Bone Marrow-derived Cells Contribute to Tumor ... · Flk-1/KDR (VEGFR-2; 14–16). We have shown...

Bone Marrow-derived Cells Contribute to Tumor Neovasculatureand, When Modified to Express an Angiogenesis Inhibitor, CanRestrict Tumor Growth in Mice1

Andrew M. Davidoff,2 Catherine Y. C. Ng,Peggy Brown, Margaret A. Leary,William W. Spurbeck, Junfang Zhou,Edwin Horwitz, Elio F. Vanin, andArthur W. NienhuisDepartments of Surgery [A. M. D., C. Y. C. N., W. W. S., J. Z.] andHematology/Oncology [P. B., M. A. L., E. H., E. F. V., A. W. N.], St.Jude Children’s Research Hospital, and Department of Surgery,University of Tennessee College of Medicine [A. M. D., W. W. S.],Memphis, Tennessee 38105

ABSTRACTInhibition of tumor-induced neovascularization ap-

pears to be an effective anticancer approach, althoughlong-term angiogenesis inhibition may be required. Analternative to chronic drug administration is a genetherapy-mediated approach in which long-term in vivoprotein expression is established. We have tested thisapproach by modifying murine bone marrow-derivedcells with a gene encoding an angiogenesis inhibitor: asoluble, truncated form of the vascular endothelialgrowth factor receptor-2, fetal liver kinase-1 (Flk-1). Mu-rine bone marrow cells were transduced with a retroviralvector encoding either truncated, soluble Flk-1 (tsFlk-1)together with green fluorescent protein (GFP) or GFPalone. Tumor growth in mice challenged 3 months aftertransplantation with tsFlk-1-expressing bone marrowcells was significantly inhibited when compared with tu-mor growth in control-transplanted mice. Immunohisto-chemical analysis of tumors in each group demonstratedcolocalization of GFP expression in cells staining withendothelial cell markers, suggesting that the endothelialcells of the tumor-induced neovasculature were derived,at least in part, from bone marrow precursors. Theseresults suggest that long-term expression of a functionalangiogenesis inhibitor can be generated through gene-

modified, bone marrow-derived stem cells, and that thisapproach can have significant anticancer efficacy. Modi-fying these cells seems to have the added potential benefitof targeting transgene expression to the tumor neovascu-lature, because bone marrow-derived endothelial cell pre-cursors seem to be recruited in the process of tumor-induced angiogenesis.

INTRODUCTIONAngiogenesis, the formation of new capillary blood ves-

sels, is required for the growth and spread of cancer (1–3). Thecorollary to this is that inhibition of tumor-induced neovascu-larization may be an effective anticancer approach that could bebroadly applicable because most, if not all, malignancies seemto be angiogenesis-dependent (4–6). Although an increasingnumber of studies have demonstrated the efficacy of recombi-nant antiangiogenic proteins in murine tumor models, a genetherapy-mediated approach to their delivery has a number ofpotential advantages (7–9). (a) Angiogenesis inhibition is likelyto be a cytostatic therapy; therefore, long-term delivery of theseagents, as can be achieved through a gene therapy-mediatedapproach, may be necessary. (b) Difficulties with protein pro-duction and maintenance of function, especially when “scalingup” for clinical trials, may be avoided by in situ expression inhost tissues. And (c), continuous low levels of these proteins, aswould be generated from gene-modified cells, may be the op-timal delivery schedule (10).

Gene therapy strategies for tumor antiangiogenesis have, infact, already been tested in a number of different murine tumormodels, and this approach has had some success. Most of thesestudies have used either retroviral vector producers, nakedDNA, or adenoviral vectors as the angiogenesis inhibitory gene-delivery vehicles (9). Unfortunately, retroviral vector producersmay be impractical for human use, and only transient expressionof the various proteins was established by the latter two ap-proaches in which, after an initial delay, tumor growth oftenresumed. This probably occurred because antiangiogenic ther-apy is only cytostatic; tumor growth may resume once therestrictions of angiogenesis inhibition are removed. Therefore,long-term expression of angiogenesis inhibitors is likely to berequired for sustained anticancer efficacy. Alternative gene ther-apy approaches are needed to ensure long-term expression ofthese proteins. In addition, however, the effects of chronicangiogenesis inhibition on physiological, angiogenesis-depen-dent processes, such as healing and fertility, will also have to becritically evaluated.

The use of bone marrow-derived cells as targets fortransduction and, therefore, the source of angiogenesis inhib-itor expression is intriguing for several reasons. (a) Trans-duction of bone marrow-derived stem cells with retroviralvectors has been shown to be feasible and results in long-term

Received 2/19/01; revised 6/11/01; accepted 6/18/01.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 This work was supported by Grant 94-000 from the Assisi Foundationof Memphis, Grant IRG-87-008-09 from the American Cancer Society,Cancer Center Support CORE Grant P30 CA 21765, and AmericanLebanese Syrian Associated Charities (ALSAC).2 To whom requests for reprints should addressed, at Department ofSurgery, St. Jude Children’s Research Hospital, 332 N. Lauderdale,Memphis, TN 38105. Phone: (901) 495-4060; Fax: (901) 495-2176;E-mail: [email protected].

