Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP … · Metabolism Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP

Metabolism

Extracellular ATP a New Player in CancerMetabolism: NSCLC Cells Internalize ATP In Vitroand In Vivo Using Multiple Endocytic MechanismsYanrong Qian1, XuanWang1,2,3,Yunsheng Li1,Yanyang Cao1,2,3, and Xiaozhuo Chen1,2,3,4,5,6

Abstract

Intratumoral extracellular ATP concentrations are 1000 timeshigher than those in normal tissues of the same cell origin.However, whether or not cancer cells use the abundant extracel-lular ATPwas unknownuntil we recently reported that cancer cellsinternalize ATP. The internalized ATP was found to substantiallyincrease intracellular ATP concentration and promote cell prolif-eration and drug resistance in cancer cells. Here, using a nonhy-drolyzable fluorescent ATP (NHF-ATP), radioactive and regularATP, coupled with high and low molecular weight dextrans asendocytosis tracers and fluorescence microscopy and ATP assays,cultured human NSCLC A549 and H1299 cells as well as A549tumor xenografts were found to internalize extracellular ATP atconcentrations within the reported intratumoral extracellular ATPconcentration range. In addition to macropinocytosis, both cla-thrin- and caveolae-mediated endocytosis significantly contributeto the ATP internalization, which led to an approximately 30%(within 45 minutes) or more than 50% (within 4 hours) increase

in intracellular ATP levels after ATP incubation. This increasecould not be accounted for by either purinergic receptor signalingor increased intracellular ATP synthesis rates in the ATP-treatedcancer cells. These new findings significantly deepen our under-standing of the Warburg effect by shedding light on how cancercells in tumors, which are heterogeneous for oxygen and nutritionsupplies, take up extracellular ATP and use the internalized ATP toperformmultiple previously unrecognized functions of biologicalimportance. They strongly suggest the existence of ATP sharingamong cancer and stromal cells in tumors and simultaneouslyidentify multiple new anticancer targets.

Implications: Extracellular ATP is taken up by human lung cancercells and tumors via macropinocytosis and other endocytic pro-cesses to supplement their extra energy needs for cancer growth,survival, and drug resistance, thus providing novel targets forfuture cancer therapy. Mol Cancer Res; 14(11); 1087–96. �2016 AACR.

IntroductionUpregulated glycolysis in cancer, the Warburg effect, has been

studied for almost one century (1–5). However, the biologicalreasons for the upregulation are still debated (6–8), and the effectis far from fully understood (9). One of the major controversiesrelated to the effect is the role of ATP synthesis. Warburg thoughtthat cancer cells' mitochondria were defective, and cancer cellswere forced to use glycolysis for ATP synthesis even under nor-moxic conditions (2–4). Cancer research in recent two decadesindicates that the Warburg effect is near-universally present incancer, while mitochondria in cancer cells function normally in

most cases examined (9). Other metabolic studies conclude thatATP is not a limiting factor in cancer cells (10), and there is noshortage of ATP in cancer as a whole (3, 10). On the other hand,such conclusions have been made by averaging the ATP synthesisrates among heterogeneous cancer cell populations in tumors. Inreality, due to uneven and dynamic angiogenesis as well as rapidbut constantly changing growth and proliferation rates, a largeand variable portion of cancer cells in a tumor is hypoxic, whereasothers are normoxic (11–13). The latter can synthesize twice asmuch ATP than the former (9). Thus, the conclusion that there isno shortage of ATP in cancer is correct as an average result but doesnot accurately reflect the real ATP status in individual cancercells. It has been documented that cancer cells, such as A549 cells(14–16), and stromal cells, such as T lymphocytes (17, 18), releaseATP. In addition, intratumoral extracellular ATP levels (ATP levelsin tumor interstitial space) have been found to be 103 to 104

times higher than those in normal tissues of the same cell origin(19–22), but cancer cells were not known to take up extracellularATP. The destination and function of high intratumoral extracel-lular ATP has been completely unknown until we recentlydescribed the internalization of extracellular ATP in cancer cellsbymacropinocytosis (23).We also reported that extracellular ATPnot only elevates intracellular ATP levels, but also significantlypromotes cancer cell growth, proliferation, and survival rates.Furthermore, extracellular ATP induces resistance of cancer cells toanticancer drugs such as tyrosine kinase inhibitors (TKI; ref. 23).On the basis of all these observations, we proposed an "ATP-sharing" model to explain how ATP is exchanged among cancer

