tumor and stem cell biology cancer research adipose...

3718

Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-1865

Tumor and Stem Cell Biology
Cancer
Research

Adipose-Derived Mesenchymal Stem Cells as Stable Sourceof Tumor Necrosis Factor–Related Apoptosis-InducingLigand Delivery for Cancer Therapy

Giulia Grisendi1, Rita Bussolari1, Luigi Cafarelli1, Istvan Petak7, Valeria Rasini1, Elena Veronesi1, Giorgio De Santis2,Carlotta Spano1, Mara Tagliazzucchi3, Helga Barti-Juhasz7, Laura Scarabelli1, Franco Bambi8,Antonio Frassoldati1, Giulio Rossi5, Christian Casali6, Uliano Morandi6, Edwin M. Horwitz9,Paolo Paolucci4, PierFranco Conte1, and Massimo Dominici1

Abstract

Authors' ARespiratoryand PathoPathologic6Chest SuEmilia, MoCancer ReBank and9DivisionChildren'sSchool of M

Note: SupResearch O

CorresponHematologand Reggi39-59-422unimore.it.

doi: 10.115

©2010 Am

Cancer R

Dow

Adipose-derived mesenchymal stromal/stem cells (AD-MSC) may offer efficient tools for cell-based genetherapy approaches. In this study, we evaluated whether AD-MSC could deliver proapoptotic molecules forcancer treatment. Human AD-MSCs were isolated and transduced with a retroviral vector encoding full-lengthhuman tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a proapoptotic ligand that inducesapoptosis in a variety of human cancers but not normal tissues. Although several studies have documented theantitumor activity of recombinant human TRAIL, its use in vivo is limited by a short half-life in plasma due to arapid clearance by the kidney. We found that these limitations can be overcome using stably transduced AD-MSC, which could serve as a constant source of TRAIL production. AD-MSC armed with TRAIL targeted avariety of tumor cell lines in vitro, including human cervical carcinoma, pancreatic cancer, colon cancer, and,in combination with bortezomib, TRAIL-resistant breast cancer cells. Killing activity was associated with ac-tivation of caspase-8 as expected. When injected i.v. or s.c. into mice, AD-MSC armed with TRAIL localizedinto tumors and mediated apoptosis without significant apparent toxicities to normal tissues. Collectively, ourresults provide preclinical support for a model of TRAIL-based cancer therapy relying on the use of adipose-derived mesenchymal progenitors as cellular vectors. Cancer Res; 70(9); 3718–29. ©2010 AACR.

Introduction

Mesenchymal stromal/stem cells (MSC) have gained inter-est as promising tools for cancer therapy because wild-type(WT) and gene-modified (GM) bone marrow (BM) MSC mayexert an antitumor potential (1–6). Whereas BM has been thefirst recognized source of MSC (7), adipose tissue represents avalid reservoir of mesenchymal progenitors (8). Adipose tissue

ffiliations: 1Department of Oncology, Hematology andDiseases, 2Plastic Surgery Unit, 3Department of Laboratorylogy, 4Department of Mother and Child, 5Department ofAnatomy and Forensic Medicine, Section of Pathology, andrgery Division, University-Hospital of Modena and Reggiodena, Italy; 7Department of Pathology and Experimentalsearch, Semmelweis University, Budapest, Hungary; 8BloodCellular Therapy Unit, Meyer Hospital, Firenze, Italy; andof Oncology/Blood and Marrow Transplantation, TheHospital of Philadelphia and The University of Pennsylvaniaedicine, Philadelphia, Pennsylvania

plementary data for this article are available at Cancernline (http://cancerres.aacrjournals.org/).

ding Author: Massimo Dominici, Department of Oncology,y and Respiratory Diseases, University-Hospital of Modenao Emilia, Via del Pozzo 71, 41100 Modena, Italy. Phone:-2858; Fax: 39-59-422-3341; E-mail: massimo.dominici@

8/0008-5472.CAN-09-1865

erican Association for Cancer Research.

es; 70(9) May 1, 2010

Researcon Februarycancerres.aacrjournals.org nloaded from

can be obtained in relevant amount and easily processed torelease large numbers of adipose-derived MSC (AD-MSC; refs.9, 10). Similarly to BM-MSC, AD-MSCs are particularly suitablefor cell gene therapy approaches because they can be expan-ded and then transformed by several vectors (11–13).Starting from this background, we wanted to test whether

AD-MSC could represent an efficient vehicle to deliver tumornecrosis factor (TNF)–related apoptosis-inducing ligand(TRAIL). TRAIL is a promising anticancer death ligand witha sequence homology to TNF and FasL (14). It is a type IImembrane-bound (MB) protein that can be processed by cys-teine protease to generate a soluble ligand (15). Both MB pro-tein and soluble ligand can rapidly induce apoptosis in avariety of cancers, sparing normal cells (16). TRAIL mediatesthe apoptotic effect binding to its death receptors (DR), ashomotrimer, particularly on TRAIL-R1/DR4 and TRAIL-R2/DR5 activation, a protein complex, causes caspase-8 activa-tion, triggering apoptosis (17–19). Although several studieshave shown the antitumor activity of recombinant TRAIL(rTRAIL), its in vivo use is limited due to short half-life inplasma (20). To overcome these limitations, we generate sta-bly modified AD-MSC to obtain cellular vehicles for a tar-geted and constant TRAIL delivery. We here originallydescribe the efficiency of human AD-MSC armed with TRAILto induce apoptosis in several tumor types and, particularly,in a human cervical carcinoma model.

h. 15, 2019. © 2010 American Association for Cancer

http://cancerres.aacrjournals.org/

Adipose Stromal/Stem Cells as TRAIL Vehicles


Materials and Methods

Cell lines and primary tumor cells. HeLa cells (AmericanType Culture Collection, LGC srl) were cultivated in DMEM(Life Technologies) with 10% fetal bovine serum (Hyclone),1% glutamine (200 mmol/L), and 1% penicillin-streptomycin(104 IU/mL and 10 mg/mL; Euroclone). Primary tumor speci-mens were obtained after informed consent from patientswith histologically documented lung cancer. Tumor cell sus-pensions were obtained as previously reported (21). For mor-phologic analyses, trypsinized cells were spun onto slide andstained by standard H&E staining.Isolation of TRAIL cDNA, vector production, and AD-

