of EVs involved in cell-to-cell communication via the exchange of miRNAs, mRNAs, proteins and other molecules. We herein characterized AS-EVs from both the VM and Str and addressed the effect of the Ccl3 vs. degenerative conditions (MPP+). We have characterized at the ultrastructural level AS-EVs by electron microscopy, gaining insight about dimension and concentration. We observed that the basal EV secretion rate is conserved between VM and Str, while the response to Ccl3 treatment is specific in different brain area. In particular, AS from Str release a higher number of EVs after Ccl3 treatment, in sharp contrast with the secretion rate of VM that is stable at the chemokine treatment. Moreover, these changes in the EV secretion rates are reflected on AS morphology, in absence of any influence on cellular viability and proliferation. In fact, Ccl3-treated AS show much more membrane protrusions only in the Str, reflecting the increased secretive activity. Also MPP+ treatment affects the EV secretion, that differs from Ccl3-EVs, suggesting the presence of different class/cargoes in response to specific stimuli. Moreover, both electron microscopy and nanoparticle tracking analysis revealed the presence of a conspicuous population of EVs in the size range of exosomes (~100 nm). The presence of exosomes in AS-EVs was further supported by western blotting analysis of exosomal markers (Cd63, Cd9, Alix1) that were enriched in AS-EVs, while cellular markers were retained in cells. We also investigated the presence of RNA species by qPCR, finding both mRNAs and miRNAs associated with EVs. This in-depth characterization is preliminary to our next steps of investigation that aim to evaluate, in vitro and in vivo, the impact of AS-EVs on target cells relevant in PD. Preliminary data show that AS-EV treatment improve viability and decrease apoptosis rate of MPP+-treated neuronal cells. Overall, our findings suggest a possible role for AS-EVs in facilitating glial-neuron communication and so their reparative/regenerative potential. [1] F. L’Episcopo et al., “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, vol. 41, no. 2, pp. 508–527, 2011. [2] F. L’Episcopo et al., “Wnt/β-catenin signaling is required to rescue midbrain dopaminergic progenitors and promote neurorepair in ageing mouse model of Parkinson’s disease,” Stem Cells, vol. 32, no. 8, pp. 2147–2163, 2014.

POSTER PRESENTATIONS

A reversible immune-capture strategy for purification of extracellular

vesicles from biological samples

D. Brambilla1, R. Vago2, M. Chiari1

1) Istituto di Chimica del Riconoscimento Molecolare (ICRM) CNR, Milan, Italy

2) Università Vita-Salute San Raffaele, Milan, Italy Extracellular vesicles (EVs) are membrane vesicles secreted by cells into bodily fluids that take part in cell-to-cell communication processes. The recent discovery that their membrane and cargo composition may reflect the composition of the cell of origin has increased the interest towards the potential role of Evs as biomarkers in human diagnostics[1]. In view of their potential use for detection of pathologies including cancer, infectious diseases and neurodegenerative disorders, various EVs purification methods have been developed. Among them there are ultracentrifugation, size based techniques (e.g. size-exclusion chromatography), precipitation, microfluidic-based techniques and immune-capture strategies. All these methods present advantages and disadvantages. For example, ultracentrifugation, the gold standard for EVs isolation, is time consuming, produces samples that might be contaminated by cell debris and moreover is not suitable for high throughput applications[2]. Immune-capture techniques represent a promising strategy since on the surface of EVs there are many proteins and receptors that can act as antigens for specific antibodies. If on one side this method offers the possibility of phenotyping the different subclasses of EVs, on the other hand releasing vesicles from antibodies is challenging. To date strongly acidic conditions and/or detergents are used to release immune-captured EVs, but this may result in low release yields and in vesicles damage, both conditions that may skew the downstream analysis. Here we present an innovative reversible immune-capture protocol for EVs purification starting from biological samples. This method exploits magnetic beads functionalized with antibodies directed against the tetraspanin CD63. Unlike other commercially available kits, the antibodies are anchored on the beads through DNA-directed Immobilization. Using this approach, the antibody is tagged with ssDNA, while the surface of magnetic beads is functionalized with the complementary DNA strand. In this strategy, the antibody is immobilized on the surface through the specific interaction between the two DNA strands. The intrinsic reversible nature of the oligonucleotide spacer can be then used to detach the antibody-vesicle complex from the surface by applying an appropriate stimulus. Using this protocol, EVs can be effectively purified from human blood plasma as confirmed by Nano Tracking Analysis and Western Blot. Released vesicles can also be recaptured on a microarray slide functionalized with anti-tetraspanins antibodies. In conclusion we demonstrated the feasibility of reversible immune-capture for EVs purification from biological samples. This approach is advantageous since it combines the specificity given by antibody mediated recognition of the target with the easy recovery of EVs from the beads. Supported by INDEX: European Union’s Horizon 2020 research and innovation programme under grant agreement No 766466

References [1] B. Gold et al. (2015) “Do circulating tumor cells, exosomes, and circulating tumor nucleic acids have clinical utility?” J Mol Diagn., 17(3):209–224. [2] P. Li et al. (2017) “Progress in exosome isolation techniques” Theranostics, 7(3):789-804.

Characterization and gene expression analysis of serum-derived extracellular vesicles in primary aldosteronism

J. Burrello1, C. Gai2, M. Tetti1, T. Lopatina2, M.C. Deregibus2, F. Veglio1, P.

Mulatero1, G. Camussi2, S. Monticone1.

1 - Division of Internal Medicine and Hypertension Unit, Department of Medical Sciences, University of Torino, Torino, Italy.

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

Circulating extracellular vesicles (EVs) are nano-particles mainly released by inflammatory, platelet and endothelial cells and involved in inter-cellular communication; their characteristics reflect the activity of parental cells. Compared to essential hypertensives (EH), patients with primary aldosteronism (PA) display an increased prevalence of target organ damage and cardiovascular events, which may at least in part depend on the endothelial dysfunction observed in these patients. Considering serum EVs as surrogate markers of endothelial cell function, we hypothesized that they could be one of the key mediators of endothelial dysfunction and aldosterone-mediated cardiovascular injury. Therefore, the aims of the present study were: (1) to characterize serum-derived EVs in patients with PA, compared to controls with essential hypertension (EH) and to normotensive (NT) healthy volunteers, (2) to analyse their mRNA cargo, and (3) to examine their potential functional effect in endothelial cell function in vitro. Serum EVs were isolated from 12 patients with PA and 12 with EH, matched by sex, age and blood pressure, and compared with 8 NT controls. EVs were characterized by nanoparticle tracking analysis (NTA), fluorescence-activated cell sorting (FACS) analysis and gene expression qRT-PCR array profiling. Angiogenesis and apoptosis assays were performed on human microvascular endothelial cells to explore EVs functional significance At NTA, EVs concentration was 2.2 times higher in PA patients compared to EH controls; a significant correlation between EV number and serum aldosterone and potassium levels was identified. FACS analysis demonstrated patients with PA presented a higher absolute number of endothelial-derived EVs compared to EH and NT controls. Through EV mRNA profiling, 15 up-regulated and 4 down-regulated genes in PA patients compared to EH were identified; moreover, EDN1 was expressed only in patients with PA. Micro-array platform was validated by qRT-PCR on 4 genes (CASP1, EDN1, F2R, HMOX1), involved in apoptosis, inflammation and endothelial dysfunction. PA-derived EVs were particularly enriched in CASP1 and EDN1 transcripts, suggesting their involvement in the development of endothelial dysfunction displayed by these patients. Consistenly, after curative unilateral adrenalectomy, EVs number and expression of CASP1 and EDN1 significantly decreased in PA patients. In addition, the incubation with PA-derived EVs reduced angiogenesis and induced apoptosis on endothelial cells in vitro. In conclusion, we systematically characterized for the first time circulating EVs in patients with PA. EVs might not only represent a marker of endothelial dysfunction, but also contribute themselves to aldosterone-associated vascular injury and accelerated target organ damage in PA patients.

An Innovative approach for the detection of circulating tumor and extracellular vesicles DNA mutations

S. Galbiati1, F. Damin2, N. Soriani1, V. Burgio3, M. Ronzoni3, D. Brambilla2, M. Ferrari1, M. Chiari2.

1Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy.

2Institute of Chemistry of Molecular Recognition, National Research Council of Italy, Milan, Italy. 3 Dipartimento di Oncologia Medica, IRCCS Ospedale San Raffaele, Milan, Italy.

Despite advances in screening and therapeutics, colorectal cancer (CRC) continues to account for the third most common cause of cancer‑related death in men and women in many countries. Colorectal cancers evolve by a reiterative process of genetic diversification and clonal evolution. A number of signal transduction pathway components have been evaluated to determine their potential to serve as prognostic and/or predictive biomarkers in patients with mCRC. Changes in the DNA sequence (mainly somatic point mutations) are the most frequent class of variants associated with the development of solid tumors and span common oncogenic events, such as KRAS, NRAS and BRAF mutations. The molecular profile of CRC is routinely assessed in surgical or bioptic samples. Genotyping of CRC tissue has inherent limitations; a tissue sample represents a single snapshot in time, and it is subjected to spatial selection bias owing to tumor heterogeneity. Repeated tissue samples are difficult to obtain and cannot be used for dynamic monitoring of the disease progression and response to therapy. It is now established that in biological fluids such as blood, urine, saliva, cerebrospinal fluid, pleural effusions it is possible to detect cancer causing genomic alterations by analyzing circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) or extracellular vesicles (EVs). CTCs have been the focus of liquid biopsy longest efforts, ctDNA comes next, and EVs are the most recent. We dedicated our study on exploiting either circulating tumor DNA (ctDNA) or extracellular vesicles DNA to genotype metastatic colorectal tumors and monitor clonal evolution during treatment. The passive release of cell free DNA into the bloodstream from apoptotic or necrotic cells is dependent on the location, size, and vascularity of the tumor, perhaps accounting for the variability in cell free DNA levels often observed. Exosomes are vesicles with lipid bilayer membranes that enables them to carry and transfer molecules and genetic materials to distant sites. Therefore, they can potentially play a pivotal part in inducing metastasis. Combining information derived from ctDNA and Evs offers a unique opportunity to enrich our understanding of cancer biology, tumor evolution, and therapeutic efficacy and resistance. Genetic variants were determined from liquid biopsies at the diagnosis for medication planning (T0) and following the tumor genetic evolution during treatment phase (T1 and T2) with the objective of allowing therapy response prediction and monitoring. We have selected a statistically significant cohort of patients with metastatic colorectal cancer with known mutations detected on tissue biopsy at the moment of the diagnosis. The pipeline used in our project consists of the use of a customized microarray-based assay for the simultaneous detection of single mutations in different oncogenes (KRAS, NRAS e BRAF) to identify the target mutation followed by droplet digital PCR (ddPCR) to determine the fractional abundance of the mutated allele. Our preliminary results show that this combined approach is suitable for routine mutations detection in clinical practice. The microarray platform allows for a rapid, specific and sensitive detection of the most common mutations suitable for high-throughput analysis without costly instrumentation while the ddPCR consents an absolute quantification of the mutated allele in a longitudinal observational study on patients undergoing targeted therapy.

Acute exercise modulates the release of circulating extracellular

vesicles: tissue-, gender- and BMI-related differences

Hoxha M.1, A.E. Rigamonti1, S.G. Cella1, A. De Col2, Mariani J.1, Rota F.1, S. Cicolini2, G. Tringali2, R. De Micheli2, A. Sartorio2, Bollati V1

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

2. Istituto Auxologico Italiano, Experimental Laboratory for Auxo-endocrinological Research, Verbania and Milan, Italy

Exercise is recognized to evoke multisystemic adaptations that, particularly in obese subjects,

reduce body weight, improve gluco-metabolic control, counteract sarcopenia and lower the risk of

cardiometabolic diseases. Understanding the molecular mechanisms of exercise-induced benefits

is of great interest due to the ensuing therapeutic implications against obesity. We characterized

extracellular vesicles (EVs) in obese (F/M=8/8; age=21.0±8.5 yrs, BMI=37.9±6.0 kg/m2) and

normal-weight (F/M=4/4; age=25.1±8.2 yrs, BMI=20.9±1.5 kg/m2) subjects who underwent a

moderate-intensity (60% VO2max for 30 min or until exhaustion) exercise on a treadmill. Blood

samples were drawn before, at the end and during post-exercise recovery period. EVs were

analyzed by Nanosight and flow cytometry after labelling with the following markers: CD14+

(monocyte), CD61+ (platelet), CD62E+ (activated endothelium), CD105+ (resting endothelium),

HERVW+ (human endogenous retrovirus W), SCG+ (muscle) and FABP+ (adipose tissue). After

exercise, 100-200 nm EVs significantly decreased (p <0.01), and a significantly higher release of

these EVs was observed in normal-weight than obese subjects (p=0.025). If considering the 30-

130 nm size range, we observed a significant lower release of EVs in females than males (p

<0.01). The 130-700 nm EVs significantly decreased (p=0.016), with a higher release in females

than males (p=0.05). CD61+ EVs significantly decreased in all subjects (p=0.02), while SCG+ EVs

were increased (p=0.06). In conclusion, acute exercise induces changes in the release of plasma

EVs, which are tissue-, gender- and BMI specific, suggesting that the exercise-related benefits

might depend upon a complex interaction of tissue, endocrine and metabolic factors.

CD133+ urinary extracellular vesicles in the urine of acute and chronic glomerular damaged patients

Veronica Dimuccio1, Maria Felice Brizzi2, Licia Peruzzi3, Enrico Cocchi3, Fabrizio Fop4, Maddalena Gili2, Sara Gallo2, Giovanni Camussi2 and Benedetta Bussolati1

1Department of Molecular Biotechnology and Health Sciences and 2Department of Medical Sciences, University of Turin, Italy; 3Pediatric Nephrology Unit, Regina Margherita Children's Hospital, Città della Salute e della Scienza di Torino, Turin, Italy. 4Renal Transplantation Center, "A. Vercellone", Division of Nephrology Dialysis and Transplantation, Città della Salute e della Scienza di Torino, Turin, Italy. CD133 is a progenitor/stem cell marker that is found at high levels in urinary extracellular vesicles (uEVs) of healthy subjects. In previous work [1] its concentration was found to be significantly decreased in the urine of patients undergoing renal transplant with a slow graft function recovery. In the same patients the concentration of CD133 increased to healthy controls level in the following recovery period. The aim of the present work is to evaluate the level of CD133 in the uEVs of patients with acute and chronic glomerular damage. For this purpose a group of paediatric acute glomerulonephritis (AGN) patients and a group of type 2 diabetic (T2D) patients were considered respectively as acute and chronic renal damage. Healthy subjects matched for sex and age were considered as controls. The level of CD133 in uEVs was evaluated by cytofluorimetric analysis, through the use of 4µm latex beads. An additional in vitro test was performed on renal progenitor CD133+ cells, mimicking the diabetic condition using high concentration of glucose and human serum albumin (HSA), in order to check the expression of CD133 in cells and in their derived EVs. Results showed that the level of CD133 was significantly decreased in the acute phase of damaged AGN patients, while it was restored in the subsequent recovery period. CD133+ uEVs were confirmed to be significantly decreased also in diabetic patients, mainly in those who had higher renal impairment as stated by high albuminuria. Moreover the in vitro test showed that the treatment with a combination of high glucose and HSA induced a significant decrease of CD133 both in renal progenitor cells and in their derived EVs. In conclusion the present work showed that the level of CD133 in urinary EVs reflect the degree of renal damage, reporting the injury mainly affecting the glomerular compartment. As they can also be released by CD133+ progenitor cells, their decrease in urine can also reflect an impairment of the progenitor cells niche, that over time could lead to a chronic disease. References: [1] Dimuccio V, Ranghino A, Barbato LP, Fop F, Biancone L, Camussi G, Bussolati B. Urinary CD133+ extracellular vesicles are decreased in kidney transplanted patients with slow graft function and vascular damage. PLoS One (2014). doi: 10.1371/journal.pone.0104490.

Measuring microvesicles and exosomes by conventional flow

cytometry: dream or reality?

D. Lucchetti 1, C. Ricciardi-Tenore1, F. Colella 1, A. Battaglia 2, R. De Maria 1, A. Sgambato 1,3, A.

Fattorossi 2. 1 Fondazione Policlinico Universitario A. Gemelli-IRCCS, Rome, Italy; Institute of General

Pathology, Università Cattolica del Sacro Cuore, Rome, Italy 2 Department of Obstetrics and Gynecology, Fondazione Policlinico Universitario A. Gemelli-

IRCCS, Rome,Italy; Università Cattolica del Sacro Cuore, Rome, Italy 3 IRCCS-Referral Cancer Center of Basilicata (CROB), Rionero in Vulture (PZ), Italy

Extracellular vesicles (EVs) are released by a large variety of cells and include two main subgroups plasma membrane-originated microvesicles (100–1,000 nm, MVs) and endosome-derived exosomes, which are generally smaller in size (30–100 nm, EXOs). EVs are detectable in intercellular environment as well as in many biological fluids, including blood. Flow cytometry (FC) applied to EVs fascinates researchers in various clinical settings for the possibility to analyze antigens expressed on EVs in a rapid and reproducible manner. Side scatter (SSC) is often used in the FC of EVs. However, SSC signal intensity has a strong dependence on the relationship between the size of the particle and the wavelength of the incident laser light. The CytoFLEX S cytometer (Beckman Coulter) is able to measure SSC off the violet laser (405 nm, VSSC) to better resolve individual EVs. To obtain EVs from HT29 and Caco2 cancer cells, the cell culture medium was ultracentrifuged at 17000g for MVs isolation and at 110000g for EXOs purification. The correct isolation was proved by dynamic light scatter and transmission electron microscopy analysis. The Megamix-Plus FSC beads of different sizes (100, 300, 500 and 900nm) emitting a FITC-like fluorescent light served to establish the correct gate to visualize EVs in a VSSC/fluorescence dot plot. A threshold was established on the VSSC signal that identified a VSSC/FITC region that included events producing a VSSC signal ~half decade lower than that generated by the smallest beads (100 nm). EVs were stained with the general markers Calcein-AM green and Calcein-AM violet. Immunophenotyping was performed using anti-CD63 and anti-CD9 monoclonal antibodies (moAbs) to stain EXOs, and anti- phosphatidylserine (PS) moAb to stain MVs. Binding specificity was checked by isoclonic control. Calcein-AM staining allowed identifying MVs and EXOs. As expected, EXOs were found below the 100nm VSSC threshold (low VSSC events). However, a large proportion of EXOs, was found above the 100nm VSSC threshold (high VSSC events). Dilution experiments showed that the high-VSSC events represented EXOs aggregates (probably due to isolation method) and swarming. Swarming affected also low VSSC events. Swarming was confirmed by the observation that mixing EXOs samples stained with either Calcein-AM green or Calcein-AM violet generated a double positive population due to coincidental green and violet EXOs in the flow chamber. The same experiments performed using MVs showed that swarming affected also MVs recognition, although to a lesser extent. Importantly, the anti-CD63 and -CD9 moAbs specifically stained EXOs above the 100nm threshold region and failed to stain EXOs below the 100nm threshold suggesting insufficient number of moAb binding sites on single EXOs. This hypothesis was proven by mimicking particles in the EXOs size range using anti-mouse IgG-coated 50nm-beads. These beads were reacted with fluorochrome-conjugated mouse IgG and failed to generate measurable fluorescence signal. Consistent with a larger number of moAb binding sites, MVs generated measurable fluorescence signal following incubation with the anti-PS moAb. We demonstrate that the CytoFLEX S conventional flow cytometer can resolve single MVs but not single EXOs over the background noise, and the analysis is always plagued by swarming recognition. Fluorescence signals generated by fluorochrome-conjugated moAbs on single EXOs are not strong enough to be detected, thereby making it questionable EXOs immunophenotyping. References [1] Lucchetti D. et al. Differentiation Affects the Release of Exosomes from Colon Cancer Cells and Their Ability to Modulate the Behavior of Recipient Cells. Am J Pathol. 2017 [2] Lucchetti D, et al Extracellular Vesicles in Oncology: Progress and Pitfalls in the Methods of Isolation and Analysis. Biotechnol J. 2019

Serum exosomal miRNA profiling of malignant pleural mesothelioma

and early diagnosis: a massive parallel sequencing approach E. Casalone1,2, B. Pardini1,2, G. Birolo1,2, S. Guarrera1,2, A. Allione1,2, M. Sculco3, M. Betti3, D.

Ferrante4,5, C. Casadio6, D. Mirabelli7,8, M. Mencoboni9, C. Magnani4,5,10, I. Dianzani3,10, G. Matullo1,2,10,11

1Italian Institute for Genomic Medicine, IIGM, Turin

2Dep. of Medical Sciences, Univ. of Turin, Turin 3Dep. of Health Sciences, Univ. of Piemonte Orientale, Novara

4Dep. of Translational Medicine, Univ. of Piemonte Orientale, Novara 5Cancer Epidemiology Unit, CPO-Piemonte, Novara

6 Thoracic Surgery Unit, AOU Maggiore Della Carità, Novara 7Cancer Epidemiology Unit, Department of Medical Sciences, Univ. of Turin, Turin

8Cancer Epidemiology Unit, CPO Piemonte, Turin 9Oncology Unit, Villa Scassi Hospital, Genoa

10Interdepartmental Center for Studies on Asbestos and Other Toxic Particulates "G. Scansetti", Univ. of Turin, Turin

11Medical Genetics Unit, AOU Città della Salute e della Scienza, Turin Malignant Pleural Mesothelioma (MPM) detection, particularly at early stages, remains a great challenge owing to lack of specific biomarkers. In recent years much attention has been focused on the role of exosomal microRNAs (miRNAs) as they show tumour-specific expression profiles. Indeed, miRNAs from MPM cells or patient serum, have been proposed as new potential non-invasive biomarkers of disease. We aim at identifying MPM-specific exosomal serum miRNA expression profiles in a case-control study. Serum samples were collected from 30 MPM asbestos exposed patients and 30 asbestos-exposed cancer-free controls, recruited from the areas of Casale Monferrato, Novara and Alessandria (Italy). Exosomes isolation was performed by using the ExoQuick precipitation solution (System Biosciences), RNA samples were processed using NEB Next Small RNA Library Prep Set for Illumina (New England Biolabs). A massive parallel sequencing (MPS) approach has been used to characterise exosomal miRNAs expression profiles. Differentially expressed miRNAs analyses have been performed (Bioconductor DESeq) controlling for age, sex, and asbestos exposure. MPS revealed 162 differentially expressed miRNAs (adjusted p-value) observing consistent results with previous studies. Moreover, 328 species of small non coding RNAs were found de-regulated in cases versus controls. Our results will be replicated in an additional and independent Italian cohort of MPM patients. To assess the potentiality of miRNAs as biomarkers of risk and early diagnosis, we will extend the analyses to a prospectively collected sample in the context of the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. We expect to define a panel of miRNAs to improve early detection and to set up an MPM risk-score for asbestos-exposed subjects. Investigating miRNA signatures in surrogate tissues may be a useful alternative to reduce invasiveness of biopsies, and reducing health care costs for detection. Moreover, early MPM detection could anticipate interventions at potentially treatable stages, increasing survival rates. The identification of diagnostic biomarkers for MPM may contribute to understanding the molecular mechanisms underlying the disease development and progression. Supported by the Fondazione Italiana per la Ricerca sul Cancro AIRC grants (2015-2017 and 2018-2022)

Focusing on a quantification and characterization of extracellular

vesicles from blood in brain tumors

Alberti G. (1)*, Sánchez C. M. (2)*, Campanella C. (1), Cappello F. (1), Marcilla A.(2,3)

1 Department of Biomedicine, Neuroscience and Advanced Diagnostics (BIND), Section of Human

Anatomy, University of Palermo, 90127 Palermo, Italy 2 Área de Parasitologia, Dept. Farmacia y Tecnologia Farmaceutica y Parasitologia, F. Farmacia,

Universitat de València, Burjassot (Valencia), Spain

3 Joint Unit of Endocrinology, Nutrition and Clinical Dietetics, Instituto de Investigación Sanitaria-La Fe, Valencia, Spain

Molecular chaperones are required to maintain the proteome in a folded and functional state. Chaperones are structurally and functionally normal but participate in pathways that favor disease, as a tumorigenesis (1). The incidence of brain tumors is increasing rapidly and surgery is the first therapeutic intervention to safeguard the patient's life. The prognosis is poor even after surgical resection, followed by post-operatory chemo- and radio-therapy (2). Tumor-secreted extracellular vesicles (EVs) are critical mediators of intercellular communication between tumor cells and stromal cells in local and distant microenvironments. EVs play an essential role in both primary tumor growth and metastatic evolution. This concept suggests new diagnostic strategies in which the primary targets are chaperones. The purpose of this study is to search for particular chaperones, in terms of presence and / or absence, level of expression and distribution in the tumor. The presence and level of HSP60, HSP27 and HSP10 in exosomes isolated by blood samples obtained from patients with cancer before and after ablative surgery were investigated. For each patient, blood samples were collected after one week, after one month, and after three months of surgery, and processed for plasma isolation. EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 7–10, while the bulk of the plasma proteins were present in fractions 15–20. Vesicle markers peaked in fractions 7–10. Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations’ purity, using different approaches such as Nanoparticle Tracking Analysis (NTA), and Transmission Electron Microscopy (TEM). NTA analysis was used to determine the concentration of particles in each fraction and vesicle markers peaked in fractions 7–9. Electron microscopy focused on various criteria, including size ranging from 30 to 150 nm, confirmation of spherical morphology. Western Blotting analysis was also performed to verify the presence and the levels of those HSPs. Data regarding exosomal fractions assessment by standard methods (TEM) and WB analysis (for Alix) confirmed their identity. Hsp60 levels showed no significant changes in EVs from the same patients after surgery. Our work provided evidences about presence and levels of the main chaperonins involved in regulation of brain tumors, which could be useful in detecting the disease and monitoring its progression. For this reason, we hypothesize that chaperonins could be good candidates as biomarkers for brain tumors.

AM was supported by the Conselleria d’Educació, Cultura i Esports, Generalitat Valenciana, Grant Prometeo 2016-0156 (Valencia, Spain).

Keywords: HSP60, HSP27, HSP10, exosomes, brain tumor, biomarkers

References

(1) Campanella et al, Cancer 2015, 121:3230-3239 (2) Alexiou et al, J Neurosurg Pediatr 2013, 12:452-457

Plasma derived exosomal microRNAs profile: new potential biomarkers for Alzheimer’s Disease (AD) diagnosis

M. Serpentea, C. Fenoglioa , M. D’Ancaa, M. Arcaroa, E.Oldonic, A. Arighia, A. Cattaneob, L. Porrettib, E. Scarpinia and D. Galimbertia

a University of Milan, “Dino Ferrari" Center, Fondazione Ca’ Granda, IRCCS Ospedale Maggiore Policlinico, Milan, Italy. bClinical Chemistry and Microbiology Laboratory, Flow Cytometry Service, Fondazione Ca’ Granda, IRCCS Ospedale Maggiore Policlinico, Milan, Italy c Laboratory for Neuroimmunology, KU Leuven University, Leuven, Belgium

The aim of the study was to isolate and characterize exosomes in plasma of AD patients and healthy controls in order to detect specific microRNAs signature as potentially peripheral AD biomarkers. In particular, we focused on a specific subpopulation of plasma exosomes derived from neurons (NDEs). We isolated total plasma exosomes from 20 AD patients and 20 controls by using ExoQuick exosome precipitation solution (SBI). Total exosomes were enriched for neural sources by immunoprecipitation with anti-L1CAM antibody and analyzed by FACS, Transmission Electron Microscopy (TEM) and Nanosight Nanoparticle Tracking Analysis (NTA). Total and NDEs miRNAs levels were determined by RT-qPCR using TaqMan OpenArray technology in a QuantStudio 12K system (Thermo Fisher Scientific). A panel of 754 miRNAs was analyzed, and 75 of them reached the optimal expression quality score. MiR-146a-5p, miR-23a-3p, miR223-3p, were expressed in both exosomes categories, but only in the total fraction a statistical significant deregulation was observed (P= 0,03; P=0,02; P= 0,0002). MiR-1260a, miR-1-3p, miR-190a-5p (P=0.02), miR-448, miR-628-3p and miR-653-5p were expressed exclusively in NDEs. These preliminary results demonstrated that plasma exosomes, especially NDEs, are easy detectable in biological fluids and are an enriched source of microRNAs. We observed deregulated expression levels of microRNAs, already associated with neurological diseases, supporting their role in the pathogenesis of AD. Moreover, our data showed a specific expression microRNAs pattern in NDEs, that could likely represent reliable early peripheral biomarker for AD diagnosis. Nevertheless, further studies are required to confirm these preliminary data.

A first study of bovine plasma Extracellular Vesicles: towards advanced monitoring

of inflammation in periparturient dairy cows.

Andrea Zendrini1, Paolo Bergese2,3, Annalisa Radeghieri2,3, Serena Ducoli2, Tatiana Pilipenko2, Paolo Ajmone

Marsan1, Erminio Trevisi1, Andrea Minuti1

1Dipartimento di Scienze animali, della nutrizione e degli alimenti – DIANA, Università Cattolica del Sacro Cuore, Piacenza, Italia.

2Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, Brescia, Italia.

3CSGI - Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Firenze, Italia.

Extracellular Vesicles (EVs) from mammals other than homo sapiens are gaining ever increasing attention

and are expected to impact public health and general economy1,2,3 in the next few years. Indeed, EVs

promise to be exploited as multiplexed fluid biopsy biomarkers to monitor animal welfare (e.g. prion

proteins spreading through EVs4 and “mad cow disease”), which is a major issue in animal husbandry, since

it directly involves food quality and safety.

Our overall interest is to explore EVs for advanced monitoring of the puerperium period of dairy cows,

which is often characterized by increased disease rate and metabolic stress. This, in turn, negatively affects

welfare milk production and fertility, with subsequent losses in productivity5.

The strategy of this study is to compare plasma EV miRNAome of healthy and diseased periparturient cows,

at different days from calving. To this aim, we will employ previously biochemically characterized plasma

samples stored in a biobank.

Methods concerning separation and characterization of EVs from bovine plasma have scarcely been

explored. Moreover, bovine plasma collection and storage are commonly performed with procedures

designed to preserve biophysical parameters instead of EV content (e.g. glycemia or enzymes).

In our contribution we will report on: i) investigation of the pre-analytical variables that may affect stored

plasma EV composition and properties and ii) the specific protocols we developed for proper separation

and characterization of EVs from bovine plasma.

Preliminary results of EV miRNA analysis around parturition will be also presented.

1. Arntz, O. J. et. Al. “Oral administration of bovine milk derived extracellular vesicles attenuates arthritis in

two mouse models”. (2015) Mol. Nutr. Food Res., 59: 1701-1712. doi: 10.1002/mnfr.201500222

2. Pieters B.C. et al. “Commercial cow milk contains physically stable extracellular vesicles expressing

immunoregulatory TGF-β”. PLoS One. 2015;10(3):e0121123. Published 2015 Mar 30. doi:

10.1371/journal.pone.0121123

3. Parry H.A., Mobley C.B., Mumford P.W., et al. Bovine Milk Extracellular Vesicles (EVs) Modification Elicits

Skeletal Muscle Growth in Rats. Front Physiol. 2019;10:436. Published 2019 Apr 16.

doi:10.3389/fphys.2019.00436

4. Hartmann A, Muth C, Dabrowski O, Krasemann S, Glatzel M. Exosomes and the Prion Protein: More than

One Truth. Front Neurosci. 2017;11:194. Published 2017 Apr 19. doi:10.3389/fnins.2017.00194

5. Trevisi E, Minuti A. Assessment of the innate immune response in the periparturient cow. Res Vet Sci.

2018 Feb;116:47-54. doi: 10.1016/j.rvsc.2017.12.001. Epub 2017 Dec 5.

How do epithelial and stromal cells affect endometrial gene expression

in tissue and biofluids?

