A Small Interfering RNATargetingVascular Endothelial ...wing’s sarcoma is a primitive neuroectodermal tumor that most often affects children and young adults in the first two decades

A Small Interfering RNATargetingVascular Endothelial GrowthFactor Inhibits Ewing’s Sarcoma Growth in aXenograft Mouse ModelHui Guan,1 Zhichao Zhou,1HuaWang,2 Shu-FangJia,1Wenbiao Liu,3 and Eugenie S. Kleinerman1

Abstract Angiogenesis plays an essential role in tumor growth andmetastasis and is a promising therapeu-tic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogene-sis aswell as in angiogenesis.TC71human Ewing’s sarcoma cells overexpressVEGF,with a shift inisoform production from membrane-boundVEGF189 to the more solubleVEGF165. Transfection ofTC71cells with a vector-basedVEGF targeted small interfering RNA expression system (VEGFsi)inhibitedVEGF165 expression by 80% andVEGF165 protein production by 98%, with no alterationin VEGF189 expression. Humanmicrovascular endothelial cell proliferation and migration inducedby conditioned medium fromVEGFsi-transfected TC71cells was significantly less than that in-duced by conditioned medium fromTC71cells and control vector-transfectedTC71cells. Further-more, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressingTC71cells was significantly less than that of parental or control vector-transfected cells.Vessel densityas assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed byNorthern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsialso resulted in tumor growth suppression.When inoculated into the tibias of nude mice,VEGFsi-expressingTC71cells inducedosteolytic bone lesions that were less severe than those inducedbycontrol groups. These data suggest that targetingVEGF165 may provide a therapeutic option forEwing’s sarcoma.

Ewing’s sarcoma is a primitive neuroectodermal tumor thatmost often affects children and young adults in the first twodecades of life. It is the second most common malignant bonetumor and accounts for 10% to 15% of all primary bone tumors(1). Patients presenting with localized disease have a signifi-cantly better chance of survival than patients who present withmetastases (2). Despite the use of multimodal therapy(chemotherapy, radiation therapy, and surgery), the long-termdisease-free survival rate of Ewing’s sarcoma patients is stilldisappointingly low, particularly in the high-risk groups (3, 4).The identification of new therapeutic targets is therefore needed.

Angiogenesis has been specifically linked to increased growthand metastatic potential in human tumors (5). Althoughnumerous growth factors are involved, vascular endothelialgrowth factor (VEGF), particularly VEGF-A, has been shown toplay a pivotal role in tumor angiogenesis (6). Binding of VEGF-A to its receptors induces mitogenesis and chemotaxis of

normal endothelial cells and increases vascular permeability, allof which contribute to new vessel formation and tumor growth(7). VEGF also contributes to neovascularization by mobilizingbone marrow–derived endothelial progenitor cells (8). To date,five isoforms of human VEGF have been identified (VEGF121,VEGF145, VEGF165, VEGF189, and VEGF206; ref. 9). Increasedlevels of VEGF expression have been found in most humantumors, including those of the lung, gastrointestinal tract,kidney, thyroid, bladder, ovary, and cervix (10). Pediatrictumors have also been shown to be very vascular, with a highproliferation rate (11).

Several lines of evidence point to a role for VEGF in thepathogenesis of Ewing’s sarcoma. We recently reported elevatedVEGF expression in three of four Ewing’s sarcoma cell lines andin human primary tumor specimens (12).4 Serum VEGF levelswere found to be significantly higher in children with Ewing’ssarcoma than in healthy controls (11, 13). These elevated VEGFlevels declined after tumor regression (11, 13). High levels ofVEGF165 and VEGF121 expression were detected by immunohis-tochemical analysis in 17 of 31 Ewing’s tumor samples taken atinitial biopsy (14). Positive staining for VEGF at the time ofdiagnosis correlated with a poor prognosis (14, 15). Finally,EWS-ETS, the specific oncoprotein in Ewing’s sarcoma, has beenshown recently to be a transcription factor for the VEGFpromoter, stimulating the expression of VEGF (14). Takentogether, these data indicate that inhibiting the expression orfunction of VEGF may lead to improvements in disease outcome.

