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Neural Stem Cell-Mediated Delivery ofIrinotecan-Activating Carboxylesterases to Glioma:Implications for Clinical Use

MARIANNE Z. METZ,a,* MARGARITA GUTOVA,a,* SIMON F. LACEY,b YELENA ABRAMYANTS,a TIEN VO,a

MEGAN GILCHRIST,a REVATHISWARI TIRUGHANA,a LUCY Y. GHODA,a MICHAEL E. BARISH,a

CHRISTINE E. BROWN,c JOSEPH NAJBAUER,a PHILIP M. POTTER,d JANA PORTNOW,e

TIMOTHY W. SYNOLD,f KAREN S. ABOODYa,g

Key Words. Cancer therapy • Carboxylesterase • CPT-11 • Glioma • Immunogenicity • Irinotecan •Microdialysis • Neural stem cells

aDepartment of Neurosciences,bClinical ImmunobiologyCorrelative Studies Laboratory,cDepartment of CancerImmunotherapeutics andTumor Immunology,eDepartment of MedicalOncology and ExperimentalTherapeutics, fDivision of Clinicaland Molecular Pharmacology,and gDivision of Neurosurgery,Beckman Research Institute ofthe City of Hope, Duarte,California, USA; dDepartment ofChemical Biology andTherapeutics, St. Jude Children’sResearch Hospital, Memphis,Tennessee, USA

*Contributed equally.

Correspondence: Marianne Z. Metz,B.S., Department of Neurosciences,Beckman Research Institute of theCity of Hope, 1500 East DuarteRoad, Duarte, California 91010, USA.Telephone: 626-256-4673,ext. 65714; Fax: 626-471-7371;E-Mail: mmetz@coh.org; orKaren S. Aboody, M.D., Departmentof Neurosciences and Division ofNeurosurgery, Beckman ResearchInstitute of the City of Hope, 1500East Duarte Road, Duarte, California91010, USA. Telephone: 626-471-7177; Fax: 626-301-8857; E-Mail:kaboody@coh.org

Received December 13, 2012;accepted for publication July 19,2013; first published online inSCTM EXPRESS October 28,2013.

©AlphaMed Press1066-5099/2013/$20.00/0

http://dx.doi.org/10.5966/sctm.2012-0177

ABSTRACT

CPT-11 (irinotecan) has been investigated as a treatment for malignant brain tumors. However,limitations of CPT-11 therapy include low levels of the drug entering brain tumor sites and systemictoxicities associatedwith higher doses. Neural stem cells (NSCs) offer a novelway to overcome theseobstacles because of their inherent tumor tropism and ability to cross the blood-brain barrier, whichenables them to selectively target brain tumor sites. Carboxylesterases (CEs) are enzymes that canconvert the prodrug CPT-11 (irinotecan) to its active metabolite SN-38, a potent topoisomerase Iinhibitor. We have adenovirally transduced an established clonal human NSC line (HB1.F3.CD) toexpress a rabbit carboxylesterase (rCE) or a modified human CE (hCE1m6), which aremore effectiveat converting CPT-11 to SN-38 than endogenous human CE. We hypothesized that NSC-mediatedCE/CPT-11 therapy would allow tumor-localized production of SN-38 and significantly increase thetherapeutic efficacy of irinotecan. Here, we report that transduced NSCs transiently expressed highlevels of active CE enzymes, retained their tumor-tropic properties, andmediated an increase in thecytotoxicity of CPT-11 toward glioma cells. CE-expressing NSCs (NSC.CEs), whether administeredintracranially or intravenously, delivered CE to orthotopic human glioma xenografts in mice. NSC-delivered CE catalyzed conversion of CPT-11 to SN-38 locally at tumor sites. These studies demon-strate the feasibility of NSC-mediated delivery of CE to glioma and lay the foundation for transla-tional studies of this therapeutic paradigm to improve clinical outcome and quality of life in patientswith malignant brain tumors. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:983–992

INTRODUCTION

A major challenge in treatment of solid tumors isthe lack of tumor-specific delivery of drugs andachieving sufficiently high drug concentrationswithin the tumor while minimizing the toxic sideeffects to normal tissues [1, 2]. Treatment ofbrain tumors, either primary or secondary me-tastases to the brain, poses additional challengesbecause the delivery of therapeutic drugs is im-peded by the blood-brain barrier (BBB), whichoften results in subtherapeutic levels of drug inthe cerebral compartment [3–6]. Neural stemcell-mediated enzyme prodrug therapy (NMEPT)is an emerging field that holds promise for treat-ment of many invasive types of cancer, includingglioma, neuroblastoma, medulloblastoma, met-astatic breast cancer, and melanoma [7–15]. Ap-proaches similar to NMEPT but using mesenchy-mal stem cells (MSCs), induced pluripotent stemcells (iPSCs), or other types of stem cells are also

under investigation [16, 17]. A key characteristicof many of these stem cells is that they exhibittumor-specific migration to distant tumors lo-cated intracranially or extracranially [18].

Many therapeutic genes, including those en-coding prodrug-activating enzymes, have beenintroduced into neural stem cells (NSCs), MSCs,and iPSCs. Prodrug-activating enzymes enableconversion of an inactive prodrug to active drugat tumor sites [7, 13, 14, 17, 19]. This approachallows for production of tumor-localized chemo-therapy, which makes it possible to maximize theconcentration of the tumor-toxic drug whilesparing normal tissues. NSC-mediated cytosinedeaminase (CD)/5-fluorocytosine (5-FC) therapywas developed for treatment of glioma as a first-generation enzyme/prodrug system that servedas proof of concept in a recently completed safe-ty/feasibility recurrent glioma clinical trial(ClinicalTrials.gov identifier NCT01172964) [19].In the second-generation CE/CPT-11 enzyme/

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prodrug system (the subject of the current report), the CE en-zyme is secreted by the tumor-tropic NSCs, providing a largerradius of action. CPT-11 has low but significant penetration ofthe central nervous system (CNS) and is converted to the highlypotent topoisomerase I inhibitor SN-38 following activation bythe CE-secreting NSCs (NSC.CE). Because the half-maximal inhib-itory concentration (IC50) value for SN-38 in glioma cells is in the5–100 nM range, it is likely that therapeutically effective levels ofactive metabolite can be achieved in the tumor milieu followingNSC.CE and CPT-11 administration. Furthermore, becauseCPT-11 is already approved for the treatment of several solidtumors in humans (including colon cancer and neuroblastoma)and demonstrates excellent antitumor activity, even modest im-provements in drug activation may yield significant increases indrug efficacy. The studies presented in this report sought to val-idate these hypotheses.