Criscuoli M.1, Pavone V. 1, Ghelardi C. 1, Zocco D. 2, Luddi A. 1, Piomboni P. 1, Zarovni N. 2

1 Department of Molecular and Developmental Medicine, University of Siens

2 Exosomics Spa

Introduction

Endometrium is a complex tissue with self-renewing properties, normally undergoing cyclic

modifications regulated by ovarian steroids divided into proliferative and secretory phase. The

transcriptomic profile of the endometrium is influenced by other endometrial cell types (glandular

epithelial and stromal) in both physiological and pathological conditions. Each cell type has its

unique gene expression profile and together, they have mutual paracrine effects, partially mediated

by EVs. They grow in different proportions in a cycle-dependent manner. To evaluate the

endometrium status, several invasive or expensive techniques are currently employed, such as

immunohistochemistry (IHC) on tissue biopsy, cytology and imaging. Development of protocols for

the isolation of EVs from novel biological sources is an extremely attractive means to surrogate

endometrial biopsies. These novel protocols may enable the identification and sensitive detection

of specific endometrial EV biomarkers for diagnostic solutions in reproductive medicine,

endometriosis or cancer.

Methods

Samples: primary endometrial culture (stromal and epithelial cells), urine from heathy donors; Size

exclusion chromatography (SEC); Nanoparticle Tracking Analysis (NTA), BCA assay, ELISA, HS

Qubit, ddPCR, FACS for EVs and EV markers quantification and characterization.

Results

We show that stromal and epithelial cells, as well as respectively released EVs, contribute to

endometrial gene expression in tissue and in biofluids, as urine, in a different manner. We also

provide new evidence that urine is a surrogate biofluid suitable for the detection of endometrial EV

biomarkers in secretory phase of menstrual cycle. Using pre-selected antibody panels, we identify

specific endometrium EV binding antibodies in relevant in vitro models. Coupling immune-isolation

to pre-analytical protocols for urine processing and sample quality testing enables detection of a

panel of endometrial genes in urine-recovered EVs.

Summary/Conclusion

Overall, the study may provide a tool for non-invasive monitoring of the functional status of the

endometrium, supporting biomedical niches such as assisted fertilization and diagnosis of

endometriosis.

Funding

ENDEvor POR Region Tuscany (identification of the project) and Exosomics R&D Programme

Heat Shock Proteins exosomal localization and levels in non-tumoral

and tumoral thyroid tissues

F. Rappa 1, C. Caruso Bavisotto 1,2, R. Barone 1, A. Marino Gammazza1, F. Bucchieri 1, E. Conway de Macario 2,3, A. Macario JL2,3, C. Cipolla 4, G. Graceffa 4, D. Cabibi 5, C. Campanella 1,2, F.

Farina1, F. Cappello 1,2

1.Section of Human Anatomy, Department of Biomedicine, Neuroscience and Advanced

Diagnostics, University of Palermo, Palermo, Italy.

2. Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy.

3. Department of Microbiology and Immunology, School of Medicine, University of Maryland at

Baltimore-Institute of Marine and Environmental Technology (IMET), Baltimore, Maryland., USA.

4. Department of Surgical Oncological and Oral Sciences, University of Palermo, Palermo, Italy.

5. Pathology Institute, Department BG. D’Alessandro^, University of Palermo, Palermo, Italy.

Exosomes (EXs) secreted from cells to the extracellular environment play an important role in intercellular communication in normality and pathology. In particular, EXs contribute to cancer progression through the modulation of tumor microenvironment. Heat Shock Proteins (Hsps) are implicated in the development of carcinogenic process because they interact with many metabolic and biomolecular mechanisms of cancer cells [1]. Different studies shown that they can be released by tumors cells and that the mechanism of release is mediated by EXs pathway. In this study we performed an immunomorphological and biomolecular analysis to investigate Hsp27, Hsp60, Hsp70, Hsp90 levels expression profile in thyroid tissue and in plasma samples from patients undergoing thyroidectomy. We studied two groups of patients, one surgically treated for benign goiter, another surgically treated for papillary carcinoma. Samples of thyroid tissue of benign goiter and papillary carcinoma were used for IHC analysis. Two blood samples from each subjects were taken, the first one day prior to the surgical removal of the thyroid, and the second, one week after, on the day of medication. These blood samples were used for exosomal isolation. The immunoistochemistry shown an overexpression of Hsp27, Hsp60 and Hsp90 in the PTC cases comparison with peritumoral tissue and with goiter samples. Hsp70 expression level has not shown significant difference between two groups studied. In particular Hsp27, Hsp60 and Hsp90 were visible at cytoplasmic and membrane levels. The membrane localization of these HSP suggested their release in tumor microenviroment. The exosomes, obtained from the plasma, were analyzed by Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), and Atomic Force Microscopy (AFM) in order to estimate the exosomes size/diameter and morphology. After, we performed Western Blotting analysis for Alix and CD81, as exosomal markers, and for Hsp27, Hsp60 and Hsp90. The levels of Hsp27, Hsp60 and Hsp90 in the exosomes of patients with PTC before surgery were significantly higher than in the exosomes from the same patients after surgery. The data obtained shown that, as demonstrated in human large bowel cancer [2], the Hsp levels studied increased in PTC specimens respect to goiter specimens.

References: [1] Rappa et al. (2012) HSP-molecular chaperones in cancer biogenesis and tumor therapy: an

overview. Anticancer Res. 32:5139-50.

[2] Campanella et al. (2015) Heat shock protein 60 levels in tissue and circulating exosomes in

human large bowel cancer before and after ablative surgery. Cancer 121:3230-9.

doi:10.1002/cncr.29499.

Profiling Extracellular Vesicles is a specific diagnostic tool for Parkinson disease

E. Vacchi1,2, J. Burrello3, S. Bolis4, A. Burrello5, L. Barile2,4, G. Vassalli 2,4,6, A. Kaelin-Lang1,2,7,8, G.

Melli1,2,7 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. Division of Internal Medicine and Hypertension Unit, Department of Medical Sciences, University of Torino, Torino, Italy

4. Cellular and Molecular Cardiology Laboratory and Laboratory for Cardiovascular Theranostics, Cardiocentro Ticino Foundation, Lugano, Switzerland

5. Department of Electrical, Electronic and Information Engineering, University of Bologna, Bologna, Italy

6. Department of Cardiology, Molecular Cardiology Institute, University of Zurich, Zurich, Switzerland 7. Neurology Department, Neurocenter of Southern Switzerland, Ente Ospedaliero Cantonale,

Lugano, Switzerland 8. Department of Neurology, Inselspital, Bern University Hospital, University of Bern, Bern,

Switzerland Background: Parkinson disease (PD) is the second most frequent neurodegenerative disorder of the elderly, characterized by the progressive loss of dopaminergic neurons in the substantia nigra and it still lacks an effective causal therapy. There is an urgent need for an early biomarker that can identify patients at the beginning of disease or at risk of developing PD to maximize the impact of the treatment. Extracellular vesicles (EVs) have several characteristics that make them excellent potential biomarkers: 1) they carry cell-type specific proteins and nucleic acids reflecting the pathogenic intracellular processes; 2) they cross the blood-brain barrier so that CNS-derived EVs can reach the peripheral blood; 3) they can be immuno-phenotyped to enrich for CNS-derived EVs to have specific information on neurological diseases. Main aims of the study are: (1) to investigate differences in EVs protein expression between PD patients, healthy control (HC) and subjects with atypical parkinsonism (AP); (2) to identify candidate biomarkers among EVs surface membrane-proteins. Methods: Plasma samples were collected from 27 PD, 19 HC and 18 AP (9 with possible alpha-synucleinopathies (AP-SYN) and 9 with possible tauopathies (AP-TAU)). Demographic data, medical history, therapy, disease duration and clinical scales were recorded in a database including MDS-UPDRS, Hoehn and Yahr scale, Beck Depression Inventory, MOCA, RBD scale, olfactory test. Quantification of EVs was performed by nanoparticle tracking analysis (NTA). EVs surface-protein expression was characterised by an antibody-coated bead-based multiplex flow cytometric assay. Based on differential EVs-surface markers expression, two diagnostic models were built and validated through supervised learning algorithms: linear discriminant analysis (LDA) and random forest. Results: NTA analysis revealed higher EVs concentration in PD vs HC and AP-TAU, but not vs AP-SYN; while no differences were observed in EVs diameter between groups. Among EVs-surface markers, 15 of 37 were differently expressed in PD vs HC, 12 in AP-SYN vs HC and 4 in AP-TAU vs HC. Of these, the majority of EVs-surface markers were linked to inflammatory response. At univariate analysis, respectively 11, 6 and 3 EVs-surface markers were significantly associated with the diagnosis of PD, AP-SYN and AP-TAU. The reliable diagnostic performances was confirmed for each single candidate biomarker through ROC curve analysis, comparing each pathological group vs HC (AUC range 0.713-0.843 for PD, 0.743-0.865 for AP-SYN and 0.743-0.795 for AP-TAU). Finally, the combination of all markers differently expressed correctly detected 84.8% of PD, 100% of AP-SYN and 89.3% of AP-TAU in LDA model (validation between 71.4% and 78.6%), whereas random forest model correctly detected 97.8% of PD, 100% of AP-SYN and 85.7% of AP-TAU (validation between 82.1% and 89.3%). Conclusions: In PD the total number of EVs are increased and surface markers involved in cellular and humoral immune responses signature, are significantly more expressed. Notably the expression pattern of EVs markers distinguish PD from healthy subjects and AP.

Microvesicles as promising biomarker of frailty

Marta Giannini1,2, Matteo Bordoni1, Orietta Pansarasa1, Daisy Sproviero1, Stella Gagliardi1, Maria Chiara Mimmi1,3, Annalisa Davin3, Antonio Guaita3, Cristina Cereda1

1 Genomic and post-Genomic Center, IRCCS Mondino Foundation, Pavia, Italy 2 Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy

3 Laboratory of Neurobiology and Neurogenetic, Golgi-Cenci Foundation, Abbiategrasso (MI), Italy Frailty is a geriatric syndrome characterized by the reduction of functional reserves and the decreased resistance to stress factors resulting from the cumulative decline of multiple physiological systems that cause vulnerability and adverse consequences. Older people with frailty have an increased risk of adverse outcomes including disability, hospitalization, nursing home admission and mortality [1]. Several mechanisms related to senescence and cellular inflammation are involved in frailty, as suggested by several works [2, 3]. Identifying new biomarkers could help in the diagnosis of frailty syndrome; therefore, the identification of possible biomarkers is fundamental. Aim of this work is the evaluation of microvesicles (MVs) as possible frailty biomarkers. A group of non-frail subjects and a group of frail subjects were subdivided according to the frailty index of Rockwood influenced by functional status, diseases, physical and cognitive deficits, psychosocial risk factors and geriatric syndromes. The MVs were isolated from the plasma of the two groups and characterized by two panels to detect receptors linked to inflammation and senescence. The inflammation panel includes: CD3, CD4, CD8, CD163, CD197, TLR2 and TLR4, CD40 and CD120b. The senescence panel includes: CD221, CD182 (IL8R), CD126 (IL6R). The samples were analyzed using the FACS Canto II (BD Biosciences, USA) flow cytometer using calibration beads (Submicron Bead Calibration Kit, 0.2 μm-1 μm). The MVS were analyzed for size and counts using NTA software (version 2.2, NanoSight). The concentration of MVs between the two groups did not show a statistically significant difference either by flow cytometer analysis or by nanosight analysis. Furthermore, nanosight analysis did not show a significant difference in the size of the MVs between the two groups. A pilot study carried out on a subgroup of the starting samples (14 frail vs 14 non-frail) showed a significant increase in the CD221 + marker in the frail group compared to the non-frail (median non-frail = 0.12 and brittle median = 0.78; Mann Whitney test ** p value = 0.0015), while no significant differences were found in the markers CD3, CD4, CD163 and CD197. Preliminary results obtained from the analysis of a subgroup of the samples (5 frail vs 5 non-frail) also showed a significant increase in the markers TLR2 +, TLR4 +, CD40 +, CD120b +, CD126 + correlated to the condition of frailty. The significant presence of CD221 + MVs in frail elderly people suggests that IGFR may be used as a potential biomarker of senescence. Supported by Cariplo Foundation with the project “Association between frailty trajectories and biological markers of aging” (FrailBioTrack 2017-0557) References

[1] Clegg A, Young J, Iliffe S, Rikkert MO, Rockwood K. Frailty in elderly people. Lancet. 2013 Mar 2;381(9868):752-62. doi: 10.1016/S0140-6736(12)62167-9. [2] Cesari M, Calvani R, Marzetti E. Frailty in Older Persons. Clin Geriatr Med. 2017 Aug;33(3):293-303. doi: 10.1016/j.cger.2017.02.002. [3] Soysal P, Stubbs B, Lucato P, Luchini C, Solmi M, Peluso R, Sergi G, Isik AT, Manzato E, Maggi S, Maggio M, Prina AM, Cosco TD, Wu YT, Veronese N. Inflammation and frailty in the elderly: A systematic review and meta-analysis. Ageing Res Rev. 2016 Nov;31:1-8. doi: 10.1016/j.arr.2016.08.006.

Isolation and characterization of extracellular vesicles (EVs) derived

from human coelomic fluid

Sabrina Picciotto(1), Giorgia Adamo(1), Daniele Romancino(1), Valentina Cigna(3), Aurelio Maggio(2), Antonino Giambona(2), Antonella Bongiovanni(1) and Francesco Picciotto(3)

(1) Institute for Biomedical Research and Innovation (IRIB), National Research Council of

Italy (CNR), Palermo, Italy (2) Hematology Unit for Rare Diseases of the Blood and Blood-forming Organs, Laboratory

for Molecular Diagnosis of Rare Diseases and Prenatal Diagnosis Villa Sofia-Cervello Hospital, Palermo, Italy

(3) Prenatal Diagnosis Unit, Villa Sofia-Cervello Hospital, Palermo, Italy Human coelomic fluid (CF) is the first dynamic and complex fluid of the gestational sac; being of embryonic derivation, it is used for early prenatal diagnosis of hemoglobinopathies and other genetic diseases1. The biological features of CF have not yet been explored due to difficulties in the sampling procedure, which is carried out between seven and nine weeks of gestation by transvaginal sonography. CF’s protein profile shows changes throughout the first trimester of pregnancy, which could reflect physio-pathological alterations affecting the embryo or the mother, and may be related to particular types of pathologies. In this view, the identification and characterization of CF-derived extracellular vesicles (CF-EVs) may help in setting up an innovative, very early prenatal screening method. EVs are now considered to be an additional mechanism for intercellular communication, allowing cells as well as organisms to exchange proteins, lipids and genetic material. Recent results indicate that transportation of the intracellular proteins via EVs contributes to various types of disease. In the context of a disease, EVs can be enriched with specific pathological molecules, and for this reason can be used to identify “biomarkers” for early diagnosis or to monitor the prognosis. The present study on CF-EVs could add new insight regarding the crosstalk between fetal and maternal cells and could pave the way for the use of CF-EVs as biomarkers for the early identification of fetal pathologies. After celocentesis, a quantitative fluorescent analysis (QF-PCR) of short tandem repeats (STRs) was performed on fetal cells derived from CF. This analysis was carried out to evaluate monogenic disorders and maternal contamination, and was based on the identification of several markers for chromosomes 13, 18, 21, X and Y. In parallel, CF-EVs were obtained from several CF samples using an optimized differential ultracentrifugation protocol. The number and size distribution of CF-EVs were analyzed by Nanoparticle Tracking Analysis (NTA). Further, immunoblot analyses are performed to identify and establish specific biomarkers of CF-EVs. The characterization of the CF samples demonstrated the presence of CF-EVs. The investigation of CF-EV properties could prove useful in the future as a very early and reliable prenatal screen. References: 1. Giambona A, Makrydimas G, Leto F, Damiani G, Jakil MC, Picciotto F, Renda D, Fiorino R, Renda MC, Schillaci G, Gueli-Alletti D, Nicolaides KH, Maggio A. Feasibility of DNA diagnosis of haemoglobinopathies on coelocentesis. Br J Haematol. 2011 Apr;153(2):268-72.

Monocytes- but not platelets-derived microvesicles from healthy donors and CAD patients increase endothelial adhesiveness.

Maria Talmon1, Marta Brambilla2, Paola Canzano2, Luigia G Fresu1, Sandra Brunelleschi1, Elena

Tremoli2, Marina Camera2

1Department of Health Sciences, School of Medicine, University of Piemonte Orientale, Via Solaroli, 17 - 28100 Novara (Italy)

2Centro Cardiologico Monzino I.R.C.C.S., Milan, Italy.

BACKGROUND. Plasma microvesicles (MVs) are recognized as both biomarkers and mediators of endothelial dysfunction in many pathological conditions, including cardiovascular diseases. Two main subtypes were identified to be responsible of mediating this inflammation message: monocytes (Mo)- and platelets (PLT)-derived MVs, although the specific contribution of each of them is not well defined. AIM. We aim to investigate the role of Mo- and PLT-derived MVs setting an in vitro model of MVs generation from monocytes and platelets isolated from healthy donors and CAD patients, as such or in a stimulated inflammation environment. This strategy allowed us to separately investigate the role of the two MVs subtypes in triggering endothelial adhesiveness of leukocytes, a key event to support immune cell infiltration, which is a cardinal process of inflammation. METHODS. Mos and PLTs were isolated from healthy donors and CAD patients and cultured without stimuli or in presence of LPS (1 or 10 µg/ml, for 16h at 37°C) in the case of monocytes and of ADP (10 µM) or collagen I (4 or 10 µg/ml), or epinephrine (10 µM), or arachidonic acid (1 mM) or TRAP-6 (20 µM, for 30 min at 37°C) in the case of platelets. MVs were counted and characterized by flow cytometry (FACS) for expression of platelets activation markers, such as Tissue Factor (TF), P-selectin, CD41 or monocytes CD16 and CD14 markers to define the subsets defined as classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14+CD16+).Once having set the optimal conditions to generate Mo- and PLT-MVs, human vascular endothelial cells (hECV) were incubated with the obtained microvesicles and peripheral blood mononuclear cell (PBMC) adhesiveness after MVs challenge were evaluated. RESULTS. In healthy subjects, Mo-MVs significantly augmented after stimulus with LPS (~0.7-fold change) while the number of PLT-MVs raised after stimulus with ADP and TRAP-6 (~2-fold change, both), while the other stimuli did not determine a significant increase. Interestingly, FACS analysis showed that stimulation, in all the conditions tested, determined an increase in number of annexin V+ and TF+ MVs, without affecting their relative composition. Given these observations MVs from monocytes and platelets of CAD patients were generated upon stimulation with LPS and TRAP-6, respectively. Unstimulated monocytes from CAD patients produced a significant higher amount of CD16+ MVs (~2-fold change) compared to those from healthy subjects. This data reflects the reported increase of non-classical monocytes subpopulations associated to acute or chronic inflammation and, particularly, in CAD patients. MVs derived from PLT of CAD patients differ from that of healthy both as number and percentage composition: in fact, CAD patients’ platelets produced a significantly higher number of PLT-MVs expressing a significant augmented rate of TF, CD41 and P-Selectin both at rest and after stimulus. As seen for Mo-MVs also CAD PLT-MVs reflects the inflammatory status of patients. Finally, we assessed the Mo- and PLT-MVs effect on endothelial cells adhesiveness. The challenge of endothelial cells with microvesicles showed that Mo-MVs derived but not PLT-MVs were able to increase the adhesiveness. Mo-MVs from LPS stimulated healthy monocytes and from CAD in both conditions significantly augmented the number of PBMC adhering on stimulated endothelium. Interestingly, the effect of incubation with unstimulated CAD MVs was significantly higher compared with the healthy unstimulated ones and was completely superimposable to their stimulated counterpart. CONCLUSION. Mo-MVs, increasing the endothelial adhesiveness, pave the way for an augmented infiltration. Interestingly, the effect of CAD unstimulated Mo-MVs was comparable to the healthy Mo-MVs generated by LPS stimulus suggesting that an altered inflammation status was essential to generate MVs triggering an endothelial activation.

Development of exosomes based non-invasive technique for early detection of Parkinson’s disease

K. Rani, E. Singh, S. Kumar1, V. Goyal2, F. Nikolajeff3 , S. Kumar1

1Department of Biophysics, AIIMS, New Delhi 2 Department of Neurology, AIIMS, New Delhi

3Department of engineering sciences, Uppsala University, Sweden

Parkinson’s disease (PD) is the second most common chronic progressive neurodegenerative disease worldwide with limited effective treatment options to arrest the disease progression and hence has a huge burden on health care system [1]. The main pathology is the loss of dopaminergic neurons leading to dopamine depletion. It shows a wide variation in prevalence rates, probably due to lack of proper diagnostic tools to detect it in the early stages and where there may be more effective options to halt the disease progression. Till date, no biomarker based tests are available for the effective diagnosis of PD. Recent findings of efflux of α‑synuclein and other proteins from CSF to peripheral blood incorporated into exosomes are showing promising results suggesting role of exosomes in pathophysiology of PD [2]. We intend to develop easy and cost effective exosomes isolation from human biofluids and validation of neuronal exosomes and α- synuclein oligomerization. This work is first in kind to develop a noninvasive method to extract the exosomes from the patient samples and standardize the method to quantify exosomes level by nanotracking analysis (NTA).

A total of 18 PD patients were recruited. All the PD patient’s physical and neurological examinations were in accordance with the United Kingdom Parkinson’s Disease Society Brain Bank criteria [3]. This study was approved by the ethics committee of AIIMS, India. Unstimulated saliva was collected. The salivary exosomes were isolated using PEG percipitation method. Western blot analysis was performed with antibodies for exosomal marker (CD63), neuronal origin exosomes marker (L1CAM) and phospho α-syncluein. Nanoparticle tracking analysis (NTA) was used to quantify the concentration of salivary exosomes.

Salivary exosmes wers isolated and confirmed in both PD pateints and healthy control by western blot using anti CD63 antibody. In addition, western blot validation also revealed that expression of neuronal origin exosomes marker L1CAM (p=0.0001) and phospho α-synuclein (p=0.0001) were found to be significantly higher in PD patients than in healthy controls of salivary exosomes. NTA based quantification also showed higher concentration of salivary exosomes in PD patients as compared to healthy control (p=0.0001). These result indicates that salivary exosomes may help in establishing exosomes as a candidate marker for PD detection at pre-symptomatic stage as well as an easy tool to objectively measure disease progression for drug efficacy studies, all with a fully non-invasive salivary exosome-based technique. References

[1] Poewe W, Seppi K, Tanner CM, et al. Parkinson disease. Nat Rev Dis Prim. 2017; 3:17013.

[2] Shi M, Liu C, Cook TJ, et al. Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson's disease. Acta Neuropathol. 2014; 128(5):639-50.

[3] Marsili L, Rizzo G, Colosimo C. Diagnostic Criteria for Parkinson's Disease: From James Parkinson to the Concept of Prodromal Disease. Front Neurol. 2018;9:156.

Development of extracellular vesicle-targeting tools for novel liquid

biopsy approaches in lung cancer

Diogo Fortunato1, Mattia Criscuoli1, Laura Bianciardi1, Davide Zocco1, Natasa Zarovni1

1Exosomics SpA, Siena, Italy

Mounting clinical evidence suggests that liquid biopsy may revolutionize the way cancer patients

are currently managed. Timely and accurate diagnosis is fundamental for effective cancer

prevention and treatment. Within this context, our study aims to assess and reinforce unique and

complementary advantages of extracellular vesicle (EV)/exosome-based approaches, through

identification and quantitative detection of non-small cell lung cancer (NSCLC) EV biomarkers.

Current technology and methods for exosome isolation from complex biological samples (i.e.

plasma), have shown to be unreliable. There is a need to substantially improve them to enable

multiparameter EV analysis. Therefore, in addition to EV-biomarker discovery, we are testing

plasma processing and pre-analytical tools, devices and optimized immunoaffinity protocols that

tackle fundamental obstacles, such as complex matrix effects.

We aim to provide an EV immunocapture approach with enough sensitivity, specificity and

robustness for clinical grade diagnostic applications.

We have been applying physical, enzymatic and immunological treatments for plasma pre-clearing

to compare downstream purification steps, mainly focused on size-exclusion chromatography

(SEC) and immunocapture strategies. By performing spike-and-recovery experiments using

fluorescently labelled EVs, we determine efficiencies of recovery at the particle number level.

Additionally, we characterize EV subpopulations by nano flow cytometry (nFC), ELISA,

nanoparticle tracking analysis (NTA), western blot and digital droplet PCR (ddPCR).

By applying plasma pre-analytical treatments, we were able to enrich samples in EV content, as

well as to characterize these enriched EV subpopulations. Our ultimate goal is to combine pre-

analytical protocols that optimally counteract complex matrix effects in plasma, hence improving

the use of screened, proprietary antibodies to increment EV yield by affinity-isolation approaches

and facilitate the analysis of enriched EV subpopulations, relevant for early-stage NSCLC

detection.

This project is funded under the Marie Skłodowska-Curie grant agreement No. 765492 “ELBA - European Liquid Biopsies Academy” and internal Exosomics R&D Funds.

Extracellular vesicles as graft biomarkers to monitor lung transplantation in the context of extracorporeal photopheresis

Mario Barilani1, Giuseppe Buono1, Ilaria Righi2, Lorenzo Rosso2,3, Mario Nosotti2,3, Lorenza Lazzari1 1Laboratory of Regenerative Medicine – Cell Factory, Department of Transfusion Medicine and Hematology, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy 2Thoracic Surgery and Lung Transplantation Unit, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy 3University of Milan, Milan, Italy In the medical practice, lung transplantation is the last therapeutic option for end-stage pulmonary failure, when other treatments are no longer effective [1]. Yet, only 15-20% of the multi-organ donors have suitable lungs. Furthermore, clinical complications may rise after organ retrieval following ischemia–reperfusion lung injury, such as primary grafts dysfunction or chronic lung allograft dysfunction. Currently, clinical parameters implemented to assess the quality of the graft have failed to evaluate tissue damage at the cellular level and to predict transplantation outcome. Therefore, we focused our attention on extracellular vesicles (EV) as innovative, non-invasive biomarkers urgently needed to assess lung quality and monitor organ engraftment. Moreover, EV were addressed in the context of extracorporeal photopheresis (ECP), which is a treatment normally used for graft-versus-host disease. During ECP, peripheral blood white blood cells (WBC) are isolated, exposed to 8-methoxypsoralen photosensitizing agent, and subsequently treated with ultraviolet radiation before reinfusion into the patient, causing massive WBC apoptosis. Two cohorts of patients receiving (n=10) or not (n=10) ECP are currently being enrolled in the study. Research activities involving human subjects complied the Declaration of Helsinki. Informed consent and local ethics committee approval were obtained. The ongoing study is showing presence of extracellular vesicles (EV) of different sizes in bronchoalveolar lavage (BAL) and plasma of both donors (n=3) and recipients (n=3), as analyzed by nanoparticle tracking analysis (Nanosight NS300, Malvern). EV presented highly polydispersed size distributions in a 50-1000 nm range. Different kinetics of EV production were observed in the recipients (108-1010 particles/mL range): BAL samples showed peaks within 72 hours post-transplant and a subsequent decreasing trend, whereas plasma samples showed a general increasing trend. EV have been analyzed for antigen expression with MACSplex bead-based assay. The expression of EV-associated markers CD63, CD9 and CD81 was detected. Furthermore, a dramatic upregulation of platelet, endothelial and T cell markers (CD62P, CD41b, CD42a, CD31, CD56, CD69, CD29, HLA-ABC) was observed in ECP-treated patients compared to the control group. A further correlation with lung transplantation outcome will be evaluated at the conclusion of the follow-up. The identification of specific EV kinetics patterns and antigen signatures represents a promising approach to define biomarkers useful for thoracic surgeons who want to manage in advance complications associated to lung transplantation. Supported by Ricerca corrente 2016-2018, Minister of Health, Italy. References [1] Rosso, L., Zanella, A., Righi, I., Barilani, M., Lazzari, L., Scotti, E., Gori, F., Mendogni, P., 2018. Lung transplantation, ex-vivo reconditioning and regeneration: state of the art and perspectives. Journal of Thoracic Disease 10(Suppl 20): S2423–S2430.

Urinary exosomes in the idiopathic nephrotic syndrome: a pilot study

L.Santorelli1, W. Morello1,2, E. Barigazzi1, F. Raimondo1, B. Crapella2, L. Ghio2, C. Tamburello2,

G. Montini2 and M. Pitto1

1. School of Medicine and Surgery, University of Milano - Bicocca, Milano, Italy

2. Paediatric Nephrology, Dialysis and Transplant Unit, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milano, Italy

Growing interest regards the use of minimally invasive “liquid biopsies” to identify new biomarkers. Urinary exosomes (UE) are nanovesicles released into urine by cells facing the urinary space. Their molecular composition depends upon the type of the cell of origin, providing a fingerprint, capable to monitor its status. Their presence in urine makes them readily accessible, giving the possibility to investigate pathological conditions especially related to kidney [1]. While exosome research has flourished, few studies have specifically targeted the role of UE in the Nephrotic Syndrome (NS). As a rule, NS is a manifestation of many underlying renal disease processes. However, in children NS is most often Idiopathic (INS), constituting the major childhood glomerular disease, with an incidence of 2-7/100,000 children <16 years old. Response to initial treatment with corticosteroids is an indicator of long-term prognosis, as resistant patients often present progressive disease [2]. The aim of the study is to verify the feasibility to use UE of INS patients as a source of predictive markers of response to corticosteroid treatment, and/or for clarifying the disease etiopathogenesis and/or the pharmacoresistance. Thus, we investigated the UE protein content of a paediatric cohort of 30 patients, classified in three clinical classes, according to the corticosteroids response: steroid-Dependent, (D), steroid-Sensitive (S) and steroid-Resistant (R). Firstly, a characteristic SDS-PAGE protein profile emerged to be associated with each class, which preserves its peculiarity also when compared to UE protein content of non-INS patients (orthostatic proteinuria, hereditary tubulopathies) and healthy age-matched controls. Through a hierarchical clustering, we confirmed the imprints provided by the gel analysis: the UE of INS patient cohort divided according to the clinical classification. Secondly, we also pinpointed different levels of specific glomerular membrane proteins (PgP, CD10, Podocin), described as involved in the INS development. Moreover, we observed differential immunoblotting bands significantly varied among the three classes, especially in the case of CD10. These evidences confirmed the feasibility to use a UE approach to intercept the pathophysiological differences underlying response to therapy. We are currently proceeding in the study performing mass spectrometric analysis to more in depth comprehend the emerged differences in the UE proteome of the pilot cohort. Simultaneously, we are enlarging the sample size in order to develop a robust classification model, able to use UE as predictive parameter of long term prognosis to treatment response. References:

[1] Morrison, E.E., Bailey M., Dear J.W., 2016 Renal extracellular vesicles: from physiology to clinical application. The Journal of Physiology; (20):5735-5748. [2] Pasini A., Benetti E., Conti G., Ghio L., Lepore M., Massella L5, Molino D6, Peruzzi L7, Emma F5, Fede C3, Trivelli A., Maringhini S., Materassi M1., Messina G., Montini G., Murer L., Pecoraro C., Pennesi M., 2017. The Italian Society for Pediatric Nephrology (SINePe) consensus document on the management of nephrotic syndrome in children: Part I - Diagnosis and treatment of the first episode and the first relapse. Italian Journal of Pediatrics; 43:41

Concomitant cfDNA and EVs isolation from plasma for the accurate detection of

biomarkers in liquid biopsies

Vera Mugoni 1,*, Vito D’Agostino 1,*, Orsetta Quaini 1, Yari Ciani 1, Michela Notarangelo 1,

Antonella Ferro 2, Orazio Caffo 2, Caterina Nardella 1, Francesca Demichelis 1,3

1 Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento,

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

3 Englander Institute for Precision Medicine, New York Presbyterian Hospital- Weill Cornell Medicine, New York, USA

*These authors contributed equally to this work

Tumor cells – derived material that circulates in the peripheral blood, such as cell free DNA (cfDNA)

and extracellular vesicles (EVs), represents an invaluable source of biomarkers from liquid biopsies.

