Intranasal Delivery of Mesenchymal Stem Cells Significantly Extends Survival of Irradiated Mice with Experimental Brain Tumors

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1 originl rticle The Americn Society of Gene & Cell Therpy see pge xxx Intrnsl Delivery of Mesenchyml Stem Cells Significntly Extends Survivl of Mice with Experimentl Brin Tumors Irin V Blysnikov 1, Melnie S Prsol 1, Sherise D Ferguson 1, Yu Hn 1, Atique U Ahmed 1, Mrgrit Gutov 3, Alex L Tois 1, Devkumr Mustfi 2, Esther Rincón 1, Lingjio Zhng 1, Kren S Aoody 3 nd Mciej S Lesnik 1 1 Deprtment of Surgery, The Brin Tumor Center, The University of Chicgo, Chicgo, Illinois, USA; 2 Deprtment of Rdiology, The University of Chicgo, Chicgo, Illinois, USA; 3 Deprtment of Neurosciences, Beckmn Reserch Institute of the City of Hope, Durte, Cliforni, USA Tretment options of gliolstom multiforme re limited due to the lood rin rrier (BBB). In this study, we investigted the utility of intrnsl (IN) delivery s mens of trnsporting stem cell sed ntigliom therpeutics. We hypothesized tht mesenchyml stem cells (MSCs) delivered vi nsl ppliction could imprt therpeutic efficcy when expressing TNF-relted poptosis-inducing lignd (TRAIL) in model of humn gliom. 111 In-oxine, histology nd mgnetic resonnce imging (MRI) were utilized to trck MSCs within the rin nd ssocited tumor. We demonstrte tht MSCs cn penetrte the rin from nsl cvity nd infiltrte intrcrnil gliom xenogrfts in mouse model. Furthermore, irrdition of tumor-ering mice tripled the penetrtion of 111 In-oxine leled MSCs in the rin with fivefold increse in cereellum. Significnt increse in CXCL12 expression ws oserved in irrdited xenogrft tissue, implicting CXCL12-dependent mechnism of MSCs migrtion towrds irrdited gliom xenogrfts. Finlly, MSCs expressing TRAIL improved the medin survivl of irrdited mice ering intrcrnil U87 gliom xenogrfts in comprison with nonirrdited nd irrdited control mice. Cumultively, our dt suggest tht IN delivery of stem cell sed therpeutics is fesile nd highly efficcious tretment modlity, llowing for repeted ppliction of modified stem cells to trget mlignnt gliom. Received 11 June 213; ccepted 21 August 213; dvnce online puliction 31 Decemer 213. doi:1.138/mt INTRODUCTION Gliolstom multiforme (GBM) is the most common nd ggressive form of primry rin tumor. Ptient prognosis is poor even with ggressive interventions including surgicl resection nd rdition. Tumors typiclly recur fter tretment nd the medin survivl time following dignosis is ~15 months. 1,2 The lood rin rrier (BBB) limits the ility of systemiclly delivered nticncer phrmceuticls to rech the rin, hence complicting the tretment of GBM due to lck of ccessiility to the tumor ed. Direct delivery of chemotherpeutic drugs to the tumor site, through methods such s convection-enhnced delivery, llows for high concentrtion of the drug t the pproprite loction. However, this method is invsive, risks dmging surrounding norml rin tissue, nd t present, remins to e fully optimized for clinicl pplictions. 3 Previous work hs demonstrted tht stem cells, specificlly neurl stem cells (NSCs) nd mesenchyml stem cells (MSCs), hve tropism for rin tumors. 4,5 This property hs generted much interest in utilizing stem cells s vehicles for trgeted drug delivery. As is the cse in CNS drug delivery, stem cell delivery is lso hmpered y the presence of the BBB. Becuse of the BBB, few stem cells rech the rin following intrvenous delivery nd hve propensity to ccumulte in the lungs or other orgns. 6,7 Intr-rteril delivery hs een shown to deliver lrger numers of cells to the rin compred with intrvenous delivery; 7 9 however, this method hs lso een ssocited with high incidence of mortlity nd impired cererl lood flow in rts. 9,1 Attempts hve een mde to increse the efficiency of systemic delivery y disrupting ll or portions of the BBB, 11 ut this could potentilly leve the CNS vulnerle to toxins or infection. Recent pulictions hve explored the nsl system s novel stem cell delivery route to the rin. MSCs delivered into the nsl cvity hve een shown to migrte through the cririform plte nd into rin tissue vi the olfctory nd trigeminl pthwys. 12 Not only were stem cells locted in vrying nd reltively remote regions of the rin such s the cereellum, ut the delivery of MSCs ppered to hve therpeutic effect in niml models of Prkinson s disese nd ischemic rin injury. 13,14 NSCs hve lso een shown to penetrte into mouse rin nd rech the tumor ed in experimentl gliom models fter intrnsl (IN) ppliction. 15 Thus, ccumulting evidence suggests tht IN delivery of stem cells might e vile pproch for tretment of CNS pthology. Moreover, complictions ssocited with intrvsculr delivery, such s ostruction y the BBB, pulmonry emolism nd infrctions could lso e voided using this pproch. In ddition, IN delivery offers prcticl dvntge over direct Correspondence: Irin V Blysnikov, The Brin Tumor Center, The University of Chicgo, 5841 South Mrylnd Ave, MC 326, Chicgo, Illinois, USA. E-mil: ilysn@surgery.sd.uchicgo.edu 14 vol. 22 no. 1, jn. 214

