Three-Dimensional Cell Culture-Based Screening Identifies ... · Identifies the Anthelmintic Drug...

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Models and Technologies Three-Dimensional Cell Culture-Based Screening Identies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer Wojciech Senkowski 1 , Xiaonan Zhang 2 , Maria Hagg Olofsson 2 , Ruben Isacson 3 , Urban Hoglund 3 , Mats Gustafsson 1 , Peter Nygren 4 , Stig Linder 2,5 , Rolf Larsson 1 , and Ma rten Fryknas 1 Abstract Because dormant cancer cells in hypoxic and nutrient-deprived regions of solid tumors provide a major obstacle to treatment, compounds targeting those cells might have clinical benets. Here, we describe a high-throughput drug screening approach, using glucose-deprived multicellular tumor spheroids (MCTS) with inner hypoxia, to identify compounds that specically target this cell population. We used a concept of drug reposi- tioningusing known molecules for new indications. This is a promising strategy to identify molecules for rapid clinical advancement. By screening 1,600 compounds with documen- ted clinical history, we aimed to identify candidates with unforeseen potential for repositioning as anticancer drugs. Our screen identied ve molecules with pronounced MCTS-selec- tive activity: nitazoxanide, niclosamide, closantel, pyrvinium pamoate, and salinomycin. Herein, we show that all ve compounds inhibit mitochondrial respiration. This suggests that cancer cells in low glucose concentrations depend on oxida- tive phosphorylation rather than solely glycolysis. Importantly, continuous exposure to the compounds was required to achieve effective treatment. Nitazoxanide, an FDA-approved antiproto- zoal drug with excellent pharmacokinetic and safety prole, is the only molecule among the screening hits that reaches high plasma concentrations persisting for up to a few hours after single oral dose. Nitazoxanide activated the AMPK pathway and downregu- lated c-Myc, mTOR, and Wnt signaling at clinically achievable concentrations. Nitazoxanide combined with the cytotoxic drug irinotecan showed anticancer activity in vivo. We here report that the FDA-approved anthelmintic drug nitazoxanide could be a potential candidate for advancement into cancer clinical trials. Mol Cancer Ther; 14(6); 150416. Ó2015 AACR. Introduction The cancer drug development process has become increasingly costly and inefcient resulting in few new highly effective drugs reaching the market yearly (1). New strategies for drug discovery are therefore urgently needed. One such strategy is drug reposi- tioning in which a new indication for an existing drug is identied. Using this approach, approved, discontinued, or withdrawn drugs with unrecognized anticancer activity can be rapidly advanced into clinical trials, because much of the required documentation already exists. The availability of compound libraries containing drugs with documented clinical use makes unbiased screening for repositioning candidates an attractive approach. During the past decades, most screening approaches for iden- tication of new cancer drug candidates have used cell-free assays for detection of specic interactions with known molecular targets (2). However, there has been a renewed interest in drug screening focused on compound-induced phenotypic changes in live cells (3, 4). Monolayer two-dimensional (2D) cultures of human tumor cell lines have been the predominant models in these efforts. However, these models do not mimic the complex patho- physiological conditions present in solid tumors (5, 6). Moreover, most of the standard chemotherapy drugs identied by using 2D models target proliferating cells. In recent years, it has been shown that dormant cells, residing in areas far from blood vessels are, at least partially, responsible for cancer drug treatment failure (7). These cells are highly resistant to standard cytotoxic compounds and, due to poor drug penetration, are also insufciently exposed to the treatment (7). Therefore, tumor cells grown in three-dimensions (3D) have been suggested to provide a more clinically relevant model. The multicellular tumor spheroid (MCTS) is one such 3D model. MCTS are known to closely simulate the tumor microenviron- ment with respect to glucose, oxygen, and lactate distribution, resulting in gene expression and phenotypic changes similar to those observed in vivo (5, 810). MCTS not only simulate the harsh conditions present in poorly vascularized tumors but also facilitate simultaneous evaluation of the penetration properties of investigated compounds (11, 12). Moreover, if uniform and equal in size, MTCS can be used for drug activity comparisons (8, 13). We have previously shown that compounds identied in 1 Department of Medical Sciences, Division of Cancer Pharmacology and Computational Medicine, Uppsala University, Uppsala, Sweden. 2 Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden. 3 Adlego Biomedical AB, Stockholm, Sweden. 4 Department of Radiology, Oncology and Radiation Sciences, Division of Oncology Uppsala University, Uppsala, Sweden. 5 Department of Medical and Health Sciences, Linkoping University, Linkoping, Sweden. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Ma rten Fryknas, Uppsala University, Klinisk Farmako- logi, ing 61 4tr, Dag Hammarskjolds Vag 18, 751 85 Uppsala, Sweden. Phone: 46 186112931; Fax: 46 18519237; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-14-0792 Ó2015 American Association for Cancer Research. 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  • Models and Technologies

    Three-Dimensional Cell Culture-Based ScreeningIdentifies the Anthelmintic Drug Nitazoxanide asa Candidate for Treatment of Colorectal CancerWojciech Senkowski1, Xiaonan Zhang2, Maria H€agg Olofsson2, Ruben Isacson3,Urban H€oglund3, Mats Gustafsson1, Peter Nygren4, Stig Linder2,5, Rolf Larsson1, andMa

    �rten Frykn€as1

    Abstract

    Because dormant cancer cells in hypoxic and nutrient-deprivedregions of solid tumors provide a major obstacle to treatment,compounds targeting those cells might have clinical benefits.Here, we describe a high-throughput drug screening approach,using glucose-deprived multicellular tumor spheroids (MCTS)with inner hypoxia, to identify compounds that specificallytarget this cell population. We used a concept of drug reposi-tioning—using known molecules for new indications. This is apromising strategy to identify molecules for rapid clinicaladvancement. By screening 1,600 compounds with documen-ted clinical history, we aimed to identify candidates withunforeseen potential for repositioning as anticancer drugs. Ourscreen identified five molecules with pronounced MCTS-selec-tive activity: nitazoxanide, niclosamide, closantel, pyrviniumpamoate, and salinomycin. Herein, we show that all five

    compounds inhibit mitochondrial respiration. This suggeststhat cancer cells in low glucose concentrations depend on oxida-tive phosphorylation rather than solely glycolysis. Importantly,continuous exposure to the compounds was required to achieveeffective treatment. Nitazoxanide, an FDA-approved antiproto-zoal drug with excellent pharmacokinetic and safety profile, is theonly molecule among the screening hits that reaches high plasmaconcentrations persisting for up to a few hours after single oraldose. Nitazoxanide activated the AMPK pathway and downregu-lated c-Myc, mTOR, and Wnt signaling at clinically achievableconcentrations. Nitazoxanide combined with the cytotoxic drugirinotecan showed anticancer activity in vivo. We here report thatthe FDA-approved anthelmintic drug nitazoxanide could be apotential candidate for advancement into cancer clinical trials.MolCancer Ther; 14(6); 1504–16. �2015 AACR.