MSC transduction. Full-length human TRAIL gene(NM_003810.2) was amplified from cDNA isolated by ExpandHigh Fidelity Taq (Roche), as described (22). The followingprimers containing XhoI and EcoRI sites were used:5 ′ -ATGGCTATGATGGAGGTCCA-3 ′ ( forward) and5′-CCCCGGAAAAATCAACCGATT-3′ (reverse). A bicistronicmurine stem cell virus–derived retroviral vector (pMIGR1)encoding for green fluorescent protein (GFP) was modified,including the amplified full-length human TRAIL cDNA.The resulting vector was defined as MIGR1-TRAIL-GFP,whereas the empty MIGR1-GFP vector was used as control.Retrovirus production was performed by the FLYRD18 pack-aging cell lines, as published (23). After approval by localEthical Committee, AD-MSCs were obtained from individualsperforming liposuction for aesthetic purposes and processedas reported (8). AD-MSCs were then transduced by virus-con-taining media from either FLYRD18-TRAIL or FLYRD18-GFP(both ∼1 × 106 transducing units/mL), as described (23). Theobtained AD-MSC lines were defined as AD-MSC TRAIL andAD-MSC GFP, respectively.Fluorescence-activated cell sorting analysis. Cells were

stained with PE-anti-TRAIL, PE-anti-TRAIL-R1/DR4, PE-anti-TRAIL-R2/DR5 (Biolegend), and isotype controls (BectonDickinson). Intracellular staining on transduced AD-MSCand controls was performed with BD Cytofix/Cytoperm kit(Becton Dickinson) using the PE-anti-TRAIL antibody.ELISA. TRAIL was measured by Quantikine Human TRAIL/

TNFSF10 kit (R&D Systems) according to instructions.Apoptosis and caspase-8 activation assays. HeLa cells

were seeded, and after 12 h, either AD-MSC TRAIL or AD-MSC GFP were added at different T:E ratios (1:1, 1:2, and1:5). Activity of AD-MSC TRAIL was evaluated by propidiumiodide (PI; Sigma) staining after 24 and 48 h by fluores-cence-activated cell sorting (FACS) gating on GFP-negativecells. Soluble rTRAIL (up to 20 μg/mL; PeproTech, Inc.)was used as positive control. Experiments were performedat least thrice. Isolated primary lung tumor cells were cocul-tured with either AD-MSC TRAIL or AD-MSC GFP at 1:5 T:Eratio, and tumor cell death was assessed as described above.Caspase-8 activation was measured by FACS with the

CaspGLOW Red Active Caspase-8 Staining kit (BiovisionResearch, Inc.) coculturing either AD-MSC TRAIL or AD-MSC GFP with HeLa (T:E ratio: 1:5 for 8 h) using as negativecontrol the caspase inhibitor Z-VAD-FMK (10 μmol/L). Inaddition, cocultures of HeLa cells with either AD-MSC TRAIL

www.aacrjournals.org

Researcon Februarycancerres.aacrjournals.org Downloaded from

or AD-MSC GFP were established using a 0.4-μm Transwellsystem (Corning, Inc.) to separate the two populations (T:Eratios: 1:1, 1:2, and 1:5). For inhibition studies, cocultures ofHeLa cells and AD-MSC TRAIL (T:E ratio: 1:1) were treatedwith 0.8, 1.6, 3.2, and 6.4 μg/mL of neutralizing anti-humanTRAIL antibody (PeproTech). HeLa viability was tested byFACS after PI staining.Animal studies. Eight- to 10-wk-old NOD.CB17-Prkdcscid/J

male and female mice (Charles River) were kept in accor-dance with guidelines under approved protocols. Six groupsof mice (n ≥ 3/each) were considered as follows: (a) s.c. flankinjected once with 1 × 106 AD-MSC GFP; (b) s.c. flank injectedonce with 1 × 106 AD-MSC TRAIL; (c) s.c. flank injected oncewith 2 × 105 HeLa; (d) s.c. flank injected with 2 × 105 HeLaand, as soon as an appreciable tumor burden appeared(15–20 d), treated with multiple (n = 3) biweekly intratumorinjections of 106 AD-MSC GFP; (e) tumor injected as in (d)but treated with multiple (n = 3) biweekly intratumor injec-tions of 106 AD-MSC TRAIL; ( f ) tumor injected as in (d) buttreated with multiple (n = 3) biweekly tail i.v. injections of 106

AD-MSC GFP; and (g) tumor injected as in (d) but treatedwith multiple (n = 3) biweekly tail i.v. injections of 106

AD-MSC TRAIL.AD-MSC TRAIL toxicity was assessed in groups (a) and (b),

particularly considering liver (24). Parameters such as survi-val, weight, and serum liver enzymes by spectrophotometer(Cobas C501, Roche Diagnostic) were recorded. In groups (c)to (g), weights were weekly measured and tumor sizes werecalculated as reported (25): volume = length × width2/2. After60 d, animals were sacrificed, liver was harvested, and tumorwas excisedPCR. GFP-marked AD-MSCs were monitored in excised

and processed tumors by PCR using GFP (5′-GTAAACGGC-CACAAGTTCAG-3′ and 5′-TGTCGGCCATGATATAGACG-3′;DQ768212) and glyceraldehyde-3-phosphate dehydrogenase(GAPDH; 5′-GCAGTGGCAAAGTGGAGATT-3′ and 5′-GCA-GAAGGGGCGGAGATGAT-3′; XM_973383) primer pairs.Histology. Histochemistry was performed as reported (26).Statistics. Data are expressed as the mean ± SD. A two-

tailed P value of ≤0.05 from Student's t test was considered sta-tistically significant by Excel 2003 software (Microsoft, Inc.).

Results

AD-MSC can be genetically modified to express highlevels of TRAIL. AD-MSCs were transduced by vector en-coding for full-length human TRAIL (MIGR1-TRAIL-GFP)and with control vector (MIGR1-GFP). In both cases,>95% of GM AD-MSCs were obtained (Fig. 1A, left col-umn). FACS analyses show that WT AD-MSC and AD-MSC GFP do not constitutively express TRAIL; in contrast,gene modification of AD-MSC with TRAIL-encoding vectorallows a relevant protein expression by surface and intra-cellular stainings (Fig. 1A, middle and right columns). Be-cause TRAIL in nature can be released as soluble ligand(14), we wanted to evaluate whether this soluble formcould be also produced by AD-MSC TRAIL. A time courseexperiment (Fig. 1B), started with confluent culture of

Cancer Res; 70(9) May 1, 2010 3719



Grisendi et al.