www.aacrjournals.orgClin Cancer Res 2005;11(7) April 1, 2005 2662

Authors’ Affiliations: 1Division of Pediatrics and Departments of 2CancerBiology and 3Surgical Oncology, University of Texas M.D. Anderson CancerCenter, Houston, TexasReceived 6/22/04; revised12/14/04; accepted12/23/04.Grant support: Kayton Fund, Lindner Fund, and NIH core grant CA16672.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.Requests for reprints: Eugenie S. Kleinerman, Division of Pediatrics, Universityof Texas M.D. Anderson Cancer Center, Unit 87, 1515 Holcombe Boulevard,Houston, TX 77030. Phone: 713-792-8110; Fax : 713-794-5042; E-mail:[email protected].

F2005 American Association for Cancer Research. 4 Unpublished data.

CancerTherapy: Preclinical

RNA-mediated interference is a conserved gene silencingmechanism that recognizes dsRNA as a signal to trigger thesequence-specific degradation of homologous mRNA (16). Itwas first studied in Caenorhabditis elegans and plants in 1995.RNA-mediated interference was adapted for work with mam-malian cells in 2001, with the discovery that the introduction ofsmall interfering RNA (siRNA) <30 nucleotides long inmammalian cells avoids the induction of an IFN response thatactivates protein kinase R (17). The high efficiency andspecificity of RNA-mediated interference has made it a powerfuland widely used tool for the analysis of gene function. In thisreport, we used a vector-based siRNA expression system, whichovercomes the limitations of transience and high cost insynthetic siRNAs, to specifically inhibit VEGF165 expression inEwing’s sarcoma cells. The siRNA that we synthesized inhibitedEwing’s sarcoma tumor growth in a nude mouse model.

Materials and Methods

Expression plasmids. siRNA expression vector pSilencer2.1-U6 hygrowas purchased from Ambion (Austin, TX). siRNA-expressing plasmidstargeting human VEGF (VEGFsi) were constructed according to themanufacturer’s instructions. Briefly, four pairs of cDNA oligonucleotidestargeting human VEGF mRNA at different locations were synthesized byIntegrated DNA Technologies (Coralville, IA). Each pair of oligonucleo-tides was annealed at 90jC for 3 minutes, cooled to 37jC, and incubatedfor 1 hour. The annealed dsDNA oligonucleotides were ligated betweenthe BamHI and HindIII sites on the pSilencer2.1-U6 hygro vector. Thecontrol vector (si) was constructed by inserting a sequence that expressesa siRNA with limited homology to sequences in the human and mousegenomes. The targeted VEGF sequences were VEGFsi-2: TCATCAC-GAAGTGGTGAAG; VEGFsi-4: GTGGTGAAGTTCATGGATG; VEGFsi-6:GTTCATGGATGTCTATCAG; VEGFsi-7: GATAGAGCAAGACAAGAAA;and si control: CTACCGTTGTTATAGGTGTCTCTTGAACACCTATAA-CAACGGTAGT. All inserted sequences were verified by DNA sequencing.

Cell culture and transfection. TC71 human Ewing’s sarcoma cellswere cultured as described previously (12). MDA-MB-231 human breastcancer cells were obtained from American Type Culture Collection

(Manassas, VA) and cultured in DMEM with 10% fetal bovine serum.Human microvascular endothelial cells (HMVEC) were purchased fromCambrex (East Rutherford, NJ) and grown in microvascular endothelialcell medium (5% fetal bovine serum in endothelial basal medium with12 Ag/mL bovine brain extract, 10 Ag/mL human epidermal growthfactor, 1 Ag/mL hydrocortisone, and 1 Ag/mL GA-1000). HMVECs(passage 3 or 4) that were f80% confluent were used for mostexperiments. All cells were free of Mycoplasma , as screened by MycoplasmaPlus PCR Primer Set (Stratagene, Inc., La Jolla, CA), and verified to be freeof pathogenic murine viruses (National Cancer Institute-FrederickCancer Research & Development Center, Frederick, MD). Transfectionwas done with Superfect (Qiagen, Valencia, CA) as directed by themanufacturer and selected in hygromycin B (Invitrogen Life Technolo-gies, Carlsbad, CA) containing medium at 400 Ag/mL for TC71 cells and200 Ag/mL for MDA-MB-231 cells. Stable transfected cell clones weretested for VEGF expression by Northern blotting or ELISA. VEGFsi-7-transfected TC71 cell clone 7-1 (TC/VEGFsi) and control vector-transfected TC71 cell clone (TC/si) were used for the in vivo experiments.

