Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2′3′-cAMP

Exosome secretion by cells is a complex, poorly understood process. Studies of exosomes would be facilitated by a method for increasing their production and release. Here, we present a method for stimulating the secretion of exosomes. Cultured cells were treated or not with sodium iodoacetate (IAA; glycolysis inhibitor) plus 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor). Exosomes were isolated by size-exclusion chromatography and their morphology, size, concentration, cargo components and functional activity were compared. IAA/DNP treatment (up to 10 µM each) was non-toxic and resulted in a 3 to 16-fold increase in exosome secretion. Exosomes from IAA/DNP-treated or untreated cells had similar biological properties and functional effects on endothelial cells (SVEC4-10). IAA/DNP increased exosome secretion from mouse organ cultures, and in vivo injections enhanced the levels of circulating exosomes. IAA/DNP decreased ATP levels (p < 0.05) in cells. A cell membrane-permeable form of 2′,3′-cAMP and 3′-AMP mimicked the potentiating effects of IAA/DNP on exosome secretion. In cells lacking 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; an enzyme that metabolizes 2′,3′-cAMP into 2′- and 3′-AMP), effects of IAA/DNP on exosome secretion were enhanced. The IAA/DNP combination is a powerful stimulator of exosome secretion, and these stimulatory effects are, in part, mediated by intracellular 2′,3′-cAMP.


IAA/DNP stimulates exosome secretion in vitro.
were isolated from cancer cell lines UMSCC47, PCI-13 and MEL526, which were treated with 0, 1 or 10 µM IAA/DNP for 72 h. All cell cultures showed a concentration-dependent increase of exosomes in the conditioned medium quantified by BCA protein assays and expressed in µg as total exosomal protein as shown in Fig. 1A-C. Treatments of UMSCC47 cells with 10 µM IAA/DNP revealed a 3-fold increase of exosome secretion after 6 h, an almost 6-fold increase after 12 h and ≥10-fold increase for cellular treatments longer than 48 h. (Fig. S1). Results were validated by qNano, which measures particle size and concentrations (Fig. 1D-F). The particle size in fraction #4 of all three cell lines www.nature.com/scientificreports www.nature.com/scientificreports/ measured by qNano ranged from approximately 60 to 160 nm, and no alterations in exosome size were observed comparing exosomes derived from treated or untreated cells ( Fig. 2A,B). Exosomes isolated from cells treated with increasing concentrations of IAA/DNP showed similar morphology by TEM ( Fig. 2A). All exosomes carried TSG101, which indicated their origin from the endocytic compartment of the parent cell (Fig. 2C).
To measure biological activity of exosomes derived from treated or untreated cells, functional studies with SVEC4-10 lympho-endothelial cells were performed. Exosomes were internalized by SVEC4-10 cells within 4 h, and the same concentration of exosomes derived from treated or untreated cells had similar functional activity. SVEC4-10 cells internalized slightly more exosomes derived from cells treated with 1 µM IAA/DNP (Fig. 3C). The migration of SVEC4-10 cells was similarly stimulated by exosomes from treated or untreated cells (Fig. 3D,E).

IAA/DNP combination stimulates exosome secretion ex vivo.
To investigate further the stimulatory effects of IAA/DNP on exosome secretion, tissue explants (kidneys) were harvested from C57BL/6 mice and cultured for 48 h in the presence or absence of IAA/DNP. Some kidneys were minced and other kidneys were left intact. Intact kidneys also received injections of IAA/DNP at three sites using a syringe. IAA/DNP caused a concentration-dependent increase of exosome release from tissue explants into culture medium. The concentration of 10 µM IAA/DNP was found to be most effective for the intact and minced tissues, whereas the tissue explants treated with IAA/DNP injections already responded to 5 µM IAA/DNP ( Fig. 4A-C). Similar TSG101 levels were detected in all exosome samples regardless of the concentration of IAA/DNP used (Fig. 4D).

IAA/DNP stimulates exosome secretion in vivo.
In vitro and ex vivo results were validated by injecting IAA/DNP into mice. Based on the amount of body fluid of mice, a dose of 0.195μmoles of IAA/DNP was used to provide an initial concentration of 10 μM in the body fluids. Another group of mice received a 5-fold higher dose (0.975μmoles). The injections did not affect the weight of the animals and did not alter their behaviour or induced signs of stress or pain (Fig. 4F). Both doses of IAA/DNP stimulated the levels of circulating exosomes in the blood  www.nature.com/scientificreports www.nature.com/scientificreports/ compared to control mice (Fig. 4E). Kidneys and livers of mice were harvested and cultured for 48 h after 14 days of treatment with IAA/DNP. Notably, exosome levels were elevated in both tissue types in a dose-dependent manner (Fig. 4G,H).

