H OH OH
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Stable Isotope-Resolved Metabolomics Shows Metabolic Resistance to Anti-Cancer Selenite in 3D Spheroids versus 2D Cell Cultures Teresa W.-M. Fan 1, *,† , Salim S. El-Amouri 1,† , Jessica K. A. Macedo 1 Huan Song 1 ID , Teresa Cassel 1 and Andrew N. Lane 1 ID 1
2
* †
ID
, Qing Jun Wang 2 ,
Center for Environmental and Systems Biochemistry, Markey Cancer Center, and Depart of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY 40506, USA;
[email protected] (S.S.E.-A.);
[email protected] (J.K.A.M.);
[email protected] (H.S.);
[email protected] (T.C.);
[email protected] (A.N.L.) Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY 40506, USA;
[email protected] Correspondence:
[email protected]; Tel.: +1-859-218-1028 These authors contributed equally to this work.
Received: 9 May 2018; Accepted: 6 July 2018; Published: 10 July 2018
Abstract: Conventional two-dimensional (2D) cell cultures are grown on rigid plastic substrates with unrealistic concentration gradients of O2 , nutrients, and treatment agents. More importantly, 2D cultures lack cell–cell and cell–extracellular matrix (ECM) interactions, which are critical for regulating cell behavior and functions. There are several three-dimensional (3D) cell culture systems such as Matrigel, hydrogels, micropatterned plates, and hanging drop that overcome these drawbacks but they suffer from technical challenges including long spheroid formation times, difficult handling for high throughput assays, and/or matrix contamination for metabolic studies. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles that enable spheroid formation and growth via magnetizing cells. M3DB spheroids have been shown to emulate tissue and tumor microenvironments while exhibiting higher resistance to toxic agents than their 2D counterparts. It is, however, unclear if and how such 3D systems impact cellular metabolic networks, which may determine altered toxic responses in cells. We employed a Stable Isotope-Resolved Metabolomics (SIRM) approach with 13 C6 -glucose as tracer to map central metabolic networks both in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells without or with an anti-cancer agent (sodium selenite). We found that the extent of 13 C-label incorporation into metabolites of glycolysis, the Krebs cycle, the pentose phosphate pathway, and purine/pyrimidine nucleotide synthesis was largely comparable between 2D and M3DB culture systems for both cell lines. The exceptions were the reduced capacity for de novo synthesis of pyrimidine and sugar nucleotides in M3DB than 2D cultures of A549 and PANC1 cells as well as the presence of gluconeogenic activity in M3DB spheroids of PANC1 cells but not in the 2D counterpart. More strikingly, selenite induced much less perturbation of these pathways in the spheroids relative to the 2D counterparts in both cell lines, which is consistent with the corresponding lesser effects on morphology and growth. Thus, the increased resistance of cancer cell spheroids to selenite may be linked to the reduced capacity of selenite to perturb these metabolic pathways necessary for growth and survival. Keywords: selenite; cancer metabolism; A549; PANC1; 3D spheroids
Metabolites 2018, 8, 40; doi:10.3390/metabo8030040
13 C
6 -glucose
tracer; 2D cell cultures;
www.mdpi.com/journal/metabolites
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1. Introduction It is now clear that mammalian cells grown as two-dimensional (2D) cultures on rigid and treated surfaces may exhibit different behavior and functional properties (such as drug sensitivity) from those in the native microenvironment or in three dimensional (3D) cultures i.e., spheroids and organoids [1–4]. This could be attributable to the lack of cell–cell and cell–extracellular matrix (ECM) interactions as well as the unrealistic gradients of O2 , nutrients, growth factors, and treatment agents in 2D cultures [1]. Three-dimensional spheroids are self-assembled compact aggregates of single cell types to enable extensive cell–cell and cell–ECM interactions, whereas 3D organoids add the interactions with other cell types [3,5]. They can circumvent the drawbacks of 2D cell cultures in terms of ECM formation, cell–cell interactions, and physicochemical environment. Spheroids can be generated from mono-cultures spontaneously or in an appropriate matrix such as Matrigel, collagen, hyaluronan, or synthetic polyethylene glycol [5–10], while organoids are typically formed from live tissues or multiple cell types in Matrigel or collagen [5,9,11–13]. Both spheroids and organoids more closely mimic the natural tissue organization than 2D cultures, while retaining similar experimental flexibility [10,14]. They have been shown to display altered gene expression profiles including those metabolism-related, nutrient oxidation capacity, metastatic capacity induced by proline catabolism, O2 consumption/extracellular acidification, and drug response compared with the 2D cell culture counterparts [1,3,4,7,15–18]. However, our knowledge is still very limited regarding the functional or metabolic mechanisms underlying these differences. Although multiple scaffolds (e.g., Matrigel, hydrogel) or scaffold-free (hanging drop, micropatterned/U-shape cell-repellent plates) 3D cell culture systems have been developed for culturing cancer cell spheroids [3,5,19], they suffer from technical challenges such as long spheroid formation times with variable efficiency and/or difficult handling for high throughput assays [1]. For metabolomic studies, there are added problems of matrix contamination and/or limitations in scaling-up. Magnetic 3D bioprinting (M3DB), a more recent development in 3D culture system, can overcome these difficulties. This technique utilizes nanoparticles composed of gold, iron oxide, and poly-L-lysine to magnetize cells, followed by spheroid assembly under mild magnetic forces [1]. Spheroids are formed reproducibly within minutes to hours in cell repellent plates and are biocompatible for various functional assays including toxicity testing [1,20–26]. This matrix-free method is particularly amenable for Stable Isotope-Resolved Metabolomic (SIRM) studies [2,27–37] as securing spheroids with magnets enables rapid tracer medium change, sampling at different time points or removal during metabolic quenching. Here, we demonstrate the utility of coupling the M3DB culturing with SIRM in exploring the metabolic mechanism that underlies the increased resistance to anti-cancer sodium selenite in 3D spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells versus their 2D cell counterparts. 2. Results 2.1. Three-Dimensional A549 and PANC1 Spheroids Are More Resistant to Selenite Than Their 2D Cell Counterparts We have shown previously that sodium selenite (a chemopreventive agent [38,39]) is toxic to A549 cells in 2D cultures with 24 h IC50 (half maximal inhibitory concentration) at 6.25 µM [40]. Here we determined the 26 h IC50 of selenite in 2D PANC1 cells to be 10 µM. We also investigated the dose-dependent effect of selenite on the growth of A549 and PANC1 M3DB spheroids. As shown in Figure 1A, selenite had a complex effect on the growth of both spheroid types after 1- and 2-day of treatment. There appeared to be at least two populations of cells with one more resistant than the other. The IC50 for neither population could be determined after 1-day treatment. In addition, at doses ≤50 µM, selenite slightly stimulated the overall growth of both spheroid types at 1-day, but not 2-day, of treatment. However, selenite inhibited the growth (Figure 1A) and disrupted spheroid structures (Figure 1B) of both sensitive and resistant populations alike after 3 days of treatment. The complexity of
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the dose-dependence curves made it difficult to estimate the IC50 for the 1-day treatment. We were able to determine as Metabolitesthe 2018,IC 8, x50 for the sensitive A549 spheroids as 16.7 µM at 3-day and PANC1 spheroids 3 of 16 9.4 and 13.7 µM at 2- and 3-day, respectively (Table 1). The 2-day IC50 for the sensitive A549 spheroids and PANC1 spheroids as 9.4was and not 13.7as µM at 2-defined and 3-day, (Table 1). The IC50 for was estimated at 46 µM, which well duerespectively to insufficient points in 2-day the dose-response the sensitive A549 spheroids was estimated at 46 µM, which was not as well defined due to curve. Suffice to say, the spheroid cultures of A549 and PANC1 cells displayed overall less growth insufficient points in the dose-response curve. Suffice to say, the spheroid cultures of A549 and inhibition than the 2D counterparts. PANC1 cells displayed overall less growth inhibition than the 2D counterparts. In addition, we measured the the ROS production asasa afunction selenitedose doseininA549 A549 and In addition, we measured ROS production functionof oftime time and and selenite PANC1 spheroids, as shown in Figure 1C. Selenite induced a burst ofofROS 10–20 µM and PANC1 spheroids, as shown in Figure 1C. Selenite induced a burst ROSproduction production atat10–20 after 1µM dayafter of treatment in both in A549 PANC1 spheroids. ThisThis burst persisted, 1 day of treatment bothand A549 and PANC1 spheroids. burst persisted,albeit albeitto to aa lower extent, for upoftotreatment 3 days of in treatment in A549 not in spheroids. PANC1 spheroids. In addition, ROS extent,lower for up to 3 days A549 but not inbut PANC1 In addition, ROS production did notwith commensurate with growth inhibition in A549 appeared did notproduction commensurate growth inhibition in A549 spheroids but spheroids appeared but to be related to to be reduced related to reduced growth and/or cell death in PANC1 spheroids after 3 days of treatment. Based on growth and/or cell death in PANC1 spheroids after 3 days of treatment. Based on these data, we chose these data, we chose 24 h of 10 µM selenite treatment, which gave a burst of ROS in both spheroid 24 h of 10 µM selenite treatment, which gave a burst of ROS in both spheroid types, in subsequent types, in subsequent 13C6-glucose-based SIRM experiments on the spheroid cultures. The results 13 C -glucose-based SIRM experiments on the spheroid cultures. The results were compared with those 6 were compared with those obtained from the 2D culture experiments performed with IC50 doses of obtained fromi.e., the6.25 2D µM culture experiments doses of selenite, i.e., 6.25 µM for A549 50 design selenite, for A549 and 10 µMperformed for PANC1with cells. IC This should maximize differential and 10metabolic µM for PANC1 Thisspheroid design should maximize differential metabolic responses between responsescells. between (with the resistant cell population presumably unaffected) and(with 2D cellthe cultures. spheroid resistant cell population presumably unaffected) and 2D cell cultures.
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Figure 1. Dose- and time-dependent growth inhibition and ROS production of A549 and PANC1
Figurespheroids 1. Dose-byand time-dependent growth inhibition and ROS production of A549 and PANC1 selenite. A549 or PANC1 spheroids were formed, cultured, and assayed for growth spheroids selenite. A549 or PANC1 spheroids were formed, cultured, and assayed growth usingby a live cell stain PrestoBlue in 384-well plates as described in Materials and Method. The for slope using of a live cell stain PrestoBlue inof384-well plates as described in Materials and Method. The the time-dependent reduction PrestoBlue was calculated for each well, averaged, and plotted as slope 50 function of selenitereduction concentrations in (A); n = 5was per treatment. These to estimate ICplotted of the atime-dependent of PrestoBlue calculated for data eachwere well,used averaged, and and percentage of sensitive cell population in n Table by treatment. data fitting (see Materials and Methods). In as a function of selenite concentrations in (A); = 5 1per These data were used to estimate (B),percentage example images (10× magnification) of spheroids after 3 days 0 or 50 µMMaterials selenite treatment. IC50 and of sensitive cell population in Table 1 by data of fitting (see and Methods). Scale bars are 400 µm. In (C), time- and selenite dose-dependent production of reactive oxygen In (B), example images (10× magnification) of spheroids after 3 days of 0 or 50 µM selenite treatment. species (ROS) by A549 and PANC1 spheroids was measured by dichlorofluoroscein (DCF) Scale bars are 400 µm. In (C), time- and selenite dose-dependent production of reactive oxygen fluorescence. n = 3 per data point. species (ROS) by A549 and PANC1 spheroids was measured by dichlorofluoroscein (DCF) fluorescence. n = 3 per data point. Table 1. IC50 of selenite for A549 and PANC1 spheroids after 2 and 3 days of treatment. Spheroids Treatment Days IC50 (µM) % Sensitive a R2 Table 1. IC50 of selenite for A549 and PANC1 spheroids after 2 and 3 days of treatment. b A549 2 (46) 53 0.997 A549 3 16.7 ± 0.6 81 0.998 Spheroids Treatment Days IC50 (µM) % Sensitive a R2 PANC1 2 9.4 ± 0.2 32 0.999 A549 53 0.997 (46)±b0.9 PANC1 32 13.7 92 0.995 a
a
A549 3 16.7 ± 0.6 81 0.998 b not well-determined. Percentage PANC1 of the sensitive 2 cell population 9.4 ±(cf. 0.2Figure 1A); 32 0.999 PANC1 3 13.7 ± 0.9 92 0.995
Percentage of the sensitive cell population (cf. Figure 1A); b not well-determined.