Northern blot analysis. Cultured cells or tumor tissue was lysed inTrizol reagent (Life Technologies, Inc., Grand Island, NY). Total RNAwas purified according to the manufacturer’s instructions. VEGF andtopoisomerase IIa (topo IIa) gene expression was determined asdescribed previously (18). Densitometric analysis was done usingPersonal Densitometer SI (Molecular Dynamics, Sunnyvale, CA) andadjusted by glyceraldehyde-3-phosphate dehydrogenase internalcontrol.

Vascular endothelial growth factor protein quantitation. TC71, TC/si,and TC/VEGFsi cells (2.5 � 105) were seeded into 24-well plates. Freshmedium was added after overnight culture. The cultured supernatantswere collected 24 hours later and centrifuged at 13,000 rpm for 10minutes to eliminate cellular fragments. Phenylmethylsulfonyl fluoridewas then added to the cultured supernatants at 2 mmol/L and stored at�20jC. The cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent for 1 hour and lysed in equalamounts of DMSO (Sigma Chemical Co., St. Louis, MO). The relativecell density was assessed by A450 nm. VEGF protein concentration wasquantified using an anti-human VEGF165 ELISA kit (R&D Systems,Minneapolis, MN) according to the manufacturer’s protocol.

Migration assay. Cultured supernatants from TC71, TC/si, and TC/VEGFsi cells were collected. Transwells (Costar, Cambridge, MA) were

www.aacrjournals.org Clin Cancer Res 2005;11(7) April 1, 20052663

Fig. 1. Effect of VEGFsi on VEGF expression inTC71Ewing’s sarcoma or MDA-MB-231breastcancer cells.A ,TC71or MDA-MB-231cells werestably transfected with differentVEGFsi constructs.VEGFmRNAwas quantified by Northern blotanalysis. Densitometric analysis was done and therelative expression for each bandwas normalizedwith glyceraldehyde-3-phosphate dehydrogenase(GAPDH). B, differentTC71cell clones stablytransfectedwithVEGFsi-7 or the control vector wereassessed forVEGF165 andVEGF189 expression byNorthern blotting. C, cultured supernatants fromdifferentTC71clones stably transfected withVEGFsi-7 or the control vector were collected andassayed for VEGF by ELISA.

siRNATargetingVEGFin Ewing’s Sarcoma

pretreated with serum-free medium at 37jC for 1 hour before seeding

with HMVECs at 1 � 105 per well in 100 AL endothelial basalmedium with 0.1% fetal bovine serum. The transwells were then

inserted into 24-well plates containing 600 AL conditioned medium

and incubated at 37jC for 6 hours to allow HMVEC cells to migrate.Cells on the upper side of the filter were removed with cotton swabs.

Migrated cells on the lower side of the filter were fixed and stainedwith H&E. The number of migrated cells was counted under a

binocular microscope.

Proliferation and cytostasis assay. HMVEC cells (3 � 103) wereseeded into 96-well cell culture plates and allowed to adhere for 5 hours

before the addition of conditioned medium from either TC71, TC/si, orTC/VEGFsi cells. The proliferative activity was determined 48 hours

later by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

as described previously (19). Cytostasis was also quantified 48 hoursafter adding different concentrations of trastuzumab.