2 The Americn Society of Gene & Cell Therpy Therpeutic Effect of Intrnslly Delivered Stem Cells in Mouse Model of Gliom intrcrnil ppliction of stem cells into resection cvity during surgery or convection-enhnced delivery, since it might llow for multiple tretment regimens nd cn lso e utilized in ptients with inoperle tumors. In this study, we exmined if MSCs delivered vi the nsl cvity cn rech intrcrnil humn gliom xenogrfts in mice nd e therpeuticlly relevnt when expressing TNF-relted poptosis-inducing lignd (TRAIL). TRAIL hs een shown to promote poptosis in vriety of cncers including gliom, 16 with miniml or no effect on norml cells. 17 The therpeutic efficcy of stem cells modified to express TRAIL hs een previously demonstrted in gliom. 18,19 However, in these studies the delivery method of the stem cells to the rin ws limited either to injection vi til vin or to direct intrcrnil inocultion. IN delivery of therpeutic stem cells is potentilly dvntgeous tretment modlity ecuse it represents noninvsive, nontoxic delivery method; once optimized, it cn llow for repeted ppliction of therpeutic stem cells lone or in comintion with trditionl tretment methods such s rdition nd chemotherpy. Reltive luciferse ctivity 3, P =.5 25, Left Right 2, 15, 1, 5, 11% 1%.4%.6% 2 hrs Dys fter injection Control Tumor RESULTS MCSs migrte in the rin of nimls ering intrcrnil gliom xenogrft It hs een previously shown tht MSCs disply tropism to solid tumors. 2 In this study, we first confirmed tht MSCs re cple of migrting towrds intrcrnil gliom xenogrfts. Figure 1 shows tht MSCs-fluc injected in the left hemisphere re cple of migrting quickly towrds the right, tumor-ering side of the rin. On verge, 11% of implnted MSCs were detected in the right hemisphere s soon s 2 hours following MSC inocultion. Numer of MSCs remined stedy t the 24-hour time point ut significntly decresed in oth hemispheres through dy 11 (P <.5). Migrtion of MSCs towrds the tumor-ering side of the rin ws further confirmed using live niml imging. Figure 1 shows distinct pttern of MSCs migrtion from the injection (left) site towrd the right side of the rin in tumor ering ut not control nimls. Thus, collectively this dt demonstrte tht MSCs re cple of migrting towrds intrcrnil tumor in mouse model. MSCs penetrte into mouse rin fter IN inocultion Next, we investigted if MSCs could penetrte nd e trcked in the rin fter IN dministrtion. Live niml imging ws performed fter ppliction of MSCs-fluc into the nsl cvity of the nimls. Figure 2 shows the presence of fluc signl in the nsl cvity of oth control nd tumor-ering nimls. A significnt decrese in signl intensity in the nsl cvity ws oserved from dy to dy 1, nd therefore, imging required longer exposure times for detection of MSCs on following dys. No significnt dvncement of MSCs into the rin eyond the oritl rches ws detected t vrious time points either in control or tumor-ering nimls using this technique. Tking into considertion the limittions of this method nd in order to demonstrte tht MSCs re le to dvnce in the rin of the nimls fter IN ppliction, the rins of nimls were removed 3 hours fter IN inocultion of MSCs-fluc nd imged for the presence of fluc nd green fluorescent protein (GFP) signl (tumors expressed GFP). Figure 2 shows imges of Dy 4 Figure 1 MCSs migrte in the rin of nimls towrds intrcrnil gliom xenogrft. () Quntittive luciferse ssy of migrtion of hmscs-fluc in the rin of nude mice. MSCs-fluc were inoculted into the left hemisphere of rin of nimls with U87 gliom xenogrfts estlished in the right hemisphere of rin. Luciferse ctivity in the left nd right hemispheres of rin ws mesured t vrious time points to determine dynmics of MSCs migrtion towrd the tumor-ering side of the rin. () Imging of MSCs-fluc migrtion within the rin of control nd tumor-ering mice. Arrow shows site of injection of MSCs in the left hemisphere of the rin. T-tumor injection site. Dt re presented s men + SEM. Pired t-tests were pplied to exmine differences in reltive luciferse ctivity etween the left nd right side of the rin t ech time point (n = 4/group). Unpired t-tests were utilized to exmine chnges in luciferse ctivity from dy to dy 11 in ech side of the rin. P <.5, P <.1. MSCs, mesenchyml stem cells. MSCs in the nsl cvity of the (pnel A) live niml nd luciferse signl over the olfctory uls nd frontl loes of the (pnel B) rin of tumor-ering niml. No signl ws detected in control, phosphte-uffered sline (PBS) treted nimls. Figure 2 (pnel C) shows the locliztion of xenogrft tumors in the right hemisphere of oth nimls. Finlly, pnels D nd E in Figure 2 show tht MSCs re lso cple of rpid penetrtion into the rin of mice fter IN inocultion in control, nontumor-ering nimls in greement with previous report showing penetrtion MSCs in the rin fter IN ppliction in rts. 12,13 Distriution of 111 In-oxine leled MSCs in the rin fter IN delivery This prt of the study ws designed to quntittively evlute the ility of MSCs to penetrte into the rin of tumor-ering nimls fter IN delivery. Figure 3 shows tht MSCs treted with 111 In-oxine for 1, 2, nd 3 minutes were leled t similr T Moleculr Therpy vol. 22 no. 1 jn

3 Therpeutic Effect of Intrnslly Delivered Stem Cells in Mouse Model of Gliom The Americn Society of Gene & Cell Therpy Dy 1 Dy (1 hour) No tumor PBS MSCs Tumor No tumor Tumor Dy 6 Dy 3 A B C 12 D PBS MSCs PBS MSCs No tumor Figure 2 MSCs penetrte into mouse rin fter IN inocultion. () Live niml imging fter intrnsl inocultion of MSCs-fluc. Control (no tumor) nd U87 gliom-ering nimls were imged live t vrious time points fter IN ppliction of MSCs-fluc. () Imging of MSCs-fluc in the rin of the control nd tumor-ering nimls fter intrnsl inocultion. In order to demonstrte the dvncement of MSCs into the rin fter IN ppliction, the rins of nimls were removed 3 hours fter IN inocultion of MSCs-fluc nd imged for the presence of fluc nd GFP signl corresponding to U87 cells. (A) live niml imging without (left) nd with inoculted MSCs (right); (B) imging of rin without (left) nd with inoculted MSCs (right); (C) imging of U87-GFP tumor in the right hemisphere of the rin; (D) imging of MSCs in control, nontumor-ering nimls; (E) imging of rin of nontumor nimls for GFP signl. MSCs, mesenchyml stem cells. level, therefore 2 minutes leling time point ws chosen for following experiments. Tking into considertion the short hlflife of 111 In-oxine (2.8 dys), we determined the sensitivity t which MSCs could e potentilly detected within the rin 24 hours fter leling. Figure 3 shows tht rdioctivity corresponding to s little s one leled cell could e detected using this method. It hs previously een shown tht MSC tropism to tumor is incresed following irrdition of the tumor. 