3720


AD-MSC TRAIL, allows to detect soluble TRAIL startingfrom 6 hours (126.8 ± 18.4 pg/mL), and further analysesat 12, 24, and 48 hours show a constant release of solubleTRAIL (up to 366.4 pg/mL).Having shown that AD-MSC TRAIL express the desired

protein, we evaluated whether forced TRAIL production couldbe followed by death of AD-MSC themselves. Thus, we inves-tigated TRAIL-R1/DR4 and TRAIL-R2/DR5 expression on WTAD-MSC, uncovering that these cells lack TRAIL-R1/DR4 andshow low (<26%) TRAIL-R2/DR5 (Fig. 1C). These findings werevalidated by PI staining (at 24 and 48 hours), which revealsno differences (P > 0.08) in cell death between confluent WTAD-MSC, AD-MSC GFP, and AD-MSC TRAIL (Fig. 1D). AnnexinV staining performed to detect early apoptosis (SupplementaryFig. S1A) further indicates that∼85 ± 4% of AD-MSCs are refrac-

Cancer Res; 70(9) May 1, 2010


tory to TRAIL expression, prompting their use in our cancergene therapy approach.AD-MSCs are not affected by retrovirus transduction

and TRAIL expression. AD-MSCs were then analyzed forknown surface antigens and differentiation potentials (27).As shown in Supplementary Fig. S2A, WT and GM AD-MSCexpress high level of CD90, CD105, CD73 lacking of CD45,CD34, and CD14. In addition, adipogenic and osteogenicdifferentiation assays indicate that gene modifications donot affect main AD-MSC differentiation pathways (Supple-mentary Figs. S2B and C and S3A and B). Collectively, thesedata indicate that the mentioned cell manipulations do notperturb the main AD-MSC features.AD-MSCs expressing TRAIL display an in vitro antitu-

mor activity in cancer cell lines. HeLa cells have been

Figure 1. AD-MSCs expressing TRAIL survive to its apoptosis-inducing function. A, FACS analyses of WT AD-MSC (top), AD-MSC transduced with GFPvector only (middle), and AD-MSC transduced with vector coding for TRAIL (bottom). GFP fluorescence is detected in all GM AD-MSC, whereas TRAILexpression is exclusively present in AD-MSC TRAIL, both as surface and as intracellular protein. B, ELISA measuring TRAIL released in culture byconfluent AD-MSC GFP versus AD-MSC TRAIL at different time points (P < 0.01). C, FACS analyses of TRAIL-R1 and TRAIL-R2 on AD-MSC. D, TRAILtoxicity on AD-MSC at 24 and 48 h (PI staining by FACS).

Cancer Research





selected by FACS according to their high expression ofTRAIL-R1/DR4 and TRAIL-R2/DR5 receptors as predictivemarkers for TRAIL sensitivity (Fig. 2A). Moreover, a concen-tration-dependent effect of rTRAIL on HeLa has been testedto optimize their sensitivity (Supplementary Fig. S1B).Coculture experiments were then performed; at 24 hours,

AD-MSC TRAIL induce HeLa death represented by reductionof adherent HeLa cells and the appearance of cellular debris.These features are even more prominent at 48 hours (Fig. 2B,arrows). The native fluorescence of transduced AD-MSCallows a clear distinction between target and effector cellseither by fluorescence microscopy (Fig. 2B) or by FACS(Fig. 2C). Gating on GFP-negative cells (HeLa), we are ableto detect relevant amount (≥70%) of PI-positive cells onlyin coculture where AD-MSC TRAIL are present (Fig. 2C, bot-tom). To quantify cell death at 24 and 48 hours, different T:Eratios were assessed using AD-MSC TRAIL and AD-MSC GFPas control. As seen in Fig. 2D, cytotoxicity on HeLa cells isdetectable only in coculture with TRAIL-producing AD-MSC starting from the 1:1 ratio (P < 0.01). At 24 hours, thiseffect increases significantly with lower T:E ratios (P <0.003) and is even more prominent than rTRAIL (P = 0.002).Interestingly, cytotoxicity persists up to 48 hours and is mar-ginally affected by an increase of AD-MSC TRAIL number (P >0.22). For all the considered ratios, AD-MSC GFP do not causeHeLa death, indicating a specific action of AD-MSC TRAIL.Having established optimal coculture conditions of our

gene therapy approach, we wanted to test other two celllines known to be sensitive to rTRAIL (28, 29). Thus, pan-creas (BxPc3) and colon adenocarcinomas (LS174T) werecocultured with AD-MSC TRAIL using AD-MSC GFP andrTRAIL as controls (Supplementary Fig. S4A and B). At 24and 48 hours, AD-MSC TRAIL are able to induce a compa-rable or even superior apoptosis than rTRAIL (20 μg/mL).In addition, analogous approaches were performed onBT549 (breast cancer) and IMR32 (neuroblastoma), knownrTRAIL-resistant cell lines (30, 31). As expected, AD-MSCTRAIL were unable to induce a significant apoptosis, under-lining the high specificity of this strategy (SupplementaryFig. S4A and B).AD-MSC TRAIL specifically induce apoptosis by cell-to-

cell contact through caspase-8 activation. To confirm thatHeLa mortality is specifically due to antitumoral effect ofTRAIL-expressing AD-MSC, anti-TRAIL antibody was dilutedin coculture media (Fig. 3A). Starting from 1.6 μg/mL, we ob-serve a significant (P < 0.02) reduction of HeLa cell death, andthis effect is more prominent at the highest concentration(6.4 μg/mL) where HeLa death is comparable with control(P = 0.08).To additionally validate at the intracellular level the

specificity of TRAIL effect, we investigate caspase-8 activa-tion. HeLa cells were cocultured with either AD-MSC TRAILor AD-MSC GFP at 1:5 ratio for 8 hours both in the pre-sence and in the absence of a known caspase inhibitor,Z-VAD-FMK (32).Gating on GFP-negative cells, we are ableto identify and selectively analyze the amount of caspaseactivation in HeLa cells only. As shown in Fig. 3B (left, bot-tom), the majority (78.3 ± 2.5%) of HeLa cells reveal an ac-



tivated caspase-8 after 8 hours of coculture with AD-MSCTRAIL (black line), whereas caspase-8 activation is undetect-able in the presence of the caspase inhibitor (Fig. 3B, left,bottom, gray line; P = 0.005). On the contrary, either in thepresence or in the absence of the inhibitor, caspase-8 activa-tion is undetectable in coculture with AD-MSC GFP (Fig. 3B,left, top; P = 0.003).We have previously shown that AD-MSC TRAIL express