One prodrug-activating enzyme that has been extensivelystudied is carboxylesterase (CE), which converts the prodrugCPT-11 (irinotecan) to SN-38, a potent topoisomerase I inhibitor[20–23]. Humans express several isoforms of CE in the liver(hCE1, hCE2) and intestinal tract (hiCE), but CPT-11 is poorly ac-tivated by these endogenous CEs (typically less than 5% ofCPT-11 is converted to SN-38). Two other forms of CE, a rabbitliver enzyme (rCE) and a modified human enzyme (hCE1m6),have been developed and demonstrate much more efficient con-version of CPT-11 to SN-38 [24–27]. In particular, hCE1m6 wasgenerated using structure-guided mutagenesis. hCE1m6 is �70-fold more efficient at activating CPT-11 than the wild-type hCE1and is comparable in activity to the intestinal isoform hiCE [24].

We adenovirally transduced a clinically relevant human NSCline, HB1.F3.CD [19], to secrete rCE (NSC.rCE) or hCE1m6 (NSC.hCE1m6) in order to evaluate each enzyme’s potential utility inNMEPT. We compared NSC.rCE versus NSC.hCE1m6 in regard to(a) retention of tumor tropism, functional activity of CE, and cy-totoxicity to cancer cells in the presence of CPT-11; (b) delivery offunctional CE to orthotopic glioma in vivo; (c) increased conver-sion of CPT-11 to SN-38 in the presence of CE-expressing NSCs atthe tumor site in a dose- and time-dependent manner; and (d)immunogenicity. Here, to the best of our knowledge, we presentthe first demonstration of NSC-mediated delivery of functionallyactive hCE1m6 to glioma and localized in vivo production of ac-tive SN-38 at the brain tumor site. These results support furthertranslational development of CE-expressing NSCs in combinationwith CPT-11 for the clinical treatment of brain tumors and poten-tially other cancers.

MATERIALS AND METHODS

Neural Stem Cell CultureFor these studies, we used the v-myc-immortalized, humanclonal HB1.F3.CD NSC line (clone 21), which is genetically andfunctionally stable, nontumorigenic, and minimally immuno-genic [19, 28, 29]. HB1.F3.CD NSCs were thawed and cultured inDulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% heat-inactivated fetal bovine serum and 2 mM L-glu-tamine for 3 days (37°C, 6% CO2) in T-175 tissue culture flasksprior to adenoviral transduction. Control nontransducedHB1.F3.CD cells were cultured under identical conditions. Ad-enoviral stock for each virus type, AdrCE [20] or AdhCE1m6 [24],was diluted in media and added to cells to give a final multiplicity

of infection (MOI) of 20. NSCs were incubated for 18–24 hours,washed with phosphate-buffered saline (PBS), trypsinized, and re-suspended in fresh culture medium. NSCs were then plated for var-ious assays, or used in animal experiments. For CE activity assays,cells were grown for 96 hours in culture, after which mediacontaining CE were collected, cleared by centrifugation, andstored frozen at �80°C until use. In some experiments, NSCswere labeled with Feraheme (ferumoxytol; AMAG Pharma-ceuticals, Lexington, MA, http://www.amagpharma.com)(Fehe; 100 �g/ml) as previously reported [30], followed byadenoviral transduction with AdrCE or AdhCE1m6 (MOI � 20).

Expression and Purification of RecombinantCarboxylesterasesAll CEs used in degranulation assays were obtained by expressionin Sf21 insect cells using baculoviral vectors [24, 31]. All proteinswere functionally active and �95% pure [27, 31, 32].

Carboxylesterase Activity AssayCE enzyme activity was measured by conversion of o-nitrophenylacetate substrate to o-nitrophenol and determined by spectro-photometry at 420 nm [33, 34].

Boyden Chamber Cell Migration AssayIn vitro cell migration/chemotaxis assays were conducted using24-well cell culture plates with polycarbonate inserts (8 �m poresize; Millipore, Billerica, MA, http://www.millipore.com) as de-scribed previously [35]. Conditioned media were prepared byadding serum-free media to cultured U251 human glioma cells.Conditioned media from glioma cells were collected after 48hours and added to the lower chambers of 24-well plates (600�l). Suspensions of NSCs in serum-free culture media containing5% bovine serum albumin (BSA) were added to the upper cham-bers (105 cells per 400 �l), and after incubation for 4 hours at37°C, migrated cells were detached from the lower surface of theinsert by treatment with Accutase (eBioscience, San Diego, CA,http://www.ebioscience.com) and counted using the Guava Via-Count assay (Guava Technologies, Hayward, CA, http://www.guavatechnologies.com). Cell migration assay controls includedDMEM with 5% BSA (negative control).