Western blotting. Cells were pipetted into 100 mm dishes. When

cells reached 80% confluence, cell lysate was collected and proteinconcentrations were determined using the Bio-Rad protein assay kit

(Bio-Rad Laboratories, Hercules, CA). The protein (50 Ag) was boiled

for 5 minutes before being loaded onto a 7.5% SDS-polyacrylamide geland then transferred to a nitrocellulose membrane (Amersham,

Piscataway, NJ). The specific protein bands were detected withmonoclonal anti-human HER-2/neu (Ab-3; Oncogene, San Diego,

CA) and h-actin antibody (Sigma Chemical) using the enhanced

chemiluminescence Western blotting analysis system (Amersham)according to the manufacturer’s instructions. Densitometric analysis

was done, and values were normalized with h-actin loading control.Plasmid/polyethylenimine formulations. Polyethylenimine (25 kDa,

branched form, Aldrich Chemical, Milwaukee, WI) was prepared at a

concentration of 0.1 mol/L in water. A polyethylenimine/plasmidmixture (1.29:1 polyethylenimine/DNA weight ratio) was prepared asdescribed previously (20) by slowly adding the plasmid to thepolyethylenimine solution while vortexing vigorously. The solution

was then allowed to incubate at room temperature for 15 to 20 minutesbefore use.

In vivo studies. Four- to 5-week-old specific pathogen-free athymic(T-cell deficient) nude mice were purchased from Charles RiverBreeding Laboratories (Kingston, MA). TC71, TC/si, and TC/VEGFsiEwing’s sarcoma cells in mid-log-growth phase were harvested bytrypsinization. Single-cell suspensions (2 � 106 cells in 0.1 mL HBSS)were injected s.c. into the nude mice. The tumors were measured every4 days with a caliper, and the diameters were recorded. Tumor volumewas calculated by the formula: a2b/2, where a and b are the two

maximum diameters. When tumors reached 2 � 2 cm, the duration ofsurvival was recorded, the mouse was euthanized, and the tumor tissuewas collected for analysis of CD31, apoptosis, basic fibroblast growthfactor (bFGF), platelet-derived growth factor-h (PDGF-h), transforminggrowth factor-h (TGF-h), and interleukin (IL)-8 using immunohisto-chemical analysis and terminal deoxynucleotidyl transferase�mediateddUTP nick end labeling.

For the VEGFsi gene therapy experiments, 2 � 106 TC71 cells wereinjected s.c. into nude mice. Three days later, when the tumors werepalpable, the mice were divided into three groups. Group 1 micewere used as untreated controls. Group 2 received intratumor injections(20 Ag/mouse) of polyethylenimine/control vector twice weekly forvarious times. Group 3 received intratumor injections with 20 Agpolyethylenimine/VEGFsi twice weekly as described for group 2. Tumorsize was measured and tumor tissue was examined as described above.


Fig. 2. Effect ofVEGF siRNA on HMVEC cell chemotaxis and proliferation.Cultured supernatants fromTC71,TC/si, andTC/VEGFsi were collected and assayedfor their ability to induce HMVEC cell migration and proliferation. A , HMVEC cellmigrationwas quantified after 6 hours as described in Materials andMethods.*, P < 0.01, compared withTC/si supernatants.B, HMVEC cells were incubated withthe indicated supernatant for 48 hours. Cell proliferationwas assessed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. *, P < 0.01,compared withTC/si supernatants.

Fig. 3. Effect of VEGFsi on HER-2/neu and topo IIa expression. Total protein orRNAwas extracted fromTC71,TC/si, or TC/VEGFsi cells.A , HER-2/neu proteinwas quantified byWestern blotting. B, topo IIa expression was quantified byNorthern blotting.


To assess bone tumor formation, 2 � 105 TC71, TC/si, or TC/VEGFsiclone 7-1 cells were injected into the right tibia under anesthesia. Threeweeks later, digitized radiographic images were taken using a MX-20Specimen Radiograph System (Faxitron X-ray Co., Wheeling, IL). Agrading system for bone lysis with numerical values ranging from 0 to 4was used to determine the extent of bone destruction (21). A grade of 0represented no bone lysis; grade 1 was minimal but visible bone lysiswithin the medullary canal; grade 2 was moderate bone lysis in themedullary canal with preservation of the cortex; grade 3 was severebone lysis with cortical disruption; and grade 4 was massivedestruction.