    IntroductionThe cancer drug development process has become increasingly

    costly and inefficient resulting in few new highly effective drugsreaching the market yearly (1). New strategies for drug discoveryare therefore urgently needed. One such strategy is drug reposi-tioning inwhich anew indication for an existing drug is identified.Using this approach, approved, discontinued, orwithdrawndrugswith unrecognized anticancer activity can be rapidly advancedinto clinical trials, because much of the required documentationalready exists. The availability of compound libraries containingdrugs with documented clinical usemakes unbiased screening forrepositioning candidates an attractive approach.

    During the past decades, most screening approaches for iden-tification of new cancer drug candidates have used cell-free assaysfor detectionof specific interactionswith knownmolecular targets(2). However, there has been a renewed interest in drug screeningfocused on compound-induced phenotypic changes in live cells(3, 4). Monolayer two-dimensional (2D) cultures of humantumor cell lines have been the predominant models in theseefforts. However, these models do not mimic the complex patho-physiological conditions present in solid tumors (5, 6).Moreover,most of the standard chemotherapy drugs identified by using 2Dmodels target proliferating cells. In recent years, it has been shownthat dormant cells, residing in areas far from blood vessels are, atleast partially, responsible for cancer drug treatment failure (7).These cells are highly resistant to standard cytotoxic compoundsand, due to poor drug penetration, are also insufficiently exposedto the treatment (7).

    Therefore, tumor cells grown in three-dimensions (3D) havebeen suggested to provide a more clinically relevant model. Themulticellular tumor spheroid (MCTS) is one such 3D model.MCTS are known to closely simulate the tumor microenviron-ment with respect to glucose, oxygen, and lactate distribution,resulting in gene expression and phenotypic changes similar tothose observed in vivo (5, 8–10). MCTS not only simulate theharsh conditions present in poorly vascularized tumors but alsofacilitate simultaneous evaluation of the penetration propertiesof investigated compounds (11, 12). Moreover, if uniform andequal in size, MTCS can be used for drug activity comparisons(8, 13). We have previously shown that compounds identified in

    1Department of Medical Sciences, Division of Cancer Pharmacologyand Computational Medicine, Uppsala University, Uppsala, Sweden.2DepartmentofOncology-Pathology,Karolinska Institute,Stockholm,Sweden. 3Adlego Biomedical AB, Stockholm, Sweden. 4Departmentof Radiology,Oncology and Radiation Sciences, Division of OncologyUppsala University, Uppsala, Sweden. 5Department of Medical andHealth Sciences, Link€oping University, Link€oping, Sweden.

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

    Corresponding Author: Ma�rten Frykn€as, Uppsala University, Klinisk Farmako-

    logi, ing 61 4tr, Dag Hammarskj€olds V€ag 18, 751 85 Uppsala, Sweden. Phone:46 186112931; Fax: 46 18519237; E-mail: [email protected]

    doi: 10.1158/1535-7163.MCT-14-0792

    �2015 American Association for Cancer Research.

    MolecularCancerTherapeutics

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    on June 14, 2020. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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  • 3D-model screens are distinct from those found using 2Dmodels(14, 15). Thus, tumor cells in 3D models are not necessarilyalways drug resistant, opening a possibility for identification ofmore active cancer drugs if large-scale 3D-based drug screeningcould be pursued.

    There are numerousMCTS formationmethods (14, 16–20) butmost are not suited for high-throughput screening (HTS); they areeither costly, difficult to handle in a large scale or the spheroidsoften vary in size and number. Thus, MCTS-based screeningefforts performed so far have used small drug libraries, notexceeding a few hundred compounds (14, 15, 17, 21).

    Here, we present a novel MCTS model system and illustrate itssuitability forHTS.We applied themodel for screening a library of1,600 clinically tested compounds, making hit compounds suit-able for drug repositioning. By comparing the activity in ourMCTS model with that in 2D cultures, compounds with 3D-specific activity were identified and further evaluated in a 3D-based clonogenic assay. Thefivemost activehits identifiedwere allcompounds reported to target mitochondria, supporting ourprevious finding that respiration is an attractive target in solidtumors (22).

    Among the hit compounds nitazoxanide, an FDA-approvedantiprotozoal drug with an excellent pharmacokinetic and safetyprofile, was selected for further evaluation and demonstratedstrong antitumor activity in vivo when combined with a standardchemotherapeutic agent.

    Materials and MethodsReagents

    Antibodies to 4EBP-1 (#9452), phospho-4EBP-1 (#9459), p70-S6K (#9202), phospho-p70-S6K (#9205), AMP-activated proteinkinase (AMPK; #2532), phospho-AMPK (#4188), caspase-3(#9664S), and Wnt Signaling Antibody Sampler Kit (#2915) werepurchased fromCell Signaling Technology.Antibody to c-Myc (#sc-40) was purchased from Santa Cruz Biotechnology. Antibody tob-actin (#A5316) was purchased from Sigma-Aldrich. AntibodiestoKi-67(#IR626)andCD44 (#M7082)werepurchased fromDakoSweden AB. Pimonidazole staining kit was purchased fromHypox-yprobe. JC-1 was purchased from Sigma-Aldrich. Pharmakon1600 (1,600 compounds) was purchased from MicroSource Dis-covery Systems Inc. Hit compounds: closantel, niclosamide, nita-zoxanide, pyrvinium pamoate, and salinomycin; and standardcytotoxic compounds: doxorubicin andoxaliplatinwere purchasedfrom Sigma-Aldrich. Tizoxanide was purchased from CaymanChemicals. All compounds were dissolved in DMSO. DMSOconcentration incell cultureduring screeningdidnot exceed0.32%.

    Cell cultureHCT116 GFP and HT-29 GFP (human epithelial colon carci-

    noma cell lines constitutively expressing green fluorescent pro-tein) were purchased from Anticancer Inc. in 2009 and 2014,respectively. HCT116 andHT-29 cell lineswere obtained from theATCC in 2009. The cell banks performed authentications by shorttandem repeat analysis. No further authentication was performedin our laboratory. All experiments with purchased cell lines wereperformedwithin 6month after resuscitation. Cells were culturedin McCoy's 5A Modified Medium (Sigma-Aldrich) þ v/v 10%inactivated fetal calf serum, antibiotics (streptomycin 50 mg/mLand penicillin 60 mg/mL) and 2 mmol/L L-glutamine at 37�C in5% CO2.

    Spheroid formationSpheroids were formed from HCT116 GFP or HT-29 GFP cells

    for 7 days without medium change. In 50 mL of fresh medium,10,000 cells per well were plated into 384-well F-bottom UltraLow Attachment plates (Corning) using Biomek 2000 (BeckmanCoulter). Todecrease liquid evaporation, plateswere coveredwithhumidified MicroClime Environmental Microplate Lids (Lab-cyte). To induce cell aggregation, plates were placed in a position,in which corner P1 (bottom-left) was located lower than othercorners of the plate. Plates were incubated in this "tilted" positionin 37�C, humidified atmosphere containing 5% CO2 for 3 hours.Subsequently, plates were placed on the laboratory rocker (Vari-Mix Platform Rocker; Thermo Scientific), laying on the P1-P24edge in a way that angle between rocker's shelf and plate's bottomwas around 85�. The rocker was set to the following settings: 3hours stationary, 15-minute rocking at a speed 5 rpm and max-imum rocking angle 48�. The plates were incubated on the rockerin 37�C for 4 days. After this, they were incubated in 37�C for 3days in a "tilted" position.