Cytotoxicity AssaysHuman glioma cell lines (U87, U251, SJ-G2) and early passageglioma cells derived from biopsy/surgery materials from gliomapatients (PBT017, PBT018, PBT028) [36] were used as targets incytotoxicity assays. Cells were placed into 96-well plates (3,000–5,000 cells per well, in triplicate) in 100 �l of culture media perwell and cultured for 24 hours. The following day, CPT-11, dilutedin untransduced culture media (as a control) or conditioned me-dia from CE-expressing NSCs, was added (final concentrations of0–1,000 �M). Cells were incubated with drug for 4 hours, afterwhich media were aspirated and replaced with drug-free freshmedia, and cells were grown for an additional 96 hours. The cellnumber was measured by sulforhodamine B (Sigma-Aldrich, St.Louis, MO, http://www.sigmaaldrich.com) or WST-1 assays(Roche Applied Science, Indianapolis, IN, https://www.roche-applied-science.com), using spectrophotometric determinationat 570 or 450 nm, respectively. NSCs and NSC.CEs were also ex-posed to CPT-11 (at concentrations of 0–10 �M) in standardcytotoxic assay as described above. Cytotoxicity of CPT-11 toNSC.CEs was also measured in the presence of 1 ng/ml colchicine

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in the culture media. NSC.CEs were detached from the flask andviability was measured using Guava EasyCyte. Data were plottedusing GraphPad Prism 5 software and expressed as a percentageof untreated controls. Mean values � SD of triplicate measure-ments are shown.

Liquid Chromatography-TandemMass SpectrometryAssay for CPT-11 and SN-38Liquid chromatography-tandem mass spectrometry (LC-MS/MS)analysis was performed using a Waters Acquity HPLC system(Milford, MA, http://www.waters.com) interfaced with a WatersQuattro Premier XE Mass Spectrometer. High-performance liq-uid chromatography (HPLC) separation was achieved using aSynergi Hydro-RP 4 �m 150 � 2.0 mm analytical column (Phe-nomenex, Torrance, CA, http://www.phenomenex.com) pre-ceded by a Phenomenex C18 guard column. The column temper-ature was maintained at 30°C, and the flow rate was 0.4 ml/minute. The mobile phase consisted of A (20 mM ammoniumacetate buffer, pH 3.5) and B (acetonitrile). The following gradi-ent program was used: 20% B (hold, 0–3 minutes), 20%–68% B(3–6 minutes), 68% B (hold, 6–6.2 minutes), 68%–20% B (6.2–6.3 minutes), 20% B (hold, 6.3–8 minutes). The total run timewas 8 minutes. The electrospray ionization source of the massspectrometer was operated in positive ion mode with a cone gasflow of 80 liters/hour and a desolvation gas flow of 700 liters/hour. The capillary voltage was set to 0.6 kV, and the cone andcollision cell voltages were optimized to 60 V and 36 eV for CPT-11, 48 V and 26 eV for SN-38, and 45 V and 23 eV for camptoth-ecin (internal standard). The source temperature was 125°C, andthe desolvation temperature was 450°C. A solvent delay pro-gram was used from 0 to 4.7 minutes and from 6.1 to 8 minutesto minimize the mobile phase flow to the source. MassLynx (Wa-ters Corp., Milford, MA, http://www.waters.com) version 4.1software was used for data acquisition and processing. Positiveelectrospray ionization of CPT-11, SN-38, and camptothecin pro-duced abundant protonated molecular ions (MH�) at m/z587.31, 393.21, and 349.15, respectively. Fragmentation ofthese compounds was induced under collision-induced dissocia-tion conditions and acidic mobile phase. The precursor3product ion combinations at m/z 587.313124.14 for CPT-11,393.213349.20 for SN-38, and 349.153305.11 for camptoth-ecin were used in multiple-reaction monitoring mode for quan-titation. Under optimized assay conditions, the retention timesfor CPT-11, SN-38, and camptothecin were 5.25, 5.43, and 5.62minutes, respectively.

Animal Models and Intracranial Human GliomaXenograftsPlasma Es1e esterase-deficient severe combined immunodefi-cient mice (Es1e/SCID) were used in in vivo studies. These micehave plasma CE levels comparable to those in humans [37]. Micewere housed in an Association for Assessment and Accreditationof Laboratory Animal Care (AALAAC)-accredited facility and weregiven food and water ad libitum. U251.eGFP.ffLuc human gliomacells (2 � 105 cells per 2 �l) were implanted stereotactically intothe frontal lobe (1 mm rostral and 2 mm right of the bregma, 2.5mm initial depth, retracted to 2.25 and 2.0 mm; 0.667 �l injectedat each depth). HB1.F3.CD NSCs were labeled with Fehe ultras-mall superparamagnetic iron oxide nanoparticles (100 �g/ml)and adenovirally transduced with hCE1m6 (MOI � 20) as de-scribed previously [30, 38]. Three days after tumor cell implan-

tation, HB1.F3.CD.hCE1m6-Fehe NSCs were intracranially (i.c.)injected caudolateral to the tumor (1 � 105 cells per 2 �l) usingthe same volume and depth as for glioma cells. Mice were eu-thanized 4 days after NSC implantation. Another group of micewas given intravenous (i.v.) injections of Fehe-labeled NSCs (1 �106 cells per 200 �l). Mice were treated for 3 days with CPT-11(25 mg/kg), 4 days after NSC injection, and the treatment wasrepeated once. Mice were euthanized 10 days after the sec-ond treatment. Brains were embedded in paraffin, and serial10-�m horizontal sections were processed for hematoxylinand eosin (H&E) histology, Prussian blue staining, and immu-nohistochemistry for the detection of NSCs and CE enzymeafter CPT-11 treatment in vivo [9].

