[CANCER RESEARCH 55, 2240-2244, June 1, 1995]

Advances in Brief

Angiogenesis-directed Implantation of Genetically ModifiedEndothelial Cells in Mice1

John O. Ojeifo, Reza Forough,2 Soonmyoung Paik, Thomas Maciag, and James A. Zwiebel3

Department of Medicine, Division of Hematology and Oncology [J. O. O., S. P., J. A. Z.]. Department of Pathology [J. A. Z.], and the Vincent T. Lombardi Cancer ResearchCenter [J. O. O., S. P., J. A. Z.¡,Georgetown University Medical Center, Washington, DC 20007, and Jerome H. Holland Laboratory for the Biomedicai Sciences, American RedCross. Rockville, Maryland 20855 ¡R.F.. T. M.¡

Abstract

By virtue of their location within blood vessels and their ability toexpress foreign genes, endothelial cells are attractive vehicles for thedelivery of therapeutic molecules in vivo. We wished to determine whetheri.v.-injected, genetically modified endothelial cells can become incorpo

rated into sites of active angiogenesis in vivo. To do so, we studied the fateof i.v.-injected, ¿acZ-expressing human umbilical vein endothelial cells in

athymic nude mice bearing lethally irradiated NIH 3T3 murine fibroblastcells transfected with a sp-hst/KS3:fibroblast growth factor-1 chimera thatforces the secretion of the angiogenic protein, fibroblast growth factor-1.Following i.v. injection, ¿acZ-labeled human umbilical vein endothelialcells accumulated at sites of fibroblast growth factor-1-induced angiogenesis, persisting for at least 4 weeks. These results suggest that i.v.-admin-

istered, genetically modified endothelial cells can migrate into and survivewithin an angiogenic site. This strategy may be useful for delivery oftherapeutic molecules to sites of pathological angiogenesis during tumormetastasis.

Introduction

A number of endothelial cell properties make these cells attractiveas vehicles for the delivery of therapeutic recombinant molecules invivo: (a) endothelial cells synthesize and secrete many differentproteins (1); (b) they can sustain a high level of transgene expression(2, 3); and (c) they exist in the interface of blood and tissues withinthe different vascular environments of the body and are ideally positioned for efficient delivery of therapeutic gene products to achievelocal or systemic effects. Thus, implantation of GMEC4 within the

extensive surface area of microvascular beds may permit the deliveryof pharmacologically significant levels of a therapeutic agent at sitesof tumor angiogenesis. We sought to develop a delivery systemwhereby GMEC are incorporated into newly forming microvascularbeds that have been induced by an angiogenic growth factor. Wehypothesized that GMEC injected i.v. might become lodged in sites ofactive angiogenesis where the cells would proliferate and becomeincorporated into a newly developing microvascular network. GMECintroduced this way remain in constant contact with the blood andshould be free to settle in angiogenic sites associated with tumordeposits that might exist anywhere throughout the body. To address

Received 2/24/95; accepted 4/24/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants HL32348 and HL44336 (to T. M.) and by NIH

Grants HL48334 and CA59311 (to J. A. Z.). A preliminary report was presented at theAmerican Society of Hematology, December, 1992.

2 Present address: Department of Surgery, University of Washington, Seattle, WA

98195.3 To whom requests for reprints should be addressed, at Georgetown University

Medical Center, 3800 Reservoir Road, NW, Washington, DC 20007.4 The abbreviations used are: GMEC, genetically modified endothelial cells; FGF-1,

acidic fibroblast growth factor; HUVEC, human umbilical vein endothelial cells; FBS,fetal bovine serum; ADA, adenosine deaminase; X-gal, 4-Cl-5-Br-3-indolyl-j3-D-galacto-pyranoside.

these hypotheses, we induced neoangiogenesis in athymic nude micewith s.c. implants of FGF-1-secreting mouse fibroblasts. /acZ-trans-

duced HUVEC that were injected via the tail vein of athymic nudemice became incorporated into the neovessels that formed at the sitesof FGF-1-induced neovascularization.