In the last few years, several protocols have been designed to optimize the effective collection and

integrity preservation of cfDNA or EVs for subsequent molecular analysis. However, the simultaneous

isolation of diverse tumor–derived circulating components from a single aliquot of plasma is still

challenging. By starting from few milliliters of plasma, we proved a novel methodological approach for

the isolation of both cfDNA and EVs from the same plasma aliquot. Specifically, we took advantage of

a recently published nickel – based EVs isolation (NBI) protocol1 that allowed the recovery of cfDNA

after EVs isolation. We proved the quality of the isolated cfDNA by bioanalyzer assay and digital

polymerase chain reaction (PCR). On the other side, we assessed the recovery of the EVs by counting

the number of the isolated EVs, detecting the expression of specific biomarkers and analyzing the

nucleic acids contained in the core of the EVs.

By performing a comparison of the data obtained from liquid biopsies of healthy donors’ and breast

cancer patients’, we demonstrated the applicability of combined cfDNA and EVs isolation method for

detecting tumor - derived circulating materials. The efficacy of the combined cfDNA and EVs isolation

approach was also compared with specific methods for cfDNA -only or EVs-only isolation.

Our data unlock new perspectives for the implementation of liquid biopsies in the clinic and the

consequent advanced diagnosis of cancer, monitoring of the disease and the overall progression of

precision medicine in oncology.

References

1. Notarangelo, M. et al. Ultrasensitive detection of cancer biomarkers by nickel-based isolation of

polydisperse extracellular vesicles from blood. EBioMedicine 43, 114–126 (2019).

Extracellular vesicles interaction with model membranes

F. Perissinotto1, B. Senigagliesi1, V. Rondelli2, L. Almasy3, L. Bottyan3, D. Merkel3, H. Amenitsch4, B. Sartori4, K. Pachler5, M. Mayr5, M. Gimona5, E. Rohde5, P. Parisse1, L. Casalis1

1Elettra Sincrotrone Trieste, s.s 14 km 163.5 in Area Science Park, 34149, Italy 2Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano,

Italy 3Budapest Neutron Center, Budapest, Hungary 4Graz University of Technology, Graz, Austria

5GMP Unit, Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria

Extracellular vesicles (EVs) are a potent intercellular communication system. Within a lipidic bilayer, such small vesicles (diameter ranging from 30 to few hundreds nanometers) transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. They have been proposed as biomarkers for several diseases and as optimal candidates for therapeutic applications, due to their small size and specific biological functions. Nonetheless, since their isolation, quantification and biophysical and biochemical characterization are challenging tasks, the understanding of the complex network of EVs/cell interaction is still incomplete. Here we propose a combination of Atomic Force Microscopy and Small Angle X-Ray and Neutron Scattering (SAXS and SANS)) for the careful characterization of isolated vesicles and for the analysis of their interaction with model membranes, in form of liposomes and supported lipid bilayers. Our analysis reveals a strong interaction suggesting the opening and compartmentation of EVs derived bilayers inside supported lipid bilayers.

The saturation degree of membrane lipids modifies the lipid signature of

extracellular vesicles released by HuH7 hepatocarcinoma cells

S. Buratta1 Y. Shimanaka2, Eva Costanzi1, L. Urbanelli1, K. Sagini1, S. Giovagnoli3, F. Morena1, H. Arai2 and C. Emiliani1

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

2 Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan. 3 Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.

Extracellular vesicles (EVs) are membrane-limited particles released by cells during physio-pathological conditions, involved in cellular communication. Recent in vivo and in vitro studies demonstrate that lipotoxic hepatocytes release ceramide-enriched EVs, which activate macrophage chemotaxis [1, 2]. These proinflammatory EVs are considered a link between lipotoxic ER stress conditions and the macrophage recruitment to the liver [1, 2]. So far, lipid profile changes in EVs released under lipotoxic conditions have not been investigated, despite the evidence that EVs shuttle many membrane-derived bioactive lipids playing crucial role in several processes, including inflammation [3]. In this study, we evaluate how the saturation degree of membrane lipids affects the release and the structural and biochemical properties of EVs from HuH7 cell line. In particular, EVs were isolated by differential centrifugation form cell culture media of HuH7 cells treated with lipotoxic palmitate (PA), a cellular model germane to non-alcoholic fatty liver disease, or Δ9 desaturase inhibition (SCD1i). Since membrane lipid saturation induces ER stress, HuH7 cells were also treated with Thaspigargin (Tg), a conventional ER stress inducer, and with oleate (OA), a nontoxic monounsaturated fatty acid, as control. All treatments were performed in serum-free medium containing 0.1% free fatty acids-BSA. EVs were characterized by biophysical analyses and Immunoblotting. Lipids from EVs and their parental cells were analysed by LC/MS-MS. The concentrations and the period of treatment with fatty acids or Tg or SCD1i were chosen on the basis of previous studies on HuH7 cells [1, 2]. Firstly, we confirmed that these experimental conditions did not decrease cell viability and demonstrated that all treatments, except OA, induced ER stress. SEM images showed the characteristic cup-shaped morphology and size of small EVs. NTA analyses demonstrated that treatment of HuH7cells with Tg or PA induced an increase in EV release. Immublotting analyses revealed that EVs from HuH7 cells expressed positive EV markers (CD63, Alix and Flot-1), whose levels were higher in PA-derived EVs. Phospholipid class analyses showed that EVs have a peculiar composition as compared to their parental cells, with a higher percentage of phosphatidylserine accompanied by a lower percentage of phosphatidylethanolamine respect to cells. The comparison between treated-derived EVs and control EVs revealed specific differences in phospholipid class distribution and in the levels of numerous molecular species in each phospholipid class. The raw data from LC-MS/MS were first processed and normalized based on the controls. 217 significant features for cells and 101 for EVs with a threshold of p < 0.05 by one-way analysis of variance (ANOVA) followed by post-hoc analyses using Fisher’s least significant difference method (Fisher’s LSD) and Tukey’s Honestly Significant Difference (Tukey’s HSD) were obtained. Through principal component analysis (PCA), the main lipid variations among the five EVs groups were identified. The Heat Map scores grouped the HuH7-derived EVs in separate clusters. These results suggest that under increased membrane saturation and/or ER stress conditions cells released EVs carrying a particular lipid cargo. Ongoing experiments will demonstrate whether the changes in lipid profile of EVs from treated cells modify their cellular uptake. References [1] Kakazu, E., Mauer, A.S., Yin, M., Malhi, H. 2016. Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner. J Lipid Res. 57, 233-245. [2] Hirsova, P., Ibrahim, S.H., Krishnan, A., Verma, V.K., Bronk, S.F., Werneburg, N.W., Charlton, M.R., Shah, V.H., Malhi, H., Gores, G.J., 2016. Lipid-induced signaling causes release of inflammatory extracellular vesicles from hepatocytes. Gastroenterology 150, 956-967. [3] Sagini, K., Costanzi, E., Emiliani, C., Buratta, S., Urbanelli, L., 2016. Extracellular vesicles as conveyors of membrane-derived bioactive lipids in immune system. Int J Mol Sci. 9, pii: E1227.

Efficacy of sponge-like dressing containing MSC lyo-secretome in a

wound healing murine model

Bari Elia1, Perteghella Sara1,2, Marrubini Giorgio1, Mastracci Luca3,4, Grillo Federica3,4, Grisoli Pietro1, Nardini Marta4, Mastrogiacomo Maddalena5, Sorlini Marzio2,6, Torre Maria Luisa1,2

1 University of Pavia, Department of Drug Sciences, Pavia, Italy 2 PharmaExceed S.r.l., Pavia, Italy

3 University of Genoa, Department of Surgical Science and Integrated Diagnostics, Genoa, IT 4 University of Genoa, Department of Experimental Medicine, Genoa, IT

5 University of Genoa, Department of Internal Medicine, Genoa, IT 6 University of Applied Sciences and Arts of Southern Switzerland, Manno, Switzerland

Adipose mesenchymal stem cells (MSCs)-secretome have been recently proved to be a promising tool for skin regeneration and wound healing applications [1]. Starting from this evidence, we investigate the wound healing efficacy of lyo-secretome-loaded alginate sponge-like membrane in a murine model; freeze-dried MSC-secretome (lyo-secretome) has been obtained by a scalable GMP-compliant production process [2,3]. Supernatants from MSCs were purified by ultrafiltration, added of mannitol and freeze-dried. Lyo-secretome and low viscosity sodium alginate (1% w/v) were solubilized in water and then freeze-dried, obtaining sponge-like membranes (2×106 cell equivalents each one); alginate membranes without lyo-secretome were prepared to be used as control. Two skin lesions per mice were produced: one was treated with the lyo-secretome-based membrane, the other with alginate membrane (control). At 3, 7 and 21 days after surgery, the animals were euthanized and the region of each lesion was sampled for histological analysis; early, mature and total collagen deposition, granulation tissue amount, acute and chronic inflammation have been scored. For each mouse and for each parameter considered, the score of the control lesion was subtracted from the score of the treated lesion, and the median was calculated. For median values lower than 0 the effect is more evident in control-wounds, while for median values higher than 0 the effect is prevalent in treated-lesions. Statistical data analysis was carried out using the Kruskal-Wallis and Mann-Whitney tests. Logarithmic lowering of the bacterial population was calculated for lyo-secretome against the main pathogenic agents of skin infections. Both treated and control wounds are characterized by a significant acute inflammatory process quantifiable on the 3rd day. After 7 days the median value for acute inflammation is significantly higher than 0, while after 21 days, it is not significantly different from 0. An earlier induction of acute inflammation leads to an acceleration of the healing process, which is confirmed, after 7 days, for the treated-wound, by the reduction of the wound area, the prolonged presence of granulation tissue, the greater collagen deposition and the faster collagen maturation. Chronic inflammation was more pronounced in controls at the 3rd day, while after 7 days it was more prevalent in treated-lesions. In the long term, no significant differences for chronic inflammation between treated and control wounds were observed. Finally, lyo-secretome showed anti-bacterial effect against E. coli, K. Pneumoniae, P. aeruginosa and C. albicans. Results suggest that, in this ulcer murine model, lyo-secretome-treated lesions healed faster than the control ones, without any complications or infections. Therefore, lyo-secretome may be a ready-off-the-shelf medicinal product effective for wound healing applications.

References

[1] Collawn SS et al. J Cell Sci Ther 5:165 (2014) [2] Bari E et al. Cells 7(190) (2018). [3] Bari E et al. Nanomedicine 14(6), 753-765 (2019).

LIPOSOMAL VESICLES AS POSSIBLE IRON CARRIERS

M. Battistelli Department of Biomolecular Sciences (DiSB), University of Urbino Carlo Bo, Italy E-mail: michela.battistelli@uniurb.it In the last decades, deficiency of macro- and micronutrients was considered as a serious problem associated with the increase in the human population. Micronutrient malnutrition causes blindness and anemia (even death) in more than half of the world's population, especially among women of reproductive age, pregnant and lactating women, and preschool children. In particular, iron deficiency is one of the most frequent health problems, affecting approximately one third of the entire population. Its request may not be satisfied with a regular diet, due to a low iron bioavailability and to dietary factors that limit its absorption. Iron supplementation is considered a global economic and effective strategy for prevention and control of anemia, in particular, during pregnancy [1]. Therefore, there is the need to develop new experimental protocols to improve its bioavailability. Liposomes [2] are vesicular structures with double-layer lipid membrane, which are proved to be particularly interesting in the biomedical field as a delivery system of pharmaceutically active substances [3]. In this study, ultrastructural analyses have been carried out to verify if liposome dehydration process induces structural alterations of lipid membranes, which could compromise carriers integrity and function. Morphological integrity of Biofer and Lifervit (two new commercial iron carriers), both in liquid form and in dried powder, has been investigated by means of transmission electron microscopy [4]. Moreover, liposome interaction with U937 cell line, a well-known phagocytosis human model, has been studied. Both compounds revealed a good stability and the ability to penetrate into cells, interacting with cytoplasmic organelles without inducing, at least apparently, any ultrastructural damage and toxicity. Therefore, Biofer and Lifervit do not cause cellular damage, and for that they can be considered potential candidates in iron vehiculation. Further studies are in progress to evaluate their interaction with intestinal cell models. References 1. Kamau MW et al. BMC Public Health 2018;18:580. 2. Tamjidi F et al. Innov Food Sci Emerg Technol 2014,26:366-74. 3. Keshari SA et al. Pharmanest, 2014;5:2019-33. 4. Battistelli M et al. Microsc Res Tech. 2018;81:1295-1300.

Exploiting human amniotic fluid stem cells-extracellular vesicles to

unlock endogenous mechanisms of cardiac repair and regeneration

A. Costa1,*, C. Balbi1,2,*, K. Lodder3, T. van Herwaarden3, L. Pascucci4, D. Reverberi5, A. Palmeri6, P. De Biasio6, M.J. Goumans3, A.M. Smits3, L. Barile2, S. Bollini1. *First joint contribution

1Dept. of Experimental Medicine, University of Genova, Genova, Italy;

2Molecular and Cell Cardiology Laboratory, CardioCentro Ticino, Lugano, Switzerland; 3Dept. of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands;

4Department of Veterinary Medicine, University of Perugia, Perugia, Italy; 5Molecular Pathology, IRCCS Ospedale Policlinico San Martino, Genova, Italy;

6Dept. of Obstetrics and Gynaecology, IRCCS Ospedale Policlinico San Martino, Genova, Italy.

The human amniotic fluid stem cell (hAFS) secretome has been previously shown to exert significant cardioprotective effects in vitro on cardiomyocytes and cardiac progenitor cells during doxorubicin exposure[1]; as well, we have recently described hAFS-derived extracellular vesicles (hAFS-EV) endowed with remarkable pro-survival and proliferative paracrine potential in vitro, via the direct transfer of their microRNA (miRNA) cargo[2]. Here, we further analysed the hAFS-EV potential to enhance endogenous cardiac repair and regeneration in a preclinical mouse model of myocardial infarction (MI); we also evaluated gestational age influence on EV from II trimester cells (fetal hAFS) over III trimester hAFS (perinatal hAFS) in terms of yield and miRNA content. c-KIT+ hAFS were isolated as described before1, from left-over samples of amniotic fluid of II trimester amniocentesis (fetal hAFS) and from clinical waste fluid of III trimester scheduled C-section delivery (perinatal hAFS). hAFS were stimulated under 1% O2 to enrich their cell-conditioned medium (hAFS-CM)1,2. hAFS-EV were obtained by serial ultracentrifugation of hAFS-CM2. A preclinical MI mouse model was treated with a single intra-myocardial injection of fetal hAFS-CM versus hAFS-EV, soon after ligation of the left anterior descendant coronary artery; cardiac repair and regenerative mechanisms were evaluated at 7- and 28-days post injury. Fetal versus perinatal hAFS-EV were characterized via BCA assay, nanoparticle tracking analysis, transmission electron microscopy, flow cytometry and by real time qRT-PCR for miRNA expression. Fetal hAFS-CM and hAFS-EV equally enhanced cardiac repair and endogenous reactivation of epicardial progenitor cells with stimulation of resident cardiomyocyte cell cycle re-entry; hAFS-CM retained higher pro-angiogenic potential, while hAFS-EV particularly sustained cardiac function in the long term. We also detected a positive trend of enrichment for regenerative miRNAs (miR-146a, miR-210, miR-199a-3p) within the myocardial tissue at 3 hours following MI and fetal hAFS-EV administration. Preliminary results suggest that perinatal hAFS-EV seem comparable to fetal ones in terms of yield and miRNA content. In conclusion, the hAFS secretome may represent an appealing source for future cardiac regenerative medicine; in particular, fetal hAFS-EV recapitulated most of the hAFS-CM cardio-active paracrine effects, with stronger capacity in rescuing cardiac function, but with lower angiogenic influence. Further experiments to assess whether perinatal hAFS-EV may have the same capacity are currently ongoing. References [1] E. Lazzarini et al., “The human amniotic fluid stem cell secretome effectively counteracts

doxorubicin-induced cardiotoxicity.,” Sci. Rep., vol. 6, no. 1, p. 29994, Sep. 2016. [2] C. Balbi et al., “First Characterization of Human Amniotic Fluid Stem Cell Extracellular

Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential,” Stem Cells Transl. Med., vol. 6, no. 5, pp. 1340–1355, May 2017.

This work contributes to the COST Action CA17116 “International Network for Translating Research on Perinatal Derivatives into Therapeutic Approaches (SPRINT)”, supported by the EU Framework Programme Horizon 2020

Human Corneal Endothelial Cell regeneration induced by the treatment with Mesenchymal Stem Cell-derived Extracellular Vesicles

L. Buono1, M. De Iuliis2,3, B. Bussolati1 and R. Nuzzi2,3

1Department of Molecular Biotechnology and Health Sciences, University of Torino, Turin, Italy 2S.C.U. Ophthalmology Unit, "City of Health and Science" University Hospital, Via Cherasco 23,

10126 Turin, Italy 3Department of Surgical Sciences, University of Turin, Corso Dogliotti 14, 10126

Turin, Italy.

Human corneal endothelial cells (HCECs) have two major functions: barrier and pump function

which help to maintain visual function. However, HCECs are notorious for their limited proliferative

capability in vivo and are therefore prone to corneal endothelial dysfunction that eventually may

lead to blindness. At present, the only method to cure corneal endothelial dysfunction is by

transplantation of a cadaver donor cornea [1]. Due to the global shortage of donor corneas, it is

important to find alternative strategies to induce regeneration of damaged corneal endothelial cells.

The aim of this work was to evaluate the effect of extracellular vesicles deriving from bone marrow-

mesenchymal stem cells (MSC-EVs) on HCECs dysfunction.

Methods: We isolated HCECs from discarded corneas of patients undergoing corneal

transplantation (N=18 patients). Cells were characterized for expression of HCECs markers

(ATP1A1 and ZO-1). We set up two models of HCEC damage induced by serum deprivation, (2%

FBS for 24 hours) or oxidative stress (10uM H2O2 for 3 hours). HCECs were treated with MSC-

EVs. We evaluated cell viability, wound healing ability and the genetic pathways regulated by the

treatment.

Results: The treatment with different doses of EVs (5-20x103 MSC-EV/cell) induced an increase in

the proliferation rate of HCECs in both damage models. The treatment also increased the

percentage of live cells. The wound healing assay showed a faster repair of the wound after

HCECs treatment with MSC-EVs. Three pathways induced by stress conditions, (ATF4,

CASPASE-3 and CASPASE-7) were up-regulated by the induction of the two types of damage.

Treatment with MSC-EVs down-regulated these pathways in the serum deprivation model;

whereas no effect was observed in the H2O2 damage.

These results highlight the already well-known pro-regenerative potential of MS-derived EVs in a

new biological model. MSC-EVs, indeed, induced proliferation, survival and migration of human

corneal endothelial cells, inducing the recovery of damage pathways at RNA level.

References

[1] Tan DT, JK Dart, EJ Holland and S Kinoshita. (2012). Corneal transplantation. Lancet 379:1749–1761.

[2] Yamashita K, Inagaki E, Hatou S, Higa K, Ogawa A, Miyashita H, Tsubota K, Shimmura S. Corneal

Endothelial Regeneration Using Mesenchymal Stem Cells Derived from Human Umbilical Cord. Stem Cells

Dev. 10.1089/scd.2017.0297

A millifluidic advanced in vitro model of glomerular filtration to test the

regenerative effect of Mesenchymal stem cell-derived extracellular

vesicles

Linda Bellucci1, Benedetta Bussolati1

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

Novel therapies are urgently needed to address the rising incidence and prevalence of acute kidney

injury (AKI) and chronic kidney disease (CKD). Recent studies have shown that Mesenchymal stem

cell-derived extracellular vesicles (MSC-EV) therapy improved renal outcomes in several animal

models of AKI and CKD, including ischemia-reperfusion injury, drug/toxin-induced nephropathy,

renovascular disease, ureteral obstruction, and subtotal nephrectomy [1]. However, to better

investigate the molecular mechanisms of pathology and regeneration, we need real human organ–like devices that not only use human cells but also mimic 3D architecture and flow conditions within

real human organs [2]. The aim of our work is to evaluate the regenerative potential of MSC-EV in a

millifluidic in vitro 3D model of glomerulus, used to reproduce pharmacological damage and

reperfusion injury.

Methods: we set a millifluidic in vitro 3D model of glomerular filtration, a three-layers structure

composed by human-GEC and human-POD-SV 40 cell lines, and the basement membrane in

between coated with a matrix of Collagen type IV. The barrier thus formed is set up inside a

bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. Once

designed this device, we reproduced different pathological conditions examining co-cultures in term

of viability, microscopy and filtration. Finally, we tested MSC-EVs as a possible therapy, introducing

them in the dynamic system.

Results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit,

realizing co-cultures of h-GEC and h-POD in bioreactors subject to continuous reperfusion. We set

two types of renal damage: one pharmacologic (doxorubicin) and a hypoxia-reperfusion injury. we

are currently evaluating the regenerative capacities of MSC-EV, entering them directly into the

dynamic co-culture system. Furthermore, we were able to see the presence of EVs inside the cells

by confocal microscopy.

I can conclude that this is a functional model of glomerulus, usable to recreate different disease

models; I observed that EVs are able to penetrate cells (both GEC and Podocytes) in dynamic

conditions, it suggests that they could have an interesting therapeutic potential.

References

[1] Nargesi AA, Lerman LO, and Eirincorresponding A, 2017. Mesenchymal stem cell-derived

extracellular vesicles for kidney repair: current status and looming challenges. Stem Cell Res Ther.

2017; 8: 273.

[2] Kim S and Takayama S, 2015. Organ-on-a-chip and the kidney. Kidney Res Clin Pract. 2015 Sep; 34(3): 165–169.

miRNA enrichment of mesenchymal stromal cells extracellular vesicles

enhance their renal pro-regenerative potential.

Marta Tapparo1*, Stefania Bruno1*, Federica Collino2, Gabriele Togliatto1, Maria Chiara Deregibus3,

Paolo Provero4, Sicheng Wen5, Peter J Quesenberry5 and Giovanni Camussi1

1 Department of Medical Sciences, University of Torino, Torino, Italy 2 Department of Biomedical Sciences and Paediatric Research Institute “Citta della Speranza”, University of Padova,

Padova, Italy. 3 2i3T Scarl, University of Torino, Torino, Italy.

4 Department of Molecular Biotechnology and Health Sciences, University of Torino ,Torino, Italy . 5 Division of Hematology/Oncology, Brown University , Rhode Island Hospital, Providence, Rhode Island.

Mesenchymal Stromal Cells (MSCs) are extensively applied in the field of regenerative medicine and in the last years, many studies demonstrated that their therapeutic effects were mainly exerted by the secretion of bioactive molecules (RNAs, proteins, lipids) that can be directly released in the local microenvironment or packaged in extracellular vesicles (EVs) [1]. These paracrine factors, including EVs, released by MSCs were shown to be effective in the recovery of kidney dysfunction. Different preclinical models of have been used to demonstrate the efficacy of MSC-EVs in the amelioration of acute kidney injury (AKI) and in preventing the progression at the chronic stage [2]. Among the shuttled molecules, miRNAs have a relevant role in the pro-regenerative effects of EVs and are a promising target for therapeutic interventions. We previously demonstrated that the deregulation of miRNAs, by Drosha-knockdown in MSCs, inhibits the regenerative potential of MSCs and of their derived EVs in a murine model of AKI [3] suggesting a critical role of miRNAs in the renal recovery. By bioinformatics analyses, we identified a group of putative miRNAs involved in renal regeneration based on the predicted interactions of miRNAs present inside MSC-EVs with the genes modulated during AKI treatment with MSC-EVs. Moreover, this list was implemented with miR-486-5p that was highly expressed in the exosomal fraction of MSC-EVs [4].Of notice, the selected miRNAs (miR-486-5p, miR-10a-5p, miR-29a-3p) were found to be downregulated in EVs obtained by Drosha knock-down MSCs which were ineffective in glycerol induced AKI [3]. In order to test the role of these miRNA in AKI recovery we set up a method to increase their expression inside MSC derived EVs and to assess the capacity of modified-EV to contribute to renal regeneration in vivo, in a model of acute kidney injury induced by glycerol. To this purpose, MSCs were transiently transfected by electroporation with specific miRNA mimics. miR-127 and miR-486 were more enriched than miR-29a in cells and consequently in their EVs, suggesting differences in efficiency of transfection for different miRNAs. MSC-EVs obtained from electroporated cells were characterized by Nanosight and FACS analysis showing no differences with naïve EVs. In vitro proliferation assay experiments indicated that EVs engineered with miRNAs maintained their pro-proliferative effects. In vivo experiments showed that engineered EVs used at the effective dose (165x106 particles) did not allow to see any significant improvement and for what concerned miR-10a and miR-486 enriched EVs even a worsening was observed. However, when a suboptimal dose was used (82.5 x 106

particles), engineered EVs were more effective than naïve MSC-EVs showing a significant improvement of renal function and morphology. These data demonstrate that it is possible to modify the miRNA content of MSCs and of their EVs and suggest that modify the miRNA EV content could change the dose range for therapeutical intervention. References

1. Heldring, N.; Mager, I.; Wood, M.J.A.; Le Blanc, K.; Andaloussi, S.E. Therapeutic potential of multipotent mesenchymal stromal cells and their extracellular vesicles. Hum Gene Ther. 2015, 26, 506-517.

2. Grange, C.; Iampietro, C.; Bussolati, B. Stem cell extracellular vesicles and kidney injury. Stem Cell Investig. 2017, 16, 4:90.

3. Collino, F.; Bruno, S.; Incarnato, D.; Dettori, D.; Neri, F.; Provero, P.; Pomatto, M.; Oliviero, S.; Tetta, C.; Quesenberry, P.J.; Camussi, G. AKI Recovery Induced by Mesenchymal Stromal Cell-Derived Extracellular Vesicles Carrying MicroRNAs. J Am Soc Nephrol, 2015, 26(10), 2349-2360.

4. Bruno, S.; Tapparo, M.; Collino, F.; Chiabotto, G.; Deregibus, M.C.; Soares Lindoso, R.; Neri, F.; Kholia, S.; Giunti, S.; Wen, S.; Quesenberry, P.; Camussi, G. Renal Regenerative Potential of Different Extracellular Vesicle Populations Derived from Bone Marrow Mesenchymal Stromal Cells. Tissue Eng Part A, 2017, 23, 1262-1273

Canine lyo-secretome for osteoarticular regeneration in veterinary

medicine: pilot-production and in vitro efficacy

Mocchi Michela1, Perteghella Sara1,2, Dotti Silvia3, Villa Riccardo3, Bertocchi Luigi3, Conti Vilma4, Ramoni Roberto4, Del Bue Maurizio5, Sorrenti Milena1, Catenacci Laura1,

Bari Elia1 and Torre Maria Luisa1,2, Grolli Stefano4

1 University of Pavia, Department of Drug Sciences, Pavia, Italy 2 PharmaExceed S.r.l., Pavia, Italy

3 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy 4 Department of Veterinary Medical Science, University of Parma

5 Freelance Veterinary Medical Doctor Regenerative medicine is traditionally related to the use of mesenchymal stem/stromal cells (MSCs) and its safety and efficacy are nowadays proven. Nevertheless, the future goes beyond the MSCs limits maintaining their advantages, moving towards the so-called secretome and/or the extracellular vesicles (EVs). This is well established for human therapy, whereas, in canine veterinary medicine is not fully yet demonstrated (1). In this work we used a validated GMP-compliant process, previously developed for human (2), to produce freeze-dried secretome (lyo-secretome) from canine adipose-mesenchymal stem/stromal cells (AD-MSCs). AD-MSC, tenocytes and chondrocytes have been isolated from a one-year-old Cavalier King female dog and expanded till the third passage. AD-MSC-secretome release was obtained by Fetal Calf Serum starvation and supernatant was purified by ultrafiltration, added with mannitol and then freeze-dried. Physico-chemical characterization and FU quality controls for injectable pharmaceutical dosage forms have been performed: Sterility Classical test, Bacterial Endotoxin (LAL test) and Mycoplasma (NAT test). Lyo-secretome effect on metabolic activity (MTT test) of AD-MSC, tenocytes and chondrocytes has been determined in a dosage range of 0-200x102 cell equivalents/mL of serum-free medium. Results are reported as mean values ± standard deviation, n=3. Lyo-secretome average protein content was 27.46 ± 0.27 µg/mg (BCA-Protein Assay Kit) and average lipid content was 9.52 ± 0.83 µg/mg with (Nile Red fluorescence measure). DSC analysis revealed that the lyophilization process was concluded successfully, without any interactions between components, thus confirming the amorphous nature of secretome. FTIR spectra analysis showed amino picks corresponding to the presence of proteins. Picks were found at 1653 cm-1 due to the presence of amide I, and at 1539 cm-1 for the presence of amide II. Moreover, absorbance bands nearly at 1457 cm-1 and 1380 cm-1 were related to CH2 and CH3 groups, confirming protein and lipid presence. Vesicles’ morphology has been determined by scanning transmission electron microscopy (SEM), confirming that EVs maintained their integrity and spherical structure. Vesicles’ particle size was determined by nanoparticle tracking analysis (NTA), demonstrating sizing lower than 101.1 ± 6.1 nm. Results of lyo-secretome activity on tenocytes, chondrocytes and AD-MSCs indicate, for all the considered cell types, a dose-response improvement, expressed as percentage of Odds Ratio, reaching 85% at higher concentrations of lyo-secretome, considering 10% FBS as a positive control (100% of OR). In conclusion, this work paves the way to lyo-secretome use for canine tendon and chondrocyte regeneration, thanks to a direct activity on tenocytes/chondrocyte as well as on resident tissue MSCs. References 1. Jezierska-Wozniak K; Nosarzewska D; Tutas A; Mikolajczyk A; Oklinski M; Jurkowski MK. Use of adipose tissue as a source of mesenchymal stem cells. Postepy Higieny I Medycyny Doswiadczalnej. 2010;64:326-32. 2. Bari E; Perteghella S; Di Silvestre D; Sorlini M; Catenacci L; Sorrenti M, et al. Pilot Production of Mesenchymal Stem/Stromal Freeze-Dried Secretome for Cell-Free Regenerative Nanomedicine: A Validated GMP-Compliant Process. Cells. 2018;7(11).