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4 of Due 16 2.2. 2.2. The The Higher Higher Selenite Selenite Resistance Resistance of of 3D 3D A549 A549 and and PANC1 PANC1 Spheroids Spheroids Is Is Not Not Due to to Less Less Se Se Accum Accum Than Than the the 2D 2D Counterparts Counterparts
2.2. The Higher Selenite ResistanceOne of 3D A549 andquestion PANC1 Spheroids Is higher Not Dueselenite to Less Se Accumulation One immediate immediate question about about the the higher selenite resistance resistance in in the the 3D 3D spheroids spheroids was was Than the 2D Counterparts due due to to less less Se Se accumulation accumulation in in their their biomass biomass than than that that of of the the corresponding corresponding 2D 2D cells. cells. To To add add
question, question, we performed parallel parallel selenite selenite in treatments treatments of of A549 A549 and and PANC1 cells cells as as 2D 2D and and 3D 3D One immediate question aboutwe theperformed higher selenite resistance the 3D spheroids wasPANC1 if this is cultures cultures for for total total Se Se analysis analysis by by ICP-MS. ICP-MS. We We found found that that total total Se Se in in 3D 3D spheroids spheroids were were hig hig due to less Se accumulation in their biomass than that of the corresponding 2D cells. To address this that that in in 2D 2D cultures cultures for for both both A549 A549 and and PANC1 PANC1 cells cells (sum, (sum, Figure Figure S1). S1). The The higher higher Se Se accumu accumu question, we performed parallel selenite treatments of A549 and PANC1 cells as 2D and 3D spheroid 3D 3Dby biomass biomass was wasWe aa result result of ofthat elevated elevated selenite uptake from fromwere the the media media for for A549 A549 cells cells but but not not for fo cultures for total Se analysis ICP-MS. found total selenite Se in 3Duptake spheroids higher than cells cells (medium (medium uptake, uptake, Figure Figure S3). S3). The The latter latter suggests suggests enhanced enhanced loss loss of of absorbed absorbed sele sel that in 2D cultures for both A549 and PANC1 cells (sum, Figure S1). The higher Se accumulation volatilization volatilization [41] in in 2D 2Duptake over over 3D 3D PANC1 PANC1 cells. cells. By By analyzing analyzing Se Se in in polar polar extracts, protein protein extr extr in 3D biomass was a result of elevated[41] selenite from the media for A549 cells but not extracts, for residue residue (after (after polar polar and and protein protein extractions) extractions) of of A549 A549 and and PANC1 PANC1 cells, cells, we we found found that that the the Se S PANC1 cells (medium uptake, Figure S3). The latter suggests enhanced loss of absorbed selenite via followed followed the the order order residue residue > > protein protein > > polar polar fractions fractions for for both both 2D 2D and and 3D 3D cultures. cultures. It It is is inter inter volatilization [41] in 2D over 3D PANC1 cells. By analyzing Se in polar extracts, protein extracts, and note note that that Se Se was was enriched enriched relatively more more in in we the thefound soluble soluble (polar (polar and and protein) protein) fractions fractions of of 2D 2D residue (after polar and protein extractions) of A549 relatively and PANC1 cells, that the Se content cultures cultures for for both both A549 A549 and and PANC1 PANC1 cells. cells. It It is is possible possible that that some some form(s) form(s) of of Se Se in in these these followed the order residue > protein > polar fractions for both 2D and 3D cultures. It is interesting contribute contribute to tomore higher higher toxicity in in the the 2D 2D cell cell cultures [40]. In In any any rate, there there was was no no straigh straigh to note that Se was enriched relatively intoxicity the soluble (polar andcultures protein)[40]. fractions of rate, 2D than relationship between selenite selenite uptake uptake and and observed observed differences differences in infractions 2D 2D and and 3D 3D cell cell toxicity. toxicity. 3D cultures for both A549 relationship and PANC1between cells. It is possible that some form(s) of Se in these contribute to higher toxicity in the 2D cell cultures [40]. In any rate, there was no straightforward 2.3. 2.3. Glycolysis Glycolysis and and the the Krebs Krebs Cycle Cycle Respond Respond Less Less to to Selenite Selenite in in A549 A549 and and PANC1 PANC1 Spheroids Spheroids than than in in relationship between selenite uptake and observed differences in 2D and 3D cell toxicity. Cell Cell Counterparts Counterparts
2.3. Glycolysis and the Krebs CycleIn Less to Selenite6.25 in A549 PANC1 Spheroids than Their 2D InRespond SIRM SIRM experiments, experiments, 6.25 µM µMand selenite selenite attenuated attenuated net net in growth growth and and induced induced morph morp Cell Counterparts changes changes in in A549 A549 cells cells grown grown in in 2D 2D cultures cultures (Figure (Figure S2A). S2A). Such Such changes changes were were less less evide evide In SIRM experiments, corresponding 6.25 µM selenite3D attenuated net induced morphological changes corresponding 3D spheroids spheroids at atgrowth 10 10 µM µM and selenite selenite (Figure (Figure S2B). S2B). Likewise, Likewise, 10 10 µM µM selenite selenite elicite elicite in A549 cells grown in 2D cultures (Figurein Such changes were less evident the corresponding perturbations perturbations inS2A). morphology morphology and and proliferation proliferation in in 2D 2Din(Figure (Figure S3A) S3A) than than 3D 3D cell cell cultures cultures of o 3D spheroids at 10 µM selenite (Figure S2B). Likewise, 10 µM selenite elicited greater in (Figure (Figure S3B), S3B), which which is is consistent consistent with with the the presence presence of of perturbations aa higher higher population population of of resistant resistan morphology and proliferation in 2D (Figure thancultures, 3D cell cultures of PANC1 (Figure S3B), which spheroid spheroid than than in inS3A) 2D 2D cell cell cultures, as as described described above. above. Consistent Consistent with with the the PrestoBlue PrestoBlue aa is consistent with the presence of (Figure a(Figure higher1A), population of resistant cells in spheroid than in 2D cell growth growth 1A), the the mitotic mitotic index index (as (as indicated indicated by by the the PCNA PCNA fluorescence) fluorescence) was was redu redu cultures, as described above. Consistent with the PrestoBlue assay for growth (Figure 1A),spheroids. the mitoticThe days days of of 100 100 µM µM selenite selenite treatment treatment in in both both A549 A549 and and PANC1 PANC1 spheroids. The growth growth inhibi inhib index (as indicated by the accompanied PCNA fluorescence) was reduced by 3(as days of 100 µM treatment accompanied by by increased increased necrosis necrosis (as indicated indicated by by selenite the the RIP-1 RIP-1 fluorescence) fluorescence) in in both both A A in both A549 and PANC1 spheroids. The growth inhibition PANC1 PANC1 spheroids spheroids (Figure (Figure S2). S2). was accompanied by increased necrosis (as indicated by the RIP-1 fluorescence) in both A549 and PANC1 (Figure These These phenotypic phenotypic differences differences were werespheroids accompanied accompanied by by S2). differential differential metabolic metabolic responses responses to to 13 13 These phenotypic differences were accompanied by differential metabolic responses to selenite between between 2D 2D cell cell and and 3D 3D spheroid spheroid cultures. cultures. Figure Figure 22 compares compares the the extent extent of of C C66-glucose -glucose ( 13 C -glucose (13 C -Glc) between 2D cell and 3D spheroid cultures. Figureglycolysis 2 compares thethe extent ofcycle transformation transformation through through glycolysis and and the Krebs Krebs cycle between A549 A549 in 2D 2D culture culture and and as as ss 6 between 6 in transformation through glycolysis and the between treatments A549 in 2D culture spheroids in respectively. in in response response to toKrebs 24 24 hhcycle of of selenite selenite treatments at at 6.25 6.25and µM µMasand and 10 10 µM, µM, respectively. Si Si response to 24 h of seleniteperturbations treatments at to 6.25 µMglycolytic and 10 µM, respectively. Significant perturbations perturbations to the the glycolytic activity activity were were evident evident in in selenite-treated selenite-treated 2D 2D cells cells based based to the glycolytic activity were evident in selenite-treated 2D the cells based on the enhanced release enhanced enhanced release release of of 1313C C33-lactate -lactate into into the medium medium (Figure (Figure 2(I-D)) 2(I-D)) and and lower lower fractional fractional enrich enric 13 13C of 13 C3 -lactate into the medium (Figure and (3; lower fractional enrichment in intracellular intracellular intracellular C2(I-D)) 33-pyruvate -pyruvate (3; Figure Figure 2(I-B)). 2(I-B)). In In contrast, contrast, there there was was no no effect effect of of sel sel 13 C -pyruvate (3; Figure 13 13 13 13 13 2(I-B)). In contrast, was effect of selenite on C3 -pyruvate C C33-pyruvate -pyruvate (Figure (Figurethere 2(II-B)) 2(II-B)) and andnothe the stimulation stimulation of of C C33-lactate -lactate release release was was relatively relatively low low 3 13 C -lactate release was relatively lower from spheroid cultures (Figure 2(II-B)) and the stimulation spheroid spheroidofcultures cultures (Figure (Figure 2(II-D)) 2(II-D)) than than from from 2D 2D cultures cultures (Figure (Figure 2(I-D)); 2(I-D)); indicating indicating 3 (Figure 2(II-D)) than from perturbation 2D cultures (Figure 2(I-D)); indicatingin a spheroids lesser perturbation of selenite to perturbation of of selenite selenite to to glycolysis glycolysis in spheroids than than in in 2D 2D cells. cells. Likewise, Likewise, the the selenite selenite glycolysis in spheroids thanchanges in 2D cells. Likewise, the selenite-induced in the Krebs cycle changes in in the the Krebs Krebs cycle cycle was was also also much muchchanges less less in in spheroids spheroids than than in in was 2D 2D cells, cells, as as evidence evidence 13 13C also much less in spheroids than reduced in 2D cells, as evidenced muchof perturbation to the much much reduced perturbation perturbation to to by the thethe extent extent ofreduced C incorporation incorporation into into the the Krebs Krebs cycle cycle me me 13 extent of C incorporation including into the Krebs cycle metabolites, including citrate and2(II-E,II-F) α-ketoglutarate (αKG) including citrate citrate and and α-ketoglutarate α-ketoglutarate (αKG) (αKG) (Figure (Figure 2(II-E,II-F) versus versus Figure Figure 2(I-E,I-F), 2(I-E,I-F), respe resp (Figure 2(II-E,II-F) versus Figure 2(I-E,I-F), In particular, the fractional enrichment of In In particular, particular, the therespectively). fractional fractional enrichment enrichment of of 1313C C44-- (red (red boxes) boxes) and and 1313C C33-citrate -citrate (green (green boxes) boxes) 13 C - (red boxes) and 13 C -citrate (green boxes) showed prominent differences between the two culture prominent differences differences between between the the two two culture culture types types (Figure (Figure 2(I-E,II-E)). 2(I-E,II-E)). As As 1313C C44-citrate -citrate is is a 4 3 prominent 13 13 13 types (Figure 2(I-E,II-E)). of As C4 -citrate is a marker of pyruvate dehydrogenase (PDH)-initiated of pyruvate pyruvate dehydrogenase dehydrogenase (PDH)-initiated (PDH)-initiated Krebs Krebs cycle cycle activity activity [42] [42] while while C C33-citrate -citrate resu res 13 C -citrate results from pyruvate carboxylase (PCB)-initiated Krebs Krebs cycle activity [42] while pyruvate pyruvate carboxylase carboxylase (PCB)-initiated (PCB)-initiated Krebs Krebs cycle cycle reactions reactions [34], [34], these these differences differences su s 3 cycle reactions [34], these differential differences suggest a differential effect of selenite on both canonical and differential effect effect of of selenite selenite on on both both canonical canonical and and anaplerotic anaplerotic Krebs Krebs cycle cycle activities activities anaplerotic Krebs cycle activities in A549 spheroids versus 2Dprominent cells. Other prominentin ineffect the on spheroids spheroids versus versus 2D 2D cells. cells. Other Other prominent differences differences indifferences the the selenite selenite effect on the the two two cultu cult selenite effect on the two culture types involved Glumetabolism. and GSH metabolism. extent of enrichment involved involved Glu Glu and and GSH GSH metabolism. The The extent extentThe of of enrichment enrichment in in 1313C C22-Glu -Glu and and -GSH -GSH (re (re 13 in C2 -Glu and -GSH (red boxes;2(I-I,I-J,II-I,II-J)) Figure 2(I-I,I-J,II-I,II-J)) was much less attenuated in spheroids Figure Figure 2(I-I,I-J,II-I,II-J)) was was much much less less attenuated attenuated in in spheroids spheroids than than in in 2D 2D cells. cells. than in 2D cells. The 13 C atom-resolved atom-resolved tracing tracing shown shown in in Figure Figure 22 (●, ( , ●) ) indicates atom-resolved tracing shown in Figure (●, ●) indicates that that these these these two two two 1313C C isotopologue isotopologue 13 C isotopologues can be derived from the Krebs cycle reactions initiated by PDH-, PCB-, or both.
