Assessment of ATP Metabolism to Adenosine by Ecto-Nucleotidases Carried by Tumor-Derived Small Extracellular Vesicles

Background Immunosuppression is a hallmark of cancer progression. Tumor-derived small extracellular vesicles (sEV), also known as TEX, produce adenosine (ADO) and can mediate tumor-induced immunosuppression. Methods Here, the ATP pathway of ADO production (ATP◊ADP◊AMP◊ADO) by ecto-nucleotidases carried in sEV was evaluated by a novel method using N6-etheno-ATP (eATP) and N6-etheno-AMP (eAMP) as substrates. The “downstream” N6-etheno-purines (ePurines) were measured by high performance liquid chromatography with fluorescence detection (HPLC-FL). Results Human melanoma cell-derived TEX (MTEX) metabolized eATP to N6-etheno-ADP (eADP), eAMP and N6-etheno-Adenosine (eADO) more robustly than control keratinocyte cell-derived sEV (CEX); due to accelerated conversion of eATP to eADP and eADP to eAMP MTEX and CEX similarly metabolized eAMP to eADO. Blocking of the ATP pathway with the selective CD39 inhibitor ARL67156 or pan ecto-nucleotidase inhibitor POM-1 normalized the ATP pathway but neither inhibitor completely abolished it. In contrast, inhibition of CD73 by PSB12379 or AMPCP abolished eADO formation in both MTEX and CEX, suggesting that targeting CD73 is the preferred approach to eliminating ADO produced by sEV. Conclusions The noninvasive, sensitive, and specific assay assessing ePurine metabolism ± ecto-nucleotidase inhibitors in TEX enables the personalized identification of the ecto-nucleotidase primarily involved in ADO production in patients with cancer. The assay could guide precision medicine by determining which purine is the preferred target for inhibitory therapeutic interventions.


INTRODUCTION
Suppression of adaptive and innate immune responses is a recognized hallmark of cancer progression [1].In this regard, tumor-derived small extracellular vesicles (sEV), otherwise known as tumorderived exosomes or TEX, play a key role in tumor-induced suppression of immune effector cells in cancer and in promotion of tumor growth [2,3].TEX are small virus-size sEV derived from the endocytic compartment of tumor cells [4].They are present in all body uids of patients with cancer, circulate freely and serve as a communication network between malignant and nonmalignant cells [5,6].As sEV are secreted by normal and malignant cells, TEX represent a subset of total circulating sEV that can be identi ed by a unique surface protein pro le that mimics that of parent cancer cells [7,8].TEX exert direct immunoregulatory effects on immune cells, re-program functions of normal tissue cells located in the tumor-microenvironment (TME) and by autocrine or paracrine mechanisms support tumor growth [9,10].
TEX are potentially useful as biomarkers of tumor progression and of immune responses or as potential predictors of response to cancer therapies [11,12].
Numerous in vitro and in vivo studies in animal models demonstrate autocrine effects of TEX on tumor growth, tumor resistance to chemotherapies and establishment of metastases [13][14][15].Via juxtacrine or paracrine signaling in the TME, TEX are known to alter functions of mesenchymal stem cells, broblasts, endothelial cells and immunocytes [16,17].TEX-mediated changes in recipient cells are the result of receptor-ligand interactions on the cell surface and/or up-take by recipient cells of signaling proteins or micro-RNAs (miRs) carried by TEX [18,19].As TEX circulate freely delivering pro-tumor and anti-immune response signals to a broad variety of cells, they represent a major mechanism of tumor immune escape.
Among various immunosuppressive molecular pathways utilized by TEX, the adenosinergic pathway is emerging as a major contributor to TEX-induced suppression of immune cells and a promoter of tumor growth and metastasis [20,21].TEX-derived adenosine (ADO) inhibits T-cell activation and proliferation through A 2A Rs [22].Our studies show that of 20 different purines measured, ADO is the most abundant purine in TEX [23].Thus, intra-vesicular ADO carried by TEX, which are present in high numbers (e.g., 10 12 /mL) in cancer patients' plasma, would be e ciently delivered to near or distantly located recipient cells in the TME.Therefore, TEX may be a major source of immunosuppressive ADO, as well as ADO mediating pro-tumor activities in patients with cancer.