Engineered exosomes for the treatment of metastatic malignancies

F. Villa1, R. Tasso1,2, A. Profumo3, S. Bollini1,2, R. Quarto1,2

1 U.O. Cellular Oncology, IRCCS Ospedale Policlinico San Martino, Genova, Italy; 2 Department of Experimental Medicine, University of Genova, Genova, Italy;

3U.O. Biopolymers and Proteomics Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy Tumor metastases represent the main cause of mortality in neoplastic patients. This is due to the still incomplete understanding of biological and molecular mechanisms underlying the induction of the metastatic process, along with the meager effectiveness of current therapeutic strategies only addressing the cellular component of metastases. Therefore, the development of innovative strategies offering specificity and therapeutic efficacy with minimal systemic toxicity represents a major clinical goal. The composition of the extracellular matrix (ECM) generated by the tumor microenvironment represents a neglected source of potential therapeutic targets. In particular, the onco-fetal variant of fibronectin, called ED-B, is abundantly expressed at tumor ECM level and is specifically recognized by the L19 antibody. Indeed, ED-B can be considered an ideal target for immunotherapy, thanks to its abundance, accessibility and stability1. Starting from these assumptions, we propose an innovative approach that exploits the intrinsic properties of exosomes as a cell-derived natural vehicle to safely and specifically transport encapsulated chemotherapeutic agents for the treatment of tumors and metastatic sites. In particular, our experimental strategy involves the use of engineered exosomes as bio-medical "trojan horses", endowed with a portion of the L19 antibody exposed on the membrane through a safe and innovative method. This will make them specifically targeted toward the oncofetal ED-B variant in the neoplastic ECM, thus delivering and releasing the chemotherapeutic drug only after their lysis or internalization with minimal side effects. Briefly the exosomes, derived from human fresh frozen plasma (FFP) of healthy donors are isolated and purified through a standard protocol of serial ultracentrifugation. The chemotherapy drug is uploaded inside the vesicles by electroporation, which is a broadly-used and efficient methodology, since it does not alter the integrity and biological properties of exosomes. As anti-neoplastic molecule we selected Doxorubicin due to its broad anti-tumor spectrum and intrinsic fluorescence, which allows us to easily evaluate encapsulation efficiency in the engineered exosomes by fluorimetry analysis. In order to functionalize the exosome membranes with the L19 antibody, we propose a simple and non-invasive method based on a particular lipid anchor DMPE-PEG-Maleimide, where the DMPE tail integrates into the phospholipid double layer and therefore physically anchors the entire construct on the outer surface of the exosomal membrane. To firmly bind the L19 antibody to the lipid anchor we take advantage of the formation of the strong thioether bond (RSR) resulting from the reaction from the double reactive bond of the maleimide molecule with the thiol groups on the cysteine tail residues of the L19 antibody itself. The specificity and the anti-tumor efficacy of the engineered exosomes will be validated in vitro and in vivo in a mouse model of breast cancer-induced bone metastasis. Our innovative approach could establish the basis for the development of a new therapeutic platform, spendable in different types of solid tumors, that would bring about a significant improvement in the clinical management of cancer and metastatic disease. References 1Borsi L et al, Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin. Int J Cancer, (2002); 102(1):75-85.

Tomato fruit-derived nano vesicles: isolation and characterization

R. Bokka1, T. Silvestre1, A. Ruggiero2, S. Raccosta3, T. Csizmadia4, I. Fiume1, A.P. Ramos-Juarez1,5, M. Moubarak1,6, M. Yahklef1, C. Stanly1, G. Antonucci1, G. Batelli2,

S. Grillo2, M. Manno3, G. Pocsfalvi1 1Extracellular Vesicles and Mass Spectrometry Group, Institute of Biosciences and

BioResources, National Research Council, Napoli, Italy 2Institute of Plant Genetics Group, Institute of Biosciences and BioResources, National

Research Council, Portici, Italy 3Institute of Biophysics, Institute of Biosciences and BioResources, National Research

Council, Palermo, Italy 4Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University,

Budapest, Hungary 5Department of clinical and experimental medicine, University of Campania Luigi

Vanvitelli, Caserta, Italy 6Department of Horticulture, Faculty of Agriculture, University of Damanhour,

Damanhour, Egypt Plants have been shown to secrete extracellular vesicles (EVs) like structures that have been isolate from a number of edible plants such as ginger, grape, sunflower, rice, citrus, etc. Different characterization methods have shown that these structures are similar to mammalian EVs. Other studies showed that plant vesicles are involved in the transport of different cargo materials inside and outside the cell.[1] Especially fruit-derived vesicles raised considerable interest due to their high potential as a resource of future micro and nano-scale delivery vectors for pharmaceutical, nutraceutical and cosmeceutical applications.[2] In nanoTOM, a Horizon 2020 MSCA-IF project, we focus on the isolation of nanovesicles from tomato (Solarium lycopersicum) fruit and apply them as nanovectors for nutraceuticals incorporation. We have chosen tomato fruit because it is one of the most important crops in south Italy and also because it gives a convenient opportunity for future large-scale production of nanovesicles (NVs). Here, we present our first results on the isolation and characterization of different vesicle populations from the fruits two tomato variants, Redsetter and Microtom. To the best of our knowledge no previous studies have been reported in the characterization of tomato fruit-derived vesicles. Thus, we have set–up a method that employs extraction buffers used in food industry and applied it in our ultracentrifugation-based protocols to isolate micro (MVs) and NVs enriched fractions. The protocol was fine tuned to eliminate the co-purifying pectins. For the isolation of different NV sub-populations we employed continuous sucrose gradient ultracentrifugation. Protein biocargo profiles of MVs and NVs were characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). To obtain morphological images we have applied transmission electronic microscopy (TEM). The size distribution and particle concentrations of different NV and MV populations were measured by dynamic light scattering (DLS). To identify vesicular proteins and to compare the biocargo of the two variants shotgun proteomics was applied. SDS-PAGE profiles of MVs and NVs were show different patterns for Redsetter and Microtom. Bioinformatics-based data mining confirms the presence of different transmembrane and lipid-bound as well cytosolic proteins including different transporters and enzymes. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 798576. References [1] Pocsfalvi, G., Turiák, L., Ambrosone, A., Del Gaudio, P., Puska, G., Fiume, I., Silvestre, T. Vékey, K., 2018. Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular vesicle populations. Journal of Plant Physiology 229, 111-121. [2] Zhang, M., Viennois, E., Xu, C., Merlin, D., 2016. Plant derived edible nanoparticles as a new therapeutic approach against diseases. Tissue Barriers 4(2), 1134415.

Mesenchymal stem cell derived extracellular vesicles ameliorate fibrosis in a chronic model of kidney disease

Sharad Kholia1, Maria Beatriz Herrera Sanchez1, Massimo Cedrino1, Elli Papadimitriou1, Marta Tapparo1, Maria Chiara Deregibus1, Maria Felice Brizzi1, Ciro Tetta2 and Giovanni

Camussi1

1University of Torino, Torino, Italy and 2Unicyte AG, Oberdorf, NW, Switzerland

Abstract

Limitations in therapeutic strategies for preventing the progression of chronic kidney disease (CKD) to end stage renal disease has always been a drawback on improving patient

recovery and well-being overall(1,2). As a result, it has become a burden on healthcare

systems worldwide and therefore imperative that a solution be found to alleviate this problem. Aristolochic acid (AA) induced nephropathy is a model of CKD characterised by

inflammation, tubular injury and interstitial fibrosis(2). Extracellular vesicles derived from

mesenchymal stem cells (MSC-EVs) have been reported to exhibit therapeutic properties in

various disease models including kidney injury(3). In the present study, we aimed to

investigate the effects of MSC-EVs on tubular regeneration and interstitial fibrosis in an AA induced mouse model of CKD. NSG mice were injected with MSC-EVs three days after administering AA on a weekly basis for four weeks. Mice inoculated with AA significantly lost weight over the four week period together with deterioration in kidney function. Preliminary data revealed that treatment with MSC-EVs not only prevented weight loss but also reduced tubular necrosis, interstitial fibrosis, as well as curtailed the infiltration of CD45 positive immune cells and fibroblasts which were elevated post AA induced injury. Kidneys were also subjected to molecular analyses to evaluate the regulation of pro-fibrotic genes. MSC-EVs significantly downregulated AA induced upregulation of the pro-fibrotic genes α-Sma, Tgfb1 and Col1a1. Furthermore, fibrosis gene array analyses revealed an upregulation of 35 pro-fibrotic genes in mice injured with AA. Gene array analyses of MSc-EV treated mice are still ongoing. These results therefore suggest that MSC-EVs could play a regenerative and anti-fibrotic role in CKD through the transfer of biologically active cargo that regulates the disease both at a protein and genetic level.

References 1. Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, Eggers P, et al. Prevalence of

chronic kidney disease in the United States. JAMA (2007) 298(17):2038-47. 298/17/2038 [pii];10.1001/jama.298.17.2038 [doi]

2. Debelle FD, Vanherweghem JL, Nortier JL. Aristolochic acid nephropathy: a worldwide problem. Kidney Int (2008) 74(2):158-69. S0085-2538(15)53279-1 [pii];10.1038/ki.2008.129 [doi]

3. Bruno S, Grange C, Collino F, Deregibus MC, Cantaluppi V, Biancone L, et al. Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury. PLoS One (2012) 7(3):e33115. 10.1371/journal.pone.0033115 [doi];PONE-D-11-21867 [pii]

Adipose stem cell-derived extracellular vesicles prevent muscle damage

and inflammatory cell recruitment in a mouse model of CLI: role of

neuregulin 1

Federico Figliolini1, Andrea Ranghino

2, Cristina Grange

2, Massimo Cedrino

1, Marta Tapparo

2,

Claudia Cavallari1, Andrea Rossi

2, Gabriele Togliatto

2, Maria Vittoria Gugliuzza

2, Giovanni

Camussi2 and Maria Felice Brizzi

2

12i3T Scarl University of Turin,

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

Objective: Critical hind limb ischemia (CLI)[1] is a severe consequence of peripheral artery disease. Surgical treatment does not prevent skeletal muscle impairment or improve long-term patient outcomes [2, 3]. The present study investigates the protective/regenerative potential and the mechanism of action of adipose stem cell-derived extracellular vesicles (ASC-EVs) in a mouse model of CLI.

Approach and results: We demonstrated that ASC-EVs exert a protective effect on muscle damage by acting both on tissue microvessels and muscle cells. The genes involved in muscle regeneration were up-regulated in the ischemic muscles of ASC-EV-treated animals. MyoD expression has also been confirmed in satellite cells. This was followed by a reduction in muscle function impairment in vivo. ASC-EVs drive myoblast proliferation and differentiation in the in vitro ischemia/reoxigenation model. Moreover, ASC-EVs have shown an anti-apoptotic effect both in vitro and in vivo. Transcriptomic analyses have revealed that ASC-EVs carry a variety of pro-angiogenic mRNAs, while proteomic analyses have demonstrated an enrichment of neuregulin 1 (NRG1). A NRG1 blocking antibody used in vivo demonstrated that NRG1 is relevant to ASC-EV-induced muscle protection by preventing the recruitment of inflammatory cells and improving vascular growth. Finally, bioinformatic analyses on 18 molecules that were commonly detected in ASC-EVs, including mRNAs and proteins, confirmed the enrichment of pathways involved in vascular growth and muscle regeneration.

Conclusion: This study demonstrates that ASC-EVs display pro-angiogenic and skeletal muscle protective properties that are associated with their NRG1/mRNA cargo. We therefore propose that ASC-EVs are a useful tool for therapeutic angiogenesis and muscle regeneration.

This work was supported by grant No. 071215 from Unicyte to 2i3T. Associazione Italiana per la Ricerca sul Cancro (AIRC) project IG 2015.17630 to MFB

References

[1] Olin JW, White CJ, Armstrong EJ, Kadian-Dodov D, Hiatt WR. Peripheral Artery Disease. N Engl J Med. 2016. doi:10.1016/j.jacc.2015.12.049 [2] Vartanian SM, Conte MS. Surgical Intervention for Peripheral Arterial Disease. Circ Res. 2015;116(9):1614-1628. doi:10.1161/CIRCRESAHA.116.303504. [3] Thukkani AK, Kinlay S, Hospital MB, Divisions C. Endovascular intervention for peripheral artery disease. Circ Res. 2015. doi:10.1161/CIRCRESAHA.116.303503.

VES4US, a Horizon 2020-Future and Emerging Technology project:

use of the invertebrate animal model C. elegans to study the bioactivity of EVs from natural extracts

P. Santonicola1, N. Mazzarella1, M. Paglione1, A. Camera1, S. Visone1, C. Stanly1, A. Cusimano2, D. Romancino2, V. Martorana3, R. Noto3, R. Carrotta3, G. L. Liguori4, A.

Kisslinger6, K. Landfester7, S. Morsbach7, A. Iglic9, V. Iglic9, L. Corcuera8, P. Arosio10, M. Manno3, N. Touzet5, A. Bongiovanni2 and E. Di Schiavi1

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

Napoli, Italy (2) Institute for Biomedical Research and Innovation (IRIB), CNR, Palermo, Italy;

(3) Institute of Biophysics, Palermo, CNR, Italy; (4) Institute of Genetics and Biophysics (IGB), CNR, Napoli, Italy;

(5) Institute of Technology Sligo (ITSligo), Sligo, Ireland; (6) Institute of Experimental Endocrinology and Oncology (IEOS), CNR, Napoli, Italy;

(7) Max-Planck Institute for Polymer Research, Mainz, Germany;

(8) Zabala Innovation Consulting, Navarra, Spain; (9) Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia;

(10) ETH Zurich Institute for Chemical and Bioengineering, Zurich, Switzerland.

VES4US is a new European project funded by the Horizon 2020-Future and Emerging Technology (FET) Open program, which aims to develop an innovative platform for the efficient production of extracellular vesicles (EVs) from a renewable biosource (https://ves4us.eu). We have selected Caenorhabditis elegans as an alternative in vivo model to test in vivo the bio-activity of EVs for the project. C.elegans is increasingly and successfully being used to assess the pharmacological and toxic effects of drugs and natural extracts. In fact it represents a valuable model organism to study human diseases for a number of reasons: the ease and speed of genetic methods to identify and characterize new mutants; the conservation of proteins and pathways with humans, with more than 70% of disease genes presenting a nematode ortholog; a simplified nervous system, which has allowed in many occasions to understand the cellular and molecular mechanisms that underlie neurodegeneration and to develop appropriate leading compounds; the body transparency which offers the advantage of visualizing individual neurons in living worms, while the biological processes take place; finally, the fact that the use of invertebrates has fewer ethical concerns for the public and is highly supported by the E.U. (Resolution on the protection of animals used for scientific purposes, 5/05/2009). C. elegans has also emerged as one of the most convenient and versatile model to evaluate the effects and biological functions of nanomaterials and EVs. We started determine the possible neuroprotective effects of natural extracts and to this aim we are using several models of neurodegenerative diseases (Parkinson, Alzheimer, Huntington, Spinal muscular atrophy) obtained in C.elegans. By doing so we successfully set up C. elegans-based assays to test in vivo the EV features and identified several drugs, natural extracts and natural compounds that prevent the neurodegeneration observed in those models [1, 2]. Many of these natural extracts are enriched in EVs and our aim is to determine whether EVs play a role in neuroprotection. Once isolated, we will test EVs uptake, their biodistribution, the biological activity and toxicology.

Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 801338 and from

Telethon Grant 2016 GGP16203. References [1] Gallotta I, Mazzarella N, Donato A, Esposito A, Chaplin JC, Castro S, Zampi G, Battaglia GS, Hilliard MA, Bazzicalupo P, Di Schiavi E, 2016. Neuron-specific knock-down of SMN1 causes neuron degeneration and death through an apoptotic mechanism. Hum Mol Genet. 2016 Jun 15;25(12):2564-2577. [2] Mazzarella N, Giangrieco I,Visone S, Santonicola P, Achenbach J, Zampi G, Tamburrini M, Di Schiavi E, Ciardiello MA. Green kiwifruit extracts protect motor neurons from death in a spinal muscular atrophy model in Caenorhabditis elegans. Food Sci. Nutr. in press.

Metabolome profiles and bioactivity assessments of different Citrus fruits-derived vesicle populations

Ambrosone Alfredo1, Mariaevelina Alfieri1, Christopher Stanly2, Immacolata Fiume2, Gabriella

Pocsfalvi2

1Department of Pharmacy, University of Salerno

2Extracellular Vesicles and Mass Spectrometry Group, Institute of Biosciences and BioResources,

National Research Council of Italy

Plant-derived nanovesicles are involved in plant cell communication and in cross-kingdom

relationships among plants and animals/fungi. Vesicles isolated from edible plants have been

proposed as promising therapeutic agents for inflammation and cancer [1]. Recently, we described

biophysical and protein biocargo of micro (MVs) and nano vesicles (NVs) fractions isolated by

differential ultracentrifugation from the fruit juices of four Citrus species (C. sinensis, C. limon, C.

paradisi and C. aurantium) [2]. Here, Citrus-derived MVs and NVs were further evaluated for their

ability to influence the growth of tumour cells. Viability assays carried out on a panel of different solid

cancer cell lines showed that they specifically inhibit cancer cell proliferation, without affecting the

growth of non-cancer cells. Vesicles obtained from grapefruit (C. paradisi) had the highest inhibitory

effects on cancer cell proliferation and were selected for subsequent analysis. In order to shed light on

the molecular mechanism underlying the anti-neoplastic activity exerted by MVs and NVs isolated

from C. paradisi, we evaluated the expression of different molecules involved in the proliferation

pathway. Our results showed a modulation of ERK and AKT signalling especially in melanoma cells. In

order to gain knowledge about the biological activity of grapefruit-derived vesicles, we investigated

their metabolomic profile by gas chromatography-mass spectrometry (GC-MS). A total of about 100

peaks were detected from grapefruit samples of which about half were tentatively identified. GC-MS

metabolomics reveals similar but distinct profiles for MV and NV fractions containing different organic

acids (oxalic and citric acid) including amino acids, fatty acids, and carbohydrates as the most

prominent components. This study successfully demonstrates the potential of metabolomic analysis in

non-mammalian EV research. Moreover, it shows that grapefruit vesicles can be a rich source of lead

molecules in drug discovery based on their capability to create unique and diverse chemical

structures. Finally, our findings reveal the molecular signatures of Citrus-derived vesicles and

substantiate the important role that plant edible vesicles may play in new anticancer strategies.

[1]. Zhang, M., Viennois, E., Xu, C., & Merlin, D. (2016). Plant derived edible nanoparticles as a new

therapeutic approach against diseases. Tissue barriers, 4(2), e1134415.

doi:10.1080/21688370.2015.1134415

[2]. G. Pocsfalvi, L. Turiák, A. Ambrosone, P. del Gaudio, G. Puska, I. Fiume, T. Silvestre, K.Vékey

Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular

vesicle populations, Journal of Plant Physiology, Volume 229, 2018, Pages 111-121,

Therapeutic role of stem cell derived extracellular vesicles in

hyperglycemia-induced endothelial injury

C. Gai1,#, M.A.C. Pomatto1, M. Tapparo1, T. Lopatina1, G. Camussi1, M.F. Brizzi1

1 Department of Medical Sciences, University of Torino, Italy

# presenting author

Background: Adipose and bone marrow-derived mesenchymal stem cells are two populations of multipotent adult stem cells with immunosuppressive, anti-inflammatory, and regenerative properties. It has been previously described that extracellular vesicles (EVs) derived from stem cells possess pro-regenerative and pro-angiogenic abilities. Hyperglycemia is a pathological condition affecting diabetic patients. Long-term effects of hyperglycemia are endothelial dysfunction and vascular lesions leading to diabetic microangiopathy and impaired wound healing.

The aim of the present study was to evaluate whether stem cell-derived EVs would inhibit endothelial cells microangiopathy-like dysfunctions induced by hyperglycemia and would ameliorate wound healing process. The second objective was to perform a bioinformatic analysis of the miRNA and protein content of stem cell-derived EVs in order to identify molecules involved in their biological effect.

Methods: We set up an in vitro hyperglycemic model by culturing human microvascular endothelial cells in hyperglycemic constant or intermittent conditions for 7 days, to mimic a chronic damage. At day 5, endothelial cells were incubated with adipose and mesenchymal stem cell-derived EVs or vehicle alone for 48 hr. At day 7, we evaluated apoptosis, oxidative stress, migration, and capillary-like formation ability on Matrigel. Moreover, we induced diabetes in mice by streptozotocin administration and we generated diabetic ulcer by a mechanic punch. Then we treated the wound with both kind of EVs and followed up the wound closure for 15 days. Finally, we analyzed EV miRNA and protein cargo and performed a bioinformatic analysis.

Results: Intermittent and constant high glucose models significantly decreased endothelial cell proliferation, increased the percentage of apoptotic cells, promoted oxidation of intracellular proteins, and reduced cell migration and capillary-like structure formation. Treatment with both kinds of EVs significantly restored proliferation, inhibited apoptosis and oxidation, and restored migration and capillary-like formation in vitro. Preliminary results on diabetic mice models showed an increased rate of wound closure with adipose stem cell -derived EV and a mild amelioration with mesenchymal stem cell -derived EV. Protein and miRNA carried by EVs significantly target pathways involved in cell cycle and proliferation, motility and angiogenesis, and apoptosis.

Conclusions: The results of the present study demonstrate that adipose and bone marrow mesenchymal stem cell-derived EVs may inhibit the endothelial dysfunction induced by high glucose concentration, which mimics diabetic microvascular injury. Probably, these effects are due to the transfer of their miRNA and protein cargo to recipient cells. However, the bioinformatic analysis highlighted that both kind of EV share a pattern of miRNA and proteins, which explain their regenerative effect on endothelial damage. Some proteins and miRNA are differentially expressed among the two kind of EV. This partially explain the different results obtained in vivo on diabetic

ulcers, suggesting that the therapeutic effect of stem cells derived EV can differ depending on their cargo and on the condition of target cells.

GMP validation of production process for freeze-dried secretome from bone marrow mesenchymal stem/stromal cells

Bari Elia1 , Katia Mareschi 2,3, Elena Marin3, Aloe Adamini,3,Laura Castello3, Alessandra

Mandese3,Perteghella Sara1,4, Torre Maria Luisa1,4, Ivana Ferrero3.  

1 University of Pavia, Department of Drug Sciences, Pavia, Italy 2 Department of Public Health and Pediatric Sciences, Medical School, University of Turin, Turin, Italy. 3 Pediatric Onco-Hematology, Stem Cell Transplantation and Cell Therapy Division, City of Science and Health of Turin, Regina Margherita Children Hospital, Turin, Italy. 4 PharmaExceed S.r.l., Pavia, Italy MSC-secretome is composed of a heterogeneous pool of bioactive agents, including cytokines, chemokines and growth factors, partially encapsulated in a phospholipidic bilayer, as extracellular vesicles (EVs) [1]. Although MSC-secretome efficacy has been widely established in numerous preclinical models, limitations regarding its large-scale good manufacturing practice (GMP) isolation, standardization, characterization and formulation still remain an issue. Here, we validated a GMP integrated process of ultrafiltration and lyophilization able to isolate and freeze-dry Bone Marrow MSC-secretome. The aseptic validation process was projected to validate the process in GMP conditions as illustrated in the flowchart.

MSCs were cultivated for 2 passages, detached after the supernatant collection and analyzed for cell number, viability, identity, and sterility according to GMP Pharmacopeia requirements [2-4]. Lyo-secretome average protein content was 14.98 ± 0.330 µg/mg (BCA-Protein Assay Kit) and average lipid content was 4.62 ± 0.266 µg/mg with (Nile Red fluorescence measure). Vesicles’ particle size was determined by nanoparticle tracking analysis (NTA), demonstrating sizing lower than 105.6 ± 7.1 nm. The product was not cytotoxic up to concentrations of 25 mg/ml (on human fibroblasts, chondrocytes and nucleus pulposus cells by MTT assay) and was blood compatible up to 150 mg/ml. In conclusion, this work describes the production process of clinical-grade MSC-secretome to be used in regenerative medicine for different clinical applications. References [1] Bari E., et al. Cells. 2018;7(11). [2] The Rules Governing Medicinal Products in the European Union, Volume 4, Good Manufacturing Practice, Medicinal Products for Human and Veterinary Use. Ann Rev Mar Sci 2001, 15. [3] European Pharmacopoeia: European Directorate for the Quality of Medicines & HealthCare (EDQM). 9th edition; 2017.[4] Guidelines on Good Manufacturing Practice specific to Advanced Therapy Medicinal Products, 22/11/2017.

ExoLat: a nutraceutical formulation enriched with beneficial exosomes from milk for relieving intestinal inflammation

S.Mecocci1, L.Pascucci1 G.Chillemi2, P. Del Porto3 , F.Ascenzioni3, S.Capomaccio1,

A.Valentini2 and K.Cappelli1.

1Dipartimento di Medicina Veterinaria, Università degli studi di Perugia, Perugia, Italy. 2Dipartimento per l’Innovazione Nei Sistemi Biologici, Agroalimentari e Forestali, Università della Tuscia, Viterbo, Italy. 3Dipartimento di Biologia e Biotecnologie C. Darwin, Università di Roma la Sapienza, Roma, Italy.

Extracellular vesicles (EVs) act as signal mediators between distant cells and/or tissues through antigenic stimuli and epigenetic modifications mediated by miRNAs carried within the EV. They act as signal mediators between cells and/or tissues through different mechanisms involving their complex cargo [1]. Exosomes (EXs) are a class of EVs that can be obtained also from dietary sources, for example milk. Milk EXs can enter the intestinal cells by endocytosis and protect their labile cargos against harsh conditions in the intestinal tract [2]. Our overall goal is to use milk Exs as beneficial agents to improve inflammatory conditions that are typical of IBD (Inflammatory bowel disease) in order to create a nutraceutical product (ExoLat) that could be eventually commercialized. We have two significant advantages by the use of milk: being vastly available and being food, than it would be easier to obtain administration approval. The achievement of this goal requires extend characterization of milk Exs at morphological, molecular and functional level. In the first step of the study, we evaluated methods for isolation, since milk is a complex matrix particularly enriched in proteins that make this step critical. We excluded commercial kits for scarce recovery and low purification as well as ultracentrifugation (100,000xg) that, after evaluation with CONAN assay, delivered low purity vesicles pellet caused by protein contamination. We finally chose to execute some preliminary centrifugation steps aimed at deleting fat globules on top of the solution and pelleted material. Then, we ultra-centrifuged the supernatant on sucrose gradient of fifteen 350 l fractions from 2M to 0,4M on 700 l 2,5M. Afterward, we recovered phases of 450 l and we subjected them to 100.000xg ultracentrifugation for exosomal pellet collection. Western blot analysis was performed to localize Exs within the gradient testing the different fractions for the EX-associated markers (CD81, CD63, TSG101, Calnexin). The fractions from 6 to 9 were found to be those containing the EXs, on which nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis are in progress. This will allow us to obtain a structural characterization of these vesicles and to evaluate the presence of contaminant molecules such as proteins. At the same time, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis are being carried out in order to assess morphological features and confirm the expression of specific surface markers. After that, the purified exosomes will be submitted to the molecular characterization of the nucleic acids content by next generation sequencing. References

[1] Golan-Gerstl R, Elbaum Shiff Y, Moshayoff V, Schecter D, Leshkowitz D, Reif S.

Characterization and biological function of milk-derived miRNAs. Molecular Nutrition & Food

Research 2017;61:1700009. doi:10.1002/mnfr.201700009.

[2] Benmoussa A, Lee CHC, Laffont B, Savard P, Laugier J, Boilard E, et al. Commercial Dairy Cow Milk microRNAs Resist Digestion under Simulated Gastrointestinal Tract Conditions. J Nutr 2016;146:2206–15. doi:10.3945/jn.116.237651.

Thrombogenicity of extracellular vesicles of different origins and

settings

Carla Tripisciano1, René Weiss1, Tanja Eichhorn1, Victor U. Weiss2, Viktoria Weber1

1Christian Doppler Laboratory for Innovative Therapeutic Approache in Sepsis, Danube

Univesity Krems, Krems, Austria 2 Vienna University of Technology, Department of Analytical Chemistry, Vienna, Austria

The ability to trigger or support blood coagulation has been recognized for extracellular vesicles

(EVs) depending on the state and type of the cell they originate from. We have previously

reported on the role of phosphatidylserine (PS)-exposing vesicles released by platelets from

healthy donors in supporting thrombin generation [1]. Here, we compare EVs from unstimulated

cells derived from blood products and from in vitro settings regarding their pro-coagulant activity.

Blood-derived EVs were harvested from medical grade platelet and red blood cells units, and

monocytic EVs were obtained from cell cultures by sequential centrifugation. After removal of

cells and cell debris (2,500 g), supernatants were centrifuged at 20,000 g to collect vesicle-

containing pellets. EV fractions were characterized with respect to protein content, particle size

and number (nanoparticle tracking analysis), exposure of PS and cell markers (flow cytometry;

lactadherin, LA, for PS, CD41 for platelets, and CD235a for red blood cells) and expression of

other proteins, such as CD63, Alix, α-Actinin1 (Western blotting, WB). Pro-coagulant activity of

EVs was assessed by measuring the generation of thrombin over time (TGA, Technoclone).

Blood product-derived EVs supported thrombin generation to a higher extent than monocytic

EVs. In platelet and red blood cell-derived EVs, no indication for the presence of coagulation

factors IX and X was found by WB. Removal of soluble factors by size exclusion

chromatography (qEV Izon Science) increased the pro-coagulant ability of EVs, due to sample

standardization according to protein content. Digestion of surface-exposed proteins by mild

protease treatment had no negative impact on the thrombogenicity of EVs from blood products,

showing that no membrane-associated factor was responsible for their enhanced pro-coagulant

potential. By comparing all EV fractions after protease treatment, thrombin peaks levels were

comparable. Only when samples were standardized to counts of LA+ EVs (flow cytometry),

platelet EVs generated significantly higher amounts of thrombin than monocytic EVs.

In conclusion, EVs enriched from different settings contain different levels of soluble

contaminants which, however did not contribute to thrombin generation in our experimental

settings. Membrane-associated proteins did not seem to be responsible for enhanced

coagulation, suggesting quantitative differences in the number of exposed PS residues.

Alternatively, the presence of smaller EVs in blood-derived samples may lead to an

underestimation of the overall PS exposing vesicles.

Supported by the Christian Doppler Society (Christian Doppler Laboratory for Innovative

Therapeutic Approache in Sepsis)

References

[1] Tripisciano, C., Weiss, R., Eichhorn, T., Spittler, A., Heuser, T., Fischer, M.B., Weber, V., 2017. Different Potential of Extracellular Vesicles to Support Thrombin Generation: Contributions of Phosphatidylserine, Tissue Factor, and Cellular Origin. Scientific Reports 7:6522.

