Cell Culture
The human B cell lines, Nalm6 and RN95 were cultured for this study. Nalm6 was purchased from the cell bank of Pasteur Institute in Iran. RN95 was established in-house (Aberuyi et al., 2022) (25) and authenticated (national patent number: 100281). Cells were grown in RPMI1640 (Bio Idea, Tehran, Iran) containing 1% penicillin/streptomycin and 1% L-glutamine. Culture media were supplemented with 20% and 10% heat-inactivated FBS (Bio Idea, Tehran, Iran) for Nalm6 and RN95, respectively. cells were then incubated at 37°C in a humidified atmosphere of 5% CO2.
Patients and sampling
Sampling was achieved with full written informed parent’s consent. Peripheral blood was obtained from children with acute lymphoblastic leukemia who referred to the Sayed-ol-Shohada Hospital (Isfahan, Iran) and age matched healthy donors. 3 patients and healthy donors entered the screening phase of the study. The number of patients and controls in validation phase was 30 and 10, respectively. The study was conducted in accordance with the Declaration of Helsinki, and permitted by the Ethics Committee of the University of Isfahan (agreement number: IR.UI.REC.1398.010). Samples were transferred to the University on ice for preanalytical procedures. Plasmas were isolated from the whole blood and stored at -70℃ freezer.
Exosome extraction
Patient samples
10 ml peripheral blood was obtained from the cases involved in the screening phase. In order to harvest platelet-free plasma (PFP), samples were centrifugated twice at 2500 g for 15 min at RT. PFP was diluted in equal volume with PBS and then centrifugated at 12000 g for 45 min at 4°C.Then, supernatant was harvested and filtered through 0.22 µm filters (Millipore, Burlington, MA). Filtered supernatant was subjected to ultracentrifugation twice at 110000 g for 2 h using an ultracentrifuge (Beckman Coulter, Brea, CA). The supernatant was removed and the pellet was resuspended in PBS. The tube was filled with PBS and subjected to ultracentrifugation for 70 min at 110000 g. Exosomes were pelleted at the final centrifugation step and were resuspended in 1ml PBS. The extracted exosomes were filtered using 0.22 µm filter and stored at –80°C in 200 µl aliquots.
500 ul plasma was used as starting material for isolating exosomes from each sample incorporated in the validation phase. Exosomes were isolated using ExoQuick TM (System Biosciences, Palo Alto, CA) according to the manufacturer protocol with minor modification. Briefly, samples were centrifuged at 3000 g for 15 min. For each sample, the Supernatant was transferred to a sterile vessel and filtered using 0.22 syringe filter. 63 µl ExoQuick solution was added to 500 ul clarified plasma. Samples were inverted completely and then incubated at 4°C overnight. The mixture was centrifuged at 1500 g for 30 and 5 min, respectively, at RT in order to pellet the exosomes. The pellet was then resuspended in 200 μl of PBS for downstream analysis (26, 27).
Culture media
In the screening phase, exosomes of Nalm6 and RN95 cell lines were isolated by differential ultracentrifugation according to the protocol described by Thery et al (2006) (28). 40 ml conditioned media were used as biological fluids for extracting exosomes. Each cell line was grown in regular complete media to a high-density cell population in logarithmic phase. Then, regular complete media was substituted thoroughly with FBS-free complete media for exosome production. After 24h Nalm6/RN95, and 48h RN95 cultivation in exosome production media, supernatant was harvested. Different centrifuge condition was applied to the aforementioned supernatant. At first supernatant was centrifuged at 300 g for 10 min at 4°C in polypropylene centrifuge tubes by fixed angle rotor centrifuge (Gyrozen, Daejeon, Korea). Cleared supernatant underwent centrifugation at 2000 g at 4°C for 10 min. After discarding the pellet, supernatant underwent centrifugation for another round, at 10,000 g at 4°C for 30 min. Subsequently upper supernatant was transferred to a new tube and ultracentrifuged at 100,000 g for 70 min with swinging-bucket rotors (Beckman coulter, Brea, CA). In the subsequent washing step, EVs were washed with PBS and the final EV particles were resuspended in 1 ml PBS. After filtering EV particles using 0.22 µm filter. Aliquots of exosomes were stored at - 80°C for further analysis.