Although tumor cells produce and release ADO into extracellular space, ADO is rapidly (within seconds) metabolized and quickly depleted.On the other hand, ADO preformed in tumor cells, packaged, and delivered to recipient target cells by TEX is protected and functionally active [22,23].Also, ectonucleotidases involved in ADO production, CD39 and CD73, are present on the surface of TEX and are able to convert ATP to ADO [21].Thus, in addition to intra-vesicular ADO, TEX are equipped with CD39 which converts ATP to ADP and ADP to AMP and with CD73 which metabolizes AMP to ADO [22].TEX appear to sustain immunosuppressive levels of ADO near target cells by utilizing e cient ecto-enzymatic machinery to manufacture ADO in the extracellular compartment close to cell surface receptors for ADO.
Since TEX are equipped to generate ADO in the extracellular compartment and thus effectively contribute to tumor-induced immune suppression, the ability to measure adenosinergic activity of TEX in patients with cancer might serve as a useful guide to therapy selection and prognostic considerations.Our preliminary data suggest that levels of TEX-mediated adenosinergic activity differ among cancer patients [23].Thus, the development of a rapid, sensitive, and speci c assay that could guide precision medicine for each patient using TEX is a rational objective.We describe here a novel approach to assessing activity of the ATP pathway (ATP ◊ ADP ◊ AMP ◊ ADO) mediated by ecto-nucleotidases residing on the TEX surface and how to selectively block the ecto-nucleotidases mediating extracellular ADO production in every cancer patient.

MATERIALS and METHODS
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% CO 2 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 Scienti c, Waltham, MA) previously depleted of extracellular vesicles by ultracentrifugation at 100,000xg for 3h.Cells were cultured in 150 cm 2 cell culture asks containing 25 ml of the culture medium.Each ask was seeded with 4x10 6 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 asks 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 ltrated using a 50mL syringe and a 0.22µm bacterial lter.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).
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 ddH 2 O 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, ve 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 ow cytometry using FITC Annexin-V (ANXV) Apoptosis Detection Kit (BD Biosciences, #55647, Jose, CA) in a Cyto ex ow 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-lter (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 10 10 /mL) versus CEX (4.26 x 10 10 /mL).
Assessment of N 6 -etheno-ATP and N 6 -etheno-AMP metabolism by sEV.Cell line-derived sEV were incubated at 37 C in 60µl of PBS with N 6 -etheno-ATP (eATP) or N 6 -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 C for 90sec to denature ecto-enzymes, centrifuged at 13,000rpm at 4 C and diluted 10-fold before analysis of N 6 -etheno-Purines (ePurines, BioLog Life Science Institute, Hayward, CA) including N 6 -etheno-ADP (eADP), eAMP and N 6 -etheno-Adenosine (eADO).ePurines were quanti ed using high pressure liquid chromatography with uorescence 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 (coe cient of variation, < 2%) and accuracy (excellent match between assay values versus known concentrations of standards) of this assay system [27].Speci city was con rmed 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 con rmed that: 1) ePurines are metabolized by ecto-nucleotides with an e ciency 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 speci c 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 speci c 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 30 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 signi cance.Values are means and SEMs.