2870 Vol. 7, 2870–2879, September 2001 Clinical Cancer Research

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transgene expression from mature bone marrow-derived cellsin mice (11). (b) Because bone marrow transplantation is astandard part of treatment for many high-risk malignancies inchildren, this approach could, ultimately, be readily incorpo-rated into existing pediatric treatment protocols. And (c), theendothelial cells incorporated into tumor-induced new bloodvessels may, in fact, be derived from precursor cells of bonemarrow origin (12), and modifying these cells to express aninhibitor of endothelial cell activation may prevent endothe-lial cell precursor differentiation and/or create a milieu ofangiogenesis inhibition at the sites where tumors are trying toinduce neovascularization.

VEGF3 is one of the primary tumor-expressed endothe-lial cell mitogens (13). The activity of this ligand can beinhibited by a soluble, truncated form of one of its receptors,Flk-1/KDR (VEGFR-2; 14 –16). We have shown previouslythat autocrine expression of truncated, soluble Flk-1 (tsFlk-1)from either ex vivo or in situ gene-modified neuroblastomacells results in a significant inhibition of local angiogenesisand tumor growth in vivo (17, 18). The purpose of this studywas to determine whether gene-modified bone marrow cellsexpressing this angiogenesis inhibitor could, through para-crine delivery of this angiogenesis inhibitor, provide thesame antitumor efficacy.

MATERIALS AND METHODSCell Lines. The murine neuroblastoma cell line NXS2

(19), provided by Dr. R. Reisfeld (La Jolla, CA), was main-tained in DMEM (Mediatech, Inc., Herndon, VA) supple-mented with 10% fetal bovine serum (Summit Biotechnol-ogy, Ft. Worth, CO), 100 units/ml penicillin-100 �g/mlstreptomycin (Life Technologies, Inc., Grand Island, NY),and 2 mM L-glutamine (Life Technologies, Inc). GP�E86(3T3-based retroviral packaging cell line; ATCC, Manassas,VA), SK-NEP-1 (human Wilms’ tumor cell line; AmericanType Culture Collection), and 293T cells (human embryonickidney cells expressing SV40 large T antigen; Ref. 20) weremaintained in medium similar to that of NXS2. HUVECswere obtained from Clonetics (Walkersville, MD) and main-tained in Endothelial Growth Medium.

Making of Retroviral Vector Plasmids and ProducerCells. Retroviral vector plasmids based on the murine stemcell virus and encoding either tsFlk-1 linked via an internalribosomal entry site to GFP or GFP alone were constructed asdescribed previously (17). Briefly, cDNA for the truncated,soluble VEGF receptor was provided by Dr. Pengnian Lin(Durham, NC). The tsFlk-1 cDNA was contained within anAdExFlk.6His plasmid that has been described previously (16).A 2.3-kb HindIII-BamHI fragment containing the tsFlk-1transgene was excised from this vector and ligated into

pSP72 (Promega, Madison, WI), cut with HindIII and BamHIto make p72-mFlk. A 2352-bp EcoRI-XhoI fragment fromp72-mFlk was then ligated into MSCV-I-GFP cut with EcoRIand XhoI. MSCV-I-GFP is a retroviral expression plasmidthat contains the MSCV long terminal repeat driving expres-sion of enhanced GFP (CLONTECH, Palo Alto, CA). Theresulting retroviral expression plasmid, MSCV-tsFlk-1-I-GFP, contains the GFP gene linked to an internal ribosomalentry site from the encephalomyocarditis virus 3� of thetsFlk-1 cDNA. The MSCV-long terminal repeat that acts as apromoter in this construct is not an endothelial cell promoterand, therefore, both tsFlk-1 and GFP will be expressed in alltransduced cells.

GP�E86 retroviral packaging cells were then trans-duced with conditioned medium containing high-titer, vesic-ular stomatitis virus-G-pseudotyped MSCV-tsFlk-1-I-GFP orMSCV-I-GFP vector particles supplemented with 6 �g/mlPolybrene (Sigma Chemical Co. Chemical, St. Louis, MO).These conditioned media were derived by cotransfection of293T cells with the respective retroviral vector plasmids andtwo helper plasmids, one containing the gag and pol retro-viral genes and the other containing the gene for the vesicularstomatitis virus-G envelope, as described previously (17).Transduced GP�E86 cells were then sorted by FACS(Becton Dickinson, Bedford, MA) to delete untransducedcells. Once expanded, viral production from these cells wasdetermined by titering conditioned medium on NIH 3T3 cells(American Type Culture Collection).