2.3. 2.3.Glycolysis Glycolysisand and the theKrebs KrebsCycle Cycle Respond Respond Less Lessto toSelenite Selenite in inA549 A549 and andPANC1 PANC1 Spheroids Spheroids than than in inTheir Their 2D 2D Cell Cell Counterparts Counterparts In In SIRM SIRM experiments, experiments, 6.25 6.25 µM µM selenite selenite attenuated attenuated net net growth growth and and induced induced morphological morphological changes changes in in A549 A549 cells cells grown grown in in 2D 2D cultures cultures (Figure (Figure S2A). S2A). Such Such changes changes were were less less evident evident in in the the Metabolites 2018, 8,corresponding 40 5 of 16 corresponding 3D 3D spheroids spheroids at at 10 10 µM µM selenite selenite (Figure (Figure S2B). S2B). Likewise, Likewise, 10 10 µM µM selenite selenite elicited elicited greater greater Metabolites 2018, 8, 2018, x 5 of 16 5 of 16 Metabolites 8, x perturbations perturbations in in morphology morphology and and proliferation proliferation in in 2D 2D (Figure (Figure S3A) S3A) than than 3D 3D cell cell cultures cultures of of PANC1 PANC1 (Figure (Figure S3B), S3B), which which initiated is is consistent consistent with with the the presence presence of of This aa higher higher population population of of resistant resistant cells cells in in derived from the Krebs cycle reactions by on PDH-, PCB-, or both. agrees with This agrees with differential selenite initiated effect canonical and anaplerotic Krebsagrees cycle the activities derived from thethe Krebs cycle reactions by PDH-, PCB-, or both. This with the spheroid spheroid than than in in 2D 2D cell cell cultures, cultures, as as described described above. above.described Consistent Consistent with with the the PrestoBlue PrestoBlue assay assay for for differential selenite effect on canonical and anaplerotic Krebs cycle activities above. described above. differential selenite effect on canonical and anaplerotic Krebs cycle activities described above. growth growth (Figure (Figure 1A), 1A), the the mitotic mitotic index index (as (as indicated indicated by by the the PCNA PCNA fluorescence) fluorescence) was was reduced reduced by by 33 days of of 100 100 µM µM selenite selenite treatment treatment in in both both A549 A549 and and PANC1 PANC1 spheroids. spheroids. The The growth growth inhibition inhibition was was I.days 2D Cells II. 3D Spheroids I. 2D Cells II. 3D Spheroids accompanied accompanied by by increased increased necrosis necrosis (as (as indicated indicated by by the the RIP-1 RIP-1 fluorescence) fluorescence) in in both both A549 A549 and and Glycolysis PANC1 PANC1 spheroids spheroids (Figure (Figure S2). S2). Glycolysis Glycolysis C -lactate Glycolysis * C -lactate C -lactate * C -lactate * C B B C differential * These phenotypic phenotypic differences differences were were accompanied accompanied by differential metabolic metabolic responses responses to to selenite selenite C BThese B by C * * * DD 1 A 1 P * 1P D A 1 DD 1P A 1 1 1 13 13C 13 13C 1P D A 1 1 1 between between 2D cell and and 3D 3D spheroid spheroid cultures. cultures. Figure Figure 2 2 compares compares the the extent extent of of C 6 6 -glucose -glucose ( ( C66-Glc) -Glc) Pyruvate 1 1 Pyruvate 1 2D 1 cell 3 Pyruvate 3 3 Pyruvate 3 3 3 6P 6 3 3 6P Lactate 6 6P CO 6 Lactate 6 P transformation CO Lactate A549 6 CO CO transformation through through glycolysis glycolysis and and the the Krebs Krebs cycle cycle between between A549 in in 2D 2D culture culture and and as as spheroids spheroids Lactate C -Glc F6P CO CO C -Glc F6P E 1 C -Glc F6P E 1 C -Glc F6P E 1 Acetyl CoA E 1 Acetyl CoA *** * Acetyl CoA Acetyl CoA Citrate *** * in in response response to to 24 24 h h of of selenite selenite treatments treatments at at 6.25 6.25 µM µM and and 10 10 µM, µM, respectively. respectively. Significant Significant Citrate 1 Citrate 1 1 Citrate * 1 1 K * 1 1 K 1 4 ** 4 K 4 4 K 4 selenite-treated 4 perturbations perturbations to to the the glycolytic glycolytic activity activity were were evident evident in in selenite-treated 2D 2D cells cells based based on on the the 4 6 4 Asp 6 Oxaloacetate Isocitrate Asp F Oxaloacetate 6 Isocitrate Asp 6 Oxaloacetate Isocitrate Asp F Oxaloacetate Isocitrate F 13 13C F 1 enhanced enhanced release release of of C 3 3 -lactate -lactate into into the the medium medium (Figure (Figure 2(I-D)) 2(I-D)) and and lower lower fractional fractional enrichment enrichment in in 1 CO 1 CO 1 CO CO 4 4 Krebs 13 13 4 αKetoglutarate Glu Malate In 4 Krebs αKetoglutarate Glu intracellular C C33-pyruvate -pyruvate (3; (3; Figure Figure 2(I-B)). 2(I-B)). In contrast, contrast, there thereαKetoglutarate was was1 no no effect effect of of selenite selenite on on Malate intracellular Krebs Glu Malate Krebs 1 αKetoglutarate Glu Malate 1 1 1 1 Cycle Cycle 1 1 Cycle Cycle 13 13C 13 13C 1 5 C 33-pyruvate -pyruvate (Figure (Figure 2(II-B)) 2(II-B)) and and the the stimulation stimulation of of C 3 3 -lactate -lactate release release was was relatively relatively lower lower from from 5 H 1 5 5 1 H 5 5 H 1 5 CO H CO5 4 CO 4 CO 4cultures 4 spheroid spheroid cultures cultures (Figure (Figure 2(II-D)) 2(II-D)) than than from from 2D 2D cultures (Figure (Figure 2(I-D)); 2(I-D)); indicating indicating aa lesser lesser Succinyl CoA Fumarate I Succinyl CoA Fumarate I Succinyl CoA Fumarate I Succinyl CoA Fumarate I ** ** Succinate perturbation perturbation of of selenite selenite to to glycolysis glycolysis in in spheroids spheroids than than in in 2D 2D cells. cells. Likewise, Likewise, the the selenite-induced selenite-induced Succinate J J J 1 Succinate 1 Succinate J JJ 1 G G 1 *** G Glu-GSH Glu-GSH *** cells, ** changes changes in in the the Krebs Krebs cycle cycle was was also also much much less less in inGspheroids spheroids than than in in 2D 2D cells, as as evidenced evidenced by by the the ** 5 Glu-GSH ** 5 Glu-GSH ** 5 5 13 13 * much much reduced reduced perturbation perturbation to to the the extent extent of of C C incorporation incorporation into into the the Krebs Krebs cycle cycle metabolites, metabolites, * including including citrate citrate and and α-ketoglutarate α-ketoglutarate (αKG) (αKG) (Figure (Figure 2(II-E,II-F) 2(II-E,II-F) versus versus Figure Figure 2(I-E,I-F), 2(I-E,I-F), respectively). respectively). FigureFigure 2. Glycolysis and theand Krebs respond less to less selenite inselenite A549inspheroids than inthan their Figure 2. Glycolysis Glycolysis and thecycle Krebs cycle respond less in A549 spheroids than in their 2. the Krebs cycle respond to to selenite A549 spheroids in2D their 2D 13 13C 13 13C In InA549 particular, particular, the thespheroids fractional fractional enrichment enrichment of of C 44-- (red (red boxes) boxes) and and C33-citrate -citrate (green (green boxes) boxes) showed showed cell counterparts. cells and were extracted for polar metabolites, which were 2D cell counterparts. A549 spheroids were extractedfor forpolar polarmetabolites, metabolites, which which were were cell counterparts. A549 cellscells andand spheroids were extracted 13 13 prominent differences differences between between the themetabolites two twoby culture culture types types (Figure (Figure 2(I-E,II-E)). 2(I-E,II-E)). As C C44-citrate -citrate is is aa marker marker 13C isotopologues 13 3-lactate 13 C isotopologues 13 As of various metabolites IC-UHRFT-MS and for quantified for theprominent quantified for the of various by and -lactate isotopologues of various metabolites by IC-UHRFT-MS IC-UHRFT-MS andCfor for 13C C3-lactate quantified for the 13C 13 13C of of pyruvate pyruvate dehydrogenase dehydrogenase (PDH)-initiated (PDH)-initiated Krebs Krebs cycle cycle activity activity [42] [42] while while C 3 3 -citrate -citrate results results from from 1 13 1 13 H-NMR, as described in the Materials and Methods. Oxidation of C6of -Glc via glycolysis (Lac) by (Lac) by 1H-NMR, H-NMR, as described described in the and Oxidation C -Glc via glycolysis glycolysis as in the Materials Materials and Methods. Methods. Oxidation of 13C 66-Glc via (Lac) by 13 pyruvate carboxylase carboxylase (PCB)-initiated (PCB)-initiated Krebs Krebs cycle cycle reactions [34], [34], metabolites these these differences suggest suggest aa 13 Cmetabolites and Krebs traced along with the fractional distribution of relevant Creactions labeled in differences 13C labeled andcycle Krebsispyruvate cycle traced along with thefractional fractional distribution relevant labeled and Krebs cycle isistraced along with the distribution ofofrelevant metabolites in 13C3anaplerotic 13 differential differential effect effect of of selenite selenite on on both both canonical canonical and and anaplerotic Krebs Krebs cycle cycle activities activities in in A549 A549 controlcontrol versus selenite-treated 2D cells (I) and spheroids (II), except for -lactate as µmoles/g in control versus selenite-treated cells(I)(I)and andspheroids spheroids (II), (II), except except for -lactate as versus selenite-treated 2D2D cells for 13C C33-lactate as µmoles/g µmoles/g 13 13 spheroids spheroids versus versus 2D 2D cells. cells. Other Other prominent prominent differences differences in in the the selenite selenite effect effect on on the the two two culture culture types types protein. Not allNot possible isotopologues are shown. Numbers in X-axis are those C atom eachin protein. Not all possible possible isotopologues are shown. shown. Numbers in X-axis X-axis areof those of 13C Cinatom atom in each each protein. all isotopologues are Numbers in are those of 13 13C involved involved Glu Glu and and GSH GSH metabolism. metabolism. The The extent extent of of enrichment enrichment in in C 22-Glu -Glu and and -GSH -GSH (red (red boxes; boxes; isotopologue. →, ↔ , and ---> indicate irreversible, reversible, and multi-step reactions, isotopologue. → , ↔ , and —> indicate irreversible, reversible, and multi-step reactions, respectively; isotopologue. →, ↔ , and ---> indicate irreversible, reversible, and multi-step reactions, 13 13C Figure Figure 2(I-I,I-J,II-I,II-J)) 2(I-I,I-J,II-I,II-J)) was was much much less less attenuated attenuated in in spheroids spheroids than than in in 2D 2D cells. cells. The The C respectively; double dashed line depicts plasma or mitochondrial membrane; numbers in X-axis refer double dashed line depicts plasma or mitochondrial membrane; numbers in X-axis refer to those respectively; double dashed line depicts plasma or mitochondrial membrane; numbers in X-axis refer 1313 122 12 1313 13 13C of C atoms in each isotopologue of metabolites. : C; , : respective C fate via the 1st turn C atoms in each isotopologue of metabolites. ●: C; ●, ●: respective C fate via the 1st to those of atom-resolved atom-resolved tracing tracing shown shown in in Figure Figure 2 (●, (●, ●) ●) indicates indicates that that these these two two C isotopologues isotopologues can can be be to those of 13C atoms in each isotopologue of metabolites. ●: 12C; ●, ●: respective 13C fate via the 1st 0.6 0.4 0.2
0
1
2
3
4
0.6
0
1
2
3
1.0 0.8
0
0.4
0.0 4
0
1
2
3
0.2
0.4
3
0.00
1
2
3
2
4
1.0
0
1
2
0.8
Ctl SeO3
0.6 0.4 0.2
3
4
1.0
Fumarate
1.0 0.8
0.4 0.2
0.0
1
2
3
GSH
0.8
Ctl SeO3
0.6
0.6
0.04
0.4
0.02
0.2
0.00
Ctl 1.0 SeO3
0
1
0
0.0
0.0 4
0
1
2
3
4
0
1
2
3
4
0.0
0.4
0.04 0 1 0.02
0.2
0.00
0.6
2
3
3
4
1
2
4
13C
5
6
2
0.7
2
6
Ctl SeO3
Asp
2
0.6
0
0.4 0.3 0.2 0.1 0.0
1
2
3
4
2
3 0.04
0
1
2
3
0.6
Ctl SeO3
5
Lac
0.2
1.0
αKG
0.8
0
1
0.6
2
3
0.0
1.0
αKG
Ctl 5 6 0.8 SeO3
4
0.4
Ctl SeO3
0.6 0.4 0.2
0
1
2
2
3
4 0.0 5
0
0
1
2
3 0.04
3
4
5
1.0
2
0
1
2
3
Fumarate1.0
4
0.4 0.2
1.0
Fumarate
Ctl 0.8 SeO3
0.6
0.4
3
4
5
0.2
1
2
3 0.04
0.0
0
1
2
3
4
0
Ctl SeO3
1
Glu
0.8
0.4
0.0
0.6
Ctl 1.0 SeO3
2
0.6
0.2GSH
0.8
0.6 0.4
Glu
0.8
Ctl 1.0 SeO3
GSH
0.8
Ctl SeO3
0.6
0.2
0
2
Ctl SeO3
0.2
0.0
1
Malate
0.4
0.8
0.00 2
0
Ctl SeO3
0.2
0.8
1.0
0.05
1
30
0.3
50.0 6
4
Ctl SeO3 0.6
0.4
0.02
0.4 0.2
4
Malate
0.04
0.6
60
0.4
0.2
1
90
0.5
0.1
0
Media
120
Citrate
0.6
0.5
0.4
Lac
Ctl 0.7 SeO3
Citrate
0.6
2
mmole/g protein
mmole/g protein
Fraction
Fraction
Fraction
Fraction
4
0.0
3
30
3
Ctl SeO3
150
5
Glu
0
0.05
0
3
0.2
0.8
GSH0.00Ctl SeO3
0.2
0.8
60
2
2
Ctl 1.0 SeO3
0.02
0.4
3
0.00
0.04
0.6
2
0.05
Glu 2
0.8
Fumarate
0
2
13
0.4 0.2
0
4 0.0
1.0
Fraction
0.4
Ctl SeO3
0.0
0.6
1
0.8
0.4
αKG0.2
0
6
Ctl 1.0 SeO3
0.6
0.02
0.8
0.0
Fraction
2
Fraction
1
Ctl 1.0 SeO3
3
0.6
2
Fraction
0
0.8
0.02 4 5 6
2
50.0 6
Asp
0.6
0.2
0.0
4
Fraction
Fraction
Fraction
0.4
3
Ctl SeO3
Malate
0.8
0.6
2
0.8
Ctl 1.0 SeO3
Malate
0.8
1
0.2
4
2
1.0
1
1.0
αKG
1
0.2
0
13
0.4
5 0.06
0.2
0.0
0.0
0
Ctl SeO3
0.2
0.0
100
Citrate
0.8
0
13
Media180
90
13C
0
1.0
3
3 0.0
2
3
0.4
Fraction
Ctl SeO3
2
2
120
Fraction
0.6
Citrate
0.8
1
1
3
Ctl SeO3
150
Fraction
6
1.0
0
180
Fraction
0.2
2
Fraction
Fraction
0.4
2
Ctl SeO3
Asp
0.8
0.6
6
0.4 0.2
0.0
0
13
Ctl SeO3
0.6
Fraction
5
Fraction
4
13
Ctl 1.0 SeO3
Asp
0.8
3
0.4
Fraction
2
0.2
Fraction
1.0
1
Fraction
0
6
P
5 0.06
13
DH
4
H
3
PD
2
PCB
1
PCB
0
0.4
200
13C
13C
100
0.2
0.0
300
200
3
Lactate
0.8
Fraction
2
2
0.2
1.0
Fraction
1
1
Ctl SeO3
Fraction
0
0
Ctl SeO3
0.4
H PD
0.0 3
0.6
0.6
H
2
3
0.8
Media 0.6
F6P
PD
1
2
Ctl SeO3
0.5 0.4 0.3 0.2 0.1 0.03
PCB
1
1.0
Lactate
0.8 Pyruvate
0.7
Ctl 0.6 SeO3
Fraction
0
0
F6P
1.0
Pyruvate0.8
PCB
0.0 3
0.8
Ctl SeO3
400
300
3
Fraction
2
Media
Ctl SeO3
Fraction
1
1.0
13
Ctl SeO3
400
0.2
0.0
0.4
0.4
3
Fraction
0
0.6
0.6
Fraction
0.0
13
Ctl SeO3
mmole/g protein
Fraction
0.2
0.2
Lactate
0.8
Fraction
0.2
0.2
1.0
Fraction
0.4
F6P
0.4
0.4
SeO3
Fraction
0.8
0.6
Fraction
Ctl SeO3
0.6
Ctl SeO3
0.6
mmole/g protein
1.0
Fraction
Fraction
0.8
F6P
0.4
Lactate PyruvateCtl
0.8
0.8
Fraction
Ctl SeO3
Fraction
1.0
Ctl SeO3
0.6
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
1.0
Pyruvate 1.0
0.8
Fraction
1.0
Ctl SeO3
0.6 0.4 0.2
4 0.0 5
0
1
2
3
4
5
0.2
0
1
2
3
4 0.0 5
0
1
2
3
4
5
ofthe theof pyruvate dehydrogenase (PDH)pyruvate carboxylase (PCB)-initiated Krebs cycle;cycle; ME: turn ofturn pyruvate dehydrogenase (PDH)ororpyruvate carboxylase (PCB)-initiated Krebs cycle; the pyruvate dehydrogenase (PDH)or pyruvate carboxylase (PCB)-initiated Krebs malic enzyme; αKG: αketoglutarate; GSH: glutathione; *: q (false discovery rate) ≤ 0.05; **: q ≤ 0.01; ME: malic enzyme; αKG: αketoglutarate; GSH: glutathione; *: q (false discovery rate) ≤ 0.05; **: q ≤ ME: malic enzyme; αKG: αketoglutarate; GSH: glutathione; *: q (false discovery rate) ≤ 0.05; **: q ≤ 6 −6 n − ***: ≤ 0.005; ****: q5≤× 510 0.01; ***: q ≤q ***: 0.005; q ≤****: . n=3.=2 2oror3.3. 0.01; q ≤ ****: 0.005; q× ≤ 5.10 × =102.−6nor
Similarly, selenite distinctly impacted glycolysis and the Krebs cycle activity inactivity PANC1 cell2D cell Similarly, selenite distinctly impacted glycolysis and thethe Krebs cycle activity in PANC1 Similarly, selenite distinctly impacted glycolysis and Krebs cycle in2D PANC1 2D 13C cultureculture (Figure 3I) versus spheroids (Figure 3II). At 10 µM, selenite significantly decreased 13 (Figure 3I) versus spheroids (Figure 3II). 3II). At 10 selenite significantly decreased C cell culture (Figure 3I) versus spheroids (Figure AtµM, 10 µM, selenite significantly decreased 13C-lactate in the labeling inlabeling Krebs cycle metabolites and increased theincreased amount of 2Dthe cells 13 C 13 C-lactate 13C-lactate labeling in Krebs cycle cycle metabolites and increased the amount of excreted in 2Dincells in Krebs metabolites and theexcreted amount of excreted the but had effect thelittle spheroids. Thethe reduced enrichment by selenite in 13C2in -Asp box, 13 C 13C2(red but had little effect in theeffect spheroids. The reduced enrichment byenrichment selenite (redFigure box, Figure 2Dlittle cells but in had in spheroids. The reduced by -Asp selenite in 2 -Asp 13 13 3(I-K))3(I-K)) and C 2-/ 13 C 4-citrate (red box, Figure 3(I-E); produced in the first and second Krebs cycle turn, 13 13 13 andFigure C2-/ C 4-citrate (red C box, 3(I-E);(red produced in the 3(I-E); first and second Krebs (red box, 3(I-K)) and C4 -citrate box, Figure produced in thecycle firstturn, and 2 -/ Figure 13C3-Asp and respectively indicated disrupted PDH-initiated Krebs Krebs cycle activity while while that inthat 13C3-Asp and respectively [42]) indicated disrupted PDH-initiated cycle PDH-initiated activity in cycle second[42]) Krebs cycle turn, respectively [42]) indicated disrupted Krebs activity 13C3-citrate could result from and perturbed PCB-initiated Krebsfrom cycleperturbed reactions (green(green box, Figure 13perturbed 13C3-citrate could from PCB-initiated Krebs cycle reactions box, while that in 13 C3result -Asp C3 -citrate could result PCB-initiated KrebsFigure cycle 13C2-Glu and -GSH (red box, Figure 3(I-I,I-J)) 3(I-K,I-E)). Again, the reduced enrichment of by selenite 13 13 3(I-K,I-E)). Again, box, the reduced C2-Glu -GSH (red box, Figure selenite reactions (green Figure enrichment 3(I-K,I-E)). of Again, theand reduced enrichment of 3(I-I,I-J)) C2 -Glu by and -GSH is consistent with attenuated PDHand/orand/or PCB-mediated Krebs cycle activities. However, these these is consistent with3(I-I,I-J)) attenuated PCB-mediated Krebs cycle activities. However, (red box, Figure by PDHselenite is consistent with attenuated PDHand/or PCB-mediated selenite-induced clearlyclearly observed in 2D cells 3(I-E,I-I–I-K)) wereobserved diminished selenite-induced perturbations in 2D(Figure cellsperturbations (Figure 3(I-E,I-I–I-K)) were diminished in Krebs cycleperturbations activities. However, theseobserved selenite-induced clearly in in 2D cells spheroids (Figure 3(II-E,II-I–II-K)). spheroids (Figure 3(II-E,II-I–II-K)). (Figure 3(I-E,I,J,K)) were diminished in spheroids (Figure 3(II-E,I,J,K)).
