completeregressionofmetastaticrenalcellcarcinomaby ...using the adeno-x rapid titer kit (clontech)....

Cancer Therapy: Preclinical

Complete Regression of Metastatic Renal Cell Carcinoma byMultiple Injections of Engineered Mesenchymal Stem CellsExpressing Dodecameric TRAIL and HSV-TK

Sae Won Kim1, Su Jin Kim1, Sang Hoon Park1, Hyun Gul Yang1, Moon Cheol Kang1, Young Woo Choi1,Seong Muk Kim2, Sin-Soo Jeun2, and Young Chul Sung1

AbstractPurpose:Durable complete remission ofmetastatic renal cell carcinoma (RCC) has rarely been achieved

with current treatment modalities. To solve this problem, alternative therapeutic options with high efficacy

and minimal side effects are strongly needed.

Experimental Design:Mesenchymal stem cells (MSC)were engineered to coexpress dodecameric TRAIL

and herpes simplex virus thymidine kinase (MSC/dTRAIL-TK). The antitumor effects of MSCs expressing

dTRAIL (MSC/dTRAIL) or HSV-TK alone (MSC/TK) andMSC/dTRAIL-TKwere compared withmurine RCC

cells using in vitro coculture system and in vivo experimental lung metastasis model. The effects of different

doses and schedules of engineered MSCs on mice survival were also evaluated.

Results: MSC/dTRAIL-TK exerted stronger apoptotic response in Renca cells than did MSC/TK or MSC/

dTRAIL after ganciclovir (GCV) treatment. In vivo imaging results suggest that MSCs reside longer in the

lungs of metastatic tumor-bearing mice, compared with that of control mice, regardless of genetic

engineering. In addition, MSC/dTRAIL-TK treatment followed by ganciclovir administrations significantly

decreased the number of tumor nodules in the lung, to a greater degree than MSC/dTRAIL or MSC/TK, and

led to a prolonged survival. More importantly, the antimetastatic effect of MSC/dTRAIL-TK was markedly

enhanced by repeated injections but not by increased dose, and resulted in 100% survival of tumor-bearing

mice after three injections.

Conclusion: Sequential combination gene therapy usingMSC/dTRAIL-TK achieved long-term remission

of metastatic RCC without noticeable toxicity. Our findings provide an innovative therapeutic approach to

completely eradicatemetastatic tumors by simple, repeated administrations ofMSC/dTRAIL-TK.ClinCancer

Res; 19(2); 415–27. �2012 AACR.

IntroductionRenal cell carcinoma (RCC) is the eighth most common

type of cancer and the sixth leading cause of cancer-related

death (1, 2). Approximately 30% of patients with RCCpresent with metastatic disease and the 5-year survival rateof these patients is less than 10% (3, 4). Despite the recentadvances in understanding genetic and epigenetic eventsinvolved in the metastasis of RCC, current therapies haveshown limited success in improving the overall survival ofpatients with metastatic RCC. As RCC generally possessesinherent resistance to chemotherapy and radiotherapy (5),other treatment modalities, such as cytokine-based immu-notherapy or targeted therapy usingmultikinase inhibitors,have been actively investigated in the clinic. Immunothera-pies using interleukin-2 (IL-2) or IFN-a have shown ben-eficial effects in some clinical settings, and even completeremissions in small cohort of patients using high-dose IL-2,but low response rates and systemic toxicities have limitedtheir clinical use (6). Tyrosine kinase inhibitors, includingsunitinib and sorafenib, have substantially improved theoverall survival of patients with RCC, but complete remis-sion has rarely been achieved (7). Therefore, there remains aneed to develop potent therapeutic agents for metastaticRCC that can induce high response rates with minimal sideeffects, and most importantly, durable complete responses.

Authors' Affiliations: 1Division of Molecular and Life Science, IntegrativeBioscience & Biotechnology, Pohang University of Science and Technol-ogy (POSTECH), Pohang, Gyungbuk; and 2Department of Neurosurgery,Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Repub-lic of Korea

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

S.W. Kim and S.J. Kim contributed equally to this work.

Corresponding Authors:Young Chul Sung, Division of Molecular and LifeSciences, Integrative Bioscience & Biotechnology, Pohang University ofScience and Technology (POSTECH), San 31, Hyoja-dong, Nam-ku,Pohang 790-784, Republic of Korea. Phone: 82-54-279-2294; Fax: 82-54-279-5544; E-mail: [email protected]; and Sin-Soo Jeun, Depart-ment of Neurosurgery, Seoul St.Mary'sHospital, TheCatholic University ofKorea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Republic of Korea.Phone: 82-2-2258-7535; Fax: 82-2-3482-1853; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-12-1568

�2012 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 415

on February 14, 2020. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 30, 2012; DOI: 10.1158/1078-0432.CCR-12-1568

http://clincancerres.aacrjournals.org/

Mesenchymal stem cells (MSC) have recently emerged aspotential therapeutic agents and cellular vehicles for gene/drug delivery, and their therapeutic efficacy is currentlybeing assessed in various disease models including inflam-matory and autoimmune diseases and cancer. Two uniqueproperties of MSCs provide them advantages over othercellular delivery vehicles, especially in themetastatic setting:(i) tumor tropic capabilities of MSCs enable target-specificdelivery of therapeutic agents (8), which can be especiallybeneficial when surgical resection is not indicated becauseof the metastatic dissemination of cancer cells to otherorgans, and (ii) hypoimmunogenic properties of MSCsallow sustained release of therapeutic molecules withouteliciting unwanted immune response, even in the allogeneicor xenogeneic settings (9). The efficacy and safety of MSCsas delivery vehicles were shown in preclinical modelsof RCC, in which human bone marrow–derived MSCstransduced with adenovirus encoding IL-12 (10) or thoseloaded with conditionally replicative oncolytic adenovirus(11) were used to generate tumor site-specific antitumoreffects in subcutaneous and orthotopic xenograft models,respectively.