1The Edison Biotechnology Institute, Ohio University, Athens, Ohio.2Department of Biological Sciences, Ohio University, Athens, Ohio.3InterdisciplinaryGraduateProgram inMolecular andCellular Biology,Ohio University, Athens, Ohio. 4Department of Biomedical Sciences,Ohio University, Athens, Ohio. 5Heritage College of Osteopathic Med-icine, Ohio University, Athens, Ohio. 6Department of Chemistry andBiochemistry, Ohio University, Athens, Ohio.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Correspondence Author: Xiaozhuo Chen, Department of Biomedical Sciences,Heritage College of Osteopathic Medicine; The Edison Biotechnology Institute,Ohio University, 101 Konneker Research Center, 172Water Tower Dr, Athens, OH45701. Phone: 740-593-9699; Fax: 740-593-4795; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0118

�2016 American Association for Cancer Research.

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cells and/or among stromal and cancer cells in tumors (8).According to this model, cancer cells use macropinocytosis andpossibly other endocytic processes to take up large amounts ofextracellular ATP to perform important biological functions.

To test the hypothesis, as a first step to validate the ATP-sharingmodel,we use anonhydrolyzablefluorescent ATP (NHF-ATP) as asurrogate for ATP to monitor the internalization of ATP intohuman non–small cell lung cancer (NSCLC) cells in vitro, ex vivo,and in vivo. High and low molecular weight fluorescent dextrans(HMWFD and LMWFD) as well-accepted and widely usedmolec-ular tracers for macropinocytosis (23, 24) and other endocytoses(25), respectively, are also used. Themajor objectives of this studyare to (i) demonstrate that ATP internalization is common inNSCLC cells; (ii) identify new ATP internalizationmechanisms inaddition to macropinocytosis; and (iii) determine whether ATPinternalization also occurs in tumors to show the in vivo relevanceand potential medical importance of the phenomenon. Theresults of these studies will shed light on the important questionof how the intratumoral extracellular ATP is used to promotetumorigenesis.

Materials and MethodsHuman NSCLC cells A549 and H1299 and nontumorigenic

lung cells NL-20 were from ATCC. NHF-ATP was from JenaBioscience, "high" and low molecular weight fluorescent TMR-dextrans (HMWFD and LMWFD) were from Invitrogen. Endocy-tosis inhibitors IPA-3, nystatin, filipin, and chlorpromazinehydrochloride (26, 27), purinergic receptor inhibitor suramin(28), and calcium chelator BApTA (29) were from Sigma.

ATP/dextran internalization assayThe study was performed as described previously (23) with

some modifications. Briefly, cancer cells and nontumorigeniclung cells NL-20 were grown on cover slips, then serum starvedfor 18 hours, washed with PBS, and incubated with serum-freeDMEM containing 10 mmol/L NHF-ATP or 8 mg/mL HMWFDor LMWFD in the presence or absence of endocytosis inhibitorsIPA-3, filipin, nystatin, or chlorpromazine at 37�C for varioustimes. After incubation and removal of the ATP solution and PBSwashes, cell-growing coverslipswerefixedwithparaformaldehydefor 10 minutes and then mounted with Gold Antifade Reagentwith DAPI (Invitrogen). Twelve to 24 hours after mounting, fixedcells were examined and photographed with fluorescence micros-copy (ECLIPSE E600, Nikon). Fluorescent images were analyzedand quantified using ImageJ (NIH, Bethesda, MD) as describedpreviously (23, 24). For each condition, 50 to100 cell imageswereanalyzed to quantify their number of fluorescent vesicles and thenaveraged. Cells without inhibitor treatment were used as controls,and their average number was assigned a relative value of 100%,andother treated cells were normalized by and comparedwith thecontrols.