LC-MS/MS Measurements of CPT-11 and SN-38 inTumor-Bearing BrainEs1e/SCID mice were injected i.c. with U251.eGFP.ffLuc gliomacells (2 � 105 cells per 2.5 �l), using the same coordinates asdescribed above. HB1.F3.CD.hCE1m6 cells were i.c. injected cau-dolateral to the tumor (5 � 105 cells per 2 �l) using the samevolume and depth as for glioma cells. Three days later, mice weregiven 3.75 or 7.5 mg/kg CPT-11 via tail vein injection. Mice (n �3) were euthanized 1 or 4 hours after drug administration. Brainswere harvested and quartered, and concentrations of CPT-11and SN-38 were determined by LC-MS/MS. Comparisons weremade between tumor-bearing and non-tumor-bearing areas ofthe brain.

Histochemistry, Immunohistochemistry, and PrussianBlue StainingSections (10 �m thick) were processed by H&E staining. En-hanced green fluorescent protein (eGFP) immunohistochemistrywas performed using an anti-eGFP antibody (Abcam, Cambridge,MA, http://www.abcam.com) and detected by 3,3-diaminoben-zidine (DAB). Immunohistochemical detection of hCE1m6 wascarried out using sodium citrate (pH 6) antigen retrieval for 30minutes at 95°C, rabbit anti-human liver CE antibody (generatedin the laboratory of Dr. Philip Potter; final concentration, 1.6�g/ml), biotinylated goat anti-rabbit antibody (Vector Laborato-ries, Burlingame, CA, http://www.vectorlabs.com), VectastainABC-Elite Kit (Vector Laboratories), and DAB. Detection of Fehe-labeled NSCs was performed using Prussian blue staining withthe Accustain Iron Stain Kit (Sigma-Aldrich) as described previ-ously [10].

Microdialysis StudiesMicrodialysis cannulae were placed stereotactically into thebrains of non-tumor-bearing nu/nu rats, followed by a 2-weekrecovery period. For administration of NSCs (HB1.F3.CD,HB1.F3.CD.hCE1m6, and HB1.F3.CD.rCE), cells were resus-pended in sterile PBS and i.c. injected (2.5 � 105 NSCs) throughthe cannula, after which the dummy catheter was replaced withthe microdialysis probe (CMA12; CMA, Solna, Sweden, http://www.microdialysis.com). This was then perfused with artificialcerebrospinal fluid (CMA) at a rate of 0.5 �l/minute for 24 hours.After equilibration, CPT-11 (20 mg/kg) was administered via thetail vein, and dialysate and plasma samples were collected andstored for analysis. Dialysate was collected over a single 20-hourinterval after the CPT-11 dose. Whole blood for plasma isolationwas collected from tail vein bleeds of each rat at 0.5, 2, and 20hours after each CPT-11 dose. Concentrations of CPT-11 and

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SN-38 in dialysate and plasma were determined by LC-MS/MS asdescribed above.

Degranulation AssaysPeripheral blood mononuclear cells (PBMCs) were isolated from20 healthy volunteers, using standard Ficoll density gradient cen-trifugation, and cryopreserved. Thawed aliquots of cells wereallowed to rest overnight in T-cell media (RPMI supplementedwith 10% fetal calf serum and 2 mM L-glutamine). PBMCs wereplated (1 � 106 cells per milliliter) in 48-well flat-bottomed tissueculture plates, and purified hCE1, hCE1m6, or rCE was added(final concentration, 1 �g/ml). For positive control, a mixture ofphorbol myristate acetate (PMA; 50 ng/ml) and phytohemagglu-tinin (PHA; 1 �g/ml) was added to the plated PBMCs. Fluoresceinisothiocyanate-conjugated antibodies against CD107a andCD107b (Pharmingen/BD Biosciences, San Diego, CA, http://www.bdbiosciences.com) were added to all wells prior to theaddition of 1 �l of monensin-containing protein transport inhib-itor (GolgiStop; Becton, Dickinson and Company, Franklin Lakes,NJ, http://www.bd.com). Cells were then incubated (5 hours,37°C, 5% CO2), washed, and labeled with fluorescently conju-gated antibodies against CD3, CD4, CD56, CD16, CD14, andCD19 prior to analysis with a Gallios flow cytometer (BeckmanCoulter, Fullerton, CA, http://www.beckmancoulter.com).Monocytes and B cells that expressed CD14 and CD19 wereexcluded. Degranulation of PBMCs was measured in the pop-ulation of CD4�, CD8� and natural killer (NK) cells in the pres-

ence of hCE1, hCE1m6, rCE, or mixture of PMA and PHA (pos-itive control) [39].

RESULTS

HB1.F3.CD NSCs Retain Their Characteristics AfterTransduction With rCE or hCE1m6In all our experiments, we used the v-myc immortalized, humanclonal HB1.F3.CD NSC line, which is genetically stable, nontu-morigenic, and minimally immunogenic [19]. In this study,HB1.F3.CD NSCs were transduced with adenovirus, expressingCE enzymes rCE or hCE1m6. To determine whether the charac-teristics of HB1.F3.CD cells are affected by CE transduction, wecompared CE-transduced NSCs with untransduced cells for via-bility, growth kinetics, tumor tropism, and expression of func-tional rCE or hCE1m6 enzymes in vitro. rCE- or hCE1m6-trans-duced NSCs secreted functional CE enzymes into the culturemedia, as was measured by spectrophotometry assays (Fig. 1A).We detected similar activities for each enzyme in conditionedmedia derived from NSC.CEs. Neither viability nor growth kinet-ics of cells were affected by rCE or hCE1m6 expression (�90%viability, data not shown). It is important for future clinical usethat no significant differences were detected in the tumor tro-pism of NSC.CEs compared with nontransduced NSCs in vitro(Fig. 1B). We also determined that both secreted CEs (rCE andhCE1m6) had similar abilities to sensitize human glioma cells to