Herpes simplex virus thymidine kinase (HSV-TK)–medi-ated suicide gene therapy has been widely accepted strategyfor cancer. HSV-TK converts the nontoxic nucleoside analogganciclovir (GCV) into a toxic triphosphorylated form,which can subsequently induce the apoptosis of HSV-TK–transduced cells as well as that of rapidly dividingbystander cells (12).Onepotential drawback of this strategyis that it fails to destroy slow-proliferating or quiescenttumor cell population, which may cause tumor recurrence.To achieve complete eradication of malignant tumor andprevent tumor relapse, HSV-TK/GCV therapy must be usedin combination with other therapeutic agents that can

eliminate residual tumor cells while enhancing the efficacyof suicide gene therapy.

TRAIL is a homotrimeric, type II transmembrane proteinthat induces apoptosis through both intrinsic and extrinsicdeath pathways (13). Because TRAIL is known to induceselective apoptosis on transformed cells without affectingnormal cells, it has been regarded as a promising candidatefor the treatment of cancer. Clinical applications of recom-binant TRAIL, however, have been hampered by its short invivo half-life and intrinsic instability (14). To overcome itslimited efficacy, viral vectors or MSCs were engineered toexpress TRAIL (15, 16), or agonist monoclonal antibodiestargeting TRAIL receptors (17) were developed. One of thepotential disadvantages of agonistic antibodies, proposedby a recent study, is that their bivalent feature may reducetherapeutic efficacy and mimicking trimeric structure ofnative TRAIL is crucial for eliciting full biologic activity ofagents (18). Another strategy to enhance therapeutic effectsof TRAIL is the addition of other treatment modalities forcombination therapy. A previous study showed that thecombination of agonistic anti-TRAIL receptor antibodywith adenovirus-mediated cytosine deaminase/5-fluorocy-tosine suicide gene therapy produces additive antitumoreffects in vivo using human glioma and pancreatic carcino-ma xenograft model (19). It was also found that TRAIL canenhance the efficacy of HSV-TK/GCV therapy by augment-ing both target and bystander killing effect (20), furthersupporting the potential clinical use of combined treatmentwith HSV-TK/GCV gene therapy and TRAIL-based therapy.

In thepresent study,we analyzed the therapeutic effects ofMSC-mediated combination gene therapy of TRAIL andHSV-TK inmurine Renca RCC experimental lungmetastasismodel. This model was designed to recapitulate postoper-ative condition in human patients who have not yet devel-oped macroscopically detectable metastatic tumors in thelung—the most common site for RCC metastasis (21). Theuse of MSCs is especially advantageous in such settings, astumor tropic capacity of MSCs allows them to search anddestroy undetectable tumor cells. Furthermore, the use ofsyngeneic transplantable model enabled proper tumor–host interaction to take place. As RCC (also Renca) isregarded as an immunogenic tumor, the induction oftumor-specific immune responses by MSC therapy wasanother important parameter for evaluation of therapeuticefficacy. After establishing in vivomodel system, we assessedantitumor effects of engineered MSCs coexpressing dode-cameric TRAIL andHSV-TK (MSC/dTRAIL-TK). dTRAIL wasused inplace of conventional trimeric TRAIL (tTRAIL) basedon its superior apoptosis-inducing ability. EngineeredMSCs were found to be specifically localized in the lungupon in vivo administration, in which secreted TRAIL andHSV-TK in conjunction with ganciclovir acts to exertenhanced killing activity against target tumor cells. Moreimportantly, multiple injections of MSC/dTRAIL-TK at alow dose exhibited stronger antimetastatic effects thansingle injection at a high dose, resulting in 100% survivalof tumor-bearing mice after triple injections. Althoughclinical application of optimistic results from animal

Translational RelevanceAmajor hurdle in the treatment ofmetastatic renal cell

carcinoma (RCC) is the lack of therapeutic agents thatcan induce durable complete remission without causingsystemic toxicities. In this study, we achieved completeelimination of established metastatic RCC in 100% ofmice bymultiple injections of xenogeneic mesenchymalstem cells (MSC) coexpressing dodecameric TRAIL(dTRAIL) and herpes simplex virus thymidine kinase(HSV-TK) followed by ganciclovir (GCV) administra-tions. The use of MSCs as a delivery vehicle allowed site-specific secretion of therapeutic molecules into tumorsites, thereby minimizing any side effects. Moreover, thecombination of dTRAIL and HSV-TK/GCV causedincreased apoptosis of tumor cells as well as MSCs,resolving safety concerns about malignant transforma-tion of therapeutic stem cells. Our findings propose asafe and effective strategy to achieve long-term remis-sions ofmetastatic RCCby simple, repeated injections ofMSCs coexpressing dTRAIL and HSV-TK.

Kim et al.

Clin Cancer Res; 19(2) January 15, 2013 Clinical Cancer Research416




models should be taken cautiously, this study shows thatmultiple injections of MSC/dTRAIL-TK could be an attrac-tive therapeutic approach for completely curing RCCpatients with distant metastasis.

Materials and MethodsCellsRat bone marrow–derived MSCs were prepared as previ-

ously described (9). MSCs were cultured in low glucoseDulbecco’s modified Eagle’s medium (Welgene) supple-mented with 10% FBS and 1� antibiotic-antimycotic (Invi-trogen). MSCs, in passages 5 through 6, were used for allexperiments. RENCA murine renal carcinoma cell line and4T1murine breast carcinoma cell line were purchased fromAmerican Type Culture Collection and maintained inRPMI-1640 (Welgene) with 10% FBS and antibiotic-anti-mycotic (Invitrogen).