ATP measurementCancer cells grown in 96-well plates were incubated with

serum-containing DMEM supplemented with ATP at variousconcentrations in the presence or absence of endocytosis inhibi-tors/siRNA at 37�C for different durations. After incubation, cellswere washed, lysed, and their intracellular ATP levels measuredwith a 96-well luminescence reader (Turner BioSystems) using aluciferase-based ATP Assay Kit as described previously (23). The

measured ATP levels of the untreated samples (negative controls)in the relative luminescence units were assigned a relative value of100%, and all othermeasured ATP levels were normalized by andcompared with the control.

Radioactive ATP assay[g-32P]-ATP (3,000 Ci/mmol and 10 mCi/mL, PerkinElmer)

was mixed with regular ATP at a final concentration of 2mmol/Land incubated with A549 cells grown in 6-well plates for varioustimes in a 37�C incubator with 5% CO2. After incubation, theATP solution was removed and cells were thoroughly washedwith PBS and then lysed. Cell lysates were either untreated orcentrifuged at 13,000 rpm, 4�C, to remove insoluble cell debrisbefore measuring the retained radioactivity using a scintillationcounter (Beckman). This study was to ascertain that ATP actuallyenters NSCLC cells without being associated with the plasmamembrane.

Purinergic receptor signaling inhibitor studyA549 cells in DMEM supplemented with 1 mmol/L ATP were

treated with 50 mmol/L suramin or 1 mmol/L BApTA. The treatedcells were kept in a 37�C incubator under 5% CO2 for varioustimes. After incubation, cells were lysed and their ATP levelsmeasured as described above in the "ATP measurement" section.

siRNA knockdown studyA549 cells were transiently transfected with siRNA specific

against mRNA of p21-activated kinase 1 (PAK1), an importantprotein involved in macropinocytosis (30, 31). The transfectionwas done by following the company's instructions. ScrambledsiRNA served as a mock control. Thirty-six hours after the trans-fection, 1 mmol/L extracellular ATP was added to the cells for2 hours. Cells were then lysed for their intracellular ATP levelmeasurement. Cell samples without ATP treatment were lysed fortheir protein level measurement of PAK1 by Western blots.Scrambled siRNA was used as a negative control.

Metabolic rate measurementsGlycolytic or mitochondrial OXPHOS rates of 30,000 A549

cells treated with or without ATP at different concentrations weremeasured continuously with anXFe 24 Extracellular Flux Analyzer(Seahorse Bioscience) in the assay medium containing DMEM,sodium chloride (143 mmol/L), glucose (10 mmol/L), pyruvate(1mmol/L), andGlutaMAX (2mmol/L). ATPwas dissolved in theassay medium and adjusted for pH with NaOH. These rates werereported as the extracellular acidification rate (ECAR) for glycol-ysis and oxygen consumption rate (OCR) for mitochondrialOXPHOS.

Tumor studyA549 tumors were grown on nude mice by subcutaneous

injection of 5 � 106 A549 cells into flanks of mice. When 200to 500 mm3 in size, tumors were injected with either DMEM(vehicle), 8mg/mLHMW, or LMWdextran with or without NHF-ATP (100 mmol/L) in DMEM with a volume of approximately50 mL using 1 CC syringes with 27 G needles. Four tumors wereinjected for each condition. Five minutes after injection, injectedmice were euthanized, and the tumors were surgically removedand immediately processed. The entire process from the begin-ning of injection to tumor freeze in OCT lasted about 7 to 8

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minutes.More than 30 slices per tumorweremounted, examined,and analyzed as described previously (23, 24). Briefly, photo-graphed fluorescent (NHF-ATP positive) cells in tumor slices werequantified using ImageJ for the number of fluorescent vesiclesinside each cells, and at least 100 NHF-ATP–positive cells perpositive condition were quantified and then averaged. The tumorprocessing and analysis were performed as the ex vivo proceduredescribed in Supplementary Information. Animal studies wereperformed in accordance to policies of NIH and Ohio UniversityIACUC.

Statistical analysisFor bioassays of cancer cell lines, each experimental condition

was performed in triplicate and repeated at least once. All data arereported as mean � SD and analyzed using Student t test or one-way ANOVA whenever appropriate. P < 0.05 was consideredsignificant.

