suppression of lung tumor growth and metastasis in mice by ...suppression of lung tumor growth and...

Suppression of Lung Tumor Growth and Metastasis in Mice byAdeno-AssociatedVirus-Mediated Expression of VasostatinKe Xia Cai,1Lai YingTse,3 Carly Leung,1Paul K.H. Tam,2 Ruian Xu,3,4 andMai Har Sham1

Abstract Purpose: Angiogenesis inhibitors have strong therapeutic potential as antitumor agents insuppressing tumor growth and metastatic progression.Vasostatin, the N-terminal domain ofcalreticulin, is a potent angiogenesis inhibitor. In this study, we determined the effectiveness ofvasostatin delivered by recombinant pseudotype adeno-associated virus 2/5 (rAAV2/5-VAS)as a gene therapy approach for lung cancer treatment.Experimental Design:We used rAAV2/5 to deliver vasostatin intratumorally or systemically indifferent mouse lung tumor models 1 subcutaneous, orthotopic xenograft, and spontaneousmetastasis lung tumormodels.The therapeutic efficacyof rAAV2/5-VASwasdeterminedbymoni-toringtumorvolume,survivalrate,anddegreeofneovascularizationafter treatmentinthesemodels.Results: Mice bearing subcutaneous tumor of rAAV2/5-VAS pretreated Lewis lung carcinomacells showed >50% reduction in primary tumor volume and reduced spontaneous pulmonarymetastases. The tumor-suppressive action of rAAV2/5-VAS in subcutaneous human lung tumorA549 xenograft correlatedwith a reducednumberof capillary vessels in tumors. In the orthotopicxenograft model, rAAV2/5-VAS suppressed metastasis of A549 tumors to mediastinal lymphnodes and contralateral lung. Furthermore, treatment of immunocompetent mice in thespontaneous lung metastases model with rAAV2/5-VAS after primary tumor excision prolongedtheir median survival from 21to 51.5 days.Conclusion:Our results show the effectiveness of rAAV2/5-VAS as an angiogenesis inhibitor insuppressing tumor growth during different stages of tumor progression, validating the applicationof rAAV2/5-VAS gene therapy in treatment against lung cancer.

Angiogenesis is a major feature of tumor growth andmetastasis. As such, targeting the tumor neovasculature is anattractive strategy for effective cancer therapy. A number ofendogenous angiogenesis inhibitors have been identified andtested in preclinical models (1), and several inhibitors,including endostatin and angiostatin, have been introducedinto clinical trials (2). Although existing antiangiogenesistherapies show lower toxicity than conventional treatments,such as radiotherapy, they are often associated with limitedtumor regression and other clinical effects (3). Therefore, mucheffort has been focused on identifying novel angiogenesisinhibitors and evaluating new approaches to maximize theeffects of antiangiogenesis therapies (4).

Vasostatin, a 180–amino acid fragment from the N-terminaldomain of calreticulin, is a potent endogenous angiogenesisinhibitor (5). Daily administration of recombinant vasostatin, asmall soluble molecule, was shown to reduce xenograft growthin mice (5, 6). Repeated vasostatin gene delivery by DNA i.m.injection or adenoviral vectors i.t. injection also suppressedsubcutaneous tumor growth in mice (7, 8). The biosafety ofvasostatin has been recognized, as vasostatin treatment did notaffect physiologic angiogenesis at tumor-inhibiting doses (9).However, previous reports on the therapeutic effects of vaso-statin were based solely on subcutaneous tumor models,whereas the key challenge in cancer therapy lies in eradicatingmetastases at the subclinical and advanced stages, wherein thetumor microenvironment affects induction of tumor angiogen-esis (10). Given the strong therapeutic potential of thismolecule, evaluation of the efficacy of vasostatin in morerelevant orthotopic and metastatic models of tumorigenesis isduly warranted.The method used for delivering angiogenesis inhibitors

greatly affects their efficacy (3, 11). Maintaining sustainedlevels of angiogenesis inhibitors is crucial for prolongedsuppression of angiogenesis. To target tumor angiogenesis,gene therapy vectors are required to offer long-term expressionwithout vector-associated toxicity or immunity. Adeno-associ-ated viruses (AAV) have been previously used with acceptablesafety (12, 13) and are attractive vectors for angiogenesisinhibitor gene transfer as they offer stable and persistent geneexpression in transduced cells (14). Recombinant AAV (rAAV)vectors are effective in using normal tissues as a platform for

Cancer Therapy: Preclinical

Authors’Affiliations: Departments of 1Biochemistry and 2Surgery, Li Ka ShingFaculty of Medicine and 3Institute of Molecular Biology,The University of HongKong, Pokfulam, Hong Kong, China and 4Institute of Molecular Medicine, HuaqiaoUniversity, Fujian, ChinaReceived 8/8/07; revised10/11/07; accepted10/11/07.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: Supplementary data for this article are available at Clinical Cancer ResearchOnline (http://clincancerres.aacrjournals.org/).Requests for reprints: Mai Har Sham, Department of Biochemistry, Li Ka ShingFaculty of Medicine, The University of Hong Kong, 3rd Floor Laboratory Block,21 Sassoon Road, Pokfulam, Hong Kong, China. Phone: 852-28199195;Fax: 852-28551254; E-mail: [email protected].