Bone Marrow Cell Transduction and Transplantation.Retroviral transduction of murine bone marrow cells wasperformed as described previously (11). Briefly, bone mar-row was harvested by flushing the femurs and tibias of either8- to 12-week-old female A/J mice (Jackson Laboratory, BarHarbor ME) or C.B-17 SCID mice (Charles River Labora-tory, Wilmington, MA) with 2% heat-inactivated FCS(Hyclone, Logan, UT) in PBS,2 days after i.p. injection of150 mg/kg 5-fluororacil (Pharmacia, Kalamazoo, MI). Themarrow cores were dissociated by flushing through a 21-gauge needle, and the cells were counted after red-cell lysis.Marrow cells were then stimulated for 48 h with 20 ng/mlmouse IL-3, 50 ng/ml human IL-6, and 50 ng/ml mouse stemcell factor (R & D Systems, Minneapolis, MN) in DMEMsupplemented with 15% heat-inactivated FCS. Bone marrowcells were subsequently cocultured with irradiated (1200cGy; Cesium-137 source; Gammacell 40 Exactor; MDSNordion, Inc., Ontario, Canada) viral producer cells using theabove culture medium supplemented with 6 �g/ml Poly-brene. This coculture was performed on gelatin-coated platesto prevent detachment of the irradiated producer cells. Forty-eight h later, nonadherent bone marrow cells were gentlyrinsed off the viral producer-cell monolayers, pelleted, andresuspended in PBS for transplantation. Two million gene-modified bone marrow cells were then administered via tailvein to recipient female A/J or C.B-17 SCID mice 8 –12weeks of age that had received 975 or 375 cGy total bodyirradiation, respectively.

Quantification of Systemic tsFlk-1 and VEGF Expres-sion. Conditioned medium from NXS2 cells that had beentransduced directly with the MSCV-tsFlk-1-I-GFP retroviral

3 The abbreviations used are: VEGF, vascular endothelial growth factor;Flk-1 fetal liver kinase-1; HUVEC, human umbilical vein endothelialcell; GFP, green fluorescent protein; MSCV, murine stem-cell virus;FACS, fluorescence-activated cell sorter; SCID, severe combined im-munodeficient; VEGFR-2, vascular endothelial growth factor recep-tor-2.

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vector was used as the source for tsFlk-1 protein. 6-Histidine-labeled tsFlk-1 was purified from the medium using Ni2�-NTA-beads (Qiagen, Santa Clarita, CA) as described previ-ously (17). Purity of protein recovery was evaluated byCoomassie Blue staining, and tsFlk-1 concentration was de-termined by the Bio-Rad Protein Assay using BSA as astandard. Western blot analysis was performed as describedpreviously (17) using a goat antimouse VEGFR-2 (R & DSystems) primary antibody. The level of tsFlk-1 expressionin the serum was quantified using an ELISA assay. Ninety-six-well plates (Nunc) were incubated overnight at 4°C withthe anti-VEGFR-2 (R & D Systems) antibody (2.5 �g/ml)dissolved in 0.1 M sodium bicarbonate buffer (pH 10.6). Thenext day, the wells were washed with PBS and blocked with3% BSA/2% sucrose in PBS at room temperature for 30 min.Then serum samples (diluted 1:500 with 1% BSA in PBS)were added and incubated at room temperature for 1 h. Thenthe wells were washed with 1% BSA/0.05% Tween 20 in PBSand biotinylated anti-VEGFR-2 (50 �g/ml 1% BSA in PBS)was added. After 1 h, streptavidin-conjugated alkaline phos-phatase was added, and subsequent detection was achievedusing a chromogenic substrate from CytImmune Sciences(College Park, MD) according to the manufacturer’s instruc-tions. Levels were quantified by comparing sample absorb-ance values to a standard curve generated by recombinanttsFlk-1 protein dissolved in naı̈ve mouse serum.

Quantification of systemic VEGF expression was per-formed on mouse serum using a murine VEGF ELISA kit(Quantikine; R & D Systems) according to the manufacturer’sinstructions.

Analysis of Endothelial Cell Precursor Frequency.FACS analysis was performed to compare the frequency ofendothelial cell precursors in the tsFlk-1- and the GFP-trans-planted cohorts of mice. Peripheral blood was collected inEDTA from four mice in each group. After RBC lysis, primarystaining was performed with either a monoclonal antibodyagainst mouse VE-cadherin, CD144, (PharMingen, San Diego,CA), which was used as an endothelial lineage marker, or ratIgG2a� (PharMingen), which served as an isotype control. Thesewere diluted 1:2 and 1:400 respectively. A PE-conjugated anti-rat antibody (1:10; Caltag Laboratory, Burlingame, CA) wasused as the secondary antibody. Analysis of CD144-positivecells was then performed by FACS after gating for monocytesize.

Serum Inhibition of VEGF-stimulated HUVEC Migra-tion. Endothelial cell migration assays were performed asdescribed previously (17). Endothelial Growth Medium supple-mented with VEGF (10 ng/ml; R & D Systems) along withserum (5%) from tsFlk-1- or GFP-transplanted mice was placedin the bottom wells, and HUVECs (passage 5) were added to theupper chambers. The plates were incubated for 6 h at 37°C with5% CO2 to allow the cells to migrate. Nonmigrated cells on theupper surface of the filter were removed by wiping with a cottonswab. Then the cells were fixed with 10% formalin, stained withHarris’ hematoxylin, and, after washing, the migrated cells werecounted. The assays were run in triplicate.