Adenoviruses and transductionThe DNA sequences encoding dTRAIL, tissue plasmin-

ogen activator (tPA) secretion signal sequence, surfactantprotein D (SPD) dodecamerization domain, and extra-cellular domain of human TRAIL were linked into a DNAcassette, as shown in Fig. 1A. As a control, the isoleucinezipper (ILZ) trimerization domain was used to constructtTRAIL (22). To construct a DNA cassette encoding bothdTRAIL and HSV-TK, an internal ribosome entry site wasinserted between dTRAIL and HSV-TK. All codons oftransgenes were optimized to increase expression levels.Recombinant replication-deficient adenoviruses withouttransgene (rAd/Mock) or encoding enhanced GFP (rAd/EGFP), tTRAIL (rAd/tTRAIL), dTRAIL (rAd/dTRAIL), HSV-TK (rAd/TK), or both dTRAIL and HSV-TK (rAd/dTRAIL-TK), were produced using the AdEasyTM Vector System(QBioGene) as previously described (23) and titratedusing the Adeno-X Rapid Titer Kit (Clontech). All rAdconcentrations used for the transduction of cells areprovided as the multiplicity of infection (MOI) in pla-que-forming unit per cells (PFU/cell). For MSC transduc-tion, a mixture of 50 MOI of rAd and 50 mmol/L of Fe3þ

was preincubated in serum-free medium at room tem-perature for 30 minutes, and then infected into MSCs for30 minutes. Transduction efficiency of adenovirus inMSCs was about 80%.

Apoptotic protein expression and apoptosis signalingassayToquantify the level of secreted TRAIL fromMSC/dTRAIL

or MSC/tTRAIL, culture supernatants were analyzed byELISA using purified anti-human TRAIL antibody (Pepro-tech) and biotinylated anti-human TRAIL antibody (Pepro-tech), according to the manufacturer’s instructions. Tomeasure the concentration of TRAIL secreted from MSC/dTRAIL-TK or MSC/dTRAIL upon ganciclovir treatment,culture supernatants were harvested every day and changedwith addition of fresh ganciclovir. Collected supernatantwere subjected to ELISA as described earlier.

To analyze the gene expression of HSV-TK, reverse tran-scriptionPCRassaywas conducted usingprimers for codon-optimized HSV-TK cDNA, (forward, 50-GCCTTCGAC-CAGGCCGCTAG-30 and reverse, 50-CCATGCCGTGGGG-TCCATCG-30).

To analyze expression of apoptotic signaling molec-ules, tumor cells transduced with rAd/mock, rAd/dTRAIL,rAd/TK, or rAd/dTRAIL-TK were lysed in radioimmuno-precipitation assay (RIPA) buffer [25 mmol/L Tris–HClpH 7.6, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP-40,1% sodium deoxycholate, 0.1% SDS, 1 mmol/L dithio-threitol, 1 mmol/L phenylmethylsulfonylfluoride (PMSF),and 1 mmol/L Na3VO4] supplemented with CompleteProtease Inhibitor Cocktail Tablets (Roche). After titration,protein expression was analyzed by conventional Westernblot analysis using mouse anti-human TRAIL antibody(Peprotech), caspase-3, caspase-8, PARP, cytochromec (Cell Signaling Technology), or b-actin antibody (SantaCruz Biotechnology).

In vitro viability assayTo measure the effects of dTRAIL or tTRAIL on MSC

viability, MSC were transduced with different doses (0,10, 50, and 100 MOI) of rAd/mock, rAd/tTRAIL, or rAd/dTRAIL and allowed to grow for 3 days. Cell viability wasmeasured by a CellTiter 96 AQueous One Solution CellProliferation assay (Promega). Percentage survival was cal-culated as [the absorbance at 490 nm (A490 nm) of MSC/dTRAIL or MSC/tTRAIL � A490 nm of blank wells]/(A490 nm

of MSC/mock � A490 nm of blank wells) � 100.To evaluate ganciclovir cytotoxicity on engineered MSCs,

various concentrations of ganciclovir (0, 10, 30, and 100mmol/L) were used to treat MSC/dTRAIL-TK, MSC/dTRAIL,MSC/TK, and MSC/mock. Cell viability was measured atday0, 1, 3, 5, and7andpercentage survivalwas calculated asdescribed earlier.

In vitro coculture assayMSC/tTRAIL, MSC/dTRAIL, and MSC/Mock were label-

ed with 20-mmol/L carboxyfluorescein succinimidyl ester(CFSE, Invitrogen), and then directly cocultured withRENCA or 4T1 tumor cells at a ratio of 1:1 for 48 hours.All floating and adherent cells were harvested, stainedwith 5 mL of allophycocyanin-conjugated Annexin V (eBio-science) in 100 mL binding buffer, and then analyzed byGallios flow cytometer (Beckman Coulter) to determinethe proportion of apoptotic tumor cells. In a separateexperiment, CFSE-labeled MSC/dTRAIL-TK, MSC/dTRAIL,MSC/TK, and MSC/Mock were cocultured with RENCAcells at a ratio of 1:1 for 48 hours. After further incubationwith 100mmol/L of ganciclovir for 48 hours or 72hours, theproportion of apoptotic cells was analyzed as describedearlier.

Animal model and in vivo MSC migration assayFemale BALB/C (5–7-week old) mice were purchased

from Jackson Laboratory. To establish experimental lungmetastases, 5 � 105 RENCA cells were intravenously

Complete Cure of Metastatic Tumor by Engineered MSCs

www.aacrjournals.org Clin Cancer Res; 19(2) January 15, 2013 417




Figure 1. Relative expression levels and apoptotic potential of tTRAIL and dTRAIL expressed by engineered MSCs. A, schematic diagram of DNA constructsencoding EGFP, tTRAIL, dTRAIL, dTRAIL-TK, or thymidine kinase. tPA-S, tissue plasminogen activator signal sequence; IRES, internal ribosome entry site. B,TRAIL expression ofMSC/tTRAIL andMSC/dTRAILwas analyzed byWestern blot analysis using cell lysates. C,MSCswere transducedwith increasing titersof rAd/tTRAIL or rAd/dTRAIL in the presence or absence of Fe3þ (50mmol/L) for 30minutes. TRAIL expression levelwas analyzedbyELISA, using supernatant,at 24 hours after transduction. ��, P < 0.01. D, At 3 days after transduction of MSCs with rAd/tTRAIL or rAd/dTRAIL, MSC cell viabilities were determined byCellTiter 96 Aqueous One Solution cell proliferation assay. Data represent average � SEM of 3 independent experiments. E and F, MSC/tTRAIL or MSC/dTRAIL were cocultured with Renca (E) or 4T1 cells (F) at a ratio of 1:1 for 48 hours. The percentages of apoptotic cells were then quantified by Annexin Vstaining. Data represent average percentage � SEM of 3 independent experiments.