Ves4US, a Horizon 2020-Future and Emerging Technology project: Quality management based approach

Giovanna L. Liguori (1), Christopher Stanly (2), Giorgia Adamo (3), Samuele Raccosta(4) Darja Božič (5), Antonella Cusimano (3), Daniele Romancino (3), Rita Carrotta (4) , Vincenzo Martorana (4), Rosina Noto (4), Katharina Landfester (6), Blanca Rodriguez (7), Svenja Morsbach (6), Paolo Arosio (8), Elia Di Schiavi (2), Laura Corcuera (7), Nicolas Touzet (9), Mauro Manno (4), Gabriella Pocsfalvi (2), Ales Iglic

(6), Veronika Iglic (6), Antonella Bongiovanni (3) and Annamaria Kisslinger (10)

(1) National Research Council of Italy (CNR), Institute of Genetics and Biophysics (IGB), Napoli, Italy;

(2) CNR, Institute of Biosciences and BioResources (IBBR), Napoli, Italy; (3) CNR, Institute for Biomedical Research and Innovation (IRIB), Palermo, Italy;

(4) CNR, Institute of Biophysics, Palermo, Italy; (5) Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia;

(6) Max-Planck Institute for Polymer Research, Mainz, Germany;

(7) Zabala Innovation Consulting, Navarra, Spain; (8) ETH Zurich Institute for Chemical and Bioengineering, Zurich, Switzerland;

(9) Institute of Technology Sligo (ITSligo), Sligo, Ireland; (10) CNR, Institute of Experimental Endocrinology and Oncology (IEOS), Napoli, Italy

In recent years, the scientific world has been clearly experiencing a revolution: the attention of the scientific and social community is not focused solely on the final results, but also on the process utilized and other related issues such as the reliability, safety, and efficacy of the discoveries, the research integrity and the efficient and effective use of resources. Quality management might provide key tools to ensure valuable, robust and dependable outcomes, within a framework of best practice. Nowadays, different quality models have been developed in different countries and institutions and a quality approach represents an added value for research project proposals submitted for funding (1). Here we report the quality management approach developed inside the European project funded by the Horizon 2020 - Future and Emerging Technology (FET) Open program, entitled “Extracellular vesicles from a natural source for tailormade nanomaterials”-“Ves4US” (https://ves4us.eu). The Ves4US consortium is a highly interdisciplinary and interconnected network that involves 9 teams from different institutes, covering different research disciplines in a joint effort to develop an innovative platform for the efficient production of extracellular vesicles (EVs) from a natural source, and their use as safe and efficient tailor-made nanocarriers. Due to their high level of heterogeneity, specific issues arise when working with EVs, that need to be addressed to assure reliability and reproducibility of scientific data as well as the possibility of manufacturing at large scale. Therefore, one of the tasks of the project is specifically devoted to the definition of a Quality management system (QMS) fundamental for the identification and diffusion of guidelines and best practice (2), standardization of procedures, reproducibility and validation of the scientific results. We will also take advantage of Quality methodologies such as Failure Mode and Effect Analysis (FMEA) and Design of Experiments (DoE) (3) specifically applied in research for the analysis and optimization of scientific procedures. The Ves4US QMS might be an operating model for quality management of research projects based on the development of radically new technologies of the future, in which interdisciplinarity, innovation and interconnection are key features.

Supported by the European Union’s Horizon 2020 research and innovation program (GA No 801338)

References: (1) Bongiovanni A., Colotti G., Liguori G.L., Di Carlo M., Digilio F.A., Lacerra G., Mascia A., Cirafici A.M., Barra

A., Lanati A. and Kisslinger A. Applying Quality principles and Project Management methodologies in biomedical research: a public research network's case study. (2015) AQA 20, 203–213. (2) Digilio F.A., Lanati A., Bongiovanni A., Mascia A., Di Carlo M., Barra A., Cirafici A.M., Colotti G., Kisslinger

A. Lacerra G., Liguori G.L. Quality-based model for life sciences research guidelines. AQA 21,221–230 (2016). (3) Mancinelli S., Zazzu V., Turcato A., Lacerra G., Digilio F.A., Mascia A., Di Carlo M., Cirafici A.M., Bongiovanni

A., Colotti G., Kisslinger A., Lanati A. and Liguori G.L. Applying Design of Experiments methodology to PEI toxicity assay on neural progenitor cells. Mathematical Models in Biology Springer (2015).

Tiotropium inhibits proinflammatory microparticle generation by human bronchial and endothelial cells

Tommaso Neri1, Valentina Scalise1, Ilaria Passalacqua1, Chiara Sanguinetti1,

Stefania Lombardi2, Laura Pergoli3, Valentina Bollati3, Roberto Pedrinelli1, Pierluigi Paggiaro1, Alessandro Celi1

1Centro Dipartimentale di Biologia Cellulare Cardiorespiratoria, Università di Pisa

2SSD Analisi Chimico Cliniche, Ospedale Apuane Toscana Nordovest, Massa 3Epidemiology, Epigenetics and Toxicology Lab, Università degli Studi di Milano

Tiotropium is a muscarinic antagonist that reduces the risk of acute exacerbations of chronic obstructive pulmonary disease, possibly through an as yet incompletely characterized anti-inflammatory activity. We hypothesized that muscarinic activation of bronchial epithelial cells and endothelial cells causes the release of proinflammatory microparticles (MP) and that tiotropium inhibits the phenomenon. MP generation was assessed by a functional assay, by flow cytometry and by NanoSight technology. Immortalized bronchial epithelial cells (16HBE) and umbilical vein endothelial cells (HUVEC) were treated with acetylcholine. Intracellular calcium concentration, extracellular regulated kinase phosphorylation and chemokine content in the conditioned media were assessed by commercial kits. Acetylcholine (Ach; 1mM) causes significant MP generation that is completely inhibited by tiotropium (Tio;50pM) [for 16HBE 0.42±0.02(baseline), 0.72±0.39(Ach), 0.17±0.02(Tio+Ach) mM PS; for HUVEC 0.40±0.04(baseline), 1.07±0.49(AcH), 0.23±0.10(Tio+Ach) nM PS]. These results were confirmed by FACS analysis and Nanotrack analysis (data not shown). MP generated by Ach-stimulated cells increase the synthesis of proinflammatory mediators in an autocrine fashion [for 16HBE: IL-8 (46,63±15,74 pg/mL vs 188,2±49,56 pg/mL for unstimulated and MP-stimulated 16HBE cells, respectively; p<0.05 paired t-test; for HUVEC: IL-8 (118±33 pg/mL vs 240±75 pg/mL) and MCP-1 (503±702 pg/mL vs 1422±1874 pg/mL), for unstimulated and MP-stimulated HUVEC, respectively; p<0. 05 for both comparisons, paired t-test]. Ach-induced upregulation of MP generation is significant inhibited by an inhibitor of extracellular regulated kinase phosphorylation (PD98059; 1μM) and by a phospholipase C inhibitor (U73211; 1μM) [For 16HBE: 0,21±0,07(baseline), 0,34±0,10(Ach), 0,21±0,06(PD98059+Ach), 0,16±0,06(U73122+Ach); for HUVEC 0,29±0,05(baseline), 0,46±0,15(Ach), 0,28±0,05 (PD98059+Ach), 0,26±0,06(U73122+Ach)]. Tiotropium blocks both extracellular regulated kinase phosphorylation [for 16HBE: 0,445(baseline) 0,723(Ach), 0,490(Tio+Ach) ratio total ERK/pERK; for HUVEC: 0,576(baseline) 0,821(Ach), 0,547(Tio+Ach) ratio total ERK/pERK] and calcium mobilization [For 16HBE: 0.46±0.01(baseline), 0.64±0.04(Ach), 0.56±0.03(Tio+Ach) [Ca2+](RFU); for HUVEC 0.37±0.01(baseline), 0.53±0.02(Ach), 0.33±0.01(Tio+Ach) [Ca2+](RFU]], consistent with the hypothesis that the drug prevents MP generation through inhibition of these critical pathways. These results might contribute to explain the effect of tiotropium in reducing acute exacerbations of chronic obstructive pulmonary disease.

Active enzyme sialidase NEU3 is present on small extracellular vesicle

surface

Lucia Paolini1,4, Miriam Romano1,4, Paola Fusi2, Matilde Emma Forcella2, Flavia Orizio1, Sara Busatto3, Annalisa Radeghieri1,4, Roberto Bresciani1, Paolo Bergese1,4, Eugenio Monti1

1Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, Brescia, Italia

2 Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano Bicocca, Milano, Italia 3Department of Transplantation Medicine; Department of Physiology and Biomedical Engineering,

Mayo Clinic, Jacksonville, FL, USA 4CSGI - Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Firenze, Italia

Sialidases are glycohydrolases that remove terminal sialic acid residues from oligosaccharides, glycolipids, and glycoproteins. The plasma membrane associated sialidase NEU3 is involved in the fine-tuning of sialic acid-containing glycans directly on the cell surface. The enzyme is engaged in both physiological and pathological events such as cell to cell communication, cell to extracellular matrix interaction and signal transduction [1]. Considering the relevance of Extracellular Vesicles (EVs) in a variety of biological events we decided to analyze the presence of NEU3 in two different EV subtypes (small and medium EVs) in the cellular medium of HeLa cell engineered to express, upon induction, the murine form of NEU3. Using Western blot, dot blot analysis and measuring sialidase activity, we have demonstrated that the active enzyme NEU3 is associated with small EVs and localizes on the external leaflet of these nanovesicles [2]. Different studies demonstrated that NEU3 is highly expressed in neoplasms including renal, ovarian and colorectal cancer [3] thus we investigated the topology of human endogenous NEU3 on EVs in human epithelial colorectal adenocarcinoma cells line (Caco-2). NEU3 activity on the surface of small EVs is expected to modify their external composition with relevant consequences on their glycocalyx formation, modeling and net charge, therefore influencing their recognition by target cells. The presence of NEU3 on the small EV surface represent a useful marker for the detection of these nanovesicles and a tool for improving our understanding of the biology of these important extracellular carriers in physiological and pathological conditions. References [1] Miyagi, T., Yamaguchi K., (2012) Mammalian sialidases: Physiological and pathological roles in cellular functions. Glycobiology 22(7), 880–896, doi:10.1093/glycob/cws057. [2] Paolini, L., Orizio, F., Busatto, S., Radeghieri, A., Bresciani, R., Bergese, P., Monti, E. (2017) Exosomes Secreted by HeLa Cells Shuttle on Their Surface the Plasma Membrane-Associated Sialidase NEU3. Biochemistry. 5,56(48), 6401-6408, doi: 10.1021/acs.biochem.7b00665. [3] Forcella, M., Mozzi, A., Stefanini, F. M., Riva, A., Epistolio, S., Molinari, F., Merlo, E., Monti, E., Fusi, P., and Frattini, M. (2018) Deregulation of sialidases in human normal and tumor tissues. Cancer Biomark 21, 591-601, doi: 10.3233/CBM-170548.

Characterisation of human umbilical cord mesenchymal stromal

cells and their derived extracellular vesicles as a first approach to

assess effects on monocyte-derived macrophages

T. Borges da Silva1, C. David1, A. Taylor1, B. Wilm1, N. Liptrott1, P. Murray1

1University of Liverpool, Institute of Translational Medicine, United Kingdom

The study of new therapeutic strategies for kidney disease treatment is of crucial importance,

as the only available options now are dialysis and kidney transplantation. In this context,

macrophages can be a valuable tool to promote tissue regeneration, as they can be polarised

towards an anti-inflammatory state in which they stimulate angiogenesis, extracellular matrix

remodelling and cell proliferation [1]. Mesenchymal stromal cells (MSCs) have been reported

to shift macrophage polarisation from a pro-inflammatory to an anti-inflammatory phenotype

[2]. Nevertheless, the administration of cells can trigger immune reactions and the risk of cell

differentiation and potential tumour formation is always present. Therefore, this study aims to

investigate the role of extracellular vesicles (EVs) derived from human MSCs in macrophage

polarisation. MSCs were cultured in four different types of supplementation (conventional

foetal bovine serum, exosome-depleted foetal bovine serum and presence or absence of basic

fibroblast factor for both serums) and the metabolic state of cells was assessed through Cell

Titer-Glo Assay. No differences were found in the morphology of cells, their metabolic state

and the expression of mesenchymal surface markers. The protocol for EV isolation consisted

in depriving cells from serum for 24, 48 and 72 hours and used several centrifugation steps in

order to remove cell debris and dead cells and ultracentrifugation. There was no difference

regarding size of particles between groups, but concentration of vesicles was clearly impacted

by period of serum deprivation. Also, the expression of EV markers was affected by slight

changes in the protocol, suggesting that the process of isolation of EVs can change the

expression of important markers. Preliminary results of co-incubation of extracellular vesicles

with monocyte-derived macrophages during 48 and 72 hours suggest that EVs may enhance

the expression of CD14 in M1-like macrophages. In conclusion, differences in medium do not

seem to affect cell morphology and metabolism, nevertheless FGF impacts the expression of

mesenchymal surface markers. The population of EVs obtained relies not only in the medium

used to culture cells, but also greatly on the protocol of isolation. More experiments are needed

in order to determine the potential immunomodulatory effects of MSC-derived EVs in human

monocyte-derived macrophages.

Supported by the Wellcome Trust

References

1. Williams, T.M. et al. (2010) Macrophages in renal development, injury, and repair. Semin Nephrol 30 (3), 255-67. 2. de Witte, S.F.H. et al. (2018) Immunomodulation By Therapeutic Mesenchymal Stromal Cells (MSC) Is Triggered Through Phagocytosis of MSC By Monocytic Cells. Stem Cells 36 (4), 602-615.

Ovarian Aging is Associated with Enhanced Release of Exosomes in

Human Follicular Fluid influencing the miRNA profile

R. Battaglia1, P. Musumeci2, M. Ragusa1, D. Barbagallo1, M.E. Vento3, M. Purrello1, C. Di Pietro1

1Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics G.

Sichel, University of Catania, Catania, Italy.

2Department Physics and Astronomy, University of Catania, Catania, Italy

3IVF Unit, Cannizzaro Hospital, Catania, Italy

Advanced reproductive aging determines transcriptome and proteome modifications in the ovarian

follicles resulting in a dramatic decrease of oocyte competence and reproductive potential. In the

female, in fact, not only the number but also the quality of oocytes progressively declines with the

increase of maternal age.

Previous experiments from our laboratory demonstrated that human follicular fluid (FF) contains

exosomes and identified miRNA exosome cargo able to control pathways involved in meiosis

resumption and epigenetic modifications [1]. Besides these observations, we hypothesized that

exosome-mediated transport represents a communication mechanism among follicular somatic

cells and between somatic cells and the oocyte, able to drive follicle development and oocyte

maturation [2]. Recently, we investigated miRNA expression profiles in human FF, comparing the

transcriptome of older and younger women, to explore the relationship between the alteration of

pathways affecting oocyte quality in reproductive aging and the altered miRNA expression. We

found that miRNA expression profile in FF shows significant differences related to aging that could

partially depend on the different number of exosomes present in FF from older women. In fact, we

demonstrated that follicular cells from older women secrete twice as many exosomes than younger

ones. First, we calculated the different distribution of miRNAs inside FFs, without considering the

distribution of miRNAs within the exosomes (global Fold Change, gFC), then the different

distribution of miRNAs inside the exosomes (exosomal Fold Change, eFC). Comparing gFC and

eFC, we found 46 miRNAs having the same expression trend, 18 miRNAs exclusively up-regulated

in FF (gFC) and 34 miRNAs exclusively down-regulated inside exosomes (eFC). We conclude that

the increase of exosome number in FF leads to a different miRNA expression profile in FF of aged

women that could explain the reduced oocyte competence related to the aging. Moreover, the

increase of exosome secretion in older women, causing not only a different FF miRNA signature in

aging, but also of the different molecules (e.g., mRNA, long non-coding RNAs) that are cargo of FF

exosomes, could explain why still today reproductive aging represents a complex biological

problem.

References

1) Santonocito M, Vento M, Guglielmino MR, Battaglia R, Wahlgren J, Ragusa M, Barbagallo D, Borzì P, Rizzari S,

Maugeri M, Scollo P, Tatone C, Valadi H, Purrello M, Di Pietro C. Molecular characterization of exosomes and their

microRNA cargo in human follicular fluid: bioinformatic analysis reveals that exosomal microRNAs control pathways

involved in follicular maturation. FertilSteril. 2014,102:1751-61.

2) Di Pietro C. Exosome-mediated communication in the ovarian follicle. J Assist Reprod Genet. 2016; 33:303-311.

Review.

Citrus clementina derived vesicles: A quick glance on membrane

transporters

C. Stanly1, R. Bokka1, M. Moubarak1,3, G. Antonucci1,4, A.P. Ramos-Juarez1,5, I. Fiume1, L. Turiák2 and G. Pocsfalvi1

1. EVs-MS Research Group, Institute of Biosciences and BioResources, National Research Council of Italy, Italy

2. MS Proteomics Research Group, Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Hungary

3. Faculty of Agriculture, University of Damanhour, Egypt 4.Department of Biology, University of Federico II, Napoli, Italy

5.Department of Clinical and Experimental Medicine, University of Campania Luigi Vanvitelli, Caserta, Italy

Fruit-derived vesicles are a topic of interest in various fields of EVs research, specifically in drug delivery studies due to their efficient cellular uptake and lack of adverse immune reaction.[1],[2] Fruit vesicles are extremely complex and contain both intra and extracellular vesicles classes. Recent progress in mass spectrometry-based proteomic analyses of secreted nanovesicles has resulted in the identification of several vesicular proteins which provide us with essential clues towards understanding the mechanisms involved in vesicle cargo sorting, membrane trafficking and biogenesis.[3] In this study, we aim to describe a method for isolating nanovesicles (NVs) from the sac-cells of Citrus clementina using a double-sucrose/D2O cushion ultracentrifugation with differential solubilisation and characterize their protein content. The vesicle size-distribution and particle concentration were determined using Nano-particle tracking analysis. Purified vesicles were lysed and their protein biocargo was profiled using SDS-PAGE and shotgun proteomics. In-solution digestion based proteomics and bioinformatics studies were done to study the protein biocargo and its functional characteristics, respectively. On further investigation into our bioinformatics data, we found that much of the protein content of these vesicles corresponds to membrane transporters that facilitate transport of molecules across the membrane. Moreover, high number of different enzymes including hydrolases, oxyreductases were also found in these vesicles. The proteomic results obtained in this study were compared to our previous study on vesicles isolated from four different citrus species[1], C. sinensis, C. limon, C. paradisi and C. aurantium and also with the existing EV datasets.

References

[1]. Pocsfalvi, G. et al. Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular vesicle populations. J. Plant Physiol. (2018). doi:10.1016/j.jplph.2018.07.006

[2]. Stanly, C., Fiume, I., Capasso, G. & Pocsfalvi, G. Isolation of exosome-like vesicles from plants by ultracentrifugation on sucrose/deuterium oxide (D2O) density cushions. in Methods in Molecular Biology (2016). doi:10.1007/978-1-4939-3804-9_18

[3]. Pocsfalvi, G. et al. Mass spectrometry of extracellular vesicles. Mass Spectrometry Reviews (2016). doi:10.1002/mas.21457

Hypoxia and ototoxic drugs modulate exosome biogenesis and release in cochlear pericyte

Elisa Ghelfi1, Fernanda Estevan2, Emil Millet1, Magda Bortoni1, Yohann Grondin1, Rosalinda

Sepulveda1,2, Rick Rogers1

1Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA. 2Universidade de Sao Paulo, Sao Paulo, Brazil

3Universidad Autonoma de Nuevo León, Monterrey, Mexico. Introduction: Hearing loss is a global problem. The World Health Organization estimates that worldwide 21% in the working adult and 15.7% of children and young-adults are at risk for hearing loss, due to occupational and recreational unsafe level of noise-exposure [1, 2, 3]. Spiral ligament (SL) pericytes are potential therapeutic targets for intervention to the inner ear. SL pericytes surround the endothelial cells (EC) in the cochlear microvasculature in a 1:1 ratio, the highest EC/pericytes ratio in the body. SL pericytes control important functions such as the maintenance of the Blood Labyrinth Barrier, signaling pathways to ECs and modulation of microvessels wall permeability. Oxidative stress has been established as the key mechanism in the etiology of sensorineural hearing loss by noise and ototoxic drugs. Gentamicin (GTM) and cisplatin (CIS) are ototoxic drugs used worldwide for the treatment of infections and cancer respectively. The drugs are also used as a model for studying noise induced hearing loss (NIHL). Hypoxia induced by loud noise has been related to hearing disorders. Extracellular vesicles (EVs) have been detected in cochlear explants from rats treated with ototoxic drugs GTM and CIS. Aim: We investigated whether ototoxic conditions modify pathways of EVs biogenesis, release and EVs concentration by SL pericytes. Methods: SL pericytes from

ImmortoMouse® were incubated for 24 hours with GTM, CIS and intermittent hypoxia (IH). EVs were isolated using differential centrifugation. NanoTraker Analysis was used for quantification. Marker proteins quantification was determined by immunoblotting. Results: Ototoxic drugs significantly reduced cell number and EVs released in the cell culture supernatant. IH doubled the concentration of EVs released without increasing cell number. Cell viability and cell proliferation were affected by GTM and CIS but not IH. ESCRT proteins Alix and syntenin were unchanged while the protein Vps4, involved in ending the vesicles formation, decreased in IH. ARF6 a protein playing a role in EVs biogenesis and microvesicles release was significantly modified by ototoxic drugs and IH. Rab11a involved in MVBs transport and fusion to the plasma membrane increased in GTM an IH treated cells. Conclusion: Ototoxic drugs and IH modulate MVBs’ ESCRT-dependent pathway, ESCRT-independent pathway, EVs biogenesis, EVs release and concentration in cell culture. Supported by: Office of Naval Research (ONR) Grants: N00014-16-1-2411/2966/2822 References:

[1] The Lancet, Editorial, vol 387; 2016), [2] World Health Organization Media Center 2015; accessed October 2018 http://www.who.int/mediacentre/news/releases/2015/ear-care/en/ ). [3] Nelson DI, Nelson RY, Concha-Barrientos M, Fingerhut M.; Am J Ind Med. 2005;48(6):446-58.

APOPTOTIC BODY: A PARTICULAR MEMBER OF THE

EXTRACELLULAR VESICLE FAMILY?

S. Salucci, S. Burattini, E. Falcieri, M. Battistelli

Department of Biomolecular Sciences (DiSB), University of Urbino Carlo Bo, Italy E-mail: michela.battistelli@uniurb.it All the organisms produce extracellular vesicles: normal and dying cells release, indeed, membrane-

bound vesicles such as exosomes, microvesicles, and apoptotic bodies [1, 2].

These can encapsulate small portions of the subjacent cytosol, creating a heterogeneous

population of phospholipid-walled vesicles, that play an important role in intercellular

communication, immune response, angiogenesis and signal transduction, in both physiological and

pathological conditions [3].

These structures are referred to as extracellular vesicles, but also as microparticles, microvesicles,

microsomes, lipid vesicles, apoptotic blebs or exosomes, depending on the basis of their biogenesis

or release pathways.

EVs are characterized by their size (30–100nm for exosomes, 100–500nm diameter for the larger

microvesicles, and 500nm-2µm for apoptotic bodies), by their cells of origin including

megakaryocytes, platelets, red blood cells, endothelial cells and others, and by their intravesicular

contents. Their inner contents depend upon their cells of origin and can include double stranded

DNA, mRNA, microRNA, adhesion integrins, growth factors, protease inhibitors, P-selectin

glycoprotein ligand-1 (PSGL-1), ceramides, metabolites and other.

Exosomes, whose diameters range from 40 to 120 nm, are formed by the fusion between

multivesicular bodies and the plasma membrane.

Microvesicles bud directly from the plasma membrane, show a 100 – 500nm size, and contain

cytoplasmic material.

Dying cells, however, release 500 nm–2 μm vesicular apoptotic bodies, that can be more abundant

than exosomes or MVs under specific conditions and appear variable in size, structure and

composition.

While micro-vesicles can operate as ‘safe containers’ mediating inter-cellular communication,

apoptotic bodies appear after the disassembly of an apoptotic cell into subcellular fragments. The

formation of apoptotic bodies is an important process downstream apoptotic cell death, considered

a hallmark of apoptosis. In This work we evaluated the morpho-functional features of apoptotic

bodies.

References

1. Raposo G, Stoorvogel W.. J Cell Biol. 2013;200:373–383.

2. Maas SL, Breakefield XO, Weaver AM. Trends Cell Biol. 2017;27:172–188.

3. Hauser P, Wang S, Didenko VV. 2017;1554:193-200

Inhibition of Cholesterol transport: implications in caveolin-1 trafficking between plasma membrane and exosomal secretion Daniela Peruzzu1, Zaira Boussadia2, Lucia Bertuccini3, Federica Fratini3, ,Paola Matarrese1, Maria Carollo3, Mario Falchi5, Francesca Iosi3, Carla Raggi6, Isabella Parolini7, Maria Cristina Gagliardi1, Alessandra Carè1, Massimo Sargiacomo4, Katia Fecchi1

1Center for Gender-Specific Medicine, 2National Center for Drug Research and Evaluation, , 3Major equipments and core facilities, 4National Center for Global Health, 5Center AIDS Research and Development, 6National Center for the control and evaluation of medicine 7Dept. Oncology and Molecular Medicine; Italian National Institute of Health Rome, Italy.

Abstract

Caveolin-1 (Cav1), is a fundamental constituent of caveolae, whose functionality and structure are

strictly dependent on cholesterol. In the present work, the U18666A inhibitor was used to study the

role of cholesterol in Cav-1 trafficking, investigating its implications in the endolysosomal

degradative-secretory system in a human metastatic melanoma cell line (WM266-4). We

demonstrated that U18666A causes a shift of Cav-1 from the plasma membrane to the

endolysosomal compartment without altering caveolae resistance to TX-100. The endolysosomal

system is involved, through the Multi Vescicular Bodies (MVBs), in the formation and release of

exosomes (EVs). We found that the inhibitor induces an increase in the production of EVs with

chemical-physical characteristics similar to control EVs but with a different protein composition

(lower expression of Cav1 and increase LC3II). Furthermore, we found that U186666A induces a

significant increase of mitochondrial ROS production and an increase of mitochondrial membrane

potential. Finally, functional assays demonstrated a lower migration and invasion capacity of

treated cells. Taken together, these results indicate that the blockage of cholesterol transport,

determining the internalization of Cav1, may modify the exosomes secretory pathways through an

increased fusion between autophagosomes and MVBs, inducing a secretion of the contents of

these organelles to maintain cellular homeostasis and ensure correct cellular functionality.

EV-associated DNA: a role in inflamm-aging?

Massimiliano Bonafè1,2, Sabrina de Carolis1,2, Gianluca Storci1,2

1 Department of Experimental, Diagnostic and Specialty medicine, University of Bologna, Italy;

2 Center for applied biomedical research, CRBA, S. Orsola-Malpighi Hospital, Bologna, Italy;

ABSTRACT

Aging is associated with the progressive activation of the systemic inflammatory response that has

been named inflamm-aging [1]. In the recent past, a wealth of literature put forth the primary role of

cytoplasmic/extracellular DNA in systemic inflammation and inflammatory diseases [2]. Moreover,

several investigations reported on the presence of genomic and mitochondrial DNA into

extracellular vesicles (EVs) [3, 4]. The biological significance of such EV-associated DNA is still

matter of debate: some authors propose the hypothesis that it ensues the discharge of cytoplasmic

nucleic acids that otherwise would trigger the inflammatory/interferon response, while others

propose a role of EV-associated DNA in in the horizontal transfer of genetic material [5, 6]. In the

recent past we have explored the presence of DNA of various origins (genomic, mitochondrial and

viral DNAs) in EVs obtained from cell lines and biofluids [7, 8] Our data pinpoint that EV DNA cargo

can be transferred to target cells and that EV-DNA is able to modulate the inflammatory/interferon

response. On one hand we observed that the amount of telomeric EV-DNA correlates with the

onset of an anti-inflammatory profile in recipient cells. On the other we found that the presence of

viral (e.g HPV) DNA in EV is associated with the induction of the inflammatory response.

Intriguingly, even circular DNA, enriched in ribosomal sequences, takes part to the EVs DNA

payload. Our data show that EV-associated DNA is biologically active and may play a role in

inflamm-aging that currently represents the major liability factor for a wide range of age-related

diseases.

[1] Franceschi C, Bonafè M, et al. Inflamm-aging. An evolutionary perspective on immunosenescence Ann N Y Acad Sci. 2000 Jun;908:244-54. [2] Storci G, De Carolis S, Olivieri F, Bonafè M. Changes in the biochemical taste of cytoplasmic and cell-free DNA are major fuels for inflamm-aging. Semin Immunol. 2018 Dec;40:6-1 [3] Thakur BK, Zhang H, Becker A, et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 2014June;24(6):766–769 [4] Guescini M, Genedani S, Stocchi V, et al. Astrocytes and Glioblastoma cells release exosomes carrying mtDNA. J Neural Transm (Vienna). 2010January;117(1):1–4 [5] Takahashi A, Okada R, Nagao K, et al. Exosomes maintain cellular homeostasis by excreting harmful DNA from cells.Nat Commun. 2017 May 16;8:15287 [6] Asada K, Ito K, Yui D, Tagaya H, Yokota T. Cytosolic Genomic DNA functions as a Natural Antisense Sci Rep. 2018 Jun 4;8(1):8551. doi: 10.1038/s41598-018-26487- [7 De Carolis S, Pellegrini A, Santini D et al., Liquid biopsy in the diagnosis of HPV DNA in breast lesions Future Microbiol. 2018 Feb;13:187-194 [8]. Sansone P, Savini C, Kurelac I, et al Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer. Proc Natl Acad Sci U S A. 2017 Oct 24;114(43):E9066-E9075.

Role of extracellular vesicles in the muscle niche during aging

Andrea Cioccoloni, Serena Maggio, Paola Ceccaroli, Emanuela Polidori, Vilberto Stocchi and Michele

Guescini

Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via I Maggetti, 26, 61029 Urbino, Italy

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

Sarcopenia is a disease where muscle results inflamed, and satellite cell (SC) pool is reduced. SCs are

involved in muscle fiber remodelling/repair and self-maintenance. During aging, SCs lose their

functions, probably due to altered levels of soluble factors present in their “niche”. Raising data suggest that SCs modulate the niche releasing these factors free or packaged within extracellular

vesicles (EVs). SCs lie within a microenvironment defined “niche,” where they secrete molecules to communicate with surrounding cells. These factors can be free or packaged within extracellular

vesicles (EVs) which are very conserved membranous structures with a size of 30-100 nm, their

membrane is very similar to cell membrane and their content consists of different kind of

macromolecules (nucleic acids, cytokines and transcriptional factors).

During aging SCs lose their functions, probably due to altered levels of soluble factors present in the

muscle “niche”. Recent studies have reported several changes in EV secretion and composition during aging thus also EVs have been suggested to be part of the SASP.

The aim of this work was to investigate the role of EVs in the alteration of satellite cell niche during

aging, therefore we cultured two C2C12 cell clones, defined as “old” and “young”, for more than 40 and less than 15 passages, respectively, to stimulate the senescent process in the old one. In our

conditions, old myoblasts showed delayed and more disorganized myotube formation, and lower

myogenic index than the young ones. This picture was confirmed by gene expression analysis that

showed a derangement of the transcription profile of the genes involved in proliferation (Pax7,

ccnd1), differentiation (MyoG, Myf-6, Id1, Id3) and inflammation (IL-6, IKB, TNFα) in the old myocytes. Furthermore, it was studied the effect of the EVs secreted by old cells on differentiating

young myoblasts and on immune cells, which are commonly present in the niche. Our results

suggest that old EVs have a negative impact on the myogenic process, and, more interesting, we

observed that all treated cells exhibit a significant inflammation process.

EXOSOMES AND MICROVESICLES UNDER THE LENS OF THE

SUPER RESOLUTION MICROSCOPY

G. Anselmi1

1Alfatest srl, Rome, Italy

E-mail: giulia.anselmi@alfatest.it

Exosomes and microvesicles play an important role in many biological processes.

From understanding their function and origin, it is likely that a number of

exosome-based diagnostic platforms will follow. In addition, because of their role

in cell-to-cell communication and their inherent ability to cross biological barriers

and be internalized within the cell with a high degree of specificity, exciting

possibilities for exosomes as therapeutic agents and drug delivery vehicles

emerge. In order to realize their full potential, adequate tools to study their

function and structure must be available. To date, the rate of advancement within

the field has been rapid, but with this increased understanding it has become

apparent that current tools for studying these exciting biological structures have

significant limitations.