Metabolites 2018, 8, 40
6 of 16
Metabolites 2018, 8, x Metabolites 2018, 8, x
6 of 16 6 of 16
Metabolites 2018, 8, x
II.reactions 3D Spheroids derived from the Krebs cycle initiated by PDH-, PCB-, or both. This II. 3D Spheroids differential selenite effect on canonical and anaplerotic Krebs cycle activities describ Glycolysis
1
2
3
4
Fraction
0.8 0.6 0.4
Fraction
1.0
CTL SeO3
0.6
Fumarate
0.4
** ** G
** **0 1G 2
0.0
0.2 0.0
0
1
2
3
3 4
4
Glu-GSH
5
5
0.8 0.6
1
2
1.0 0.8
0.4
** **SeO3
0.4 0.2 0.0
0.2
J ** ** 0 1 2
3
3
0.6 0.4 0.2 0.0
1.0 0.8 0.6 0.4
4
0
1
2
3
4
5
CTL SeO3
0.5
1
0.0
0
norm. µmoles 13C
2
0.4
3
0 1 2 3 0.2 4 5 Isocitrate 0.0
0
4
5
0.6
6 1
0.0
2
3
0.5
20.43
CO2 0.3 0.2 0.1
1
Fumarate
1
0.5
4
5
6
Ctl SeO3
0.8
0
1
6P F6 0.1 CO2 13C -Glc F6P 0.0 06 1 2 3 4 5 6
Glu 4 5
Ctl 1SeO3 2 3
11
Glu
1
0.2
Fraction
Ctl SeO3
0.6 0.4
C
0.2
2
0.0
3
0
1 Pyruvate 3
SeO3
0.3
K
0.4
1SeO3 P
B
FCtl
αKG
0.4
40.2 5
1.0 CO Asp 0.0 2 αKetoglutarate 0.8 0 0.6
Ctl
1αKG
A 0.6
6 0.7
Lactate
0.8
Ctl SeO3
CO2
Acetyl CoA 1
1
1 3
2
3
Lactate 1
Citrate
0.7 0.6
Fraction
0.6
0.7
1.0
Pyruvate
H
norm. µmoles 13C
1
*
F6P
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
PD
13C
SeO3 Ctl 0.8 SeO3
Fraction
P
Ctl E
PCB
0.0
1.0
1
0
Isocitrate
0
Ctl SeO3
Glycolysis
Krebs 1 * Cycle αKetoglutarate *Krebs αKG Cycle 0.02 1 5
0.2
0
0.0
0.1
100
0.2
E
0.1
0.2
II. 3D Spheroids
0.0
0.4
Fraction
*
0.3
0.2
1.0
Fraction
Media
0.2
D
D
0.4
0.0
0.3 Citrate
Media
0.6 SeO3
Fraction
Fraction
SeO3
Fraction
Fraction
Fraction
H
PD
DH
PCB
PCB
Citrate Ctl
Ctl SeO3
0.4
1
0.4
0.6
Citrate
0.4 0.5
3-lactate
0.8
1.0
3-lactate
Ctl Media SeO3 3-lactate
0.8 Ctl
0.6
6
0.5 0.4 0.3 0.2 0.1 0.0
4
54
0.7 Asp Ctl Oxaloacetate 0.0CO Glu F 0.6 0 21 2 3 54 1 Isocitrate SeO3 5 4 0.5 0.7 0.00 0.4 Glu 1 2 3 4 1 SuccinylCO CoA Fumarate 0.6 ICtl 2 SeO3 0.7 4 0.3 CO 0.8 0.5 2 Fumarate 0.6 0 1 2 3 4 5 GSH Ctl 0.4 0.2 0.7 2 3 4 Succinyl CoA Fumarate 4 I SeO3 Ctl 0.5 0.6 0.1 0.7 0.3 0.8 SeO3 αKetoglutarate Malate Krebs Succinate Glu 0.7 0.5 GSH JCtl 0.4 Fumarate 0.6 0.2 0.80.0 11 0.4 0 1Malate 2 3 4 5 0.3 SeO3 Ctl G 1 0.5 0.6 0.1 0.3 0.6 0.2 Ctl SeO3 Cycle 0.5 Succinate 0.4 0.0 0.2 J 1 0.4 0.1 Glu-GSH 0 1 2 3 SeO3 4 5 0.3 0.1 G 0.4 1 5 0.3 0.0 5 54 0.0 0.2 H 0 1 2 3 0.2 0 1 2 3 4 5 0.2 0.1 Glu-GSH 0.1 CO2 4 5 Glu 0.0 Ctl 0.0 SeO3 0.0 0 1 2 3 4 0 1 2 3 4 5 0 1 2 3 4 Fumarate 0.04 Succinyl CoA 1.0 I
0
Isoci
0.4 0.2 0.0
0
Malate
4
4
Malate
SeO3
Ctl 4H SeO3
Malate
0.5
Citrate
E
0.6
3
3
Citrate 4300 D 200
6
H6
0.1 0.0
H
3
Fraction
Fraction
0.3
5
2
Fraction
0.4 Malate
4 1
1
0.5
0.8
2
2
400
1
Oxaloacetate 4 6
4
C1
1 3
13C
1
1
3
1
1
Ctl Malate Citrate
0.3 0.2
COCoA Acetyl 2
Acetyl CoA
2
1
Fraction
H 0.7
4
5 Ctl SeO3
C
0
1 Lactate CO2 3 Lactate 1
1.0
4
0.2 0.4 Oxaloacetate 0.1
0.0
0
1
0
1 3
Asp Lactate Oxaloacetate 1
0.6
0.2
0.0
3
0.6
CTL ** **SeO3I I ** ** 0 1 2 3 4 3
F6P
3
0.7
Glu
2
0.2
PD
Fraction
15 1
Malate 1
1.0
Acetyl CoA
Asp Glu
5 4
0
5
Glu
2
2
6 P Pyruvate 1 CO2 3
6 PLactate F6P 0.8 6-Glc Ctl CO SeO3 2 0.6
6-Glc SeO3 0.4
SeO3
2
5
45
0.8
0.0
4
Fraction
Fraction
norm. µmoles 13C
norm. µmoles
0
Fraction
Fraction
13C
CO2
1.0
1
1 1.0
0.6 Succinyl0.8CoAGSH CTL SeO3 0.4 1.0 0.6 Succinate 0.2 GSH CTLJ
Succinate 1 Glu-GSH
CTL SeO3
0.2
0.0
4
4
Glu
0.4
SuccinylCO CoA 2
Fumarate Fumarate Fumarate
0.8
K
0.2
5
4
4
4 1.0
0.6
αKetoglutarate
Ctl SeO3 1 2 3
3
* Ctl
B1
1.0
Fraction
0
0
2
0
Pyruvate 1 2 3
131.2C
1.2
Fraction
3
0.0
0.8
1
6
6
0
13C
C
Fraction
0.2
1.0
5
0
5
1 0.0 4 0 1 1 Asp 1 Pyruvate 0 1 2 3 344 3
CO 0 21
F6P
0.0
CO2 αKetoglutarate 0.0Asp
1
13C F 6-Glc
0.2
0.2
0.0
BKCtl
K1
0.2 0 0.1
0.1
6 P0.0
Asp
0.0
B
Fraction
** **0 1H 2
1
0.0
0.4
0.0
CTL 0.3 SeO3 0.2
CTL
6
0.4 0.5
0.2
Fraction
** ** H
CTL SeO3
0.2
CO0.62
F 6SeO3
αKG
AspC
0.6
0.6 0.2 0.4 0.3 0.5
4
Ctl SeO3 13
0.7
Ctl SeO3
0.4
0.8 Ctl Lactate 0.7 SeO3 0.6 0.5 Ctl Lactate 0.4 SeO3 0.3 0.2 0.1 0.0
Krebs Cycle
αKe
1
5
1.0 0.8
Fraction
1
0.6
13C Pyruvate 3 4 5 6 6
2
3
0.0
0.8 0.7 0.6 0.5 0.4 0.3 30.2 0.1 0.0
Fraction
Malate
CTL Malate SeO3
Isocitrate
αKG
0.8 1.0
1 0.8 2 3 0.4 4 5
0
0.6
2
Fraction
Malate
0.8
1P
1
1
1.0
0.4 0.2
A 1
0.0
*0
1 P0.4
1
1.0
0.4
0.0
*0
0.0
0.7
0.6
0.0
1
0.0
CTL
0.2
Krebs Krebs Cycle Cycle
0.4
0SeO3
0 1 2 3 4 5 6 Isocitrate
0.2
0.6
1P 1 Cells I. 2D
SeO3
0.4
0.8
A
Ctl
PyruvateSeO3
0.6
Fraction
0.4
0.4
4
100
Glycolysis
0.1 F6P
0.1
0.2
0
Ctl SeO3
0.6 Ctl
Fraction
0.6
Fraction
Fraction
0.8
0.6
Fraction
Malate
1.0
1.0
4
D
100
0.6
CTL 1.0SeO3 Ctl SeO3 0.8
0.2 0.0
0.8
*
** **** F6P E **** **1**2 3 4 5 6 A 0** 0.4
Citrate
0.2 0.0
** **
*
DMedia
0.8
Fraction
1
0.4
** **E
Ctl SeO3
200
Fraction
4
*** 0.8
Fraction
Fraction
1 Citrate 1 Citrate 4 1 Oxaloacetate 4 6 6 Oxaloacetate 1
Asp
4
0.6
200
Citrate
0.6
Acetyl CoA
300
1.0
1.0
PCB
4
1 Lactate 3 Lactate 1
Acetyl CoA
1
Asp
4
3
3
Fraction
1
2
2
Fraction
0
1
*** 2 3
2
1 33
Fraction
0
0.0
1
CO2
1
400
1
Fraction
0.0
0.2
F6P
SeO3
** ** SeO3 ** ** ** ** ** **
3
0 C
1.0
Glycolysis F6P
3-lactate
Ctl Media -lactate 300 3 SeO3
13C
Fraction
0.4
K
0.0
400
C
Fraction
0.2
0.4
CTL SeO3
Fraction
0.4
0.6
K Asp CTL
0.2
Fraction
0.6
LactateSeO3
0.4
Fraction
Fraction
Fraction
0.8
0.8
0
6 P Pyruvate 1 CO2 3
13C
CTL
0.6
0.6
B 1 2 30.2 1 0.0 Pyruvate 1 2 3 0 0
6 6-Glc6 P F6P
13 CTL C6-Glc Asp
1.0
0.0
0.0
0.8
Fraction
0 1 2 3 4 5 6
1.0
1P 6
13C
0.0
0.4 1P
B
CTL SeO3
H
0.2
0.2
0.2
1
A
0 1 2 3 4 5 6
1.0
PyruvateSeO3
0.4
PD
0.0
1
CTL
0.6
0.6
Lactate
0.8
P
0.2
0.4
*** A
0.8
DH
0.6
F6P
Fraction
0.8 0.6 CTL 0.4 SeO3
1.0
***
F6P
1.0
Pyruvate
0.8
PCB
0.8
CTL SeO3
PCB
Fraction
1.0
Fraction
1.0
Pyruvate
0.8
1.0
mmole/g protein
1.0
Glycolysis Glycolysis
Fraction
I. 2D Cells I. 2D Cells
0.6
Succiny 1.0
Figure 3. Glycolysis and the Krebs cycle respond less to selenite in PANC1 spheroids than in their 2D Figure Glycolysis and Krebs cycle respond less to selenite PANC1 spheroids than their Succinate Succinate J 1 Figure 3. 3. Glycolysis and thethe Krebs cycle respond less to selenite in in PANC1 spheroids than inin their 2D2D 1 G G cell counterparts. Extraction of polar metabolites and their analysis are as described in Figure 2, so Glu-GSH cell counterparts. Extraction of polar metabolites and their analysis in Figure 2, so are Glu-GSH are as described ** 5 cell counterparts. Extraction of polar metabolites and their analysis5 are as described** in Figure 2, so are all symbols and abbreviations. (I) Metabolite responses in 2D cultures;* (II) metabolite responses areallallsymbols symbolsand andabbreviations. abbreviations.(I) (I)Metabolite Metaboliteresponses responsesinin2D 2Dcultures; cultures;(II) (II)metabolite metaboliteresponses responsesin in 3D cultures. in3D 3Dcultures. cultures. Figure 2. Glycolysis and the Krebs cycle respond less to selenite in A549 spheroids tha cell counterparts. A549 cells spheroids were extracted We also noted two clear metabolic differences in PANC1 2D and cell and spheroids, regardlessforof polar metabolites, Wealso also noted two clear metabolic differences in13PANC1 and spheroids, regardless We noted two clear metabolic differences PANC1 2D132D cell cell andof spheroids, regardless C isotopologues various metabolites byofIC-UHRFT-MS and fo forinthe the selenite treatment. One was thequantified higher enrichment in 13 C3-fructose-6-phosphate (F6P) in 13C3C of selenite the selenite treatment. higher enrichment in -fructose-6-phosphate (F6P) in the treatment. OneOne waswas the the higher enrichment in -fructose-6-phosphate (F6P) in 1 13C6-Glc v 3 13 H-NMR, as described in the Materials and Methods. Oxidation of (Lac) by spheroids (Figure 3(II-A)) than in 2D cells (Figure 3(I-A)). F6P can be produced from13 C3-pyruvate 13CC spheroids (Figure 3(II-A)) thaninin2D 2Dcells cells(Figure (Figure 3(I-A)). F6Pcan canbe beproduced fromdistribution -pyruvate spheroids (Figure 3(II-A)) than 3(I-A)). F6P from 3-pyruvate cycle is traced along fractional of relevant 13C labeled m 13produced via gluconeogenesis [35]. The other wasand theKrebs higher enrichment in thewith C1the -isotopologues of3 fumarate, 13 13 via gluconeogenesis[35]. [35].The The other was the