ResultsA549 cells internalize NHF-ATP through bothmacropinocytosis and nonmacropinocytotic processes

When NHF-ATP (green) was added together with HMWFD(red) to A549 cells, they colocalized in vesicles inside A549 cells asdemonstrated by the yellowish color of the merged vesicles(merged image of inlets in Fig. 1A). This indicates that NHF-ATPwas internalized along with HMWFD by macropinocytosis asHMWFD is a macropinocytosis tracer (23–25). Interestingly, notall internalized NHF-ATP colocalized with HMWFD as indicatedby the presence of some green-colored vesicles (Fig. 1A), suggest-ing that NHF-ATP might also be internalized by some nonma-cropinocytic processes. This was supported by an inhibitor studyinwhich a large and constant fraction of intracellular ATP increasewas not blocked by a macropinocytosis inhibitor IPA-3 at

50 mmol/L at 1, 4, and 24 hours (Fig. 1B), the maximum con-centration of IPA-3 used in the literature.

NHF-ATP acts similarly to ATP in the ATP internalizationprocess

We went on to address the question whether NHF-ATP ordextrans acts similarly to regular ATP in the internalizationprocess. This is very important as NHF-ATP or dextrans can beused in place of ATP only if they behave like ATP in the process.

Regular ATP added to cell culture media induced a time-dependent intracellular ATP increase in A549 cells, and theincrease was reduced by IPA-3 in a short-term study (Fig. 2A).This again indicates the mechanism of the intracellular ATPincrease involves macropinocytosis. This was further confirmedby a time course study in which HMWFD and NHF-ATP showedqualitatively similar internalization profiles (Fig. 2B, quantifica-tions of the internalized HMWFD or NHF-ATP are shown inSupplementary Fig. S1). Corresponding dose-dependent reduc-tions for HMWFD and NHF-ATP were also observed in IPA-3–treated A549 cells (Fig. 2C), indicating that macropinocytosisplays an important role in the internalization mechanism forboth HMWFD and NHF-ATP. Therefore, for studying macro-pinocytosis-mediated ATP internalization, HMWFD can partial-ly substitute NHF-ATP. The time- and dose-dependence of ATPinternalization was further supported by the radioactive ATPstudy, in which the intracellular radioactivity of g-32P–labeledATP-treated A549 cells steadily increased over the course of 2hours, and the increase was proportional to the radioactive ATPadded to the cell culture media (ratios ¼ 1:3:10, Fig. 2D). TheATP internalization profile demonstrated by the radioactive ATPwas confirmed with a similar profile of regular ATP (Supple-mentary Fig. S2A). Consistent with fluorescence microscopyresults showing that internalized HMWFD- and NHF-ATP–con-taining vesicles were located in the cytoplasm of cancer cells,

Figure 1.

A549 cells internalize NHF-ATP andNHF-ATP colocalizeswith HMWFD. A, A549 cells were incubated with both10 mmol/L NHF-ATP (green) and HMWFD (red) for30 minutes and then examined with fluorescencemicroscopy and quantified by ImageJ. Inlets showcolocalization of the two molecules. Contour of each cellwasdrawnbased onphotos of the samebutoverexposedcells. B, intracellular ATP levels in A549 cells treated withorwithout extracellular ATP or IPA-3, amacropinocytosisinhibitor. �� , P < 0.001.

NSCLC Tumors Endocytotically Engulf ATP

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increased intracellular ATP or radioactivity was also shown to beassociated with the soluble cytoplasmic cell component (Sup-plementary Fig. S2B and S2C). These indicate that ATP or NHF-ATP actually entered the cytoplasm without being simply asso-ciated with the cell plasma membrane.

Macropinocytosis is upregulated in NSCLC cellsWhen HMWFD was used to treat A549 and H1299 cells in

the presence or absence of IPA-3, it was found that both celllines internalized HMWFD at relatively high levels (Fig. 3A).Also, IPA-3 treatment led to substantially reduced internaliza-tion (Fig. 3A), indicating that both cell lines show the pheno-type of macropinocytosis and internalize HMWFD (ATP). Incomparison, nontumorigenic NL-20 lung cells displayed muchlower HMWFD internalization (Fig. 3B), consistent with ourprevious results that extracellular ATP did not induce large

intracellular ATP increase in NL-20 cells as compared with theA549 cells (23).