We hereby introduce the concept of single-molecule and super-resolution

techniques and their application within vesicle research. Improving access to

high-resolution, high-content imaging data within the study of extracellular

vesicles is a primary goal of the Nanoimager. We discuss how these exciting

developments in imaging can expand the knowledge and overcome the barriers

with traditional characterisation techniques such as Flow Cytometry (FC),

Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing

(TRPS), as well as purification techniques such as ultra-centrifugation, and

affinity and size exclusion chromatography.

The complex cargo of Streptomyces coelicolor extracellular vesicles

T. Faddetta1, G. Renzone2, E. Rimini1, F. Amato3, C. Miccichè3, R. Caruana4, L. Modica de Mohac5, G. Nasillo4, G. Buscarino3,4, S. Agnello3,4, M. Licciardi1,4, L. Botta6, A. Palumbo

Piccionello1, A. Scaloni2, G. Gallo1,4, A. M. Puglia1

1 Department of Biological Chemical and Pharmaceutical Sciences and Technology (STEBICEF), University of Palermo, Italy

2 Proteomic and Mass Spectrometry Laboratory, ISPAAM, CNR (National Research Council), Napoli, Italy

3 Department of Physics and Chemistry, 3 University of Palermo, Italy 4 Advanced Technologies Network (ATeN Center), University of Palermo, Italy

5 Department of Internal Medicine and Medical Specialties (DIMIS), University of Palermo, Italy 6 Department of Civil, Environmental, Aerospace, Materials Engineering (DICAM), University of

Palermo, Italy

Extracellular vesicles (EVs) are produced by cells in all domains of life. Bacterial EVs carry

macromolecules and metabolites and are involved in cell communication, competition and survival.

EVs have been characterized in many Gram-negative bacteria and, more recently, in several Gram-

positive bacteria.

Streptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-

positive bacteria that undergoes a complex life cycle (vegetative mycelium, aerial mycelium and

spores) and produces an enormous repertoire of bioactive metabolites and extracellular enzymes

[1]. This bacterium, when grown on solid medium, produces macroscopic blue-pigmentated

exudates, known as blue-droplets, containing EVs [2]. In our laboratory a density gradient

ultracentrifugation-based protocol was applied to purify EVs from S. coelicolor liquid cultures. The

presence of EVs was revealed by Transmission Electron Microscopy, Scanning Electron Microscopy

and Atomic-Force Microscopy. Moreover, Dynamic Light Scattering analysis confirmed the particle

sizes ranging between 100 and 200 nm and Raman microscopy highlighted that actinorhodin

antibiotic is associated with EVs. Electrophoretic analyses revealed that vesicular cargo includes

proteins and nucleic acids. Liquid Chromatography Tandem-Mass Spectrometry analysis revealed

a total of 166 protein species involved in cell metabolism, molecular processing and transport.

Interestingly, some proteins such as cytochromes, bacterioferritin and the spore-associated protein

SapA are inside vesicles and some other proteins, such as ribosomal and stress response proteins,

are associated to EV surface as suggested by protease accessibility assay coupled with proteomics.

Moreover, EVs contain metabolites such as antibiotics, vitamins, aminoacids, aminoacid precursors

and components of carbon metabolism. RNAseq and DNA sequencing are ongoing to determine the

nucleic acid vesicular cargo.

This study, revealing the presence of EVs in liquid medium cultivations for the first time, may shed

light on the role of the extracellular vesicles in the physiology and development of S. coelicolor.

References [1] Bentley S. D et al., (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417 (6885):141-147. [2] Schrempf H. and Merling P. (2011) Extracellular Streptomyces vesicles: amphorae for survival and defence. Microbial Biotechnology 4(2): 286–299.

Protocol optimisation to study the glycoproteome of urinary exosomes

E. Barigazzi, L. Santorelli, M. Pitto and F. Raimondo

School of Medicine and Surgery, University of Milano - Bicocca, Milan, Italy

Protein N-glycosylation is a biologically important post-translational modification (PTM), often altered on both glycosites and glycans. Its aberrant changes are closely associated with several diseases, including renal dysfunction and urologic malignancies [1]. In this context, Urinary Exosomes (UE), nanovesicles representing a molecular snapshot of the parental cell, are enriched in glycosylated renal proteins; in particular, their surface presents a complex glycoprotein pattern that can be considered a specific glycan signature [2]. While it is widely known the connection between UE and the pathophysiologic status of the origin cell, the characterisation of their glycoprotein content requires the development of new analytical procedures. Current MS approaches for the UE glycoproteome investigation are limited by the low amount of UE proteins; furthermore, the presence of Uromodulin (UMOD), the most abundant glycoprotein in urine, hampers the UE recovery and the study of the other glycoproteins. These evidences arise a double technical challenge: primarily, the reduction of UMOD contamination, and secondly the search of a suitable strategy to investigate the UE glycoproteome. Thus, we tested different experimental procedures, starting from normal urine specimens, in order to improve the sample preparation and achieve a better MS analysis. First, we performed the UE isolation through multistep differential centrifugations, trying to reduce UMOD presence by exploiting some chemical-physical features of this protein (oxidative state, solubility, size). The best compromise between the UE yield and the UMOD depletion emerged to be the addition of ZnSO4 to frozen urine specimens prior to UE purification. Then, we set up an optimised N-Glyco-FASP (Filter Aided Sample Preparation), a recently developed MS approach based on lectin-enrichment able to capture UE glycoproteins [3]. We compared the efficiency of different types of filters (Microcon vs Amicon) in order to improve the UE peptides and glycopeptides recovery. Despite the small amount of starting sample (urine volume < 40 ml; UE proteins < 100 μg), we confirmed the correct glycopeptides enrichment, highlighting that our combined approach is suitable for the study of the UE glycoproteome. It would be interesting to glyco-characterise UE from patients affected by different type of kidney pathologies (Renal Cell Carcinoma and Idiopathic Nephrotic Syndrome), to bring out the remarkable impact of this PTM within the pathological mechanism of these diseases. References: [1] Taniguchi, N., Kizuka, Y., 2015. Glycans and cancer: role of N-glycans in cancer biomarker,

progression and metastasis, and therapeutics. Advances in Cancer Research. 126: 11-51. [2] Williams, C, Royo, F., Aizpurua-Olaizola, O., Pazos, R., Boons, G., Reichardt, N., Falcon-Perez, J. M.,

2018. Glycosylation of extracellular vesicles: current knowledge, tools and clinical perspectives. Journal of Extracellular Vesicles. 7(1): 1442985.

[3] Deeb, S., Cox, J., Schmidt-Supprian, M., Mann, M., 2014. N-linked Glycosylation Enrichment for In-

depth Cell Surface Proteomics of Diffuse Large B-cell Lymphoma Subtypes. Molecular and Cellular Proteomics. 13(1): 240-251.

Moonlighting proteins in Extracellular Vesicles: do Hsp70 and

Alpha-enolase share common localization and function?

Giovanni Perconti, Francesca Costantini, Daniele Romancino, Antonella Bongiovanni, Patrizia

Rubino, Agata Giallongo

Istituto per la Ricerca e l'Innovazione Biomedica, CNR, Palermo

Moonlighting proteins (MPs) exhibit more than one physiologically relevant function at distinct cellular

and extracellular sites. A large number of MPs are cytoplasmic chaperones and enzymes that can

play a different role on the cell surface. Among them Hsp70 and alpha-enolase, a chaperone and a

glycolytic enzyme respectively, are both intracellular and surface MPs commonly overexpressed in

different tumor types and present in extracellular vesicles (EVs). Surface alpha-enolase acts as

receptor for plasminogen and this enolase activity has been directly linked to tumor invasion and

metastasis [1, 2], likewise surface Hsp70 is a recognized marker of tumor cells [3]. In breast cancer

cells, Hsp70 is a molecular partner of alpha-enolase and we have shown that the protein-protein

interaction is involved in the surface localization of the enzyme, which lacks a canonical localization

signal [4]. Moreover, in breast cancer cells both proteins are overexpressed in the plasma membrane

in response to inflammatory stimuli, namely LPS and EGF [4,5], and are released in EVs when cells

are exposed to cytokines, such as TGF-β1, CCL2, and TNF-α [5]. It has been shown that alpha-

enolase-exposing EVs enhance cancer cell migration and invasion [5]. On the other hand, Hsp70 on

the surface of EVs elicits different immune responses, mainly immunosuppression, contributing to

tumor immune escape. In particular, the interaction between cell toll-like receptor 2 (TLR2) and

Hsp70 exposed on the tumor-derived small EVs or exosomes results in the activation of myeloid-

derived suppressive cells (MDSC) and the consequent inhibition of adaptive and innate immunity

[6]. Similarly, EVs from microorganisms, like protozoa or bacteria, modulate host immune response

[7]. Despite both alpha-enolase and Hsp70 surface localizations are promoted by factors associated

with tumor progression, and their exteriorization contributes to cell invasion and immunoregulation,

the functional relationship between these proteins in EVs is still unknown. We propose that Hsp70

and alpha-enolase at the surface of EVs may cooperate to modulate the activity of the immune

system using overlapping mechanisms. To this end, we generated a cellular model ectopically

expressing epitope-tagged alpha-enolase and Hsp70 and we verified the presence of these proteins

in the EVs, than we performed preliminary experiments to investigate the localization of alpha-

enolase and Hsp70 in the vesicles lumen and/or surface. These data may contribute to elucidate the

mechanisms by which EVs transfer materials or induce signaling in immune cells and to ascertain

novel EV surface markers.

References

[1] Hsiao, K. C. et al. 2013. Surface α-enolase promotes extracellular matrix degradation and tumor metastasis and represents a new therapeutic target. PLoS One. Jul 19; 8(7) [2] Principe M et al 2015. Targeting of surface alpha-enolase inhibits the invasiveness of pancreatic cancer cells. Oncotarget. May 10; 6(13) [3] Stangl, S. et al. 2011. Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc. Natl. Acad. Sci. U. S. A. 108, 733–8 [4] Perconti et al. 2017. Pro-invasive stimuli and the interacting protein Hsp70 favour the route of alpha-enolase to the cell surface. Sci Rep. Jun 19; 7(1) [5] Didiasova et al. 2015. STIM1/ORAI1-mediated Ca2+ Influx regulates Enolase-1 exteriorization. J Biol Chem. May 8; 290(19) [6] Jessica Gobbo et al. 2015. Restoring anticancer immune response by targeting tumor-derived exosomes with a HSP70 peptide aptamer. J Natl Cancer Inst. Nov 22; 108(3) [7] Li S. et al. 2018. Extracellular Vesicles secreted by Neospora caninum are recognized by Toll-Like Receptor 2 and modulate host cell innate immunity through the MAPK signaling pathway. Front Immunol. Jul 24; 9:1633

Myocardial hypoxic stress mediates functional cardiac microvesicle release

A. Anselmo1, D. Frank2, L. Papa3, C. Viviani Anselmi3, F. Clemente1, E. Di Pasquale3,5, R.

Hinkel6,7, R. Kozlik-Feldmann7, F. Freudenthal7, M. Mazzola,8, M. Miragoli3,9, M. Vacchiano3,10, S. Serio3, S. Betocchi PhD11, M.C. Ferrari 12, A. Eusebio12, A.A. Puca13, V. Christiansen4, S. Freitag-Wolf4, N.Frey4, A. Dempfle14, A. Moretti7, K.L Laugwitz7, C. Briguori14, G. Esposito11, C. Kupatt6,7,

G.Condorelli3,5,10

1 Flow Cytometry Core, Humanitas Clinical and Research Center, Rozzano, Milan, Italy. 2DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Kiel, Germany.

3Department of Cardiovascular Medicine, Humanitas Clinical and Research Center, Rozzano, Milan, Italy 4University Hospital Schleswig- Holstein, Campus Kiel, Germany

5 Institute of Genetics and Biomedical Research, National Research Council of Italy, Rozzano, Milan, Italy. 6 DZHK (German Centre for Cardiovascular Research), partner site Munich, Germany. 7 Medizinische Klinik und Poliklinik Technical University of Munich, Munich, Germany.

8 Division of Cardiac Surgery, Department of Surgery, Università degli Studi di Verona, Verona, Italy. 9 Department of Medicine and Surgery, University of Parma, Parma, Italy.

10 Humanitas University, Rozzano, Milan, Italy. 11Department of Cardiology, University Federico II, Naples, Italy.

12 Department of Orthopedics and Rehabilitation, Humanitas Research Hospital, Milano, Italy. 13 Department of Medicine and Surgery, University of Salerno, Baronissi, Italy.

14 Hemodynamic Unit, Clinical Mediterranea, Naples, Italy.

Microvescicles (MVs) are 100–1000-nm-sized particles, budded from the plasma membrane and

released by many cell types into the plasma. Increased MV release has been associated with human

disease, e.g., atherosclerosis, but their role in cardiopathies is unknown. Here, we describe a flow

cytometry-based method for the characterization of cardiomyocyte (CM)-derived MVs, using

CD172a, an antigen expressed on human CMs. CD172a+ MVs contained the cardiac-specific

protein troponin T and two cardiac-enriched microRNAs, miR-1 and miR-133. Myocardial stress

was found to be a major determinant of MV release from CMs: indeed, hypoxia induced the release

of MVs from in vitro human CMs, and patients with different types of cardiopathies had increased

levels of CD172a+

MVs in plasma. Functionally, CD172a+

MVs isolated from stressed CMs had a

positive inotropic effect on unstressed CMs through ceramide mediated signals.

Moreover, aortic stenosis patients with higher plasma MVs levels had a more favourable prognosis

to transcatheter aortic valve replacement than those with lower plasma levels. In summary, we have

defined a method for isolating and measuring CD172a+

cardiac MVs, demonstrated the release

mechanism and providing evidence for their prognostic potential in cardiac disease.

Plasma exosomal-miRNAs in lung cancer are associated to pro-

tumorigenic and immunosuppressive microenvironment

Orazio Fortunato1, Viviana Bornaghi1, Mattia Boeri1, Massimo Moro1, Ugo Pastorino2 and Gabriella Sozzi1

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

INTRODUCTION: Exosomes are small vesicles released by all cell types that contain a specific subset of functional biomolecules, such as microRNAs (miRNAs). It has widely demonstrated that exosomal miRNAs (exo-miRNAs) from cancer cells can manipulate the tumor microenvironment regulating the gene expression of recipient cells. We previously identified a three levels risk classifier (MSC) based on 24 plasma-miRNAs associated with lung cancer development and prognosis. The aim of this study was to investigate the potential role of exo-24 miRNAs as mediators of pro-tumorigenic features in the lung microenvironment. MATERIAL AND METHODS: Exosomes were isolated from plasma of heavy-smoker individuals

with high (MSCpos) or low (MSCneg) risk of lung cancer by differential centrifugation method. The expression of exosomal markers (CD9, CD63, CD81 and Alix) was assessed by western blot and flow cytometry, and miRNA levels was analyzed by dPCR. In vitro and in vivo analysis were used to assess the biological effect of plasma exosomes on different type of recipient cells, such as endothelial cells (HUVECs) and human macrophages. Purity of exosomes isolated was confirmed by sizing using NTA and TEM analysis and expression of exosome-enriched proteins. RESULTS: Concerning plasma samples, exosome fraction of 27 plasma samples of individuals with different plasma MSC risk profiles (19 heavy-smokers volunteers and 8 lung cancer patients), were analyzed and the presence of all the 24 miRNAs composing the MSC within exosomes were confirmed. Moreover, by comparing miRNAs CTs in plasma and in their respective CT in exosomes, a good degree of correlation (Pearson’s R=0.90, p for Pearson’s R <0.001) was observed, indicating that miRNAs expression in exosomes could reflect miRNA levels in total plasma. Interestingly, miR-133a, miR-197, miR-320 and miR-660 were enriched in exosomes fraction whereas miR-451 and miR-140-3p were present mostly outside exosomes. Accordingly to plasma modulation using dPCR, we found 9 miRNAs upregulated, 9 miRNA down-regulated, 1 miRNA not modulated in high-risk individuals (MSCpos) resulting in a 80% of correlation between plasma and exosomes. Interestingly, exo-MSCpos increase the ability of HUVEC to form tubular structures compared to exo-MSCneg. Furthermore, exo-MSCpos promote vessels leakness by the analysis of rhodamine-dextran. In vivo co-culture of exo-MSCpos-treated HUVEC with A549 lung cancer cells resulted in an increase of tumor growth compared to exo-MSCneg-treated HUVEC. Modulation of mir-126 in HUVEC demonstrated that this miRNA is implicated in the proangiogenic modulation of HUVEC phenotype by exo-MSCpos. Exo-MSCpos subjects were added on cultured macrophages and induced M2 polarization, evaluated as increase of IL-10 expression and the presence of more CD163 and CD206 positive cells. Conditioned medium of exo-MSCpos treated macrophages stimulated also the formation of tubular structures by HUVEC on matrigel suggesting a possible role in angiogenesis. Mir-320 over-expressing macrophages recapitulate exosomes-treated phenotype suggesting the involvement of this miRNAs in immunosuppressive modulation of these cells. CONCLUSION: These data show that plasma exosomes of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of extracellular vesicles in lung cancer development.

MiR-214 in tumor-stroma cell interactions

F. Orso1,2,3, F. Virga1,2,4, D. Dettori1, 2, M. Paradzik1,2, D. Baruffaldi1,2, A. Massa1, 2, K Mareschi5,6, M. Van Wynendaele1, 2, E. Bolli1,2, F. Cavallo1,2, M. Forni1, L. Salmena7, F. Fagioli5,6, M. Mazzone4, P.

P. Pandolfi8, D. Taverna1,2,3

1Molecular Biotechnology Center (MBC), 2Dept. Molecular Biotechnology and Health Sciences and 3Center for Complex Systems in Molecular Biology and Medicine at the University of Torino, Italy;

4Lab of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium;5Department of Public Health and Pediatric Sciences, Medical School, University of Turin,

Turin, Italy; 6Pediatric Onco-Hematology, Stem Cell Transplantation and Cell Therapy Division, City of Science and Health of Turin, Regina Margherita Children Hospital, Turin, Italy;7Princess

Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada;8Cancer Research Institute, Beth Israel Deaconess Cancer Center, Dept. Medicine and Pathology, Beth Israel

Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

MicroRNAs (miRs) are small non-coding RNAs that act as negative regulators of gene expression

and control tumor progression. We previously demonstrated that miR-214 is upregulated in

malignant melanomas and triple negative breast tumors and promotes metastatic dissemination by

affecting a complex pathway including transcription factors and adhesion molecules as well as the

anti-metastatic miR-148b. Importantly, tumor dissemination can be reduced by blocking miR-214

function or increasing miR-148b expression and further impaired by simultaneous interventions,

thus suggesting that the miR-214/miR-148b axis can be exploited for miR-based therapeutic

approaches. Interestingly, we evidenced that miR-214 is highly expressed in various stroma cells

and it correlates with a stroma signature in human tumors. Moreover, we found miR-214 in stroma

cells and observed that miR-214 expression in tumor cells is strongly influenced by stroma cells. In

order to explore the function of miR-214 in the crosstalk between tumor and stroma cells, we

evaluated tumor formation and progression in immunocompetent mice carrying a total-body miR-

214 deletion (miR-214KO mice) or overexpression (miR-214OVER mice) crossed with MMTV-

PyMT mice or used as recipients for tumor transplants. While growth of endogenous or

transplanted tumors was similar for all animals, metastasis formation in distant organs was

diminished in miR-214KO mice while increased in miR-214OVER mice. We are now investigating

the mechanism leading to altered aggressiveness. We have evidence showing increased

epithelial-mesenchymal transition and inflammation in the primary tumor and increased

macrophage recruitment in distant organs (before metastasis formation) when enhanced

dissemination is observed, therefore suggesting the relevance of stromal miR-214 in tumor

progression.

RAB7 regulates small extracellular vesicle secretion: insights from cisplatin resistance

F. Guerra1, A. Paiano1, G. Girolimetti2, D. Migoni1, F.P. Fanizzi1, G. Gasparre2 and C. Bucci1

1Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Via Provinciale Lecce-Monteroni 165, 73100 Lecce, Italy;

2 Department of Medical and Surgical Sciences (DIMEC), Medical Genetics Unit, University

Hospital S. Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy;

RAB7 is a small GTPase belonging to the Rab family with multiple key roles in cellular physiology. Indeed, RAB7 controls transport to late endocytic compartments and regulates late endocytic organelle biogenesis, lysosomal positioning and functions, trafficking and degradation of several signaling receptors [1]. Recently, defects in lysosomal function have been implicated in drug resistance. In fact, it was demonstrated that lysosomes are able to sequestrate multiple chemotherapeutic agents reducing accessibility of these drugs to their target sites [2]. In contrast, it was also hypothesized that sequestration of drug into lysosomes, in particular cisplatin, may also induce lysosomal damage and apoptosis [3]. In fact, lysosome-dependent cell death represents a form of regulated cell death initiated primarily by lysosomal membrane permeabilization (LMP). Therefore, it has been speculated that impairment of the lysosomal compartment, determining reduced cisplatin sequestration, protects cells from LMP and induces the chemoresistant phenotype [2]. On the basis of these previous observations, we decided to analyze the role of RAB7 in cisplatin chemoresistance, being RAB7 the master regulator of lysosomal biogenesis. We have observed that cisplatin-resistant cell lines are characterized by a downregulation of RAB7 compared to their chemosensitive counterparts. This downregulation is accompanied by a reduction in the amount of lysosomal proteins and in the number and size of lysosomes, and by impairment of acidification. Importantly, we demonstrated that silencing of RAB7 by RNA interference in chemosensitive cell lines determined occurrence of chemoresistant phenotype. Coherently, overexpression of RAB7 decreased drug resistance in chemoresistant cells. These data suggest that RAB7 is directly involved in the acquisition of chemoresistance. In order to establish the mechanism involved in RAB7-mediated chemoresistance we measured the amount of cisplatin present in chemoresistant and in chemosensitive cells and we found that in chemoresistant cells was significantly lower. As it was previously hypothesized that chemoresistant cells might export cisplatin through exosomes, a type of extracellular vesicles (EVs) derived from fusion of MVBs (Multivesicular Bodies) with the plasma membrane [2], we analyzed EV production in chemoresistant and chemosensitive cells. We found, in chemoresistant cells, increased secretion of CD9- and CD81-positive extracellular vesicles, suggesting that they could be responsible for cisplatin efflux. Furthermore, we demonstrated that overexpression of RAB7 increases intracellular accumulation of cisplatin and it decreases secretion of CD9- and CD81-positive EVs, suggesting that the production of EVs is regulated by RAB7. Further experiments are ongoing in order to demonstrate how RAB7 controls EV formation. References [1] Guerra, F.; Bucci, C. 2016. Multiple roles of the small GTPase Rab7. Cells, 5, E34. [2] Duvvuri, M.; Krise, J.P. 2005. Intracellular drug sequestration events associated with the emergence of multidrug resistance: A mechanistic review. Front. Biosci. 10, 1499–1509. [3] Safaei, R.; Larson, B.J.; Cheng, T.C.; Gibson, M.A.; Otani, S.; Naerdemann, W.; Howell, S.B. 2005. Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells. Mol. Cancer Ther. 4, 1595–1604.

Flow cytometry characterization of extracellular vesicles in human health and disease

Claudia Maria Radu1,2, Elena Campello2, Erica Bazzan3, Cristiana Bulato2, Marina Saetta3,

Manuel Cosio3, Paolo Simioni2.

1Department of Women's and Children's Health, University of Padua, Italy 2 Department of Medicine Thrombotic and Hemorrhagic Diseases Unit, University of Padua, Italy 3 Department of Cardiac, Thoracic, and Vascular Sciences, University of Padua, Italy The term Extracellular Vesicles (EVs) is a more generic term used for all secreted vesicles including exosomes, microparticles and apoptotic bodies. EVs are released from cell membranes in response to cellular activations, cell injury and apoptosis and are characterized by a phospholipid bilayer membrane. MVs contain functional cytoadhesions molecules, bioactive phospholipids, cytoplasmic components and various antigens that are characteristic of the state of the originating cells and the type of stimuli. Several studies in the cell-cell communication have proven that EVs are able to transfer information by miRNA, mRNA and DNA to the recipient cells1. Flow cytometry remains one of the widely used methods for MVs analysis to investigate the potential of EVs to be biomarkers of disease 2. Conventional flow cytometry, however, it has many limitations, because of the size ranges of MPs and inability of most flow cytometers to detect particles <0.5 μm. However, standardization of pre-analytical and analytical assays for isolating, count and fluorescent labeling of MVs remains a challenge3. In this study was evaluated CytoFLEX flow cytometry (Beckman Coulter) to enumerate and analyzed the different size of MVs using a blend of fluorescent beads with known sizes (Gigamix) and relatives amount. CytoFLEX is equipped with three laser: red (638nm), blue (488 nm) and violet (405 nm). To discriminate the different sizes of MVs (100-900nm) the violet side scatter (VSSC) of the violet laser was used as trigger signal to discriminate the noise. Used as trigger signal the VSSC the noise was significant lower in comparison to the normally 488 nm SSC when beads were used as standardization reference. Therefore, could be design a reproducible collection gate for MVs count. Plasma from healthy subjects and with different pathological disorders were collected and analyzed in in triplicate in the same experimental analysis to evaluate the reproducibility of data. The VSSC are more sensitive, resulting in higher particle resolution. Gigamix solution is recommended for the standardization for MVs measurement. The intra and inter tests data were reproducible with the same samples. CytoFLEX could be a flow cytometer which could be reduced the inter-laboratory variability in the detection of MVs size. 1Jella K. et al. (2018) Vaccines. Exosomes, their biogenesis and role in inter-cellular communication, tumor microenvironment and cancer immunotherapy. 2 Campello E. et al. (2016) Biomark Med. Microparticles as biomarkers of venous thromboembolic events. 3 Van Der Pol E. et al. (2018) J Thromb Haemost. ISTH-SSC-VB Working group. Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation.

New generation flow cytometry to measure circulating microvesicles in cancer

E. Campello1, C.M. Radu1,2, L. Spiezia1, S. Rampado3, C. Bulato1, R. Bardini3, P. Simioni1

1Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua, Italy 2Department of Women's and Children's Health, University of Padua, Italy 3Department of Surgery, Oncology and Gastroenterology, University of Padua, Italy

Cancer is a prothrombotic state, with an increased prevalence of venous thromboembolism (VTE). Plasma microvesicles (MVs) are sub-micron-sized vesicles derived from activated or apoptotic cells that may play an important role in the development of VTE 1. MVs have been proposed as possible mediators of cancer-associated hypercoagulability. Tissue Factor-bearing MVs (TF-MVs) in plasma may contribute to cancer-associated thrombosis2. We conducted a longitudinal cohort study to measure the trend of different type of circulating MVs in gastro-intestinal cancer using a new-generation flow-cytometer and a functional tissue factor (TF)-dependent assay. Patients with a first diagnosis of gastro-intestinal cancer who underwent surgery were consecutively enrolled. Blood samples were collected at baseline, 7 days, 1 and 6 months after surgery. Flow cytometry was performed using a CytoFLEX flow cytometer equipped with a more sensitive Side Scatter using the violet laser (405 nm) resulting in higher particle resolution. To detect the different sizes of MVs was used a new kind of fluorescent beads of variant diameters for a best resolution between the different size of MVs and background noise exclusions for setting MVs size ranges and to design a reproducible collection gate for MVs count. According to beads diameter three gate were created to detect big (0.5-0.9 μm), small (0.2-0.4 μm), and nano MVs (0.1-0.2 μm). MVs were further characterized by surface staining: total annexin-positive (phosphatidylserine [PS]+), total annexin-negative (PS-), annexin+CD62E+ (endothelial MVs), annexin+CD227+ (pancreatic and gastric tumour MVs) or annexin+CD326+ (colon tumour MVs). MV-associated TF activity was measured by a functional assay. Ninety three patients (25 pancreatic, 33 colon and 35 gastric-esophageal cancer) were enrolled. The majority of MVs detected were of small size and PS-. Pancreatic cancer showed increased MVs and MVs-TF activity compared with other cancer subtypes. Interestingly, MVs-TF activity correlated with big and small PS+ (R 0.559 p 0.0001), endothelial (R 0.28 p 0.028) and big tumour MVs (R 0.26 p 0.03), not with MVs PS-. Baseline endothelial MVs and MVs-TF activity were significantly associated with surgical radicality and cancer severity (OR 1.19 [1.04-1.36] and 1.30 [1.05-1.6], respectively). Baseline MVs-TF activity was associated with venous thromboembolism occurrence (HR 2.38 [1.81-4.11]). Annexin V did not detect total MVs. However, only PS+, endothelial and bigger tumour MVs conveys the procoagulant activity. Tissue Factor-bearing MVs (TF+MVs) in plasma may contribute to cancer-associated thrombosis. Baseline MVs-TF activity and endothelial MVs are independent predictors of advanced disease and incomplete surgical resection. Cancer-associated thrombosis was associated with TF procoagulant activity more than MVs number. 1 Campello E. et al. (2011) Thrombosis Research. Endothelial, platelet, and tissue factor-bearing microparticles in cancer patients with and without venous thromboembolism 2 Campello E. et al. (2016) Modulating thrombotic diathesis in hereditary thrombophilia and antiphospholipid antibody syndrome: a role for circulating microparticles?

Interleukin-17A affects extracellular vesicles release and cargo in human keratinocytes

Giorgio Mangino1, Marco Iuliano1, Silvia Carlomagno1, Nicoletta Bernardini1,2, Paolo Rosa1, Maria Vincenza Chiantore3, Nevena Skroza1,2, Antonella Calogero1,4, Concetta Potenza1,2, Giovanna

Romeo1,5

1Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy 2Dermatology Unit “Daniele Innocenzi”, Fiorini Hospital, Terracina, Italy 3Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy 4Istituto Chirurgico Ortopedico Traumatologico, ICOT, Latina, Italy 5Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy

Background: psoriasis is a chronic inflammatory skin disease caused by the excessive secretion of inflammatory cytokines. Deregulation of the interleukin-23/-17 axis allows the activation of Th17 lymphocytes and the reprogramming of keratinocytes proliferative response, thereby inducing the secretion of cyto-/chemokines and antimicrobial peptides. Currently, one anti-IL-17 biological agent (i.e. Secukinumab) is approved for the treatment. Psoriasis-associated inflammation also affect systemic functions associated with extracutaneous manifestations. Beside cell-to-cell contacts and release of cytokines, hormones and second messengers, cells communicate each other through the release of extracellular vesicles containing DNA, RNA, microRNAs and proteins. It has been reported the alteration of extracellular vesicles trafficking in several diseases, but there is scarce evidence of the involvement of extracellular vesicles trafficking in the pathogenesis of psoriasis. Objective: the main goal of the study was to characterize the release, the cargo content and the capacity to transfer bioactive molecules of extracellular vesicles produced by keratinocytes following recombinant IL-17A treatment if compared to untreated keratinocytes. Methods: a combined approach of standard ultracentrifugation, RNA isolation and Real Time RT-PCR techniques was used to characterize extracellular vesicles cargo. Flow cytometry was used to quantitatively and qualitatively analyze extracellular vesicles and, in association with structured illumination microscopy, to evaluate cell-to-cell extracellular vesicles transfer. Results: we report that the treatment of human keratinocytes with IL-17A significantly modify the extracellular vesicles cargo and release. Vesicles from IL-17A-treated cells display a specific pattern of mRNA which is abrogated by Secukinumab neutralization. Extracellular vesicles are taken up by acceptor cells irrespective of their content but only those derived from IL-17A-treated cells enable receiving cells to express psoriasis-associated mRNA. Conclusion: the obtained results imply a role of extracellular vesicles in amplifying the pro-inflammatory cascade induced in keratinocyte by pro-psoriatic cytokines.

