Cell cultures
An immortalized human bone marrow-derived mesenchymal stem cell (MSC) line (3A6) was a present from Dr. Shih-Chieh Hung of Veterans Normal Hospital-Taipei (Taipei, Taiwan) [32] and was characterised and utilized in our earlier research [33, 34]. Daoy, U87-MG, PC3, and NIH/3T3 cell traces had been initially obtained from American Kind Tradition Assortment (ATCC, Manassas, VA, USA). The ONS76 cell line was initially obtained from Japanese Assortment of Analysis Biosources Cell Financial institution (NIBIOHN, Osaka, Japan). Daoy-MYCN, a extremely metastatic by-product of Daoy that overexpresses MYCN, was beforehand constructed by means of the service of the Nationwide RNAi Core Facility (Academia Sinica, Taipei, Taiwan) [7]. HEK293FT and HEK293A cell traces had been initially obtained from Invitrogen (ThermoFisher Scientific, Waltham, MA, USA). MSCs had been cultured in low-glucose Dulbecco’s modified Eagle medium (DMEM) (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Cytiva) and 1% penicillin-streptomycin (PS) (Cytiva). Daoy and U87-MG cells had been cultured in Eagle’s minimal important medium (Cytiva) supplemented with 10% FBS and 1% PS. ONS76 and PC3 cells had been cultured in RPMI 1640 medium (Cytiva) supplemented with 10% FBS and 1% PS. HEK293 cells had been cultured in high-glucose DMEM (Cytiva) supplemented with 10% FBS, 1% PS, and 1% non-essential amino acids (Cytiva). All cell traces had been incubated at 37 °C in 5% CO2 and had been routinely passaged upon < 80% confluence for the 3A6 cell line and upon ~ 90% confluence for the opposite cell traces.
Plasmid development and gene manipulation
Complementary (c)DNA of human Lamp2 variant B was bought from OriGene (cat. no. RC200456, Rockville, MD, USA). A a number of cloning web site was designed close to the N-terminus of the Lamp2 protein to insert a nucleotide sequence coding for the specified peptide, offered in Supplementary Desk S1. Then, your complete Lamp2 with the inserted sequence was cloned into the pLAS3w.Pbsd plasmid to supply lentiviruses utilizing a protocol of the Nationwide RNAi Core Facility (Academia Sinica, Taipei, Taiwan), HEK293FT cells, and the Maestrofectin transfection reagent (Omics Bio, Taipei, Taiwan), all in accordance with the producers’ protocol. Lentiviral transduction was carried out on MSCs, adopted by choice with 2.5 µg/mL blasticidin to ascertain steady traces.
Extracellular vesicle (EV) isolation
MSCs had been cultured beneath regular circumstances to achieve round 90% confluency. Then, cells had been washed with 1× phosphate-buffered saline (PBS) to take away traces of the outdated medium and had been then replenished with serum-free medium. After 48 h, conditioned medium was collected in a 50-mL centrifugation tube and centrifuged at 3000 ×g for 20 min to pelletize useless cells. The supernatant was filtered utilizing a 0.22-µm Millex syringe filter (cat. no. SLGVR33RS, Merck Millipore, Burlington, MA, USA) to exclude bigger particles. Subsequent, the filtered supernatant was concentrated 2–3-fold utilizing a Vivaspin Turbo 15 5000 MWCO ultrafiltration column (cat. no. VS15T12, Sartorius, Göttingen, Germany), after which was subjected to exosome isolation utilizing an exoEasy Maxi Equipment (cat. no. 76064, Qiagen, Hilden, Germany) following the producer’s directions. Within the ultimate step, EVs had been eluted in 1 mL of elution buffer per column. Lastly, the eluate was buffer-exchanged with 1× PBS and concentrated to the specified quantity utilizing a Vivaspin Turbo 4 3000 MWCO ultrafiltration column (cat. no. VS04T91, Sartorius). Roughly 1011 EVs had been remoted and recovered from ~ 2 × 107 MSCs cultured in 4 p100 dishes.
Nanoparticle monitoring evaluation (NTA) and zeta potential evaluation
The particle dimension and focus analyses of EV samples was carried out with a NanoSight NS300 (Malvern Panalytical, Malvern, UK) geared up with a inexperienced laser and a CMOS digicam following the producer’s directions. Earlier than measurement, samples had been diluted with 1× PBS to attain a measured particle focus inside the 108–109 particles/mL vary. Zeta potential measurement was performed with a Zetasizer Nano ZSP (Malvern Panalytical). For every pattern, knowledge acquired from not less than three repetitions had been analyzed utilizing the producer’s software program.