Murine Tumor Models. Heterotopic neuroblastomaswere established in syngeneic female A/J mice by s.c. injec-tion of 106 NXS2 tumor cells in 200 �l of PBS. Wilms’

tumor xenografts were established by injecting 2 � 106

SK-NEP-1 cells s.c. in C.B-17 SCID mice. Tumor measure-ments were performed in two dimensions with calipers twiceweekly, and volumes were calculated as width (2) �length � 0.5. All mice had undergone bone marrow trans-plantation previously, with cells that had been modified toexpress either tsFlk-1 together with GFP, or GFP alone.These experiments were performed in accordance with aprotocol approved by the Institutional Animal Care and UseCommittee of St. Jude Children’s Research Hospital.

Immunohistochemistry. Tumors were excised from ex-perimental mice, placed in Tissue Freezing Medium (TriangleBiomedical Sciences, Durham, NC) and snap-frozen in liquidnitrogen. Six-�m sections were fixed with cold acetone, rinsedtwice with PBS, and blocked with 3% hydrogen peroxide inPBS for 12 min. The samples were washed three times with PBSand incubated for 10 min at room temperature with a proteinblocking solution consisting of PBS (pH 7.5) and containing 5%normal horse serum and 1% normal goat serum. Excess block-ing solution was drained, and the samples were incubated for18 h at 4°C with a 1:100 dilution of monoclonal rat anti-CD31antibody (PharMingen). Then the samples were rinsed fourtimes with PBS and incubated for 60 min at room temperaturewith the appropriate dilution of peroxidase-conjugated antiratIgG. The slides were rinsed with PBS and incubated withdiaminobenzidine (Research Genetics, Huntsville, AL). Thenthe sections were rinsed three times with distilled water andcounterstained.

Stained tumor sections were scanned at low power, and theareas of greatest CD31-positive density were chosen for quan-tification of intratumoral vessel density. The number of individ-ual brown-staining endothelial cells or clusters in these areaswere then counted at �400. Microvessel density counts, in twoareas/section, were determined by two independent, blindedobservers.

Fluorescent staining was performed as described above,except that slides were fixed in 4% paraformaldehyde, andblocking was done with 10% normal goat serum/0.1% BSAbut without hydrogen peroxide. The rat antimouse CD34antibody (PharMingen) was used at a 1:200 dilution, as wasthe rabbit anti-GFP antibody (Molecular Probe, Eugene, OR).Immunohistochemical detection of GFP in tumor sectionsseemed to be more sensitive and equally specific as comparedwith direct tissue fluorescence in demonstrating GFP expres-sion. Controls in which only the secondary antibodies wereadded were routinely performed to exclude nonspecific cross-reactivity of the secondary antibodies. Alexa Fluor goat anti-rabbit (Molecular Probe) and Cy3 donkey antirat (JacksonImmunoResearch, West Grove, PA) secondary antibodieswere also used at 1:200 dilutions.

Quantification of Intratumoral tsFlk-1 Expression.Tumors were excised from tsFlk-1- and GFP-transplantedmice and protein lysates made by homogenizing tumor spec-imens using a Dounce (Kontes, Vineland, NJ) homogenizerin 3 ml of lysis buffer [25 mM Tris-HCl, 150 mM HCl, 0.5%NP40, 0.5% sodium deoxycholate, 0.2% SDS, 1.0 mg ofPefabloc SC (Boehringer Mannheim, Indianapolis, IN), and1 protease inhibitor tablet (Boehringer Mannheim)]/1.0 g oftissue. The homogenates were then incubated on ice for 30

2872 Bone Marrow Cells Contribute to Tumor Vasculature



min and centrifuged at 10,000 � g for 10 min at 4°C. Thesupernatants were then recentrifuged, collected, and frozen at�70°C for later use. Total protein levels were determined foreach specimen using the Bradford Assay (Bio-Rad). Intra-tumoral levels of tsFlk-1 were quantified by ELISA as de-scribed previously.

Statistical Analyses. Results are reported as means �SE. Student’s t test was used to analyze statistical differencesamong groups in the HUVEC migration assays, microvesseldensity determination, VEGF expression levels, and between-time-points tumor-growth curves. A P � 0.05 was considered tobe statistically significant.

RESULTSTransplantation of Mice with Gene-modified Bone

Marrow Cells. Retroviral vector producer cells that gener-ated replication incompetent ecotropic retrovirus were madebased on the MSCV-tsFlk-1-I-GFP plasmid using GP�E86packaging cells. The tsFlk-1 protein encoded for by thisexpression cassette had been shown previously to be a func-tional inhibitor of endothelial cell activation in vitro but tohave no direct effect on tumor cell growth in vitro (17, 18).Additional producer cells were made that generatedGFP-only retroviral vectors to serve as controls. Each ofthese producer cell lines generated replication-defectiveretrovirus at a titer of 5 � 105 infectious units/ml. Musdunni (American Type Culture Collection) coculture wasperformed to confirm the absence of replication-competentretrovirus. Ex vivo transduction of bone marrow-derived cellswas performed by coculture of these cells with the retroviralvector-producer cells. After transplantation, successful en-graftment was confirmed by the presence of a normal com-plete blood count for A/J mice transduced with each of thevectors. The complete blood count remained normal for eachgroup of transplanted mice (Table 1), some of which werefollowed for up to 1 year after bone marrow transplantation.Although there was some concern that the process of engraft-