Ultracentrifugation versus size exclusion chromatography: in vitro autocrine effect of canine and feline mammary tumor-derived

extracellular vesicles

A. Sammarco1, V. Moccia1, M. Romano2, V. Dolo3, L. Cavicchioli1, S. Ferro1, A. Radeghieri2,4, P. Bergese2,4, V. Zappulli1

1Department of Comparative Biomedicine and Food Science, University of Padua, Padua,

Italy 2Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy 3Department of Medicine, Health and Environment Sciences, University of L’Aquila, Italy

4CSGI, Research Center for Colloids and Nanoscience, Florence, Italy Mammary tumors are among the most frequent tumors in intact female dogs and cats. These tumors are often malignant and have a high tendency to metastasize, especially in the feline species, and they show features that can resemble aggressive human breast cancer. Extracellular vesicles (EVs) are involved in tumorigenesis and have been described for their role in increasing migration, invasion, and metastases in HBC. The choice of the EV isolation/purification strategy is very important in functional studies to establish that an effect is due to EVs and not to other co-isolated materials. Among others, ultracentrifugation (UC) and size exclusion chromatography (SEC) have been largely used and UC has been for long time the most commonly used primary EV concentration technique. However, the main disadvantage of this method is the co-pelleting of high molecular weight protein complexes and the potential partial loss of EV integrity (and function) due to ultra-centrifugal force. Other EV isolation methods, such as SEC, allow to achieve better specificity of EVs or EV subtype separation. The aim of this study was to evaluate the autocrine effect of canine and feline mammary tumor-derived EV formulations comparing two different EV isolation methods (UC vs SEC). A cell proliferation assay as well as cancer cell migration and invasion assays were performed on canine mammary carcinoma (CIPp) and feline mammary carcinoma (FMCp) cell lines treated with CIPp- and FMCp-derived EVs, respectively. Unconditioned medium (UCM)-derived pelleted material was always included as negative control. EVs were obtained from CIPp and FMCp by both UC and SEC and characterized by western blot. Nanoparticle Tracking Analysis, Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) were used to quantify and visualize EVs. EV formulations isolated by UC and by SEC were also compared for purity using the COlorimetric NANoplasmonic (CONAN) assay and the BCA protein assay. TEM and AFM revealed a high protein background in the UC pelleted materials and confirmed EV isolation both by UC and SEC. Large spherical molecular complexes mimicking EVs were observed by AFM also in UCM. CONAN assay confirmed a higher EV purity of SEC fractions. BCA assay showed a higher protein content in the EV pellet obtained by UC when compared to EVs purified by SEC. EVs purified by SEC induced a higher proliferation rate (p<0.05) in both cell lines when compared to EVs isolated by UC. This effect could be due to the highest purity, preservation, and functionality of EVs isolated by SEC. CIPp treated with CIPp EVs and FMCp treated with FMCp EVs showed a higher migration (p<0.0001) in the wound healing assay with no differences between UC and SEC. The effect of UC-derived EV formulation on cell migration might be partially associated with the co-isolated soluble proteins. Further analyses are on-going to characterize the observed protein complexes recently ascribed by other authors to lipoproteins complexes (i.e. exomers), that could mimic EV morphology. These results show that methods for pure isolation of EVs and subsequent characterization of EVs and co-isolated materials are a key aspect to be set, particularly for functional studies.

Assessment of activinA effect on b-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell vesiculation

G. Cricrì1, L. Pergoli2, F. Portale1, A. Fallati1, G. Bedini1, N. Di Marzo1, A. Biondi1, V. Bollati2, E. Dander1, G. D’Amico1

1Centro Ricerca Tettamanti, Pediatric Dep, University of Milano-Bicocca, Fondazione MBBM,

Monza, Italy 2EPIGET-Epidemiology, Epigenetics and Toxicology Lab, Department of Clinical Sciences and

Community Health, University of Milano, Milan, Italy

B-Cell Precursor-Acute Lymphoblastic Leukemia (BCP-ALL) is largely the result of a growing number of cooperating genetic and epigenetic aberrations that corrupt hematopoietic developmental pathways and ultimately lead to uncontrolled production of malignant B lymphoid precursor cells within the bone marrow (BM) niche. BCP-ALL can occur at any age; however, it mainly affects children where it is by far the most common malignancy, with a peak incidence around 2-5 years of age. Despite cure rate exceeds 85%, it remains the leading cause of childhood cancer-related mortality. In the BM niche, stromal and leukemic cells dialogue through soluble factors, cell-to-cell contact and extracellular vesicles (EVs) which are increasingly recognised as modes of local as well as distal intercellular communication. The limited-leukemia EVs studies have highlighted a possible role in leukemia development and progression, particularly in the context of acute myeloid and chronic lymphocytic leukemia. Notably, EVs’ contribution in BCP-ALL pathogenesis has not been fully explored. In the context of BCP-ALL, we recently identified ActivinA, a TGF-β family member, which confers aggressiveness to leukemic cells by promoting their invasive and motility properties through the increase of their intracellular calcium content. In view of ActivinA property to positively regulate cytosolic calcium and actin polymerization, which are associated to EVs formation, secretion and cargo sorting, we evaluated whether this molecule is able to influence EVs release by leukemic cells. Therefore, we optimized a method to isolate and characterize EVs from BCP-ALL cells by using the 697 cell line cultured in serum-free medium for 24-48h in presence or in absence of ActivinA (50 ng/mL) (n=8). Only cells with a viability percentage higher of 80% were used for EVs evaluation. Cell culture media were analyzed with Nanoparticle tracking analysis (NTA) to determine EVs concentration (particle/mL) and size distribution. Total EVs (30–700 nm), exosomes (30–150 nm) and microvesicles (151–700 nm) have been classified. We demonstrated that 697 cell line induced the production of both EVs populations. Notably, ActivinA treatment increased the mean concentration of exosomes derived from 697 (range: 60%, p<0,008 and 30%, p<0,016 at 24h and 48h, respectively), compared to unstimulated condition. The microvesicle mean concentration was nearly significant after 24h of ActivinA stimulation (p=0,055), whereas at 48h there was an increase (range: 30%, p<0,016) compared to unstimulated condition. Moreover, a dedicated fraction of cell culture media was ultracentrifugated and the obtained EV preparations were characterized by flow cytometry. In order to evaluate the integrity of EVs, we used 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE). In addition, each CFSE-stained sample was incubated with specific antibodies to verify the membrane expression of CD19 (microvesicles from leukemic cells), CD9 and CD63 (exosomes). Preliminary data indicated that 697 cell line was able to produce both the population of microvesicles expressing CD19 and of exosomes expressing CD9 and CD63. Furthermore, ActivinA did not alter the ratio of these two EVs populations. We also characterized the protein content of EVs derived from 697 before and after ActivinA treatment. Through the BCA assay, we determined the concentration of proteins present in the compartment of 697-derived EVs. We observed that EVs released by 697 contain proteins at both the analyzed timepoints and ActivinA stimulation did not alter their protein content. Overall, we demonstrated that ActivinA positively regulates BCP-ALL cell vesiculation. A better comprehension of EVs’ role in the leukemia-stroma crosstalk may provide new therapeutic targets that should allow eradication of leukemic cells without adversely affecting healthy hematopoiesis.

Circulating microRNAs enriched in nanoscale extracellular vesicles are

consistent with tumour growth in brain cancer preclinical models

L. Guglielmi1, C. Musa2, M. Porru3, B. Cardinali2, C. Di Pietro2, V. Palmieri4, M. Papi4, F. Scavizzi2, M. Raspa2, C. Leonetti3 A. Felsani5, G. Falcone2, and I. D’Agnano1

1Institute of Biomedical Technologies, CNR, Segrate, Italy 2Institute of Cell Biology and Neurobiology, CNR, Monterotondo, Italy

3Regina Elena Cancer Institute, Rome, Italy 4Università Cattolica Sacro Cuore, Rome, Italy

5Genomnia, Bresso, Italy

The causes of brain tumours in adults remain largely unknown. Glioblastoma Multiforme (GBM), the most malignant primary tumour of the brain, is associated with an average survival from diagnosis of only about 1 year. Brain metastases account for more than one-half of all intracranial tumours in adults. Therefore, the identification of new biomarkers remains a major challenge to integrate prognosis and improve diagnosis for brain tumours. The relative stability, accessibility and efficient detection of miRNAs in body fluids suggest their use as “liquid biopsy”-based biomarkers for tumour progression. miRNAs packed into tumour-released nanoscale extracellular vesicles (nEVs) have been shown to contribute to tumour establishment and metastatic spread, suggesting not only diagnostic/prognostic but also therapeutic target potential.

In order to identify miRNAs to be used as biomarkers for brain tumour diseases, we analysed the expression profiles of circulating miRNAs in preclinical models of intracranial human metastatic melanoma and glioblastoma in nude mice. Thus, extracellular miRNAs have been studied in the nEVs purified from culture cell supernatants and from the plasma of mice bearing tumour to brain. The isolated nEVs were counted and have been characterized by FACS, for the presence of surface EV specific markers (e.g. CD81, CD9) and by TEM for morphology. Profiling human miRNA expression in nEVs by next-generation sequencing we identified a selected group of human specific miRNAs contained in the nEVs, which were released by the human tumour cells injected in the mice brain and that could be related to the growth of tumours.

Extracellular vesicles from tumor-derived endothelial cells transform recipient cells and

favortumor progression. Enrica Favaro, Tatiana Lopatina, Benedetta Bussolati, Tiziana Martone, Renato Romagnoli,

Giovanni Camussi, Maria Felice Brizzi.

ABSTRACT Head and neck cancer has a high rate of recurrence and metastasis. The tumor mass consists

of cancer cells surrounded by inflammatory cells, vascular cells and stromal cells, also

defined tumor microenvironment (TME). The role of TME intumor expansion and

progressionhas been extensively documented. Among cells contained in the TME

mesenchymal stem cells are included. Mesenchymal stem cells secrete a variety of growth

factors, which could promote tumor angiogenesis, enhance epithelial-mesenchymal transition,

and induce stem-like properties allowing cancer stem cells to expand and survive. Moreover,

a bidirectional cross-talk among cells contained in TME contribute to these processes. Besides

growth factors the release of extracellular vesicles (EVs) has been reported in TME. EVs

released in TME contribute to both local and distant malignant transformation andto the

expansion of cancer stem cells. EVs participate to this process mainly by transferring to

recipient cells their cargo consisting in proteins, lipids, nucleic acids and other molecules. In

this study the role of EVs released by tumor-derived endothelial cells (TEC-EVs) obtained

byhead and neck cancer metastasis (HNC) was evaluated. In particular, the cross-talk between

TEC-EVs and adipose-derived mesenchymal stem cells (ASC) was investigated. We found that

TEC-EVs regulated gene expression in ASC as well as their biological functions. At this regard,

we demonstrated that ASC treated withTEC-EVs highly express the cancer stem cell marker

ALDH. As the results of such pre-conditioning ASC treated with TEC-EVs (ASCind) release EVs

(ASCind-EVs) able to promote tumor cell proliferation, growth factor secretion, as well as

invasion and sphere formation. In vivo the tumor masses formed bytumor cellspretreatedwith

ASCind-EVs,are significantly increased. These data indicate that the cross-talk between TEC-

EVs andASC contained in TEM could reprogram non-tumor cells and favortumorexpansion.

Extracellular vesicles from melanoma cells and their potential role in immune escape and cancer progression as a consequence of the MHC

class II signalling

Francesca Costantini and Giovanna Barbieri. IRIB-CNR (ex Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”), Via Ugo La Malfa

153, 90146 Palermo, Italy.

Melanoma, is one of the most aggressive cancers worldwide, its incidence rate increases rapidly in western population and unfortunately it is responsible of about 20% overall cancer mortality as well as of approximately 80% skin cancer related mortality. Indeed, the aggressive metastatic trend of melanoma is associated to the increase of the cellular dissemination and is a consequence of the tumour cells detachment from primary tumour as well as to the interaction between tumour cells and cellular blood components. Melanoma cells also secrete in their microenvironment microvesicles and exosomes, that for their nanoscale size, can circulate and interact with immune cells, tumour cells and with different cell types localized in advancing tumour front and in distant tissues. Indeed, the extracellular vesicles secreted by melanoma cells play a main role in the metastatic progression of melanoma regulating the immune cell functions and modifying the tumour microenvironment. Furthermore, the metastatic progression of melanoma is associated to the constitutive expression of Major Histocompatibility Complex (MHC) class II molecules that are constitutively expressed in almost 50% of metastatic lesions and therefore are a marker of poor prognosis in melanoma. Therefore, the aim of our work was to understand the consequences on the extracellular vesicles secreted by melanoma cells, of the MHC class II mediated signalling. Indeed, the MHC class II molecules are signalling receptors whose engagement leads to the activation of several signalling pathways. In particular, we showed in the extracellular vesicles secreted by two class II constitutive expressing melanoma cell lines, that the MHC class II mediated signalling increases the expression of HLA-DR, ICAM and MCAM adhesion receptors, FasL and STAT3 signalling proteins and also increases the proteolytic activity of some members of the matrix metalloproteinases (MMPs) family. Interestingly, the tumour microvesicles and exosomes secreted by the MHC class II stimulated melanoma cells, through the expression of these proteins and the activation of the proteolytic activity of MMP family members, could have immune suppressive function inducing the apoptosis of activated T cells and the inhibition of dendritic cells differentiation and could be implicated in tumour cells invasion of the surrounding tissues and also in the host-tumour communication. The increased amount of these receptors and signalling proteins in the extracellular vesicles as well as of the MMP gelatinases activity, would act in concert to enhance the breakdown and the remodelling of the extracellular matrix, thus enhancing the tumour cell invasion of microenvironment and the metastatic progression of melanoma cells. Therefore, our results suggest that the MHC class II mediated signalling plays a new role to promote melanoma progression enhancing, through the extracellular vesicles secreted, the metastatic dissemination of melanoma cells as well as inhibiting the immune response in tumour microenvironment.

Normal human fibroblasts activated by extracellular vesicles

affect neighbouring cells’ behaviour.

I. Giusti1, S. D’Ascenzo1, E. Eleuteri1, V. Dolo1.

1 Department of Life, Health and Environmental Sciences, University of L’Aquila,

Via Vetoio, Coppito 2, 67100 L’Aquila, Italy

Introduction: Fibroblasts in the tumor microenvironment have been proven to actively participate in

tumor progression; they can be “educated” by tumor cells, acquiring an activated state [1]. These

activated cells, identified as Cancer Associated Fibroblasts (CAFs) can, in turn, remodel the tumor

stroma to be more advantageous for cancer progression [2].

Aim of this work was to verify if normal human fibroblasts, activated into a CAF-like state by small

and large extracellular vesicles (EVs) released from the human ovarian cancer cell line CABA I,

could modulate the behavior of neighbouring cells.

Methods: Fibroblasts’ activation into a CAF-like phenotype triggered by CABA I EVs administration

was confirmed by cells morphology, marker expression and their increased biological activities such

as proliferation, motility and invasiveness. To verify if fibroblasts modify their secretome after EVs

treatment, untreated and EVs-treated cells were incubated for 24 h to produce the corresponding

conditioned media (secretome). The untreated and EVs-treated cells’ secretome ability to stimulate

neighbouring cells (unactivated fibroblasts, endothelial cells, tumor cells) was analysed by

proliferation (XTT test), motility and invasion (Boyden Chamber) assays.

Results: Small and large EVs induce a morphological changes and markers expression (α-SMA,

TIMP-2 and uPAR) typical of CAFs; they also modulate biological activities of CAFs-like fibroblasts

inducing motility and invasiveness. Small EVs seem to be more effective than large ones.

Secretome of small/large EVs-activated fibroblasts was not able to modulate proliferation of

unactivated fibroblasts, endothelial cells or cancer cells. It stimulated, instead, motility of unactivated

fibroblasts and tumor cells (about 2 and 2.2 fold respectively), invasiveness of tumor cells (2.5 fold)

and tube formation in endothelial cells. No differences were observed in effectiveness of secretome

from small or large EVs activated fibroblasts.

Conclusions: Ovarian cancer cells can modulate fibroblasts’ behaviour through the release of EVs,

activating them to a CAFs-like state. EVs activated fibroblasts, in turn, affect neighbouring cells’

behaviour to be more advantageous for cancer progression.

[1] Kalluri R. The biology and function of fibroblasts in cancer. Nat Rev Cancer. 2016;16(9):582-598.

Kuzet SE, Gaggioli C. Fibroblast activation in cancer: when seed fertilizes soil. Cell Tissue

Res. 2016;365(3):607-619.

[2] Dasari S, Fang Y, Mitra AK. Cancer Associated Fibroblasts: Naughty Neighbors That Drive

Ovarian Cancer Progression. Cancers (Basel). 2018 Oct 29;10(11). pii: E406.

Inflammatory role of extracellular vesicles transported sphingolipids in

Cystic Fibrosis

A. Zulueta1, V. Peli1, M. Dei Cas1, M. Colombo2, R. Paroni1, M. Falleni3, A. Baisi4, V. Bollati5, R.

Chiaramonte2, E. Del Favero6, R. Ghidoni1, A. Caretti1

1Biochemistry and Molecular Biology Lab., Health Sciences Department, University of Milan, Milan, Italy

2Laboratory of Experimental Medicine and Pathophysiology, Health Sciences Department, University of Milan, Milan, Italy

3Pathology Division, Health Sciences Department, University of Milan, San Paolo Hospital Medical School, Milan, Italy

4Thoracic Surgery Unit, Health Sciences Department, University of Milan, San Paolo Hospital Medical School, Milan, Italy

5EPIGET LAB, Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy

6Department of Medical Biotechnology and Translational Medicine, University of Milan, Via Fratelli Cervi 93, Milan, Italy

Ceramide is emerging as one of the players of inflammation in lung diseases. However, data on its

inflammatory role in Cystic Fibrosis (CF) as part of the extracellular machinery driven by lung

mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are missing.

We obtained an in vitro model of CF-MSC by treating control human lung MSCs with a specific CFTR

inhibitor (I-172). We characterized EVs populations derived from MSCs (ctr MSCs) and CF-MSCs

(CF-EVs) by means of Nanoparticle Tracking Analysis (NTA) and Laser light scattering analysis.

Furthermore, we analyzed their sphingolipid profile by LC-MS/MS.

To evaluate their immunomodulatory function, we treated an in vitro human model of bronchial

epithelial cell line derived from a CF patient (IB3 cell line), with both EVs populations.

Our data show that the two EVs populations differ for the average size, amount, and rate of uptake.

CF-EVs display higher ceramide and dyhidroceramide accumulation as compared to control EVs,

suggesting the involvement of the de novo biosynthesis pathway in the parental CF-MSCs. Higher

sphingomyelinase activity in CF-MSCs, driven by inflammation-induced ceramide accumulation,

sustains the exocytosis of vesicles that export new formed pro-inflammatory ceramide.

Our results suggest that CFTR dysfunction associates with an enhanced sphingolipid metabolism

leading to an increase in the content of ceramide. This in turn promotes the release of CF-EVs that

export the excess of pro-inflammatory Cer to the recipient cells, thus contributing to maintain the

chronic, unresolved inflammatory status of CF.

NGS analysis of cfDNA and exosomal dsDNA extracted from colon cancer patient’s plasma

Taverna Simona1, Galvano Antonio2, Castiglia Marta2, Barraco Nadia2, Duro Giovanni1, Bazan Viviana2, Russo Antonio2

1Institute of research and innovation in Biomedicine (IRIB), National Research Council, Palermo, Italy.

2Section of Medical Oncology, Department of Surgical, Oncological and Oral Sciences, University of Palermo, Palermo, Italy.

Colorectal cancer (CRC) is one of the most frequent malignant neoplasms in the world, with greater mortality in developing countries. The colorectal carcinogenic evolution is marked by the succession of well-known phases characterized by the progressive accumulation of genetic and epigenetic mutations in addition to histological transformation. Several reports have recently shown that liquid biopsy can be a potential new alternative to traditional tissue biopsy, thanks to its wealth of genetic material as circulating tumor DNA (ctDNA), circulating-free DNA (cfDNA), exosomes and circulating microRNAs. In 2017, in an innovative and still unique study in the field of liquid biopsy, the diagnostic accuracy was compared, in the identification of the KRAS gene mutation, the cfDNA and the exosomal dsDNA extracted from the plasma of patients with pancreatic ductal adenocarcinoma. The exosomal dsDNA allows identifying mutations with greater accuracy than the cfDNA in localized and locally advanced carcinomas [53]. The biological explanation of this result seems to be linked to the origin of the two forms of DNA, while exosomes are released into the blood by cells actively; the ctDNA represents epiphenomena of necrosis and apoptosis that occur especially in the advanced stages of cancer. The aim of our study is the comparison between the genetic information carried by cfDNA and exosomal dsDNA of colon cancer origin, with a view to their use in clinical practice. CfDNA and exosomal dsDNA were collected, by plasma of 10 patients with colon cancer, at 2 different time points (T1 and T2). The cfDNA and exosomal dsDNA were obtained from plasma using commercial kit, and were analyzed by Next Generation Sequencing (NGS). The NGS analysis was conducted on IonS5 platform with Ion AmpliSeq Cancer Hotspot Panel v2 used to perform the library preparation procedure. This panel contains 207 primer pairs in a single tube and allows investigating hotspot regions of 50 oncogenes and tumor suppressor genes, with extensive coverage of KRAS, BRAF and EGFR genes. The analysis of the data and the annotation of the variants were conducted with Ion Reporter Software. We reported a representative case in which emerged an interesting result from the comparison between exosomal dsDNA and cfDNA through NGS analysis. The sequencing of cfDNA extracted from colon cancer patient's plasma to T1 and T2 revealed 5 and 8 non-synonymous somatic mutations respectively. The KRAS gene mutation was detected in the T2 sample, but not T1. The same analysis was performed on exosomal dsDNA: at T1 17 missense mutations were identified, with a high average allelic frequency (VAF = 81). KRAS mutation was identified in T1 exosomal dsDNA according to neoplastic tissue mutational status. Interestingly, KRAS gene mutation was detected in cfDNA only at T2. The case under report has shown that exosomal dsDNA is able to intercept KRAS gene mutation before than cfDNA. Clinically, it could allow defining disease recurrence and the secondary resistance phases. The mentioned diagnostic anticipation could be explained by the ability of exosomes to protect from degradation their cargo of proteins and nucleic acids. On the contrary, since the cfDNA in the blood is subjected to continuous degradation and denaturing processes, a longer time is required to identify mutated genes to exceed the sensitivity limit of the NGS.

Exosomes from ankylosing spondylitis patients modulate macrophage

immunophenotype towards M2 polarization

C. Corrado1, S. Fontana1, F. Monteleone1, L. Saieva1, A. Conigliaro1, V. Verdi1, G. Guggino2, F.

Ciccia3, R. Alessandro1

1University of Palermo, dept. of Biomedicine, Neurosciences and Advanced Diagnostics, Palermo, Italy

2 University of Palermo, dept. Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties, Palermo, Italy

3University of Campania L. Vanvitelli, Napoli, Italy

Ankylosing spondylitis (AS) is a chronic inflammatory disease that primarily affects the spine and sacroiliac joints leading to bone fusion and loss of spine mobility; there is not a cure but only therapies to relieve pain. The pathological process starts with inflammatory activation (early stage) while the advanced stage is characterized by new bone formation [1- 3]. Macrophages are classified in two subsets, according to their functions and phenotype: M1, with a pro inflammatory phenotype (CD68+) and M2, with an anti-inflammatory phenotype (CD163+/CD206+). Recent data demonstrated that macrophages isolated from AS patients show an M2 phenotype [4]; moreover, treatment of peripheral monocytes with synovial fluid of Spondiloarthritis’ patients induces macrophage polarization toward M2 phenotype [5]. Exosomes are now considered as an integral part of the intercellular microenvironment and may act as immune modulators with immunosuppressive or immune-activating effects by delivering proteins or nucleic acid to recipient cells [6]. Increasing experimental evidences are emerging about the role of exosomes in joint diseases [7, 8]; to our knowledge, there are no data about their involvement in AS progression. In this study, for the first time, we demonstrated, by FACS analysis, that in non-polarized macrophages (M0), the treatment with exosomes isolated from AS patients (AS-exo) upregulates the expression of surface markers of M2-like phenotype (CD163 and CD206). Interestingly, quantitative SWATH MS proteomic analysis allowed to identify 18 proteins differentially expressed (at least ± 2 fold) between AS-exo and exosomes isolated from plasma of healthy donors (H-exo); Gene Ontology enrichment analysis, performed by using STRING v. 11, showed that these proteins are specifically involved in leukocyte mediated immunity, humoral immune response and innate immune response. Moreover, among these 18 proteins, the STRING network analysis allowed to identify paraoxonase/ arylesterase 1 (PON1) and haptoglobin (HP) as direct interactors of CD163, CD204, CD206, markers of M2 phenotype. These two proteins, enriched in AS-exo in comparison to H-exo, are described in literature to be involved in regulation of mechanisms driving M2 polarization [9, 10]. Future analysis of RNA and protein content of macrophages M0 treated with AS-exo will allow us to better clarify the molecular mechanism involved in M2 polarization. In conclusion, our studies show that AS-exo has an active role, through M2 polarization, in defining the immune response during AS progression. References [1] Braun J. and Sieper J. 2007. Lancet 369, 1379–90. [2] Dougados M, Baeten D. 2011. Lancet 377, 2127–37. [3] Ciccia F. Guggino G. et al. 2018. Arthritis Rheumatol. 2018. 70(12), 2003-2013

[4] Zhao J, Yuan W. et al. 2017. APMIS 125, 1070–1075. [5] Vandooren B., Noordenbos T. et al. 2009. Arthritis & Rheumatism 60(4), 966–975. [6] Corrado C., Raimondo S. et al. 2013. Int. J. Mol. Sci. 2013, 14, 5338-5366. [7]Kolhe R., Hunter M. et al. 2017. Sci Rep. 17(7), 2029. [8] Domenis R., Zanutel R. et al. 2017. Mediators Inflamm. 2017, 4814987. [9] Aharoni S., Aviram M. et al. 2013. Atherosclerosis (2), 353-61. [10] Chistiakov DA., Bobryshev YV. et al. 2015. J Cell Mol Med. 19(6), 1163-73.

Colon cancer cell-derived exosomes modulate macrophage immunosuppressive phenotype associated to PD-L1 expression

M. Pucci, L. Saieva, G. Buscemi, C. Zichittella, R. Alessandro, S. Fontana

1 University of Palermo, Dep. of Biomedicine, Neurosciences and advanced Diagnostics, Palermo,

Italy

Tumor-associated macrophages (TAMs) are a prominent component of cancer microenvironment having a key role in promoting tumor progression [1]. Several studies have demonstrated that TAMs phenotypically and functionally correspond to M2-polarized macrophages known to mediate anti-inflammatory response [2]. Moreover, it was shown that TAMs have immunosuppressive phenotype associated with the expression of programmed cell death ligand 1 (PD-L1) [3]. It is well known that tumor-derived exosomes (TDEs) play a pivotal role in cell-cell communication in the local tumor microenvironment modulating the properties and the behavior of surrounding cells such as TAMs. Even if several studies demonstrated the ability of TDEs to induce M2-like macrophage polarization, few data are available about their involvement in regulating the expression of PDL-1 in TAMs. The aim of the current study has been to examine the effect of exosomes derived from colon cancer cells (SW480) on macrophage function. With this purpose, non-polarized macrophages (M0) differentiated from THP-1 cells were co-cultured with exosomes derived from SW480 cells (SW480exos). Our results indicate that after 48h treatment, exosomes can significantly upregulate the expression of surface markers of M2-like phenotype (CD163 and CD206) and as well as of PD-L1, inducing macrophages to acquire a markedly immunosuppressive phenotype. In parallel, we found that SW480exos were also able to induce a significant increase of interleukin 6 (IL6) expression at both mRNA and protein level. By using an IL6 antagonist and investigating the modulation of STAT3 signaling pathway (shown to regulate PD-L1 and cytokine secretion as well), we think to characterize the molecular mechanism through which exosomes modulate the macrophage phenotype. Finally, since the consequence of PD-1/PD-L1 axis activation is the T cell dysfunction, we assessed the ability of macrophages treated with TDEs to affect vitality of T cells. According to the results described above, we found that CD3+ T cells co-cultured with M0 macrophages treated with SW480exos showed a significant increase of their apoptotic rate in comparison to those grown in presence of no-treated macrophages. Further analyses will be performed to evaluate if M0 macrophages treated with TDEs can also affect the activation status of T cells. Cumulatively, these preliminary data suggest that within local colon cancer microenvironment TDEs can act as positive modulators of the immunosuppressive status of TAMs, actively promoting the immunotolerance necessary to favor tumor growth and progression.

References [1] Yang L and Zhang Y Journal of Hematology & Oncology, 10(1):58, 2017. [2] Mantovani A, Sozzani S et al. Trends Immunol 23:549–555, 2002. [3] Hartley GP, Chow L et al. Cancer Immunol Res.6(10):1260-1273, 2018.

HIV-1 myrNefSF2 protein induces the release of stat-1 and -2 activating factors and affects the extracellular vesicles production in plasmacytoid

dendritic cells.

A. Aiello1, F. Giannessi1, Z. A. Percario1, K. Fecchi2, M. Carollo3, M. Sargiacomo2, E. Affabris1

1 Dept.of Sciences, University Roma Tre, Rome, Italy 2 Global Health Center, Istituto Superiore di Sanità, Rome, Italy.

3Core Facilities Cytometry Unit, Istituto Superiore di Sanità, Rome, Italy

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset specialized in type I interferon (IFN) production that play a pivotal role in HIV infection. They seems to be involved in HIV pathogenesis by contributing dichotomously to both immune activation and immunosuppression [1]. The protein Nef is a known virulence factor of Human Immunodeficiency Virus type 1 (HIV-1) that acts as molecular adaptor inside infected cells and it has an important impact on the chemo-cytokine network. Importantly, Nef can be transferred to uninfected cells through extracellular vesicles (EVs) [2; 3], which have been recently recognized as potent vehicles of intercellular communication in both physiological and pathological conditions, including HIV infection. In this work we investigated the effects induced by Nef protein in uninfected pDCs. As experimental model, we used a human plasmacytoid dendritic cell line that was treated with a highly purified myristoylated recombinant Nef protein generated from HIV-1 SF2 allele (myrNefSF2). Confocal microscopy and western blot analyses were performed to verify internalization of the protein and to evaluate activation of pDCs, respectively. To characterize EVs production, we used a commercially available BODIPY®-C16 fatty acid to label the cells. The fluorescent EVs released from the cells were examined and quantified as reported by Sargiacomo and colleagues [4]. We observed that myrNefSF2 is internalized by about 50% of pDCs and induces the tyrosine phosphorylation of STAT1 and STAT2 protein after 3 hours and the expression of ISG15, an IFN inducible gene. This result suggests that pDCs stimulated by Nef release cytokines, including type I or type III IFN because only these two classes of cytokines are able to induce tyrosine phosphorylation of STAT2. Moreover, we observed that supernatant from pDCs treated with myrNefSF2, even if depleted of extracellular vesicles, can induce STAT1 tyrosine phosphorylation only after 30 minutes of cell treatment, suggesting that Nef induces the release of STAT1 and -2 activating factors that are not associated with EVs. Finally, we observed that Nef is incorporated only in the small (size < 200 nm) but not in the medium/large EVs (size > 200 nm) released by the treated cells and it reduces the production of small EVs by about 40%. Further studies are in progress to treat pDCs with exosomes containing Nef protein in order to analyse the cell response. Supported by grants from MIUR - Departments of Excellence- 2017 - legge 232/2016 - art.1, commi 314-337 awarded to Dept. of Science - University Roma Tre - Rome - Italy for 2018-2022 References:

1. Aiello A, Giannessi F, Percario ZA, Affabris E. The involvement of plasmacytoid cells in HIV infection and pathogenesis. Cytokine Growth Factor Rev. 2018;40. doi:10.1016/j.cytogfr.2018.03.009

2. Muratori C, Cavallin LE, Krätzel K, et al. Massive Secretion by T Cells Is Caused by HIV Nef in Infected Cells and by Nef Transfer to Bystander Cells. Cell Host Microbe. 2009;6(3):218-230. doi:10.1016/j.chom.2009.06.009

3. Arenaccio C, Chiozzini C, Columba-Cabezas S, et al. Exosomes from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Cells License Quiescent CD4+ T Lymphocytes To Replicate HIV-1 through a Nef- and ADAM17-Dependent Mechanism. J Virol. 2014;88(19):11529-11539. doi:10.1128/jvi.01712-14

4. Coscia C, Parolini I, Sanchez M, Biffoni M, Boussadia Z, Zanetti C, Fiani ML, Sargiacomo M. Generation, Quantification, and Tracing of Metabolically Labeled Fluorescent Exosomes. Methods Mol Biol. 2016;1448:217-35. doi: 10.1007/978-1-4939-3753-0_16.