F2008 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-07-1930

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producing secretory proteins, allowing persistent expression ofthe transgene product. They have been successfully used fordelivery of angiogenesis inhibitors for cancer therapy indifferent murine models (15–17). The most extensively studiedAAV serotype 2 (AAV2) vector has a low transduction efficiencyin polarized airway epithelia (18). However, cross-packagingAAV2 vector genome into AAV5 capsid, which results inpseudotyped rAAV2/5 vector that share a similar safety profileas AAV2, can mediate efficient gene transfer to the lung(14, 19).In this study, we evaluated the efficacy of vasostatin delivered

by the rAAV2/5 viral vector (rAAV2/5-VAS) in subcutaneous,orthotopic xenograft, and spontaneous metastasis models oflung cancer. These mouse models serve to simulate differentaspects of tumor growth, allowing assessment of vasostatingene therapy during different stages of lung cancer develop-ment. In an ex vivo setting, we showed that rAAV2/5-mediatedexpression of human vasostatin from highly metastatic Lewislung carcinoma could reduce neovascularization during tumorinitiation, resulting in suppression of primary tumor growthand subsequent pulmonary metastasis. The antitumor effect ofrAAV2/5-VAS was further confirmed by treating preexistingxenografts in nude mice at subcutaneous and orthotopictransplantation sites. During advanced tumor stages, long-termexpression of vasostatin after intratracheal delivery was shownto prolong the survival of immunocompetent mice which haveundergone surgery for primary tumor removal. This is the firstreport demonstrating the antimetastatic effects of vasostatin onorthotopic xenograft and metastatic tumor models. Ourfindings show that rAAV2/5-mediated vasostatin is an effectivecandidate for lung cancer gene therapy.

Materials and Methods

Cells and animals. A549 human lung adenocarcinoma cells, Lewislung carcinoma (LLC) cells, MS1 mouse pancreatic endothelial cells,and HEK293 cells were obtained from American Type CultureCollection.

C57B6 and BALB/c nude mice were obtained from the LaboratoryAnimal Unit of University of Hong Kong. All animal procedures wereapproved by the Ethics Committee of University of Hong Kong anddone according to institutional guidelines.Preparation of rAAV stocks. Vasostatin cDNA was cloned into

pAAV-2 expression vector under the regulation of cytomegalovirusenhancer, chicken h-actin promoter, and a woodchuck hepatitis B virusposttranscriptional regulatory element to boost expression levels (20).Recombinant AAV1/2, AAV2/2, AAV2/5, and AAV2/8 stocks weregenerated by a three plasmid, helper virus-free packaging method (21)and purified on a CsCl gradient. The titer of rAAV vectors wasdetermined by real-time PCR analysis as described previously (22).Control rAAV-eGFP vectors were prepared using identical method.Transgene expression in vitro. LLC cells were infected with eGFP

reporter virus (MOI, 104) rAAV2/1, rAAV2/2, rAAV2/5, rAAV2/8 for72 h. eGFP expression was analyzed by flow cytometry (Beckman-Coulter). Infection was determined as the percentage of cells expressingthe viral-derived eGFP. HEK293 cells were infected with rAAV2/5-VASor rAAV-eGFP for 60 h (MOI, 104). Protein extracts from cell lysates andconcentrated conditioned medium were analyzed by Western blot andprobed with monoclonal antibody against human calreticulin (Stress-gen Biotechnologies), followed by horseradish peroxidase–conjugatedsecondary antibody and enhanced chemiluminescence detection(Amersham Pharmacia).

LLC spontaneous pulmonary metastasis model. A highly metastaticsubline of LLC (hm-LLC) were generated by three rounds of serialin vivo passage and recovered from spontaneous lung metastasis aftersubcutaneous inoculation of LLC cells. After in vitro expansion tosufficient numbers, cells were frozen until use for in vivo implantation.

For ex vivo treatment experiments, hm-LLC cells were infected with

rAAV2/5-VAS or rAAV2/5-eGFP overnight before inoculation.

Seven-week-old male C57B6 mice were anesthetized and received

subcutaneous injection with 100 AL cell suspension (1 � 106 cells)

from rAAV2/5-VAS, rAAV2/5-eGFP–pretreated, or naive cells. Two sets

of experiments were done. In the first set, mice were sacrificed 7 days

after tumor cell inoculation. The tumors were excised together with

adjacent skin from the underlying tissues, and feeding tumor blood

vessels along the undersurface of the surrounding dermis were

quantified by counting. The tumors were snap-frozen for RNA

extraction or fixed in 4% paraformaldehyde for histologic and

immunohistochemical analysis. In the second set of experiment, tumor

growth was monitored regularly. Tumor volume was measured with a

digital caliper and calculated using the formula 0.52 � a � b2, wherein

a and b are the largest and smallest diameters, respectively. Animals

were sacrificed 21 days after tumor cell implantation. Lung samples

were collected and fixed in 4% paraformaldehyde, and tumor nodules

on the lung were counted under a dissecting microscope.In intratracheal delivery experiments, 5 � 106 hm-LLC cells were

injected s.c. into 7-week-old male C57B6 mice. Primary ectopic tumorsreached a diameter of 5 mm in f1 week. Animals were anesthetized,and the entire subcutaneous tumors, together with surrounding skin,were surgically excised. After wound closure, animals were randomizedinto three groups and intratracheally given 2 � 1011 viral particles ofrAAV2/5-VAS, rAAV2/5-eGFP, or PBS. Animals were humanely euthan-ized when they showed the inability to reach food or water, showedsymptoms of emaciation, dehydration, or a 20% decrease in normalbody weight. Four mice from each group were sacrificed 30 days afterviral delivery or humanely euthanized, and lung tissues were harvestedfor histologic and immunohistochemical examination. The rest of themice were sacrificed at the end of the experiment (10-week posttreat-ment), and their lungs were examined.Subcutaneous A549 xenograft model. A549 human lung adenocar-