Figure 1. HB1.F3.CD neural stem cells (NSCs) transduced with adenoviral vectors encoding rCE or hCE1m6 produce functional enzymes,retain tumor tropism, and enhance killing of glioma cells. (A): Enzymatic activities of rCE and hCE1m6 secreted by HB1.F3.CD NSCs transducedwith adenovirus encoding rCE or hCE1m6. (B): Tumor tropism of CE-expressing HB1.F3.CD NSCs to conditioned media from U251 glioma cells,as detected by Boyden chamber cell migration assay (5% BSA was used as negative control). (C):Dose-response of PBT018 human glioma cellsto CPT-11 and SN-38 alone and to CPT-11 in the presence of rCE or hCE1m6 expressed by NSCs. Mean values � SD of triplicate measurementsare shown. Abbreviations: BSA, bovine serum albumin; hCE1m6, modified human carboxylesterase; NSC.CD, parental neural stem cell lineexpressing Escherichia coli cytosine deaminase; NSC.hCE1m6, neural stem cells expressing a modified human carboxylesterase; NSC.rCE,neural stem cells expressing rabbit carboxylesterase; o-NP, o-nitrophenol; rCE, rabbit carboxylesterase.

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CPT-11 (Fig. 1C; Table 1), whereas no differences were detectedin the CPT-11 toxicities in the assays that used culture media(DMEM) compared with media collected from NSCs transducedwith null adenoviral vector. Indeed, for all six glioma cell linestested, significant reductions in the IC50 values (�70–1,200-fold) for CPT-11 were observed, consistent with the hypothesisthat exogenous expression of CE results in increased intracellularlevels of SN-38 in drug-treated glioma cells. As an example, theIC50 of CPT-11 in the PBT018 cell line derived from a glioma bi-opsy decreased by 1,280-fold after incubation with conditionedmedia derived from NSCs transduced with adenovirus-express-ing either rCE or hCE1m6, as compared with IC50 values forCPT-11 alone (Table 1).

Pharmacokinetics of CPT-11 Conversion to SN-38 in thePresence of rCE and hCE1m6 In VitroOur pharmacokinetic studies showed a time-dependent increaseof SN-38 concentration with a concomitant decrease in CPT-11concentration in the presence of CE enzymes in vitro (Fig. 2). Tomeasure direct conversion of CPT-11 to SN-38, we used an LC-MS/MS assay for CPT-11 and SN-38. CPT-11 at a concentration of3 or 15 �M was added to conditioned media collected fromtransduced NSC.CEs or untransduced NSCs, followed by incuba-tion at 37°C, 6% CO2. Media samples were collected over 96hours. Both enzymes were similar in their ability to convertCPT-11 to SN-38 (Fig. 2A, 2B), consistent with previous reportsusing purified CEs [24]. These data also indicated that the CEpresent in the media collected from NSC.CEs was stable over 96hours and that CPT-11 in the absence of CE was not converted toSN-38 in control samples.

Expression of hCE1m6 in Mouse Brain by Fehe-LabeledNSC.CEs In VivoPlasma Es1e esterase-deficient severe combined immunodeficientmice (Es1e/SCID) were used in in vivo studies to distinguish endog-enous esterase activity in these experiments. These mice haveplasma CE levels comparable to those in humans [37].U251.eGFP.ffLuc human glioma xenografts were established in thefrontal lobes of mouse brains by stereotactic injection (Fig. 3A, H&E,eGFP). Three days after tumor cell implantation, Feraheme-labeledNSC.hCE1m6 cells were injected intracranially (i.c.), caudolateral tothe tumor (1 � 105 cells per 2 �l) site using the same volume anddepth as for glioma cells. Mice were euthanized 4 days after NSCimplantation. Brains were embedded in paraffin, and serial 10-�mhorizontal sections were processed for H&E histology and Prussianblue and immunohistochemical staining using anti-green fluores-cent protein and CE antibodies. Prussian blue staining of adjacentbrain sections revealed that Fehe-labeled NSCs were distributed inand around glioma tumor xenografts 4 days after NSC administra-

tion (Fig. 3A, Prussian blue). Typically, NSCs were detected at thetumor edge (Fig. 3A, Prussian blue), although some were presentwithin the tumor (Fig. 3A, Prussian blue, arrow). CE was detected atthe tumor edge, colocalizing with Prussian blue-labeled NSCs (Fig.3A, carboxylesterase).

A second group of U251 tumor-bearing mice received i.v. injec-tions of Fehe-labeled NSC.hCE1m6 followed by CPT-11 treatment(25 mg/kg per day for 3 days). The treatment with NSC.CE andCPT-11 was repeated once the following week. Mice were eutha-nized on day 21 after tumor implantation, and their brains wereprocessed for immunohistochemical staining to detect the pres-ence of CE enzyme after CPT-11 treatment. Intravenously injectedNSC.CEs showed distribution in and around the glioma tumor xeno-grafts (Fig. 3B, Prussian blue). Feraheme-labeled NSCs were de-tected at the tumor edge and tumor center using Prussian bluestaining (Fig. 3B, Prussian blue), whereas CE expression was demon-strated in the tumor xenograft in adjacent sections stained with CEantibodies (Fig. 3B, carboxylesterase).

We and others have shown that NSCs do not divide in vivo[19, 40]. In order to determine whether CPT-11 and/or SN-38

Table 1. Sensitivity of human glioma cells to CPT-11 and SN-38, and increased cytotoxicity of CPT-11 in the presence of rCE or hCE1m6

IC50 (�M) Fold decrease in IC50 of CPT-11

Glioma cells CPT-11 SN-38 rCE � CPT-11 hCE1m6 � CPT-11 rCE � CPT-11 hCE1m6 � CPT-11

U87 201 � 82 0.152 � 0.031 2.16 � 1.14 0.34 � 0.06 93 591U251 42 � 3 0.102 � 0.040 0.06 � 0.00 0.085 � 0.005 700 494SJ-G2 16.5 � 10.6 0.009 � 0.003 0.08 � 0.07 0.097 � 0.001 206 170PBT017 13.3 � 3 0.020 � 0.007 0.04 � 0.03 0.032 � 0.023 325 406PBT018 32 � 0 0.024 � 0.005 0.025 � 0.006 0.025 � 0.01 1,280 1,280PBT028 31 � 13 0.037 � 0.012 0.063 � 0.002 0.049 � 0.01 489 629

Abbreviations: hCE1m6, modified human carboxylesterase; IC50, half-maximal inhibitory concentration; rCE, rabbit carboxylesterase.

