Cell lines. Human melanoma cell line Mel524 and human keratinocyte cell line HaCaT were obtained from the ADCC and used for preparation of melanoma cell-derived sEV (MTEX) and control keratinocyte cell-derived sEV (CEX). The cell lines were grown at 37°C in an atmosphere of 5% CO2 in air. Cultures were routinely tested and found to be mycoplasma free. Cells were cultured in RPMI-1640 medium, 1% (v/v) penicillin/streptomycin and 10% (v/v) heat-inactivated fetal bovine serum (FBS, ThermoFisher Scientific, Waltham, MA) previously depleted of extracellular vesicles by ultracentrifugation at 100,000xg for 3h. Cells were cultured in 150 cm2 cell culture flasks containing 25 ml of the culture medium. Each flask was seeded with 4x106 cells and following 72h of incubation, supernatants were collected, while the cells were harvested using 2 mL of TrypLE Express (Gibco, Grand Island, NY) and washed in serum-containing medium. For subsequent passages, cells were re-seeded in new flasks using the cell numbers described above Supernatants were collected for isolation of TEX and CEX.
Collection of cell line supernatants for sEV isolation. Cell culture supernatants were combined, and a 50mL aliquot of cell culture supernatant was centrifuged at room temperature for 10min at 2,000xg to sediment cells and cell fragments. Supernatants were transferred to new tubes for centrifugation at 10,000xg at 4°C for 30min. Supernatants were collected and filtrated using a 50mL syringe and a 0.22µm bacterial filter. Afterwards, aliquots of supernatants were concentrated to 1mL by using Vivacell 100 concentrators (Sartorius Corporation, Bohemia, NY) at 2,000xg[24].
sEV isolation by size exclusion chromatography (SEC). sEV isolation by SEC was previously described and is routinely used in our lab[25]. An aliquot (1 ml) of concentrated supernatant was loaded on a 10 cm-long Sepharose 2B column and was eluted with phosphate-buffered saline (PBS). Individual 1 ml fractions were collected. Fraction #4 containing the bulk of non-aggregated morphologically intact sEV was harvested, concentrated using 100,000 MWCO Vivaspin 500 centrifugal 450 concentrators (Sartorius Corporation) and evaluated for protein, vesicle size and number, molecular content and sEV functions[25].
Transmission electron microscopy (TEM). TEM of sEV was performed at the Center for Biologic Imaging, the University of Pittsburgh as previously described[25]. Freshly isolated sEV were placed on copper grids coated with 0.125% Formvar in chloroform and stained with 1% (v/v) uranyl acetate in ddH2O. A JEM 1011 microscope was used for sEV visualization. TEM showed particle morphology consistent with sEVs (see Fig. 1A).
Western blot analysis. To concentrate isolated sEV, 0.5 mL 100K Amicon Ultra centrifugal filters (EMD Millipore, Burlington, MA) were used for centrifugation at 4,000xg. Vesicle aliquots were lysed with Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) and separated using 4–15% SDS/PAGE gels. Each lane was loaded with 10µg of fraction #4 protein. After transfer from gels to the polyvinylidene fluoride (PVDF) membranes, proteins were detected using antibodies specific for CD81(ThermoFisher, MA5-13548), ALIX (ThermoFisher, MA5-32773), calnexin (Cell Signalling, #2433), CD39 (Santa Cruz #33558) and CD73 (Abcam #81720). Immunodetection by blotting showed the protein profile consistent with sEVs (see Fig. 1B).
NanoSight measurements. The concentration and size distribution of sEV were measured by nanoparticle tracking analysis (NTA) using NanoSight 300 (Malvern, UK). The vesicles were diluted in ddH2O and then the video image was captured at a camera level of 14. The captured videos were analyzed using NTA software, maintaining the screen gain and the detection threshold at 1 and 5, respectively. To determine mean particle size/concentration in each sample, five consecutive measurements were obtained and averaged. NanoSight measurements yielded particles sizes consistent with sEV (see Fig. 1c).
Protein concentration. Protein concentrations of sEV were determined by using a BCA protein assay (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions.
Functional activity. The ability of isolated sEV (10µg protein) to induce apoptosis of Jurkat T cells during a 6-hour co-incubation was measured by flow cytometry using FITC Annexin-V (ANXV) Apoptosis Detection Kit (BD Biosciences, #55647, Jose, CA) in a Cytoflex flow cytometer (Beckman, Indianapolis, IN) as previously described[26]. Flow cytometry indicated that isolated sEV were functionally active (data not shown).