cinoma cells (5 � 106) resuspended in 100 AL PBS were injected intothe dorsal flank of 6-week-old to 8-week-old male BALB/c nude mice.When tumor size reached f50 mm3, mice were randomized into threegroups (n = 6 per group). Particles (2 � 1011 vector genomes) ofrAAV2/5 encoding eGFP or vasostatin in PBS or PBS were injected intotumor under anesthesia. Tumor growth was monitored regularly for upto 30 days, and volume was calculated as described previously. Tumorswere harvested for further analysis.Orthotopic A549 xenograft model. A549 cells were resuspended in

cold PBS with 1 mg/mL Matrigel (BD Biosciences) and concentrated to5 � 107 cells/mL; 20 AL inoculum of cells were injected into the leftlung parenchyma of 6-week-old male BALB/c nude mice (n = 8 pergroup) as described (23). Tumor-bearing mice were treated 1 week afterinoculation with 2 � 1011 vector genomes of virus of rAAV2/5-VAS orrAAV2/5-eGFP by intratracheal administration. Animals were sacrificed50 days after tumor cell implantation. Lung samples were harvested forfurther examination, and the mass of enlarged lymph nodes wasquantified.Analysis of in vivo gene expression by reverse transcription–

PCR. Total RNA from mice tissues was extracted by Trizol (Invitro-gen), and first-strand cDNA was synthesized with Superscript II(Invitrogen) and tested for the expression of vasostatin and CD105with the following primer pairs: Vas-F (5¶-AACTCGAGCCCGC-CATGCTGCTATCC), WPRE-R (5¶-GTTCCGCCGTGGCAATAG) andCD105-F (5¶-CGCTTCAGCTTCCTCCT), CD105-R (5¶-CGATGCTGT-GGTTGGT). PCR conditions were 45 s each at 94jC, 55jC, and 72jCfor 32 cycles. 18S rRNA was used as internal control.Endothelial cell proliferation and tube formation assays. MS1 mouse

pancreatic endothelial cells were used for assessing the biological


www.aacrjournals.orgClin Cancer Res 2008;14(3) February1, 2008 940



activity of the secretory product mediated by rAAV2/5. Conditionedmedium were harvested from LLC cells 60 h after infection with rAAV2/5-VAS or rAAV2/5-eGFP (MOI, 104). MS1 cells were seeded onto96-well plates at 2 � 103 cells per well. After 24-h incubation, themedium was replaced by conditioned medium with the exception ofcontrol culture. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assays were done at 24 h, 48 h, and 72 h, and cell viabilitywas determined according to the formula relative cell viability =(A570-690 of conditioned medium–treated cells)/(A570-690 of controlcells). Three independent experiments were done in triplicates foreach treatment.

The tube-like network formation of MS1 cells was assessed onMatrigel after mixing cells with conditioned medium. MS1 cells (2 �104) were seeded in triplicates into Matrigel-precoated 96-well plates for8 h in the presence of CM/rAAV2/5-VAS or CM/rAAV2/5-eGFP.Formation of tubes was examined, and images were captured usingan inverted microscope.Histology, immunohistochemistry, and TUNEL assay. Lung and

tumor samples were fixed, paraffin-embedded, and sectioned (5 Am)for histologic analysis. For immunohistochemical analysis, primaryantibodies, including calreticulin (1:300, Stressgen Biotechnologies),CD34 (1:300, Santa Cruz), Ki-67 (1:300, Lab Vision), a-smooth muscle

actin (a-SMA; 1:1,000, Sigma), and E-cadherin (1:500, Calbiochem)were used. Sections were subsequently incubated with horseradishperoxidase–conjugated secondary antibodies for 1 h. Staining wasvisualized using a 3,3¶-diaminobenzidine kit (Zymed) and counter-stained with hematoxylin. For double immmunofluorescence of CD34and a-SMA staining, FITC or Cy3-conjugated secondary antibodies(Sigma) were used.

TUNEL assay was done using In situ Cell Death Detection kit (Roche)according to manufacturer’s instructions. Cell nuclei were stained withpropidium iodide.Analysis of tumor microvessel density, Ki-67 proliferation index, and

apoptotic index. Quantitative analysis of images was conducted usingImage-Pro Plus analysis software (Media Cybernetics). Microvesseldensity (MVD) was assessed after CD34 staining by the method definedby Weidner and coworkers (24). Tumor sections were scanned by lightmicroscopy at low power field (�40), and three fields with the mostintense neovascularization (hotspots) were selected. Microvessel countsof hotspots were done at high power field (hpf; 200�). The mean valueof three hotspots was taken as the MVD, which was expressed asnumber of microvessels per hpf. For Ki-67 proliferation and apoptoticindices, positively stained cells and nuclei were counted on a minimumof 10 randomly selected �400 hpf from representative tumor sections.

Fig. 1. rAAV-mediated transgene expression and secretion of vasostatin with bioactivity on endothelial cell culture. A, flow cytometric analysis of transduction efficiencyof LLC with different serotypeAAV vectors encoding eGFP at 72 h postinfection (MOI, 104; n = 3). Bars, SE. B,Western blot analysis showing vasostatin expression incultured medium by rAAV2/5-VAS ^ infected cells (lanes 2 and 4) but not rAAV2/5-eGFP ^ infected HEK293 cells (lanes1and 3). C, cell viability as measured by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on MS1cells incubated with conditioned medium from rAAV2/5-VAS (!) and rAAV2/5-eGFP(o) transduced LLC cells at 24, 48, and 72 h. MS1cell viability was significantly reduced after 48-h incubation with rAAV2/5-VAS conditioned medium. Bars, SD; n = 3;*, P = 0.039 (48 h) and 0.0027 (72 h). D, endothelial cell tube formation assay showed interference of network assembly of MS1cells on presolidified Matrigel inconditioned medium from rAAV2/5-VAS ^ transduced LLC cells (right) compared with rAAV2/5-eGFP conditioned medium (left).

rAAV-Vasostatin GeneTherapy for Lung Cancer




Indices were calculated as number of (Ki-67 or TUNEL) positive cellsdivided by total cell count.Statistical analysis. Student’s t test was used to compare the values

or indices between different groups. Kaplan-Meier survival curves werecompared using the log-rank test in GraphPad Prism.