PAK1 is known to be involved in macropinocytosis (30, 31).siRNA knockdown of the PAK1 gene resulted in the near completeelimination of the PAK1 protein in the knockdown cells and anapproximately 50% reduction of the intracellular ATP level ele-vation induced by extracellular ATP (Fig. 3C), consistent with theresult of IPA-3 inhibition (Figs. 1B and 3A). This result verifies therole of macropinocytosis in ATP internalization.

NSCLC cells also use clathrin- and caveolae-mediatedendocytoses to internalize ATP/dextran

We went on to address the question whether other nonmacro-pinocytotic endocytoses are also involved in ATP internalization.Fluorescent microscopy using NHF-ATP and/or HMWFD/LMWFD revealed that in H1299 cells, NHF-ATP not only

Figure 2.

Time-dependent and macropinocytosis-mediated internalization of NHF-ATP/dextran or radioactive ATP. A549 cells were incubated with either regular ATP,NHF-ATP, HMWFD, or radioactive ATP for various times with or without macropinocytosis inhibitor IPA-3. A–D, after incubation, cells were either lysed forintracellular ATP (A) or radioactive ATP measurement (D), or fluorescence microscopic examination for NHF-ATP/dextran (Dex) internalization (B) orNHF-ATP/dextran internalizations in the presence of increasing amount of IPA-3 (C). �� , P < 0.01, �� , P < 0.001.

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colocalized with HMWFD but also with LMWFD (Fig. 4A), whichis internalized by both macropinocytosis and nonmacropinocy-totic endocytoses. Furthermore, IPA-3 reduced HMWFD internal-izationwithout affecting LMWFD internalization asmuch in bothA549 and H1299 cells (Supplementary Fig. S3), suggesting that aportion of the LMWFD-associated internalization was mediatedby nonmacropinocytosis endocytosis.

Additional studies revealed that inhibitors blocking eithercaveolae- and clathrin-mediated endocytosis reduced intracellu-lar ATP level elevation induced by extracellular ATP in A549 cells(Fig. 4B) and H1299 cells (Fig. 4C), indicating that both types ofendocytoses, in addition to macropinocytosis, contributed to theATP elevation. These results were further confirmed by the fluo-rescence microscopy study using the same inhibitors (Fig. 4D, itsquantifications are shown in Supplementary Fig. S4).

Purinergic receptor signaling, glycolysis, or mitochondrialOXPHOS does not significantly contribute to intracellular ATPincrease

Purinergic receptor (PR) signaling is ATP dependent (28, 29),calcium release dependent (32, 33), and cell metabolism regu-latory (29, 32). To rule out the possibility that the intracellularATP increasewas partiallymediated by PR signaling, 50mmol/L ofsuramin, a PR inhibitor targeting P2 receptors (28), or BApTA, aCa2þ chelator (29) and general PR inhibitor, was used individ-ually to treat A549 cells incubated in ATP-containing DMEM.

Intracellular ATP levels were not significantly affected by eithersuramin (Fig. 5A) or BApTA (Fig. 5B).

Metabolic rate study using Seahorse analysis showed that afteraddition of extracellular ATP at 0.5 and 1 mmol/L, there weresmall and temporary increases of glycolysis rates, which lasted nomore than 30 minutes (Fig. 5C). Similar changes were observedfor the mitochondrial OXPHOS rates (Fig. 5D). These rateincreases were not large or long enough to account for theextracellular ATP-induced intracellular ATP level elevation, fur-ther supporting the conclusion drawn from other studies that theelevationwas not caused by potential PR-mediated ATP synthesis,but by ATP internalization.

A549 tumors internalize NHF-ATP ex vivo and in vivoFluorescencemicroscopy revealed that with an incubation time

of 8 minutes for ex vivo and 7 to 8 minutes for in vivo, NHF-ATPcolocalized with the HMWFD or LMWFD inside of tumor cells(Fig. 6A and B). IPA-3 treatment reduced internalization of NHF-ATP and its colocalized dextrans (Fig. 6B), indicating that A549tumors internalize ATP by mechanisms including macropinocy-tosis. When LMWFD (red) and NHF-ATP (green) were coinjectedinto A549 tumors grown on nude mice, a portion of these twomolecules colocalized as shown by the presence of yellowishintracellular vesicles (Fig. 6C). On the other hand, pure greenvesicles were also observed, indicating that some of the LMWFDwas not internalized by macropinocytosis. Similar in vivo results

Figure 3.