this molecule both as MB protein (Fig. 1A) and as solubleligand (Fig. 1B). Because both TRAIL forms can exert an anti-tumoral activity (14), we sought to evaluate the mechanismby which AD-MSC armed with TRAIL could mediate theirapoptotic effect. Thus, an in vitro coculture assay has beenestablished using a Transwell system preventing cell-to-cellcontact so that HeLa cells, separated from AD-MSC TRAIL,could be induced to death exclusively by soluble TRAIL.Although apoptosis can still be detected in the AD-MSCTRAIL coculture at 1:5 ratio versus HeLa cells alone (P =0.01), the lack of contact significantly reduces the antitumoractivity by AD-MSC TRAIL (P > 0.06), indicating the cell-to-cell contact as the main mechanism by which AD-MSCTRAIL induce apoptosis (Fig. 3C).Bortezomib sensitizes a TRAIL-resistant breast cancer

cell line to AD-MSC TRAIL. Bortezomib cooperates withrTRAIL to induce apoptosis in TRAIL-resistant tumorsmainly by upregulation of TRAIL receptors (33). To investi-gate whether bortezomib could be combined with AD-MSCTRAIL, we selected BT549 as cell line resistant to bothrTRAIL and TRAIL cell delivery (Supplementary Fig. S4).We confirmed that BT549 can survive up to 20 μg/mL ofrTRAIL (Supplementary Fig. S5A) and lack of TRAIL-R1/DR4 and TRAIL-R2/DR5 (Supplementary Fig. S5B, top). Wethen uncovered that a 12-hour treatment with bortezomib(10 nmol/L) induces a significant increase of TRAIL-R2/DR5 (P = 0.02), with no significant effect on TRAIL-R1/DR4(Supplementary Fig. S5B, bottom). We also asked whetherbortezomib could upregulate TRAIL-R2/DR5 expression inAD-MSC, inducing their death with negative effect on possi-ble combinations of drug and cell therapy. Interestingly,bortezomib upregulates TRAIL-R2 receptor (from 20% upto 73% after 24 hours of treatment; P = 0.01), and this changeis followed by an increase in cell death versus WT AD-MSC(P = 0.02; Supplementary Fig. S5C and D). Nevertheless, themajority (>85%) of AD-MSC TRAIL survive to the treatment(Supplementary Fig. S5D).Having considered these findings, BT549 cells were trea-

ted with bortezomib and cocultured with AD-MSC TRAIL.At 48 hours, the combination of AD-MSC TRAIL and borte-zomib versus bortezomib alone (Supplementary Fig. S5E)significantly increases apoptosis (P = 0.01), indicating thatbortezomib can be successfully combined with a cell-basedTRAIL delivery for resistant tumor cells.AD-MSC TRAIL induce apoptosis in primary tumor cells.

To further challenge our cell therapy approach in a primarytumor model, we focused on lung cancer as leading cause ofdeaths (34). Four distinct lung tumor specimens wereprocessed. Of those, the best in vitro performance was ob-served in cells obtained from a patient affected by signet ring

Cancer Res; 70(9) May 1, 2010 3721



Grisendi et al.

3722


Figure 2. AD-MSCs producing TRAIL exert a potent cytotoxic effect on a target tumor cell line. A, FACS analyses of TRAIL receptor (TRAIL-R1/DR4and TRAIL-R2/DR5) expression on HeLa cells. B, in vitro cultures of HeLa alone as control (CTRL; top), HeLa with AD-MSC GFP (middle), andAD-MSC TRAIL (bottom) visualized by both phase-contrast and GFP filter fluorescence microscopy at 1:5 T:E ratio. Scale bar, 200 μm. At 24 h,AD-MSC TRAIL exert a cytotoxic effect on HeLa cells as shown by the presence of cellular debris in culture medium (left; arrows), and this effect iseven more prominent at 48 h (right). C, representative FACS plot of PI staining detecting cell death induced by AD-MSC TRAIL at 48 h. Apoptoticcells were identified gating on (red arrow) GFP-negative cells (HeLa). D, cell death by PI staining on gated GFP-negative HeLa cells in coculturewith AD-MSC TRAIL, AD-MSC GFP, rTRAIL, and controls. Different T:E ratios have been tested showing significant cytotoxic effects of AD-MSCTRAIL versus AD-MSC GFP both at 24 and 48 h (P < 0.016). Cytotoxicity is maintained up to 48 h (right) when the lowest ratio (1:5) is comparablewith rTRAIL at 20 μg/mL (P = 0.9).

Cancer Res; 70(9) May 1, 2010 Cancer Research

Research. on February 15, 2019. © 2010 American Association for Cancercancerres.aacrjournals.org Downloaded from




cell carcinoma (SRCC). The histologic staining of the startingtumor specimen typically revealed the positivity for TTF-1,CK7, and the lack of CDX2 and CK20 (data not shown; refs.35, 36). Starting from 4 days after isolation, SRCC in vitrogenerated cell clusters that rapidly reached the confluence(Fig. 4A, left and middle). Tumor population was constituted



by elements with the classic signet ring jeweler aspect cha-racterized by abundant intracellular mucin and a crescenticnucleus displaced at the cell periphery, as displayed by H&Estaining of cultured cells (Fig. 4A, right).We first investigated the presence of TRAIL receptors on

primary SRCC population revealing the exclusive presence

Figure 3. AD-MSC TRAILspecifically induce apoptosis bycaspase-8 activation throughcell-to-cell contact. A,neutralization of TRAIL-inducedapoptosis by anti-human TRAILantibody diluted in cocultures ofHeLa cells and AD-MSC TRAIL at1:1 T:E ratio. HeLa viability isassayed by PI staining. UntreatedHeLa cells were used as control.B, FACS analyses at 8 h ofcaspase-8 activation on gatedGFP-negative HeLa cells incoculture with AD-MSC TRAIL andAD-MSC GFP as control (1:5; T:E).Left, bottom, representativehistogram showing thecaspase-8 activation on HeLa cellsafter AD-MSC TRAIL coculture(black line). The addition of specificcaspase-8 inhibitor(Z-VAD-FMK, 10 μmol/L)counteracts TRAIL activityinhibiting caspase-8 activation(gray line). Top, caspase-8activation is undetectable also incoculture of HeLa cells andAD-MSC GFP with or withoutcaspase inhibitor. Columns, meanpercentage of caspase-8 activationon HeLa cells in coculture(1:5 ratio) with AD-MSC TRAIL orAD-MSC GFP both with (w) andwithout (w/o) specific inhibitors(P = 0.003); bars, SD. C, HeLa cellswere cocultured in Transwell (TW)plates with either AD-MSC GFP orAD-MSC TRAIL and tested forapoptosis by PI staining.