Results

rAAV2/5 viral vector–mediated expression of biologically activevasostatin in vitro. Human vasostatin cDNA encoding theN-terminal fragment of human calreticulin (residues 1-180)was inserted into a rAAV2 expression vector cassette. To obtainoptimal transgene expression for lung cancer treatment, wetested the transduction efficiency of different pseudoserotypedvirion combinations in LLC cells. The vectors were packagedusing the eGFP reporter gene, and transduction efficiencies ofthe serotypes rAAV2/2, rAAV2/1, rAAV2/5, and rAAV2/8 in LLCculture were compared. By flow cytometric analysis, rAAV2/5showed the highest transduction rate of 63.5% (Fig. 1A). Theability of rAAV2/5 vector to mediate protein expression andsecretion was tested by infecting HEK293 cells with rAAV2/

5-VAS vector. Western blot analysis using anticalreticulinantibody showed the production and secretion of vasostatininto culture medium f60 h after rAAV2/5-VAS infection(Fig. 1B). Protein expression was not detected in either celllysates or conditioned medium from rAAV2/5-eGFP–trans-duced HEK293 cells.We next examined the biological activity of secreted vaso-

statin mediated by rAAV2/5 viral vectors in vitro . Theantiangiogenic activity of vasostatin was assessed by evaluatingthe viability of MS1 endothelial cells cultured in the presence ofconditioned medium from rAAV2/5-VAS transduced LLC cells.There was significant inhibition of MS1 proliferation in rAAV2/5-VAS conditioned medium compared with control rAAV2/5-eGFP conditioned medium in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Fig. 1C). Thisinhibitory effect was specific for endothelial cells, as treatmentwith rAAV2/5-VAS conditioned medium on A549 lung adeno-carcinoma cells did not reduce cell growth (data not shown).Using tube formation assay to test the effect of vasostatin onin vitro angiogenesis, rAAV2/5-VAS conditioned medium wasshown to inhibit tube-like network formation of MS1 cells onMatrigel precoated plates, whereas tube formation was ob-served in MS1 cells incubated in rAAV2/5-eGFP conditionedmedium (Fig. 1D). Thus, expression of rAAV2/5-encodedvasostatin was able to inhibit angiogenesis in vitro .rAAV2/5-vasostatin expressed from hm-LLC cells suppressed

subcutaneous tumor growth and metastasis. To test whetherrAAV2/5-VAS can serve as an antiangiogenesis factor andsuppress tumor growth in vivo , we s.c. injected hm-LLC cells,which had been preincubated with rAAV2/5-VAS, rAAV2/5-eGFP, or PBS, into C57B6 mice. As an aggressive tumormodel, the growth of tumor mass in control groups (PBS orrAAV2/5-eGFP– treated cells) was rapid, whereas tumorgrowth from rAAV2/5-VAS–treated hm-LLC cells was delayedand became palpable much later than control virus treatment.As a result, the mean tumor volume in mice with rAAV2/5-VAS–treated hm-LLC cells was significantly reduced to lessthan half of control tumors by 16 to 18 days after inoculation(Fig. 2A).As hm-LLC cells can readily metastasize from the subcutane-

ous primary tumor site to the lung, we evaluated the ability ofvasostatin-producing hm-LLC cells to modulate metastaticprogression. The pulmonary metastatic burden of the treatedmice was assessed by counting the number of tumor noduleson the surface of the lung at the conclusion of the experiment(day 21). Mice in control groups displayed numerousdistinguishable pulmonary metastatic nodules (average of20), whereas mice bearing rAAV2/5-VAS–treated hm-LLC cellsshowed fewer visible tumor nodules (average of 8). Thereduction of surface nodules by >2-fold with rAAV2/5-VAStreatment (Fig. 2B) showed that vasostatin not only efficientlysuppressed subcutaneous tumor growth but also reducedsubsequent spontaneous lung metastasis.Vasostatin from transduced hm-LLC cells suppressed tumor-

induced neovascularization. To determine whether suppressionof primary and metastatic tumor growth in mice was a directresult of the antiangiogenic activity of viral-encoded vasostatin,the formation of new blood vessels induced by tumor wasassessed after subcutaneous inoculation of tumor cells pretreatedwith rAAV2/5-VAS or rAAV2/5-eGFP. Tumors were excised withthe adjacent dermis at day 7 after tumor cell inoculation. Tumors

Fig. 2. Suppression of subcutaneous tumor growth and metastasis by rAAV2/5-VAS. A, vasostatin expressed by rAAV2/5-VAS ^ transduced hm-LLC cellssuppressed the growth of subcutaneous hm-LLC tumors.The volumes ofsubcutaneous tumors initiated from hm-LLC cells pretreated with PBS (5), rAAV2/5-eGFP (o), or rAAV2/5-VAS (!; MOI, 5 � 104) were estimated at the indicatedday after inoculation. Bars, SD; n = 7; *, P = 0.041 (day16) and 0.0005 for rAAV2/5-VAS versus rAAV2/5-eGFP pretreated tumor. B, pulmonary metastatic burdenwas assessed by counting the number of tumor nodules on the lung surface at21-d postinoculation. rAAV2/5-VAS ^ mediated expression of vasostatin inhibitedthe spontaneous metastasis of subcutaneous hm-LLC tumors. Bars, mean; n = 5;*, P = 0.045.