IAA/DNP causes a non-toxic energy depletion in cultured cells.
The numbers of dead cells in the culture medium measured indirectly by LDH assays showed low levels of LDH in the culture medium up to concentrations of 10 µM IAA/DNP (Fig. S2). However, 15 µM IAA/DNP led to a very slight, but statistically significant (p = 0.033), increase in LDH. Therefore, 10 µM was used as the highest concentration of IAA/DNP in subsequent assays and was considered as a non-toxic dose.
To further characterize the effects of IAA/DNP, HPLC was used to quantify levels of ATP, ADP and AMP after treatment of cells with 0, 1 and 10 µM IAA/DNP. Data were normalized to account for differences in cell number between conditions. In cultured cells, IAA/DNP decreased ATP levels ( Fig. 5A) but increased AMP levels (Fig. 5C), and these effects were concentration dependent. ADP levels were not affected by IAA/DNP treatment (Fig. 5B). Calculating the energy status of the cells using the formula (ATP + 1/2 ADP)/(ATP + ADP + AMP) revealed a significant drop of the energy charge (Fig. 5D).
To test the toxicity of IAA/DNP, SVEC4-10 were cultured in the presence of IAA/DNP for 48 h followed by 48 h of culture in the regular growth medium. This led to exosome levels which were comparable to those in untreated cells, indicating that the treatment with IAA/DNP is reversible and non-toxic (Fig. 3A,B).

Stimulation of exosome secretion by IAA/DNP is augmented by AMPK inhibition and attenuated by A 2B R antagonism. Stimulation of exosome production by IAA/DNP was significantly augmented
by an inhibitor (dorsomorphin) of AMP-activated protein kinase (AMPK) (p < 0.05, Fig. 5E).