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injected into the lateral tail vein. To examine the in vivodistribution of intravenously injected MSCs, 5� 105 CFSE-labeledMSCswere injected into the tail veinof na€�ve controlormetastatic tumor-bearingmice.Micewere sacrificed at 24hours after MSC injection, and then various organs (lung,liver, spleen, heart, kidney, and brain) were isolated. Eachorgan was minced, treated with collagenase, and preparedfor flow-cytometric analysis. CFSE-positive cells in eachorgan were analyzed using a Gallios flow cytometer (Beck-man Coulter). To analyze the localization of MSCs in thelung, MSCs were labeled with 5 mmol/L CM-DiI (MolecularProbes) and 5 � 105 cells were intravenously injected intona€�ve control mice or tumor-bearing mice on day 7 aftertumor injection. Mice were sacrificed on day 14 and lungtissues were embedded in paraffin blocks for histologicanalysis. The labeled MSCs were visualized using a ZeissAxiovert1135 microscope and AxioVision3 software. For invivo imaging, MSCs were labeled with NEO-LIVE Magnox-ide 675 (BITERIALS) and intravenously injected into na€�veor metastatic tumor-bearing mice on day 7 after tumorinjection. Because ventral images can produce abdominalautofluorescence (24), dorsal images ofmice were obtainedusing Maestro (CRi) after being anesthetized. All MSCmigration-related assays described earlier were conductedin the absence of ganciclovir administration.

In vivo antitumor assaysTo investigate antitumor effects of engineered MSCs in

metastatic renal cell carcinoma model, mice were intrave-nously injectedwithMSC/Mock,MSC/TK,MSC/dTRAIL, orMSC/dTRAIL-TK (5 � 105 cells) on day 1 or 7 after RENCAcell injection. Then, 50 mg/kg of ganciclovir was intraper-itoneally injected for 7 consecutive days starting on day 0, 1,2, or 3 afterMSC treatment.Onday14 after tumor injection,lungs of tumor-bearing mice were isolated, stained withpicric acid, fixed in acetic acid solution, and the number ofmetastatic tumor nodules on the lung surface was counted.Survival of tumor-bearing mice was monitored up to 100days posttumor injection.To investigate the effects of different doses of MSC/

dTRAIL-TK on the survival of metastatic tumor-bearingmice, MSC/dTRAIL-TK (5 � 105 cells, 1 � 106 cells, or1.5 � 106 cells) or MSC/EGFP (5 � 105 cells) were intra-venously injected into tumor-bearing mice on day 7 aftertumor injection. Antitumor effects of sequential therapywere evaluated by repeating second and third injections of 5� 105 cells MSC/dTRAIL-TK at 2-week interval. Ganciclovirwas administered for 7 consecutive days starting on day 2after each MSC/dTRAIL-TK administrations. Survival oftumor-bearing mice was monitored up to 125 days post-tumor cell injection.

StatisticsAll data were expressed as average � SEM. To measure

the statistical differences between groups, a 2-tailed Studentt test was used. For all statistical tests, a P value of less than0.05 was considered to be statistically significant.

ResultsDodecameric form of TRAIL induces higher levels oftumor cell apoptosis than the trimeric form of TRAIL

To investigate the effects of the dodecamerization ofTRAIL on its apoptosis-inducing activity, we generated rAdexpressing either dTRAIL or tTRAIL, as described in Materi-als and Methods (Fig. 1A). The expressions of dTRAIL andtTRAIL protein from transduced MSCs were confirmed byWestern blot analysis (Fig. 1B). When MSCs were trans-duced with rAd/dTRAIL or rAd/tTRAIL, the inclusion offerric ion (Fe3þ) significantly enhanced the expression ofboth dTRAIL and tTRAIL by 10- to 100-fold. The amount ofsecreted TRAIL from MSC/dTRAIL in culture supernatantwas about 3-fold higher than that from MSC/tTRAIL (Fig.1C). As the titer of rAd increased, the expression levels ofTRAIL also increased, but the viability of MSCs was nega-tively affected by the viral doses of greater than 100 MOI(Fig. 1D). Decreased MSC viability was unlikely to becaused by high levels of secreted TRAIL, as the extent ofdecrease in cell survival was comparable between MSC/tTRAIL and MSC/dTRAIL despite the difference in theirTRAIL induction. This result is supported by a previousobservation that MSCs are resistant to TRAIL-mediatedapoptosis (25). On the basis of these results, 50 MOI ofrAd in the presence of Fe3þ was determined to be theoptimal condition for MSC transduction, which did notaffect the phenotype of MSCs (Supplementary Fig. S1).