from animals treated with rAAV2/5-VAS–transduced hm-LLCcells seemed pale and flat compared with tumors obtainedfrom control animals (Fig. 3A). The tumor-feeding vessels alongthe undersurface of the surrounding dermis were significantlyreduced in tumors from rAAV2/5-VAS–pretreated hm-LLC cells(Fig. 3B), indicating a reduction in angiogenic potential. ThePBS or rAAV2/5-eGFP pretreated hm-LLC cells formed tumorswith multiple large feeding blood vessels (7.8 F 0.9 and 8 F0.8 vessels, respectively), whereas tumors from rAAV2/5-VASpretreated hm-LLC cells displayed significantly fewer (3.2 F 0.8vessels) and narrower blood vessels. Histologic analysis showedmultiple lumen-like formations containing RBC present in thetumors of control groups (Fig. 3D, a and b), demonstratingmature blood vessel formation. Tumor vascularity was observedin almost the entire tumor with peripheral vessels penetratingthroughout control tumors. However, in rAAV2/5-VAS pre-

treated tumors, vascularity was observed only at the tumorperiphery. Expression of CD105, an activated endothelial cellmarker, was shown to be significantly reduced in rAAV2/5-VASpretreated tumors compared with control tumors (Fig. 3C).Fewer host endothelial cells migrated into rAAV2/5-VAS–pretreated tumors as indicated by the relative level of CD105transcripts in tumors at this stage, thus confirming rAAV2/5-VAS suppression of tumor growth through inhibition ofangiogenesis. In addition, we examined the expression ofE-cadherin, a cell adhesion molecule which negatively corre-lates with the invasive potential of tumor cells (25). rAAV2/5-VAS pretreated tumors showed strong positive signals(Fig. 3D, f), which may be associated with lower metastaticpotential, whereas weak signals were observed in controltumors (Fig. 3D, d and e), suggesting an inherent ability ofthese cells to metastasize.

Fig. 3. Suppression of tumor-induced neovascularization by rAAV2/5-VAS ^ transduced hm-LLC cells. A, tumors displayed varying degrees of feeding tumor vesselsalong the undersurface of the surrounding dermis at 7-d postinoculation. B, quantification of blood vessels feeding to tumors pretreated with rAAV2/5-VAS, rAAV2/5-eGFP,or PBS. Bars, SE; n = 5; *, P = 0.0001for rAAV2/5-VAS versus rAAV2/5-eGFP tumors. C, relative expression of CD105 in tumor tissues by semiquantitative reversetranscription ^ PCR. CD105 mRNA levels are presented as ratio of CD105 PCR band intensity to control 18S rRNA. Bars, SE; n = 3; *, P = 0.0217 for rAAV2/5-VAS versusrAAV2/5-eGFP tumors. Gel images are presented in Supplementary Fig. S1. D, histologic and immunohistochemical analysis of tumors at 7-d postinoculation. Sectionsof tumor tissues from mice receiving PBS (a, d), rAAV2/5-eGFP (b, e), and rAAV2/5-VAS (c, f) pretreated hm-LLC cells stained with H&E (top) or probed with E-cadherinantibody (bottom). Magnification, 200� (a-c) and 400� (d-f).





Intratumoral injection of rAAV2/5-VAS inhibited subcutaneouslung cancer xenograft growth. We further assessed the thera-peutic potential of rAAV2/5-VAS on a subcutaneous lung cancerxenograft model by intratumoral delivery of rAAV2/5-VAS,rAAV2/5-eGFP, or PBS. The subcutaneous model was estab-lished by implantation of A549 cells (5 � 106) subcutaneousinto the dorsal flank of nude mice, and tumor growth wasmonitored for up to 1 month after treatment. Vasostatin geneexpression was confirmed by reverse transcription–PCR intumors receiving rAAV2/5-VAS (Fig. 4B). Treatment with 2 �1011 vector genomes of rAAV2/5-VAS retarded subcutaneousxenograft progression as mean tumor volumes were signifi-cantly reduced in mice receiving rAAV2/5-VAS compared with

those receiving PBS or rAAV2/5-eGFP (Fig. 4A). Histologicanalysis of tumor samples revealed massive apoptosis in largeextended areas in tumor sections obtained from rAAV2/5-VASgroup compared with those from control groups (rAAV2/5-eGFP or PBS; data not shown). The proliferation index(proportion of Ki67-positive cells) of tumors treated withrAAV2/5-VAS was reduced compared with control group (meanindex of 50.2 F 10.7% versus 37.5 F 4.3%; Fig. 4C, a). Incontrast, f2-fold increase in apoptosis (proportion of TUNEL-positive cells) was detected (41.7 F 7.9% versus 18.4 F 2.0%;Fig. 4C, b) in rAAV2/5-VAS–treated tumors compared withcontrol mice (Fig. 4C, b). To determine whether the poorgrowth of rAAV2/5-VAS–treated subcutaneous xenografts was