ABSTRACT

HIV-1 NefSF2 protein increases vesicles production in thp-1 human monocytic cells differentiated with pma

F. Giannessi1, A. Aiello1, Z.A Percario1, K. Fecchi2, M. Carollo3, M. Sargiacomo2 and E. Affabris1

1 Dept.of Science, University Roma Tre, Rome, Italy

2Global Health Center, Istituto Superiore di Sanità, Rome, Italy. 3Core Facilities Cytometry Unit, Istituto Superiore di Sanità, Rome, Italy

Extracellular vesicles (EVs) are lipid bilayer-enclosed entities containing proteins and nucleic acids that mediate intercellular communication both in physiological and pathological conditions [3]. EVs resemble enveloped viruses in both structural and functional aspects. In full analogy with viral biogenesis, some of these vesicles are generated inside cells and once released into the extracellular milieu, are called “exosomes”(size 30-100nm), whereas others bud from the plasma membrane and are generally referred to as “microvesicles”(size >100nm) [5]. Since now there aren’t specific markers universally recognized to discriminate EVs for their biogenesis process, therefore the EVs were classified using physical characteristic like size, density and biochemical composition. The vesicles smaller than 200nm are named small EVs(sEVs) while the vesicles bigger than 200nm are named medium/large EVs (m/lEVs)[4]. Nef, a protein codified by Human Immunodeficiency Virus (HIV), is a virulence factor that acts as an adaptor molecule inside the infected cell. It regulates viral production and induces immunoevasion, in order to create a positive environment for viral spreading. Importantly, it can also be transferred to uninfected cells through exosomes [2]. In this study we examined if recombinant myristoylated HIV-1 NefSF2 (myrNefSF2) was able to affects the production of vesicles in THP-1 cells differentiated with PMA. THP-1 monocytic cells, differentiated with PMA, were treated with myrNefSF2 and its mutants. It is known in literature that THP-1 cells assume the phenotype of macrophages, a primary target of natural viral infection, when treated with PMA (phorbol 12-myristate 13-acetate). We used commercially available BODIPY®-C16 fatty acid to label the cells producing fluorescent vesicles that we examined and quantified as reported by Sargiacomo and colleagues [1]. The content of the vesicles was studied using Western Blot analysis. We observed that cell treatment with myrNefSF2

increases about twice the production of sEVs but not that of m/lEVs. The analysis of the vesicular production induced by cell treatment with Nef mutants suggests that some of its conserved domains influence sEVs production. In addition, all the Nef proteins used in this work are incorporated into the sEVs fraction but not in the m/lEVs fraction. Moreover, the produced sEVs, once used to treat the cells, can be internalized by differentiated THP-1 cells. Further studies are in progress to better understand the process and to analyze the effects induced by the vesicles containing myrNefSF2. Supported by grants from MIUR - Departments of Excellence - 2017 - legge 232/2016 - art.1, commi 314–337 awarded toDept. of Science - University Roma Tre - Rome - Italy for 2018–2022

References

[1] Coscia C, Parolini I, Sanchez M, Biffoni M, Boussadia Z, Zanetti C, Fiani ML, Sargiacomo M. Generation, Quantification, and Tracing of Metabolically Labeled Fluorescent Exosomes. Methods Mol Biol. 2016;1448:217-35. [2] Percario Z A , Ali M, Mangino G, Affabris E. Nef, the shuttling molecular adaptor of HIV, influences the cytokine network, Cytokine & Growth Factor Reviews,Volume 26, Issue 2,2015,Pages 159-173,ISSN 1359-6101 [3] Schorey JS, Cheng Y, Singh PP, Smith VL. Exosomes and other extracellular vesicles in host-pathogen interactions. EMBO Rep. 2015 Jan;16(1):24-43 [4] 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;7(1):1535750. Published 2018 Nov 23. [5]Wurdinger T, Gatson NN, Balaj L, Kaur B, Breakefield XO, Pegtel DM. Extracellular vesicles and their convergence with viral pathways. Adv Virol. 2012;2012:767694.

Strawberry nanovesicles as allergen vehicles: A preliminary proteomic

investigation.

G. Antonucci1,3, C. Stanly1, R. Bokka1, M. Moubarak1,2, A. P. Ramos1,4, I. Fiume1, I. Giangrieco1, M. A. Ciardiello1 and G. Pocsfalvi1

1. EVs-MS Research group, Institute of Biosciences and BioResources, National Research Council (CNR), Napoli, Italy

2. Faculty of Agriculture, University of Damanhour, Egypt 3. Department of Biology, University of Federico II, Napoli, Italy

4. Department of clinical and experimental medicine, University of Campania Luigi Vanvitelli, Caserta, Italy

Strawberry (Fragaria species) is one of the most popular fruits that is consumed in large quantities all around the world for its delicious taste. Besides being delicious, they have numerous beneficial properties such as high content of vitamin C and represent a valuable aid in reducing low density lipoproteins like cholesterol. [1] Eating strawberry can be a delightful experience but for people who have strawberry-related allergies it could cause a range of symptoms. The role of extracellular cellular vesicles (EVs) in intercellular communication is well documented. [2] They are known to carry proteins, lipids and nucleic acids and other molecules from one cell to another and, in certain cases, nanovesicles isolated from edible sources are even known to take part in interspecies communication. [3] Here we present for the first time the isolation of nanovesicles from Fragaria ananassa. The nanovesicles were isolated using ultracentrifugation-based methods. The protein contents of the vesicle-enriched samples were quantified and the protein profiles were monitored on SDS-PAGE analyses. In-solution protein digestion and LC-MS/MS-based shotgun proteomics analysis was performed to study the protein contents of the micro (MVs) and nanovesicles (NVs) enriched fractions. The analysis of our proteomic data provided evidence of the presence of a major allergen known in strawberry, Allergen Fra a 1 (Q5ULZ4.2) molecular mass 11113 Da. The NV fraction were further purified by double sucrose cushion /D2O ultracentrifugation and differential solubilisation method using 50 mM Tris-HCl pH 8.5 to confirm if the allergens were co-purified during vesicle isolation or if they are associated to the vesicles. In-solution digestion and shotgun proteomics analysis still revealed the presence of Allergen Fra a 1 in the purified NV fraction as well. Further studies needed to confirm if our findings are specific to the strawberry samples. In addition, our findings raise the question of nanovesicles could be vehicles of allergens? References 1. J.M., A.-S. et al. One-month strawberry-rich anthocyanin supplementation ameliorates

cardiovascular risk, oxidative stress markers and platelet activation in humans. J. Nutr. Biochem. (2014). doi:10.1016/j.jnutbio.2013.11.002

2. Pérez-Bermúdez, P., Blesa, J., Soriano, J. M. & Marcilla, A. Extracellular vesicles in food: Experimental evidence of their secretion in grape fruits. Eur. J. Pharm. Sci. (2017). doi:10.1016/j.ejps.2016.09.022

3. Pocsfalvi, G. et al. Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular vesicle populations. J. Plant Physiol. (2018). doi:10.1016/j.jplph.2018.07.006

IL-3R blockade on tumor derived endothelial cells impairs solid tumor formation and metastasis by modifying the cargo of extracellular

vesicles (EVs)

Tatiana Lopatina, Cristina Grange, Victor Navarro, Giusy Lombardo, Massimo Cedrino, Marta Tapparo, Giovanni Camussi, and Maria Felice Brizzi

Department of Medical Science, University of Turin, Italy

We have previously demonstrated that IL-3 receptor (IL-3R) blockade in tumor derived endothelial cells (TEC) induced the release of extracellular vesicles (anti-IL-3R-EVs) promoting the regression of in-vivo TEC-derived neovessels. In the present study, anti-IL-3R-EV effects were evaluated on tumor cell growth. Different tumor cell lines have been evaluated in vitro. Among them only MDA-MB-231 cells were inhibited in term of proliferation and migration upon anti-IL-3R-EV treatment. Moreover, anti-IL-3R-EV treatment increased the number of apoptotic cells, reduced MDA-MB-231 sphere formation and the expression of Twist. In the in vivo experiments anti-IL-3R-EV treatment induced regression of tumor vascular tree, as well as the number of liver and lung metastasis, as shown by human HLA-I staining. Histologic analysis demonstrated that -catenin and Twist were reduced in tumors obtained by animals treated with anti-IL-3R-EVs. In addition anti-IL-3R-EVs reduced lung metastasis generated by intravenous injection of MDA-MB-231 cells. Proteomic analysis on EVs demonstrated that anti-IL-3R-EVs were depleted of -catenin, VEGF, and TGF. In addition, EV miRnomic analysis demonstrated that IL-3R blockade reduced the expression of miR-24 and increase the expression of miR-214 in anti-IL-3R-EVs. In vitro and in vivo experiments using EVs depleted of miR-24 recapitulate the anti-IL-3R-EV effects on tumor cell migration, Twist expression and lung metastasis. Overall, these data provide the proof of concept that IL-3R blockade on TEC interferes with the pro-tumorigenic effects of their released EVs.

Raman spectroscopy unveils specific differences in plasma derived EVs

from ALS patients

C. Morasso1, D. Sproviero2, M. Giannini2,3, O Pansarasa2, R. Vanna1, A. Ricciardi1, C. Mimmi2, S. Gagliardi2, A. Bonizzi4, F. Corsi1,4, C. Cereda2

1) Laboratory of Nanomedicine and Molecular Imaging, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy

2) Genomic and post-Genomic Center, IRCCS Mondino Foundation, Pavia, Italy 3) Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy 4) Department of Biomedical and Clinical Sciences “Luigi Sacco”, University of Milan, Milano,

Italy.

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease, is characterised by the rapid progressive degeneration of the upper and lower motor neurons [1]. Study of EVs, extracellular vesicles (MVs, microvesicles and EXOs, exosomes) from plasma of ALS patients have the advantage of being relatively noninvasive and capable of monitoring neuronal and other brain cell health. A particular interest in plasma derived EVs of ALS patients is rising in the literature [2], but still, there is a lot to be known.

Raman spectroscopy is a relatively new approach that, by analysing of the inelastic scattering of light emitted by a sample when is irradiated with a Laser light, can provide in few minutes a complete biochemical characterization of EVs without using any particular reagent or without the need to target a predefined molecule. Besides, as most of the modern Raman instrument are equipped with a microscope set up, the laser light used can be focused on an area of few squared microns and thus the analysis can be performed on a very small amount of sample. For these reason recently a few group tested the possibility of using Raman spectroscopy in order to obtain a general overview of their complex biochemical composition [3,4]. The aim of this study was to characterize MVs and EXOs from plasma of ALS patients and CTRLs by Raman spectroscopy.

In our study, extracellular vesicles were extracted from the plasma of 20 subjects with ALS and 20 healthy controls by ultracentrifugation. Vesicles were dried on top of a CaF2 slide and Raman spectra were acquired using a 633 nm laser line.

The results obtained show that vesicles extracted by patients with ALS are characterized by a specific biomolecular pattern that differs from the one of HC mostly on the peaks relative to aromatic amino-acids such as phenylalanine, nucleic acid content and lipids. This result suggest that the analysis of exosomes could be a potential plasmatic biomarker for ALS disease.

[1] Morgan, S, Orrell, RW. 2016 Pathogenesis of amyotrophic lateral sclerosis. Br Med Bull; 119, 87–98. 10.

[2] Sproviero, D., La Salvia, S., Giannini, M., et al., 2018. Pathological proteins are transported by extracellular vesicles of sporadic Amyotrophic Lateral Sclerosis patients. Front Neurosci.; 12, 487.

[3] Krafft, C., Wilhelm, K., Eremin, A., 2017. A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. Nanomedicine 13(3), 835-841.

[4] Gualerzi, A., Niada, S., Giannasi, C., et al. 2017. Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells. Scientific Report 7(1), 9820.

Implications of synergistic mitochondrial and lysosomal impairment on EV secretion

F. Guerra1, A. Picca2,3, R. Beli1, M. Mitruccio 1, G. Girolimetti4, G. Gasparre4, T. Cocco5, E.

Marzetti3 and C. Bucci1

1Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Via Provinciale Lecce-Monteroni 165, 73100 Lecce, Italy;

2 Catholic University, Institute of Internal Medicine and Geriatrics, 00168 Rome, Italy; 3Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, 00168 Rome, Italy;

4 Department of Medical and Surgical Sciences (DIMEC), Medical Genetics Unit, University

Hospital S. Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy; 5 Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari

“A. Moro”, 70122 Bari, Italy;

Emerging evidences suggest a key role of the crosstalk between mitochondria and lysosomes in cellular physiology and its dysregulation in neurodegenerative disease [1]. In fact, it is known that mitochondrial impairment influences lysosomal function and viceversa. We recently demonstrated alterations of the late endocytic pathway in primary fibroblasts from a patient affected by juvenile Parkinson’s Disease, harboring frameshift mutation in mitochondrial DNA (mtDNA). In these cells, we observed, besides impairment of mitochondrial biogenesis and decrease of mitochondrial mass and membrane potential, accumulation of enlarged lysosomes, reduced autophagic flux, decreased Cathepsin D maturation and inhibited lysosomal activity. We also discovered that this synergist impairment of both organelles causes growth arrest and premature senescence [2]. In this scenario, it is possible to imagine the vicious circle in which damaged mitochondria induce lysosomal dysfunction and lysosomal dysfunction does not allow elimination of damaged mitochondria by autophagy. Nevertheless, the cells can remove unwanted materials through the release in the extracellular medium of specialized vesicles [3]. Thus, we hypothesize that mitochondrial and lysosomal crosstalk dysfunction can cause secretion of extracellular vesicles (EVs) containing mitochondrial particles and/or mitochondrial proteins contributing to inflammation, a feature of Parkinson’s pathogenesis and/or progression. To demonstrate this hypothesis, we have collected sera from 20 patients (with a diagnosis of PD according to the Queen Square Brain Bank criteria) and 20 control individuals (sex- and age-matched people without any signs of parkinsonism or potential premotor symptoms) and we started to analyze EVs. Preliminary data indicate that EVs contain specific mitochondrial proteins with different patterns in patient and control cells. It is known that mitochondrial-derived vesicles (MDVs) are normally delivered to multivesicular bodies (MVBs) and late endosomes to reach then lysosomes for degradation, and they are part of mitochondria quality control mechanisms. Transport of MDVs to MVBs explains the presence of mitochondrial proteins in exosomes and this process could be altered in Parkinson’s diseases and in the case of lysosomal dysfunction. We are now analyzing the type and content of EVs of patient and control sera and we are quantifying mediators of the inflammatory response to find a possible correlation. In conclusion, we believe that secretion of EVs containing mitochondrial proteins could cause Parkinson’s disease inflammatory response, which induces dopaminergic neurodegeneration, and finally loss of dopaminergic neurons, and we are investigating mechanisms of formation and secretion of EVs in Parkinson’s disease. References [1] Plotegher, N., Duchen, M.R. 2017. Crosstalk between Lysosomes and Mitochondria in Parkinson's Disease. Front Cell Dev Biol 5, 110. [2] Guerra, F., Girolimetti, G., Beli, R., Mitruccio, M., Pacelli, C., Ferretta, A., Gasparre, G., Cocco, T., Bucci, C. 2019. Synergistic Effect of Mitochondrial and Lysosomal Dysfunction in Parkinson's Disease. Cells 8(5), E452. [3] Baixauli, F., López-Otín, C., Mittelbrunn, M. 2014. Exosomes and autophagy: coordinated mechanisms for the maintenance of cellular fitness. Front. Immunol. 5, 403.

A potential role for extracellular vesicles in smooth muscle cells-

endothelial cells communication during human pulmonary arterial

hypertension

Ilaria Passalacqua1, Julie Rodor2, Raghu Bhushan3, Fernando de la Cuesta2, Andrew H. Baker2

1 Centro Interdipartimentale di Biologia Cardiorespiratoria, Azienda Ospedaliera Pisana, Università di Pisa, Pisa

2 Centre for Cardiovascular Science, Queen’s Medical Institute, University of Edinburgh, Edinburgh, Scotland

3 Yenepoya Research Centre, Yenepoya University, Deralakatte, Mangalore, India

Pulmonary arterial hypertension (PAH) is a pathology identified by acute remodelling of distal pulmonary arteries, right ventricular dysfunction, and increased pulmonary vascular resistance that promotes heart failure. Additionally, heritable PAH is caused by a mutation that results in impairments of the TGFβ superfamily signalling pathway. Previously regarded as a largely untreatable disease, recent studies have utilised TGFβ1 treatment of Human Pulmonary Ar-terial Smooth Muscle Cells (HPASMCs) to mimic in vitro PAH mechanisms behind the dis-ease. Although extracellular vesicles (EVs) have been shown to influence the vascular envi-ronment, their role remains obscure for many pathologies. Recent work in our laboratory demonstrated that EVs are involved in communication between vascular cells during PAH. The aim of this project has therefore been to try and assess whether EVs from HPASMCs treated with an excess of TGFβ1 are involved in transporting functional cargo to Human Arte-rial Endothelial Cells (HPAECs) and whether this cargo may affect them within the vascular microenvironment of PAH. In order to demonstrate the transfer of mRNA and its translation into protein during EVs uptake, we optimised the Cre-loxP method for use in primary cell cul-tures. Using this method, we were able to determine whether HPAECs had uptaken circulating EVs during PAH-simulated HPASMC cell cultures. The method works simply by enclosing Cre proteins upon donor HPASMC EVs while recipient cells (HPAECs) show whether Cre+ EVs have been taken up by switching from red to green fluorescence. Single-stranded low-input RNAseq was used to characterise HPASMC-EVs cargoes. Fluorescence-activated cell sorting of GFP out of DsRed positive cells (ratio of 1.53±0.26%) demonstrated that using our Cre-loxP system we could observe the transfer and translation of Cre-mRNA into protein from HPASMC-EVs to HPAECs. Interestingly, this transfer was related to TGF-β1 treatment which resulted in the marked increase of EV uptake from HPAEC but not an increase in the number of EVs released by HPASMCs. We also analysed the cargoes present in EVs from control and TGFβ1 treated HPASMCs measuring 2417 differentially expressed transcripts in HPASMC-EVs. Among these, a subset of 759 RNAs was found to be significantly enriched in Control EVs when compared to their donor cells. Furthermore, EVs from TGFβ1 treated cells showed 90 differential transcripts when compared to Control EVs. Gene Ontology Enrichment Analysis showed that these transcripts were typically associated with cellular differentiation, migration, and response to wounding: all mechanisms associated with PAH development. Preliminary experiments show that after treating HPAECs with HPASMC-EVs, their cellular phenotype changed slightly, including some of common endothelial markers; Although, this behaviour needs to be investigated further. In summary, the present results obtained using our in vitro system, demonstrate that HPASMC-EVs release functional cargoes that can differentially reg-ulate several transcripts that are critical for the remodelling of the vasculature during PAH development. However, more research on these preliminary results needs to be undertaken before the role of HPASMCs-EVs during PAH is more clearly understood.

Osteosarcoma-derived exosomes and their role in bone remodelling

and angiogenesis.

L. Raimondi1, A. De Luca1, A. Gallo2, V. Costa1, N. Cuscino2, M. Manno3, V. Carina1, D. Bellavia1, A. Conigliaro4, R. Alessandro4, P.G. Conaldi2 and G. Giavaresi1.

1 IRCSS ISTITUTO ORTOPEDICO RIZZOLI, Bologna, Italy.

2 IRCCS ISMETT, Department of Research, Palermo, Italy.

3 National Research Council of Italy, Institute of Byophysics, Palermo, Italy.

4 Department of Biomedicine, Neuroscience and Advanced Diagnostics (Bi.N.D.), Section of Biology and Genetics, University of Palermo, Palermo, Italy.

Osteosarcoma (OS) is a primary malignant bone tumor, characterized by high degree of malignancy, early metastasis and poor prognosis. Bone microenvironment provides growth and survival signals crucial for OS initiation and rapid progression. Communications inside tumor microenvironment are guaranteed through multiple ways and cancer-derived exosomes strongly foster tumor cell dissemination and early events in metastasis. In our study, we evaluated the contribute of OS-derived exosomes (OS-EXs) in the establishment of the tumor niche, focusing our attention on the effects induced in bone remodelling and angiogenesis. OS-EXs promoted osteoclasts differentiation and sustained bone resorption. Furthermore, we provide evidences that OS-EXs strengthened tumor angiogenesis via tube formation assay of Huvec cells and induced angiogenic markers expression. Otherwise, exosomes from human primary osteoblast did not induce the same effects, both for bone remodeling and angiogenesis. By performing small RNA sequencing through NGS Illumina platform we analyzed the miRNA cargo from OS-EXs and their parental cells; notably, we found a unique molecular profile of exosomal miRNAs. Indeed, bioinformatic analysis by DIANA-mirPath revealed the involvement of the exosomal miRNAs with various biological processes and carcinogenesis. Finally, we focused our attention on two miRNAs: miR-148a and miR-21-5p, whose expression increased after OS-EXs treatment in the pre-osteoclastic Raw264.7 cells and Huvec cells. Their enforced expression in the target cells induced similar effects to those observed after OS-EXs treatment. Nevertheless, transfection with miR-148a and miR-21-5p inhibitors in target cells, previosuly treated with OS-EXs, counteracted the effects described above. Overall, our data highlighted a key role of OS-EXs inside tumor niche, also by a specific packaging of miRNAs.

Role of osteosarcoma-derived extracellular nanovesicles in angiogenesis

F. Perut ,1 L. Roncuzzi,1 N. Zini,2 N. Baldini1,3

1IRCCS Rizzoli Orthopaedic Institute, Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Bologna, Italy.

2 CNR – National Research Council, Institute of Molecular Genetics, Bologna, Italy. 3University of Bologna, Department of Biomedical and Neuromotor Sciences, Bologna, Italy

Angiogenesis is a pivotal process in osteosarcoma (OS) development and progression [1]. Angiogenesis involves a number of different players among which extracellular nanovesicles (EVS) have been recently proposed as efficient cargo of pro-angiogenic mediators. A common feature of solid tumors is their acidic microenvironment, resulting from an increased energetic metabolism [2]. Recently, we have demonstrated that extracellular acidosis contributes to chemoresistance of OS cells [3]. In cancer other than sarcoma, tumor induced extracellular acidity has been associated with an increased exosomes release and uptake [4]. In this study, we investigated the role of human OS-derived EVs on tumor angiogenesis, and the influence of acidity of tumor microenvironment in this process. EVs were isolated by differential centrifugation of culture media from 143B OS cells grown at different pH (6.5 or 7.4). EVs size (30–100 nm) and morphology was verified by transmission electron microscopy. The release of EVs by OS cells was significantly increased at acidic compared to neutral pH (p = 0.002). After 24 h, EVs were internalized by HUVEC cells. OS-derived EVs significantly promoted the tubulogenesis in vitro (p=0.034), and induced new blood vessel growth on chicken chorioallantoic membrane (CAM) in vivo. The lengh of vessels and the number of branch points was significantly higher on CAM treated with EVs derived from OS cells cultured at acidic pH (p=0.018 and p=0.0026). Angiogenesis-related proteins (i.e. SerpinE1, TIMP1, Thrombospondin -1, uPA, VEGF, PTX3, CD105) and pro-angiogenic miRNA (i.e. miR21-5p, miR26a-5p, miR106a-5p and miR92a-3p) were detected in OS-derived EVs. Our findings demonstrated that EVs released by OS cells present a specific repertory of proteins and miRNAs associated with angiogenesis. OS-derived EVs induce angiogenesis, both in vitro and in vivo and this activity is increased by the acidity of tumor microenvironment. Supported by AIRC (IG 2014—ID15608 and IG 2018—ID21403 to N.B.) and by The Italian Ministry of the Health, Financial Support for Scientific Research (5 per 1000 2016 to N.B.). References [1] Zhu, L.; McManus, M.M.; Hughes, D.P. Understanding the Biology of Bone Sarcoma from Early Initiating Events through Late Events in Metastasis and Disease Progression. Front. Oncol. 2013, 17, 230. [2] Corbet, C.; Feron, O. Tumour acidosis: From the passenger to the driver’s seat. Nat. Rev. Cancer 2017, 17, 577–593. [3] Avnet, S.; Lemma, S.; Cortini, M.; Pellegrini, P.; Perut, F.; Zini, N.; Kusuzaki, K.; Chano, T.; Grisendi, G.; Dominici, M.; et al. Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance. Oncotarget 2016, 7, 63408–63423 [4] Parolini, I.; Federici, C.; Raggi, C.; Lugini, L.; Palleschi, S.; De Milito, A.; Coscia, C.; Iessi, E.; Logozzi, M.; Molinari, A.; et al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J. Biol. Chem. 2009, 284, 34211–34222.

Acute myeloid leukemia cells dysregulate normal hematopoiesis by releasing extracellular vesicles

I. Laurenzana1, S. Trino1, D. Lamorte1, M. Santodirocco2, L. Di Mauro2, P. Musto3, L. De Luca1 and

A. Caivano1

1 Laboratory of Preclinical and Translational Research, IRCCS Centro di Riferimento Oncologico della Basilicata (CROB), Rionero in Vulture, PZ, Italy

2 Transfusion Medicine Unit, Puglia Cord Blood Bank, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy

3 Hematology and Stem Cell Transplantation Unit, IRCCS CROB, Rionero in Vulture, PZ, Italy

Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal disorder of

hematopoietic stem/progenitor cells (HSPCs). During leukemogenesis, AML cells progressively

occupy and likely alter bone marrow (BM) niche where normal HSPCs reside. Despite recent

improvements in therapeutic approaches, only a fraction of AML patients is cured. Therefore, it is

crucial to identify new molecular mechanisms driving the AML pathogenesis.

Extracellular vesicles (EVs) are bi-layer membrane particles that shuttle a complex molecular

cargo which can be transferred to target cells and alter their functions. Tumor derived EVs promote

a tumor-supporting environment in non-malignant cells favoring cancer proliferation.

We investigated the effect of leukemia derived-EVs (LEVs) on healthy umbilical cord blood HSPCs

to explore their potential role in the induction of leukemic like-phenotype.

EVs were isolated from AML cell lines by centrifugation steps and analyzed for AML surface

markers by flow cytometry. Next, they were co-incubated with healthy CD34+ sorted cells. After 24

h, cell count, apoptosis, HSPC–specific cluster of differentiation markers, CXCR4 expression,

colony-forming unit and migration assays were assessed.

Our data showed: i) LEVs displaying specific AML surface markers; ii) a similar number of CD34+

cells in LEV treated-HSPCs, including HSCs, compared with controls; iii) no differences in

apoptosis between the two groups; iv) an increase of multipotent progenitor (MPP) associated with

a reduction of lymphoid-primed multipotent progenitors (LMPP) and multipotent lymphoid

progenitors (MLP) in treated cells; v) an increase of common myeloid progenitors (CMP), together

with reduction of granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid

progenitors (MEP) in treated group; vi) a reduction of colony-forming ability, including erythroid and

granulocytic colonies, in treated cells; vii) decreased levels of CXCR4 in LEV treated-HSPCs, that

were accompanied by reduced HSPC migration mediated by SDF-1.

Altogether, this study suggests that AML derived-EVs can modify BM niche by deregulating normal

hematopoiesis and by promoting an increase of MPP and CMP. Furthermore, they seem to induce

less attraction of HSPCs in BM niche by interfering with SDF1/CXCR4 axis. Strategies to block EV

production and secretion, as well as EV induced-reprogramming, could be a novel exciting

therapeutic approach in AML.

The role of extracellular vesicles in the removal of aggregated TDP43

responsible for ALS/FTD diseases

Elena Casarotto1, Daisy Sproviero2, Stella Gagliardi2, Barbara Tedesco1, Riccardo Cristofani1,

Veronica Ferrari1, Marta Chierichetti1, Paola Rusmini1, Mariarita Galbiati1, Cristina Cereda2, Poletti

Angelo1, Valeria Crippa1,

1 Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Department of excellence 2018-2022, Università degli Studi di Milano Via Balzaretti 9, 20133 Milano (Italy)

2 Mondino Foundation – IRCCS, Via Mondino 2, 27100 Pavia (Italy)

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two related neurodegenerative diseases. ALS is caused by the death of both upper and lower motoneurons, while FTD is characterized predominantly by circumscribed atrophy of the frontal and temporal lobes. ALS and FTD overlap each other. This is demonstrated by the presence of cognitive and behavioral dysfunction in up to 50% of ALS patients and by the presence of frontotemporal atrophy in patients with ALS. Moreover, these diseases are both characterize by the presence of TAR DNA binding protein 43 (TDP43) inclusions in affected cells. These inclusions, observed in 97% of patients with ALS and 50% of patients with FTD, are composed by TDP43 and its C-terminal fragments of 35 kDa (TDP35) and 25 kDa (TDP25). These fragments are highly aggregation-prone and probably neurotoxic. Thus, their removal is protective for cells. The mechanism responsible for the clearance of aggregates and misfolded proteins is the intracellular protein quality control (PQC) system. It consists of molecular chaperones/co-chaperones and the degradative pathways. PQC controls the folding status of proteins and prevents the aggregation of misfolded proteins by refolding them or degrading. Recent data demonstrated that also extracellular secretory pathway, represented especially by exosomes (EXOs) and microvesicles (MVs), might be involved in the removal of misfolded proteins from affected cells. Thus, we evaluated the role of EXOs and MVs in the secretion of TDP43 and its C-terminal fragments, using neuronal cell models. We used ultracentrifugation, that allowed us to separate MVs from EXOs on the basis of their dimension. Then we analyzed them through i) Nanoparticle Tracking Analysis (NanoSight) to establish their number and sizes, and ii) western blot analysis, to characterize their protein content. Our preliminary results show that TDP43, TDP35 and TDP25 are all secreted, mainly by MVs. In particular, we found that MVs are enriched of insoluble forms of TDPs and also of superoxide dismutase 1 (SOD1), another ALS-related protein. Finally, both in EXOs and in MVs, we observed the presence of some important PQC-components, suggesting an interplay between the two pathways. GRANTS: Fondazione Cariplo, Italy (n. 2017_0747); Università degli Studi di Milano e piano di sviluppo

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