Transmission electron microscopy (TEM)
TEM was carried out by means of the service of the Imaging Core of Taipei Medical College Core Facility Middle utilizing an HT-7700 TEM (Hitachi Excessive-Applied sciences, Tokyo, Japan). A normal unfavorable staining protocol was utilized to stain the EV samples. Briefly, 10 µL EVs in 1× PBS had been loaded onto a TEM grid for two min earlier than being largely eliminated with a micropipette. Subsequent, 10 µL of a 2% uranyl acetate answer was utilized onto the grid for two min and gently eliminated. Lastly, 10 µL of double-distilled (dd)H2O was utilized and shortly eliminated. The grid was air-dried for 10 min with no therapy with glow discharge earlier than TEM picture seize. Aside from EV buildings, an uneven staining distribution and synthetic distinction variations of the untreated floor could also be noticed within the background of samples ready by this technique.
EV labeling
For in vitro assays, roughly 5 × 1010 EVs had been labeled in a single response of the ExoGlow Membrane EV Labeling Equipment (cat. no. EXOGM600A-1, System Biosciences, Palo Alto, CA, USA) following the producer’s protocol. As advisable by the producer, the ExoQuick-TC Exosome Precipitation Resolution (cat. no. EXOTC10A-1, System Biosciences) was used to make sure maximal elimination of free dye, adopted by low-speed centrifugation of 1000 ×g at 4 °C for 60 min to precipitate the labeled EVs, which had been gently washed and resuspended in 1× PBS. All labeled EVs had been subjected to an NTA to re-determine EV numbers earlier than use in downstream assays.
EV transfection with siRNAs
The LOXL1-AS1-targeted siRNA #1196 (si1196) and the non-targeted siRNA (siNC) had been bought from GenePharma (Shanghai, China). Non-targeted fluorescein isothiocyanate (FITC)-conjugated siRNA (siNC-FITC) was bought from Santa Cruz Biotechnology (cat. no. sc-36869, Dallas, TX, USA). Sequences of those synthesized siRNAs are offered in Supplementary Desk S2. In complete, 5 × 109 EVs and a pair of.5 µL of 10 µM siRNA had been transfected per response utilizing an ExoFect siRNA/miRNA Transfection Equipment (cat. no. EXFT200A-1, System Biosciences) following the producer’s protocol. After a 1-h incubation step, 2 µL of 1 µg/mL RNase A (cat. no. 11119915001, Roche, Basel, Switzerland) was added to the response for 30 min at 37 °C to degrade the untransfected siRNAs, adopted by clean-up steps offered by the transfection equipment to acquire a complete of 200 µL transfected EVs.
Fluorescence-based quantification
Absorbance measurement of EV samples and EV-treated cells. Primarily based on NTA outcomes of labeled EVs, the quantity of every pattern was adjusted with 1× PBS to attain an equal EV focus between samples. Then, 100 µL of pattern was loaded right into a black 96-well plate adopted by absorbance measurement at 465/645 nm (ExoGlow) or 488/580 nm (FITC). Subsequent, cells had been seeded in black 96-well plates at 2500 cells/properly in a single day. Previous medium was changed with 2.5 × 107 labeled EVs (~ 104 EVs/cell) blended in 100 µL 1× Hank’s balanced salt answer (HBSS) and incubated for 1 h at 37 °C. Then, cells had been gently washed thrice with 1× PBS, adopted by absorbance measured at 465/645 nm (ExoGlow). All absorbance measurements had been carried out with a microplate reader (CLARIOstar Plus, BMG Labtech, Ortenberg, Germany).
Move cytometry. The E1-3 peptide (FSRPAFL) [31] and a non-cell penetrating (NC) peptide (YDEEGGGE) [35] had been synthesized and conjugated with the fluorescent label N-rhodamine (cat. no. IS025, Cloud-Clone, Katy, TX, USA) (Supplementary Desk S1). Cells in regular monolayer tradition had been gently indifferent utilizing Accutase (BD Biosciences, Franklin Lakes, NJ, USA) till they’d fully separated into single cells, after which had been washed thrice with 1× PBS. In complete, 106 cells suspended in 100 µL of 1× PBS had been supplemented with 1 µg peptide and incubated for 1 h at 37 °C. After being washed thrice with 1× PBS to take away unbound peptides, cells had been subjected to circulate cytometry (BD FACSCanto II, BD Biosciences) to measure the fluorescence depth of PE: rhodamine. Move cytometric knowledge had been analyzed utilizing FlowJo software program vers. 10.10 (BD Biosciences).