2,21 With this in mind, we next investigted the distriution of 111 In-oxine leled MSCs in the rin of tumor-ering control nd irrdited nimls. Figure 3c shows the distriution of 111 In-oxine leled MSCs in the olfctory uls; the left, nontumor-ering hemisphere of the rin; the right, tumor-ering hemisphere of the rin, nd in the cereellum of control nd irrdited nimls. No difference in the presence of MSCs ws detected in the olfctory uls of control nd irrdited nimls, wheres the presence of MSCs ws two- (P =.3, n = 4) nd threefold (P =.57, n = 4) higher in the left nd right hemispheres of irrdited nimls respectively. Furthermore, we oserved pproximtely fivefold (P =.3, n = 4) increse in the presence of MSCs in the cereellum of irrdited nimls. On verge, the totl numer of MSCs penetrting in the rin of irrdited nimls ws 2.8 times higher (P =.28, n = 4) thn in control nonirrdited tumor-ering nimls (Figure 3d). 111 In-oxine ws lso detected in other orgns E Tumor (Figure 3e); specificlly, the highest quntities were in the lung nd stomch. Thus, these dt quntittively vlidte nd further indicte tht MSCs re cple of penetrting into the rin of mice fter IN inocultion nd their penetrtion is significntly enhnced y irrdition. Detection of MSCs in the tumor ed fter IN inocultion In order to further loclize MSCs within the rin, MSCs were leled with micron-size prmgnetic iron oxides (MPIOs) efore IN delivery to tumor-ering mice. Supplementry Figure S1 demonstrtes the efficiency of MSCs leling with MPIOs t vrious cell-to-prticle rtios. The rtio of 1:15 generted out 6% of positive cells in llophycocynin chnnel in the sence of toxicity (dt not shown) nd, therefore, ws chosen s optiml for stem cell leling. Supplementry Figure S1 shows the presence of flsh red fluorescent eds in leled MSCs counterstined with 4,6-dimidino-2-phenylindole (DAPI) (right pnel); the left pnel shows control, nonleled cells. Next, we IN inoculted MPIOs leled MSCs in control nd tumor-ering mice. Prussin lue stining ws performed to detect iron prticles in multiple rin tissue sections from nimls collected t dy one nd three following IN stem cell inocultion. Prussin lue stining ws detected in ll nimls tht hd received MPIOs-leled MSCs (Figure 4; pnels B F). The vst mjority of the positive signl ws detected in immedite proximity to or within the tumor ed of control nd irrdited nimls. Single clusters of positive stining were detected in other rin regions (dt not shown). Collectively, these dt indicte tht stem cells re cple of migrting to intrcrnil tumor when delivered IN in control nd irrdited nimls. Trcking of MSCs in the rin fter IN injection using mgnetic resonnce imging MPIOs were previously utilized to trck the migrtion of vrious cell types in vivo, including stem cells In order to chieve sufficient resolution to visulize leled MSCs within the mouse rin, multi-slice high resolution T 2 weighted, Rpid Acquisition with Relxtion Enhncement spin echo imges nd multi-slice T 1 weighted Fst Low Angle Shot (FLASH) grdient echo sequences were cquired in oth coronl (Figure 4; pnel B) nd xil plnes of the mouse rin (pnel C). Figure 4 demonstrtes the pth of migrtion of MPIO-leled MSCs towrds the tumor (red rrows) on sequentil T1-weighted imges of nonirrdited (pnels C,D) nd irrdited nimls (pnels E G). In order to identify the tumor mss, sequentil T2-weighted imges of n irrdited niml were tken nd presented in Figure 4 (pnels H J). The signl induced y MPIOs is shown y red rrows. The yellow rrow indictes the tumor mss. MPIO-induced signl ws more pprent in irrdited nimls. This signl ppered in the sl region nd sequentilly extended towrd the tumor. This result ws confirmed on T2-weighted imges. On xil imges of the rin of n irrdited niml (Figure 4c; pnels A E nd Supplementry Video S1), the MPIO-induced signl ppers s drk spot in the sl region of the rin, visulized in ll sequentil imges nd finlly ppers in the re corresponding to the loction of the tumor (Figure 4c; pnel E). Animls undergoing mgnetic resonnce imging (MRI) vol. 22 no. 1 jn. 214

4 The Americn Society of Gene & Cell Therpy Therpeutic Effect of Intrnslly Delivered Stem Cells in Mouse Model of Gliom minutes hours fter leling 2 minutes minutes Cell numer Cell numer d 8, 6, 4, 2, Control e Control Figure 3 Rdioleling of MSCs with 111 In-oxine nd their distriution in the rin nd other orgns fter IN delivery. () Leling of MSCs with 111 In-oxine. MSCs were incuted with 111 In-oxine t 1, 2, nd 3 minutes in CO 2 incutor t 37 C in order to determine n optiml leling time. () Sensitivity of ssy. In order to determine if the level of MSCs leling with 111 In-oxine is sufficient to detect low numer of cells, MSCs were titrted from 1 to 1 6 cells per tue nd the rdioctivity in corresponding smples ws mesured using gmm counter. (c) Distriution of MSCs through vrying prts of the rin of control nd irrdited tumor-ering nimls. 