To examine the cytotoxic effects of TRAIL-expressingMSCs on tumor cells, CFSE-labeled MSC/dTRAIL, MSC/tTRAIL, and MSC/mock (control) were cocultured withRENCA mouse renal cell carcinoma or 4T1 mouse breastcancer cell lines and the proportion of apoptotic cells wasdetermined. MSC/dTRAIL induced stronger apoptoticresponses in RENCA and 4T1 cells, which were known tobe sensitive to TRAIL-mediated apoptosis (Fig. 1E and F).MSC/dTRAIL treatment resulted in higher level of apoptosisin RENCA and 4T1 cells than MSC/tTRAIL treatment. It isworth noting that a half dose of MSC/dTRAIL exertedstronger killing activity against Renca cells than MSC/tTRAIL (Supplementary Fig. S2).

dTRAIL and HSV-TK exert combinatory effects inapoptosis induction of both transduced MSCs andtarget tumor cells

To evaluate the combinatorial effects of dTRAIL andHSV-TK in killing of transducedMSCs as well as bystander tumorcells, we first generated MSCs coexpressing dTRAIL andHSV-TK (MSC/dTRAIL-TK) or HSV-TK alone (MSC/TK).The expression of HSV-TK was confirmed by reverse tran-scription PCR (Fig. 2A). The cytotoxic effects of gancicloviron engineered MSCs were analyzed by measuring theviability and TRAIL secretion from transduced cells. Asshown in Fig. 2B, ganciclovir treatment induced the celldeath of thymidine kinase–expressing MSCs (MSC/dTRAIL-TK and MSC/TK) in a dose-dependent manner butnot in dTRAIL-expressing MSCs. Furthermore, MSC/dTRAIL-TK were found to be more susceptible to






ganciclovir-induced apoptosis thanMSC/TK. As 100-mmol/L ganciclovir could achieve nearly complete destruction ofMSC/dTRAIL-TK after 7 days of treatment (percentage ofremaining MSCs ¼ 1.02% � 0.58% for MSC/dTRAIL-TKand 6.94% � 1.74% for MSC/TK), this dosage was selectedfor the following in vitro coculture experiment. Consistent-ly, TRAIL secretion from MSC/dTRAIL-TK was significantlyreduced by ganciclovir treatment but not from MSC/dTRAIL (Fig. 2C).

To investigate whether the combination of dTRAIL andHSV-TK could exhibit enhanced apoptogenic effects onbystander tumor cells, CFSE-labeled engineered MSCs werecocultured with RENCA cells for 48 hours and treated with100 mmol/L of ganciclovir for additional 48 or 72 hours. Atboth time-points, MSC/dTRAIL-TK exhibited stronger apo-ptotic effects than the sum of effects from MSC/TK andMSC/dTRAIL in RENCA cells (Fig. 3A). It is also worthnoting that decreasing the number of MSC/dTRAIL-TK by75% in coculture still induced similar level of tumor cellapoptosis to that ofMSC/TK (datanot shown). Aprior studyalso showed that TK-expressing MSCs could induce celldeathof 10 timesmore tumor cells completely viabystandereffect in coculture assay (26). Fluorescencemicroscopy alsoindicated that the highest level of RENCA cell death wasobserved in coculture with MSC/dTRAIL-TK (Fig. 3B). Toinvestigate themolecularmechanisms involved in the com-binatory action of dTRAIL and HSV-TK, we analyzed theexpression levels of apoptosis-related signaling molecules,including caspase-3, caspase-8, cytochrome c, and PARP.Expression levels of all the molecules tested were signifi-cantly increased by coexpression of dTRAIL and HSV-TK(Fig. 3C), which is consistent with the previous reportthat concerted activation of caspases is responsible for the

cooperative antitumor effects between HSV-TK/GCV andTRAIL (20).

MSC/dTRAIL-TK significantly regresses metastatictumors and prolongs mice survival, to a greater extentthan MSC/dTRAIL or MSC/TK

To evaluate tumor site-specific localization of MSCsand their duration of residence in vivo, several experimen-tal approaches were used. First, CFSE-labeled engineeredMSCs (E-MSC that represents MSC/dTRAIL-TK) wereintravenously injected into na€�ve or tumor-bearing miceat day 1 or 7 after RENCA cell administration. Twenty-fourhours after MSC injection, a greater number of engineeredMSCs were detected in the lungs of tumor-bearing micethan those of na€�ve mice (Fig. 4A). In addition, a higherfrequency of na€�ve or engineered MSCs was detected in thelungs of mice bearing 7-day-old tumors than those with1-day-old tumors. These results suggest that higher levelsof tumor-derived chemokines in the lung may cause moreMSCs to be attracted to the tumor sites. Second, in vivoimaging analysis was conducted to assess the duration ofMSC residence in the lung. As shown in Fig. 4B, muchstronger fluorescent signals emitted by labeled MSCs weredetected in the lungs of tumor-bearing mice than in thoseof na€�ve mice throughout the entire observation period.Finally, to determine whether MSCs could migrate pre-ferentially toward tumor nodules within the lung tissue,the localization of labeled MSCs was investigated byhistologic analysis on day 7 after MSC injection. Interest-ingly, a greater number of DiI-labeled MSCs was found inthe vicinity of tumor nodule sites compared with non-nodule sites in the lungs of tumor-bearing mice (Fig. 4C).Similar results were reported by a previous report that

Figure 2. Effects of ganciclovir (GCV) treatment on cell death and TRAIL expression in engineered MSCs. A, expression of HSV-TK in engineered MSCs wasanalyzed by reverse transcription PCR at 48 hours after transduction of MSCs. B, various concentrations (0, 10, 30, or 100 mmol/L) of ganciclovir were treatedto engineered MSCs, and then cell viability was measured by CellTiter 96 Aqueous One Solution cell proliferation assay. C, TRAIL secretion by engineeredMSCs was analyzed at indicated time-points after ganciclovir treatment using ELISA assay. Data represent average � SEM of 4 independent experiments.