Confocal fluorescence microscopy. Roughly 2.5 × 105 cells/properly had been seeded on a coverslip in a six-well plate in a single day. Then, cells had been handled with 2.5 × 109 labeled EVs (~ 104 EVs per seeded cell) properly blended in both 2 mL 1× HBSS for 1–3 h of incubation or 2 mL tradition medium for 16 h of incubation. Then, coverslips had been gently washed thrice with 1× PBS, fastened with 4% paraformaldehyde in 30 min, rewashed thrice with 1× PBS, and mounted on a glass slide utilizing the ProLong Gold Antifade mountant with the DAPI DNA Stain (cat. no. P36935, Invitrogen). Pictures had been captured with a Stellaris 8 Confocal Microscope (Leica Microsystems, Wetzlar, Germany), buying a number of fluorescence channels, together with DAPI, inexperienced fluorescent protein (GFP), FITC, and Cy3 (ExoGlow). Fiji/ImageJ software program was used to quantify the fluorescent depth and analyze sign co-localization in three or 4 completely different captures per pattern.
Cell-based purposeful assays
Cytotoxicity assay. EV toxicity towards cells was assessed utilizing the WST-1 Cell Proliferation Reagent (cat. no. 11644807001, Roche) in accordance with the producer’s protocol. Briefly, cells had been seeded right into a 96-well plate at 1000 cells/properly in 100 µL of tradition medium and supplemented with completely different EV doses as indicated. After 48 h, 10 µL of WST-1 reagent was added to every properly, adopted by incubation at 37 °C for 4 h and absorbance measurement at 440/630 nm wavelengths.
EV therapy on cells. Daoy-MYCN cells had been seeded at 2.5 × 105 cells per p60 dish. The subsequent day, cells had been handled with 2.5 × 109 EVs (~ 104 EVs per seeded cell) in 3 mL of recent tradition medium for 16 h. For comparability, cells with the identical seeding density had been instantly transfected with an equal quantity of siRNA utilizing the HiPerFect transfection reagent (cat. no. 301705, Qiagen, Hilden, Germany) in accordance with the producer’s protocol. Cells had been subsequently replenished with recent medium and cultured for an additional 32 h (i.e., 48 h after therapy started). Then, cells had been collected for a reverse-transcription qualitative polymerase chain response (RT-qPCR) evaluation to find out the knockdown effectivity or subjected to downstream purposeful assays, together with wound-healing, transwell, and tumor sphere-formation assays, carried out as beforehand reported [7] and briefly described as follows.
Wound-healing assay. In complete, 2.5 × 104 EV-treated cells in 100 µL of tradition medium had been seeded into every properly of two-well tradition inserts (Ibidi, Gräfelfing, Germany) to create a non-cell hole for microscopic picture captures at indicated time factors.
Transwell migration and invasion assays. Hanging tradition inserts with 8-µm pores (Merck Millipore, Burlington, MA, USA), both non-coated for the migration assay or precoated with 50 µL of 1 mg/mL Matrigel (Life Sciences, Corning, NY, USA) for the invasion assay, had been positioned in a 24-well plate containing 1% FBS tradition medium. Every insert was seeded with 5 × 104 EV-treated cells in serum-free medium adopted by incubation for 3 h (migration) or 12 h (invasion). Migrated cells had been fastened and stained with 0.5% crystal violet for quantification by cell counting utilizing microscopic photos or by absorbance measurement of crystal violet dissolved from stained cells.
Sphere-formation assay. Roughly 4 × 104 EV-treated cells had been seeded at a really low seeding density of 2000 cells/mL in outlined sphere-forming tradition medium utilizing an ultra-low-attachment T175 flask (day 0). At days 4, 7, and 10, 5 mL of recent medium and 5 × 109 EVs had been added to every flask. Then, your complete tradition of every pattern was subjected to sphere dimension measurement and counting, RNA extraction, and protein extraction.
Mobile RNA extraction, cDNA synthesis, and RT-qPCR evaluation
Complete mobile RNA was extracted with the NC RNA extraction reagent (EBL Biotechnology, Taipei, Taiwan) after which transcribed into cDNA utilizing the PrimeScript RT reagent equipment (Takara Bio, Shiga, Japan). Then, a real-time RT-qPCR was carried out utilizing gene-specific primer pairs listed in Supplementary Desk S3, a QuantiNova SYBR Inexperienced PCR equipment (Qiagen), and LightCycler 96 (Roche), all in accordance with the producers’ directions. The two− ΔΔCt technique was utilized to calculate gene expressions, with HSPCB because the housekeeping gene for normalization.