RESULTS
Characterization of sEV isolated from cell line supernatants.Figure 1 presents results of sEV characterization performed with MTEX and CEX.The results show that the EV morphology (Fig. 1a), endocytic origin (Fig. 1b) and size (Fig. 1C) of the vesicles we isolated by ultra ltration and SEC from supernatants of the two cell lines are consistent with the sEV category.Additionally, western blots showed that these vesicles were positive for CD39 and CD73 (Fig. 1b).Also, functional assays demonstrated that sEV induced apoptosis of activated T cells in co-incubation assays (data not shown).Vesicles obtained from supernatants of MTEX and CEX EVs (Fig. 1b) and from peripheral blood of a patient with melanoma and a healthy donor (Fig. 1d) had the same characteristics.
eATP metabolism by MTEX and CEX.MTEX and CEX (6ug protein each) were incubated for 3h with 100µmol/L of eATP; then concentrations of "downstream" ePurines including eADP, eAMP and eADO were measured (Figures 2a, 2c and 2e, respectively).Notably, the concentrations of eAMP and eADO were higher in MTEX than in CEX (P=0.0393 and P=0.0061, respectively).eADP level tended to be higher in MTEX than in CEX; however, this difference was not statistically signi cant.We also calculated the ratios of downstream metabolites to upstream substrates as an index of rate of metabolism of a given ePurine.
Importantly, the (eAMP+eADO)/eADP ratio was signi cantly greater (P=0.0397) in MTEX compared with CEX (Figure 2e), and the (eADP+eAMP+eADO)/eATP ratio also tended to be greater in MTEX (Figure 2b).However, eADO/eAMP ratio was similar in MTEX and CEX (Figure 2f).These ndings support the conclusion that MTEX metabolize eATP more rapidly to eADP and metabolize eADP more rapidly to eAMP than do CEX.This results in little/no change in steady state eADP levels between MTEX and CEX, yet it increases levels of eAMP in MTEX compared to CEX.The data suggest that increased levels of eADO in MTEX are due to higher eAMP levels driving eADO production.
Inhibition of CD73 with PSB12379 or AMPCP blocks metabolism of eATP to eADO in MTEX and CEX.To test the role of CD73 in the metabolism of eATP to eADO, we used α,β-methyleneadenosine 5'diphosphate (AMPCP); a commonly used CD73 inhibitor.However, our preliminary testing showed that a concentration of AMPCP that suppressed CD73 by 83% also inhibited CD203a (ENPP-1) by 87% (Table 1).By contrast, a concentration of PSB12379, an alternative CD73 inhibitor that blocked CD73 by 98%, had no detectable effect on a panel of ecto-nucleotidases (Table 1).Although CD203a is not known to metabolize AMP to ADO, here we used both AMPCP and PSB12379 to block CD73.
Consistent with ndings reported in Fig. 2, regardless of treatment with PSB12379 (40 µmol/L) or AMPCP (400 µmol/L), 3hr incubation of vesicles with eATP (100 µmol/L) resulted in a greater increase of eAMP levels in MTEX than in CEX (Figs. 3a-3c; P = 0.0008 for effect of MTEX versus CEX with No Inhibitor versus PSB12379 groups and P = 0.0030 for effect of MTEX versus CEX with No Inhibitor versus AMPCP groups).Also, in both MTEX and CEX incubated with eATP, PSB12379 and AMPCP increased eAMP levels (Figs.3a-3c; P = 0.0001 for effect of PSB12379 versus No Inhibitor; P = 0.0026 for effect of AMPCP versus No Inhibitor).In the No Inhibitor groups, eADO levels were elevated in MTEX compared to CEX (Fig. 3d; P = 0.0061) yet were below detection limit in MTEX and CEX treated with either PSB12379 (Fig. 3e) or AMPCP (Fig. 3f).These ndings con rm that MTEX metabolize eATP more rapidly to eAMP and eADO than CEX and demonstrate that in both MTEX and CEX inhibition of CD73 abolishes the conversion of eAMP to eADO resulting in higher levels of eAMP and undetectable levels of eADO.