ment after bone marrow transplantation might be affected byangiogenesis inhibition, expression of tsFlk-1 by the bonemarrow-derived cells did not preclude engraftment of thesecells. There was also no long-term selection against thehematopoietic cells that had been modified to express Flk-1.The percentage of cells that were GFP-positive remainedstable for each of the transplant groups in each of the hema-topoietic lineages over the course of 6 months (Table 1). Thatthe percentage of GFP-positive WBCs in the Flk-1-I-GFPmice was less than in the control mice probably reflects lessefficient transduction of these cells rather than an inhibitoryeffect on WBC maturation by tsFlk-1, because the absoluteWBC counts were equivalent between the two groups andremained stable, and the transduction of RBCs and plateletswas also less efficient for the tsFlk-1-transplanted group.

Expression of a Functional Endothelial Cell ActivationInhibitor. The level of transgene expression in the sera oftsFlk-1 transplanted A/J mice was determined by ELISA. Serumlevels of Flk-1 (mean, 12.3 �g/ml � 0.7) peaked within 6–8weeks after transplant (Fig. 1) and remained stable for 1 year(data not shown). A similar level of tsFlk-1 expression wasdetected in the C.B-17 SCID mice transplanted with MSCV-Flk-1-I-GFP-transduced bone marrow.

Western blot analysis of sera from tsFlk-1-I-GFP-trans-planted mice confirmed that a protein of the correct molecularweight was being expressed (Fig. 2A). It also demonstrated thata small amount of soluble Flk-1 could be detected in sera fromthe control mice. That the tsFlk-1 protein being expressed in thetsFlk-1-I-GFP-transplanted mice maintained its endothelial cellinhibitory activity was confirmed by testing the ability of serafrom these mice to inhibit endothelial cell migration. VEGF-stimulated HUVEC migration was inhibited by sera from micetransplanted with bone marrow-derived cells expressing thetsFlk-1 protein but not by sera from GFP-transplanted controlmice (Fig. 2B).

Table 1 Hematopoietic reconstitution after bone marrowtransplantation of A/J mice with gene-modified cellsa

Retroviral vector Cell type 2 mo 6 mo

MSCV-Flk-1-I-GFP Red blood cellsb (%) 43 � 1.1 44 � 1.2GFP (%) 48 � 0.6 48 � 0.3White blood cellsc

(K/�l)8.1 � 0.3 7.5 � 0.4

GFP (%) 57 � 0.9 57 � 1.0Platelets (K/�l) 1111 � 69 1260 � 45GFP (%) 55 � 0.3 53 � 0.4

MSCV-I-GFP Red blood cellsb (%) 40 � 0.8 45 � 0.8GFP (%) 54 � 0.9 52 � 0.5White blood cellsc

(K/�l)7.4 � 0.4 7.7 � 0.6

GFP (%) 79 � 1.3 80 � 0.6Platelets (K/�l) 1054 � 69 1246 � 56GFP (%) 62 � 0.4 58 � 0.2

a n � 10 mice in each group.b Expressed as hematocrit.c No difference in cell number or percentage of GFP was observed

when the white blood cells were analyzed further for granulocytes andlymphocytes.

Fig. 1 Levels of Flk-1 protein detected in the serum of twelve A/J miceat time points after transplantation with MSCV-Flk-1-I-GFP-modifiedbone marrow cells.




Circulating Endothelial Cell Precursor Frequency andSystemic VEGF Levels. FACS analysis of peripheral bloodfrom transplanted mice for the endothelial cell surface markerVE-cadherin (CD144) was performed to determine whether theexpression of tsFlk-1 from transduced bone marrow cells hadany effect on the frequency of circulating endothelial precursorcells. This analysis revealed that the tsFlk-1-transplanted miceactually have had a slightly higher frequency of circulatingendothelial cell precursors than the control GFP-transplantedmice (0.78% � 0.23 versus 0.42% � 0.18), although thisdifference did not reach statistical significance. Similarly, theserum VEGF levels were higher in the tsFlk-1-transplanted micethan the GFP-transplanted control mice (43.11 pg/ml � 10.82versus 25.92 pg/ml � 7.19; P � 0.02).

Inhibition of in Vivo Tumor Growth. Next we soughtto determine what effect this systemic state of angiogenesisinhibition would have on in vivo tumor growth. First, we eval-uated the effect in a localized murine neuroblastoma model.NXS2 cells (1 � 106) were implanted in the s.c. space over theflank of syngeneic, immunocompetent A/J mice. These mice

had been transplanted two months before with bone marrowcells transduced with either MSCV-Flk-1-I-GFP or MSCV-I-GFP. Ten mice in each group were studied. Tumor growth overtime in this heterotopic location is shown graphically in Fig. 3A.Tumors grown in mice transplanted with truncated Flk-1-expressing bone marrow cells were 50% smaller than thosewhich developed in GFP-control-transplanted mice 25 daysafter tumor cell injection (mean volume, 602.6 mm3 � 211.2versus 1175.0 mm3 � 142.1; P � 0.02 at day 25 only). Themean intratumoral tsFlk-1 level in these tumors was 0.75 ng �0.05/�g total protein as compared with 0.08 � 0.02 ng/�g totalprotein within tumors in the GFP-transplanted mice. Immuno-