Figure 2. Kinetics of carboxylesterase-mediated conversion ofCPT-11 to SN-38 in vitro. Media were collected from rCE- or hCE1m6-transduced NSCs. (A, B): 3 �M CPT-11 (A) or 15 �M CPT-11 (B) wasadded to media containing rCE or hCE1m6 enzymes, and sampleswere collected at 4, 24, 48, 72, and 96 hours. Controls were gen-erated using media collected from nontransduced HB1.F3.CDNSCs (filled circles, CPT-11; outlined circles, SN-38). Values shownare mean � SD, for reactions performed in duplicate. Abbrevia-tions: hCE1m6, modified human carboxylesterase; rCE, rabbitcarboxylesterase.

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were toxic to nondividing NSC.CE cells in vivo, we performedexperiments mimicking the nondividing/quiescent NSC state invitro (Fig. 3C). First, Figure 3C demonstrates that dividing NSCs orNSC.CEs were sensitive to CPT-11 in vitro, with IC50 values of 1 or0.07 �M, respectively. In contrast, cells treated with CPT-11 inthe presence of 1 ng/ml colchicine (designed to inhibit cell divi-sion) were insensitive to the former agent. As demonstrated inFigure 3D, the viability of NSC.CE cells under these conditionswas not affected by addition of CPT-11 to the culture media,compared with non-drug-treated controls. This further supportsour hypothesis that in vivo, NSC.CEs will survive for extendedperiods of time, even in the presence of CPT-11.

In Vivo Microdialysis Studies of CPT-11 ProdrugActivation by CE-Expressing NSCsWe used in vivo microdialysis to determine the concentration ofSN-38 in the brains of rats that received CE-expressing NSCs. Inthese studies, nontransduced cells served as controls. NSCs wereinjected i.c. into the brains of rats, which then received i.v. injectionsof CPT-11 (20 mg/kg). Both CPT-11 and SN-38 were detected in thebrain after i.v. CPT-11 administration (Table 2). In control rats thatreceived nontransduced NSCs, the drug concentrations in the braininterstitium were less than 1% of the corresponding plasma druglevels, indicating a low degree of CNS penetration by these mole-

cules. Average plasma concentrations of CPT-11 and SN-38 in ratsthat received CE-expressing NSCs were not significantly differentfrom those that received nontransduced NSCs. However, the ratiosof SN-38 to CPT-11 concentrations in brains of rats injected withNSCs that expressed rCE (p � .02) or hCE1m6 (p � .002) were sig-nificantly higher compared with rats that received nontransducedNSCs. The SN-38/CPT-11 ratios in rats treated with rCE or hCE1m6were similar, indicating that both enzymes were equally effective atconverting CPT-11 to SN-38. In summary, although the CNS pene-tration of i.v. administered CPT-11 was low, we found fourfoldhigherconcentrationsofSN-38 in thebrainsof rats treatedwith rCE-or hCE1m6-expressing NSCs, compared with rats treated with con-trol NSCs. Furthermore, the ratios of SN-38/CPT-11 were three- tofourfold higher in the former. Taken together, these results demon-strate that CE-transduced HB1.F3.CD NSCs can express functionallyactive CEs in rat brain, and that both CE variants are equally able toconvert CPT-11 to SN-38 in vivo.

CE-Expressing NSCs Increase the Concentration of SN-38in Tumor-Bearing Brain

The accumulation of SN-38 at brain tumors in glioma-bearingmice was measured in the presence of NSC.hCE1m6 cells in com-bination with CPT-11 and compared with samples from mice that

Figure 3. HB1.F3.CD.hCE1m6-Fehe NSCs can target and deliver hCE1m6 to human glioma xenografts in the mouse brain. (A): Consecutivehorizontal brain sections from a tumor-bearing mouse that received an i.c. injection of NSC.hCE1m6-Fehe cells. (B): Serial horizontal brainsections from a tumor-bearing mouse that received an i.v. injection of NSC.hCE1m6-Fehe. H&E indicates hematoxylin and eosin-stainedhorizontal sections of brain containing glioma xenograft. The panel marked “eGFP” shows immunohistochemistry (IHC) detection of eGFP-expressing human glioma xenograft. Also shown is Prussian blue staining of carboxylesterase (CE)-expressing NSCs labeled with Fehe (blackarrows, Prussian blue staining of NSCs). Right panel (carboxylesterase) shows IHC staining for CE. NSCs stained positive for CE are indicated byblack arrows. Scale bars � 200 �m. (C): Dose-response of NSCs and CE-expressing NSCs (NSC.CEs) to CPT-11. NSCs or NSC.CEs were exposedto CPT-11 in a range of concentrations (0, 0.001, 0.01, 0.1, 1, and 10 �M) in a standard cytotoxicity assay. (D): Viability of NSC.CEs treated withcolchicine (1 ng/ml) was measured after exposure to 0, 0.1, or 1 �M CPT-11 on days 1, 3, 4, and 5. Mean values � SD of triplicatemeasurements are shown. Abbreviations: eGFP, enhanced green fluorescent protein; Fehe, Feraheme; hCE1m6, modified human carboxy-lesterase; H&E, hematoxylin and eosin; NSC, neural stem cells; NSC.rCE, neural stem cells expressing rabbit carboxylesterase.