Inhibition of CD73 with PSB12379 blocks metabolism of eAMP to eADO in MTEX and CEX.First, MTEX and CEX were incubated for 20min with 100µmol/L of eAMP in the absence and presence of PSB12379 (40 µmol/L); then eAMP and eADO were measured.In the absence of PSB12379, approximately half of the added eAMP was converted to eADO during the 20min incubation in both MTX and CEX (Figs. 4a and  4c).By contrast, in both MTEX and CEX treated with PSB12379, the added eAMP was recovered as intact, unused eAMP (Fig. 4b) and levels of eADO were below the assay detection limit (Fig. 4d).These results con rm that CD73 mediates the enzymatic conversion of eAMP to eADO in MTEX and CEX and demonstrate that CD73 activity levels are similar in MTEX and CEX.
Inhibition of tissue nonspeci c alkaline phosphatase (TNAP) with L-p-bromotetramisole (L-p-BT) does not affect the metabolism of eATP to eADP or eAMP in either MTEX or CEX.Our results are consistent with the conclusion that CD73 mediates the metabolism of eAMP to eADO in MTEX and CEX.However, the ecto-nucleotidase(s) responsible for converting eATP to eADP and eAMP in MTEX and CEX is (are) unknown.Here, we incubated MTEX and CEX with 100 µmol/L of eATP in the absence and presence of the TNAP inhibitor L-p-bromotetramisole (L-p-BT; 400 µmol/L); then eADP and eAMP were measured.L-p-BT is a commonly used TNAP inhibitor, and our preliminary testing showed that a concentration of L-p-BT that inhibited TNAP activity by 87% had little or no effect on other ecto-nucleotidases (Table 1).
L-p-BT did not affect the levels of either eADP (Figs.5a and 5b) or eAMP (Figs.5c and 5d).As observed earlier, eADP levels tended to be greater in MTEX than in CEX, and eAMP levels were signi cantly (P = 0.0038) greater in MTEX versus CEX.These results con rm that MTEX metabolize eATP more rapidly than do CEX; however, there appears to be little, if any, role for TNAP in either MTEX or CEX.
CD39 (ENTPD-1) blockade with ARL67156 inhibits the metabolism of eATP to eADP and eAMP in MTEX and CEX.ARL67156 is a commonly used CD39 inhibitor.Our preliminary screening experiments indicate, however, that while somewhat selective for CD39, ARL67156 induces considerable blockade of CD203a at concentrations that inhibit CD39 (Table 1).Therefore, we evaluated, using ARL67156 (400 µmol/L) in MTEX and CEX, the role of CD39 to metabolize eATP in the presence of AMPCP (400 µmol/L; blocks CD203a) to remove any in uence of CD203a.
ARL67156 reduced eADP (Figs.6a and 6b; P = 0.0101) and eAMP (Fig. 6c and 6d; P = 0.0082) levels in MTEX and CEX and attenuated the excessive eAMP produced in MTEX compared with CEX (Figs. 6c and  6d).These ndings support the conclusion that CD39 contributes to the metabolism of eATP to eADP and eAMP in both MTEX and CEX and that excessive CD39 activity accounts for the differential metabolism of eATP to eAMP in MTEX.
CD39 (ENTPD-1) blockade with POM-1 inhibits the metabolism of eATP to eADP and eAMP in MTEX and CEX.POM-1, like ARL67156, is also a commonly used CD39 inhibitor.Our preliminary screening experiments indicate, however, that POM-1 is a pan-ecto-nucleotidase inhibitor (i.e., a non-selective CD39 inhibitor) that induces considerable blockade of not only CD39, but also ENTPD2, ENTPD3, CD203a and TNAP (Table 1).Therefore, to evaluate the combined role of CD39, ENTPD2, ENTPD3 and TNAP, we examined the effects of POM-1 (600 µmol/L) on the ability of MTEX and CEX to metabolize eATP in the presence of AMPCP.Here, the pretreatment with AMPCP was employed to match the conditions used for assessment of the actions of ARL67156 on eATP metabolism.