111 In-oxine leled MSCs were inoculted in the rin of nonirrdited nd irrdited tumor-ering nimls. Animls were killed 24 hours fter IN inocultion nd the rin nd other orgns were collected for mesurement of 111 In-oxine ctivity. OB-olfctory uls, LB-left hemisphere of the rin, RB-right hemisphere of the rin, CEREB-cereellum. (d) Comprison of MSC presence in the whole rin of control nd irrdited nimls. (e) Distriution of MSCs through the orgns of control nd irrdited tumor-ering nimls. Dt re presented s men + SEM. Two-wy nlysis of vrince followed y Bonferroni s post hoc test hs een pplied to exmine the effect of incution time on leling efficiency of MSCs. The presence of 111 In-oxine leled MSCs in ech orgn hs een compred etween control versus irrdited nimls using Mnn Whitney tests. P <.5. MSCs, mesenchyml stem cells. Lung Hert Liver Spleen Stom c 5, 4, 3, 2, 1, Kidney Testis Blood Bone OB Control LB RB CEREB were killed nd their rins collected for Prussin lue stining. Positive Prussin lue stining in the tumor region confirmed the results of the MRI (dt not shown). These findings further confirmed the direct migrtion of IN delivered MSCs towrds the tumor through rin prenchym in control nd irrdited nimls. MSCs-expressing TRAIL kill gliom cells in vitro nd improve survivl of irrdited nimls fter IN delivery To investigte if IN delivered MSCs could e therpeuticlly eneficil, MSCs modified to express TRAIL nd irrdition were utilized s therpeutic modlities. Figure 5 shows tht MSCs- TRAIL, ut not control MSCs (vector control), express TRAIL protein t ~1 ng in cell-ound frction per cells, nd out 8 ng of protein is relesed into the culture medi during 24 hours incution. Next, we investigted the sensitivity of vrious gliom cell lines to MSCs-TRAIL. For this, U87, GBM43, GBM12, nd GBM39 gliom cells expressing fluc were cocultured either with control or MSCs-TRAIL t the rnge of 1:.5 1:1 of gliom cells to MSCs rtios for 24 hours. Figure 5 shows tht gliom cell lines possess different sensitivity to MSCs-TRAIL nd re not ffected y control MSCs. The IC 5 is expressed s the rtio of gliom cells to MSCs-TRAIL nd ws clculted to e.49,.38,.66, nd 1.19 for GBM43, GBM39, U87, nd GBM12, respectively. Next, U87 nd GBM43 gliom cell lines were chosen to further confirm tht MSCs-TRAIL cn cuse poptosis in gliom cells. Figure 5c demonstrtes 2.5- to 3-fold increse in cleved cspse-3 expression in U87 nd GBM43 gliom cell lines cocultured with MSCs-TRAIL compred with control MSCs, wheres no chnge in cleved cspse-3 expression ws detected etween untreted (PBS) nd control MSCs treted gliom cells. Lstly, we investigted the effect of IN delivered MSCs-TRAIL on the survivl of nimls ering intrcrnil U87 gliom xenogrfts. The MSCs-TRAIL group (n = 1) demonstrted 11% improvement in survivl in comprison with PBS (n = 8, P <.5), ut not with control MSCs group of nimls (Supplementry Figure S2). Irrdition tretment of control nimls resulted on verge in 28% improvement in medin survivl. The MSCs-TRAIL plus irrdition group of nimls demonstrted 45% nd on verge 13% improvement in medin survivl compred with control nonirrdited nd control irrdited nimls respectively (n = 6, P <.1) (Figure 5d). These dt indicte tht MSCs delivered IN could e therpeuticlly eneficil in intrcrnil gliom niml models. However, vrious tretment modlities nd gliom models need to e further investigted to vlidte this pproch. Chnges in CXCL12 expression in U87 gliom xenogrft tissue s potentil mechnism for incresed penetrtion of MSCs in the mouse rin fter gmm irrdition In this set of experiments, we ttempted to uncover potentil mechnisms governing the therpeutic effect of the comined Moleculr Therpy vol. 22 no. 1 jn

5 Therpeutic Effect of Intrnslly Delivered Stem Cells in Mouse Model of Gliom The Americn Society of Gene & Cell Therpy c Non irrdited A No tumor No MSCs A Dy 1 Non irrdited tumor, MSCs Dy 1 Dy 3 D E F Dy 3 Dy 3 tumor, MSCs C E F G B D H I J A B C D E Figure 4 Trcking MPIO-leled MSCs in the rin of the mice fter IN injection using MRI. () Prussin lue stining of rin tissue sections from ( c) control nonirrdited or (d f) treted with irrdition mice ering U87 xenogrfts t dy 1 nd 3 fter IN dministrtion of MPIOleled MSCs. represents negtive control stining of tissue sections from the rin of the nimls treted with PBS. f represents higher mgnifiction of e (scle r is 5 μm). The tumor mss is outlined. Arrows indicte positive Prussin lue stining. Scle r for e is 2 μm. ( c) T1- nd T2-weighted MRI imges of control nimls (no MSCs), nonirrdited or irrdited tumor-ering nimls were tken t dy 2 fter IN inocultion of MPIO-leled MSCs. shows coronl imges. (A,B) No MPIO-leled MSC-induced signl ws detected in the rin of control mouse (no MSCs, no tumor). Sequentil T1-weighted imges demonstrte the pth of migrtion of MSCs towrds the tumor in (C,D) nonirrdited niml nd (E G) irrdited niml. (H,I,J) T2-weighted sequentil imges of irrdited niml were tken to visulize the tumor nd MPIO-induced signl. Red rrows show signl corresponding to the migrting MSCs. Yellow rrow shows tumor on T2-weighted imge (J). (c) Axil T1-weighted rin imges in (A E) irrdited tumor-ering niml. MSCs, mesenchyml stem cells. tretment of irrdition nd MSCs-TRAIL following IN delivery. The expression of TRAIL receptors, DR4 nd DR5, ws nlyzed in control nd irrdited U87 gliom xenogrft tissues. GFP expressing U87 gliom tissues were removed from the rin of nimls 24 hours fter irrdition under the guidnce of fluorescent microscope nd smples were processed nd nlyzed for DR4 nd DR5 expression y quntittive PCR nd flow cytometry. Figure 6 shows tht the expression of DR4 nd DR5 mrna remined unchnged in xenogrft tissue fter irrdition in our experimentl setting. Similrly, no chnge in the expression of DR4 nd DR5 ws found on the surfce of U87 gliom cells otined from the gliom xenogrfts (Figure 6). In hs een previously shown tht certin chemokines nd cytokines secreted from the tumor ed cn ct s chemo-ttrctnt for stem cells. 