higher enrichment in the C -isotopologues of fumarate, via gluconeogenesis other was the higher enrichment in the C 1 -isotopologues of fumarate, 1 control versus selenite-treated 2D cells (I) and spheroids (II), except for 13C3-lactate malate, and Asp in spheroids (Figure 3(II-G,II-H,II-K)) than in 2D cells (Figure 3(I-G,I-H,I-K)). These malate, and Asp spheroids (Figure 3(II-G,II-H,II-K)) than in 2D cells (Figure 3(I-G,I-H,I-K)). These malate, and Asp inin spheroids (Figure 3(II-G,II-H,II-K)) than in 2D cells (Figure 3(I-G,I-H,I-K)). These Notbe allproduced possible isotopologues are shown. Numbers X-axis are those of 13C a isotopologues (tracked by ● in Figureprotein. S4A) can via the reversible reactions of in malic isotopologues(tracked (trackedby by ● in can be be produced via the reactions of malic isotopologues inFigure FigureS4A) S4A) can produced via the---> reversible reactions of enzyme malic →, ↔ andreversible indicate reversible, and multi-ste enzyme (ME). Alternatively, these 13isotopologue. C1-isotopologues can, be formed by theirreversible, condensation of 13 C -isotopologues 13 C -1,2-OAA 13Crespectively; (ME). Alternatively, these can be formed by the condensation of enzyme (ME). Alternatively, these 1 -isotopologues can be formed by the condensation of double dashed line depicts plasma or mitochondrial membrane; numbers in 1 13C2-1,2-OAA with unlabeled acetyl CoA and subsequent Krebs cycle reactions, as depicted2 in Figure 13 12 13C with unlabeled CoA and subsequent Krebs reactions, as depicted in S4B ). IfC;the C atoms in each isotopologue of Figure metabolites. ●, ●: respective 13C fa those of cycle 2-1,2-OAA withacetyl unlabeled acetyl CoAto and subsequent Krebs cycle reactions, as depicted in(●: Figure S4B (●). If the latter is the case, one would expect the fractional enrichment of 13C1-fumarate to be 13 13 turn expect of theenrichment pyruvate dehydrogenase (PDH)or1higher pyruvate (PCB)-initiated latter oneiswould expect fractional of C to beC thancarboxylase that S4B (●).isIfthe thecase, latter the case, onethe would the fractional enrichment of -fumarate to beof 1 -fumarate higher than that of 13C1-malate, which was not the case. We hypothesize that ME-mediated reactions 13 C -malate, which 13C ME: malic enzyme; αKG: αketoglutarate; GSH: glutathione; *: q (false discovery rate) ≤ was not the case. We hypothesize that ME-mediated reactions contributed at least higher than that of 1 -malate, which was not the case. We hypothesize that ME-mediated reactions 1 contributed at least in part to the production of 13C1-isotopologues −6of the Krebs cycle intermediates 13 13 . n = 2 or 3. 0.01; ***: q ≤ 0.005; ****: q ≤ 5 × 10 in part to the production C1 -isotopologues Krebs cycle intermediates in PANC1 spheroids. contributed at least in partof to the production of ofC1the -isotopologues of the Krebs cycle intermediates 0.6 0.4
0.6
0.04
0.4
0.02
0.2
0.2
0.0
0.0
0
1
2
GSH
0.8
3
4
Ctl SeO3
0.02
0.4
0.0
0
1
2
3
4
5
0.4
0.0
1
2
0.8
Ctl SeO3
0.6
0.2
0.00
0
Fumarate
0.8
0.00
0.2
Fraction
1.0
Ctl SeO3
Fraction
Fraction
0.8
Fraction
Fumarate
1.0
3
4
5
0.6 0.4 0.2
0
1
2
3
4
0.0
in PANC1 spheroids. Thus, spheroid formation led to a higher resistance to selenite toxicity in A549 or PANC1 cells, in PANC1 spheroids. Thus, spheroid formation led to a Similarly, higher resistance to selenite impacted toxicity inglycolysis A549 or PANC1 cells, selenite distinctly and the Krebs cycle activity in which was reflected respectively in atheir attenuated or to lack of changes in glycolysis, Krebs cycle, Thus, spheroid formation led to higher resistance selenite toxicity in A549 or the PANC1 cells, which was reflected respectively in their attenuated orversus lack of spheroids changes in(Figure glycolysis, the Krebs cycle, culture (Figure 3I) 3II). At 10 µM, and GSH metabolism in response to selenite. Additional occurred in PANC1 which was reflected respectively in their attenuated or lack ofmetabolic changes inrewiring glycolysis, the Krebs cycle,selenite significantl and GSH metabolism in responselabeling to selenite. Additional metabolic and rewiring occurred in PANC1 in Krebs cycle metabolites increased the amount of excreted 13C-lactat spheroids compared in with 2D cultures, most likely involving enhanced gluconeogenesis malic and GSH metabolism response to selenite. Additional metabolic rewiring occurred in and PANC1 spheroids compared with 2D cultures, most likely involving enhanced gluconeogenesis and malic but had effect in the spheroids. reduced enrichment by selenite in 13C2-Asp enzyme activity. spheroids compared with 2D cultures, mostlittle likely involving enhancedThe gluconeogenesis and malic enzyme activity. 13 13 3(I-K)) and C2-/ C4-citrate (red box, Figure 3(I-E); produced in the first and second K enzyme activity. 2.4. Pyrimidine and the Hexosamine respectively Biosynthetic Pathways Respond Less to Selenite in A549 And PANC1 [42]) indicated disrupted PDH-initiated Krebs cycle activity while that 2.4. Pyrimidine andTheir the Hexosamine Biosynthetic Pathways Respond Less to Selenite in A549 And PANC1 Spheroids Than in 2D Cell Counterparts 13C3-citrate could result from perturbed PCB-initiated Krebs cycle reactions (gr 2.4. Pyrimidine and the Hexosamine Biosynthetic Pathways Respond Less to Selenite in A549 And PANC1 Spheroids Than in Their 2D Cell Counterparts 3(I-K,I-E)). Again, the ring reduced enrichment of 13isC2-Glu and -GSH (red box, Figure 3(ISpheroids Cell precursor Counterparts As Than Asp inisTheir the 2D direct to pyrimidine synthesis, which required for cell As Asp is the direct precursor to pyrimidine ring synthesis, which is required for cell is consistent with attenuated and/or PCB-mediated Krebs cycle activities. proliferation, then askedprecursor if distinct selenite-induced changes ofPDHAsp synthesis in differential As Asp iswethe direct to pyrimidine ring synthesis, which is results required for cell proliferation, we then asked if distinct selenite-induced changes of Asp synthesis results in 3(I-E,I-I–I-K)) we selenite-induced perturbations clearly observed in 2D cells (Figure inhibition ofwe pyrimidine ring in 2D versus spheroid of A549 and PANC1 proliferation, then asked if synthesis distinct selenite-induced changes cultures of Asp synthesis results in differential inhibition of pyrimidine ring synthesis in 2D versus spheroid cultures of A549 and 13 spheroids (Figure 3(II-E,II-I–II-K)). cells. Figure 4 showsofthe C enrichment patternsin of 2D various precursors products of uridine differential inhibition pyrimidine synthesis versus spheroid and cultures of A549 and 13ring PANC1 cells. Figure 4 shows the C enrichment patterns of various precursors and products of synthesis in A549 cells and spheroids including those of the pyrimidine ring and the ribosyl 13 PANC1 cells. Figure 4 shows the C enrichment patterns of various precursors and products unit, of uridine synthesis in A549 cells and spheroids including those of the pyrimidine ring and the ribosyl i.e., ribulose/ribose-5-phosphate of theincluding pentose phosphate pathway (PPP)ring andand phosphoribosyl uridine synthesis in A549 cells and(R5P) spheroids those of the pyrimidine the ribosyl unit, i.e., ribulose/ribose-5-phosphate (R5P) ofC enrichment the pentosepatterns phosphate pathway (PPP)ofand 13 pyrophosphate (PRPP). Also tracked were the of the intermediates the unit, i.e., ribulose/ribose-5-phosphate (R5P) of the pentose phosphate pathway (PPP) and 13 phosphoribosyl pyrophosphate (PRPP). Also tracked were the C enrichment patterns of the hexosamine biosynthetic pathway (HBP), to were the synthesis of UDP-N-acetylglucosamine phosphoribosyl pyrophosphate (PRPP). Alsoleading tracked the 13C enrichment patterns of the intermediates of the hexosamine biosynthetic pathway (HBP), leading to the synthesis of (UDPGlcNAc). intermediates of the hexosamine biosynthetic pathway (HBP), leading to the synthesis of UDP-N-acetylglucosamine (UDPGlcNAc). UDP-N-acetylglucosamine (UDPGlcNAc).