POM-1 reduced eADP (Figs.7a and 7b; P = 0.0462) and eAMP (Fig. 7c and 7d; P = 0.0032) levels in MTX and CEX and attenuated the excessive eAMP produced in MTEX compared with CEX (Figs. 7c and 7d).The effects of POM-1 were nearly identical to the effects mediated by ARL67156 (compare Fig. 6 with 7).These ndings support the conclusion that CD39 contributes to the metabolism of eATP to eADP and eAMP in MEX and CEX and that excessive CD39, but not ENTPD2, ENTPD3 or TNAP activity, accounts for the differential metabolism of eATP to eAMP in MTEX.
CD203a inhibition with AMPCP does not affect the metabolism of eATP to eADP or eAMP in either MEX or CEX.Based on the results described above, it appeared that CD39 is the dominant ecto-nucleotidase in sEV that regulates eATP metabolism.Nonetheless, we considered that CD203a might be involved as well.Accordingly, we evaluated the metabolism of eATP in MTEX and CEX in the absence and presence of AMPCP (400 µmol/L; blocks CD203a).Since AMPCP also inhibits CD73 we conducted these experiments in the presence of PSB12379 (40 µmol/L; blocks CD73) to remove any in uence of CD73.AMPCP did not affect the levels of eADP (Figs.8a and 8b) or eAMP (Figs.8c and 8d).As observed earlier, eADP levels tended to be greater in MTEX compared with CEX, and eAMP levels were signi cantly (P = 0.0007) greater in MTEX than in CEX.These results con rm that MTEX metabolize eATP more rapidly than do CEX; however, there appears to be little, if any, role for CD203a in either MTEX or CEX.

DISCUSSION
The primary goal of this study was to determine whether the method utilizing ePurines and HPLC-FL we previously developed was adequately sensitive to study ATP metabolism to ADO in sEV.These vesicles were previously found to carry ADO in the lumen as well as ecto-nucleotidases on the external surface and to produce ADO [21].We reported that TEX had augmented ATP metabolism to ADO and, considering the role of ADO in cancer-induced immune suppression, wished to precisely quantitate ADO generated by TEX of different patients with cancer.The rationale for this study was supported by our earlier observations that ePurines can be measured with high sensitivity and speci city using HPLC-FL, because the etheno moiety renders ePurines uorescent [27], and this approach does not require more expensive technology such as tandem mass spectrometry.The assay we performed utilizes N 6 -etheno-ATP (eATP) and N6-etheno-AMP (eAMP) as substrates while measuring "downstream" N 6 -etheno-purines (ePurines) using high performance liquid chromatography with uorescence detection (HPLC-FL).The assay has the capacity to discriminate not only high from low adenosinergic activity, but also, by the use of speci c inhibitors, to identify the ecto-nucleotidases involved in orchestrating the metabolism of external ATP in sEV thus indicating which enzyme inhibitors would be optimal for blocking this immunosuppressive mechanism.
There are several advantages to using ePurines, rather than natural purines, to evaluate the importance of ADO produced by sEV.As we described recently, ePurines are metabolized by ecto-nucleotidases with e ciencies similar to that of corresponding natural purines, yet ePurines can be measured with much greater sensitivity and speci city using HPLC-FL because the etheno moiety renders ePurines uorescent [27].Also, because ePurines do not signi cantly cross membranes and are not conveyed into cells by nucleoside/nucleotide transporters [27], the metabolism of ePurines by EVs would only re ect metabolism by the ecto-nucleotidases with the proper orientation on the sEV surface to produce ADO in the extracellular compartment.Since ADO receptors exist on surfaces of various cells, it is the pool of extracellular ADO that is most important for inducing immunosuppression.Also, ePurines are not metabolized by "off-target" pathways, e.g., deamination, which allows for more precise estimates of the metabolism rate speci cally via the extracellular ATP ◊ ADP ◊ AMP ◊ ADO pathway.Finally, the application of inhibitors of ecto-nucleotidases carried by sEV can facilitate future clinical utility of the assay by identifying which ecto-nucleotidase(s) should be targeted for inhibition.