5,27 Bsed on this, we next used n rry-sed RT-PCR pproch to evlute the effects of the rdition therpy B C on the chemokines nd cytokines milieu t the tumor sites tht cn enhnce the migrtory cpcity of the implnted MSCs. For this, gliom tissue from three control or irrdited nimls ws pooled together nd processed for rry nlysis per mnufcturer s recommendtions. Supplementry Tle S1 summrizes chnges in gene expression ove threefold in U87 xenogrft tissue fter irrdition. The CXCL12 or stroml cell derived fctor-1 α (SDF-1 α) hs een shown to contriute to tropism of MSC towrds gliom. 28,29 In our study, CXCL12 ws identified s chemokine potentilly involved in enhnced migrtion of MSCs towrds gliom tissue fter irrdition. The chnges in expression of mrna in U87 gliom xenogrfts fter irrdition were confirmed in seprte qpcr rection (Figure 6c). ELISA ws utilized to confirm the chnges of CXCL12 expression in U87 gliom xenogrft tissue otined from three control nd three irrdited nimls (Figure 6d). In summry, our dt identify CXCL12 s chemokine potentilly responsile for enhnced migrtion of MSCs in the rin of irrdited nimls. However, further studies vlidting the role of CXCL12 in driving the migrtion of IN delivered MSCs towrds intrcrnil gliom xenogrfts still need to e performed. DISCUSSION The BBB hmpers mny therpeutic gents from reching the tumor ed, thus mking tretment of GBMs chllenging tsk. Additionlly, the invsive nture of GBMs llows them to escpe loclized therpies such s surgery. Recently, stem cells hve gined significnt interest s therpeutic crriers for the trgeted tretment of rin tumors due to their inherent tumor tropism. However, current clinicl method for delivery of stem cells relies on the plcement of cells in the resection cvity during surgery. In tht respect, IN delivery of therpeutic stem cells to the CNS is n ttrctive, noninvsive, lterntive method, llowing stem cells to ypss the BBB. Therefore, in the present study, we investigted if IN delivery of MSCs is vile pproch for the tretment of rin tumors in n experimentl mouse model. We showed for the first time tht IN delivery of TRAIL expressing MSCs cn prolong medin survivl in n niml model of orthotropic humn gliom xenogrft. In this study, we employed severl methods to demonstrte the fesiility of IN delivery of MSCs to the rin in tumor-ering nimls. First, we showed tht MSCs re cple of migrting within the rin towrds tumor in greement with previously pulished reports. 5,3,31 Next, using ioluminescent imging we demonstrted penetrtion of MSCs from the nsl cvity into the rin of mice. It is worth noting tht luciferse signl ws esily detectle in the nsl cvity nd in the rin within severl hours fter injection, ut detection of MSCs in nsl cvity t lter time points required longer exposure, suggesting rpid elimintion of MSCs. Similrly, the fst disppernce of signl corresponding to leled MSCs from the nsl cvity ws oserved in rodent model of Prkinson s disese. 32 It is likely tht fst penetrtion of MSCs from the nsl cvity into rin tissue is due to direct pssge of MSCs through the cririform plte vi the olfctory nd trigeminl pthwys In-oxine leled MSCs were lso detected in the rin of mice. Moreover, rdition tretment of tumor-ering nimls enhnced MSCs penetrtion into the rin. As prolifertion nd clernce of MSCs could differ in control nd irrdited nimls, it is unlikely tht it contriuted to the oserved effect t 24 hours vol. 22 no. 1 jn. 214

6 The Americn Society of Gene & Cell Therpy Therpeutic Effect of Intrnslly Delivered Stem Cells in Mouse Model of Gliom TRAIL concentrtion, ng/1 6 cells c % Cspse-3 positive cells Iyste Super PBS Cont MSCs MSCs-TRAIL MSCs-control MSCs-TRAIL RLU, % of control d Percent survivl U87 MSCs-cont GBM12 MSCs-cont GBM39 MSCs-cont GBM43 MSCs-cont PBS PBS 5 2 Gy MSC-cont 5 2 Gy MSCs-TRAIL 5 2 Gy U87 MSCs-TRAIL GBM12 MSCs-TRAIL GBM39 MSCs-TRAIL GBM43 MSCs-TRAIL Rtio GBM:MSCs Medin Survivl U87 GBM Dys 5 6 Figure 5 Effect of MSCs expressing TRAIL on vrious gliom cells in vitro nd survivl of nimls ering intrcrnil gliom xenogrfts fter IN delivery. () The level of TRAIL expression in MSCs ws mesured using ELISA s descried in Mterils nd Methods section. () Killing effect of MSCs- TRAIL on vrious gliom cell lines. Control or MSCs-TRAIL were cocultured with U87, GBM39, GBM43, nd GBM12 expressing fluc t 1:.5 to 1:1 of gliom to MSC rtios. At 24 hours, luciferse ctivity in gliom cells ws mesured in triplictes. Two-wy nlysis of vrince followed y Bonferroni post hoc test ws utilized to evlute the difference etween control MSCs nd MSCs-TRAIL on the toxicity in gliom cell lines. (c) Chnges in cspse-3 expression in U87 nd GBM43 gliom cell lines followed y 8-hour coculture with control or MSCs-TRAIL were evluted y flow cytometry using rit nti cspse-3 ntiody. Smples were run in triplictes. Sttisticl difference mong groups ws evluted using two-wy nlysis of vrince followed y Bonferroni post hoc test. (d) Effect of intrnslly dministered MSCs-TRAIL on survivl of mice ering intrcrnil U87 gliom xenogrfts nd treted for 5 dys with 2 Gy gmm irrdition (totl dose 1Gy), n = 6/group. Survivl curves were generted y the Kpln Meier method, Logrnk test ws pplied to determine sttisticl difference on survivl time distriution etween groups. Dt re presented s men ± SEM. P <.5, P <.1, P <.1. MSCs, mesenchyml stem cells; RLU, reltive luciferse units. mrna expression, folds chnge c CXCL12 mrna expression, folds chnge DR4 Control DR5 2 Control Control Figure 6 Effect of irrdition on expression of TRAIL receptors nd the CXCL12 in U87 gliom xenogrfts. () Expression of mrna for DR4 nd DR5 in U87 gliom xenogrft tissue of control nd irrdited mice. () Expression of DR4 nd DR5 receptors in U87 gliom xenogrft tissue of control nd irrdited mice ws nlyzed using flow cytometry, nd expressed s men fluorescence intensity (MFI). Smples were nlyzed in triplictes. (c) Expression of mrna for CXCL12 in U87 gliom xenogrft tissue of control nd irrdited mice. (d) Expression of CXCL12 protein in U87 gliom xenogrft tissue of control nd irrdited mice ws nlyzed y ELISA. Smples were nlyzed in duplictes from ech control nd irrdited mice (n = 3). Student s t-tests hve een utilized to evlute the difference etween groups. Dt re presented s men ± SEM. P <.5. d Receptor expression, MFI CXCL12 protein expression, (pg/mg) mlgg Control DR4 DR5 Moleculr Therpy vol. 22 no. 1 jn