Fig. 4. Intratumoral injection of rAAV2/5-VAS inhibits subcutaneous A549 tumor growth and angiogenesis. A, tumor volume in mice receiving i.t. injection of PBS (5),rAAV2/5-eGFP (o), and rAAV2/5-VAS (!; 2� 1011vector genomes.), respectively. Bars, SD; n = 6; P < 0.01for rAAV2/5-VAS versus rAAV-eGFP or PBS group.B, detection of rAAV2/5-VAS ^ mediated vasostatin expression in tumors by reverse transcription ^ PCR. C, histologic and immunohistochemical analysis of tumors.a, tumor proliferation index as calculated by percentage of Ki-67^ positive cells (left). Ki-67 staining (brown) of proliferating cells in representative rAAV2/5-eGFP ^ treatedand rAAV2/5-VAS ^ treated tumors. Magnification, 100�. b,TUNEL assay shows increased apoptotic cells in rAAV2/5-VAS ^ treated tumor compared withrAAV2/5-eGFP ^ treated tumor. Magnification, 200�. Tumor apoptotic index calculated as percentage ofTUNEL-positive cells (left). c, tumor MVD and mature status.Tumor MVD analysis (number of CD34 positive vessels per hpf; left). Double immunofluorescence staining for CD34 (green) and a-SMA (red) show reduced number ofblood vessels with CD34-positive endothelial cells as well as mature vessels associated with a-SMA ^ positive mural cells in rAAV2/5-VAS ^ treated tumors comparedwith rAAV2/5-eGFP ^ treated tumors. Magnification, 200�. Results for tumor proliferation index, apoptotic index, and MVD analysis represent averages of at least fourdifferent tumors from each group evaluated. Bar, SE; *, P = 0.02; **, P = 0.0047. Single channel images are presented in Supplementary Fig. S2.





due to poor blood vessel supply, we stained the tumor sectionswith antibodies against CD34 and a-SMA and measured MVD.A significant decrease in MVD (61.2 F 8.3 versus 170.8 F 26.9vessels per hpf) in mice treated with rAAV2/5-VAS was observedcompared with tumors in control mice (Fig. 4C, c). In addition,a decrease in SMA-positive mural cells associated withendothelial cells was detected in rAAV2/5-VAS–treated tumors(Fig. 4C, c). These results suggested that rAAV2/5-VAS treatmentwas effective in reducing the number of capillary vessels, as wellas inducing apoptosis, resulting in the repression of vasculartumor mass expansion.Suppression of orthotopic lung tumor growth and spread after

intratracheal administration of rAAV2/5-VAS. To simulate thegrowth and spread of human lung carcinoma, we used anorthotopic implantation model. In this model, primary tumorderived from A549 cells first developed at the inoculated site,which would then invade the visceral pleura and mediastinallymph nodes and metastasize to the contralateral lung. To

evaluate the therapeutic effects of vasostatin, viral vectors wereintratracheally given 1 week after tumor cell inoculation intothe left lung. When the experimental animals were examined at50 days after initial cell inoculation, orthotopic A549 xenograftwas found to develop in all grafted mice at the inoculated site.Mice receiving intratracheal 2 � 1011 vector genomes of rAAV2/5-VAS treatment produced substantially fewer tumors in thelungs and mediastinal lymph nodes when compared withcontrols at 50th day of postinoculation (Fig. 5A, a and b).Although rAAV2/5-VAS–treated mice showed similar localtumor volume as control mice (Fig. 5A, c), they exhibited over50% decrease in mediastinal lymph node weight (Fig. 5A, d).Gross macroscopic examination of rAAV2/5-VAS–treated miceshowed fewer nodules in their contralateral lungs than controlmice, suggesting a decrease in severity of metastases in treatedmice. The intratracheal delivery of rAAV2/5-VAS did notproduce major changes in cell proliferation of primaryorthotopic tumors (Fig. 5B). In contrast, primary tumors in

Fig. 5. Intratracheal delivery of rAAV2/5-VAS inhibits orthotopical A549 tumor angiogensis and metastasis. A, gross examination of tumor and metastasis. a and b,representative lungs of mice receiving rAAV2/5-eGFPor rAAV2/5-VAS. c, estimated volume of primary tumor at the day of sacrifice. d, mediastinal lymphnodeweight due totumor metastasis at the day of sacrifice. Bar, SE; n = 6; *, P = 0.016. B, tumor proliferation index analysis (percentage of Ki-67^ positive cells). Cell proliferation index inprimary tumor (left) are similar in two groups. Ki-67 staining of rAAV2/5-eGFP ^ treated and rAAV2/5-VAS ^ treated tumors. C, tumor MVD. Quantification of tumor MVD(left) showed significant reduction of CD34-positive cells in primary tumor from treated mice compared with controls. CD34 staining of rAAV2/5-eGFP ^ treated andrAAV2/5-VAS ^ treated tumors. Magnification, 100�. Columns, mean from at least three different tumors; bars, SE; *, P = 0.033.





mice receiving rAAV2/5-VAS had a 1.6-fold fewer number ofvessels per surface unit than those of control group (Fig. 5C).These results showed that rAAV2/5-VAS treatment via trachealadministration could retard angiogenesis in the primary tumor,delay the spread of tumor cells, and achieve an antimetastaticeffect.Increased survival rate of mice and diminished expansion of

lung metastases by intratracheal delivery of rAAV2/5-VAS. Toevaluate the antimetastasis effect and survival enhancement oflong-term rAAV2/5-VAS expression in vivo, we used a sponta-neous pulmonary metastasis model wherein hm-LLC cells wereinoculated s.c. to initiate primary tumor and subsequentpulmonary metastases. When the primary subcutaneous tumorreached f0.5 cm in the longest diameter at 1 week

postinoculation, the primary tumor was surgically removed,a procedure that resulted in accelerated metastasis developmentin the lung. When mice were sacrificed 30 days after rAAV2/5-VAS or rAAV2/5-eGFP treatment or were humanely euthan-ized during this period, the lungs were harvested for examina-tion of metastatic incidence and severity. All the mice in controlgroups exhibited massive hemothorax and developed metastaticnodules in their lungs. In control animals, large, vascularizedtumors with hemorrhages were found in majority of the lungs(Fig. 6A, a). In contrast, the lung surface of one in four micereceiving rAAV2/5-VAS was tumor-free, whereas the others hadseveral small metastasis nodules (Fig. 6A, a). Moreover, rAAV2/5-VAS treatment reduced the mean weight of lungs withmetastatic tumors by >40% (Fig. 6A, b). Histologic analysis