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received CPT-11 alone (Fig. 4A). Mice that received NSC.hCE1m6cells in combination with CPT-11 had significantly more SN-38 atthe tumor site than did mice that did not receive NSCs but re-ceived CPT-11 only (Fig. 4B). SN-38 concentrations in vivo were�20 and 31 nM 1 hour after CPT-11 administration (3.75 and 7.5mg/kg, respectively) and �4 and 8 nM 4 hours after CPT-11 ad-ministration (3.75 and 7.5 mg/kg, respectively) into mice thatreceived NSC.hCE1m6 cells (Fig. 4B). These results provide evi-dence of concentration- and time-dependent conversion ofCPT-11 to SN-38 at the tumor site, but not in normal brain tissue.It should be noted that the SN-38 concentrations measured innormal, non-tumor-bearing brain were comparable in conditionswith and without administration of NSC.CEs, and most likely re-flected drug levels present in the residual blood remaining in thecollected tissue specimens, as well as a small amount of drug inthe systemic circulation that is able to penetrate the blood-brainbarrier.

Comparison of the Immunogenic Potential of hCE1,hCE1m6, and rCETo determine whether rCE, hCE1, or hCE1m6 would be immuno-genic, we assessed the ability of these enzymes to induce de-granulation (a measure of potential immune response) in PBMCsin vitro. CD4� T cells showed lower levels of degranulation inresponse to hCE1m6 compared with rCE (n � 20, p � .028, two-tailed t test), but hCE1m6 induced greater degranulation in CD4�

T cells compared with hCE1 (n � 20, p � .028) (average levels ofdegranulation: hCE1 hCE1m6 rCE) (Fig. 5A). No statisticallysignificant difference in degranulation was observed in the CD8�

T-cell population in response to hCE1m6 compared with rCE (n�20, p � .052), but hCE1m6 induced greater degranulation com-pared with hCE1 (n � 20, p � .007) (Fig. 5B). rCE induced moredegranulation compared with hCE1 (n � 20, p � .001). No sig-nificant differences in NK cell degranulation were observed forany of the CEs (Fig. 5C). Overall, these studies indicate thathCE1m6 is less immunogenic than rCE, and therefore, hCE1m6would be the enzyme of choice for future in vivo studies that useNMEPT.

DISCUSSION

Chemotherapy for glioma and metastatic cancer often fails forseveral reasons. For previously drug-treated individuals, treat-ment failure may be due to development of tumor resistance tothe agents used. However, it has been hypothesized that even inthese instances, increasing the local concentrations of the activedrugs at the site of the tumor may yield therapeutic benefits.

Previous approaches that sought to increase drug concentra-tions at primary and metastatic tumor sites in vivo (e.g., by theuse of tumor-specific antibodies that contain either drug-activat-ing enzymes or toxic drug conjugates) have shown promise butgenerally have not provided significant improvements in clinicaloutcome [41]. The exact reasons for this are unclear, but it maybe due to low levels of antigen expressed on tumor cells, limitedinternalization of drug into the target cell, and/or multidrug re-sistance of the cancer cells [41–43]. An approach based on thetumor tropism of NSCs, coupled with the selective activation ofCPT-11 afforded by modified mammalian CEs has the potentialto overcome these obstacles.

CPT-11 is poorly activated in humans, yet it demonstratessignificant antitumor efficacy for therapy for colon cancer. How-ever, the use of CPT-11 to treat brain tumors has not been effec-tive, possibly because of inefficient transfer of the drug acrossthe BBB; brain tumors demonstrate only approximately 10% ofthe blood level of CPT-11 [44]. We hypothesize that even smallincreases in the amount of SN-38 within the tumor milieu wouldbe advantageous because tumors are sensitive to SN-38 in thenanomolar range. Furthermore, the studies described herein areclinically relevant because CPT-11 is approved by the U.S. Foodand Drug Administration for clinical use, and the HB1.F3.CD NSCline used in these studies has been used in a first-in-humansafety trial for recurrent glioma in combination with the 5-FCprodrug (ClinicalTrials.gov identifier NCT01172964). Therefore,we believe that NSC-mediated tumor-specific delivery of CE incombination with CPT-11 can be fast-tracked for clinical trial,once Investigational New Drug (IND)-enabling safety, toxicity,and efficacy studies are completed.

We compared the biology and biochemical properties of twoisoforms of CE (rCE and hCE1m6) that can efficiently activateCPT-11. Although the kinetics of CPT-11 activation by hCE1m6were reported lower than those seen for rCE [24], in our cellculture-based assays, hCE1m6 performed comparably or better,and when used in combination with CPT-11 yielded large reduc-tions in the IC50 values when tested against human glioma celllines (Fig. 1; Table 1). We found that human glioma cell lines were�70–1,200-fold more sensitive to CPT-11 in the presence ofconditioned media from CE-expressing NSCs (Fig. 1; Table 1). CEexpression did not affect NSC tumor tropism in vitro, suggestingthat NSC targeting of tumors in vivo was unlikely to be affectedby adenovirus-mediated cDNA delivery and/or exogenous geneexpression. Furthermore, because both rCE and hCE1m6 wereengineered to be secreted, detection of CE and conversion ofCPT-11 to SN-38 in cell culture media could be readily assessed.NSC-secreted CEs that accumulated in the media were stable

Table 2. Conversion of CPT-11 to SN-38 by rCE and hCE1m6 in vivo

CPT-11 (nM) SN-38 (nM) Ratio (%) p valuea

BrainHB1.F3.CD.rCE 1.08 � 0.41b 0.35 � 0.36 22.1 � 12.6 0.02HB1.F3.CD.hCE1m6 1.62 � 0.78 0.35 � 0.22 16.7 � 3.9 0.002HB1.F3.CD 1.66 � 0.96 0.09 � 0.06 6.0 � 5.0

Blood plasmaHB1.F3.CD.rCE 513.8 � 524.7 57.9 � 39.6 6.9 � 5.0 NSHB1.F3.CD.hCE1m6 703.2 � 419.9 9.8 � 5.6 1.0 � 0.9 NSHB1.F3.CD 866.7 � 403.4 24.7 � 16.2 3.2 � 2.7

Table shows the average concentrations of CPT-11 and SN-38 in brain interstitium and blood plasma 0–20 hours after administration of CPT-11.aCarboxylesterase-expressing neural stem cells versus nontransduced neural stem cells; two-tailed t test.bMean � SD, n � 6.Abbreviations: hCE1m6, modified human carboxylesterase; NS, not significant; rCE, rabbit carboxylesterase.