    Immunological stainingFollowing pimonidazole (200 mmol/L for 1 hour, for pimo-

    nidazole adducts stainings) treatment spheroids were washedwith PBS, fixed with 4% formalin in PBS, dehydrated with 70%ethanol, embedded in paraffin, and sectioned. REAL EnVisionDetection System (Dako, K5007) was used to visualize thetarget antigen. The sections were deparaffinized and micro-waved in Tris–EDTA buffer (pH 9.0) or Citrate buffer (pH6.0) to unmask the epitopes. Sections were incubated for 5minutes in peroxidaze blocking solution (Dako, S2023). Fol-lowing antibody dilutions were used: pimonidazole (1:50), Ki-67 (RTU), caspase-3 (1:100), and CD44 (1:100). After 30-minute incubation with the primary antibody at room tem-perature, the sections were washed and incubated with DakoREAL EnVision/HRP for 20 minutes, washed, and incubatedwith DAB for 10 minutes. The sections were counterstainedwith hematoxylin for 5 minutes.

    Drug screeningPharmakon 1600 drugs were transferred to spheroid plates

    using Echo Liquid Handler 550 (Labcyte), resulting in final drugconcentration of 20 mmol/L (for plates' layouts, see Supplemen-tary Materials and Methods). On days 2 to 7 of drug incubation,mean spheroid GFP fluorescence intensity was measured everyday using ArrayScan VTI Reader (Cellomics Inc.). Images wereacquired for green fluorescence and bright-field channels using5� objective and suitable filters. The average pixel intensity ofeach spheroid was quantified using the BioApplication Morphol-ogy Explorer (Cellomics Inc.). One image per well was acquired.As a secondary spheroid viability assessment method, resazurin-based TOX8 assay was performed on day 7. TOX8 solution (5 mL)was added to each well. The plate was incubated in 37�C for 4hours and then resorufin fluorescence wasmeasuredwith FLUOs-tar OPTIMA plate reader (BMG Labtech), with excitation/emis-sion filter settings 544 nm/590 nm. A compoundwas defined as ahit in GFP-based viability assay when mean spheroid GFP fluo-rescence intensity from the well containing this compound waslower than mean GFP fluorescence intensity of negative controlsby at least 3 standard deviations (SD) of negative controls' meanfluorescence intensity. A compound was defined as a hit in TOX8assay when the resorufin fluorescence intensity from the well

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  • containing this compoundwas lower than 50%ofmean resorufinfluorescence intensity of negative controls.

    For monolayer experiments, 2,500 HCT116 cells per well wereplated in 50 mL of freshmedium into 384-well cell-culture treatedplates (Nunc) and cultivated for 24 hours in 37�C before drugaddition. Screening plates' layouts were identical as in spheroidplates. Final drug concentration used was 10 mmol/L. In mono-layer experiments, cell viability was assessed after 72 hours ofincubation with drugs using fluorescence microculture cytotox-icity assay (FMCA), as previously described (23).

    Screening evaluationFor the evaluation of the assay, one spheroid plate was pre-

    pared. One hundred and fifty-two wells were treated with 20mmol/L clofazimine for 72 hours. Viability measurements wereperformed at identical time points and using the samemethods asin screening. Z-factor was calculated as recommended previously(24).

    Hit validationOne hundred and sixty two spheroid-active compounds were

    screened against spheroids in duplicates at five different concen-trations from range 2 to 32 mmol/L. Then, 41 most active com-pounds were tested in spheroid or monolayer cultures in tripli-cates at 10 different concentrations from range 0.5 to 32 mmol/L.GFP-based and TOX8 readout methods were used for viabilityevaluation in spheroids and FMCA was used for monolayercultures. Twelve 3D-selective compounds (with 3D-based IC50slower than 2D-based IC50s after 3 days of drug treatment) werechosen for further evaluation. For 3D-selective activity visualiza-tion, spheroids or monolayer HCT116 GFP cell cultures weretreated with 10 mmol/L nitazoxanide or 15 mmol/L oxaliplatin for72 hours. Every 24-hour images for 3D and 2D cultures wereacquired using ArrayScan VTI Reader (Cellomics) or IncuCyte FLR(Essen BioScience), respectively.

    Clonogenic assaySpheroids were treated with drugs for 24 to 72 hours, depend-

    ing on a particular experiment. Following the treatment, spher-oids were washed with PBS and centrifuged. Supernatant wasaspirated and 50 mL of AccuMax (PAA Laboratories GmbH) wasadded. After 30-minute incubation in 37�C, spheroids werepipetted vigorously 30 times to obtain single-cell suspensions.Ten microliters or all (for Supplementary Fig. S5) of each sus-pension was added to 3 mL of fresh medium, mixed and platedinto 6-well (35 mm diameter) Nunclon Surface plates (Nunc).Following seeding, plates were incubated in 37�C for 10 days.Then, colonies were washed with PBS, preserved with methanol,stained with 5%Giemsa dye in PBS and counted. Wells too denseto count were assumed to contain over 200 colonies. Each drugincubation variant was performed in triplicate.

    Measurements of oxygen consumptionThe Seahorse XF analyzer was used as indicated by the man-

    ufacturer (Seahorse Bioscience). Seventy thousand HCT116 cellsper well in 100 mL culture medium were plated in XF24-platecontaining blank controls. Before the measurements, mediumwas replaced with 500 mL of Seahorse assay media (1 mmol/Lpyruvate, 25 mmol/L glucose, pH 7.4) at 37�C without CO2 for 1hour. Oxygen consumption rate (OCR) values were measured byXF24 Extracellular Flux Analyzer. Oligomycin and FCCP wereused at 1 or 0.5 mmol/L, respectively.

    JC-1 staining for polarization state ofmitochondrialmembranein living cells

    Twenty-five hundred HCT116 cells per well were plated in 50mL of fresh medium into optical bottom, black 384-well cell-culture–treated plates (Nunc) and cultivated for 48 hours in 37�Cbefore drug addition. Following 2-hour drug treatment, 35 mL ofmedium was aspirated and warm staining solution added (con-taining Hoechst 33342 for staining nuclei and JC-1 in PBS). Finalconcentration of JC-1 was 2.5 mg/mL. The plate was incubated in37�C for 20 minutes, washed three times with PBS, and readimmediately in Cellomics ArrayScan VTI Reader. Images wereacquired for blue (Hoechst 33342) and red (JC-1) fluorescencechannels using appropriate filters and 20� objective. The averagepixel intensities from detected cytoplasmic spots (mitochondria)were quantified using the BioApplication Spot Detector (Cello-mics Inc.). At least 1,000 cells per well were analyzed and at least 7wells per condition were measured.

    Mitochondria recovery experimentHCT116 cells were treated with 17 mmol/L nitazoxanide. Fol-

    lowing treatment, basal OCR was measured with Seahorse XFanalyzer after 24, 72, 120, or 168 hours of incubation in drug-freemedium (cell numberwas assessed before eachmeasurement andcells were split when necessary).

    Drug exposure during glucose starvationTwenty-five hundred HCT116 cells per well were plated in

    50 mL of fresh media (DMEM containing 10% inactivated fetalcalf serum, streptomycin 50 mg/mL, penicillin 60 mg/mL, and2 mmol/L L-glutamine) with or without glucose into 384-wellcell-culture–treated plates (Nunc) and cultivated for 24 hours in37�C before drug addition. Final hit compounds were added intriplicates. Cell viability was assessed after 72-hour drug incuba-tion using FMCA.

    Western blottingSpheroids were treated with nitazoxanide or tizoxanide at five

    concentrations from range 0.1 to 10 mmol/L for 24 hours. Aftertreatment, spheroids were washed with PBS and kept in �80�Cuntil further handling. For one experiment, at least 16 spheroidsfor each drug concentration were used. Western blotting was thenperformed as described previously (22). Primary antibodies wereused at the following dilutions: b-actin (1:10,000), C-myc(1:1,000), AMPK (1:1,000), phospho-AMPK (1:1,000), p70(1:1,000), phospho-p70 (1:1,000), 4EBP1 (1:1,000), phospho-4EBP1 (1:1,000), and Wnt kit antibodies (1:1,000). Horseradishperoxidase (HRP)–conjugated anti-rabbit and anti-mouse anti-bodies were used at 1:5,000.

    In vivo experimentsSeven- to 9-week-old female NMRI nu/nu mice (Crl:NMRI-

    Foxn1nu; Charles River) were housed under standard conditions.One hundred microliters of suspension containing 5 � 106HCT116-GFP cells in culturing medium was injected into theright rear flank of each animal. When tumors reached a volume of>100 mm3, animals were randomized into control (treated withvehicle) or treatment (nitazoxanide, irinotecan, or combination)groups. Nitazoxanide (200 mg/kg; OnTarget Chemistry) wasadministered by gavage in 5 mL of 1% CMC in PBS/kg twicedaily (once daily during weekends) for 28 to 30 days. Irinotecan(40mg/kg; Actavis) was injected intraperitoneally in 10 mL/kg of

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  • 0.9%NaCl once weekly, starting on day 3. Caliper measurementsof tumor volume were performed every third day. Animals sacri-ficed before day 28 were not included in the results analysis.Nonrepresentative animals were excluded on the basis of tumorobservations (for details seeResults) or outlier test (tumors,whichvolume was belowQ1-1.5IQR or above Q3þ1.5IQR; one animalremoved from combination group). On days 28 to 30, the GFPfluorescence of the xenograft tumors was measured with a CCDcamera (IVIS, Spectrum, Caliper Life Sciences) and the imageswere analyzed for radiant efficiency in Living Image 4.2. After this,animals were terminated, tumors dissected out andweighted. Theexperiments were performed with approval of local ethical com-mittee Stockholm North (N447/12).

    Statistical analysisA one-sided unpaired t test for in vivo data analysis and Pearson

    correlation were performed using Prism software.

    ResultsScreening clinically used compounds for repositioning as solidtumor therapeutics

    The aim of this study was to evaluate whether clinically usedsubstances show unforeseen anticancer activity in an in vitro 3Dsolid tumor model. The 3D model was developed to mimic themicroenvironment in the deep parenchyma of solid tumors withrespect to hypoxia, nutrient deprivation, and low pH. MCTS wereformed in 384-well plates using GFP-labeled HCT116 coloncancer cells. They were cultivated without medium change for7 days prior to drug exposure. This is a significant variation fromconventional spheroid formation protocols, in which freshmedi-um is added during the incubation period. During the 7-day-longculture period, glucose concentration in the culture mediumdropped from 15.5 to 4.66 mmol/L and further down to 2.90mmol/L after 3 additional days (drug exposure). CorrespondingmediumpHdecreased from7.47 to 7.04 at day 7 and to 6.78 after10 days (Supplementary Fig. S1). These values closely correspondto those reported for hypoxic regions in solid tumors (25).

    The procedure resulted in the formation of approximately500 mm � MCTS with hypoxic cores (pimonidazole staining),low proliferation rates (Ki-67 staining), and central regionswith increased apoptosis (active caspase-3 staining), as shownin Fig. 1A. GFP fluorescence is, as previously reported (19), asimple, reliable, and noninvasive surrogate marker of spheroidviability, well suited for read-out in drug screening (Fig. 1B–D) aswell as dose-response and time-course experiments (Supplemen-tary Fig. S2). As a secondary read-out forMCTS viability, we used aresazurin-based assay, TOX8, previously shown suitable for suchmeasurements (20). Results obtained with these two assaysshowed a high degree of concordance (Supplementary Fig. S3).The TOX8 assay made it possible to include hits that would havebeen missed if only GFP-signal was used for hit selection, such aspyrvinium pamoate (due to their auto-fluorescence in GFP emis-sion spectrum; Supplementary Fig. S3). Both assays performedwell with Z0-factors > 0.5, as calculated according to Zhang andcolleagues (24).

    We screened the collection of 1,600 clinically active com-pounds on MCTS, followed by hit-validation (See Fig. 1F for aschematic overview and Materials and Methods for details). Inparallel, we screened this library using HCT116 cells [wild-type,previously shown to respond to treatment identically as GFP-

    labeled HCT116 cells (19)] grown as 2D monolayer cultures.Comparison of the results from the 3D- and 2D-based screensshowed that most clinically used cytotoxic drugs have onlymodest efficacy in spheroids (Fig. 1E), in agreementwith previousstudies (14, 19). Clinically used cytotoxic drugs target proliferat-ing cells, which can explain their lack of efficacy inMCTS, inwhichcell proliferation is low (Fig. 1A).

    Identification of compounds with preferential activity in MCTSA comparison of the results of the 2D and 3D drug screens (Fig.

    1E) indicated the existence of compounds with selective activitytoward spheroids. Forty-one compounds were selected on thebasis of activity in the 3D-model. To validate the results, weperformed extensive dose–response experiments using both 3Dand 2D cultures. These experiments resulted in the identificationof 12 compounds with preferential activity toward the 3D model(Supplementary Table S1). The 3D-selectivity of one of these,nitazoxanide, is presented in Fig. 2A and C. The 3D-selectiveactivity of nitazoxanide could be observed as spheroids lostviability (decreased GFP signal), at a concentration at which2D-cultured cells were actively proliferating. This pattern was insharp contrast with that of mitomycin C (Fig. 2B) and oxaliplatin(Fig. 2C).

    The 12 3D-selective compounds were subsequently challengedin a spheroid-based clonogenic assay in order to select com-pounds with specificity toward the dormant hypoxic and nutri-ent-deprived cancer cells in the MCTS. Thus, to focus on 3D-selectivity rather than absolute potency, spheroids were exposedto the compounds at their respective 2D IC50 concentrations.Followingwashout of the drugs and spheroid dispersal, single-cellsuspensions were seeded in fresh medium into 6-well culturingplates and left for 10 days to regrow. Five of the compounds,closantel, nitazoxanide, niclosamide, pyrvinium pamoate, andsalinomycin abolished colony formation (Fig. 2D).Moreover, thepreferential activity toward the 3D model was not limited to theHCT116 cell line. Similar results were observed when the com-pounds were tested in the colon carcinoma cell line HT-29(Supplementary Fig. S4). Interestingly, the five final hit com-pounds were not as active in HCT116 and HT-29 spheroids thatwere formed with addition of fresh medium during the cultureperiod (Supplementary Fig. S5). Therefore, if a standard spheroidmodel rather than our medium-stressed version had been used,these compounds would most likely not have been identified as3D-selective.

    3D-selective agents inhibit mitochondrial oxidativephosphorylation

    Because the five final 3D-selective molecules have knownmechanisms of action, we had a possibility to identify cellulartargets that confer this context-dependent vulnerability. A litera-ture review (Supplementary Table S2) revealed that all final hitcompounds target mitochondria and oxidative phosphorylation(OXPHOS), albeit by different mechanisms. Niclosamide, clo-santel, and nitazoxanide [which is rapidly converted to its activemetabolite tizoxanide (26)] are all anthelmintic compoundsthat cause uncoupling of mitochondrial membrane potential(26–31). In fact, these compounds share an identical pharmaco-phore (highlighted in red; Supplementary Table S2). Pyrviniumpamoate, also an anthelmintic drug, targets the fumarate-reduc-tase systemandhasbeenpreviously recognized for its high activityagainst spheroids and glucose-deprived cells (32, 33). The

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  • ionophore salinomycin, which has recently gained attention forits activity against cancer stemcells (CSC), has also been identifiedas an OXPHOS inhibitor (34).

    Because inhibition ofmitochondrial respiration was seeminglya common denominator among the final hit compounds, wecharacterized their effects on oxygen consumption andmitochon-drial function. The compounds were assessed at concentrations

    up to their 2D IC50s for their effects on OCR in monolayerHCT116 cells, using a known uncoupler of mitochondrial mem-brane potential (FCCP) as a positive control. Hit compoundsreported to be uncouplers, that is, nitazoxanide, niclosamide, andclosantel as well as FCCP increased OCR at low concentrations(Fig. 3A–D). However, as the concentration increased, the periodof elevated OCR became shorter and was followed by shutdown

    Control

    Treated

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    Figure 1.Assay characteristics and experimental design. A, phase-contrast microphotographs of MCTS formed for 7 days following seeding of 10,000 HCT116 GFP coloncarcinoma cells. Immunohistochemistry stainings show spheroid structure (hematoxylin); core hypoxia (staining for pimonidazole adducts formed in hypoxicconditions); limited proliferation in the MCTS core (Ki-67) and central apoptosis (caspase-3). Scale bar, 250 mm. B, bright-field/fluorescence (composite) images ofuntreated and treated (20 mmol/L clofazimine, 72-hour treatment) MCTS, 10 days after seeding. Scale bar, 500 mm. C, bright-field/fluorescence (composite)overview of a 384-well plate after a scan with an automated fluorescence microscope. Spheroids were treated with 10 mmol/L clofazimine (black in the image)or left untreated (green). Image acquisition settings were identical for each well. D, mean GFP fluorescence intensity of each spheroid from the platepresented in C was quantified and plotted. Z-factor for the experiment was 0.79, indicating an excellent performance and reproducibility of the screeningassay. E, results after testing 1,600 compounds inmonolayer cultures (2D) andMCTS (3D). Read-out in themonolayer experimentswas thefluorometricmicroculturecytotoxicity assay (FMCA) after 72 hours of continuous exposure to the compounds at 10 mmol/L (horizontal axis). Read-out in the MCTS experimentswasmean spheroidGFPfluorescence intensity (seeMaterials andMethods for details) after 72 hours of continuous exposure to the compounds at 20mmol/L (verticalaxis). Clinically used cytotoxic drugs are highlighted in yellow. F, schematic overview of the screening procedure and selection criteria that led to theselection of nitazoxanide as the final candidate for drug repurposing.

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  • of mitochondrial respiration, indicated by a rapid decrease ofOCR. Importantly, these effects were observed for the uncouplersbelow their 2D IC50 concentrations. When even higher concen-trations of the uncoupling hit compounds or FCCP were used,increase in OCR could not be detected. At drug concentrationssufficient to cause a complete shutdown of mitochondrial respi-ration, late addition of FCCP caused no increase in OCR (Fig. 3B–D, highlighted with circle).

    The effect of the remaining two hit compounds, pyrviniumpamoate and salinomycin, on OCR was different from what wasobserved for the uncouplers. Pyrvinium pamoate at the 2D IC50concentration (1.5 mmol/L) resulted in an immediate decrease ofOCR (Fig. 3E). This effect was weaker at lower concentrations andabsent at 0.1 mmol/L. An increase in OCR, as caused by uncou-plers, was not observed. Salinomycin at 1 mmol/L caused a slightincrease in OCR (Fig. 3F). However, inhibition of OCR bysalinomycin used at its 2D IC50 concentration (5 mmol/L) wasweaker than the inhibition caused by corresponding concentra-tions of uncouplers. Moreover, late addition of FCCP induced anincrease in OCR (Fig. 3F), opposite to the effects observed afterexposure to high concentrations of the uncouplers.

    We confirmed these results with JC-1 staining, a probedetecting polarization state of mitochondrial membrane. At 2DIC50 concentrations 2-hour exposure to uncouplers or pyrvi-nium pamoate resulted in the complete depolarization ofmitochondrial membrane (Fig. 3G). In contrast, exposure tosalinomycin increased the amount of red fluorescent aggregateswithin mitochondria, indicating their extreme hyperpolariza-

    tion (Fig. 3H). This effect of salinomycin has been previouslyreported (35).

    Monolayer-based characterization of mitochondrial dysfunc-tion was in agreement with time-dependent changes in pimo-nidazole staining of MCTS. After 4 or 24 hours of exposure tothe hit compounds or the uncoupler CCCP (positive control),spheroids were exposed to pimonidazole and stained forpimonidazole adducts. Four-hour exposure to the compoundsthat stimulated OCR in 2D culture (nitazoxanide, niclosamide,closantel, and salinomycin) increased the hypoxic area withinspheroids (Fig. 3H), which is consistent with elevated con-sumption of oxygen in 2D cultures (Fig. 3A–D, and F). A similareffect was observed in spheroids treated with CCCP. In contrast,exposure to pyrvinium pamoate decreased the hypoxic area inspheroids (Fig. 3H), which is in accordance with an immediatedecrease of OCR in 2D cultures (Fig. 3E). After 24-hour expo-sure to uncouplers, hypoxic areas in spheroids disappeared ordecreased substantially, indicating shutdown of mitochondrialrespiration (Fig. 3H).

    Continuous drug exposure is required for cytotoxic activity inMCTS

    After 24-hour exposure to the uncouplers, we were still able toretrieve intact spheroids, whereas exposure to salinomycin orpyrviniumpamoate for this period resulted in the loss of spheroidintegrity, suggesting earlier cell death (Fig. 3H). Therefore, weexamined the clonogenicity of dissociated spheroids in relation totimeof exposure to thefivefinal hit compounds. Continuousdrug

    Figure 2.Selection of 3D-specific hit compounds. A andB, dose–response curves for HCT116 (2,500 cells/well grown asmonolayer for 24-hour prior treatment; 2D) andHCT116GFP (10,000 cells/well grown as spheroids for 7 days prior treatment; 3D) cells exposed to nitazoxanide (A) or mitomycin (B) for 72 hours. Cell viability wasassessedwith FMCA (2D) ormeasurements ofmean spheroid GFPfluorescence intensity (3D). Results are shownasmean�SD (n¼ 3). C, comparisonof drug effectson HCT116 GFP cells in 2D and 3D cultures. Concentrations used: nitazoxanide, 10 mmol/L; oxaliplatin, 15 mmol/L. Pictures were acquired using ArrayScan VTI (3D,identical settings for all pictures) or IncuCyte FLR (2D, identical settings for all pictures) for the samewell in each treatment group. Pictures are composite images offluorescence and brightfield (3D)/phase-contrast (2D) channels. Scale bars, 500 mm. D, clonogenicity of cells from dissociated HCT116 GFP spheroids after72-hour exposure to screening hit compounds or standard cytotoxic drugs at concentrations equal to 2D IC50 values. Concentrations used: closantel, 25 mmol/L;niclosamide, 1.5 mmol/L; nitazoxanide, 17 mmol/L; salinomycin, 5 mmol/L; pyrvinium pamoate, 1.5 mmol/L; doxorubicin, 1.5 mmol/L; oxaliplatin, 15 mmol/L. Outgrowthtime was 10 days.

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  • 4 h

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    Figure 3.Characterization of effects of 3D-selective hit compounds on mitochondrial respiration. A–F, effects of FCCP (A), nitazoxanide (B), niclosamide (C), closantel(D), pyrvinium pamoate (E), and salinomycin (F) at various concentrations on OCR in 70,000 HCT116 cells, as measured by Seahorse XF analyzer. Loss ofstimulation of OCR by addition of FCCP after uncoupler-induced mitochondrial respiration shutdown is highlighted with orange circles (B–D). Final hitcompounds, oligomycin or FCCP, were added as indicated with dotted lines. Results are shown as mean � SD; (n ¼ 3). G, left, effects of the final hitcompounds at concentrations equal to their 2D IC50 values and CCCP (2.5 mmol/L) on mitochondrial membrane potential in HCT116 cells (2,500/well).Results in the graph are shown as means of JC-1 aggregates fluorescence per cell þ SD; (n � 7). Right, composite pictures from Cellomics Arrayscan VTIReader of treated HCT116 cells. Cell nuclei were stained with Hoechst 33342 and polarized mitochondria were stained with JC-1 probe. All pictureswere acquired using identical settings. Magnification used was �20. H, effects of the final hit compounds or CCCP on hypoxia within HCT116 GFPspheroids. Spheroids were formed with 10,000 cells per well for 7 days without medium change and treated with CCCP (2.5 mmol/L), nitazoxanide (3 mmol/L),niclosamide (1 mmol/L), closantel (15 mmol/L), pyrvinium pamoate (1 mmol/L), or salinomycin (2 mmol/L) for 4 or 24 hours. Spheroids were treated withpimonidazole, sectioned, and hypoxia was visualized by staining for pimonidazole adducts. Scale bar, 250 mm.

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  • exposure for 48 hours was required to achieve effective loss ofclonogenicity using mitochondrial uncouplers, whereas pyrvi-nium pamoate and salinomycin induced an earlier loss of clo-nogenicity (Fig. 4A and B). This demonstrates that cells in com-promised microenvironments can survive with impaired mito-chondrial respiration for a limited period and that mitochondriaare able to recover from exposure to the tested OXPHOS inhibi-tors. The latter was evident from assessment of OCR in drug-freemedium following exposure of HCT116 to nitazoxanide for 24hours. OCR gradually increased after removal of the drug andreturned to normal levels after 7 days (Fig. 4C). Thus, continuousdrug exposure is required to eliminate tumor regrowth potential.This is an important finding considering an optimal treatmentschedule for compounds targeting mitochondria.

    Nitazoxanide downregulates cancer signaling pathwaysIn addition to oxygen availability, glucose is an established

    limiting factor for dormant cells in MCTS (21, 22). Cellsexperiencing low nutrient concentrations would be particularlydependent on mitochondrial respiration in order to meet theirenergy demands. We therefore tested the influence of glucosestarvation on the response to the final hit compounds.MonolayerHCT116 cells cultured in medium without glucose were moresensitive to treatment with all five hit compounds than whencultured under standard glucose conditions (Fig. 5A–E). Con-versely, higher survival rates were observed for cells treated withdoxorubicin in no-glucose conditions (Fig. 5F), presumablybecause of their lower proliferation rate during glucose starvation,rendering the cells less sensitive to the DNA-damaging agent.Thus, maintaining functional mitochondrial respiration seemscritical for survival in glucose-deprived conditions.

    According to our findings, efficient eradication of cancer cells inglucose-deprived conditions through inhibition ofmitochondrial

    respiration relies on continuous drug exposure for a sustainedperiod of time. Of the final five hit compounds identified in thescreen, nitazoxanide stands out as the only one that, withoutmajor side effects, reaches high systemic concentrations after oraladministration (Supplementary Table S2; refs. 36, 37). Therefore,nitazoxanide was selected as the drug with the highest reposition-ing potential. Importantly, we verified that its active metabolitetizoxanide (which is the only species detectable in plasma afteroral administration of nitazoxanide) was as active as the parentaldrug in vitro (Supplementary Fig. S6).

    Our results suggested that impairment of mitochondrial func-tion under conditions of nutrient starvation leads to an energycatastrophe and cell death. Energy stress is a trigger for activationof AMPK. Therefore, we examined whether treatment with nita-zoxanide would result in the phosphorylation of AMPK in spher-oids. Indeed, after 24-hour exposure of HCT116 spheroids tonitazoxanide, increased levels of phosphorylated AMPK could beobserved (Fig. 5G), suggesting an increase in AMP:ATP ratio. Thisincrease was observed at 0.5 mmol/L, and was slightly weaker athigher concentrations.

    AMPK activation is associated with inhibition of mammaliantarget of rapamycin (mTOR), which is one of the downstreamtargets of AMPK (38). Twenty-four-hour exposure to nitazoxanideresulted in dose-dependent downregulation of phosphorylated4EBP1 and p70 in HCT116 spheroids (Fig. 5H), indicatinginhibition of mTOR pathway. Because treatment with nitazox-anide causes energetic stress in cancer cells, we predicted it to alsoindirectly downregulate other oncogenic pathways. Indeed, treat-ment with nitazoxanide caused a decrease in c-Myc levels andinhibition of Wnt signaling (Supplementary Fig. S6G). Notably,nitazoxanide has been previously reported to strongly reducelevels of c-Myc in cancer cell lines (39). Its inhibitory effects onWnt pathway have not been reported so far.

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    Figure 4.Final hit compounds induce spheroiddeath after prolonged exposure.A, clonogenicity of cells fromdissociated HCT116 GFP spheroidsafter 24-, 48-, or 72-hour exposure toscreening hit compounds ordoxorubicin at concentrations equal to2D IC50 values in comparison withunexposed controls. Outgrowth timewas 10 days. B, number of coloniesin each well from clonogenicityexperiment presented in A wascounted. Wells too dense to countwere assumed to contain over200 colonies. Results are shown asmean þ SD; (n ¼ 3). C, mitochondriaare able to recover from treatmentwith nitazoxanide. After treatmentwith 17 mmol/L nitazoxanide for24 hours, cells were placed in the freshmedium and baseline OCR wasmeasured with Seahorse XF analyzerafter 24, 72, 120, and 168 hours ofincubation without the drug. Resultsare shown as mean OCR valuesnormalized for cell number at eachtime point (presented as percentagesof untreated control value) þ SD;(n ¼ 3).

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  • In vivo activity of nitazoxanide in combination with a standardchemotherapeutic agent

    Because solid tumors in vivo harbor both quiescent and pro-liferating cells, we reasoned that successful treatment would likelyrely on a combination of compounds targeting these distinct cellpopulations. To test this hypothesis, we treated spheroids formedwithmediumchange (i.e., notmedium-stressed), which compriseboth quiescent and proliferating cell populations (Supplemen-tary Fig. S7A), with nitazoxanide. Seventy-two hours of contin-uous treatment did not completely inhibit colony formation

    (Supplementary Fig. S7B and S7C). Exposure to the standardcytotoxic drug irinotecan, which targets proliferating cells, did notresult in a strong inhibition of colony regrowth as well (Supple-mentary Fig. S7B and S7C). However, when both drugs were usedin combination, colony formation was strongly abrogated.These results give a rationale for using nitazoxanide in combina-tion with irinotecan rather than as a single agent for in vivoexperiments.

    Consequently, nitazoxanide and irinotecan were tested in amouse xenograft model. HCT116 GFP cells were implanted into

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    Figure 5.OXPHOS inhibition in glucose-deprived conditions leads to energetic deficit. A–F, glucose starvation sensitizes cells to treatmentwith the final hit compounds. Dose–response curves for HCT116 cells (2,500/well grown as monolayer for 24-hour prior treatment) in DMEM medium with or without glucose, treated with the final hitcompounds (A–E) or doxorubicin (F). Cell viability was assessed with FMCA. Results are shown as mean � SD; (n ¼ 3). G and H, HCT116 GFP spheroids weretreatedwith nitazoxanide for 24hours at concentrations indicated and analyzed for phospho-AMPK,AMPK (G) or phospho-4EBP1, 4EBP1, phospho-p70, p70 (H) andb-actin by Western blotting. The results are representative of at least three independent experiments.

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  • NMRI nu/nu mice. After tumor establishment (volume >100mm3), mice were exposed to either vehicle control, nitazoxanide(gavage, 200 mg/kg twice daily), irinotecan (intraperitonealinjection, 40 mg/kg once weekly), or a combination of the twocompounds. Tumor volumes were monitored for 28 days fol-lowed by tumor dissection for assessment of tumor weight.Nitazoxanide alone did not cause inhibition of tumor growth(Fig. 6A–D), reminiscent of the poor effect of the drug in prolif-erating cells in 2D culture (Fig. 2A). Consequently, the lack ofeffect of nitazoxanide as a single agentmay be due to the rapid cellproliferation in xenograft models, with volume doubling times ofjust a few days. This could indicate that a substantial fraction ofcancer cells in these models has a good access to glucose. Thissituation may not apply to slow-growing tumors in humans, inwhich doubling times can be often measured in months ratherthan days.

    The topoisomerase 1 inhibitor irinotecan, expected to targetproliferating cells, produced strong tumor growth inhibition(Fig. 6A–D). Nitazoxanide potentiated the effect of irinotecancausing a significant reduction in tumor growth comparedwith irinotecan alone (P ¼ 0.030; Fig. 6A). Similar resultswere observed after analysis of tumor weight after dissection(P ¼ 0.068; Fig. 6B) and GFP fluorescence of tumors in vivo(P ¼ 0.091; Fig. 6C and D). Interestingly, in the combinationgroup, we observed late-onset, sudden apparent volumeincrease of tumors in two individuals, which both showed verylow GFP fluorescence intensity, indicating low viable cell num-ber (Fig. 6D, framed). When dissected, one of the tumors wasfilled with viscous fluid. The other, after rapid volume increase,was ruptured and decreased from 0.73 to 0.49 mL in 2 days.Thus, the animal had to be sacrificed at that point due to ethicalreasons and the full treatment schedule could not be complet-ed. This phenomenon was exclusively observed in the combi-nation group. Because of the tumors being not representative,these animals were excluded from data analysis. However, theseobservations could indicate even more pronounced anticanceractivity of the drug combination in the two individuals. Impor-tantly, nitazoxanide caused no toxicity, such as diarrhea or skinrash, neither did it cause any change in body weight (Fig. 6E).Irinotecan, on the other hand, produced a significant decreasein body weight indicating systemic adverse effects.

    DiscussionIn this work, we present a novel approach for preclinical

    anticancer drug identification, which combines two concepts thathave been recently gaining attention in cancer research. First, high-throughput drug screening was performed using a library con-sisting of drugs that have been tested clinically and in many casesFDA-approved. This presents an opportunity to find moleculessuitable for drug repositioning and rapid advancement intoclinical trials. Second, to identify drugs targeting quiescent cancercells and mimic in vivo tumor microenvironment, we used a newMCTS model well suited for HTS. However, in contrast to mostother spheroid-based approaches, the present method did notinvolve any medium change throughout the whole spheroidculture period. This enabled us to better mimic conditionsobserved in dormant tumor regions in vivo, in terms of glucoseconcentration and pH. Our screening system demonstrated excel-lent reproducibility and capacity to test thousands of compounds.For identification of drugs selectively active against medium-

    stressed spheroids, we challenged screening hits in parallel inspheroid- and monolayer-based dose–response experiments.Interestingly, standard cytotoxic agents were preferentiallyactive against 2D cultures with only minor effects in the 3Dcounterpart while for several other compounds, we observedopposite effects. These results support the notion that cytotoxiccompounds have limited activity in 3D models, but challengethe common opinion that spheroid models are more resistantto chemicals in general.

    Lowoxygen and glucose levels in dormant tumor regions in vivoare believed to result from both poor blood supply and highglucose consumption by cancer cells. It has been established thatrapidly proliferating cancer cells shift from oxidative phosphor-ylation to aerobic glycolysis, using it as a prime pathway to fulfilltheir energy requirements, a phenomenon known as theWarburgeffect (40). However, oxidative phosphorylation has recentlybeen found to be required for cancer cell proliferation under lowglucose conditions (41). Moreover, OXPHOS was reported to behyperactive in epithelial cancer cells in situ (42). This growingbody of evidence indicates that cells inmicroenvironments whereglucose is not abundant would depend on OXPHOS rather thansolely glycolysis to meet their energetic requirements. Thishypothesis is in a good agreement with the presented results. Wefound that compounds most active in 3D cultures targeted mito-chondrial respiration by different mechanisms. This confirmedour and others' recent observations that oxidative phosphoryla-tion is an attractive target for anticancer therapy (22, 43, 44).

