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Figure S1 Exosomes do not influence the adipogenic differentiation of HC-MSCs A HC-MSCs were able to internalize NB exosomes when cultured with liposomes, PKH26-labeled exosomes isolated from several NB cell lines (10 ?g/mL) for 24 hours. Scale bar: 40 ?m B Co-culture of exosomes collected from IGR-NB8, SKNBe2C or Hela cells with HC-MSCs for 24 hours, showed that only exosomes produced by BM metastasis derived cell lines such as SKNBe2C had the ability to induce the transcription of the osteogenic genes. C Exposure to SH5YSY- or IMR32-derived exosomes (10 ?g/mL, 3 times a week) for 21 days in presence of adipogenic medium has no effect on the production of lipid drops. D Quantitation of Oil Red staining after 21 days in presence of adipogenic medium. E The mRNA level of PPARγ analyzed by Q-PCR and normalized for Gapdh expression. Values are mean±SEM of 3 indipendent experiments performed at least in duplicate: ns not significant, *P<0.05; **P<0.01; ***P<0.0001, analyzed by one-way ANOVA.Figure S2 Characterization of exosomes isolated from peripheral and BM blood plasma A NTA of 15 representative samples of exosomes isolated from control and patient peripheral and BM blood plasma. B SEM showing a population of heterogeneous-sized extracellular vesicles. Scale bar: 100 nm. C Table summarizing the minimum and the maximum size detected by SEM and the mean of three captures ±SEM determined by NTA for the 15 samples analyzed, in nanometers. D Representative immunoblots showing expression of Calnexin, CD63, CD9, TSG101, HSP90, GAPDH in exosomes isolated from plasma. E Exosomal protein quantitation (?g/?l) performed by BCA of 15 representative samples of exosomes isolated from HC and NB patients. For the characterization the representative samples have been chosen as follow: 3 samples of PB plasma from HC, 3 samples of NMBM-PB plasma, 3 samples of MBM-PB plasma, 3 samples of NMBM-BM plasma and 3 samples of MBM-BM plasma. PB, peripheral blood; BM, bone marrow.Figure S3 Evaluation of miR-375 expression in a patient before and after BM relapse. Slides were backed at 60 °C for 1 hour and half and store ON at room temperature. After denaturation at 90° C for 4 minutes, miR-375, U6 small nuclear probe (positive control) and scramble-miR probe (negative control) were added directly on permeabilized and dehydrated tissue sections. After nuclear counter staining slides were finally washed, dehydrated and analyzed by light microscopy. Here, we show representative images of a patient with localized NB at the diagnosis, who presented a negative staining for miR-375 in the BM (upper panel), which became positive for miR-375 staining after metastatic BM relapse as observed in the right and left iliac crest biopsy (middle and bottom panel). The histological expression of synaptophysin (indicating NB invasion) is also shown. Magnification 20X, Scale bar: 50 ?m.Figure S4 Expression level of RANKL, IL-6 and IL-8 in HC-, NMBM- and MBM-MSCs.A Q-PCR analysis of the relative levels of RANKL, IL-6 and IL-8 mRNA expression (2-ΔΔCt method, normalized to Gapdh mRNA level) were determined in HC-, NMBM- and MBM-MSCs. B Graphs depicting the comparison of MBM-MSCs group splitted for metastatic localization to BM (red triangles) or BONE (blue triangles) with HC- and NMBM-MSCs. The panels showed that only RANKL is significantly overexpressed in NB-derived MSCs, while a strong overexpression of both RANKL and IL-8 was observed only in MBM-MSCs. These data indicate a potential role of MSCs in osteoclastogenesis at BM metastatic level. Data are reported as means±SEM of technical triplicate, n= 7 for HC-MSCs group, n= 4 for NMBM-MSCs group and n=8 for MBM-MSCs group (n=3 for MBM-MSCs only with BM metastasis, n=5 for MBM with bone matastasis) * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 analyzed by one-way ANOVA. Supplementary TablesSupplementary Table 1. A univariable analysis keeping into account patients younger or older than 18 months, different stages (L1/L2, M), MYCN status (Amplified, not amplified/gain) and BM infiltration (No, Yes). UNIVARIABLE ANALYSISMULTIVARIABLE ANALYSISVariablesCrude OR(95% CI)pAdj*OR(95%CI)pAge at diagnosis<18 months11≥18 months3.4(0.9-12.1)0.0592.8(0.7-11.4)0.138StageL1/L2 (Ref)1M 4.7(1.5-14.8)0.007MYCNAmplified (Ref)11Not Amplified/Gain 1.1(0.3-3.5)0.8491.6(0.4-5.9)0.426BM InfiltrationNo (Ref)11Yes 3.8(1.2-11.6)0.0163.6(1.1-12.2)0.032OR, odds ratios; in bold significant p values.Supplementary Table 2. Characteristics of patients with NB evaluated for miR-375 in situ hybridizationVariablesN%INSS111182915361043456INRGSSL11423L21220M3456Age Groups (months)<181728≥184371MYCNNot amplified3253Gain915Amplified1728NA23INPC-histological classificationFavorable2743Unfavorable2135NA1220miR-375Score 03456Score 11626Score 21016INSS, International Neuroblastoma Staging System; INRGSS, International Neuroblastoma Risk Group Staging System; INPC, International Neuroblastoma Pathology Classification (the Shimada system). NA, not available.Supplementary Materials and MethodsCharacterization of exosomes from PlasmaProtein concentration was measured by bicinchoninic acid assay (BCA, Pierce, Thermo Fisher Scientific). For scanning electron microscopy (SEM), 10 ?l of EX suspension were deposited on formvar coated copper grids (Agar Scientific Ltd, UK), fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences, PA, USA) and air-dried. Grids were then attached on metal stubs, coated with chrome to a thickness of 10 nm and observed with a ZEISS - LEO 1525 (Laboratorio Universitario di NAnomateriali - University of Perugia). The NS500 nanoparticle characterization system (NanoSight) equipped with a blue laser (405 nm) was used to characterize exosome size and particle number (CNIO, Madrid).For WB exosomes were lysed with Cell lysis buffer (Cell Lysis Buffer (10X) #9803 Cell Signaling Thecnology) containing 10 mM phenylmethylsulphonyl fluoride as a protease inhibitor (PMSF 93482 Sigma). Lysates were incubated on ice for 15 min and centrifugated at 13 000 × g for 20 min at 4°C. Equal micrograms (20 ?g) of proteins quanti?ed with BCA assay (Thermo Scientific) and boiled in SDS sample bu?er (2x Laemmli Sample Buffer BIORAD cat.#161-0737), were resolved on 10% SDS-PAGE and transferred to PVDF membranes (Immun-Blot? PVDF Membrane for protein Blotting BIORAD cat.#162-0177). Blots were blocked for 1 h in PBS-T (PBS plus 0.05% Tween-20), 5% nonfat, dried milk and probed overnight at 4°C with anti-TSG101 (4A10) ab83 (Abcam), anti-CD9 (C-4) sc-13118 (Santa Cruz Biotechnology), CD63 (H-193): sc-15363 (Santa Cruz Biotechnology), anti-HSP 90 (4F10): sc-69703 (Santa Cruz Biotechnology), anti-Calnexin (E-10): sc-46669 (Santa Cruz Biotechnology), anti-GAPDH (D16H11) XP (Cell Signaling Technology?). Immunocomplexes were detected with horseradish peroxidase-conjugated species-speci?c secondary antibodies (Santa Cruz Biotechnology) followed by enhanced chemiluminescence reaction with Immobilion Western Chemiluminescence HRP substrate WBKLS0100 (Millipore). ................
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