Fig. 2 A, Western blot detection of tsFlk-1 in conditioned mediumfrom MSCV-tsFlk-1-I-GFP and MSCV-I-GFP retroviral vector-producer cells and sera from two mice transplanted with tsFlk-1 gene-modified cells and one with GFP-only gene-modified cells. B, effect ofsera from mice transplanted with tsFlk-1-expressing bone marrow cells(Flk-BMT) on VEGF-stimulated HUVEC migration, as compared withsera from nontransplanted mice (naı̈ve) and control-transplanted mice(GFP-BMT). Sera were pooled for testing from four mice in each group.Also shown are the effects of diluting the Flk-BMT sera with sera fromGFP-BMT mice at ratios of 1:2 and 2:1 and the effect of medium alone(without mouse serum) with and without VEGF.

Fig. 3 A, growth of 106 NXS2 tumor cells following s.c. injection intomice transplanted with tsFlk-1 gene-modified cells (E) or GFP-onlygene-modified cells (F; n � 10 for each group; P � 0.02). B, growth of2 � 106 SK-NEP-1 tumor cells after s.c. injection into mice transplantedwith tsFlk-1 gene-modified cells (E) or GFP-only gene-modified cells(F; n � 7 for each group; P � 0.001).




histochemical evaluation of tumor vascularity was performed toconfirm that tumor growth restriction in the tsFlk-1-bone mar-row-transplanted mice was attributable, at least in part, to theinhibition of tumor-induced angiogenesis. A greater density ofcapillary vessels was seen in tumors that grew in control GFP-transplanted mice as compared with those tumors that grew intsFlk-1-transplanted mice (mean, 25.92 � 7.19 v. 20.75 � 7.44;P � 0.1) although this difference did not reach statistical sig-nificance. Representative photomicrographs are shown in Fig. 4.An even more profound inhibition of tumor growth was ob-served in the heterotopic Wilms’ tumor xenograft model(Fig. 3B). Seven mice in each group were studied. In this model,tumors grown in mice transplanted with tsFlk-1-expressing bonemarrow cells were less than 10% the size of those whichdeveloped in GFP-control transplanted mice 42 days followingtumor cell injection (mean volume � 61.3 mm3 � 24.0 versus716.7 mm3 � 156.6, P � 0.001 for all time points after day 21).

GFP Analysis of Tumor Vasculature. Tumor growthand concordant neovascularization in mice whose bone marrowcells have been modified to express the marker protein GFPafforded us the opportunity to test the hypothesis that tumor-

induced angiogenesis is supported by bone marrow-derivedendothelial cell precursors. Tumors grown in control GFP-bonemarrow transplanted mice were evaluated by immunofluores-cence for colocalization of endothelial cell markers and GFPin tumor neovasculature. Two different antiendothelial anti-bodies (anti-CD31 and anti-CD34), with slightly different cross-reactivity profiles, were used to identify the endothelial cellswithin the tumor specimens. The vascular staining patternwithin the tumors, using each of these antibodies, was similar.Approximately 5% of these CD31/CD34-positive cells, usuallythose at the tumor periphery, also expressed GFP (Fig. 5). Othertransduced hematopoietic bone marrow-derived cells within thetumor, in addition to the endothelial cells, are also seen express-ing GFP (Fig. 5A). A similar pattern of colocalization wasobserved in the smaller tumors that grew in the tsFlk-1-transplanted mice (data not shown). The demonstration that GFPexpression localizes with expression of endothelial cell markerssuggests that these endothelial cells did, in fact, arise fromthe GFP-modified bone marrow-derived cells used for trans-plantation.

DISCUSSIONInhibition of tumor-associated angiogenesis, in which the

activated endothelial cells, rather than the tumor cells, are tar-geted, has become an increasingly popular anticancer approach.This strategy is particularly appealing for several reasons. (a) Itis likely that most, if not all, types of cancer are dependent onangiogenesis, thereby providing a common target in the treat-ment of widely heterogeneous tumor types. (b) Endothelial cellsare normal cells with a low intrinsic mutation rate and thereforeare less likely to acquire a drug-resistant phenotype than thegenetically unstable tumor cells. The development of drug re-sistant clones within a population of chemosensitive tumor cellsis often the event that ultimately causes the failure of traditionalanticancer approaches. And (c), tumor-activated endothelialcells can be selectively targeted because, with the exception ofendothelial cells activated for new vessel formation required inwound healing and reproduction, nearly all other host endothe-lial cells are quiescent.