Cancer Res; 70(9) May 1, 2010 3723



Grisendi et al.

3724


of TRAIL-R2 on the SRCC surface (Fig. 4B). Cocultureexperiments were then performed with GM AD-MSC at 1:5T:E ratio at both 24 and 48 hours, obtaining a significantamount (up to 59.9%; P < 0.005) of PI-positive cells in cocul-ture with AD-MSC TRAIL versus AD-MSC GFP (Fig. 4C). Thiseffect resulted even more prominent than the one obtainedwith 20 μg/mL of rTRAIL (P < 0.001), suggesting that, evenon primary cancer cells, TRAIL delivery by AD-MSC may bemore effective than the use of the recombinant protein.In vivo toxicology of the AD-MSC TRAIL approach.

Because TRAIL exposure and MSC gene manipulation wereassociated to side effects (24, 37, 38), we established anin vivo toxicology approach assessing the effect of AD-MSC TRAIL. Firstly, based on the reported TRAIL livertoxicity, mice were s.c. injected once into the flank with1 × 106 AD-MSC TRAIL or with 1 × 106 AD-MSC GFPalone, and serum level of alanine aminotransferase (ALT)and aspartate aminotransferase (AST) was monitored. Asseen in Fig. 5A, both AST (21 ± 1 IU/L and 63 ± 29 IU/L)



and ALT (6 ± 1 IU/L and 19 ± 10 IU/L) levels were betweenranges (39, 40) in both AD-MSC GFP and AD-MSC TRAILgroups. These data are confirmed by H&E staining(Fig. 5B), where there is no evidence of abnormal hepato-cytes and inflammatory cell infiltration. In addition, toverify whether i.v. and s.c. multiple (n = 3; 1 × 106/each)AD-MSC TRAIL and AD-MSC GFP injections might havebeen linked to toxicity, same tests were repeated, confirm-ing the lack of liver damage (data not shown). To monitorthe overall status of treated mice, weight, food intake, andbehavior were monitored. As seen in Fig. 5C, both single(left) and multiple s.c. (middle) and i.v. (right) injectionsof AD-MSC TRAIL or AD-MSC GFP are not affecting animalweight gain (P > 0.63). Similarly, food intake and behaviordid not reveal pathologic status and necropsies, performedat 60 days, did not provide signs of abnormal GM AD-MSCproliferation.AD-MSC TRAIL exert an antitumor activity in vivo.

To validate in vitro findings and having considered in vivo

h. 15, 2019. © 2010 American A

Figure 4. AD-MSCs armed withTRAIL induce apoptosis in lungprimary tumor cells. A, in vitroexpanded primary lung SRCCobserved at 4 d (left) and 7 d(middle) of culture. Scale bar,200 μm. Right, H&E staining ofisolated tumor cells. Scale bar,400 μm. B, FACS analyses ofTRAIL-R1 and TRAIL-R2expression on primary SRCC.Gray, isotypic control. C, columns,mean of cell death at 24 and48 h by PI staining on gatedGFP-negative primary SRCC incocultures with AD-MSC TRAILor AD-MSC GFP at 1:5 T:Eratio; bars, SD. *, P = 0.005;**, P = 0.00009. rTRAIL (20 μg/mL)is used as positive control andSRCC alone represents a negativecontrol.

Cancer Research

ssociation for Cancer




toxicology, xenotrasplant models of cervical carcinoma havebeen established s.c. transplanting 2 × 105 HeLa cells. Tumorburdens started to appear between 15 and 20 days from theinoculum and three doses of AD-MSC TRAIL or AD-MSCGFP (1 × 106 cells/each) were injected. AD-MSC TRAIL in-hibit tumor cell growth (P < 0.006); on the contrary, bothAD-MSC GFP and HeLa alone generate large tumors (Fig. 6A).Similarly, animals treated with multiple (n = 3) i.v. injectionsof AD-MSC TRAIL (1 × 106 cells/each; Fig. 6B) were associatedto a reduced tumor burden (P = 0.01). Interestingly, earlyphases (up to 45 days) after AD-MSC GFP injection havebeen associated to a greater tumor size versus HeLa cellsalone. However, at 60 days, this difference disappeared inboth s.c. and i.v. injected mice. To validate that tumor growthreduction was due to the presence of injected AD-MSCTRAIL, we were able to detect these cells in tumor samples(Fig. 6C), and to confirm this finding, immunohistochemicalanalyses performed on tumor sections reveal specific AD-MSC TRAIL localization (Fig. 6D).

10 In preparation.

Discussion

We here show that GM AD-MSC can be used as a powerfultool in cancer therapy to counteract cancer growth. Others



reported the use of BM and cord blood MSC as drug deliverysystems (1, 2, 41–47). Dealing with AD-MSC, these cells havebeen recently used as cancer therapy tools to vehicle pro-drug-converting enzyme (13); however, to our knowledge,our strategy represents the very first example of cancer genetherapy based on AD-MSC directly producing a potent pro-apoptotic agent, such as TRAIL.Adipose tissue has been selected as source of MSC based

on a standardized and minimally invasive procedure fromnormal subjects and from cancer patients,10 allowing theiruse in a hypothetical autologous setting.To begin with, we show that AD-MSCs without gene modi-

fication do not constitutively produce TRAIL; second, we ex-cluded the presence of DR on AD-MSC, which could affectcell survival after TRAIL autocrine production. In particular,we could not detect TRAIL-R1/DR4 and low level of TRAIL-R2/DR5 similarly to what has been described on both BMand amnion-derived MSC (48). Taken together, these dataoriginally indicate that human AD-MSC could be an idealvehicle to delivery TRAIL.Because rTRAIL potential is known as well as its capabil-

ity to selectively induce death on tumor cells, sparing

Figure 5. Safety of AD-MSC TRAILinfusions. A, serum transaminase(AST-ALT) levels in AD-MSCGFP and AD-MSC TRAIL s.c.transplanted mice. Lines representphysiologic murine ranges. Nosignificant differences in AST andALT are identified (P > 0.06).B, H&E staining of liver sectionsfrom AD-MSC TRAIL–transplantedand AD-MSC GFP–transplantedmice does not show evidence ofliver toxicity. Scale bar, 100 μm.C, weight of transplanted mice.

Cancer Res; 70(9) May 1, 2010 3725



Grisendi et al.