Fig. 6. Inhibition of spontaneous pulmonary metastasis and prolonged survival after removal of primary tumor and intratracheal delivery of rAAV2/5-VAS. A, grossexamination of lung. a, representative lungs bearing tumor nodules developed frommetastatic hm-LLC cells after surgical resection of primary tumor and intratracheal deliveryof PBS, rAAV2/5-eGFP, or rAAV2/5-VAS for 30 d. b, lung weight after metastastic tumor growth from mice sacrificed 30 d after treatment. Bar, SE; n = 3. B, H&Estaining of lung sections from mice sacrificed 30 d after intratracheal distillation of PBS (a), rAAV2/5-eGFP (b), or rAAV2/5-VAS (c, d). H&E staining revealing dormantmicrometastatic tumor cells along large vessels in the lung from long-term surviving mice sacrificed10 wk after treatment (d). Magnification, 200�. C, immunohistochemicalstaining showing vasostatin expression in lung of long-term surviving mice treated with rAAV2/5-VAS (right), but not in lung from control mice treated with rAAV2/5-eGFP(left). D, rAAV2/5-VAS treatment prolonged the survival of hm-LLC tumor ^ bearing mice after primary tumor removal (n = 6, P = 0.033).





showed that large tumors from the control group werecomposed of more heterogeneous cell populations and hemor-rhage in the central lung section (Fig. 6B, a and b). On the otherhand, minimal vascularity was observed in tumor nodules inlungs which received rAAV2/5-VAS (Fig. 6B, c and d). Upontermination of the experiment at 10 weeks after treatment,significant prolonged survival was observed in rAAV2/5-VAS–treated tumor-bearing mice (Fig. 6D).To show long-term expression of vasostatin by rAAV2/5

vectors, we did immunohistochemistry analysis on lungsections. Vasostatin expression was detected in the airwayepithelium of lungs receiving rAAV2/5-VAS for 10 weeks, butnot in lungs receiving rAAV2/5-eGFP (Fig. 6C). Long-termsurvivors did not reveal any changes in overall health exceptthat dormant micrometastases of hm-LLC cells beside the largevessel in some lungs were observed (Fig. 6B, d). No abnormal-ities were noted in gross and histologic examination of lung,liver, intestine, spleen, kidneys, and heart from rAAV2/5-VAS–treated mice. In an independent set of experiment, when weinvestigated animals 30 days after receiving intratrachealdelivery of rAAV2/5-mediated angiogenesis inhibitors, we hadconfirmed, by reverse transcription–PCR, the absence oftransgene expression in any of these organs except the lungs(data not shown). As shown in Fig. 6D, the median survivaltime was 21 days in control groups and was significantlyimproved in rAAV2/5-VAS–treated mice, with a mediansurvival of 51.5 days. These results strongly support the findingthat lung-targeted administration of rAAV2/5-VAS leads tolong-term expression of vasostatin and is effective and safe forlung cancer treatment.

Discussion

We show here, for the first time, that rAAV2/5-mediateddelivery of the angiogenesis inhibitor vasostatin is an effectiveapproach for lung cancer treatment. Vasostatin inhibited thegrowth of human lung adenocarcinoma cells in a subcutaneoustumor model and reduced spontaneous metastasis in nudemice bearing orthotopic xenografts. In addition, vasostatindelayed metastic spread in immunocompetent mice that hadundergone primary tumor resection after a single intratrachealadministration of rAAV2/5-VAS. When combined with surgery,rAAV2/5-VAS gene therapy could potentially offer long-termsurvival benefit.Success in treatment targeting angiogenesis is highly depen-

dent on the delivery method used and the ability to sustainpersistent expression of the therapeutic molecule in vivo (26).In this study, we evaluated the transduction efficiency ofdifferent combinations of vector on mouse LLC cells. Weshowed that the highest transduction efficiency was achievedwith rAAV2/5, which is in line with our previous comparison oftype 5 serotype on human A549 lung adenocarcinoma cells(27). Here, we determined that intratumoral and intratrachealdelivery of rAAV2/5 vector resulted in the synthesis of bioactivevasostatin from transduced tumor cells and mouse airwayepithelium in vivo . rAAV-mediated pulmonary transduction forsystemic administration of secretory proteins was previouslyreported to result in functional protein secretion and persis-tence in the bloodstream for over 150 days after a single dose(19). We observed vasostatin expression in the lung for at least10 weeks as evaluated upon termination of experiment. Despite

previous reports wherein reduced subcutaneous tumor growthwas achieved only through repeated administration of vaso-statin cDNA or adenoviral vectors (7, 8), we observed tumorsuppression after a single dose of i.t. injected rAAV2/5-VAS.Due to its efficiency in lung cell transduction, rAAV2/5 can bedelivered via noninvasive routes and is therefore an attractiveapproach for future application in lung cancer therapy.Previous preclinical studies of antiangiogenesis inhibitors