Gene therapy-mediated delivery of angiogenesis inhibitorsis an attractive alternative means for providing long-term ex-pression of these therapeutic proteins. In this approach, hostcells are engineered to make the antiangiogenic protein in vivoon a continuous basis, thereby obviating the need for dailyadministration of recombinant protein. A number of differenthost tissues are currently being used as targets for gene therapy-mediated delivery of therapeutic proteins. These have included,most often, skeletal muscle and liver, chosen because of theirrelatively low mitotic activity. This decreases the likelihood thatan episomal transgene will be lost during cell division, thusresulting in the potential for long-term expression. Two com-mon methods for gene-transfer include liposome or adenoviral-mediated delivery. However, the transfer of naked DNA istypically an inefficient process, whereas adenoviral-mediatedgene transfer is complicated by a host immune response totransduced target cells, resulting in transient, albeit high, trans-gene expression. Retroviral-mediated transgene integration into

Fig. 4 Comparison of the vascularity of NXS2 tumors grown s.c. inmice transplanted with GFP-only gene-modified cells (A) or tsFlk-1 �GFP gene-modified cells (B), as assessed by immunohistochemicalstaining with an anti-CD31 antibody. Original magnification, �10.




Fig. 5 Immunofluorescence staining of a tumor excised from a mouse transplanted with GFP gene-modified cells and subsequently challenged withNXS2 cells. Staining with an anti-GFP marker is shown in A (original magnification, �20), D (�63), and G (�40). Staining of the same sectionsfor the endothelial cell markers is shown in B (CD31), E (CD31), and H (CD34). Double staining (yellow) is shown in C, F, and I. The final two panelsshow anti-CD31 (J) and anti-GFP (K) staining of a tumor grown in a nontransplanted control mouse.




self-renewing stem cells is an attractive alternative for achievinglong-term transgene expression.

Bone marrow-derived stem cells are among the most ac-cessible self-renewing cells, and protocols exist for the efficient,retroviral-mediated transduction of these cells in mice. In addi-tion, we hypothesized that these stem cells might be a source ofendothelial cell precursors recruited for tumor-induced neovas-cularization. Although the dependence of tumor growth onsupportive new blood vessel formation is now widely accepted,the origin of the endothelial cells that comprise tumor neovas-culature is less certain. It had been assumed that the additionalendothelial cells required to construct these new vessels comefrom the division and proliferation of local endothelial cells.However, the existence of bone marrow-derived endothelial cellprecursors, or hemangioblasts, has recently been described (21–24). These endothelial progenitor and stem cells have beendetected in the peripheral blood and have the capacity to differ-entiate into endothelial cells in vitro and in vivo (21–24). It hasbeen suggested that endothelial cells incorporated into sites ofneovascularization, including tumor-induced new blood vessels,may, in fact, be derived from these precursor cells of bonemarrow origin (12, 25–27). These progenitors can be mobilizedfrom the bone marrow in response to ischemia and VEGF (21,26), and can be recruited in the colonization of endothelial flowsurfaces of vascular prostheses and the neovasculature associ-ated with cornea micropocket surgery (22, 25). Our study hasconfirmed that these circulating bone marrow-derived endothe-lial cell precursors are recruited to tumor-induced neovascular-ization. No GFP expression was detected in the parenchyma orvasculature of other tissues within the transplanted mice, includ-ing kidney, liver, adrenal, spleen, lung, aorta, and heart (data notshown), however, despite recent evidence to suggest to bonemarrow cells include precursors to a wide variety of tissue types(28). This is probably because, in our model system, there wasno impetus for accelerated turnover or regeneration of thesetissues.

This finding that circulating bone marrow-derived endo-thelial cells are recruited to tumor neovasculature provides ad-ditional rationale for the use of bone-marrow stem cells as thetarget in an antiangiogenic gene therapy-mediated anticancerstrategy. Because these gene-modified endothelial cell precur-sors are recruited by tumor-elaborated factors to sites of tumorgrowth, a higher local milieu of angiogenesis inhibitors is likelyestablished at the desired target sites. This observation also hasimportant therapeutic implications for the specific targeting ofother classes of anticancer agents to the tumor microenviron-ment. The relative contributions of bone marrow-derived pre-cursor cells and endothelial cells recruited from local, estab-lished blood vessels to tumor neovascularization are uncertain.Although 50–80% of hematopoietic cells expressed GFP aftertransduction and subsequent engraftment, a smaller percentageof endothelial cells of the tumor neovasculature expressed de-tectable GFP by fluorescence immunohistochemistry. This maybe attributable to a number of factors, including the inability todetect low-level GFP expression, a lower transduction effi-ciency of endothelial cell precursors, or the fact that the non-GFP-expressing endothelial cells were derived from local vas-culature. The later explanation would support the hypothesis

that both vasculogenesis and angiogenesis contribute to postna-tal, tumor-induced neovascularization. Nevertheless, the findingof GFP expression in some of the endothelial cells in the tumorneovasculature of our transplanted mice confirms the origin ofthese cells from bone marrow precursors.

This study marks the first time in which long-term expres-sion of high levels of a functional angiogenesis inhibitor hasbeen established by a gene therapy approach. Serum levels ofthe transgene peaked within 6–8 weeks after transplant andremained stable for 1 year. This finding confirmed the fol-lowing. (a) Bone marrow-derived stem cells had been success-fully transduced. (b) Expression of this angiogenesis inhibitordid not adversely affect bone marrow engraftment. (c) Despiterecent observations that have suggested a role for Flk-1 inmurine embryonic hematoangiogenesis (29), generation of ma-ture hematopoietic cells (or endothelial cells) after bone marrowtransplantation was not inhibited by the enforced expression ofthe soluble, truncated receptor from precursor cells. Perhaps thisreflects differences in embryonic hematopoiesis compared withadult hematopoiesis, or it may simply be that the level of tsFlk-1expression in the environment of the bone marrow is not highenough to effect these processes. And (d), long-term transgeneexpression can be generated by gene-modified transplantedbone marrow-derived cells.