Characterization of exosomes
NTA
Concentration and size distribution of isolated exosomes from plasma and conditioned media were detected using Nanosight NS300 particle counter (Malvern Panalytical, Malvern, UK) (29, 30). The system was equipped with the LM14 laser module with a 488 nm blue laser. All samples were diluted in order to obtain 20–100 particles in the frame (107-109 particles/ml). Laser module was removed from the device and 1 ml diluted sample was injected into the module. Five video captures were recorded by sCMOS camera for all samples with 60 second intervals. Camera level was selected on 16 for analyzing video captures. appropriate setting and detection threshold were applied to the measurements. NTA software version 3.4 build 3.4.003 was used for setting and analyzing the results.
BCA assay
Before the quantification of total protein concentration, exosomes were resuspended in PBS and cell suspensions were lysed using RIPA lysis buffer system (Santa Cruz, Dallas, TX). Equal volume of RIPA buffer containing PMSF, protease inhibitor cocktail and sodium orthovanadate in water was added to every aliquot. Calculation of total protein concentration of different aliquots were assessed using a Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer protocol. Results were measured using a plate reader (Bio Tek, Winooski, VT)
Traditional and Capillary automated Western blotting
In traditional immunoblotting, extracellular vesicles and cell suspensions were examined using different anti-human(αh)primary antibodies including rabbit-αhGM130(1:400), Hsp70(1:200), Flotillin-1 (1:200) and CD9 (1:200) (All purchased from Cell signaling, Danvers, MA), in addition to mouse-αhCD63 (Thermo Fisher Scientific, Waltham, MA) (29, 31, 32). Secondary antibodies were goat anti-rabbit and horse anti-mouse antibodies (1:5000) (Cell signaling, Danvers, MA).1 to 8 µg exosomal/cellular protein was employed for traditional immunoblotting. Extracellular vesicles and cell suspensions were resuspended in the loading buffer and boiled at 99°C for 5 min with or without β-mercaptoethanol. Protein of exosomes and cell aliquots were separated by 4–20% Gradient sodium dodecyl sulphate-polyacrylamide gel (4–20%) (Biorad, Hercules, CA) electrophoresis. Proteins were transferred onto PVDF membrane (Biorad, Hercules, CA) by Wet/Tank Blotting system (Biorad, Hercules, CA). Membranes were blocked with 5% skimmed milk in 1 × TBS/Tween solution for 1 h followed by incubation with primary antibodies for 1.5 h at RT and overnight at 4°C. After washing with 1 × TBS/Tween solution, membranes were incubated with secondary antibody. Membranes were visualized using ECL solutions (Immobilon, Temecula, CA) and the ChemiDoc system (Biorad, Hercules, CA). Image Lab Software 6.0.1 was used for quantification of protein bands.
In capillary immunoblotting (33-36), mouse-αhCD81 (1:100) (Invitrogen, Waltham, MA), rabbit-αh Flotillin-1(1:25) and rabbit-αhHsp70 (1:50) (Cell Signaling, Danvers, MA) were used as primary antibodies. Anti-rabbit and anti-mouse Detection Modules (Protein Simple, San Jose, CA) were used for secondary antibody bindings. Capillary cartridges were filled with low concentration of proteins (0.02 to 0.2 µg/µl). Protein expression was shown by capillary Wes - automated Western blotting (Protein Simple, San Jose, CA). The calculation and analysis of protein expression were done according the gel-like images acquired by the Compass for SW software (Version 4.0, Protein Simple).
Scanning Electron Microscopy (FESEM)
The morphology of exosomes was analyzed using a field emission scanning electron microscope (Brno, Czech Republic). Exosomes were resuspended and diluted in I ml of PBS and subjected to sample preparation according to the related protocol. Briefly, the copper holder was covered with glue and aluminum foil, then a drop of exosome sample was placed on it and dried in a vacuum machine. After that, images were taken after gold sputtering.
RNA extraction, cDNA synthesis and RT‑qPCR assay
Total exosomal RNAs were isolated using the Plasma/Serum RNA Purification Mini Kit (Norgen Biotek Corp, Thorold, Canada) according to the manufacturer protocol. The quality and quantity of the RNAs were measured by NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The same amount of plasma samples was applied for exosomal miRNAs extraction. Complementary DNAs (cDNAs) were synthesized using microscript microRNA cDNA Synthesis Kit (Norgen Biotek Corp, Thorold, Canada) in Mastercycler thermocycler (Eppendorf, Hamburg, Germany). Real-time PCR was performed using SYBR Green 2X Real time PCR Kit (Norgen Biotek Corp, Thorold, Canada). For normalization of Ct values, the microRNA (cel-miR-39) Spike-In Kit (Norgen Biotek Corp, Thorold, Canada) was used as an exogenous control. All PCR reactions were done in duplicates during two separate experiments using Applied Biosystems StepOnePlus real-Time PCR system (Thermo Fisher scientific, Waltham, MA). Relative expression level of miRNA was normalized against that of cel-miR-39 using the 2−ΔCt method. The comparative 2−∆∆CT method was used for data analysis and calculation of relative quantification of gene expression where ΔΔCt = Patient (Ct miR-326 - Ct Cel-miR-39) – Control (Ct miR-326 - Ct Cel-miR-39).