7 Therpeutic Effect of Intrnslly Delivered Stem Cells in Mouse Model of Gliom The Americn Society of Gene & Cell Therpy fter MSCs delivery, nd rther cn e explined y higher penetrtion of MSCs in the rin of irrdited nimls. Interestingly, we lso found fivefold increse in ccumultion of 111 In-leled MSCs in the cereellum of irrdited nimls. This finding is of importnce given the high frequency of cereellr rin tumors in children 33 nd wrrnts the evlution of IN delivery of therpeutic stem cells in experimentl model of medullolstom nd other cereellr tumors. Histologicl evlution of the rin tissues further confirmed the presence of MSCs in the gliom xenogrft nd djcent tissue of control nd irrdited nimls. The ccumulting evidence of the ility of stem cells to penetrte the rin vi the nsl cvity 12 15,32 mkes it prudent tht we re le to understnd the mechnism underlying this phenomenon. Elucidting this mechnism my llow us to increse the numer of stem cells tht rech the rin fter IN delivery. Recent reports indicte tht stem cells modified with iron prticles cn e trcked in the rin using MRI. 24,34 In our study, MSCs were leled with MPIOs in order to visulize their migrtion towrds tumor following IN delivery. Using MRI, we were le to detect MPIO-induced signls extending from the sl frontl loe towrds the tumor in control nd irrdited nimls fter IN inocultion of MSCs. To our knowledge, this is the first report utilizing MRI to confirm migrtion of IN delivered MSCs towrds gliom xenogrfts. Previously, methods of fluorescent microscopy, [H3]-thymidine nd ner-infrred live imging were utilized to trck MSCs in the rin fter IN delivery ,32 In ddition to the fct tht ech of these methods hs reltively limited sensitivity, more notly there is lck of clinicl relevnce nd pplicility. MRI studies monitoring the distriution of stem cells in the CNS will e necessry to evlute the fesiility of IN delivery of therpeutic stem cells for tretment of rin tumors nd other CNS disorders on clinicl level. In our study, we demonstrted tht IN delivered MSCs expressing TRAIL improved survivl of the nimls ering intrcrnil gliom xenogrfts in comintion with irrdition. It hs een shown tht rdition tretment cn sensitize cncer cells to TRAIL tretment nd upregultion of TRAIL receptors, DR4 nd DR5, hs een implicted in this process. 35,36 However, no chnge in the expression of DR4 nd DR5 in U87 gliom tissue upon rdition tretment ws detected suggesting tht different mechnism is responsile for the therpeutic effect of MSCs expressing TRAIL. Enhnced migrtion of MSCs towrds irrdited tumors hs een demonstrted y severl groups, 2,21,37 ut the exct mechnism of this tropism remins unknown. We found tht long with other fctors, CXCL12 (e.g., SDF-1) is upregulted fter the rdition tretment suggesting tht the SDF-1/CXCR4 xis nd other mechnisms my contriute to migrtion of MSCs into the rin nd towrds the gliom tissue in irrdited nimls. Recently, Kim nd couthors demonstrted tht IL-8 is involved in migrtion of umilicl cord lood-derived MSCs towrds irrdited U87 gliom cells in vitro. 21 In contrst, no upregultion in IL-8 gene expression in irrdited gliom xenogrft tissue ws found in our study, which might relte to differences in experimentl setting. Future studies should e crried out to elucidte the mechnisms regulting the migrtion of stem cell into the rin of tumor-ering nimls vi the nsl cvity. IN delivery of stem cells ppers to e promising pproch for therpy of CNS disese. This pproch shows clinicl nd prcticl dvntge over the intrcrnil ppliction of stem cells into resection cvity during surgery nd the convection-enhnced delivery, since it is not ssocited with the time of the surgery, is noninvsive in nture, nd llows for multiple tretment regiments. It lso cn void complictions ssocited with intrvsculr delivery s well s increse numer of stem cells delivered to the tumor site through the repeted ppliction of stem cells in nsl cvity. Moreover, this method could e lso potentilly utilized to tret ptients with inoperle rin tumors. Recently, two groups reported eneficil effect of unmodified MSCs delivered vi nsl cvity in rt models of ischemi nd Prkinson disese. 13,14 NSCs hve een shown to ccumulte in intrcrnil gliom xenogrfts fter IN delivery, 15 ut the uthors did not evlute preclinicl efficcy of IN delivered NSCs. Here, we demonstrted the therpeutic effect of MSCs-TRAIL in comintion with rdition tretment in intrcrnil orthotopic gliom model. We found tht only low numer of MSCs cn penetrte the rin vi nsl cvity in mice, which could e the limiting fctor for tretment of rin tumors vi IN delivery of stem cells. To overcome this limittion, stem cells could e mnipulted to increse their migrtion from nsl cvity into the rin y vrious mens. The overexpression of CXCR4 in MSCs hs een shown to enhnce in vivo moiliztion nd engrftment of MSCs into ischemic re of myocrdium. 38,39 Hypoxic preconditioning of MSCs hs een shown to improve their homing in ischemic re of the rin fter IN delivery. 