Materials and Methods

Cells. HUVEC were isolated from freshly obtained umbilical cords usingcollagenase digestion as described (4) and grown on dishes coated with 1.5fig/cm2 fibronectin (Upstate Biotechnology Laboratories, Lake Placid, NY) in

complete endothelial cell medium: M199 (Biofluids, Rockville, MD) with 20%heat-inactivated FBS, 2 mM glutamine, 50 /j.g/ml endothelial cell growth

supplement (Upstate Biotechnology Labs), and 15 /J-g/ml heparin (SigmaChemical Co.). Cells were passaged when confluent with 0.05% trypsin/versene (Biofluids) at a split ratio of 1:3. NIH-3T3, sp-hst/KS3:FGF-l/3T3 (5),

and PA317 retroviral packaging cells (6) were grown in DMEM with 10% FBSand 2 mM glutamine.

Retroviral Infection and Selection by Flow Cytometry. The fluoresceindigalactoside-flow activated cell sorting method (7) was used to prepare/acZ-expressing HUVEC.5 The procedure is as follows: Low passage HUVEC

(passages 2-4) were infected with amphotropic retroviral vector stock(5 X IO5 colony-forming units/ml) containing the neomycin resistance (neoR)

gene in combination with either the bacterial lacZ gene [BAG vector (8), a giftfrom Constance Cepko] or the human ADA gene (9). Following two sequentialcell sorts using fluorescein digalactoside-flow activated cell sorting, >95% of

the HUVEC transduced with the BAG vector expressed the product of lacZgene, ß-galactosidase, as demonstrated by X-Gal staining. Both /acZ-express-ing (/öcZ-HUVEC) and ADA-expressing (ADA-HUVEC) cells retained the

endothelial cell phenotype, including cobblestone morphology, uptake ofacetylated-low density lipoprotein (10), and expression of von Willebrandfactor (11). No replication-competent helper virus was detected in the lacZ-HUVEC medium after 25 passages, but ADA-HUVEC supernatant did contain

helper virus.Mouse Implants. Female athymic NCr-nu mice, 4 to 6 weeks of age, were

purchased from the National Cancer Institute Animal Program (Frederick,MD). Protocols for husbandry and experimental manipulation were approvedby the Georgetown University Animal Care and Use Committee. All micewere housed in a pathogen-free environment, and NIH-Centers for Disease

Control biosafety level 2 containment procedures were observed. Prior toimplantation, confluent dishes of sp-hst/KS:FGF-lßTi and /acZ-HUVECwere trypsin-treated and resuspended in M199 (Biofluids, Rockville, MD) with10% heat-inactivated FBS and 2 mM glutamine at a concentration of 10s andIO7 cells per ml, respectively. To establish a site of active angiogenesis, IO7

irradiated sp-hst/KS:FGF-l/3T3 cells were injected s.c. into the flank of nudemice. For i.v. tail vein injection of 2 x IO6 /ocZ-HUVEC, the animal was first

sedated with inhaled methoxyflurane.Tissue Fixation and X-gal Staining. Animals were sacrificed periodically

in groups of four for analysis. Tissues were placed in PBS (pH 7.4) containing2% (v/v) formaldehyde and 0.2% (v/v) glutaraldehyde for 60 min. Afterfixation, the tissues were rinsed three times in PBS and incubated for 24 h at4°Cin X-gal staining solution [1 mg/ml X-gal, 5 mM potassium ferricyanide,

5 mM potassium ferrocyanide, 2 mM MgCl2, 0.02% (v/v) NP40, and 0.01%

' J. Ojeifo and J. Zwiebel, unpublished observation.

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IMPLANTS OF GMEC AT ANGIOGENIC SITES

(w/v) sodium deoxycholate in PBS]. Specimens obtained from each animalwere processed separately. At the end of the incubation period, the organs wererinsed with 3% DMSO in PBS followed by PBS alone. The organs werevisualized and photographed through an Olympus SZH stereo microscope.Subsequently, tissues were fixed further at 4°Cfor 2 h in 2% paraformaldehyde

in 0.1 M PBS (pH 7.4), followed by rinsing in 0.1 M PBS and dehydration in3% sucrose in 0.1 M PBS prior to performing cryosections.