The experiments we performed comparing MTEX and CEX yielded several interesting results.First, in all sEV, the main ecto-nucleotidases participating in the "extracellular ATP to ADO pathway" are CD39 and CD73; TNAP, ENTPD2, ENTPD3 and CD203a play little if any role.The use of pharmacological inhibitors which selectively blocked individual enzymes in the pathway con rmed the involvement of CD39 and CD73 in the ATP to ADO pathway in both MTEX and CEX.
The nding that the "Extracellular ATP to ADO Pathway" is signi cantly enhanced in MTEX relative to CEX represents another important observation, which agrees with previous reports that tumor cells producing sEV which carry intraluminal ADO and actively produce ADO are the major source of immunosuppressive ADO in cancer plasma [22,28].Furthermore, enhanced enzymatic activity of CD39 converting ATP to ADP to AMP was responsible for the higher activity of the entire pathway in MTEX compared to CEX.This nding also ts well with higher ATP utilization by cancer cells and cancer-derived sEV [21].Upon the addition of eATP to sEV, eAMP and eADO, but not eADP, levels were increased approximately 2-fold more in MTEX versus CEX, suggesting that MTEX metabolize eATP more rapidly to eADP and metabolize eADP more rapidly to eAMP than do CEX.Nevertheless, metabolism of eAMP to eADO remains similar in MTEX and CEX.This is because increased production and increased utilization of eADP by CD39 normalizes eADP levels and leaves them relatively constant, while increasing eAMP levels and ADO production, which is what we observed.Since CD39 metabolizes both ATP and ADP, the enhanced metabolism of eATP in MTEX is consistent with similar eADP levels in MTEX and CEX, although eAMP and eADO levels were twice as high in MTEX compared to CEX.
Selective inhibition of CD39 with ARL67156 or pan-ecto-nucleotidase inhibition with POM-1, which inhibits multiple ecto-nucleotidases, including CD39 and at least ENTPD2, ENTPD3, TNAP and CD203a, similarly normalized the conversion of eATP to eAMP.This suggests that CD39, not the other ectonucleotidases inhibited by POM-1, mediates the accelerated utilization of eATP in MTEX.Also, inhibition of CD203a did not affect the metabolism of eATP, which further rules out the involvement of this ectonucleotidase in the metabolism of eATP in sEV.Importantly, although both ARL67156 and POM-1 normalized the metabolism of eATP in MTEX to that observed in CEX, neither inhibitor abolished eATP metabolism in either MTEX or CEX.This suggests that other, yet to be identi ed ecto-nucleotidases on the surface of sEV might also contribute to metabolism of eATP but are not responsible for the accelerated metabolism of eATP by MTEX.Our experiments rule out involvement of TNAP in this step since L-p-BT did not alter the metabolism of eATP in either MTEX or CEX.In aggregate, these experiments showed that the enhanced ATP to ADO pathway in MTEX is due to higher activity of CD39 and results in increased production of eAMP (a substrate for eADO) in MTEX.
We next considered the role of CD73 in the enhanced ATP to ADO pathway in MTEX and found that CD73 activity was not responsible for increased levels of eADO production in MTEX versus CEX.This conclusion is supported by two lines of reasoning.First, incubation with eATP yielded similar eADO-to-eAMP ratios in MTEX and CEX.Second, incubation with eAMP demonstrated equal metabolism of eAMP by MTEX versus CEX.Notably, inhibition of CD73 with either PSB12379 or AMPCP reduced eADO levels in both MTEX and CEX to below the detection limit of our assay.So, although CD73 does not account for the accelerated metabolism of eATP to eADO in MTEX, CD73 in both MTEX and CEX exclusively mediates the conversion of eAMP to eADO.These ndings suggest that regardless of the upstream source of AMP, inhibition of CD73 abolishes ADO production by sEV in both MTEX and CEX.