4 Tretment of rt MSCs with insulin-like growth fctor-1 (IGF-1) hs een shown to induce n increse in MSCs migrtion in response to SDF-1 vi CXCR4 receptor signling. 41 Thus, understnding the mechnisms which drive the migrtion of stem cells will llow for the development of new strtegies to enhnce penetrtion of stem cells into the rin vi the nsl cvity nd ultimtely efficient migrtion towrds the tumor where they cn execute their therpeutic effect. In our study, we did not investigte the dynmics of the presence of therpeutic MSCs in the tumor urden. This knowledge would e necessry for development of pproches enhncing the homing of therpeutic MSCs in the tumor tissue fter their IN ppliction. Additionlly, nsl delivery of therpeutic stem cells for tretment of rin tumors hs to e evluted in solid nd infiltrtive gliom models closely resemling ptient tumors s well s in primtes, given known difference etween the rodent s nd the humn olfctory systems, 42 to mke this pproch cliniclly sound. Conclusions Our study hs demonstrted for the first time tht noninvsive IN delivery of MSCs is promising pproch for the therpeutic tretment of experimentl gliom. Using complementing methods, we were le to demonstrte (i) migrtion of MSCs into the rin nd towrds gliom xenogrfts fter IN ppliction, (ii) MSC presence in the tumor y histologicl evlution nd y MRI, nd (iii) improvement in survivl of nimls using TRAIL modified MSCs in comintion with rdition tretment. Ultimtely, further studies utilizing vrious therpeutic modlities in different niml models re necessry to vlidte IN delivery of stem cells s recognized pproch for the tretment of experimentl rin tumors vol. 22 no. 1 jn. 214

8 The Americn Society of Gene & Cell Therpy Therpeutic Effect of Intrnslly Delivered Stem Cells in Mouse Model of Gliom MATERIALS AND METHODS Cell lines. U87MG gliom cell line expressing EGFRvIII (mutnt Epiderml growth fctor receptor) ws kindly provided y Dr Frnk Furnri (the University of Cliforni t Sn Diego, Sn Diego, CA) nd is designted elsewhere in the text s U87 gliom cell line. GBM43, GBM12, nd GBM39 ptient derived gliom cell lines expressing firefly luciferse (fluc) were kindly provided y Dr Dvid Jmes (the University of Cliforni t Sn Frncisco, Sn Frncisco, CA). Humn one mrrow derived MSCs were provided y Dr Michel Mthis (the Louisin Helth Sciences Center, New Orlens, LA). Modifictions of MSCs to express fluc (MSCs-fluc) 43 nd memrne-ound TRAIL (MSCs-TRAIL) were performed s previously descried for NSCs. 44 Luciferse ssy. Killing effect of MSCs-TRAIL on gliom cells ws mesured using luciferse ssy. Briefly, of fluc-expressing gliom cells were cocultured with MSCs-TRAIL, nd in 24 hours, cells were lysed. To quntify migrtion of MSCs towrds tumor xenogrfts, U87 cells were implnted in the right hemisphere of nude mice. One week lter, MSCsfluc were inoculted in the left hemisphere of the rin nd mice were killed t vrious time points. The left nd right hemispheres of the rin were homogenized in equl volume of lysis uffer nd clered y centrifugtion. Luciferse ctivity in the cell lystes nd homogentes ws mesured per mnufcturer s recommendtion (Promeg, Mdison, WI). MSC leling with 111 In- oxine. The procedure for leling MSCs ws performed s previously descried 45 with slight modifictions. MSCs t 1 6 /ml were mixed with 5 μci of 111 In-oxine (GE Helthcre, Pittsurgh, PA) nd incuted for 1, 2 nd 3 minutes t 37 C in 5% CO 2. After two PBS wshes, cells were counted in gmm counter. The efficiency of the 111 In-oxine leling ws clculted s percentge of the lel retined y the cells reltive to the totl dded rdioctivity. MSCs leling with MPIOs. For leling of MSCs, MPIO prticles (Bngs Lortories, Fishers, IN) were dded to out 8% confluent MSC s culture in the presence of Fugene regent (Roche Applied Science, Indinpolis, IN) for overnight incution. For fluorescent microscopy, cells grown in L-Tek chmer (Thermo Scientific, Rochester, NY) slides were fixed with 4% prformldehyde nd counterstined with DAPI nucleic cid stin (Invitrogen, Grnd Islnd, NY). Slides were nlyzed using Olympus IX7 inverted microscope nd imges were otined using MetMorph softwre (Moleculr Devices, LLC, Sunnyvle, CA). The efficiency of MSCs leling with flsh red leled MPIOs ws determined y flow cytometry s percent of positive cells. Detection of MPIO-leled MSCs with Prussin Blue stining. For Prussin lue stining, frozen 16 μm rin sections were fixed in 4% prformldehyde nd fter three wshes with PBS were stined with potssium ferrocynide trihydrte (Sigm-Aldrich, St Louis, MO) nd counterstined with nucler Fst Red stin (Vector Lortories, Burlingme, CA) s descried. 34 Flow cytometry. Gliom cells cocultured with control or MSCs-TRAIL for 8 hours were stined for cleved cspse-3 s previously descried. 44 Smples were nlyzed using BD FACSCnto-A flow cytometer (BD Biosciences, Sn Jose, CA) nd FACSDiV 6.3 softwre version 6.3 (BD Biosciences, Sn Jose, CA). PCR-sed rry. Gliom xenogrft tissue ws processed for totl RNA purifiction using the RNesy Plus kit (Qigen, Vlenci, CA). For comprison of control nd irrdited smples, 2 μg of totl RNA ws treted to remove genomic DNA nd reverse trnscried using SABiosciences protocol developed for PCR-sed rrys. Humn Cytokines/Chemokines rry ws used in this study (SABiosciences, Vlenci, CA). PCR rry rection ws performed using Bio-Rd Opticon 2 system nd dt were nlyzed using SABioscience softwre (SABiosciences). In order to verify upregultion of gene of interest, quntittive PCR ws repeted using primers purchsed from RelTimePrimers (Rel Time Primers, LLC, Elkins Prk, PA). ELISA. CXCL12 expression in gliom xenogrft tissue ws determined using humn CXCL12/SDF-1 α Quntikine ELISA Kit (R&D Systems, Minnepolis, MN). Shortly, gliom xenogrft tissue ws lysed in M-Per protein extrction regent (Thermo Scientific, Hnover Prk, IL) in the presence of hlt protese inhiitors cocktil (Pierce, Rockford, IL). All smples were equilirted to pply equl mount of protein to ELISA pltes nd ound CXCL12 ws reveled per mnufcturer s recommendtion. The mount of CXCL12 in the smples ws mesured using clirtion curve nd the concentrtion of CXCL12 per mg of protein ws determined. TRAIL expression in MSCs ws mesured s previously descried. 