Metabolites 2018, 8, 40 Metabolites 2018, 8, x Metabolites 2018, 8, x
7 of 16
0.6
0.4
0.0
1
3
4
5
0.0
0
1
0.7
NAcGN6P Ctl SeO3
G
3
6 0.6
HBP
Fraction
Fraction
0.2
2
0.5 0.4 0.3 0.2 0.1
0.0
0 1 2 3 6 7 8
*** ** **
0.0
4
1
2
3
3 5 2 1P6
1’
SeO3
0.8
D
0.6
F
0.4
0.8
3 2
0.4 0.2
0.0
0.0
0
1 0
3
5
4
5 Ring
0.8
5 6
0.6
0.2
5
1.0
4
1
E
PPP
1’
0.8
NAcGN1P 0.6 Ctl 0.4 SeO3
H
0.2 0.0
NAcGN6P
0.6
Ctl SeO3
G
0.5 0.4 0.3 0.2
PPP
0.4 0.2
4 1
1
2
0.7
UDPGlcNAc Ctl SeO3
HBP I
0.1
0 1 2 3 6 0.07 8 0 1 2 3 5 6 7 8 0 5 6 8 11 12 13 14 15
3
6 0.6 0.5
4
0
5
0.6
NAcGN1P Ctl
0.4 0.3 0.2 0.1
0 1 2 3 5 6 7 8
** ** ***
0.5
PPP PPP 1’
Ring
Ctl SeO3
0.3
I
0.2 0.1 0.0
0.6
0.0
4
0
1
3
1
1
4
0.0 0.0 0
4
Fraction
Fraction Fraction
2
4 3 5 211 6
PPP
PPP
0
1
2
1
2
3
3
0.0
4
0 1 42 3 5 4 3 5 2 6 11 211 6 1’
4
4 3 15 216 1st turn 4
PPP
0.2
0.2
0.0
0.0
0
1
2
3
4
5
0
1
0 5 6 8 11 12 13 14 15
0.6 0.5
HBP
0.3 0.2 0.0
2
6
0.5
G
0.4 0.3 0.2 0.1
0 1 2 3 4 5 6 7 8
* *
***
0.2 0.2 0.0
NAcGN6P
Ctl 0.4 UDPGlcNAc SeO3
0.1
** ** ** **
0.2
3
P HN Ac 6 1 NAcGN6P
UDPGlcNAc
0.4
2
1P
1st turn
** HN Ac ** ** ** 1 P NAcGN1P 1
SeO3 UDPGlcNAc H 0.0
5
1
P
0.0
0
** *** ** ** ** ** ** ** ** ***
F
0.6
PPP
3 2
0
3
0.0
4
0
1
2
3
4
0.0
3
4
5
0.00
1
0
2
3
5
4
*
5 Ring
0.2
1’
0.0
0
P HN Ac HN Ac 1 NAcGN6P 1 6 P NAcGN1P 1 0.6 NAcGN6P0.6 NAcGN1P 0.5
Ctl SeO3
0.4 0.3
H
0.2 0.1
Ctl SeO3
G
0.5 0.4 0.3
Ctl SeO3
HBP I
0.1
0.0
0 1 2 3 4 5 60.07 8 0 1 2 3 4 5 6 7 8 0 5 6 8 11 12 13 14 15
*
* ** *
3
6
UDPGlcNAc
0.2
2
4
*
0.0
5
0
4 1
5 6
1’
0.2
1
3 2
4 3 5 2 1P6
P
6 1 1 OMP 1 1 OMP Asp Asp 13C -Glc 13C -Glc NC-Asp P POrotate NC-Asp6 Orotate 6 P P R5P R5P 5’ P PPP PP5’ P 5’ P PPP PP5’ P 1’ 1’ 1’ 1’ PRPP PRPP CO2 CO2 1.0 1.0 1.0 Ctl CtlUTP Ctl 1.0 0.8 UTP R5P PRPP Ctl Ctl Ctl UTP UTP R5P PRPP 0.8 0.8 SeO3 SeO3 0.8 4 SeO3 0.8 SeO3 SeO3 SeO3 0.8 0.6 3 5 F 5 0.6 0.6 0.6 F 0.6 0.6 2 6 D E 6 0.4 1 D E 0.4 0.4 0.4 PPP PPP PPP *** 0.4 0.4 6
OMP
1.0
1.0
0.0
4 3 5 2 16 1
4
4
P
Fraction
Fraction
Fraction
Fraction
0
1
P HN Ac ** ** HN Ac ** ** 1 NAcGN6P 1 6 P NAcGN1P 1 Fraction
0
0.4
0.4
0.2
0.0
0.6
0.8
E
0.6
P HN Ac 6 1 NAcGN6P 0.8
PRPP
0.2
0.2
1
0.0
4
5
P
P
P
P
OMP
3 2
4 1
UTP 5 6
PPP PPP 1’
CO2 PPP
PPP
PPP
Ring
HN Ac 1 P NAcGN1P 1
0.6
NAcGN1P
0.6
0.5
Ctl
0.5
SeO3 UDPGlcNAc H
Fraction
0.4
3
Pyrimidine Synthesis
C
0.4
Fraction
D
0.6
Ctl SeO3
2
1’
1.0
0.8
1
NC-Asp P POrotate Orotate R5P 5’ P PPP PP5’ P 1’ PRPP PRPP CO2 Ctl Ctl CtlUTP R5P Ctl UTP PRPP SeO3 UTP SeO3 SeO3
PP5’ P
1’ 1.0
0
4 3 5 4 3 5 2 6 11 211 6 1’
4
Asp
-Glc NC-Asp6
R5P
4
0.6
0.2
Fraction
Ctl SeO3
1
13C
Fraction
Fraction
0.8
6
PPP
3
0.4
Ctl SeO3
Orotate
0.8
Pyrimidine B Synthesis
0.6
Fraction
R5P 5’ P
1’
3
Fraction
6-Glc
2
3 15 216 1st turn 4
1
1
Asp
2
Fraction
4
1
1.0
Ctl SeO3
NCAsp
0.8
0.2
Fraction
1st turn
1
A C
0.4 0.4
Fraction
1
0
0.2
0.2
0.0
0
4
1
3 5 211 6
0.0 0.0
4
SeO3
0.2 0.2
Fraction
3
1.0
Asp CtlCtl Orotate SeO3
0.8 0.8 0.6 0.6
B
0.4
Fraction
2
0.4
0.6
1.0 1.0
Ctl SeO3
NCAsp
0.8
0.6
Fraction
1
Fraction
1
0.2
0.2 0.2
0
4
0.4
1.0
Ctl SeO3
Asp
0.8
PyrimidineA Synthesis
C
Fraction
4
0.6
Fraction
3
Pyrimidine B Synthesis
0.4
Ctl SeO3
Orotate
0.8
Ctl SeO3
0.6
Fraction
2
Fraction
1
Fraction Fraction
0
1.0
0.4 0.4
Fraction
0.0
0.0
13C
0.4
AC
1.0
NCAsp
0.8
SeO3
0.6 0.6
B
0.2
0.2
6
0.6
1.0
1.0
Asp Ctl Ctl Orotate SeO3
0.8 0.8
Ctl SeO3
Fraction Fraction
0.4
1.0 1.0
NCAsp
0.8
Fraction
Fraction
A
0.6
1.0
Ctl SeO3
Asp
0.8
II. 3D Spheroids II. 3D Spheroids Fraction
I. 2D Cells
I. 2D Cells 1.0
7 of 16
7 of 16
0.4 0.3 0.2
0.1
0.0
0.0
*
Ctl SeO3
UDPGlcNAc
I
0.2
0.1
0 1 2 3 4 5 6 7 8
UDPGlcNAc
0.4 0.3
0 5 6 8 11 12 13 14 15
* *
Figure 4.the Pyrimidine andbiosynthetic the hexosamine biosynthetic pathways respond Figure Pyrimidine and the hexosamine biosynthetic pathways respond to selenite into selenite in A549 Figure 4. 4. Pyrimidine and hexosamine pathways respond less less to selenite in less A549 spheroids than in their 2D cell counterparts. The same polar extracts from Figure A549 spheroids 2D cell counterparts. Theextracts same from polarFigure extracts from Figure by 22 were analyzed by spheroids than inthan their in 2Dtheir cell counterparts. The same polar 2 were analyzed 13 13 13 13 13 13 CC fate of ofC6-Glc was first turn the(pentose Krebs were analyzed byCIC-UHRFT-MS. IC-UHRFT-MS. fate C6 -Glc was from the firstofPPP turn of thecycle, PPP (pentose fate of C6-Glc was followed from thefollowed first followed turnfrom of thethe Krebs cycle, IC-UHRFT-MS. pathway), pyrimidine synthesis, and HBP (hexosamine pathway) into UTP Krebs cycle,pathway), PPP phosphate (pentose phosphate pathway), pyrimidine synthesis, and pathway) HBP biosynthetic (hexosamine phosphate pyrimidine synthesis, and HBP (hexosamine biosynthetic into UTP and UDPGlcNAc (UDP-N-acetylglucosamine) along with the fractional distribution of relevant biosynthetic pathway) into UTP and UDPGlcNAc (UDP-N-acetylglucosamine) along with the fractional and UDPGlcNAc (UDP-N-acetylglucosamine) along with the fractional distribution of relevant 14 N; 14N; 14 distribution of relevant isotopologues of metabolites. : NCAsp: N-carbamoylaspartate; OMP: NCAsp: N-carbamoylaspartate; OMP: orotidine isotopologues of metabolites. ●: N; NCAsp: N-carbamoylaspartate; OMP: orotidine isotopologues of metabolites. ●: orotidine monophosphate; R5P: ribose-5-phosphate; PRPP: phosphoribosylpyrophosphate; NAcGN6P: monophosphate; R5P: ribose-5-phosphate; PRPP: phosphoribosylpyrophosphate; NAcGN6P: monophosphate; R5P: ribose-5-phosphate; PRPP: phosphoribosylpyrophosphate; NAcGN6P: N-acetylglucosamine-6-phosphate; NAcGN1P: (I) N-acetylglucosamine-6-phosphate; NAcGN1P: N-acetylglucosamine-1-phosphate. (I) Metabolite N-acetylglucosamine-6-phosphate; NAcGN1P: N-acetylglucosamine-1-phosphate. N-acetylglucosamine-1-phosphate. (I) Metabolite Metabolite responses 2D2D cultures; (II) metabolite responses in 3D cultures. All other symbols and abbreviations responses in 2D cultures; (II) metabolite in 3D cultures. Alland other symbols and responsesinin cultures; (II) metabolite responses in 3D responses cultures. All other symbols are as in Figure 2. abbreviations are as in Figure 2. abbreviations are as in Figure 2. 13C enrichment Itdifferential is clear that1313the differentialpatterns patterns of 13C2-Asp in 2D selenite-treated 2D A549 clearthat thatthe the differential enrichment patterns of1313CC22-Asp -Asp in selenite-treated selenite-treated 2D A549 ItIt isisclear CCenrichment of in A549 cells versus spheroids were maintained in the intermediates of the uracil ring synthesis such as cells versus spheroids were maintained in the intermediates of the uracil ring synthesis such as cells versus spheroids were maintained in the intermediates of the uracil ring synthesis such as N-carbamoylaspartate (NCAsp) and orotate (Figure 4(I-B,I-C) versus Figure 4(II-B,II-C), N-carbamoylaspartate(NCAsp) (NCAsp) orotate versus Figure respectively). 4(II-B,II-C), N-carbamoylaspartate andand orotate (Figure(Figure 4(I-B,I-C)4(I-B,I-C) versus Figure 4(II-B,II-C), 13C-labeled precursors into the uracil ring of UTP was 13C-labeled 13 respectively). Subsequent conversion of these respectively). Subsequent conversion of these precursors into the uracil ring of UTP was Subsequent conversion of these C-labeled precursors into the uracil ring of UTP was also differentially also differentially inhibited by selenite in 2D cells (Ring, Figure 4(I-F)) also differentially by(Ring, selenite in 2D cells (Ring, Figure 4(I-F)) versus 4(II-F)). spheroidsversus (Ring,spheroids (Ring, inhibited by seleniteinhibited in 2D cells Figure 4(I-F)) versus spheroids (Ring, Figure Figure 4(II-F)). Figure 4(II-F)). selenite In contrast, had a negligible effect on the extent of 13 C incorporation into R5P and PRPP 13C incorporation into R5P and PRPP In contrast, selenite had a negligible effect extent ofdifferential In contrast, selenite had a negligible effect on the extent of 13on C the incorporation into R5P inhibition and PRPP in both 2D cells (Figure 4(I-D,I-E)) and spheroids (Figure 4(II-D,II-E)). However, 13 in both 2D cells (Figure 4(I-D,I-E)) and spheroids (Figure 4(II-D,II-E)). differential inthe both 2D cells (Figure andof spheroids (Figure 4(II-D,II-E)). However, in incorporation of the C4(I-D,I-E)) PRPP into UTP by selenite was evident for 2Ddifferential A549However, cells 5 -ribosyl unit 13 13 in the the ofCThese 5PRPP -ribosyl unit of by PRPP into by selenite inhibition in4(I-F)) the inhibition incorporation ofincorporation the(cf.C5,5-ribosyl unit into UTP selenite was evident for was evident for (cf. 5, Figure versus spheroids Figureof4(II-F)). results suggested thatUTP the differential 2D A549 cells (cf. 5, Figure 4(I-F)) versus spheroids (cf. 5, Figure 4(II-F)). These results 2D A549 cells (cf. 5, Figure 4(I-F)) versus spheroids (cf. 5, Figure 4(II-F)). These results suggested that blockade of uracil nucleotide synthesis by selenite is most likely to be mediated via the inhibition of suggested that thethe differential blockade of uracil nucleotide synthesis by likely selenite likely to be mediated via the differential of uracil nucleotide synthesis by selenite is most to isbemost mediated via Asp production blockade by Krebs cycle and of ribosyl incorporation by reactions downstream from PRPP the inhibition of Asp production by the cycle incorporation and of ribosylbyincorporation by reactions the inhibition Asp production by the Krebs cycle andKrebs of ribosyl reactions production, e.g.,oforotate phosphoribosyltransferase. 13 downstream from PRPP production, e.g., orotate phosphoribosyltransferase. downstream from distinct PRPP production, e.g., orotate phosphoribosyltransferase. Furthermore, C enrichment patterns of the HBP intermediates, N-acetylglucosamine13C 13C Furthermore, distinct enrichment the HBP intermediates, Furthermore, distinct enrichment patterns of patterns the HBPof intermediates, 6-phosphate (NAcGN6P, Figure 4(I-G) versus Figure 4(II-G)) and N-acetylglucosamine-1-phosphate N-acetylglucosamine-6-phosphate (NAcGN6P, Figure 4(I-G) versus Figure N-acetylglucosamine-6-phosphate (NAcGN6P, Figure 4(I-G) versus Figure 4(II-G)) and (NAcGN1P, Figure 4(I-H) versus Figure 4(II-H)) between selenite-treated 2D cells and spheroids of A549 4(II-G)) and N-acetylglucosamine-1-phosphate (NAcGN1P, Figure Figure 4(I-H) Figure 4(II-H)) between N-acetylglucosamine-1-phosphate Figure 4(I-H) versus 4(II-H)) between were evident, particularly for the 13 C6(NAcGN1P, - and 13 C8 -isotopologues (respectively 6 and 8,versus Figure 4(I-G,I-H) 13 selenite-treated 2D cells and spheroids of A549 were evident, particularly for the 13C6- and selenite-treated 2D cells and spheroids of A549 evident, forthe the C6- and versus Figure 4(II-G,II-H)). It is interesting to note thewere opposite effectparticularly of selenite on enrichment 13 13C8-isotopologues (respectively 6 and2D 8, Figure 4(I-G,I-H) versus Figure 4(II-G,II-H)). and 8, Figure versus Figure 4(II-G,II-H)). It is interesting patterns of the 13 CC6(respectively -8-isotopologues versus 13 C8 6-NAcGN6P for4(I-G,I-H) both cells and spheroids. Since the synthesis of It is interesting 13C6- versus 13C8-NAcGN6P 13 13 13 13 13 to note the opposite effect of selenite on the enrichment patterns of the toCnote the opposite effect of selenite on the enrichment patterns of the C6- versus C8-NAcGN6P 8 - from C6 -NAcGN6P presumably requires C2 -acetyl CoA, the large reduction in the extent 13C8- from 13C6-NAcGN6P presumably 13Csynthesis 13C forenrichment bothspheroids. 2D cells spheroids. Since of6-NAcGN6P 13 for both 2D cells and Since the synthesis ofthe 8- 13 from presumably of C8 -NAcGN6P mayand reflect reduced synthesis of C2 -acetyl CoA via PDH and/or 13C2-acetyl CoA, the large reduction 13 13C8-NAcGN6P enrichment may reflect 13 in the extent of requires 2-acetyl CoA,activity the large in the extent of[43]) C8-NAcGN6P enrichment reflect requires Clyase ATP-citrate (ACLY) (cf.reduction Figure 2 and Figure S4A; and/or inhibition in the may acetylation 13C2-acetyl CoA via PDH and/or ATP-citrate lyase (ACLY) activity (cf. Figure 2 reduced synthesis of via reduced synthesis of 13C2-acetyl and/orwith ATP-citrate lyase of (ACLY) activity of glucosamine-6-phosphate. TheCoA former isPDH consistent the inhibition the Krebs cycle(cf. by Figure selenite2 and Figure S4A; and/or inhibition inofthe acetylation of glucosamine-6-phosphate. and Figure S4A;(cf. [43]) and/or inhibition in the acetylation glucosamine-6-phosphate. The formerin is The former is described above Figure 2). The[43]) increased fractional enrichment of 13 C6 -NAcGN6P was reflected consistent with the inhibition of the Krebs cycle by selenite described above (cf. 13 consistent the inhibitionproduct of the for Krebs by selenite described (cf. (Figure Figure 2). The Figure 2). The that of the with C6 -UDPGlcNAc bothcycle 2D cells (Figure 4(I-I)) and above spheroids 4(II-I)). 13 13 13 increased fractional of was C6-NAcGN6P was thatofof 13 C13C6-UDPGlcNAc increased fractional enrichment of enrichment C6-NAcGN6P reflected thatreflected of the in C6-UDPGlcNAc This increase could also have contribution from decreased synthesis in and/or incorporation thethe both 2D cells (Figure 4(I-I))(Figure and spheroids (Figure 4(II-I)). Thisalso increase product for bothproduct 2D cellsfor (Figure 4(I-I)) and spheroids 4(II-I)). This increase could have could also have 13C labeled UTP precursor into 13 contribution from decreased synthesis and/or incorporation of the contribution from decreased synthesis and/or incorporation of the C labeled UTP precursor into as evidenced from the large in of the13enrichment of 13C11-15 UDPGlcNAc, asUDPGlcNAc, evidenced from the large reduction in thereduction enrichment C11-15-UDPGlcNAc for-UDPGlcNAc for
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labeled UTP precursor into UDPGlcNAc, as evidenced from the large reduction in the enrichment 13 of -UDPGlcNAc for both 2D Figure cells and spheroids Figureof4(I-I,II-I)). The production of 11-15 bothC2D cells and spheroids (11–15, 4(I-I,II-I)). The(11–15, production these isotopologues requires 13 these isotopologues requires C-labeled UTPnoted (Figure 4) [43]. noted wasenrichment the lower fractional 13C-labeled UTP (Figure 4) [43]. Further was the Further lower fractional in these enrichment in these isotopologues in spheroids compared with 2D cells under control conditions, isotopologues in spheroids compared with 2D cells under control conditions, which corresponded to which to the 13 Cofenrichment patterns of the precursors of UTP (Figure 4(I-A–I-F) versus the 13Ccorresponded enrichment patterns the precursors of UTP (Figure 4(I-A–I-F) versus Figure 4(II-A–II-F)) Figure 4(II-A–II-F)) and HBP intermediates (FigureFigure 4(I-G–I-I),versus Figure 4(II-G–II-I)). Thesearesults and HBP intermediates (Figure 4(I-G–I-I),versus 4(II-G–II-I)). These results suggest lower 13 C -ribosyl incorporation and suggest a lower capacity for UTP synthesis (both in terms of the 13 13 5 capacity for UTP synthesis (both in terms of the C5-ribosyl incorporation and C-ring synthesis) 13 C-ring synthesis) and HBP (cf. also higher enrichment of the all 12 C or 0 isotopologue) in spheroids and HBP (cf. also higher enrichment of the all 12C or 0 isotopologue) in spheroids than in the 2D than in the 2D counterpart. counterpart. Similar Similar to to A549 A549 cells, cells, we we observed observed differential differential selenite selenite inhibition inhibition of of HBP HBP activity activity and and UTP UTP synthesis, but not of PPP activity and PRPP production in PANC1 2D cell culture (Figure 5I versus synthesis, but not of PPP activity and PRPP production in PANC1 2D cell culture (Figure 5I versus spheroids spheroids(Figure (Figure5II. 5II.Likewise, Likewise,UDPGlcNAc UDPGlcNAcsynthesis synthesiswas wasdifferentially differentiallyinhibited inhibitedby byselenite selenitein inthe the two PANC1 systems (Figure 5(I-I) versus Figure 5(II-I)). Moreover, intrinsically lower capacity two PANC1 systems (Figure 5(I-I) versus Figure 5(II-I)). Moreover, intrinsically lower capacity for for UTP synthesis and HBP was evident in PANC1 spheroids than 2D cells (cf. higher enrichment of 0 UTP synthesis and HBP was evident in PANC1 spheroids than 2D cells (cf. higher enrichment of 0in in Figure Figure5II 5IIversus versusFigure Figure5I). 5I). II. 3D Spheroids