Upregulated macropinocytosis in NSCLC cells. NSCLC A549 and H1299 cells and nontumorigenic lung NL-20 cells were incubated with HMWFD for 30 minuteswith or without IPA-3. HMWFD internalization was fluorescent microscopically examined and quantified by ImageJ. A, HMWFD internalization in A549and H1299 cells with or without IPA-3 and their quantifications. Dex, dextran. B, comparison of HMWFD internalization between A549 and NL-20 cells. ��, P < 0.001.C, siRNA knockdown of PAK1 resulted in a significant reduction in the extracellular ATP-induced intracellular ATP level elevation. The PAK1 knockdown isdescribed in the Materials and Methods section and is seen in the Western blot analysis of the PAK1 protein using b-actin as the loading control.






were obtained with coinjection of HMWFD and LMWFD (Sup-plementary Fig. S5). The coinjection of IPA-3 resulted in largereduction, but not total elimination, of internalization ofHMWFD or LMWFD (Fig. 6B and C), possibly by nonmacropi-nocytic endocytoses-mediated internalization.

DiscussionCancer cells, including NSCLC A549 cells, are known to release

ATP (14–16). Cancer stromal cells, such as infiltrating T lympho-cytes, also release large amounts of ATPwhen stimulated (17, 18).These may account for the unusually high intratumoral extracel-lular ATP concentrations (19–22). However, cancer cells were not

known to take up extracellular ATP. Early efforts were made toshow animal cells take up ATP (34–36), but the results wereconsidered unconvincing largely because the ATP internalizationprocess could not be visualized or conclusively inferred fromindirect experimental measurements. The dominant idea of thefield was that extracellular ATP is not taken up by cells. On theother hand, intratumoral extracellular ATP levels recently havebeen found to be at least several hundred mmol/L or thousandtimes higher than those in normal tissues of the same cell origin(19–22). What is the destination of the ATP? It was unclear as tohow or even if cancer cells use the abundant intratumoralATP until we recently showed that extracellular ATP in theform of NHF-ATP was internalized by cultured A549 cells via

Figure 4.

Three different endocytosescontribute to ATP internalization.A549 and H1299 cells were incubatedwith ATP, NHF-ATP, and HMWFD orLMWFD, in the presence or absence ofdifferent endocytosis inhibitors,for 30 minutes. After incubation, cellswere fluorescence microscopicallyexamined for compoundinternalization or measured for theirintracellular ATP levels. A,colocalization of NHF-ATP withHMWFD or LMWFD in H1299 cells.B and C, intracellular ATP levelelevation was reduced by IPA-3 ornystatin, an inhibitor to caveolae-mediated endocytosis, orchlorpromazine, an inhibitor toclathrin-mediated endocytosis inA549 (B) and H1299 cells (C).� , P < 0.05; �� , P < 0.01; �� , P < 0.001.D, fluorescent microscopy ofinternalization of NHF-ATP or LMWFDin A549 cells in the presence orabsence of endocytosis inhibitors.Quantifications of the internalizationsare shown in Supplementary Fig. S4.

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macropinocytosis (23). We further demonstrated that extracellu-lar ATP performs multiple important functions in A549 cancercells, including substantial elevation of intracellular ATP levels,promotion of cell proliferation and survival, and induction ofdrug resistance to anticancer drug TKIs (23). All these functionsappeared to be at least in part a result of direct ATP internalization.However, it was unknown whether other ATP internalizationmechanisms in addition to macropinocytosis exist or, moreimportantly, whether the phenomenon occurs in vivo.