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examined the homing of syngeneic MSCs in mice bearingRenca pulmonary metastasis. In this study, histologicanalysis of lungs showed that MSCs tended to surroundtumor nodules when their diameter is large and infiltratenodules when their diameter is small (27). Althoughxenogeneic MSCs were used in our experimental system,cells that reached the tumor nodules seemed to survivelonger than those did not, as suggested by the reportmentioned earlier.Next, we aimed to optimize the time-points for ganci-

clovir and engineered MSC treatment to maximize thetherapeutic efficacy. First, we treated mice with gancicloviron day 0, 1, 2, or 3 after MSC/dTRAIL-TK injection andanalyzed the number of metastatic tumor nodules at 14days after tumor injection. Ganciclovir treatment that wasinitiated on the same day with MSC/dTRAIL-TK adminis-tration induced moderate antitumor effects, presumably

due to rapid MSC killing (Fig. 5A). On the other hand,ganciclovir treatment initiated on day 2 maximized thecombinatory effects of dTRAIL and HSV-TK, as presentedby the fewest number of metastatic nodules, indicatingthe dependence of efficacy upon time-point of ganciclovirinjection. Thereafter, ganciclovir treatment was initiatedon day 2 after MSC/dTRAIL-TK therapy for the followingin vivo experiments. Second, we compared the antimeta-static effects of engineered MSCs (MSC/TK, MSC/dTRAIL,MSC/dTRAIL-TK, orMSC/EGFP) administered onday1or 7after RENCA cell injection. Treatment ofMSC/TK andMSC/dTRAIL, either on day 1 or 7, elicited similar antitumoractivities as shown by about a 50% decrease in the nodulenumber in comparison with MSC/EGFP–treated mice (Fig.5B). However, although statistically insignificant, MSC/dTRAIL-TK therapy on day 7 achieved more than 90%regression, showing superior antimetastatic effects

Figure 3. Combinatory effect of dTRAIL andHSV-TK on apoptotic induction in Renca cells. A, Renca cells were coculturedwith engineeredMSCs labeledwithCFSE at a ratio of 1:1 for 48 hours and then treated with 100 mmol/L of ganciclovir. At 48 hours or 72 hours after ganciclovir addition, all floating and attachedcells were harvested. The percentages of apoptotic cells were quantifiedbyAnnexin V staining. Data represent average�SEMof 4 independent experiments.B, Renca cells were labeled with Calcein-AM (green) and coculturedwith CM-DiI-labeledMSCs (red) for 48 hours, and treated with 100-mmol/L ganciclovir foranother 48 hours. Labeled cells were visualized by fluorescencemicroscopy. Scale bar, 100 mm. C, Renca cells transduced with various rAd constructs wereincubated in the presence of 100 mmol/L ganciclovir for 48 hours. Expression levels of apoptosis-related molecules (such as caspase-3, caspase-8, PARP,and cytochrome c) in Renca cells were analyzed using Western blot analysis. b-actin was used as an internal control.






compared with day 1 therapy. Therefore, engineered MSCadministration was started on day 7 after tumor injectionfor the following in vivo experiments.

After optimizing ganciclovir and engineered MSC treat-ment time-points, therapeutic efficacy of engineered MSCswere reevaluated in the survival experiment. Consistent

Figure 4. Tumor-tropism of naïve or engineered MSCs. A, at 1 or 7 days after intravenous injection of Renca cells into BALB/C mice, CFSE-labeled naïve orengineeredMSCs expressing dTRAIL and TK (E-MSC) were intravenously injected into naïve or tumor-bearing mice. Percentages of CFSE-positive MSCs inthe lungor liver tissueswere analyzedby fluorescence-activated cell sorting (FACS) at 24 hours afterMSC injection. Data representmean values observed in 2separate experiments. ��,P < 0.01. B, on day 7 after intravenous injection of Renca,magnoxide 675–labeledMSCswere intravenously injected. ThemigrationofMSCswas analyzed by in vivo imaging system under fixed exposure time at the indicated time-points afterMSC administration. Red arrows indicateMSCsdetected in the lung. On day 4 and 7 after MSC injection, lungs were harvested from naïve and tumor-bearing mice and labeled MSCs were visualized.

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with the reduction of metastatic tumor nodules, mice trea-ted with MSC/dTRAIL-TK survived for the longest period oftime (maximum survival of mice¼ 38 days for MSC/EGFP,52 days for MSC/dTRAIL, 58 days for MSC/TK, and 85 daysfor MSC/dTRAIL-TK). In the end, however, all mice even-tually succumbed to their disease (Fig. 5C).

Multiple injections of MSC/dTRAIL-TK lead to 100%survival of tumor-bearing miceAlthough the treatment of MSC/dTRAIL-TK significantly

reduced the number of metastatic tumor nodules andprolongedmouse survival, complete remission ofmetastat-ic RCC could not be achieved with a single injection of 5 �106 cells. Because the alteration of dose schedule or doseintensity often affects treatment outcome, we evaluated theeffects of increasing the dose or frequency of injections onmice survival. Administrations of 2- or 3-fold higher dosesof MSC/dTRAIL-TK (1 � 106 and 1.5 � 106 cells) inducedmarginal effects on survival. However, sequential adminis-trations of MSC/dTRAIL-TK (5 � 105 cells) in 2-weekintervals (with double and triple injections) remarkablyprolonged the survival. Surprisingly, 50% of tumor-bearingmice treated with 2 injections of MSC/dTRAIL-TK surviveduntil the end of the study. More importantly, triple injec-tions of MSC/dTRAIL-TK induced a complete cure of met-astatic tumor-bearing mice, resulting in 100% survival (Fig.6A). When the number of metastatic tumor nodules wascounted on day 60 after tumor injection, we found thatdouble injections of MSC/dTRAIL-TK significantly elimi-

nated tumors in the lungs, comparedwith a single injection,but some tumor nodules still remained in a number ofmice(Fig. 6B). On the other hand, triple injections of MSC/dTRAIL-TK completely cleared metastatic tumor nodules inthe lungs, which is consistent with the 100% survival ratesin the mice that we documented.