I. 2D Cells
4
R5P 5’ P
0.6
1.0
R5P
0.2
0.8
1
2
3
4
Fraction
0.2 0.0
NAcGN6P
G
CTL SeO3
CTL SeO3
0 1 2 3 4 5 6 7 8
3
5
3 2
4 1
5 6
1
1’
6 0.8 0.6 0.4 0.2 0.0
3
4
CTL SeO3
0.6
F
0.2
UTP
** ** ** **
3 2
4 1
UTP 5 6
1’ 0
5
NAcGN1P CTL SeO3
** **
***
5
0.6 0.4 0.2 0.0
0 1 2 3 4 5 6 7 8
Fraction
Fraction
6
2
3
1st turn
4
Asp
PPP
CO2
1.0 0.8
PPP
PPP
PPP
0.2
UDPGlcNAc
** **
CTL SeO3
UDPGlcNAc ** **
0.5 0.4 0.3 0.2
3
1
2
3
4
1
1
5
Ctl SeO3
0.4
2
3
4
5
1
2
3
4
4 1
5 6
P
P
P
P
PRPP 0.6 0.5 0.4
F
UTP
Ctl SeO3
0.3
5
0.2
0.0
3 2
4 1
UTP 5 6
1’ 0
5
PPP
CO2 PPP
PPP
PPP
ring
HN Ac 1 P NAcGN1P 1
6
0.4
HBP
3 2
1’
0.1
0
0.3 0.2 0.1 0.0
0 1 2 3 4 5 6 7 8
1
OMP
E
NAcGN6P Ctl SeO3
0
Orotate
PRPP
0.6
0.0
Pyrimidine Synthesis
4 3 5 216
PP5’ P
0.5
G
0.4
0.0
4
0.2
0
0.1 0.0
0 5 6 7 8 1112131415
2
1
P HN Ac 6 1 NAcGN6P 0.6
I
0.8
D
0.4
0.0
ring
1
1’
R5P
0.6
0
4 3 5 211 6
PPP 1.0
Ctl SeO3
C
0.6
NC-Asp
R5P 5’ P
Ctl SeO3
Orotate
0.8
0.2
0.0
4
1
13C -Glc 6
P
HN Ac 1 P NAcGN1P 1 H
1
1’
0.8
0.4
0
P
P
PRPP
PRPP
2
0.1
1
P
0.4 0.2
0.0
4
0.0
0
** **
**
2
Orotate
E
0.4
HBP
1
OMP
PP5’ P
0.6
5
P HN Ac 6 1 NAcGN6P 0.4
1
0.0
0
0.6
0
3 5 216
0.2
0.0
0.8
5
4
1’
D
0.4
4
1
PPP Fraction
Fraction
CTL SeO3
C
0.2 0.0
3
NC-Asp
1’ 0.8
2
1
Asp
1.0
1
3 5 211 6
4
13C -Glc 6
0
0.2
B
0.6
1.0
Ctl SeO3
NCAsp
0.8
NAcGN1P Ctl SeO3
H 0 1 2 3 4 5 6 7 8
Fraction
0.0
1
1st turn
B ** **
0.2
4
Fraction
6
3
0.3
1.0
Fraction
1
2
A
0.4
Fraction
1
0.5
Fraction
0
Fraction
0.0
0.4
Fraction
0.2
0.4
CTL SeO3
0.6
Fraction
** ** ** **
0.4
CTL SeO3
0.6
Pyrimidine Synthesis
Orotate
0.8
Fraction
0.6
1.0
NCAsp
0.8
Fraction
A
1.0
Fraction
Fraction
0.8
CTL SeO3
Ctl SeO3
Asp
0.6
Asp
Fraction
0.7
1.0
0.3
UDPGlcNAc
0.2
I
Ctl SeO3
UDPGlcNAc
0.1 0.0
0 5 6 7 8 1112131415
Figure 5. Pyrimidine and the hexosamine biosynthetic pathways respond less to selenite in PANC1 Figure 5. Pyrimidine and the hexosamine biosynthetic pathways respond less to selenite in PANC1 spheroids than in their 2D cell counterparts. The same polar extracts from Figure 3 were analyzed by spheroids than in their 2D cell counterparts. The same polar extracts from Figure 3 were analyzed IC-UHR FT-MS. All symbols and abbreviations are as in Figure 4. (I) Metabolite responses in 2D by IC-UHR FT-MS. All symbols and abbreviations are as in Figure 4. (I) Metabolite responses in 2D cultures; (II) metabolite responses in 3D cultures cultures; (II) metabolite responses in 3D cultures
3. Discussion 3. Discussion Selenite has been investigated extensively for anti-cancer properties in 2D cultures of different Selenite has been investigated extensively for anti-cancer properties in 2D cultures of different cancer cells types including A549 cells [40,44–51]. But virtually nothing is known about its effect on cancer cells types including A549 cells [40,44–51]. But virtually nothing is known about its effect on 3D 3D spheroids, which exhibit cell–cell and cell–matrix interactions absent from the 2D cultures. To the spheroids, which exhibit cell–cell and cell–matrix interactions absent from the 2D cultures. To the best best of our knowledge, little is known about the effects of selenite on PANC1 cells either as 2D or of our knowledge, little is known about the effects of selenite on PANC1 cells either as 2D or spheroid spheroid cultures. We showed here that selenite has a complex effect on the spheroid cultures of cultures. We showed here that selenite has a complex effect on the spheroid cultures of both A549 and both A549 and PANC1 cells including responses from a mixed population of resistant and sensitive PANC1 cells including responses from a mixed population of resistant and sensitive cells (Figure 1). cells (Figure 1). Although difficult to quantify, it is clear that spheroids were more resistant to Although difficult to quantify, it is clear that spheroids were more resistant to selenite than 2D cells selenite than 2D cells when comparing their 1-day IC50 values, respectively >100 µM versus 6.25 µM when comparing their 1-day IC50 values, respectively >100 µM versus 6.25 µM for A549 and >150 µM for A549 and >150 µM versus 10 µM for PANC1 (cf. Figure 1, Figures S2 and S3). This resistance of versus 10 µM for PANC1 (cf. Figure 1, Figures S2 and S3). This resistance of 3D spheroids to selenite 3D spheroids to selenite toxicity did not result from less Se accumulation into cell biomass (Figure toxicity did not result from less Se accumulation into cell biomass (Figure S1). A higher resistance to S1). A higher resistance to selenite toxicity with comparable growth rates was also evident for A549 selenite toxicity with comparable growth rates was also evident for A549 spheroids formed without spheroids formed without the magnetic NS (data not shown). These illustrated the influence of the the magnetic NS (data not shown). These illustrated the influence of the microenvironment, albeit microenvironment, albeit in a simple form, on cancer cells’ response to toxicants. In addition, the in a simple form, on cancer cells’ response to toxicants. In addition, the selenite toxicity for the selenite toxicity for the sensitive population of PANC1 spheroids with 1-day treatment appeared to sensitive population of PANC1 spheroids with 1-day treatment appeared to be higher than that of be higher than that of A549 spheroids (Figure 1). However, after 2 and 3 days of treatment, the opposite was evident for the selenite toxicity in PANC1 and A549 spheroids (Table 1). Nevertheless,
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A549 spheroids (Figure 1). However, after 2 and 3 days of treatment, the opposite was evident for the selenite toxicity in PANC1 and A549 spheroids (Table 1). Nevertheless, selenite was toxic to both sensitive and resistant populations of A549 and PANC1 spheroids after prolonged exposure. 13 C -Glc-based SIRM investigations on 1-day selenite treatment of A549 and PANC1 cells and 6 spheroids corroborated with the 1-day IC50 trend. Both A549 and PANC1 spheroids displayed less selenite-induced perturbations in the activities of the central metabolic pathways, including glycolysis, the Krebs cycle (Figures 2 and 3), HBP, and pyrimidine ring synthesis (Figures 4 and 5), relative to the 2D cell counterparts. These growth-related metabolic effects are unlikely to be attributed to the presence of NS since NS had a negligible effect on spheroid growth rates and selenite-induced growth attenuation, as indicated above. In addition, the relatively lesser inhibition of these pathways in PANC1 than A549 spheroids was consistent with the lower growth attenuation of the former. Together, these results are consistent with the requirement of these pathways for cell growth. It should also be noted that these metabolic changes occurred before appreciable growth inhibition was evident for A549 spheroids (cf. Figure 1 versus Figure 2), making them more sensitive indicators of selenite toxicity. Moreover, it is likely that the metabolic perturbations manifested in both 3D spheroids resulted from the response of the sensitive populations. The disruption of GSH synthesis by selenite in 2D A549 cells (Figure 2(I-J)) presumably compromises anti-oxidative defense, which can be related to the enhanced production of reactive oxygen species (ROS) and ROS-mediated cell death observed by Park et al. [49] in selenite-treated A549 cells. This could be the case for the sensitive populations in both A549 and PANC1 spheroids, as evident from the burst of ROS production in 1 day of 10–20 µM selenite treatment (Figure 1C). As such, the relatively less attenuated GSH synthesis in A549 and PANC1 spheroids than in 2D cultures could lead to less oxidative damages and better survival for the resistant populations, which could in turn contribute to the higher IC50 for selenite in A549 and PANC1 spheroids than those in the 2D cell counterparts. However, the much lower ROS production in A549 than PANC1 spheroids elicited by the 3-day treatment of selenite suggests that excess ROS was not the key to cell death in A549 spheroids (cf. Figure S2C). Two alternative possibilities warrant further investigation in terms of selenite’s toxic mechanism in A549 spheroids, e.g., (1) inhibition of glutaminolysis via enhanced GLS1 degradation [52]; (2) altered detoxification mechanism involving direct interaction of selenite with GSH to form selenodiglutathione (GSSeG) [53] and subsequent efflux of GSSeG from cancer cells [54]. Besides differential metabolic responses to selenite for spheroids versus 2D cells, we also noted their intrinsically distinct metabolic activities, i.e., reduced capacity for lactate release, UTP synthesis and HBP in both A549 and PANC1 spheroids (Figures 4 and 5), and activation of gluconeogenesis in PANC1 spheroids (Figure 3). Attenuated lactate release by A549 spheroids (Figure 2(II-D)) versus Figure 2(I-D)) could be related to the differences in growth rates (2.9 days for spheroids versus 1.2 days for 2D cells in doubling time). But, this is unlikely to be the case for PANC1 spheroids (Figure 3(II-D)) versus Figure 3(I-D)) since their doubling time (0.6 days) was shorter than 2D cells (1.8 days). Reduced UTP synthesis presumably led to a lower capacity for UDPGlcNAc synthesis in spheroids than 2D cells. UDPGlcNAc is required for O-linked N-acetylglucosamine modification (O-GlcNAcylation) of regulator proteins, including those important for cancer development and survival [55–59]. It is possible that this difference in capacity for O-GlcNAcylation of proteins between spheroids and 2D cells is involved in modulating their differential responses to selenite toxicity. Further investigations will be required to test this hypothesis. It is also foreseeable that enhanced capacity for gluconeogenesis in PANC1 spheroids could improve their survival in a hostile hypoxic tumor microenvironment, where glucose is depleted due to its increased glycolytic conversion to lactate and extracellular release of lactate [60]; the latter response was observed for selenite treatment in the more sensitive A549 spheroids (Figure 2(II-D) versus Figure 3(II-D)). Again, we plan to investigate this hypothesis in future studies. In summary, both NSCLC and PDAC cancer cell spheroids displayed much higher resistance to anti-cancer selenite toxicity than their 2D cell counterparts in terms of cell growth, which was associated with attenuated response of growth- and survival-requiring central metabolic activities. This resistance
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can be attributed, at least in part, to the presence of a resistant cell population, presumably as a result of cell–cell and cell–matrix interactions in the spheroids. These interactions also led to reduced intrinsic capacity of UDPGlcNAc synthesis in A549 and PANC1 spheroids and activation of gluconeogenesis in PANC1 spheroids, which could be involved in modulating cancer spheroid metabolism and survival. 4. Materials and Methods 4.1. IC50 Determination for Selenite Treatment of 2D PANC1 Cells IC50 for sodium selenite (Na2 SeO3 ; Sigma S-1382) in PANC1 cells were initially determined in 96-well plates and further determined in 6-well plates. For 6-well plate (Greiner Bio-one CELLSTAR® 657160), PANC1 cells were grown in DMEM (Sigma D5030; supplemented with sodium bicarbonate 3.7 g/L (pH 7.2), 2 g/L glucose, 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin) for two days (reaching 60~70% confluency) before titrating with 0, 2, 10, 20, 50, 200 µM sodium selenite for 26 h. At the end of the treatment, cell viability was determined by neutral red assay. In brief, cell media were aspirated and cells were incubated in 2 mL neutral red solution (33 µg/mL in DMEM) at 37 ◦ C and 5% CO2 for 3 h. At the end of the incubation, neutral red solution was aspirated and cells were washed with 2 mL PBS twice. After removing PBS, cells were lysed in 1 mL lysis buffer containing 50% ethanol and 1% acetic acid in water. Lysates were transferred to a 96-well plate (125 µL/well, in triplicates) and A540 was measured in a plate reader. Dose response curves were plotted using A540 with background subtraction and normalization to control cells. IC50 was deduced from curve fitting using SigmaPlot. 4.2. Spheroid Formation, Culturing, and Growth Assay A549 or PANC1 cells were grown in 6-well plates to 50–70% confluence in DMEM growth medium at 37 ◦ C/5% CO2 before treating cells with NanoShuttlesTM -PL (NS, n3D, Biosciences, Inc., Houston, TX, USA) at 1 µL/104 cells overnight according to the vendor’s protocol. NS-loaded cells were then trypsinized, counted, and seeded in a cell-repellent round-bottom 384-well plate at 1000 cells per well. We have separately determined this cell seeding density to be within the linear range of spheroid growth as a function of cell seeding density. The plate was held below a 384-well spheroid drive (n3D) initially for 15 min in the biosafety hood and followed by 45 min at 37 ◦ C/5% CO2 for spheroid formation. The drive was then removed and spheroids were allowed to grow for 3 days before medium change to DMEM containing 0 to 500 µM selenite. Spheroid growth was assayed daily using the PrestoBlue® live cell fluorescent stain reagent (Thermofisher, Waltham, MA, USA) [61], which enables in situ monitoring of cellular reducing activity. PrestoBlue® reduction was followed for 30 min at 3–5 min intervals for each well in the 384-well plate using a Cytation 3 imaging microplate reader (BioTek, Winooski, VT, USA) set at 37 ◦ C with 5% CO2 . The slope of the fluorescence versus time was used to plot the selenite dose-response curve (cf. Figure 1A), from which IC50 for the selenite treatment was estimated. IC50 values were determined by non-linear regression analysis using Kaleidagraph (Synergy Software, version 4.5) as the Hill equation or the Hill equation modified to two components (sensitive and resistant). y = ymin + D/[1 + (c/IC50 )n ] where D is the change in the number of cells, ymin is the plateau value at high concentrations, c, of the inhibitor and n is the Hill coefficient. For biphasic curves, this equation was modified as follows: y = ymin + D/[1 + (c/IC50 )n ] − a.c where a represents the slope of the “resistant” cell population.