In this study, we furthered our ATP investigation by showingthat NHF-ATP is internalized not only by NSCLC A549 cells butalso NSCLC H1299 cells via not only macropinocytosis but alsoclathrin- and caveolae-mediated endocytoses (Figs. 3 and 4).Furthermore, the internalization occurs not only in culturedcancer cells but also in tumors (Fig. 6). These results indicatethat ATP internalization is common among NSCLC cells andpresent in vivo. Compared with nontumorigenic NL-20 lung cells,macropinocytosis-mediated extracellular molecule internaliza-tion is a drastically upregulated event in NSCLC lung cancer cells(Fig. 3B). The key role of macropinocytosis in ATP internalizationwas further confirmed by the PAK1 knockdown study (Fig. 3C).The finding that NSCLC cells use upregulated macropinocytosisand other two endocytoses to internalize extracellular ATP alsosimultaneously identifies three potential targets for treatingNSCLC and other cancers that also exhibit upregulated endocy-toses. Of note, both A549 and H1299 NSCLC cells expressmutated Ras genes (37, 38), which have been implicated in theupregulation of macropinocytosis (24). Our finding is also con-sistent with the recent classification of the opportunistic uptake ofextracellular nutritional molecules as an emerging hallmark for

cancer metabolism (39) and the contribution of macropinocy-tosis in the uptake of extracellular molecules in A549 lung cancerand pancreatic cancer (40, 41).

Studies using general purinergic receptor signaling inhibitorsrevealed that overall purinergic receptor signaling is unable toaccount for the intracellular ATP level elevation or ATP internal-ization (Fig. 5). However, this result does not rule out thepossibility of the involvement of an individual purinergic recep-tor, such as P2X7 (42–45), in the elevation.

Metabolic rate analyses also rule out the possibility that the ATPelevation was due to rate increases in either glycolysis or mito-chondrial OXPHOS, providing additional supportive evidencethat the ATP elevation is caused, at least in a large part, byendocytic internalization of extracellular ATP.

These studies show that the endocytosis-mediated NHF-ATPinternalization exhibits both temporal and spatial profiles similarto those of intracellular ATP increase. They indicate that the ATPinternalization process could be mimicked and visualized usingNHF-ATP. This conclusionwas further supported by the uptake ofradioactive ATP (Fig. 2D) and the association of radioactivity withthe cytoplasmic fraction of the cells (Supplementary Fig. S2C).Furthermore, multiple endocytic processes are shown to beinvolved in internalizing ATP and HMWFD/LMWFD are knowntracers for these processes. Therefore, HMWFD and/or LMWFDcan be used alone or in combinationwithNHF-ATP tomimic andmonitor ATP's internalization processes mediated by differentendocytic pathways.

It is noteworthy that fluorescence microscopic study of thein vitro ATP internalization was done at a concentration of 10mmol/L NHF-ATP, more than 20 to 50 times lower than reported

Figure 5.

Purinergic receptor signaling onintracellular ATP levels and extracellularATP on glycolytic and OXPHOS rates.A549 cells were incubated with ATP forvarious times in the presence or absenceof suramin (A), a purinergic receptorinhibitor or BApTA (B), a calciumchelator. After incubation, cells werelysed and measured for theirintracellular ATP levels. C and D,extracellular ATP induced small andtemporary change in either ECAR(glycolytic; C) or OCR (OXPHOS) rates(D) as revealed by the Seahorseanalysis. Arrows (C and D), timeof ATP addition. �� , P < 0.001.






intratumoral ATP concentrations (19–22) but also in the toprange of extracellular ATP concentrations found in some normaltissues, such as muscle. In comparison, ex vivo and in vivo studieswere done at 100 mmol/L NHF-ATP, several folds below reportedintratumoral ATP concentrations. Positive results generated fromas low as 10 mmol/L up to 1 mmol/L ATP indicate that ATPinternalization occurs in cancer cells and tumors at awide range ofextracellular ATP concentrations. These suggest that cancer cells intumors may exhibit the flexibility for boosting their intracellularATP levels by taking advantage of the presence of extracellular/intratumoral ATP regardless its concentration by using the com-pound-independent macropinocytosis/endocytoses.

Ex vivo and in vivo studies showed that A549 tumors, likecultured A549 cells, internalized NHF-ATP using multipleendocytic pathways, substantially enhancing the in vivo rele-vance and pathologic significance of the phenomenon andjustify for its future study. Internalization of ATP is more

bioenergetically and metabolically favorable than internaliza-tion of other intratumoral extracellular molecules because ofthe exceptionally high intratumoral extracellular ATP concen-trations, and the internalization is an energy-gaining processfor recipient cancer cells. Greatly upregulated ability for engulf-ing large amount of extracellular ATP enables cancer cells tomore efficiently utilize energy from its extracellular environ-ment and maintain or change intracellular metabolism, reduc-ing their burden to synthesize needed ATP under heteroge-neous cell growth conditions. This is a previously unrecognizedprocess worthy of further scrutiny to better understand theWarburg effect and to design novel anticancer strategies target-ing ATP internalization. Our findings provide insight and apossible answer to the fundamental question raised by War-burg: How can hypoxic cancer cells have enough ATP evenwhen their ATP synthesis is shifted to glycolysis, a process thatgenerates approximately 10 times less ATP than mitochondrial

Figure 6.