suggest that treatment at early stages of tumor progression ismore effective than intervention at later stages due to thecomplexity of cellular interactions in malignant tumors(16, 28). This stage-dependent effect was also observed in ourstudy of vasostatin treatment in the different tumor models. Weobserved a delay in the initiation of angiogenesis produced byrAAV2/5-VAS–transduced hm-LLC cells in vivo . The down-regulation of tumor neovascularization by rAAV2/5-mediatedvasostatin resulted in reduced spontaneous metastatic inci-dence and expansion. This was reflected in higher levels ofE-cadherin expression in rAAV2/5-VAS–pretreated tumors,suggesting a suppression of invasive ability. As rAAV vectorsare known to persist in an episomal state or concatameric formwith low frequency of integration, the proportion of rAAV2/5-VAS– transduced tumor cells would be diluted in anexpanding population of tumor cells, which would explainthe resulting pulmonary metastasis, though much reduced,observed in mice bearing rAAV2/5-VAS–pretreated hm-LLCtumors. We also observed a reduction of CD105-positiveendothelial cells in rAAV2/5-VAS–treated tumors. Vasostatin-mediated suppression of endothelial cell proliferation in tumormay result in reduced recruitment of mural cells, which isnormally facilitated by endothelial cell secreted factors (29) andis required to stabilize nascent vessels (30). In our model,vasostatin gene delivery to preestablished subcutaneous A549xenograft in nude mice led to a significant shrinkage of thetumor volume associated with lower MVD and a low degree ofvessel maturation in tumors, indicating vasostatin treatment tobe an effective strategy in tumor growth inhibition andmetastasis.Tumor invasion and metastasis are the major causes of

treatment failure and death in lung cancer patients. Lymphnode invasion and spread into contralateral lung throughlymphatic routes presented in this model represent commonclinical features of lung cancer progression (31, 32). Theorthotopic A549 transplantation nude mice model used in thisstudy, wherein tumor cells are directly injected into normallung parenchyma resulting in solitary tumor in situ andmetastases in lung and/or mediastinal lymph nodes, providesa stringent test of vasostatin gene therapy in a pathologicenvironment. Apart from reproducing human tumor metastasispattern, the A549 orthotopic xenograft have been shown to bechemoresistant, a major problem in non–small cell lungcarcinoma chemotherapy (33). Our results showed that a singleintratracheal administration of rAAV2/5-VAS achieved systemicprotein delivery, resulting in an obvious reduction of metastaticnodules. Together with the data on rAAV2/5-VAS–treated hm-LLC cells, our results suggest that vasostatin acts during theonset of metastasis to inhibit the angiogenic switch, resulting inthe delay of tumor progression and metastasis. Primary tumorvolume, however, was not reduced in the A549 orthotopicxenograft model. Similar results had been shown using endo-statin gene therapy treatment on an orthotopic breast cancer





model, wherein endostatin secreted from muscle retardedmetastasis but not primary tumor growth (34). Angiostatin,which is induced in mice bearing LLC primary tumor, also hasinhibitory effects on metastasis (35). Combined treatment withangiostatin and endostatin was shown to be more effective inthe treatment of brain metastases than with either endostatin orangiostatin alone, demonstrating synergistic effects in combi-nation therapy (17). Because the spectrum of proangiogenicfactors differs at different tumor stages (36), combined use ofdifferent angiogenesis inhibitors may overcome the limitationsassociated with single molecule treatment.Angiogenesis provides a principal route for tumor cells to exit

the primary site to a distant site (37). In instances whereincancer has spread into the bloodstream, surgical removal ofvisible tumors is often not beneficial. Surgical excision of lungtumor raises the risk of relapse from micrometastasis due toinsufficient activity of endogenous angiogenesis inhibitors(38). For instance, an increase in systemic vascular endothelialgrowth factor levels found after pulmonary surgery and localradiation therapy results in the acceleration of the micro-metastases growth in lung cancer patients or animal models(39, 40). Administration of angiostatin in combination withradiation therapy was shown to reduce proliferation of lungmetastases from LLC primary tumors (41, 42). These endoge-nous angiogenesis inhibitors, including vasostatin, have beenshown not to perturb wound healing when maintained in thebloodstream at sustained levels (9, 43, 44). Our intratracheal

administration of rAAV2/5-VAS effectively prevented theoccurrence of spontaneous metastases and micrometastasisgrowth after surgical removal of the primary tumor, prolongingthe survival of treated animals. This may be due to long-termsustained expression of angiogenic inhibitors, which couldsuppress the growth of micrometastasis (45). In lung-directedviral administration, elevated transgene expression could beobserved in the bronchial alveolar lavages and bloodstream(46). Vasostatin in both the airway lumen and bloodstreammay establish an angiogenic defense to tumor growth,especially in the lung, which is a common site of primarytumor and metastases. Our results show that rAAV2/5-mediatedvasostatin was effective in inhibiting the expansion of earlycolonies via intratracheal administration and can offer survivalbenefit in mice after primary tumor resection. The results fromthis preclinical therapy has contributed to defining a practicaland effective approach for using rAAV2/5-VAS in combinationwith surgery in future clinical applications for human lungcancer therapy.In summary, we have provided positive data for the safety,

feasibility, and efficacy of using tumor-directed and lung-directed rAAV2/5 gene transfer of vasostatin as a potentialantiangiogenic strategy against lung cancer. The effectiveness ofvasostatin in disrupting lung tumor growth and metastasisduring various phases of tumor development provides apromising basis toward further studies and clinical applicationof rAAV2/5-VAS for lung cancer therapy.

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2008;14:939-949. Clin Cancer Res Ke Xia Cai, Lai Ying Tse, Carly Leung, et al. Vasostatinby Adeno-Associated Virus-Mediated Expression of Suppression of Lung Tumor Growth and Metastasis in Mice

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