RT-qPCR-based quantification of siRNA copy numbers
Samples of EV had been subjected to RNA extraction utilizing a Complete Exosome RNA and Protein Isolation Equipment (cat. no. 4478545, Invitrogen) following the producer’s protocol of small RNA enrichment. As the ultimate step, 100 µL of nuclease-free H2O was used to elute RNA. To find out absolutely the si1196 copy quantity in these samples, a titration of 1011 si1196 copies/µL was made, adopted by 10-fold serial dilutions to make six titration samples starting from 106 to 1011 copies/µL. Subsequent, reverse-transcription into cDNA was carried out with a designed si1196-specific stem-loop primer, adopted by RT-qPCR detection with designed primer pairs (Supplementary Determine S2A and Desk S3). A no-RNA management for the reverse-transcription response and a no-template management for the RT-qPCR had been additionally included, and confirmed very excessive Cq values because the background sign (Supplementary Determine S2B). Titrations had been used to ascertain a regular curve of Cq ~ si1196 copy quantity/µL values (Supplementary Determine S2C). The si1196 copy quantity was decided for every EV pattern and was used to calculate the copy quantity per EV, the loading effectivity, and the steadiness of si1196 (Supplementary Determine S2D). All titration and management samples had been run alongside EV samples. Your complete experiment was repeated thrice utilizing independently ready EV, titration, and management samples.
Protein extraction and Western blot evaluation
Protein extraction, willpower of protein concentrations, and pattern blotting had been carried out as beforehand described [36]. To gather EV lysates, an EV pattern was additional concentrated utilizing a Vivaspin Turbo 500 3000 MWCO PES ultrafiltration column (cat. no. VS04T91, Sartorius) till ~ 75–100 µL remained. Then, 1 quantity of home made 4× RIPA answer was added to three volumes of EVs to attain a ultimate lysate in 1× RIPA buffer. Equal quantities of protein for whole-cell lysate and EV lysate had been loaded into every lane. One other pattern of low-glucose DMEM + 0.01% FBS lysate was additionally included to evaluate contamination of FBS in EV samples. First antibodies, together with anti-β-actin (cat. no. GTX109639, GeneTex, Irvine, CA, USA), anti-Bmi1 (D20B7, cat. no. #6964, Cell Signaling Know-how, Danvers, MA, USA), anti-bovine serum albumin (BSA; cat. no. 2A3E6, Santa Cruz Biotechnology), anti-calnexin (cat. no. #2433, Cell Signaling Know-how), anti-cluster of differentiation 63 (CD63; cat. no. 10628D, ThermoFisher Scientific), anti-CD81 (cat. no. sc-7637, Santa Cruz Biotechnology), anti-CD9 (cat. no. sc-59140, Santa Cruz Biotechnology), anti-CD90 (cat. no. 13801, Cell Signaling Know-how), anti-lamin A/C (cat. no. 10298-1-AP, ProteinTech, Rosemont, IL, USA), anti-Lamp2 (cat. no. GTX103214, GeneTex), anti-Nanog (D73G4, cat. no. 4903, Cell Signaling Know-how), anti-Oct4 (cat. no. GTX101507, GeneTex), anti-Sox2 (cat. no. GTX101507, GeneTex), anti-transforming progress issue (TGF)-β2 (cat. no. MAB612, R&D Techniques, Minneapolis, MN, USA), anti-TSG101 (cat. no. 72312, Cell Signaling Know-how), and anti-V5 (cat. no. A01724, GenScript, Piscataway, NJ, USA) had been probed at a 1:1000 dilution in a single day at 4°C. Second antibodies, together with anti-mouse (cat. no. 111-035-146, Jackson ImmunoResearch, West Grove, PA, USA) and anti-rabbit (cat. no. 111-035-144, Jackson ImmunoResearch), had been probed at a 1:104 dilution for 1 h at room temperature. Blots had been visualized utilizing a chemiluminescent detection equipment (T-Professional Biotechnology, New Taipei Metropolis, Taiwan) and Amersham Imager 600 (Cytiva). The Fiji/ImageJ software program was used to quantify blot lanes.