3726


Figure 6. In vivo antitumor effect of AD-MSC TRAIL. A, tumor inhibition by AD-MSC TRAIL s.c. injected. A significant reduction of tumor burden isdetected in mice treated with AD-MSC producing TRAIL in comparison with both HeLa cells only and AD-MSC GFP. *, P = 0.006; **, P = 0.002.
Right, representative tumor specimens taken from a HeLa cell–injected mouse and mouse treated with HeLa and AD-MSC TRAIL. B, AD-MSC TRAILi.v. injections reduce tumor growth in treated mice. *, P = 0.01. C, reporter gene (GFP) amplification in tumors taken from mice treated with AD-MSCTRAIL. The GFP plasmid is the positive control (PC). MK, marker; NC, negative control. GAPDH is used as housekeeping gene. D, representativephotomicrographs of anti–GFP-stained (in red) sections obtained from mice treated with HeLa cells (left) or HeLa and AD-MSC TRAIL s.c. Scale bar,100 μm. The presence of GFP-positive cells (arrows) within tumor (T) burden confirms AD-MSC TRAIL localization.
Cancer Res; 70(9) May 1, 2010 Cancer Research





normal tissues (16), several protocols based on rTRAIL wereintroduced as cancer treatment (19). However, a suboptimalhalf-life in plasma reduces its possible therapeutic effects.Bypassing rTRAIL limitation, agonistic anti-TRAIL-DR anti-bodies were generated (17). They induce a stronger antitu-mor effect than rTRAIL after binding to specific DR andovercoming the action of decoy receptors (49). Thoughtapparently favorable, this property also implies that normalcells are no longer safeguarded by apoptosis-inhibitory me-chanisms and become more sensitive to apoptosis. More-over, anti-TRAIL-DR antibodies have a longer biologicalhalf-life than rTRAIL (21 days versus 60 minutes), potential-ly increasing the risk of side effects (16, 50).To overcome the mentioned limitations of rTRAIL and

anti-TRAIL-DR antibodies, other researchers very recentlyreported the use of GM human progenitors as tools to spe-cifically deliver TRAIL (45–47, 51, 52). Although these prelim-inary approaches provided relevant insights about strategiesto deliver TRAIL, they mainly rely on adenoviruses that retainlimits due to a subefficient gene modification of MSC lackingCAR receptors (45, 53) and to a transient transgene expres-sion (51). Taking into account these limits, we generated astable retrovirally transduced population of AD-MSC ableto constantly produce TRAIL up to 20 passages (data notshown) without signs of abnormal cellular behavior eitherin vitro or in vivo, as reported by others using adipose andmarrow MSC (2, 54).Most importantly, our data indicate that AD-MSC TRAIL

exert a robust cytotoxic effect on target cell lines and, inparticular, on HeLa cells. It has been reported that HeLacells are sensitive to rTRAIL (55); however, this is the firsttime to our knowledge that a cell therapy approach basedon TRAIL has been successfully introduced to induce cervi-cal carcinoma apoptosis. We further validate these datatesting other cell lines representative of deathly tumors,such as pancreatic and colon cancers. Moreover, in associ-ation with a TRAIL-sensitizing agent such as bortezomib,we originally induce apoptosis in a TRAIL-resistant breastcancer cell line, indicating that bortezomib could be com-bined with a cell-based TRAIL delivery to successfully targetTRAIL-resistant cancers.In addition, in vitro data dealing with cytotoxicity in-

duced by AD-MSC TRAIL against primary cancer cells in-dicate how a cell-based TRAIL delivery may be effectivefor the treatment of incurable cancers. Beside this ap-proach has been tested on a single sample, due to tech-nical issues on primary tumor cell isolation, we retain itmay represent a valid proof of concept that certainly mer-its further validation.Having shown that GM AD-MSC can simultaneously ex-

press TRAIL either as MB protein or as soluble ligand, weshow that TRAIL effect is mainly based on a cell-to-cell con-tact. Even if soluble TRAIL has been detected in the culturemedia, its low concentration and/or the lack of stable TRAILtrimerization may not be sufficient to trigger cell death, aspreviously reported (56). Moreover, the TRAIL receptor pro-file on HeLa, with a predominant TRAIL-R2/DR5 expression,further confirms that cell-to-cell contact is preferred in our



system because this receptor is preferentially activated byMB TRAIL (51, 56).Confirming the in vitro results, two different in vivo deliv-

ery models indicate that AD-MSC TRAIL inhibit cancergrowth. When directly injected into the tumor burden,AD-MSC TRAIL are integrated within its stroma and gener-ate a cytotoxic cell bundle around tumors, suggesting a non-random persistence. Similarly, when i.v. injected in a s.c.established tumor, AD-MSC TRAIL maintain an antiprolifera-tive effect. Although with a less prominent effect than s.c.injected AD-MSC TRAIL, the systemic cell infusion signifi-cantly reduces tumors. These findings originally suggest thati.v. infused AD-MSC can circulate over the lung vascular bedand migrate into s.c. growing tumors, supporting the conceptthat even AD-MSCs, similarly to other progenitors, home intotumors (1, 13, 45).We also have to report a more prominent tumor growth

in mice treated with AD-MSC GFP in comparison withHeLa alone, similarly to what has been described for WTBM-MSC (57). Interestingly, this trend decreases over timeand, in late time points, seems reversible. The reason be-hind this phenomenon is under investigation; it may bethat the ratios between AD-MSC and tumor cells can ini-tially be in favor of a proliferative burst; however, whencancer cells become prevalent, the amount of AD-MSCmay not be adequate to feed tumor. Nevertheless, the anti-proliferative effect exerted by TRAIL-producing AD-MSC isable not only to counterbalance the tumor-supportive ca-pacity of AD-MSC but also to determine a powerful inhib-itory effect.Because rTRAIL has been previously associated with liver

toxicity (24), we wanted to investigate whether tumor treat-ment with AD-MSC TRAIL may be similarly related with thisside effect. In TRAIL-treated mice, liver enzyme levels arenormal and liver histology does not provide evidence ofabnormal features.Conclusively, our data indicate that a cell therapy ap-

proach with stably genetically modified AD-MSC deliveringTRAIL alone or in combination with sensitizing agents isopening novel therapeutic opportunities for still incurablecancers.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

Ministero Italiano Istruzione Università e Ricerca grants PRIN 2006 andPRIN 2008 (M. Dominici), OTKA-T046665, ETT 576/2006, and OMFB-00287/KKK; National Office for Research and Technology Hungary (I. Petak); NIHgrant R01 HL077643 (E.M. Horwitz); Regione Emilia Romagna (P. Paolucci,P. Conte, and M. Dominici); Associazione per il Sostegno dell'Ematologia edell'Oncologia Pediatrica (P. Paolucci); and Fondazione Cassa di Risparmiodi Modena (M. Dominici).