A549 cells internalize ATP ex vivo and in vivo by multiple endocytic processes. A549 cells were used to generate tumors on nude mice. NHF-ATP was eitherincubated with surgically removed tumor (ex vivo as in A) or directly injected into tumors (as in B and C) with or without HMWFD in the presence or absence ofmacropinocytosis inhibitor IPA-3. After treatment, tumors were sliced, fixed, photographed, and quantified for internalization of NHF-ATP and/or dextrans(Dex) by ImageJ. �� , P < 0.001. A, A549 tumors internalized NHF-ATP along with HMWFD ex vivo. B, A549 tumors internalized NHF-ATP and HMWFDand the internalization was reduced by IPA-3. C, colocalization of NHF-ATP and LMWFD in A549 tumors and their internalization was reduced but noteliminated by IPA-3.

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OXPHOS? They can also provide a possible explanation for theconclusion of "cancer cells have no shortage of ATP" made byothers. The presence of high levels of intratumoral extracellularATP provides a reliable source of ATP for all cancer cells intumors. Whenever there are temporary shortages of intracellu-lar ATP, cancer cells can quickly upregulate endocytoses to takeup ATP from interstitial space to meet their metabolic andgrowth needs. Our findings also simultaneously offer a poten-tial mechanism for the survival and growth of hypoxic cancercells in tumors.

All these findings are consistent with and strongly support ourrecently proposed "ATP-sharing" model (8). It is conceivable thatATP internalization occurs in a vast number of cancer types andplays significant but previously unrecognized roles in cancermetabolism, cancer development, drug resistance, and evenmetastasis as it is an ATP-dependent process (46, 47). On the

basis of these newfindings, an improvedmodel for ATP sharing intumors is proposed (Fig. 7). These findings are also compatiblewith a recently proposed tumor development model that predictsthat cancer cells migrate within tumors to accelerate tumorigen-esis (48), as ATP is required for cellmovement andmigration. Thisis also consistent with the recent finding of reciprocal interactionsbetween mutated Kras-expressing cancer cells and stromal cells(49). It also provides new evidence to support the emerginghallmark of cancer metabolism: Cancer cells utilize the opportu-nistic uptake of extracellular nutritional/energy molecules (39),ATP in this case. The identification of extracellular ATP's roles incancer metabolism has added onemore extracellular molecule tothe list of functionally important intratumoral extracellularmole-cules for cancer metabolism. It also offers a new target for futureanticancer therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Qian, X. ChenDevelopment of methodology: Y. Qian, Y. Li, X. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Qian, X. Wang, Y. CaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Qian, X. Wang, Y. Cao, X. ChenWriting, review, and/or revision of themanuscript: Y. Qian, X. Wang, X. ChenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Qian, X. Wang, Y. Cao, X. ChenStudy supervision: X. ChenOther (animal studies): Y. Li

AcknowledgmentsWe thank Athena Chen and Emily Trzeciak for proofreading the manuscript

and Misako Hata and Qiongyu Shi for technical assistance.

Grant SupportThis work was partially supported by the Konneker Fund of Ohio

University and by a Student Enhancement Award, a Graduate Student SenateOriginal Work Grant, and a Donald Clippinger Graduate Fellowship fromOhio University (to Y, Qian).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 8, 2016; revised August 7, 2016; accepted August 17, 2016;published OnlineFirst August 30, 2016.

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Qian et al.




2016;14:1087-1096. Published OnlineFirst August 30, 2016.Mol Cancer Res Yanrong Qian, Xuan Wang, Yunsheng Li, et al. Mechanisms

Using Multiple EndocyticIn Vivo and In VitroInternalize ATP Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells

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Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP … · Metabolism Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP