F-FDG PET/CT for Monitoring the Response of Breast Cancer to mir-143-based Therapeutics by Targeting Tumor Glycolysis

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1 Cittion: Moleulr Therpy Nulei Aids () 5, e57; doi:.8/mtn..7 Offiil journl of the Amerin Soiety of Gene & Cell Therpy F-FDG PET/CT for Monitoring the Response of Brest Cner to -Bsed Therpeutis y Trgeting Tumor Glyolysis Ying Mio, Ling-fei Zhng,, Rui Guo, Sheng Ling, Min Zhng, Shuo Shi, Cheng-fng Shng-Gun, Mo-fng Liu, nd Bio Li Inresed gluose utiliztion is hllmrk of ner, nd tumor metolism is emerging s ntiner trget for therpeuti intervention. Triple-negtive rest ners TNBC re highly glyolyti nd show poor linil outomes. We previously identified hexokinse, the mjor glyolyti enzyme, s trget gene of in TNBC. Here, we developed therpeuti formultion using holesterol-modified gomir enpsulted in neutrl lipid-sed delivery gent tht loked tumor growth nd gluose metolism in TNBC tumor-ering mie when dministered systemilly. The ntionogeni effets were ompnied y redution in the diret trget hexokinse nd [ F]-fluorodeoxygluose ( F-FDG) uptke sed on positron emission tomogrphy/omputed tomogrphy. Tretment with formultion hs miniml toxi effets nd mie tolerted it well. Thus, we demonstrted tht is roust inhiitor of the Wrurg effet nd n effetive therpeuti trget for TNBC. In ddition, F-FDG positron emission tomogrphy/omputed tomogrphy n e used to speifilly monitor the response of TNBC to -sed therpeutis y trgeting tumor glyolysis. Moleulr Therpy Nulei Aids () 5, e57; doi:.8/mtn..7; pulished online August Sujet Ctegory: mirnas, sirnas nd shrnas Introdution Brest ner is the most ommon mlignny in women worldwide nd the leding use of femle ner deth in less developed ountries. Roughly 5 % of ll rest ner ses fll into the tegory of triple-negtive rest ner (TNBC), whih is hrterized y lk of gene expression for the estrogen reeptor, progesterone reeptor, nd humn epiderml growth ftor reeptor (ref. ).Clinil studies hve indited tht ptients with TNBC fter neodjuvnt nd/or djuvnt hemotherpy show signifintly higher reurrene rtes nd worse prognosis, with medin survivl of ~ yer following tretment. Unfortuntely, no genomi trget of proven therpeuti utility ginst TNBC hs een identified, primrily due to the exeptionl gene expression. Metoli reprogrmming is one of the hllmrks of ner ells. 5 Even in the presene of oxygen, tumor ells preferentilly rely on glyolysis, rther thn oxidtive phosphoryltion, for ATP genertion. This metoli nomly is ommonly referred to s the Wrurg effet (eroi glyolysis), whih leds to fster gluose metolism in ner ells nd n e used for tumor visuliztion with [ F]-fluorodeoxygluose ( F-FDG) positron emission tomogrphy/omputed tomogrphy (PET/CT) snning. This method relies on the rpid gluose onsumption of tumor tissue nd hs rod linil pplitions, inluding ssisting in the detetion, stging, nd evlution of prognosis of tumors, s well s monitoring the tumor response to therpy. 7,8 Memers of the hexokinse fmily tlyze the first ommitted nd irreversile step of gluose metolism, whih governs the diretion nd mgnitude of gluose flux. 9 Hexokinse (HK), the mjor isozyme of the hexokinse fmily, shows signifintly elevted expression in mny tumor types ompred with norml ells, suggesting its potentil s metoli trget for ner therpy. MiroRNAs (mirnas) re lss of short (~ nt) endogenous RNAs involved in the modultion of diverse rnge of iologil proesses y regulting the stility nd trnsltion of their trget mrnas.,, whih is downregulted in multiple ners rnging from oloretl to rest ners, ts s n ntionomir. We previously found tht HK is diret trget of, whih my ply n importnt role in tumor defense. However, further studies re neessry to determine whether the therpeuti delivery of to trget tumor tissues n e effiiently hieved nd to monitor its tretment effiy y using noninvsive method. In this study, we demonstrted tht hemilly synthesized mimi suppressed glyolysis y downregulting HK in MDA-MB- ells, s well s regulted the prolifertive, migrtive, nd poptoti ehvior of TNBC ells. Systemi delivery of gomir to TNBC xenogrfts drmtilly redued oth tumor growth nd F-FDG uptke sed on PET/CT. Our results demonstrte tht is roust inhiitor of the Wrurg effet nd Deprtment of Nuler Mediine, Ruijin Hospitl, Shnghi Jiotong University Shool of Mediine, Shnghi, Chin; Center for RNA Reserh, Stte Key Lortory of Moleulr Biology-University of Chinese Ademy of Sienes, Institute of Biohemistry nd Cell Biology, Shnghi Institutes for Biologil Sienes, Chinese Ademy of Sienes, Shnghi, Chin; Shnghi Key Lortory of Moleulr Andrology, Institute of Biohemistry nd Cell Biology, Shnghi Institutes for Biologil Sienes, Chinese Ademy of Sienes, Shnghi, Chin; Deprtment of Nuler Mediine, Xinhu Hospitl, Shnghi Jiotong University Shool of Mediine, Shnghi, Chin. Correspondene: Bio Li, Deprtment of Nuler Mediine, Ruijin Hospitl, Shnghi Jiotong University Shool of Mediine; 97 Ruijin Er Rod, Shnghi 5, Chin. E-mil: l@rjh.om.n. Reeived 5 July ; epted July ; pulished online August. doi:.8/mtn..7

2 F-FDG PET/CT to Monitor Response of Brest Cner to -Bsed Therpeutis promising therpeuti trget for TNBC tretment. In ddition, F-FDG PET/CT exhiits gret potentil for speifilly monitoring the response of TNBC to -sed therpeutis y trgeting tumor glyolysis. Results suppresses glyolysis y trgeting HK We first nlyzed the rte of glyolysis using gluose nd ltte ssy kits nd then mesured F-FDG uptke of MDA- MB- ells trnsfeted with mimi to evlute its effet on glyolysis. Following tretment with mimi, the ells exhiited deresed rtes of gluose onsumption nd ltte prodution, inditing tht the glyolyti pthwy ws repressed (Figure ). In ddition, in vitro dynmi F-FDG uptke ssys reveled tht the mimi-treted ells exhiited signifintly lower level of F-FDG uptke thn ontrol ells (Figure ). Furthermore, we evluted the expression of HK, whih my ply pivotl role in glyolysis. Tretment with mimi deresed oth HK mrna expression nd protein levels in MDA-MB- ells (Figure,d). These findings demonstrte tht trgets HK nd exerts n inhiitory effet on glyolysis in TNBC ells. inhiits ell growth, migrtion, nd indues poptosis The results of previous studies hve supported the role of s tumor suppressor., Indeed, we found tht introdution of signifintly inhiited the prolifertion nd migrtion of MDA-MB- ells (Figure,). Flow ytometry ssy lso demonstrted enhned ell poptosis following mirna tretment (Figure ). Colletively, these results onfirmed tht ould suppress prolifertion nd migrtion while induing poptosis in TNBC ells. Systemi delivery of gomir effetively restrined tumor growth in TNBC tumor-ering mie Next, we investigted whether the tumor suppressive funtion of intervention in TNBC ells ould e reprodued in tumor-ering mie model. One the tumor dimeter rehed ~ 5mm, holesterol-modified nd Cy-leled oligo ( gomir) enpsulted in neutrl lipid-sed delivery vehile ws introdued intrvenously one every dys over totl period of 5 dys (n = ) (Figure ). Two ontrol groups tht seprtely reeived Cy-leled mirna gomir ontrol ( gomir) or phosphte-uffered sline (PBS) were lso estlished. Suessful dministrtion of mirna gomir ws onfirmed y the detetion of lolized Cy signls in the tumors of oth mie injeted with gomir nd those reeiving Ctrl RNA gomir (Figure d, left upper pnels). As shown in Figure, the time ourse of tumor growth indited tht systemi delivery of gomir effetively inhiited tumor growth ompred with gomir group. This ws supported y tumor volume mesurements (Figure ). Immunohistohemil stining reveled drmti upregultion of the ell poptosis mrker spse- nd downregultion of the ell prolifertion mrker proliferting ell nuler ntigen in tumor tissues otined from gomir group ompred with in gomir group (Figure d, right pnels).tken together, these results demonstrte the therpeuti effiy of ginst TNBC tumors. F-FDG PET/CT in monitoring response to - sed therpy in TNBC xenogrfts The mie ering TNBC xenogrfts were further sujeted to F-FDG miropet/ct sns every 5 dys during the ourse of tretment (Figure ). No sttistilly signifint differene in the men or mximum tumor uptke of F-FDG in the tretment group ws oserved prior to the dministrtion of gomir ompred with the negtive ontrol group (seline, Dy. Figure,). After tretment for 5 dys, however, the men stndrdized uptke vlue (SUVmen) derived from the tumor uptke of F-FDG ws deresed y % in gomir group ompred with in the group tht reeived gomir s quntified y regions of interest nlysis (Figure, right pnel). Furthermore, the posttretment F-FDG SUVmen ws % lower thn the tumor uptke t seline in gomir group ( Figure, right pnels). Similrly, the post-tretment mximum stndrdized uptke vlue (SUVmx) in the tretment group showed % derese ompred with the negtive ontrol group nd % derese ompred with the pretretment seline level (Figure, left pnel). Interestingly, we oserved inresed men nd mximum tumor uptke of F-FDG in mie reeiving gomir on dy 5, followed y shrp derese of oth vlues on dys nd 5 ompred with oth ontrol groups (Figure,). Additionlly, F-FDG miropet/ct imging onfirmed tht tumor growth in mir- gomir mie ws mrkedly inhiited ompred with in the negtive ontrol group (Figure ). Moreover, oth immunohistohemil stining nd western lotting indited signifintly lower levels of HK in tumors sujeted to gomir tretment ompred with in ontrols (Figure,d). In onlusion, F-FDG PET/CT, noninvsive monitoring tehnique, is fesile for estimting the linil response to mir- -sed trgeting tumor metolism therpy. Toxiity ssessment of -sed therpy To ssess the potentil toxiity of -sed therpy in longer oserved term, nother three rndomized groups of TNBC tumor-ering xenogrfts were estlished (n = mie in eh group). Enpsulted mirna gomirs or PBS ws dministered intrvenously every dys for yles. The oserved time ws prolonged to dys, while the other therpeuti onditions remined unhnged. The toxiity ssessment ws minly performed y monitoring the ody weights of the mie every dys nd performing linil hemil tests on their lood smples olleted on dy. As ntiipted, no ehviorl hnge or signifint ody weight loss ws oserved throughout the ourse of tretment (Figure 5). formultion used slight inrese in serum ure (Figure 5), nd the levels of hemogloin, pltelets, nd red lood ells ounts in gomir group showed mild deline (Figure 5), when ompred with oth ontrol groups. Serum levels of lnine minotrnsferse nd sprtte minotrnsferse liver enzymes s well s totl holesterol were slightly elevted (Figure 5), while the numers of white lood ells were mildly deresed in oth tretment group nd negtive ontrol group (Figure 5), Moleulr Therpy Nulei Aids

3 F-FDG PET/CT to Monitor Response of Brest Cner to -Bsed Therpeutis nmol / minute per ells F-FDG uptke (%/mg) 8.5 Gluose onsum Ltte minute minute 9 minute Time of F-FDG inution minute Relt Hk mrna level.5 d Mok HK Mok β-tin Figure represses glyolysis y trgeting hexokinse (HK) in MDA-MB- ells. () Following trnsfetion with RNA oligonuleotides ( mimi or ) nd inution in low-gluose medium, the gluose metolism rtes of MDA-MB- ells were deteted using speilized kits. The left pnel shows the rtes of gluose onsumption, while the right pnel shows the rtes of ltte prodution. () repressed [ F]-fluorodeoxygluose ( F-FDG) uptke of MDA-MB- ells in n in vitro dynmi F-FDG uptke ssy. () downregulted the mrna level of HK in quntittive reverse-trnsriptse-polymerse hin retion (qrt-pcr). Mok represents the phosphte uffered sline (PBS) group. (d) inhiited protein expression of HK s evident y western lotting. Vlues represent the men ± SD of three seprte experiments. P <.5, P <., P <.. % Apop ells Relt vile ell num 8 Hours Figure regultes the prolifertion, migrtion, nd poptosis of MDA-MB- ells. () inhiited the prolifertion tivity of MDA-MB- ells in MTT ssys. () mir- effetively suppressed ell migrtion in Trnswell migrtion ssys. () signifintly indued ell poptosis in flow ytometry ssys. Vlues represent the men ± SD of three seprte experiments. P <.5, P <., P <.. when ompred with PBS group. But ll the vlues remined within norml rnges. Tken together, these results revel tht tretment t the dosge nd frequeny s used Migrt ell num 5 5 in our study showed miniml toxi effets on the tested nimls. The slight hnge in levels of liver enzymes, totl holesterol nd white lood ells ounts pper to e relted to the hemistry of gomir nd delivery gent rther thn. To further explore the iodistriution of Cy-leled mir- delivery medited y neutrl lipid-sed gents to other orgns, setions of the hert, lung, rin, liver, kidney, spleen, nd musle were exmined for fluoresene. There ws signifint mirna gomir uptke nd ytoplsmi distriution in the liver nd kidney (Figure 5). Moderte mirna gomir uptke ws oserved in hert nd musle (Figure 5). There ws smll mount of uptke in the spleen, while fint fluoresene ws distriuted in the rin nd lung (Figure 5). Disussion The finding tht ner ell prolifertion enefits from elerted gluose metolism hs prompted intense reserh efforts imed t developing therpeuti gents tht trget nd seletively suppress ertin glyolyti enzymes. It hs een hypothesized tht the est inhiitory effet n e hieved y stopping gluose flux during the initil steps of glyolysis in ner ells, whih would, in theory, disrupt ll downstrem steps nd metoli intermedites tht support tumor noli proesses. In this regrd, hexokinse, whih onverts -ron sugr moleule to the orresponding hexose phosphte, ppers to e n ttrtive

4 F-FDG PET/CT to Monitor Response of Brest Cner to -Bsed Therpeutis When tumors dimeter rehed round 5 mm F-FDG miro PET/CT sn gomir or gomir injetion Dy 5 Dy Dy Dy 5 8 Tumor volume (mm) Dy 5 d µm µm µm 9 Dys 5 Cy-RNA HE DAPI PCNA Merge Cspse- µm µm µm Figure Systemi delivery of gomir inhiits tumor growth in mouse models of triple-negtive rest ner (TNBC). () Shemti digrm of the experimentl design. For this experiment, 8-week-old femle BALB/ thymi nude mie were used for suutneous xenogrfts. When tumor dimeters rehed ~ 5 mm, Cy-leled gomir (tretment group) or Cy-leled gomir (negtive ontrol group) enpsulted in lipid-sed delivery vehile ws dministered through til vein injetions t.5 mg/kg of ody weight every dys for 5 yles. () Time ourse of TNBC tumor growth in mie treted with gomir or gomir. () Tumors were reseted from gomir nd gomir mie. (d) Cy signls were detetle in tumor tissue slies from Cy-leled mirna gomir shown in the upper left pnels. Nuleus ounterstined with,-dimidino--phenylindole on tumor tissue slies re shown in the middle left pnels. The merged imges re shown in the lower left pnels. Immunohistohemil stining is presented in the right pnels nd inluded hemtoxylin nd eosin (HE), proliferting ell nuler ntigen (PCNA) (rown), nd spse- (rown) detetion of TNBC tumor setions from the groups. Sle rs: μm. All dt re men ± SD of three seprte experiments. P <.5, P <., P <.. n = in eh group. trget for ner therpy. Indeed, n inresing numer of studies hs linked onogenesis with HK overexpression, whih serves s key meditor of eroi glyolysis nd promotes tumor growth.,5 Supporting this notion, Nissim Hy et l. demonstrted tht systemi deletion of HK in mouse models redued the tumor urden of nonsmll ell lung ner nd rest ner, suggesting the possiility of trgeting HK for tretment of rest ner. Previously, we oserved signifint inverse orreltion etween HK mrna nd levels in rest ner ptients, Moleulr Therpy Nulei Aids whih lent further evidene tht mir-55/ sde ws involved in the ontrol of glyolysis y regulting HK expression in rest ner ells. The intertion etween nd HK ws lso verified in severl other types of ners, inluding olon ner, lung ner, hed nd nek squmous ell rinom, mong others.7,7 Despite these dvnes, systemi delivery of formulted s glyolysis inhiitor for ner therpy hs not een reported. Moreover, the therpeuti evlution of mirna y moleulr imging remins reltively unexplored.

5 F-FDG PET/CT to Monitor Response of Brest Cner to -Bsed Therpeutis 5 Dy Dy 5 Dy Dy 5 HK F-FDG (SUVmx).5.5 F-FDG (SUVmen).5.5 d HK β-tin 5 Dys Dys Figure Assessment of response to -sed therpy in triple-negtive rest ner (TNBC) xenogrfts. () [ F]-fluorodeoxygluose ( F-FDG) miropet/ct imging of mie treted with gomir or gomir t dys (seline), 5,, nd 5. Representtive F-FDG miro positron emission tomogrphy/omputed tomogrphy (PET/CT) imges re shown with irles inditing xenogrfted TNBC tumors. () Quntifition of tumor uptke of F-FDG is presented s mximum stndrdized uptke vlue (SUVmx) nd men stndrdized uptke vlue (SUVmen). () Hexokinse (HK) expression of TNBC tumor setions from immunohistohemil stining. (d) HK protein levels determined y western lotting. All dt re men ± SD of three seprte experiments. P <.5, P <., P <.. The results desried herein demonstrted for the first time tht intrvenous delivery of gomir with lipid-sed formultion effetively inhiited glyolysis nd the growth of xenogrfted TNBCs. This study ws performed through systemi dministrtion, y whih effetive drug distriution to primry tumors n e hieved nd is losely relted to the linil pthwy. For mirna-sed therpeuti pplition, hemil modifitions of oligonuleotides together with n effiient delivery system re required. Aordingly, we employed hemilly synthesized nd holesterol-modified mirna gomir enpsulted into neutrl lipid-sed delivery regent to hieve higher stility nd more roust penetrtion into trget tissues. Moreover, in ontrst to sirnas or shrnas, whih generlly modulte single trnsript, mirnas my simultneously trget multiple ner-relted pthwys, leding to stronger linil enefits. 8 Besides ERK5, TLR, nd N-RAS mong others, HK ws verified s one of the onogenes sujeted to regultion in this study.,9, Colletively, ppers to e strong trget ndidte for ntitumor therpeutis. F-FDG PET/CT hs een extensively explored in wide rnge of si reserh nd linil pplitions, rnging from the ellulr nd moleulr mehnisms tht determine F-FDG uptke to the well-doumented utility in multiple diseses, prtiulrly in ner. In this study, we evluted the fesiility of using F-FDG PET/CT snning to monitor the effiy of -sed therpies. We demonstrted tht five yles of intrvenous dministrtion of gomir signifintly redued tumor uptke of F-FDG (SUVmen nd SUVmx) ompred with oth the pretretment seline nd the vlue found in mie treted with gomir. Our findings in niml models were further orroorted y immunohistohemil stining nd western lotting, oth of whih demonstrted tht HK ws suppressed in the presene of, supporting the onlusion of reent study tht HK is n independent preditor of F-FDG uptke. Consistent with previous findings inditing tht orreltion exists etween F-FDG uptke nd ellulr prolifertion, the rte of growth in gomir-treted tumors ws deresed ompred with ontrols, ompnied y the downregultion of proliferting ell nuler ntigen oserved t the end of tretment. However, this orreltion ws not oserved in the lte period of the tretment, during whih tumor growth ontinued; however, F-FDG uptke deresed nd the SUVmen vlue deresed more shrply in the negtive ontrol group. These opposing trends my e ttriuted

6 F-FDG PET/CT to Monitor Response of Brest Cner to -Bsed Therpeutis Cy-RNA Merge 5 Mok Hert Weight (g) 5 Lung Dys WBC ount ( 9 /l) 5 5 Mok Mok Pltelet ount ( 9 /l) Brin RBC ount ( /l) 5 8 Mok H (g/l) 5 Mok Liver Kidney ALT (U/l) 8 Mok CHO (mmol/l) Mok Spleen 5 AST (U/l) 5 Ure (mmol/l) 8 Musle Mok Mok Figure 5 Toxiity nlyses following intrvenous delivery of enpsulted mirna gomir in triple-negtive rest ner (TNBC) xenogrfts. Another three rndomized groups of TNBC tumor-ering xenogrfts were estlished (n = mie in eh group). When tumor dimeters rehed ~5 mm, enpsulted mirna gomirs were dministered y til vein injetion t.5 mg/kg of ody weight every dys for yles. () Body weights were mesured every dys throughout the study. () Routine lood tests nd iohemil tests were performed in norml BALB/ thymi nude mie fter tretment with.5 mg/kg of ody weight gomir or gomir or phosphte uffered sline (PBS). () Cy signls were exmined in hert, lung, rin, liver, kidney, spleen, musle tissue slies shown in the left pnels to evlute the iodistriution of Cy-leled gomir. The merged imges with,-dimidino--phenylindole nuleus ounterstined re shown in the right pnels. Sle rs: 5 μm. Dt re presented s men ± SD. to n inresed level of tumorl nerosis nd indite tht F-FDG uptke, prtiulrly the SUVmen, is not good inditor of tumor prolifertion under these irumstnes. Additionlly, Chodosh et l. reommended tht onogeni pthwy tivtion within tumor, in preferene to the rte of prolifertion is dependle preditor of F-FDG uptke Moleulr Therpy Nulei Aids

7 F-FDG PET/CT to Monitor Response of Brest Cner to -Bsed Therpeutis 7 levels. Overll, our results suggest tht F-FDG PET/CT n e used to monitor the response of TNBC to -sed therpeutis y trgeting tumor glyolysis nd for the speifi evlution of ner therpy through moleulr imging method. While progress hs een mde towrd trgeting metoli enzymes for ner therpy, onerns relted to the uneptle effets on norml ells hve een rised. Although the iodistriution of formultion ws not restrited to tumors when dministrted systemilly, ut in other norml orgns, whih might inhiit HK in oth tumors nd norml tissues, we reson tht tumor ells re the min trgets of this gent euse initilly, ompred with the reltively high expression in limited norml dult tissues, suh s dipose tissues, skeletl musles, nd the hert, HK is expressed t muh higher levels in extensive tumor tissues. Furthermore, the HK isoform, whih is struturlly nd funtionlly similr to HK, is uiquitously expressed in most norml dult tissues nd my ply ompenstory or redundnt role in the sene of HK, while the myordium nd skeletl musle my utilize ftty ids s n lternte energy sustrte s uffer. Additionlly, s oserved in our study, when dministered t the sme dose nd frequeny, hd miniml toxi effets on norml ells. In summry, we demonstrted tht systemi delivery of gomir is n effetive ntiner strtegy for trgeting tumor metolism in TNBCs. Given the link etween nd HK, F-FDG PET/CT represents n ttrtive pproh for monitoring the response to -sed therpy. Extensive nlyses re required to settle the prolem of trgeting property nd unique strtegies for evlution effiy through moleulr imging pprohes requires further explortion. Mterils nd methods Cell ulture nd trnsfetion. The humn rest ner ell line MDA-MB- ws otined from the Amerin Type Culture Colletion (ATCC, Mnsss, VA), nd the ells were ultured t 7 C, 5% CO in Duleo s modified essentil medium supplemented with % fetl ovine serum nd % peniillin/streptomyin. mimi nd srmled negtive ontrol RNA () were otined from Rio- Bio (Gungzhou, Chin).Trnsfetion ws performed using Lipofetmine (Invitrogen, Crlsd, CA) ording to the mnufturer s instrutions. For trnsfetion of the RNA oligonuleotides, 5 nmol/l mimi nd 5 nmol/l Ctrl RNA were used. Anlyses of the effets of mimi on reipient ells were performed 9 hours fter trnsfetion. RNA isoltion nd reverse-trnsriptse-polymerse hin retion ssys. Totl RNA ws extrted using TRIzol (Invitrogen) nd ws reverse-trnsried with PrimeSript RT regent (Tkr, Shig, Jpn). The DNA produt ws dded to mixture of SYBR Premix Ex Tq II (Tkr) long with HK forwrd nd reverse primers (5 -AAGG CTTCAAGGCATCTG- nd 5 -CCACAGGTCATCATAGTT CC-, respetively). Polymerse hin retion ws onduted s follows: 95 C for seonds, followed y yles t 95 C for seonds nd C for seonds. Reltive HK gene expression ws nlyzed using the Ct method nd normlized using glyerldehyde--phosphte dehydrogense s n endogenous ontrol. Cell prolifertion, trnswell migrtion, nd poptosis ssys. The following ssys were performed s previously desried., Briefly, hours fter trnsfetion with mimi or, equl numers of vile MDA-MB- ells were seeded into 9-well pltes for ell prolifertion ssys. Cell growth ws determined y MTT ssys. Experiments were rried out in triplite. Apoptosis ws determined y flow ytometry using ommerilly ville Annexin V-FITC poptosis detetion kit (Sigm, St. Louis, MO). Cell migrtion ssys were performed in -well trnswell plte with 8-μm polyethylene terephthlte memrne filters (Flon ell ulture insert; BD Biosienes, Frnklin Lkes, NJ) seprting the lower nd upper ulture hmers. Approximtely, ells were plted in the upper hmer per well in serum-free Duleo s modified essentil medium. The ottom hmer ontined Duleo s modified essentil medium with % fetl ovine serum. Cells were llowed to migrte for 8 hours. After inution, the filter ws removed nd nonmigrting ells on the upper side of the filter were dethed using otton sw. Filters were fixed with % formldehyde for 5 minutes, ells loted in the lower filter were stined with.% rystl violet for minutes, nd three rndom fields were ounted. Quntifition of the results is presented s the men ± SD. Mesurement of gluose onsumption, ltte prodution, nd in vitro F-FDG uptke. Gluose onsumption nd ltte prodution were nlyzed using gluose ssy kit (Sigm) nd ltte ssy kit (BioVision, Milpits, CA) s previously desried. In the F-FDG uptke ssy, MDA- MB- ells were ultured in gluose-free medium ontining 7 kbq/ml F-FDG. After inution for different periods of time (,, 9, nd minutes), the ells were stringently rinsed in old PBS nd then F-FDG rdiotivity ws deteted using gmm ounter. Protein onentrtion of eh smple ws determined using Brdford protein ssy kit (Promeg, Mdison, WI) with ovine serum lumin s the stndrd ording to the mnufturer s reommendtions. Reltive F-FDG uptke ws normlized ording to the respetive ell lyste protein onentrtion. Therpeuti experiments in nude mie. All niml work ws performed in ordne with the Guide for the Cre nd Use of Lortory Animls (NIH pulition nos. 8-, revised 99) nd in ordne with the institutionl ethil guidelines for niml experimenttion. Six to eight-week-old femle BALB/ thymi nude mie were used for suutneous xenogrfts. Cy-leled gomir or Cy-leled gomir ws formulted with MxSuppressor In Vivo RNA-LANCEr II, neutrl lipid-sed delivery regent (BIOO Sientifi, Austin, TX) ording to the mnufturer s instrutions. When tumor dimeters rehed ~5 mm, TNBC tumor-ering xenogrfts were rndomized into three groups nd enpsulted mirna gomirs were dministered intrvenously (i.v.) y til vein injetion t.5 mg/kg of

8 8 F-FDG PET/CT to Monitor Response of Brest Cner to -Bsed Therpeutis ody weight every dys for 5 yles. Tumor growth rtes were nlyzed y mesuring tumor length (L) nd width (W) every dys nd y lulting the volume with the formul (LW )/. The Cy-leled gomir nd Cy-leled gomir were ommerilly synthesized y RioBio (Gungzhou, Chin). MiroPET/CT imging of mie. Mie ering TNBC xenogrfts were sujeted to F-FDG miropet/ct nlysis, performed on n Inveon MM Pltform (Siemens, Munih, Germny), on dys, 5,, nd 5. The mie were nesthetized with % isoflurne in O gs for F-FDG injetion ( single injetion of. ml F-FDG with n tivity of.7 7. MBq vi til vein). At minutes fter dministrtion of the trer injetion, the mie were pled prone on the PET snner ed nd were mintined under ontinuous nesthesi during the study with.5% isoflurne in oxygen t l/min. Inveon Aquisition Workple (Siemens) ws used for snning. Mie were first sujeted to 5-min CT sn nd then to -min PET sn. Imges were reonstruted using n OSEMD lgorithm followed y MAP or Fst MAP provided y Inveon Aquisition Workple. The D regions of interest were drwn over the entire tumor guided y CT imges nd trer uptke ws mesured using Inveon Reserh Workple softwre. The SUV ws lulted s (dey-orreted tivity (kbq) per milliliter of tissue volume)/ (injeted F-FDG tivity (kbq)/ody mss (g)). Toxiity ssessment. Another three rndomized groups of TNBC tumor-ering xenogrfts were estlished (n = mie in eh group) for toxiity ssessment. The mie reeived til vein injetion of enpsulted mirna gomirs t.5 mg/kg of ody weight or PBS every dys for yles. The oserved term ws extended to dys, while the other therpeuti onditions were not hnged. The tivity level, grooming ehviors nd mie weight were oserved throughout the study. Blood smples of mie were olleted on dy for further linil hemil tests to ssess hnges in reltive vlues inluding white lood ells ounts, red lood ells ounts, hemogloin, pltelets ounts, lnine minotrnsferse, sprtte minotrnsferse, ure, nd totl holesterol. Afterwrds, mie were srified nd mjor orgns were reseted, molded with optiml utting temperture ompound, nd frozen in liquid nitrogen. Frozen setions were ut with μm thikness nd stined with,-dimidino- -phenylindole. Cy signls from hert, lung, rin, liver, kidney, spleen, nd musle were exmined y fluoresene mirosope to evlute the iodistriution of mirna gomirs. Western lotting nd immunohistohemistry. Western lotting ssys were rried out using stndrd proedures. The ntiody for HK ws from Cell Signling Tehnology (Dnvers, MA). Antiodies for horserdish peroxidse-onjugted got ntirit IgG nd β-tin were from Sigm. Immunohistohemistry for xenogrfted tumor setions ws performed using stndrd protools. The dewxed 5-mm setions were sujeted to n ntigen-demsking proedure, whih involved rief high-temperture heting of the setions immersed in itrte uffer ( mmol/l, ph.). Endogenous peroxidses were loked with.% hydrogen peroxide, nd nonspeifi inding ws loked with 5% norml got serum in.% Triton X-, Tris-uffered sline (ph 7.). Setions were then seprtely inuted with rit monolonl nti-proliferting ell nuler ntigen, nti-spse-, nd nti-hk ntiody (Cell Signling Tehnology) for hours t room temperture. After wshing with PBS, the setions were inuted with iotinylted seondry ntiody, followed y further inution with the streptvidin horserdish peroxidse omplex. The setions were then immersed in, -diminoenzidine for 5 minutes, ounterstined with % Myer s hemtoxylin, dehydrted, nd mounted in rystl mount. Sttistil nlysis. All results re presented s men ± SD. The Student s t-test ws performed to ompre the differenes etween treted groups reltive to their pired ontrols using SPSS. softwre (SPSS, Chigo, IL). P-vlues re indited in the figures ove the two groups ompred with vlue <.5 (denoted y ) onsidered s signifint (P <. nd P <.). Aknowledgments The uthors delre no onflit of interest. This work ws supported y the Ntionl Nturl Siene Foundtion of Chin (NSFC; No. 87 nd 878).. Torre, LA, Bry, F, Siegel, RL, Ferly, J, Lortet-Tieulent, J nd Jeml, A (5). Glol ner sttistis,. CA Cner J Clin 5: 87.. Crey, L, Winer, E, Vile, G, Cmeron, D nd Ginni, L (). Triple-negtive rest ner: disese entity or title of onveniene? Nt Rev Clin Onol 7: Kssm, F, Enright, K, Dent, R, Drnitsris, G, Myers, J, Flynn, C et l. (9). Survivl outomes for ptients with metstti triple-negtive rest ner: implitions for linil prtie nd tril design. Clin Brest Cner 9: 9.. Lehmnn, BD, Buer, JA, Chen, X, Snders, ME, Chkrvrthy, AB, Shyr, Y et l. (). Identifition of humn triple-negtive rest ner sutypes nd prelinil models for seletion of trgeted therpies. J Clin Invest : Hnhn, D nd Weinerg, RA (). Hllmrks of ner: the next genertion. Cell : 7.. Wrurg, O (95). On the origin of ner ells. Siene : Cheson, BD (). Role of funtionl imging in the mngement of lymphom. J Clin Onol 9: Fuster, D, Duh, J, Predes, P, Velso, M, Muñoz, M, Sntmrí, G et l. (8). Preopertive stging of lrge primry rest ner with [ F]fluorodeoxygluose positron emission tomogrphy/omputed tomogrphy ompred with onventionl imging proedures. J Clin Onol : Roey, RB nd Hy, N (). Mitohondril hexokinses, novel meditors of the ntipoptoti effets of growth ftors nd Akt. Onogene 5: Ptr, KC nd Hy, N (). Hexokinse s onotrget. Onotrget :.. Brtel, DP (9). MiroRNAs: trget reognition nd regultory funtions. Cell : 5.. Amros, V (). The funtions of niml mirornas. Nture : Chng, YY, Kuo, WH, Hung, JH, Lee, CY, Lee, YH, Chng, YC et l. (5). Deregulted mirornas in triple-negtive rest ner reveled y deep sequening. Mol Cner :.. Kent, OA, Chivukul, RR, Mullendore, M, Wentzel, EA, Feldmnn, G, Lee, KH et l. (). Repression of the /5 luster y onogeni Rs initites tumor-promoting feedforwrd pthwy. Genes Dev : Mihel, MZ, O Connor, SM, vn Holst Pellekn, NG, Young, GP nd Jmes, RJ (). Redued umultion of speifi mirornas in oloretl neoplsi. Mol Cner Res : Jing, S, Zhng, LF, Zhng, HW, Hu, S, Lu, MH, Ling, S et l. (). A novel mir-55/ sde ontrols glyolysis y regulting hexokinse in rest ner ells. EMBO J : Peshiroli, A, Gioe, A, Formos, A, Mrkert, EK, Bongiorno-Borone, L, Levine, AJ et l. (). regultes hexokinse expression in ner ells. Onogene : Fng, R, Xio, T, Fng, Z, Sun, Y, Li, F, Go, Y et l. (). MiroRNA- (mir- ) regultes ner glyolysis vi trgeting hexokinse gene. J Biol Chem 87: Gregersen, LH, Josen, A, Frnkel, LB, Wen, J, Krogh, A nd Lund, AH (). MiroRNA- down-regultes Hexokinse in olon ner ells. BMC Cner :.. Yoshino, H, Enokid, H, Itesko, T, Kojim, S, Kinoshit, T, Ttrno, S et l. (). Tumor-suppressive mirorna-/5 luster trgets hexokinse- in renl ell rinom. Cner Si : Moleulr Therpy Nulei Aids

9 F-FDG PET/CT to Monitor Response of Brest Cner to -Bsed Therpeutis 9. Yo, M, Wng, X, Tng, Y, Zhng, W, Cui, B, Liu, Q et l. (). Dier mediting the expression of nd mir-55 regultes hexokinse II ssoited ellulr response to hypoxi. Am J Physiol Lung Cell Mol Physiol 7: L89 L87.. Guo, H, Chen, Y, Hu, X, Qin, G, Ge, S nd Zhng, J (). The regultion of Toll-like reeptor y suppresses the invsion nd migrtion of suset of humn oloretl rinom ells. Mol Cner : 77.. Xu, YF, Li, YQ, Guo, R, He, QM, Ren, XY, Tng, XR et l. (5). Identifition of s tumour suppressor in nsophryngel rinom sed on mirorna expression profiling. Int J Biohem Cell Biol : 8.. Mthupl, SP, Ko, YH nd Pedersen, PL (9). Hexokinse- ound to mitohondri: ner s stygin link to the Wrurg Effet nd pivotl trget for effetive therpy. Semin Cner Biol 9: Wolf, A, Agnihotri, S, Millef, J, Mukherjee, J, Sh, N, Cirns, R et l. (). Hexokinse is key meditor of eroi glyolysis nd promotes tumor growth in humn gliolstom multiforme. J Exp Med 8:.. Ptr, KC, Wng, Q, Bhskr, PT, Miller, L, Wng, Z, Wheton, W et l. (). Hexokinse is required for tumor initition nd mintenne nd its systemi deletion is therpeuti in mouse models of ner. Cner Cell : Zho, S, Liu, H, Liu, Y, Wu, J, Wng, C, Hou, X et l. (). inhiits glyolysis nd depletes stemness of gliolstom stem-like ells. Cner Lett : Selh, M, Shwnhäusser, B, Thierfelder, N, Fng, Z, Khnin, R nd Rjewsky, N (8). Widespred hnges in protein synthesis indued y mirornas. Nture 55: Noguhi, S, Ysui, Y, Iwski, J, Kumzki, M, Ymd, N, Nito, S et l. (). Replement tretment with mirorna- nd -5 indues synergisti inhiition of the growth of humn ldder ner ells y regulting PIK/Akt nd MAPK signling pthwys. Cner Lett 8: 5.. Wng, L, Shi, ZM, Jing, CF, Liu, X, Chen, QD, Qin, X et l. (). MiR- ts s tumor suppressor y trgeting N-RAS nd enhnes temozolomide-indued poptosis in gliom. Onotrget 5: Alvrez, JV, Belk, GK, Pn, TC, Chen, CC, Blnkemeyer, E, Alvi, A et l. (). Onogene pthwy tivtion in mmmry tumors dittes FDG-PET uptke. Cner Res 7: Vnder Heiden, MG (). Trgeting ner metolism: therpeuti window opens. Nt Rev Drug Disov : Wilson, JE (). Isozymes of mmmlin hexokinse: struture, suellulr loliztion nd metoli funtion. J Exp Biol (Pt ): Jing, S, Zhng, HW, Lu, MH, He, XH, Li, Y, Gu, H et l. (). MiroRNA-55 funtions s n OnomiR in rest ner y trgeting the suppressor of ytokine signling gene. Cner Res 7: 9 7. This work is liensed under Cretive Commons Attriution-NonCommeril-NoDerivs. Interntionl Liense. The imges or other third prty mteril in this rtile re inluded in the rtile's Cretive Commons liense, unless indited otherwise in the redit line; if the mteril is not inluded under the Cretive Commons liense, users will need to otin permission from the liense holder to reprodue the mteril. To view opy of this liense, visit The Author(s) ()