    Finally, we identified nitazoxanide as an OXPHOS inhibitorsuitable for direct drug repositioning. It has been approved forhuman use, with a favorable pharmacokinetic profile and verylimited side effects (37).

    Our screen was designed to find drugs with selective activitytoward quiescent tumor regions. Therefore, nitazoxanide was notexpected to show anticancer activity as a single drug in high-proliferative tumor models. This prediction was experimentallyverified, because nitazoxanide alone did not impair xenografttumor growth. However, nitazoxanide potentiated the effect ofirinotecan in vivo. This is an important finding considering repo-sitioning nitazoxanide as an anticancer agent.Our results pave theway for further testing in syngeneic (45) or hetero-/orthotopicpatient-derived xenograft models (46) prior to clinical advance-ment. Consequently, putative clinical trials should focus on usingnitazoxanide in combination with standard chemotherapy. Oneexample for similar approach was the clinical trial, which resultedin approval of bevacizumab (Avastin), the anti-VEGF antibody,which in combination with chemotherapy prolonged mediansurvival of colorectal cancer patients (47). Notably, when used asa single agent, progression-free survival of patients treated withbevacizumab alone was shorter than in patients treated withchemotherapy alone.

    Our results showed that quiescent cancer cells are able tosurvive nitazoxanide-induced inhibition of mitochondrial phos-phorylation and recover from the treatment lasting up to 24hours. Therefore, nitazoxanide treatment regimens would haveto be designed in a way that ensures continuous high plasmaconcentration of the drug. Clinical studies indicate that nitazox-anide is generally well tolerated in single doses up to 4 g (48) andits maximal plasma concentration is reached 2 to 6 hours afteradministration (36). Average maximal plasma concentrationsafter single dose of only 500 mg exceed 6 mmol/L (36), that is,twice the nitazoxanide IC50 concentration in HCT116 spheroids

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  • (3 mmol/L). It may seem surprising that amitochondrial functioninhibitor with good systemic distribution does not induce severeadverse effects, in particular cardiac or neurological. However,

    another uncoupler of OXPHOS, dinitrophenol (DNP), has beenused with good tolerability for treatment of nutritional disordersin thousands of patients before its withdrawal from the market

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    Figure 6.In vivo activity of nitazoxanide combined with irinotecan in HCT116 GFP xenografts. A, effects of treatment with nitazoxanide (gavage, 100 mg/kg, twice daily),irinotecan (i.v., 40 mg/kg, twice weekly), or combination of both drugs on changes in tumor volume. Left, measurements during the whole study period.Results are shown as mean � SEM; (n ¼ 8–10; � , P < 0.05 vs. irinotecan, t test). Right, each data point represents endpoint (28 days) measurement of an individualtumor. Lines represent mean values for each group. P value was calculated using t test. B, tumor weight after dissection at the end of the study (day 28).Each data point represents an individual tumor. Lines represent mean values for each group.P value was calculated using t test. C, tumor radiant efficiency at the endof the study (day 28). Each data point represents an individual tumor. Lines represent mean values for each group � SEM. P value was calculated using t test.D, tumor GFP fluorescence at the end of the study. Images were acquired using CCD camera and normalized for fluorescence intensity to enable animal-to-animalcomparison. Animals from the combination group highlighted with red rectangle showed nontypical response (for details, see the text) and were notincluded in the quantitative analysis. E, changes in animal body weight during the study period. Lines represent mean values for each group. Significance wasassessed using t test. n.s., not significant.

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  • (49). Acute toxicity of DNP has been reported only when used athighdoses. Conversely, irreversible inhibitors ofOXPHOS such ascyanide or antimycin A cause death within a few minutes. There-fore, reversibility of OXPHOS inhibition seems important fordrug general toxicity.

    Treatment of HCT116 spheroids with nitazoxanide resultedin activation of AMPK pathway and inhibition of mTORpathway, indicating energetic deficit. Importantly, similarmolecular effects of mitochondrial respiration inhibition wererecently observed in drug-resistant ovarian CSC populations(44). One of the final screening hits, salinomycin, has alsobeen found to selectively target breast cancer stem cells (50).This body of evidence, together with the fact that HCT116 cellline consists mainly of CSCs [(51); interestingly, culturingHCT116 cells as spheroids increased the expression of stemcell marker CD44, as previously reported (19) and shown inSupplementary Fig. S8] could indicate a novel strategy fortargeting cancer stem cells, which is in agreement with recentreports (52–54). Taken together, our findings are in accor-dance with the hypothesis on dormant cells being CSCs, anissue that is currently intensively investigated.

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

    Authors' ContributionsConception and design: W. Senkowski, S. Linder, R. Larsson, M. Frykn€asDevelopment of methodology: W. Senkowski, X. Zhang, M. Frykn€as

    Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Senkowski, X. Zhang, M.H. Olofsson, R. Isacson,U. H€oglund, P. Nygren, R. Larsson, M. Frykn€asAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Senkowski, X. Zhang, R. Isacson, M. Gustafsson,P. Nygren, S. Linder, R. Larsson, M. Frykn€asWriting, review, and/or revision of the manuscript: W. Senkowski, X. Zhang,R. Isacson, P. Nygren, S. Linder, R. Larsson, M. Frykn€asAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Zhang, R. Isacson, M. GustafssonStudy supervision: S. Linder, M. Frykn€as

    AcknowledgmentsThe authors thank Paola Pellegrini and Angelo De Milito for help with pH

    measurements and to Jan Siljason for expert immunohistochemistry stainings.Skillful technical assistance of Lena Lenhammar, Christina Leek, and NasrinNajafi is gratefully acknowledged.

    Grant SupportThis study was supported by the Swedish Cancer Society, Swedish Founda-

    tion for Strategic Research, Swedish Research Council and the Lions CancerResearch Fund. P. Nygren was supported by Strategiska Forskningsstiftelsen(SSF) and Lions Cancerforskningsfond; S. Linder by Cancerfonden, Barncan-cerfonden, Radiumhemmets Forskningsfonder, Vetenskapsra

    �det, SSF; R. Lars-

    son by Cancerfonden, SSF, Lions Cancerforskningsfond; and M. Frykn€as byLions Cancerforskningsfond.

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

    Received September 15, 2014; revised April 9, 2015; accepted April 9, 2015;published OnlineFirst April 24, 2015.

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  • 2015;14:1504-1516. Published OnlineFirst April 24, 2015.Mol Cancer Ther Wojciech Senkowski, Xiaonan Zhang, Maria Hägg Olofsson, et al. Colorectal CancerAnthelmintic Drug Nitazoxanide as a Candidate for Treatment of Three-Dimensional Cell Culture-Based Screening Identifies the

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