Results

A site of active angiogenesis was established with implants ofmouse fibroblasts secreting FGF-1. A /¡si-derivedleader sequence hadbeen attached to the FGF-l gene to allow secretion of the FGF-1molecule (5). Implants of hst-FGF-1 -secreting mouse fibroblasts (sp-

hst/KS:FGF-l/3T3 cells) in nude mice induced rapidly growing, hem-

orrhagic tumors at the sites of implantation. These tumors led to thedeath of the animals within 2 to 3 weeks (data not shown). y-Irradi

ation of the sp-hst/KS:FGF-l/3T3 cells with 3000 cGy prevented

tumor growth yet allowed the cells to induce a brisk angiogenicresponse, as seen both macroscopically (Fig. \A ) and histologically(Fig. IB). Active angiogenesis persisted for 1 to 2 weeks and declinedrapidly thereafter. By the end of the fourth week, predominantlyfibrous tissue with a few blood vessels remained at the implant site(Fig. 1, C and D). In 5 to 10% of the animals that received irradiatedsp-hst/KS:FGF-l/3T3 cells, small hemorrhagic cysts appeared at the

sites of inoculation. No angiogenic response was observed in the

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sOFig. 1. Angiogenic activity of s.c. implants of sp-hst/KS3:FGF-l-13T3 cells in nude mice. -y-Irradiated sp-hsi/KS:FGF-l/3J3 cells (IO7; FGF-1-secreting mouse fibroblasts) or

pMexn«>/3T3 cells (mouse fibroblasts containing the expression vector without the sp-hst/KS3:FGF-l sequence; Ref. 5) were injected s.c. into the flank of athymic nude mice. Theanimals were sacrificed at varying time intervals. The entire skin was examined with an Olympic SZH stereomicroscope (A, C, and E) and histologically (B and D) for the presenceof angiogenesis. Four days after implantation (bar, 1 cm): A. sp-hst/KS:FGF-l/3Ti implant (arrow); and E, pMexn«>/3T3 implant (arrow). Twenty-eight days after implantation (bar,1 cm): C, sp-hst/KS:FGF-l/yT3 implant (arrow). Microscopic appearance of sp-hst/KS:FGF-I/3T3 implant sites: B. day 4, note profusion of microvessels (arrows); hematoxylin andeosin; bar, 50 (¿m;A Day 28, note residual fibrous tissue containing blood vessels (arrows). Masson trichrome; bar, 25 /im.

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IMPLANTS OF GMEC AT ANOIOGENIC SITES

control mice with implants of irradiated NIH/3T3 cells containingonly the plasmid vector (pMex;;iY>/NIH3T3; Fig. IE). Thus, irradiatedsp-hst/KS:FGF-l/3T3 cells induced a brisk angiogenic response of

rapid onset and of short duration.To test the hypothesis that circulating GMEC cells can become

incorporated into sites of active angiogenesis, we studied the fate ofi.v.-injected, /acZ-expressing HUVEC (/acZ-HUVEC) in sp-hst/KS:FGF-//3T3-bearing nude mice. We established tocZ-HUVEC by

transducing freshly isolated normal HUVEC with the BAG retroviralvector (8) and enriching for /acZ-expressing cells using flow cytom-etry (7). After injecting IO7 irradiated sp-hst/KS:FGF-//3T3 cells s.c.

into the flank of nude mice, we waited for 2 days to allow angiogenesis to commence and then injected 2 X IO6 /ocZ-HUVEC via the tail

vein of these animals. HUVEC containing a retroviral vector with thehuman ADA gene (9) (ADA-HUVEC) were injected i.v. to serve asX-gal staining controls. Groups of animals were then sacrificed at

varying intervals in order to follow the fate of injected cells over time.The skin, brain, lungs, liver, spleen, kidney, and heart were processedfor histology and ß-galactosidase histochemistry.

Within 5 min following injection, most blue-staining lacZ-HUVEC

appeared in the lungs (Fig. 2, A and B) but were rapidly cleared fromthis organ. Three days after /ocZ-HUVEC injection, /acZ-expressing

cells, became noticeable within the angiogenic site. The magnitude ofX-gal staining increased during the ensuing days, becoming promi

nent by 7 to 10 days (Fig. 2, C and D) and persisted for at least 4weeks. All of the mice studied at 4, 7, and 14 days after injectioncontained /«cZ-HUVEC at the site of angiogenesis, whereas three of

the seven animals studied at 28 days still had detectable staining(Table 1). The absence of staining in the remaining four animalsexamined after 28 days may have resulted from cell death orinactivation of gene expression.