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 05/21/2009; revised 02/23/2010; accepted 02/24/2010; publishedOnlineFirst 04/13/2010.

Cancer Res; 70(9) May 1, 2010 3727



Grisendi et al.

3728


References
1. Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff
M. Bone marrow-derived mesenchymal stem cells as vehicles for in-terferon-β delivery into tumors. Cancer Res 2002;62:3603–8.

2. Studeny M, Marini FC, Dembinski JL, et al. Mesenchymal stem cells:potential precursors for tumor stroma and targeted-delivery vehiclesfor anticancer agents. J Natl Cancer Inst 2004;96:1593–603.

3. Nakamura K, Ito Y, Kawano Y, et al. Antitumor effect of geneticallyengineered mesenchymal stem cells in a rat glioma model. GeneTher 2004;11:1155–64.

4. Stagg J, Lejeune L, Paquin A, Galipeau J. Marrow stromal cells forinterleukin-2 delivery in cancer immunotherapy. Hum Gene Ther2004;15:597–608.

5. Khakoo AY, Pati S, Anderson SA, et al. Human mesenchymal stemcells exert potent antitumorigenic effects in a model of Kaposi's sar-coma. Exp Med 2006;203:1235–47.

6. Maestroni GJ, Hertens E, Galli P. Factor(s) from nonmacrophagebone marrow stromal cells inhibit Lewis lung carcinoma and B16melanoma growth in mice. Cell Mol Life Sci 1999;55:663–7.

7. Friedenstein AJ, Deriglasova UF, Kulagina NN, et al. Precursors forfibroblasts in different populations of hematopoietic cells as detectedby the in vitro colony assay method. Exp Hematol 1974;2:83–92.

8. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adi-pose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–28.

9. Zuk PA, Zhu M, Ashjian P. Human adipose tissue is a source of multi-potent stem cells. Mol Biol Cell 2002;13:4279–95.

10. Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative anal-ysis of mesenchymal stem cells from bone marrow, umbilical cordblood, or adipose tissue. Stem Cells 2006;24:1294–301.

11. Gimble JM. Adipose tissue-derived therapeutics. Expert Opin BiolTher 2003;3:705–13.

12. Goudenege S, Pisani DF, Wdziekonski B, et al. Enhancement ofmyogenic and muscle repair capacities of human adipose-derivedstem cells with forced expression of MyoD. Mol Ther 2009;17:1064–72.

13. Kucerova L, Altanerova V, Matuskova M, Tyciakova S, Altaner C.Adipose tissue-derived human mesenchymal stem cells mediatedprodrug cancer gene therapy. Cancer Res 2007;67:6304–13.

14. Wiley SR, Schooley K, Smolak PJ, et al. Identification and character-ization of a new member of the TNF family that induces apoptosis.Immunity 1995;3:673–82.

15. Liabakk NB, Sundan A, Torp S, Aukrust P, Frøland SS, Espevik T.Development, characterization and use of monoclonal antibodiesagainst sTRAIL: measurement of sTRAIL by ELISA. J Immunol Meth-ods 2002;259:119–28.

16. Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor ne-crosis factor-related apoptosis-inducing ligand in vivo. Nat Med1999;5:157–63.

17. Johnstone RW, Frew AJ, Smyth MJ. The TRAIL apoptotic pathwayin cancer onset, progression and therapy. Nat Rev Cancer 2008;8:782–98.

18. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation.Science 1998;281:1305–8.

19. Duiker EW, Mom CH, de Jong S, et al. The clinical trail of TRAIL. EurJ Cancer 2006;42:2233–40.

20. Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity ofrecombinant soluble Apo2 ligand. J Clin Invest 1999;104:155–62.

21. Ponti D, Costa A, Zaffaroni N, et al. Isolation and in vitro propagationof tumorigenic breast cancer cells with stem/progenitor cell proper-ties. Cancer Res 2005;65:5506–11.

22. Kayagaki N, Yamaguchi N, Nakayama M, Eto H, Okumura K, YagitaH. Type I interferons (IFNs) regulate tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL) expression on human T cells: anovel mechanism for the antitumor effects of type I IFNs. J ExpMed 1999;189:1451–60.

23. Marx JC, Allay JA, Persons DA, et al. High-efficiency transductionand long-term gene expression with a murine stem cell retroviral vec-tor encoding the green fluorescent protein in human marrow stromalcells. Hum Gene Ther 1999;10:1163–73.



24. Jo M, Kim TH, Seol DW, et al. Apoptosis induced in normal humanhepatocytes by tumor necrosis factor-related apoptosis-inducing li-gand. Nat Med 2000;6:564–7.

25. Caltagirone S, Rossi C, Poggi A, et al. Flavonoids apigenin and quer-cetin inhibit melanoma growth and metastatic potential. Int J Cancer2000;87:595–600.

26. Dominici M, Marino R, Rasini V, et al. Donor cell-derived osteopoi-esis originates from a self-renewing stem cell with a limited regener-ative contribution after transplantation. Blood 2008;111:4386–91.

27. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for definingmultipotent mesenchymal stromal cells. The International Society forCellular Therapy position statement. Cytotherapy 2006;8:315–7.

28. Wang P, Zhang J, Bellail A, et al. Inhibition of RIP and c-FLIP en-hances TRAIL-induced apoptosis in pancreatic cancer cells. CellSignal 2007;19:2237–46.

29. Coffey JC, Wang JH, Smith MJ, et al. Phosphoinositide 3-kinase ac-celerates postoperative tumor growth by inhibiting apoptosis and en-hancing resistance to chemotherapy-induced apoptosis. Novel rolefor an old enemy. J Biol Chem 2005;280:20968–77.

30. Hopkins-Donaldson S, Bodmer JL, Bourloud KB, Brognara CB,Tschopp J, Gross N. Loss of caspase-8 expression in highly malig-nant human neuroblastoma cells correlates with resistance to tumornecrosis factor-related apoptosis-inducing ligand-induced apopto-sis. Cancer Res 2000;60:4315–9.

31. Brooks AD, Ramirez T, Toh U, et al. The proteasome inhibitor borte-zomib (Velcade) sensitizes some human tumor cells to Apo2L/TRAIL-mediated apoptosis. Ann N Y Acad Sci 2005;1059:160–7.

32. Carter BZ, Gronda M, Wang Z. Small-molecule XIAP inhibitors dere-press downstream effector caspases and induce apoptosis of acutemyeloid leukemia cells. Blood 2005;105:4043–50.