Discussion
The clinical use of exosomes both as biomarkers of disease (e.g., cancer 17 , critical illness 18 or cardiovascular diseases 19 ) and carriers of drugs and biologics 5,20 is of great current interest. One of the most crucial limitations to achieve clinical use is the purification of exosomes in sufficiently large quantities. Although isolation techniques are constantly improving and several methods have been suggested to stimulate exosome release 21 , the reported techniques only yield limited quantities of exosomes and indicate that the need for an exosome stimulant remains unmet.
Several reports in the literature describe conditions or agents, such as e.g., monensin 22 or heat 23 , which increase exosome secretion by parental cells. In general, cells which are under stress are known to increase exosome secretion: thus, oxidative stress induced by ethanol increased exosome secretion by retinal pigment epithelium cells 24 or glucose starvation enhanced exosome secretion in cardiomyocytes 25 . However, the effects of glucose deprivation on exosome release is not robust and cannot be used in vivo. Additionally, these reports did not evaluate mechanisms or provide adequate details of the increased exosome production. Nevertheless, by focusing on a potential connection between energy depletion by glucose starvation and exosome release, they provided a rationale for studying IAA/DNP. It has been reported that the combination of IAA and DNP induces a potent reduction of cellular energy charge by simultaneously blocking oxidative phosphorylation and glycolysis 26 . We reasoned that blocking cellular pathways of energy production using IAA and DNP might be an effective strategy for releasing exosomes. Indeed, as shown in this report, IAA/DNP is a powerful stimulator of exosome secretion in cultured cells and in animal models.
To the best of our knowledge, IAA/DNP is the most effective method yet discovered to stimulate exosome release, and in our hands, it is more efficacious than other commonly used stimulators of exosome secretion (Fig. S3). Datta et al. screened the effects of 4580 pharmacologically compounds on exosome release and only 6 were found to be activators of exosome biogenesis with forskolin being the most potent one (6-fold increase) 27 . Also, IAA/DNP is safe, can be used both in vitro and in vivo and works across a variety of cell lines. It might therefore accelerate exosome research and be used as a tool for the generation of exosomes in different settings. However, even though the IAA/DNP treatment increases exosome secretion and even if the effect of these exosomes does not change the cell migration assays, other differences (e.g., in composition, heterogeneity, functional effects in other potency assays) might hypothetically change and should be investigated in future studies. Also, the protein-based quantification of circulating exosomes in a complex biofluid, such as mouse plasma, may also detect co-isolated non-exosome associated proteins. In particular, LDL, VLDL and chylomicron contaminations have been reported and might contribute to the heterogeneity of exosomes isolated from plasma [28][29][30] .
The underlying mechanisms for the elevated exosome secretion after IAA/DNP treatment are summarized in Fig. 7. IAA inhibits glycolysis and DNP inhibits oxidative phosphorylation and, thereby, the combination severely suppresses energy charge. Decreased ATP and increased AMP levels in IAA/DNP-treated cells confirm energy depletion. Further, AMP accumulation is well known to trigger two processes: 1) activation of adenosine receptors (ARs) via adenosine production from AMP 14 , and 2) activation of AMPK 31 . Our experiments show that blocking A 2B Rs attenuates the effects of IAA/DNP and blocking of AMPK augments IAA/DNP effects on exosome release. Therefore, we conclude that, in part, the ability of IAA/DNP to increase exosome release is mediated via A 2B Rs; but likely activation of AMPK (known to enhance energy production) attenuates the effects of IAA/DNP. Because it is known that IAA/DNP combination increases 2′,3′-cAMP 13 , we examined the role of endogenous 2′,3′-cAMP in exosome release by using 8-Br-2′,3′-cAMP and by using CNPase knockout cells. These experiments confirmed that 2′,3′-cAMP plays a role in the IAA/DNP-mediated release of exosomes from cells. Although the mechanism by which 2′,3′-cAMP increases exosome release remains unknown, it could involve: 1) formation of adenosine 14 ; 2) inhibition of mitochondrial function by opening mPTPs 15 ; 3) formation of stress granules, which would block protein synthesis 16 ; and 4) other "direct" effects of 2′,3′-cAMP on the process of exosome secretion. Besides activating the adenosine pathway and the 2′,3′-cAMP axis there may be other effects triggered by energy depletion induced by IAA/DNP. A recently published preprint by Frühbeis et al. describes further details and reports, that knockout of CNPase decreases basal release of exosomes from oligodendrocytes. They also reported that oligodendrocyte-derived exosomes are taken up by neurons and facilitate axonal transport. Notably, exosomes from CNPase knockout cells lack the ability to support nutrient deprived neurons and to promote axonal transport 32 .
IAA/DNP is the most effective method yet discovered to stimulate exosome release that involves, at least, A 2B Rs and 2′,3′-cAMP. This method allows for a harvest of ample exosomes from various cells and may serve as a platform technology for the development of exosome-based therapies in the future. www.nature.com/scientificreports www.nature.com/scientificreports/ Cell lines. Cells lines included in this study are listed in Table 1. All cell lines were grown at 37 °C in the atmosphere of 5% CO 2 in air. Cultures were supplemented with fetal bovine serum (FBS) depleted of exosomes by ultracentrifugation at 100,000xg for 3 h. Cells were cultured in 150 cm 2 cell culture flasks using 25 mL of culture medium. Media used for cell cultures are described in Table 1. Seeding protocol was optimized for each cell type as described by us 12 . After seeding, cells were allowed to attach to the flask for 6 h, were then treated with indicated reagents and incubated for 48 or 72 h as indicated.