Of particular interest was the appearance of branched, filamentous,X-gal-stained structures in the vicinity of the angiogenic site in thethree mice that were X-gal positive 4 weeks after /acZ-HUVEC

injection (Fig. 2, £and F). This staining pattern suggested that/acZ-HUVEC were becoming incorporated into microvessels, a find

ing that was supported by the microscopic examination of the angiogenic areas. Griffonia simplicifolia I agglutinin, which stains mousebut not human endothelial cells (12), revealed a marked proliferationof mouse microvascular endothelial cells (Fig. 2G). Lectin-negative/X-gal-positive /acZ-HUVEC cells were found to participate in lumenformation along with lectin-positive/X-gal-negative mouse endothe

lial cells (Fig. 2G).Following /acZ-HUVEC injection, we also detected small numbers

of X-gal-stained cells in other tissues, including the surface of thebrain, lung, liver, and, occasionally, perinephric tissue. X-gal stainingin these sites persisted only in animals with the sp-hst/KS: FGF-1/3T3implant, suggesting that /¡.tf-FGF-lexpression may have promoted thesurvival of /acZ-HUVEC in distal sites. In pMexneo/3T3 cell-bearingmice, a few X-gal-positive cells occasionally were seen (3 of 10animals) at the site of pMexneo/3T3 cells up to 4 days after lacZ-

HUVEC injection (Table 1). Except for one mouse with focal punctateX-gal staining of the arachnoid matter, we did not find X-gal-positivecells in pMexneo/3T3-bearing late animals. Also, no X-gal-positive

cells were seen in mice that did not receive s.c. NIH/3T3 cell implants(Table 1). Thus, the presence of an angiogenic implant appears to benecessary for the survival of lacZ-HWEC cells. Finally, the specificity of X-gal tissue staining was confirmed by the finding that noX-gal-positive cells were detectable in .s/j-/!.yf/A"S:FGF-//3T3-bearing

animals that received i.v. injections of ADA-HUVEC (Table 1).

Table 1 Genetically modified HUVEC fallowing i.v. injection: X-gal stainingat the angiogenic site

Normal HUVEC were freshly isolated and transduced with either the lacZ or theADA-containing vector as described (2). In order to induce angiogenesis, athymic nudemice first received s.c. flank injections of IO7 irradiated FGF-1-secreting mouse fibro-

blasts (sp-hst/KS:FGF-1/3T3) or NIH/3T3 cells with the control plasmid vector (pMex-neo) (Ref. 5). Two days later, 2 X 10'' toc-Z-HUVEC or ADA-HUVEC were administered

to the mice via tail vein injection. At varying time intervals, groups of animals weresacrificed, and the entire skin was processed for ß-galactosidase histochemistry asdescribed previously (19). Each animal was processed separately. After X-gal staining, thetissue was visualized with an Olympus SZH stereo microscope. Relative tissue X-galstaining was scored according to the following scale: negative (-), no staining; (+), few

positive single cells; (++), few positive cell clusters; (+++), moderale number of positivecell clusters; and (++++), dense clumping of X-gal staining (see Fig. 2D) as judged byvisual inspection.

X-gal-positiveanimals(amountof X-gal staining at angiogenicsite)NonesourceIrradiated

pMexneo/3T3markerlacZlacZ2onNDdays3NDNDfollowing40/13/10HUVEC70/10/5implant140/10/5280/40/5(Trace)

Irradiated FGF-1/3T3 ADA 0/1 ND 0/5 0/5 0/5 0/5

lacZ ND 1/1 10/10 14/14 15/15 3/7

Discussion

Tumor metastasis is the main cause of death in cancer patients. Anessential step in the growth and métastasesof tumors is their ability toinduce the formation of new blood vessels through the elaboration ofangiogenic factors (13). We sought to exploit the biological characteristics of endothelial cells for therapeutic drug delivery to tumorsites throughout the body. Consequently, we hypothesized that theprocess of tumor angiogenesis might afford the opportunity for incorporating GMEC into a newly developing microvascular network anddeliver a recombinant therapeutic compound against the tumor cells.Our study demonstrates that the incorporation of circulating GMEC isenhanced at a site of active angiogenesis and that the incorporatedcells maintain transgene expression. The /acZ-transduced HUVEC

appeared to participate in lumen formation with endogenous mousemicrovascular endothelium (Fig. 2G) during the period of activeangiogenesis and persisted as linear, branching structures long afterangiogenesis had ceased (Figs. 2, E and F). Thus, the human xe-nograft exhibited endothelial cell-appropriate behavior in the foreignhost by taking part in new vessel formation. While X-gal staining