DiscussionA major unmet clinical need in the treatment of meta-

static RCC is the absence of therapeutic agent that providesdurable complete remissions with minimal toxicity. Recentstudies have proposed several strategies to achieve completeremission: (i) the selection of therapeutic agents with selec-tive toxicity toward tumor cells, (ii) the identification of themethods of delivering the agents, either in combination oras sequential single agents, and (iii) the optimization of themost effective sequence or combination treatment regimen(28). In this study, we aimed to completely eliminateestablished tumors in the lung, which is the most commonsite of metastasis for RCC, by injecting MSCs transducedwith a bicistronic adenoviral vector coexpressing dTRAILand HSV-TK. Our treatment strategy not only allows tumorsite-specific delivery of therapeutic molecules, but alsoenables the implementation of "sequential combinationgene therapy" by repeatedly challenging MSCs expressing 2different therapeutic genes. As expected, MSC/dTRAIL-TKexerted more potent antitumor effects than MSC/dTRAILand MSC/TK upon ganciclovir treatment, both in vitro and

Figure 4. (Continued ) C, CM-Dil–labeled MSCs (red) were intravenously injected into naïve mice or mice bearing 7-day-old tumor. Lungs were isolated andprepared for histologic analysis at 7 daysafterMSC injection. ImageswithCM-Dil–labeledMSCs (red) and40,6-diamidino-2-phenylindole (DAPI)-positive cells(blue) were examined by fluorescence microscopy. Tumor nodules are indicated by white dashed line. Corresponding hematoxylin and eosin (H&E)–stainedsections of each fluorescent image were also presented. T, tumor nodule; scale bar, 100 mm.






in vivo. But surprisingly, complete regression of metastaticRCCwas only observedwhenMSC/dTRAIL-TKwas injectedrepeatedly at a low dose, but not with a single injection at ahigh dose. To the best of our knowledge, this is the first

report to present 100% survival ofmetastatic tumor-bearingmice by using MSC-based gene therapy alone. Because themajority ofmetastatic tumors includingRCCare resistant tochemo- or radiotherapy, our strategies solely using MSC/

Figure 5. Relative antimetastatic effects of engineered MSCs. A, engineered MSCs were intravenously injected on day 7 after tumor injection, and thenganciclovir (GCV) treatment was initiated on day 0, 1, 2, or 3 after MSC injections for 7 consecutive days. The numbers ofmetastatic tumor nodules in the lungwere counted on day 14 after tumor injection (n¼ 10). B, engineered MSCs were intravenously injected on day 1 or day 7 posttumor injection (PTI), and thenganciclovir was treated for 7 days, starting on day 2 after MSC injection. The number of metastatic tumor nodules in the lung was counted on day 14after tumor injection (n¼ 11). Representative pictures of lungs (therapy on day 7 PTI) are shown. ��,P < 0.01. C, engineeredMSCswere injected on day 7 aftertumor injection, and then ganciclovir was treated for 7 days, starting on day 2 after MSC injection. The survival rate of tumor-bearing mice wasmonitored up to 100 days posttumor injection (n ¼ 9). These results are representative of 3 independent experiments.

Figure 6. Therapeutic effects of multiple injections of MSC/dTRAIL-TK on the survival of metastatic tumor-bearing mice. A, MSC/dTRAIL-TK or MSC/EGFP(5 � 105 cells) were injected into tumor-bearing mice once (single) or repeatedly (double or triple) over an interval of 2 weeks, starting on day 7 aftertumor injection (n¼ 10). In addition, different doses ofMSC/TRAIL-TK (5� 105, 1� 106, and 1.5� 106 cells) were injected on day 7 after tumor injection (n¼ 6).Ganciclovir was treated daily for 7 days after each MSC injection. The survival rate of tumor-bearing mice was monitored up to 125 days posttumorinjection. White arrows indicate the time-points for MSC injection. B, representative pictures of the lungs isolated on day 60 are shown. Black arrows indicatetumor nodules. These results are representative of 3 independent experiments.

Kim et al.





dTRAIL-TK, without any combinatory treatment with otheragents, can provide a new milestone in the treatment ofhard-to-cure metastatic cancer.Possible explanations for the achievement of complete

remission by repeated administrations of MSC/dTRAIL-TK may be summarized as follows: First, the augmenta-tion of TRAIL-mediated antitumor effects by constructinga dodecameric form, or a 4-trimer form, of conventionalsoluble TRAIL, partly contributed to long-term remis-sions. It has been reported that increasing the valencyof TRAIL mimics can enhance their binding affinity toTRAIL receptors and apoptogenic potential (29). Ourresult also agrees with previous work showing that arti-ficial dodecamerization of CD154 (CD40L) using SPDinduces superior B-cell activation than trimeric CD40L(30). Second, the enhanced apoptotic effects by thecombination of dTRAIL and HSV-TK/GCV may play animportant role in complete eradication of metastatictumor cells. A prior study proposed that combinatorialaction of TRAIL and TK/GCV involves mutual activationof caspases and TK/GCV–mediated mitochondrial ampli-fication of caspase activity. Because toxic ganciclovir tri-phosphate can be transferred from TK-expressing cellsto nonexpressing bystander cells via gap junctions, TRAILimproved the killing of bystander cells as well (20). Inour in vitro study, the proportion of apoptotic Rencacells induced by MSC/dTRAIL-TK was greater than thesum of those induced by MSC/dTRAIL and MSC/TK,separately. Interestingly, similar results were also foundin TRAIL-resistant and taxol-resistant human ovariancancer cell line A2780-Tax (Annexin Vþ population at24 hours post-ganciclovir treatment ¼ 7.66% � 0.55%for MSC/mock, 12.81% � 3.07% for MSC/dTRAIL,41.68% � 1.89% for MSC/TK, and 98.00% � 0.11% forMSC/dTRAIL-TK, unpublished data), suggesting thatTRAIL resistance could be overcome by combinationtherapy of TRAIL and HSV-TK/GCV in selected cancercell lines. Third, the capability of dTRAIL and HSV-TK/GCV to target different populations of heterogeneousRCC cells may also aid in accomplishing durable com-plete remission. It is well established that tumors con-sisting of heterogeneous population of cells havingdiverse molecular profiles, proliferative and metastaticpotential, differentiation capacity, and susceptibility tochemo- and radiotherapy (31). Among these cells, thecapability to initiate tumor formation and sustain tumorgrowth resides in a small subset of quiescent, slow-grow-ing populations known as cancer stem cells (CSC; ref. 32).A recent study has shown the existence of CSCs in Rencacells by isolating side population cells (33). Because HSV-TK/GCV therapy only eliminates rapidly proliferatingtumor cells, residual slow-proliferating cells may repop-ulate to form recurrent tumor. Indeed, side populationcells were found to be more resistant to HSV-TK/GCVtreatment than nonside population cells in human glio-blastoma (34). On the other hand, side populationcells were more sensitive to TRAIL than nonside popula-tion cells in human colon cancer, due to the increased