Materials and methods
Exosome isolation by mini-SEC. Processing of supernatants and exosome isolation by mini-SEC was performed as previously described 11 . Briefly, cell culture supernatants were centrifuged at room temperature (RT) for 10 min at 2000 × g, transferred to new tubes for centrifugation at 10,000 × g at 4 °C for 30 min and filtrated using a 0.22 µm bacterial filter. Afterwards, aliquots of supernatants were concentrated by using Vivacell 100 concentrators at 2000 × g. 1 mL of concentrated supernatant was loaded on a 10 cm-long Sepharose 2-B column and eluted with PBS, and individual 1 mL fractions were collected. Fraction #4 containing non-aggregated exosomes was used in subsequent assays. Our established isolation technique fulfils the criteria of the MISEV2018 guidelines and we therefore use the term 'exosomes' throughout the manuscript 33   The simultaneous inhibition of glycolysis and oxidative phosphorylation leads to energy depletion in the cells (decreased ATP levels, elevated AMP levels). As a result of the energy depletion, the cells release adenosine, which activates the A 2B receptor system which then enhances exosome release. Simultaneously, the cells release 2′,3′-cAMP, which stimulates the release of exosomes directly, but can also be a source for adenosine, which again can activate the adenosine receptor system. www.nature.com/scientificreports www.nature.com/scientificreports/ Transmission electron microscopy (TEM). TEM was performed as previously described 11 . Freshly isolated tumor-derived exosomes (TEX) or normal cell-derived exosomes 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 exosome visualization.
Tunable resistive pulse sensing (TRPS). Size distribution and concentrations of the particles in isolated exosome fractions were analyzed using tunable-resistive pulse sensing (TRPS) by qNano (Izon) as described previously 34 .
Western blot analysis. To concentrate isolated exosomes, 0.5 mL 100 K Amicon Ultra centrifugal filters (EMD Millipore) were used for centrifugation at 4000 × g. Each lane was loaded with 5 μg of fraction #4 proteins, and PVDF membranes were incubated overnight at 4 °C with a TSG101 antibody (1:1000, ab30871, Abcam, Cambridge, MA) as previously described 34 . Cell migration. Cell migration by SVEC4-10 endothelial cells (ECs) was analyzed as previously described by us 12 . Briefly, 5 × 10 4 SVEC4-10 cells were starved in serum-free media overnight and were added to the upper compartment of 24-well transwell plates with 8 µm pore diameter (Corning). Cells migrated towards serum-free medium or the medium supplemented with 10 µg exosomes derived from UMSCC47 cells treated with 0, 1 or 10 µM of IAA/DNP or 10% FBS, which were added to the lower compartment. After 6 h of incubation at 37 °C, non-migrating cells in the upper chamber were removed with cotton swabs. Migrating cells on the lower surface of the membrane were fixed in methanol and stained with 0.2% crystal violet (Sigma-Aldrich). The number of migrated cells was counted in a light microscope in six randomly selected regions of interest at 20x magnification using an Olympus BX51 microscope (Olympus America, Center Valley, PA). Isolation of exosomes from tissue explants. Kidneys were harvested from 6 week old female C57BL/6 mice in an aseptic manner and immediately cultured in 6-well plates using 5 ml of DMEM supplemented with 1% (v/v) penicillin/streptomycin for 48 h as described by Mincheva-Nilsson et al. 35 . Tissue explants were treated with indicated concentrations of IAA/DNP. The treatment was given to intact tissue explants, minced tissue or was injected with an insulin syringe (29 G × 1/2″, Exelint, Redondo Beach, CA, USA) at three different locations. Supernatant was collected by gentle aspiration including washing of the tissue and the walls of the culture vessel. Processing of supernatants and exosome isolations were performed as described above.

Uptake of exosomes by
LDH assay. LDH release of cultured cells was performed using Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific) following the manufacturer's instructions. Cells were cultured for 72 h with indicated concentrations of IAA/DNP. Extraction and quantitation of NAD+, ATP, ADP, and AMP. ATP, ADP, and AMP were measured in cells treated with 0, 1 and 10 µM IAA/DNP using high performance liquid chromatography (HPLC). The protocol for the extraction of NAD+, ATP, ADP and AMP was previously described in detail 36 . isolation of rat cnpASe+/+ and −/− PGVSMCs. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase (CNPase) knockout rats used in this investigation were generated by the MCW Gene Editing Rat Resource Program (Dr. Aron M. Geurts, Department of Physiology and Human Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI). This strain was produced by injecting a CRISPR targeting the sequence GCTACTGCCGCCGGGACATC into rat embryos. The resulting mutation was a 7 base pair deletion in exon 2. Animals were genotyped by PCR. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from kidneys of wild type (CNPase+/+) and knockout (CNPase −/−) rats using our previously described method 37 . Cells were cultured at 37 °C in the atmosphere of 5% CO 2 in air using DMEM supplemented with exosome-depleted FBS. Exosome isolation was performed as described above.
Animal study. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the NIH. The protocol (18042580) was approved by the institutional Animal Care and Use Committee of the University of Pittsburgh (Animal Welfare Assurance Number: D16-00118). Female C57BL/6 mice aged 6 to 8 weeks were purchased from Jackson Laboratories. IAA/DNP was injected intraperitoneally daily for 14 days at the concentration of 0.195 or 0.975 μmoles per 100 µl. PBS injections of the same volume served as vehicle control. Blood was collected by submandibular bleeding on days 0, 7 and 14 of the experiment. Plasma was isolated by centrifuging at 1000xg for 10 min and further processed by spinning for 30 min at 10,000 × g and filtration with a 0.22 µm filter. Next, exosomes were isolated as described above. Additionally, kidneys and livers were harvested from all animals on day 14 and were cultured for 48 h. The supernatant of organ cultures was collected and exosomes were isolated as described above.