enabled us to detect lacZ gene expression in animals for at least 4weeks, it is possible that other genetically modified cells that persistedin the animals could not be detected due to the loss of expression ofthe transgene. Additional studies using PCR amplification to detecttransgene DNA in tissues that lack X-gal staining may help to clarify

this issue.The finding of persistent, although minimal, distal X-gal-positive

staining in animals with an angiogenic implant suggests that thesurvival of those cells may have been promoted by the expression ofa FGF-1 construct that forces the secretion of FGF-1 (5). Using asensitive bioassay for FGF-1, we were unable to detect FGF-1 activityin the blood of animals with active angiogenesis.6 This finding sug

gests that the amount of FGF-1 sufficient to promote endothelial cell

survival in vivo may be less than what is required for cell proliferation.Messina et al. (14) reported that small numbers of intraarteriallyinjected, /acZ-transduced endothelial cells become incorporated into

the intact capillary wall just distal to the injection site. Our finding ofdistal lacZ staining may indicate that a similar process may be

' N. Hartmann and A. Wellstein, personal communication.

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0' s

Fig. 2. X-gal stain of tissue for ß-galactosidase expression at varying intervals following tail vein injection of /i/rZ-HUVEC. Stereomicroscopic appearance of lungs (A ) at 5 minafter /iirZ-HUVEC injection; bar, 250 ^im; and angiogenic sites at C 7 days; D, 10 days; £and F, 28 days; httr, C-fc: 150 0xm; F, KO^m. X-gal-positive cells are marked with (//TÕMV.V.Note the branching linear pattern of X-gal staining at 28 days (E and /•").Microscopic sections of ft lungs at 5 min after /ncZ-HUVEC injection, with X-gal-positivc cells (urr»vf.v);bur, 25 /j.m; and G. si>-h<;l/KS3:FCìF-l/3rT3cell implant site 9 days following injection of /«rZ-HUVEO; /w. 25 u.m. Section in 6" stained for ß-galactosidase expression with X-gal

and with Grifftmia simplicifnlia I agglutinin (GSA I) for mouse endothclial cells (12). Note participation of both X-gal-positive /«cZ-HUVEC (arrows) and GSA-l-posilivc mouseendolhelium in capillary lumen formation.

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IMPLANTS OF OMEC AT ANGIOGENIC SITES

operating in animals where we observed GMEC implantation distal tothe angiogenic site and may be promoted by the presence of extracellular FGF-1 that is below the level of detection in our assay.

Recently, Lai et al. (15) reported that immortalized brain endothe-lial cells expressing the /«t'Zgene survive, proliferate, and take part in

neoangiogenesis induced by glioma cells following intracranial ands.c. inoculation. There are a number of differences between the studyof Lai et al. (15) and the current study: (a) we used nonimmortalizedendothelial cells as compared to adenovirus EIA gene-immortalized

cells in the study by Lai et al. (15); (b) in our study, GMEC that hadbeen introduced from within the vascular compartment became incorporated into newly forming vessels, unlike the process of vasculogen-

esis reported by Lai et al. (15) whereby extravascular GMEC mergedwith new tumor-induced vessels; (c) and i.v. injection of GMEC

potentially could target even occult sites of tumor throughout thebody, whereas local injection of GMEC would be targeted to knownand accessible sites of disease.

Angiogenesis occurs infrequently once vascular development iscompleted. Subsequent new blood vessel formation may occur as partof: (a) placenta! and ovarian expansion in response to estrogen andprogesterone stimulation (16); (b) wound healing (17); (c) chronicinflammation such as rheumatoid arthritis (18); and (d) tumor growth(13). The ability of i.v.-administered GMEC to migrate into and to

express exogenous genes at an angiogenic site, as we observed in thisstudy, may enable these cells to target locations of active blood vesselformation throughout the body, such as tumor deposits or sites ofchronic inflammation.

Acknowledgments

We thank Constance Cepko for the BAG vector; Owen Blair, Alexis Brown,and Karen Creswell for their expert assistance with the flow cytometry;Alexander MacPherson for excellent technical assistance; and Marc Lippmanand Anton Wellstein for their critical review of the manuscript.

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