It does not seem that the antiangiogenic effects seen withthe approach used in this study are attributable to an inhibitoryeffect on endothelial cell generation or recruitment from thebone marrow but rather on the local steps required for angio-genesis at sites of tumor growth. Expression of this VEGFinhibitor did not diminish endothelial cell precursor frequencyin the peripheral blood of tsFlk-1-transplanted mice. In fact, thefrequency was slightly higher than in the control GFP-trans-planted mice. Consistent with this was the finding of an elevatedsystemic level of VEGF in the tsFlk-1-transplanted mice. Thismay be caused by feedback resulting from inhibition of VEGFactivity outside the environment of the bone marrow by tsFlk-1.In addition, although tumors grown in tsFlk-1-transplanted micehad fewer endothelial cells, the relative proportion of cells thatwere GFP-positive, and therefore bone marrow-derived, wasapproximately the same as that found in the tumors grown in theGFP-transplanted mice, also suggesting that the effect of tsFlk-1on these endothelial cells seems to be paracrine rather thanautocrine.

It is unclear whether the antiangiogenic effect of tsFlk-1observed in these experiments is attributable, primarily, totsFlk-1 being expressed locally either by transduced endothelialcells within the tumor neovasculature or tumor-infiltrating non-endothelial hematopoietic cells or to the high systemic levels ofthe angiogenesis inhibitor being expressed by all transducedbone marrow-derived cells. The inhibitory activity of HUVECmigration demonstrated by sera from mice transplanted withtsFlk-1-expressing cells suggests that a systemic state of angio-genesis inhibition had been established. However, a particularlyhigh concentration of tsFlk-1 was also noted locally withintumors grown in these mice. Perhaps a different vector design inwhich tsFlk-1 expression were driven by an endothelial cell-specific promoter would help to determine whether the antian-giogenic effect was caused by local or systemic expression of




the inhibitor. In addition, the use of an endothelial-specific orregulatable promoter would be useful in trying to avoid thepotential side effects of chronic systemic angiogenesis inhibi-tion. With this type of vector design, antitumor tsFlk-1 efficacy,as has been achieved with recombinant protein administration(16), but without systemic exposure, as with local administra-tion of retroviral vector producer cells (18), might be achievedlong-term.

These experiments have demonstrated that bone mar-row-derived cells contribute to tumor neovasculature andwhen modified to express the angiogenesis inhibitor tsFlk-1can restrict tumor growth in a syngeneic murine neuroblas-toma model and a Wilms’ tumor xenograft model. One of theappeals of this strategy for the treatment of pediatric canceris that it could be readily incorporated into existing treatmentschema because bone marrow transplantation is already a partof most clinical protocols for children with high-risk malig-nancies. Although transduction of human hematopoietic stemcells has traditionally not been as efficient as for murinecells, there have been recent reports suggesting that thisability may be improving (30), and that a clinical trial mightbe feasible. It is not clear why the Wilms’ tumor xenograftswere more affected by the inhibition of VEGF signaling thanthe murine neuroblastoma tumors. It may be that human cellsin murine hosts have fewer adaptive responses to this type oftherapy. Or it may reflect murine host-strain differences.Alternatively, this result may reflect a difference in angio-genic capabilities of the different tumor histologies. Perhaps,Wilms’ tumor is more susceptible to VEGF inhibition thanneuroblastoma, as has been suggested recently by Kim et al.(31). This will need to be evaluated further as antiangiogenictherapy moves forward. In addition, the efficacy of thisantiangiogenic strategy will need to be tested in orthotopictumor models. Although s.c. tumor models afford the oppor-tunity to assess antitumor efficacy continuously, it has beenshown that the angiogenic factors involved in s.c. tumorgrowth may be different from those of orthotopic tumors(32). Improvement in the antitumor efficacy of this approachis also needed, as all mice eventually succumbed to theirtumors. Either higher levels of angiogenesis inhibitor expres-sion will be required or synergy achieved by combining thisapproach with a cytotoxic antitumor modality such as chem-otherapy or immunotherapy. Of course, the effects of thisapproach on angiogenesis in such physiological processes aswound healing and reproduction would have to be evaluated.

ACKNOWLEDGMENTSWe thank Dorothy Bush, Bonnie Greer, and Adriana Nance for

their assistance with immunohistochemistry and Dr. Richard Ashmun,Ed Wingfield, and Anne-Marie Hamilton-Easton for their assistancewith FACS. We also thank Drs. Brian Sorrentino and Stephen Shochatfor their critical review of this manuscript.

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2001;7:2870-2879. Clin Cancer Res Andrew M. Davidoff, Catherine Y. C. Ng, Peggy Brown, et al. Angiogenesis Inhibitor, Can Restrict Tumor Growth in MiceNeovasculature and, When Modified to Express an Bone Marrow-derived Cells Contribute to Tumor

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