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over the time course of the experiment (96 hours) and led toenhanced CPT-11 conversion (Fig. 2). Levels of SN-38 generatedin these studies were comparable between the two forms of CEassessed. Our demonstrated production of SN-38 is consistentwith, and suggests a mode of action for, our previous discoveriesthat used rCE-expressing NSCs in combination with CPT-11 forthe effective treatment of mice bearing metastatic neuroblas-toma and breast cancer [14, 38, 45].

The results from the microdialysis experiments provideproof-of-concept and establish the ability of CE-expressing NSCsto convert CPT-11 to its toxic metabolite SN-38 in vivo. Althoughthe CNS penetration of i.v. administered CPT-11 was low, wefound fourfold higher concentrations of SN-38 and significantlyhigher SN-38/CPT-11 ratios in the brains of rats treated withNSCs. Taken together, these results demonstrate that adenovi-

rally transduced NSCs can express functionally active CEs andthat both CE variants are equally efficient at locally convertingCPT-11 to SN-38 in vivo. Of note, the SN-38 levels measured bymicrodialysis are likely an underestimate of the intracellular con-centrations, because of the rapid and preferential partitioning ofdrug into cells [46]. In contrast, our HPLC analyses of brain tissuemeasured both extracellular and intracellular SN-38 concentra-tions and confirmed significantly higher levels of SN-38 in tumorxenografts in mice treated with hCE1m6 NSCs � CPT-11 (Fig. 4B).

CONCLUSION

The studies presented here indicate that hCE1m6 secreted byHB1.F3.CD NSCs is as effective as rCE in all assays performed. Be-cause of the similar biochemical and cellular properties of the rCE

Figure 4. Conversion of CPT-11 to SN-38 by NSC.hCE1m6 in the brains of glioma-bearing mice. (A): Schematic of the experimental design forimplanting U251.eGFP.ffLuc human glioma cells and administering hCE1m6-expressing NSCs and CPT-11 (3.75 or 7.5 mg/kg). Mice that did notreceive NSCs served as controls. (B):Measurements of SN-38 concentrations in the brains of mice treated as described in (A) 1 or 4 hours afterCPT-11 administration. Data shown are mean � SD (n � 3). Abbreviations: NSC, neural stem cells; NSC.CE, CE-expressing neural stem cells;NSC.hCE1m6, neural stem cells expressing a modified human carboxylesterase.

Figure 5. Measurement of degranulation in normal donor peripheral blood mononuclear cells in response to the various carboxylesterases(CEs). Degranulation of CD4� (A), CD8� (B), and NK (C) cells in the presence of hCE1, hCE1m6, rCE, or phorbol myristate acetate/phytohe-magglutinin (positive control). Abbreviations: �ive, positive control; hCE1m6, modified human carboxylesterase; NK, natural killer; NS, notsignificant; rCE, rabbit carboxylesterase.

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and hCE1m6 enzymes, we expect in vivo therapeutic efficacy to alsobe similar. The lower immunogenicity of hCE1m6 compared withrCE supports the choice of hCE1m6 for clinical application. Thera-peutic efficacy and safety/toxicity IND-enabling studies are in prog-ress to move this treatment toward clinical trial.

ACKNOWLEDGMENTS

We thank Dr. Seung U. Kim (University of British Columbia, Vancou-ver, BC, Canada) for his generous gift of the HB1.F3.CD NSC line. Weacknowledge the technical support of Elizabeth Garcia, SorayaAramburo, Valerie V. Valenzuela, Janel Ortiz, Xiaqin Wu, and ViviTran. This work was supported by funding from the California Insti-tute for Regenerative Medicine (DR1-01421), the Rosalinde and Ar-thur Gilbert Foundation, STOP Cancer, the American Lebanese Syr-ian Associated Charities, St. Jude Children’s Research Hospital, andthe National Cancer Institute (P30-CA33572). We are grateful to Dr.Keely L. Walker (City of Hope) for editing the manuscript.

AUTHOR CONTRIBUTIONS

M.Z.M. and T.W.S.: conception and design, performance of ex-periments, collection and assembly of data, data analysis and

interpretation, manuscript writing; M. Gutova: conception anddesign, collection and/or assembly of data, data analysis andinterpretation, manuscript writing; S.F.L.: conception and de-sign, performance of experiments, collection and/or assembly ofdata, data analysis and interpretation, manuscript writing; Y.A.,T.V., and R.T.: performance of experiments, collection and as-sembly of data, data analysis and interpretation; M. Gilchrist:performance of experiments, collection and assembly of data;L.Y.G. and M.E.B.: data analysis and interpretation; C.E.B.: provi-sion of study materials, consultation on study design; J.N. andJ.P.: conception and design, data analysis and interpretation,manuscript writing; P.M.P.: provision of study materials, dataanalysis and interpretation, manuscript writing; K.S.A.: concep-tion and design, data analysis and interpretation, manuscriptwriting, final approval of manuscript.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

K.S.A. is a shareholder of and has uncompensated employment(director and officer) with TheraBiologics, Inc. L.Y.G. is an uncom-pensated cofounder, shareholder, and scientific director ofKeren Pharmaceuticals.

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