In conclusion, measurements of ePurine metabolism in sEV, with or without ecto-nucleotidase inhibitors, using HPLC-FL could guide precision medicine by identifying patients with ADO-generating ectonucleotidases in TEX and by determining how best to block ADO production by TEX.Although the relative importance of ecto-nucleotidases in TEX will likely vary among patients and may depend on the type of cancer, stage of cancer and exposure to therapeutic agents, the present results suggest that CD73, rather than CD39, inhibition might be a preferred mode of therapy in cancer patients.TNAP: Enzyme amount was 5 ng and was titrated to minimize loss of substrate over 10 min due to uncompetitive inhibition in which uncompetitive inhibitor has little effect at low substrate levels.Metabolism of N 6 -etheno-ATP (eATP) to N 6 -etheno-ADP (eADP), N 6 -etheno-AMP (eAMP) and N 6 -ethenoadenosine (eADO) by melanoma cell-derived sEV (MTEX) or control sEV derived from non-malignant keratinocytes (CEX).CEX and (6µg protein each) were incubated for 3h with eATP (100 µmol/L), and levels of eADP, eAMP and eADO were determined.Compared to CEX, eADP concentrations (A) tended to be higher in MTEX, while eAMP (C) and eADO (E) levels were signi cantly higher in MTEX.The ratio of the sum of "downstream" metabolites levels divided by the immediate "upstream" substrate level also tended to be higher for the substrate eATP (B) and was signi cantly higher for the substrate eADP (D),yet was similar for the substrate eAMP (F).The results are consistent with increased metabolism of eATP to eAMP in MTEX versus CEX, which drives increased production of eADO in MTEX.This increased conversion of eATP to eAMP, rather than increased metabolism of eAMP to eADO, accounts for the higher production of eADO from eATP in MTEX.Values represent means and SEMs.P-values are from unpaired Student's t-tests.and eAMP were determined.Since ARL67156 also blocks CD203a (see Table 1), all groups were pretreated with AMPCP which also blocks CD203a (see Table 1), thus isolating the effects of ARL67156 to CD39 inhibition.The results for eADP (Aand B) and eAMP (C and D) in the No ARL67156 versus the ARL67156 groups were analyzed by 2-factor analysis of variance (2F-ANOVA).This analysis indicated that the production of eADP (P=0.0101) or eAMP (P=0.0014)from eATP was suppressed by ARL67156 in CEX and MTEX and that the excessive metabolism of eATP to eAMP was normalized by ARL67156.However, some eATP was converted to eADP and eAMP in CEX and MTEX despite inhibition of CD39.
NS= no signi cant difference.Values represent means and SEMs.(POM-1; blocks CD39, ENTPD2, ENTPD3, CD203a and TNAP, see Table 1), and levels of eADP and eAMP were determined.All groups were pretreated with AMPCP so that the results with POM-1 were obtained under the same conditions as in the ARL67156 experiments (see Figure 6).The results for eADP (A and B) and eAMP (Cand D) for the No POM-1 versus the POM-1 groups were analyzed by 2-factor analysis of variance (2F-ANOVA).This analysis indicated that the production of eADP (P=0.0462) or eAMP (P=0.0032)from eATP was suppressed by POM-1 in CEX and MTEX and that the excessive metabolism of eATP to eAMP was normalized by POM-1.However, some eATP was converted to eADP and eAMP in CEX and MTEX despite inhibition of multiple ecto-nucleotidases.NS = no signi cant difference.Values represent means and SEMs.1), and levels of eADP and eAMP were determined.Since AMPCP also blocks CD73 (see Table 1) all groups were pretreated with PSB12379 which also blocks CD73 (see Table 1), thus isolating the effects of AMPCP to CD203a inhibition.Results for eADP (A and B) and eAMP (C and D) comparing the No AMPCP versus the AMPCP groups were analyzed by 2-factor analysis of variance (2F-ANOVA).This analysis indicated that the production of eADP or eAMP from eATP was not affected by AMPCP in either CEX or MTEX.There

Figure 1
Figures

Table 1
Effects of inhibitors on recombinant ecto-nucleotidases.