Preparation of sEV for analysis. Total sEV were concentrated using Amicon ultra-filter (100,000 MWCO) and both MTEX and CEX were prepared in PBS for HPLC-FL analysis at a protein concentration of 100µg/mL. Mean estimated particle concentrations were similar in preparations of MTEX (4.89 x 1010/mL) versus CEX (4.26 x 1010/mL).
Assessment of N 6 -etheno-ATP and N 6 -etheno-AMP metabolism by sEV. Cell line-derived sEV were incubated at 37 ⁰C in 60µl of PBS with N6-etheno-ATP (eATP) or N6-etheno-AMP (eAMP) and without or with enzyme inhibitors. Matched protein amounts (6µg), rather than matched particle numbers, were employed because protein amounts can be measured with greater accuracy than particle counts. Nonetheless, both methods of normalization would provide similar results since at equivalent protein amounts, particle numbers were similar in samples from MTEX versus CEX. High concentrations of eATP and eAMP were employed (100µmol/L) and the incubation periods (3h for eATP; 20min for eAMP) were selected in preliminary experiments to prevent substrate depletion. After incubation, samples were rapidly heat inactivated at 95 ⁰C for 90sec to denature ecto-enzymes, centrifuged at 13,000rpm at 4 ⁰C and diluted 10-fold before analysis of N6-etheno-Purines (ePurines, BioLog Life Science Institute, Hayward, CA) including N6-etheno-ADP (eADP), eAMP and N6-etheno-Adenosine (eADO). ePurines were quantified using high pressure liquid chromatography with fluorescence detection (HPLC-FL) as recently described by us in detail[27].
We previously determined and reported the sensitivity (detection limit, 1 pmol injected on column), precision (coefficient of variation, < 2%) and accuracy (excellent match between assay values versus known concentrations of standards) of this assay system[27]. Specificity was confirmed by demonstrating baseline separation of all chromatographic peaks generated from samples of a mixture of ePurines and from samples of medium conditioned by four different cell lines incubated with eATP[27]. Moreover, we confirmed that: 1) ePurines are metabolized by ecto-nucleotides with an efficiency similar to their corresponding natural substrates; 2) there is no “off-target” (non-nucleotidase-mediated) metabolism of ePurines; and 3) the metabolism of ePurines is restricted to the membrane surface, i.e., is not due to intracellular nucleotidases[27].
Selectivity assessment of ecto-nucleotidase inhibitors. Many different ecto-nucleotidase inhibitors are available for pharmacological testing of the role of specific ecto-nucleotidases in the metabolism of the extracellular ATP pathway (ATP → ADP → AMP → ADO). However, the selectivity of these inhibitors is uncertain. Therefore, before choosing a given inhibitor to probe the role of a specific ecto-nucleotidase, we tested a panel of commonly used ecto-nucleotidase inhibitors for their selectivity. In this regard, human recombinant CD39 (ENTPD-1), CD203a (ENPP-1), ENTPD-2, ENTPD-3, CD73 and TNAP (R&D Systems, Minneapolis, MN) were incubated with substrate (1 µmol/L) at 30⁰ C for 30 min (with the exception of TNAP which was incubated for 10 min with 50 µmol/L), and the product was measured by HPLC-FL. For all ecto-nucleotidases except CD73, the substrate was eATP; for CD73 the substrate was eAMP. With the exception of TNAP, the amount of each enzyme was titrated to provide complete conversion of substrate to product within the incubation time in the absence of any inhibitors. For TNAP, the amount of enzyme was titrated to minimize the loss of substrate over 10 min because many TNAP inhibitors are non-competitive, and such inhibitors have little effect at low substrate levels. The results of these preliminary studies are summarized in Table 1. The commercial sources of each inhibitor are listed in Table 1.
Statistical analysis. Statistical analysis was conducted using NCSS 2019 Statistical Software (NCSS, LLC. Kaysville, Utah). Data were analyzed with either a Student’s t-test, a 2- factor analysis of variance (2F-ANOVA) or a repeated measures 2-factor analysis of variance (repeated measures 2F-ANOVA) as appropriate. P < 0.05 was the criteria for significance. Values are means and SEMs.