Exosome administration
RN95 cells were grown in T25 flask. The culture medium was RPMI-1640 supplemented with fetal bovine serum and 1% penicillin/streptomycin. Populations of RN95 cells in high density were selected for exosome isolation. After 48 h FBS starvation in culture medium, RN95 cells were centrifuged at 200 g for 5 min and 30 ml conditioned media were collected. Exosomes were extracted from conditioned media using Exo-spin TM buffer (Cell Guidance Systems, Cambridge, UK) according to the manufacturer protocol for low-protein biological fluids. Cells were removed from the supernatant by centrifugation at 300 g for 10 min. Supernatant was transferred to a new centrifuge tube and spun at 16,000 g for 30 min to remove any remaining cell debris. Resulting supernatant was filtered through a 0.22 µm syringe filter. ½ volume of Exo-spin™ Buffer was added to filtered supernatant and mixed well by inverting, then incubated overnight at 4°C. The mixture was centrifuged at 16,000 g for 1 h. Supernatant was aspirated carefully and the exosome pellet was resuspended in 1 ml PBS and then stored in -20℃ for next use. the concentration of surface proteins was determined using Bradford assay. In two separate experiments, RN95/Nalm6 cells were cultured in 96-well tissue culture plates at a density of 10 ×104 cells in 100 µl FBS-free media per well. Then 50 µl of RN95 exosomes with different concentrations (1, ½, ¼) were added to the wells. After 48 h incubation at 37℃ in an incubator containing 5 % CO2, MTT assays were performed.
Treatment of patient primary cell with exosomes
Patient mononuclear primary cells were isolated using Ficoll–Hypaquen (inno-train Diagnostik, Clinton, NY) density gradient centrifugation method, according to the manufacturer protocol. Peripheral mononuclear cells (MNCs) were cultured in 96-well tissue culture plates at a density of 10 ×104 cellsper well, in 100 µl FBS-free media. Subsequently, cells were treated with 50 µl of RPMI with and without (as control) exosomes. Following 48 h incubation, viability assays were performed. Relative gene expression level of exosomal miR-326 was quantified using 2-ΔΔCt method.
Viability/proliferation assay
MTT assay was performed by adding10 μL of MTT solution (5 mg/mL) to each well. Following incubation at 37°C for 3 h, 100 µl supernatant was removed and formazan crystals were solubilized by adding 100 μL DMSO into each well. Absorbance was captured at 492 nm by using a stat fax-2100 microplate reader (Awareness Technology, Westport, CT).
Uptake analysis
After cell washing with PBS, 2 mg/ml DiI (CellTracker™ CM-DiI, Thermo Fisher Scientific, Waltham, MA) stock solution was prepared. Exosome concentrations were assessed using lowry method. 20 µg/µl exosome was labelled with 1mg/ml DiI and incubated in dark for 5 min at RT, then 20 min at 4℃. 100 µl labelled- exosomes were adjusted to RN95 cells in dark for 30 min at 4℃. Cell labelling was performed using 1 µg/ml DAPI (Sigma-Aldrich, Darmstadt, Germany). Cells were analyzed using fluorescence microscope (Labomed. Los Angeles, CA).
Transfection of B-ALL cell line with miR-326
RN95 cells were grown to a high density, then transferred to electroporation media. 250 nM miR-326 mimic and miRNA mimic control (scrambled) (Qiagen, Hilden, Germany) were delivered separately into RN95 cells (2 × 106) using Multiporator electroporation system (Eppendorf, Hamburg, Germany). Another group of cells (mock control) were only subjected to electroporation device. Moreover, a fourth group of cells were selected as untreated control. Cells were electroporated 1 pulse (40 µS) at 250 voltage. After 4h incubation, cell viability assay was performed.
Statistical analysis
All data were reported as mean ± standard error of mean (SEM). P < 0.05 was considered as the significant difference. Data are results of 2 independent experiments in duplicates. For comparison between two groups, unpaired t or Mann-Whitney tests were performed. Ordinary one-way analysis of variance (One-Way ANOVA) was done for multiple group comparisons. GraphPad Prism 9.3.1 (GraphPad Software, San Diego, CA) software was used for analyzing results.