33. Liu X, Yue P, Chen S, et al. The proteasome inhibitor PS-341 (borte-zomib) up-regulates DR5 expression leading to induction of apopto-sis and enhancement of TRAIL-induced apoptosis despiteup-regulation of c-FLIP and survivin expression in human NSCLCcells. Cancer Res 2007;67:4981–8.

34. Fry WA, Phillips JL, Menck HR. Ten-year survey of lung cancer treat-ments and survival in hospitals in the United States. Cancer 1999;86:1867–76.

35. Merchant SH, Amin MB, Tamboli P, et al. Primary signet-ring cell car-cinoma of lung: immunohistochemical study and comparison withnon-pulmonary signet-ring cell carcinomas. Am J Surg Pathol2001;25:1515–9.

36. Rossi G, Murer B, Cavazza A, et al. Primary mucinous (so-calledcolloid) carcinomas of the lung: a clinicopathologic and immunohis-tochemical study with special reference to CDX-2 homeobox geneand MUC2 expression. Am J Surg Pathol 2004;28:442–52.

37. Leverkus M, Neumann M, Mengling T, et al. Regulation of tumor ne-crosis factor-related apoptosis-inducing ligand sensitivity in primaryand transformed human keratinocytes. Cancer Res 2000;60:553–9.

38. Nitsch R, Bechmann I, Deisz RA, et al. Human brain-cell death in-duced by tumour-necrosis-factor-related apoptosis-inducing ligand(TRAIL). Lancet 2000;356:827–8.

39. Pal S, Sadhu AS, Patra S, Mukherjea KK. Histological vis-a-vis bio-chemical assessment on the toxic level and antineoplastic efficacy ofa synthetic drug Pt-ATP on experimental animal models. J Exp ClinCancer Res 2008;27:68.

40. Shao R, Shi Z, Gotwals PJ, Koteliansky VE, George J, Rockey DC.Cell and molecular regulation of endothelin-1 production during he-patic wound healing. Biol Cell 2003;14:2327–41.

41. Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derivedmesenchymal stem cells in the treatment of gliomas. Cancer Res2005;65:3307–18.

42. Pereboeva L, Komarova S, Mikheeva G, Krasnykh V, Curiel DT. Ap-proaches to utilize mesenchymal progenitor cells as cellular vehicles.Stem Cells 2003;21:389–404.

43. Duan X, Guan H, Cao Y, Kleinerman ES. Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 genedelivery into Ewing sarcoma tumors. Cancer 2009;115:13–22.

44. Uchibori R, Okada T, Ito T, et al. Retroviral vector-producing

Cancer Research





mesenchymal stem cells for targeted suicide cancer gene therapy.Gene Med 2009;11:373–81.

45. Kim SM, Lim JY, Park SI, et al. Gene therapy using TRAIL-secretinghuman umbilical cord blood-derived mesenchymal stem cellsagainst intracranial glioma. Cancer Res 2008;68:9614–23.

46. Sasportas LS, Kasmieh R, Wakimoto H, et al. Assessment of ther-apeutic efficacy and fate of engineered human mesenchymal stemcells for cancer therapy. Proc Natl Acad Sci U S A 2009;106:4822–7.

47. Loebinger MR, Eddaoudi A, Davies D, Janes SM. Mesenchymal stemcell delivery of TRAIL can eliminate metastatic cancer. Cancer Res2009;69:4134–42.

48. Secchiero P, Melloni E, Corallini F, et al. Tumor necrosis factor-relat-ed apoptosis-inducing ligand promotes migration of human bonemarrow multipotent stromal cells. Stem Cells 2008;26:2955–63.

49. Buchsbaum DJ, Zhou T, Lobuglio AF. TRAIL receptor-targeted ther-apy. Future Oncol 2006;2:493–508.

50. Takeda K, Kojima Y, Ikejima K, et al. Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease. Proc Natl AcadSci U S A 2008;105:10895–900.

51. Carlo-Stella C, Lavazza C, Di Nicola M, et al. Antitumor activity ofhuman CD34+ cells expressing membrane-bound tumor necrosis



factor-related apoptosis-inducing ligand. Hum Gene Ther 2006;17:1225–40.

52. Mohr A, Lyons M, Deedigan L, et al. Mesenchymal stem cells expres-sing TRAIL lead to tumour growth inhibition in an experimental lungcancer model. J Cell Mol Med 2008;12:2628–43.

53. Conget PA, Minguell JJ. Adenoviral-mediated gene transfer into exvivo expanded human bone marrow mesenchymal progenitor cells.Exp Hematol 2000;28:382–90.

54. Morizono K, De Ugarte DA, Zhu M, et al. Multilineage cells from adi-pose tissue as gene delivery vehicles. HumGene Ther 2003;14:59–66.

55. Seol DW, Li J, Seol MH, Park SY, Talanian RV, Billiar TR. Signalingevents triggered by tumor necrosis factor-related apoptosis-inducingligand (TRAIL): caspase-8 is required for TRAIL-induced apoptosis.Cancer Res 2001;61:1138–43.

56. Mühlenbeck F, Schneider P, Bodmer JL, et al. The tumor necrosisfactor-related apoptosis-inducing ligand receptors TRAIL-R1 andTRAIL-R2 have distinct cross-linking requirements for initiation of ap-optosis and are non-redundant in JNK activation. J Biol Chem 2000;275:32208–13.

57. Zhu W, Xu W, Jiang R, et al. Mesenchymal stem cells derived frombone marrow favour tumor cell growth in vivo. Exp Mol Pathol 2006;80:267–74.

Cancer Res; 70(9) May 1, 2010 3729



2010;70:3718-3729. Published OnlineFirst April 13, 2010.Cancer Res Giulia Grisendi, Rita Bussolari, Luigi Cafarelli, et al. Delivery for Cancer Therapy

Related Apoptosis-Inducing Ligand−Tumor Necrosis Factor Adipose-Derived Mesenchymal Stem Cells as Stable Source of

Updated version

10.1158/0008-5472.CAN-09-1865doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2010/04/13/0008-5472.CAN-09-1865.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/70/9/3718.full#ref-list-1

This article cites 57 articles, 19 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/70/9/3718.full#related-urls

This article has been cited by 8 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/70/9/3718To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-09-1865

http://cancerres.aacrjournals.org/content/suppl/2010/04/13/0008-5472.CAN-09-1865.DC1

http://cancerres.aacrjournals.org/content/70/9/3718.full#ref-list-1

http://cancerres.aacrjournals.org/content/70/9/3718.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/70/9/3718


tumor and stem cell biology cancer research adipose...

Documents