44 Bioluminescent imging of MSCs in vivo. In order to visulize MSCsfluc in nimls, mice were injected intr peritonelly (i.p.) with 2 μl (4.5 mg per niml) D-luciferin sodium slt (Gold Biotechnology, St Louis, MO). In ddition, 3 μl of sustrte ws plced in ech nostril to help with detection of MSCs in the niml s nsl cvity. Signl ws recorded s photon counts using Xenogen IVIS 2 system. Locliztion of gliom cells expressing GFP ws lso detected using Xenogen IVIS 2 system. Trcking MPIO-leled stem cells in the rin of tumor-ering mice using MRI. MPIO-leled MSCs (1 1 6 ) were IN inoculted in control nd tumor-ering mice s descried in the niml studies section. Two dys lter, MR imges were cquired on 33 cm horizontl ore Bruker 9.4 T smll niml scnner with Bruker console (Bruker-Biospin, Billeric, MA) equipped with 12 cm shielded grdient set with mximum strength of 6 mt/m ville through the University of Chicgo Core Fcility. Mice were nesthetized with 2% isoflurne in oxygen nd fixed in prone position during scnning. In order to chieve sufficient resolution to visulize-leled MSCs within the mouse rin, multi-slice high resolution T 2 weighted, Rpid Acquisition with Relxtion Enhncement (RARE) spin echo imges (TR/TE effective = 4/26.6 ms, field of view (FOV) = 2.56 cm, mtrix size = , slice thickness =.5 mm, numer of excittion (NEX) = 2, RARE fctor = 4) were cquired in xil nd coronl plnes. Then multi-slice T 1 -weighted A Fst Low Angle Shot (FLASH) grdient echo sequence (TR/TE = 34/3.7 ms, flip ngle = 2, FOV = 2.56 cm, mtrix size = , slice thickness =.5 mm, NEX = 4) were lso cquired in xil nd coronl plnes. Animl studies. All nimls were mintined nd cred for in ccordnce the NIH Guide for the Cre nd Use of Lortory Animls nd the Institutionl Animl Cre nd Use Committee protocol. The U87-EGFRvIII gliom xenogrfts re known to e more ggressive thn prentl U87MG gliom cells in n in vivo model 46,47 nd, therefore, were used s gliom model. Intrcrnil xenogrfts were estlished s previously descried. 44 One week lter, nesthetized nimls were plced in supine position nd nsl cvity of ech niml ws treted with totl of 1 U of hyluronidse 12 s four repeted inocultions with 5-minute intervls (3 μl in ech nostril) fter which either sterile PBS, or of MSCs (3 μl in ech nostril with 5-minute intervls) were pplied for four times. This tretment ws repeted weekly for totl four times through the course of experiment. For irrdition, the entire ody of nimls excluding the hed ws covered with led shields nd exposed to 2 Gy of dily dose of rdition for 5 consecutive dys. Twenty four hours lter, nimls were treted either with sterile PBS, or of MSCs s descried ove. All mice were followed to ssess their survivl. In order to understnd distriution of MSCs in control nd irrdited nimls, nimls were killed 24 hours lter fter IN ppliction 111 In-oxine leled MSCs (1 6 per niml); rin nd other orgns were extrcted nd rdioctivity ws mesured using gmm counter. Sttisticl nlysis. Differences etween groups were evluted using prmetric or nonprmetric Student s t-test or two-wy nlysis of vrince followed y Bonferroni s post hoc correction s pproprite. For the in vivo dt, survivl curves were generted y the Kpln Meier method Moleculr Therpy vol. 22 no. 1 jn

9 Therpeutic Effect of Intrnslly Delivered Stem Cells in Mouse Model of Gliom The Americn Society of Gene & Cell Therpy nd the log-rnk test ws pplied to compre the distriutions of survivl times. All reported P vlues were two-sided nd were considered to e sttisticlly significnt t <.5. SUPPLEMENTARY MATERIAL Figure S1. Leling of MSCs with MPIOs. Figure S2. Effect of MSCs-TRAIL on survivl of mice ering intrcrnil U87 gliom xenogrfts. Tle S1. Chnges in mrna expression for selected cytokines nd chemokines in U87 gliom xenogrft tissue of nude mice fter irrdition. Video S1. Migrtion of MPIO-leled MSCs towrds the tumor through rin prenchym. ACKNOWLEDGMENTS This work ws supported y the Els U. Prdee Foundtion (I.V.B.), ACS IL. Div. (I.V.B.) nd in prt y the NIH grnts R1 CA (M.S.L.), NIH R1 CA12293 (M.S.L.), NIH R1 NS77388 (M.S.L.), NIH U1 NS69997 (M.S.L.), y pilot reserch funding provided y the Virgini nd D. K. Ludwig Fund for Cncer Reserch vi the Imging Reserch Institute in the Biologicl Sciences Division of the University of Chicgo. K.S.A. is chief scientific officer t TherBiologics. The other uthors declre no conflict of interest. REFERENCES 1. Legler, JM, Ries, LA, Smith, MA, Wrren, JL, Heinemn, EF, Kpln, RS et l. (1999). Cncer surveillnce series [corrected]: rin nd other centrl nervous system cncers: recent trends in incidence nd mortlity. J Ntl Cncer Inst 91: Stupp, R, Mson, WP, vn den Bent, MJ, Weller, M, Fisher, B, Tphoorn, MJ et l.; Europen Orgnistion for Reserch nd Tretment of Cncer Brin Tumor nd Rdiotherpy Groups; Ntionl Cncer Institute of Cnd Clinicl Trils Group. (25). Rdiotherpy plus concomitnt nd djuvnt temozolomide for gliolstom. N Engl J Med 352: Serwer, LP nd Jmes, CD (212). Chllenges in drug delivery to tumors of the centrl nervous system: n overview of phrmcologicl nd surgicl considertions. Adv Drug Deliv Rev 64: Lee, DH, Ahn, Y, Kim, SU, Wng, KC, Cho, BK, Phi, JH et l. 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