expression of c-Myc and consequent upregulation ofTRAIL receptor DR4 (35, 36). Similarly, radioresistantCSC-like esophageal cells exhibited a higher susceptibi-lity to TRAIL therapy than parental cells (36). Theseresults suggest that the combination therapy of TRAILand HSV-TK/GCV is an optimal strategy to simultane-ously target side population and nonside populationcells. Fourth, the induction of stronger tumor-specificT-cell responses by the combination of dTRAIL andHSV-TK/GCV might have contributed to the long-termremission (IFN-g spots/106 splenocytes ¼ 81 � 15 forMSC/EGFP, 124 � 7 for MSC/dTRAIL, 173 � 17 for MSC/TK, and 222 � 20 for MSC/dTRAIL-TK). The previousevidence suggests that TRAIL-mediated tumor destructionincreases the amount of antigens that could be deliveredinto the cross-presentation pathway, ultimately leadingto enhanced antitumor immunity (4). HSV-TK/GCV ther-apy is also capable of inducing potent T cell and naturalkiller cell responses (37). As immunogenic propertiesof RCC render tumor cells relatively sensitive to immu-nologic attack, the development of strong immuneresponses may play a role in complete regression. Finally,but most importantly, the optimization of biologic doseand schedule for MSC/dTRAIL-TK treatment contributeda great deal to the accomplishment of durable completeremission. The optimal time-points for in vivo applicationof ganciclovir and MSCs were empirically determinedto provide the maximum therapeutic efficacy. We thenevaluated the effects of biologic doses and frequency ofadministration on tumor regression. No significantincrease in survival was observed after a single injectionof MSC/TRAIL-TK at 2 or 3 times higher doses. Repeatedadministration of small-divided doses of MSC/TRAIL-TK,however, markedly improved mice survival, resulting in50% survival after 2 injections and 100% survival after 3injections. Multiple injections of small-divided doses ofagents were as effective as or even more advantageousthan single injection in some occasions. For example,frequent smaller doses of bevacizumab exerted similarmodulatory effects with a single large injection on VEGF-induced vascular changes in retinal neovascular model(38). It was also reported that low-dose repeated injec-tions of adriamycin was more effective than a high-dosesingle injection in terms of improving the survival oftumor-bearing mice while lowering the drug toxicity(39). Sustained release of therapeutic molecules in thetumor microenvironment and more frequent exposure toganciclovir seemed to be crucial for the achievement ofcomplete remission in our experimental system. The poten-cy of sequential suicide gene therapy was also assessed in arecent study, in which repeated injections of human adi-pose-derived MSCs expressing cytosine deaminase::uracilphosphoribosyltransferase (with intracerebroventricular5-fluorocytosine administration) achieved 88% survivalof rats bearing intracerebral glioblastoma (40).

In conclusion, sequential administration of MSCs co-expressing dTRAIL and thymidine kinase is a safe andpotent strategy for accomplishing long-term remissions






of metastatic RCC. The application of syngeneic or alloge-neic MSCs, which aremore often used in clinic, may furtherpotentiate the therapeutic efficacy of MSC/dTRAIL-TKas more MSCs would survive at the tumor site to increasethe availability of therapy. Moreover, the incorporationof HSV-TK/GCV system enables efficient elimination ofMSCs, lowering the risk of malignant transformation ofinfused MSCs. Overall, our findings propose a novel ther-apeutic approach to achieve durable complete remissionin metastatic RCC.

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

Authors' ContributionsConception and design: S.W. Kim, S.J. Kim, S.H. Park, Y.C. SungAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): Y.C. SungAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S.W. Kim, S.J. Kim, S.H. ParkWriting, review, and/or revision of the manuscript: S.W. Kim, S.J. Kim,S.H. Park, Y.C. Sung

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.W. Kim, S.J. Kim, S.H. Park, H.G.Yang, M.C. Kang, Y.W. Choi, S.M. KimStudy supervision: S.-S. Jeun, Y.C. Sung

AcknowledgmentsThe authors thank Kwan Seok Lee at POSTECH for their devoted animal

care, Dong-Young Kim at Genexine Co. for providing recombinant adeno-virus, as well as Sang Hwan Seo and Bo Yeong Song at POSTECH forproviding technical assistance.

Grant SupportThis work was supported by grants from the Korea Science and Engineer-

ing Foundation (KOSEF) funded by the Korea government (2010K001287),theNational R&DProgram forCancer Control,Ministry ofHealth&Welfare,Republic of Korea (0820040), and the World Class University (WCU)program through the National Research Foundation of Korea funded bythe Ministry of Education, Science and Technology (R31-2008-000-10105-0or R31-10105).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 6, 2012; revised October 23, 2012; accepted October 25,2012; published OnlineFirst November 30, 2012.

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2013;19:415-427. Published OnlineFirst November 30, 2012.Clin Cancer Res Sae Won Kim, Su Jin Kim, Sang Hoon Park, et al. Expressing Dodecameric TRAIL and HSV-TKMultiple Injections of Engineered Mesenchymal Stem Cells Complete Regression of Metastatic Renal Cell Carcinoma by

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