In situ hybridization
In situ hybridization in 1-µm-sectioned formaldehyde-fixed paraffin-embedded mouse tissues was carried out utilizing an RNAScope 2.5 HD Assay-RED equipment (Superior Cell Diagnostics, Newark, CA, USA) with designed gene-specific probes for human LOXL1-AS1, human peptidyl-prolyl cis-trans isomerase B (PPIB, as optimistic management RNA), and bacterial dapB (as unfavorable management RNA) following the producer’s protocol. RNA expression was decided by automated detection of chromogenic dots utilizing Fiji/ImageJ software program and was normalized to the optimistic management RNA sign.
Immunohistochemical (IHC) staining
IHC staining had been carried out utilizing an automatic staining module (BenchMark XT system, Roche/Ventana Medical Techniques, Tuscon, AZ, USA). Briefly, tissue sections had been deparaffinized with EZ Prep (Roche/Ventana Medical Techniques) at 75 °C, warmth pretreated in Cell Conditioning 1 (CC1) for antigen retrieval at 76–100 °C after which incubated with the TGF-β2 antibody (1000x, Abnova, PAB12739) for 64 min at 37 °C after inactivation of the endogenous peroxidase utilizing UV inhibitor for 4 min at 37 °C. Slides had been then incubated with the OptiView DAB IHC Detection Equipment (Roche/Ventana Medical Techniques). Antibody binding was detected utilizing DAB as chromogen and slides had been counterstained with hematoxylin.
Animal research
Orthotopic xenograft mouse mannequin. All animal experiments had been performed beneath guidelines authorized by the Taipei Medical College (TMU) Ethics Committee (LAC-2021-0475). All animal care, monitoring, and experiments adopted tips of the TMU Laboratory Animal Middle, the place the animal examine was performed. To determine a extremely metastatic MB mannequin, 4 × 105 luciferase-tagged Daoy-MYCN cells had been inoculated into the left hemisphere of NOD/SCID mice following a beforehand described protocol [7]. Bioluminescence imaging (BLI) was carried out on day 3 and day 7 post-inoculation to verify the formation of xenograft tumors, after which imaging was carried out weekly till the endpoint. Histological research had been carried out to verify the presence and site of xenograft tumors.
Microbubble-enhanced targeted ultrasound (FUS). Microbubble-enhanced FUS was carried out equally to our earlier work [37]. Briefly, anesthetized mice had been stabilized within the inclined place utilizing a stereostatic instrument pre-mounted with an equipment comprised of an ultrasound transducer and a focal cone. The cone tip was positioned to level on the web site of tumor inoculation on the mouse head with a layer of ultrasound gel utilized to the scalp to make sure efficient transmission. Subsequent, a 150 µL/kg dose of reconstituted microbubbles (SonoVue, cat. CE101301, Bracco Suisse, Switzerland) was blended with the EV pattern and injected into the tail vein. Then, FUS was instantly carried out utilizing a 50-MHz arbitrary waveform generator (mannequin 645, Rohde & Schwarz, Munich, Germany) and a ten kHz-250 MHz 75 W amplifier (mannequin 75A250A, Amplifier Analysis, Souderton, PA, USA) at 220 mV amplitude, 1 MHz frequency, and three% responsibility cycle (30,000 cycles/s) for 60 s.
In vivo EV therapy and EV monitoring. For weekly EV therapy in mice, ~ 2 × 1010 of siRNA-loaded EVs per mouse had been injected into the tail vein, adopted by a FUS session. For in vivo EV monitoring, ~ 1.5 × 1011 EVs per mouse had been labeled with the ExoGlow Vivo EV Labeling Equipment (Close to IR) (cat. no. EXOGV900A-1, System Biosciences) following the producer’s protocol after which had been injected into the tail vein, with or with out software of FUS. Fluorescence imaging was subsequently carried out to seize photos at excitation/emission wavelengths of 768/845 nm. In all experiments, mouse organs of curiosity had been subsequently obtained for histological research.
Statistical evaluation
Knowledge are offered because the imply ± normal deviation (SD) with the distinction between two teams decided by a two-tailed Pupil’s t-test until in any other case specified within the determine legends. Correlations between two variables had been assessed utilizing a easy linear regression and Pearson’s correlation coefficients. Survival analyses had been carried out utilizing a Kaplan-Meier curve and the Gehan-Breslow-Wilcoxon take a look at. All statistical analyses had been carried out with GraphPad Prism vers. 9 (GraphPad, San Diego, CA, USA) and Microsoft Workplace Excel vers. 16 (Microsoft, Redmond, WA, USA). p < 0.05 was thought of statistically vital.