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13 C
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SIRM Experiments for 2D Cells and Spheroids
2D cell experiments were performed in 10-cm plates with cells grown to 50–70% confluence in DMEM growth medium. PANC1 (passage 11, 5 × 105 ) cells were seeded on 10-cm plates and cultured in DMEM growth medium using dialyzed, exosome-depleted FBS. Exosome-depleted FBS was prepared by ultracentrifugation at 120,000× g for 2 h followed by filtration through a sterile 0.22 µm vacuum filter. Four days after seeding, media were replaced with fresh media containing either 0.2% 12 C6 -Glc (unlabeled) or 13 C6 -Glc, without or with 10 µM SeO3 in triplicates. Aliquots of 200 µL media were collected at 0, 12 and 24 h after treatment started and spun at 3500× g for 15 min at 4 ◦ C to remove debris. Aliquots of 100 µL supernatants were subject to protein precipitation by mixing with 400 µL cold acetone, incubating at −80 ◦ C for 30 min before centrifugation at 14,000× g for 10 min at 4 ◦ C. Supernatants were aliquoted and lyophilized for NMR analysis. Such acetone extraction method efficiently precipitated proteins and recovered polar metabolites from the media, similarly as the 10% trichloroacetic acid method [40] but without adding salts (Fan, unpublished data). At the end of 24 h treatment, cells were washed in cold PBS, metabolism quenched in cold acetonitrile, and polar/non-polar metabolites extracted in acetonitrile:water:chloroform (2:1.5:1 v/v/v) as described previously [62]. Polar extracts were aliquoted and lyophilized for NMR and IC-UHR FT-MS analyses. The protein pellets were washed in methanol, dried in Vacufuge (Eppendorf, Hamburg, Germany), and re-dissolved in SDS buffer containing 62.5 mM Tris (pH 6.8), 2% SDS and 1 mM DTT using mini-pestles. Protein concentrations were measured by microBCA assay (Pierce Chemical, Dallas, TX, USA), per vendor’s protocol. Of note, the metabolic response of PANC1 cells to selenite did not depend on passage numbers or different time of the experiments. This was also the case for A549 cells provided that the passage numbers do not go beyond 30. To prepare spheroid cultures for SIRM experiments, 464 µL of NanoShuttlesTM was added for 16–18 h to the cells grown in a 10-cm plate at 70% confluency. Then, cells were washed twice with IX PBS, detached from the plate using trypsin, and counted using a hemocytometer. The trypsinized cells were resuspended in DMEM growth medium before seeding in 6-well plates at a density of 400,000 cells/well. A 6-well magnetic levitation drive was put on top of the 6-well plate containing cells for 15 min in the biosafety hood. The cells were then transferred to a 37 ◦ C/5% CO2 incubator and incubated for a further 45 min before removing the magnetic drive. Cells were then monitored daily and growth media was renewed every two days. After four days of growth, cells were treated with 10 µM selenite and 13 C6 -Glc for 24 h. Media at 50 µL aliquots were collected at 0 and 24 h after selenite/13 C6 -Glc treatment. At the end of incubation, spheroids were held with the 6-well magnetic holder during medium removal and washed twice with IX ice cold PBS and once with nanopure water, followed by quenching and extraction of polar metabolites twice each with 1.0 mL 70% methanol/well. This simultaneous quenching/extraction method is compatible with immunofluorescence analysis and gave reproducible and comparable metabolite profiles as the acetonitrile-based quenching/extraction method described above for 2D cell cultures (Fan et al., unpublished data). The method efficiently quenches the hydrolysis of high-energy metabolites as evidenced by the high energy charge ratios calculated for A549 (ca. 0.9) and PANC1 (0.98) spheroids. The polar extracts were lyophilized overnight and subjected to IC-UHR FT-MS analysis. The cell residues were extracted and analyzed for proteins as described above. Polar metabolites from the media were extracted in 80% acetone as described above. 4.4. IC-UHR FT-MS Analysis Ion chromatography-ultra high-resolution Fourier transform-MS (IC-UHR FT-MS) was performed as previously described [33]. Briefly, polar extracts were reconstituted in 20 µL nanopure water, and analyzed by a Dionex ICS-5000+ ion chromatograph interfaced to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) operating at a resolution setting of 500,000 (FWHM at m/z 200) on MS1 acquisition to capture all 13 C isotopologues. The chromatograph was outfitted with a Dionex IonPac AG11-HC-4 µm RFIC&HPIC (2 × 50 mm) guard column upstream
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of a Dionex IonPac AS11-HC-4 µm RFIC&HPIC (2 × 250 mm) column. Chromatography and mass spectrometric settings were the same as described previously [30] with an acquisition m/z range of 80 to 700. Metabolites and their isotopologues were identified by chromatographic retention times and their m/z values compared with those of the standards. Peak were integrated and the areas exported to Excel via the TraceFinder 3.3 (Thermo, Waltham, MA, USA) software package. Peak areas were corrected for natural abundance as previously described [63], after which fractional enrichment and µmoles metabolites/g protein were calculated to quantify 13 C incorporation into various metabolites. 4.5. 1 H-NMR Analysis of Medium Extracts Lyophilized medium extracts were redissolved in 35 µL D2 O containing 8.81 nmoles of d6 -DSS (2,2-Dimethyl-2-silapentane-5-sulfonate, Cambridge Isotope Laboratories, Tewksbury, MA, USA) for 1D 1 H-NMR analysis using a 1.7 mm inverse triple-resonance HCN cryoprobe on a Bruker AVANCE III NMR at 16.45 T (Bruker Corp., Billerica, MA, USA). A 1 H 90◦ pulse with solvent presaturation was used to acquire 1D 1 H spectra with a 8403 Hz spectral width, 2 s acquisition time, 4 s relaxation delay, and 512 transients. The free induction decays were Fourier-transformed with 1 Hz line-broadening and zero-filled to 131,072 points. The “peak picking” routine in the MestReNova software (Mestrelab Research S.L., Santiago de Compostela, Spain) was used to quantify the 13 C satellites of the methyl resonance of lactate, which represented 13 C3 -lactate based on the splitting patterns. The lactate peak areas were calibrated for nmoles against that of the methyl resonance of d6 -DSS and normalized to the cell protein content. The final 13 C3 -lactate content in the media was expressed as mmole/g protein. 4.6. Immunofluorescence Measurements 3D Spheroids were suspended in HistoGel (Thermo Scientific HG-4000-012). After solidification, HistoGel pellets were fixed in 10% neutral buffered formalin for 24 h, followed by paraffin embedding, sectioning at 4 µm thickness, and slide mounting. The slides were deparaffinized, rehydrated, subjected to heat-induced epitope retrieval (hier) by microwaving in sodium citrate buffer (10 mM sodium citrate, 0.05% tween 20, pH 6.0) for 10 min at a sub-boiling temperature. For immunofluorescence analysis, slides were treated with AlexaFluor™ Tyramide Superboost™ kits (Invitrogen, Carlsbad, CA, USA) as per manufacturer’s instructions, followed by primary and secondary antibody treatments as described previously [33]. The following primary antibodies were used: PCNA (PC10) mouse mAb (1:4000, Cell Signaling, Danvers, MA, USA, #2586) and RIP (D94C12) XP® rabbit mAb (1:100, Cell Signaling #3493). Slides were mounted in Prolong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific P36935) before imaging. Images were acquired at 40× magnification using a laser scanning confocal microscope Olympus Fluoview™ FV1200. 4.7. Se Analysis by Inductively-Coupled Plasma-Mass Spectrometry (ICP-MS) 2D and 3D cells were cultured in 6-well plates, as described in 4.2 and 4.3. A549 cells (passage 15) were treated with 6.25 µM (2D) or 10 µM selenite (3D) for 24 h with 200 µL media sampled at 0 and 24 h after selenite treatment. Both 2D and 3D PANC1 (passage 20) cells were treated with 10 µM selenite for 24 h and media sampled as described above. Then 10 µL aliquots of media were digested in triplicate in 0.5 mL concentrated nitric acid (Aristar Plus, VWR chemicals) at 150 ◦ C for 10 min with maximal pressure of 200 psi and power of 150 W using Discover SP microwave digestor (CEM, Matthews, NC, USA). At the end of 24 h treatment, cells were washed in cold PBS, quenched in cold 70% methanol, and extracted for polar metabolites as described above. The protein pellets were then extracted using SDS/Tris buffer and protein concentrations were measured by microBCA assay as above. Both polar and protein extracts were aliquoted in duplicates and lyophilized. The residue pellets were dried in a Vacufuge (Eppendorf) and pre-digested in 100 µL nitric acid overnight. All lyophilized extracts and predigested residue pellets were digested in 250 µL concentrated nitric acid as above and the final digests were diluted to 5% nitric acid with nanopure water. ICP-MS was preformed using an 8800 Triple Quadrupole ICP-MS (Agilent Technologies, Santa Clara, CA, USA) with a micromist
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nebulizer. Selenium was monitored in MS/MS mode at the second quadrupole as the oxide (SeO, m/z 96) following collision with oxygen flowing at 50% with integration time of 1 s. Se concentrations in the digests were calculated from a standard curve of sodium selenite (Na2 SeO3 ) in 5% nitric acid at 5 concentrations from 0.1 to 10 ppm. Supplementary Materials: The following are available online at http://www.mdpi.com/2218-1989/8/3/40/s1, Figure S1: Higher selenite resistance of A549 or PANC1 spheroids is not due to lower Se accumulation than the 2D counterparts; Figure S2: A549 spheroids respond less to selenite than 2D cultures in terms of morphology, protein content, mitotic index, and necrosis; Figure S3: PANC1 spheroids respond less to selenite than 2D cultures in terms of morphology, protein content, mitotic index, and necrosis.; Figure S4: 13 C atom-resolved tracing of the synthesis of 13 C2 -acetyl CoA and 13 C1 -isotopologues of Krebs cycle metabolites from 13 C6 -glucose. Author Contributions: T.W.-M.F. conceived, designed, and performed the experiments, analyzed and interpreted data and wrote the manuscript; S.S.E. and Q.J.W. performed experiments and contributed to manuscript writing; J.K.A.M. carried out IC-UHR-FTMS experiments, data analysis, and contributed to manuscript writing; T.C. performed Se analysis by ICP-MS and contributed to manuscript writing; A.N.L. interpreted results and co-wrote the manuscript; H.S. performed the immunofluorescence experiments and contributed to manuscript writing. Funding: This work was supported in part by NIH grants 1P01CA163223-01A1 (to ANL and TWMF), 1U24DK097215-01A1 (to RMH, TWMF, and ANL), and Redox Metabolism Shared Resource(s) of the University of Kentucky Markey Cancer Center (P30CA177558). Conflicts of Interest: The authors declare no conflict of interest.
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