Immunohistochemical analysis. Tumor sections were stained withH&E. Frozen sections were fixed with acetone, incubated in 3% H2O2 inmethanol for 10 minutes to block endogenous peroxidase, and thenincubated in 5% normal horse serum plus 1% normal goat serum in PBSfor 20 minutes to block nonspecific protein. Expression of CD31 wasdetected using a rat anti-mouse CD31 as the primary antibody(PharMingen, San Diego, CA); a goat anti-rat horseradish peroxidasewas the second antibody followed by incubation with chromogendiaminobenzidine. The expression of bFGF, PDGF-h, TGF-h, and IL-8 protein was detected by incubating tissue sections with rabbit anti-human bFGF antibody (Sigma Chemical), rabbit anti-human PDGF-hantibody, rabbit anti-human TGF-h1 antibody, or rabbit anti-human IL-8 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as theprimary antibody and horseradish peroxidase– labeled goat antibodyagainst rabbit IgG as the second antibody (Jackson ImmunoResearchLaboratory, Inc., West Grove, PA). Gill’s hematoxylin was used as acounterstain.

Apoptotic and necrotic cells were quantified using terminaldeoxynucleotidyl transferase�mediated dUTP nick end labeling stain-ing. Formalin-fixed paraffin-embedded sections were dewaxed beforebeing permeabilized with proteinase K for 15 minutes at roomtemperature. After blocking endogenous peroxidase in 3% H2O2, thefragmented DNA was labeled with biotin-16-dUTP with terminaltransferase at 4jC overnight.

Statistical analysis. Two-tailed Student’s t test was used tostatistically evaluate the tumor volumes, migration, and proliferationof HMVECs. P V 0.05 was considered statistically significant.

Results

Effect of vascular endothelial growth factor small interferingRNA on vascular endothelial growth factor expression. FourVEGF siRNA-expressing plasmids (plasmid 2, 4, 6, and 7) wereconstructed using the pSilencer2.1-U6 hygro vector to targetdifferent regions of human VEGF mRNA. TC71 Ewing’s sarcomaand MDA-MB-231 breast cancer cells were stably transfectedwith these plasmids, and the VEGF mRNA levels were measuredusing Northern blotting. As shown in Fig. 1A, VEGF expressionwas significantly inhibited by VEGFsi-6 and VEGFsi-7. In TC71cells, VEGF levels were reduced by 60% and 40%, respectively,compared with nontransfected and control vector-transfectedTC71 cells. In MDA-MB-231 cells, transfection of VEGFsi-7resulted in a 75% reduction in VEGF RNA compared with bothnontransfected and control vector-transfected cells. Transfection


Fig. 4. Effect of VEGF siRNA on Ewing’s sarcoma tumor growth in mice. A ,TC71,TC/si, orTC/VEGFsi clone 7-1cellswere inoculated s.c. intonudemice.Tumor growthwasmonitored and tumor volumewas calculated. *,P < 0.01, comparedwithTC71orTC/si tumors.B, mice were euthanized when the tumor reached 2� 2 cm indiameter. Survival curve was calculated as the percentage of surviving mice on theindicated days.C, mice received a s.c. injection ofTC71cells.Three days later, micewere divided into three groups and given intratumoral injections of polyethylenimine(PEI)/si, polyethylenimine/VEGFsi-7, or no treatment.Tumor size was measuredat various time points. *,P < 0.01, comparedwith the untreated or polyethylenimine/si group.D, survival curve for mice treated as described in C.


of plasmids 2 and 4 resulted in no significant alteration of VEGFRNA expression in either cell line. Single-cell clones of TC71cells transfected with plasmid 7 were then isolated and VEGFmRNA levels were examined by Northern blotting (Fig. 1B).VEGF165 expression in clones 7-1 and 7-17 was inhibited by75% and 80%, respectively, compared with wild-type andcontrol vector-transfected cells. By contrast, expression ofVEGF189 was not altered. VEGF protein production was >98%lower in clones 7-1 and 7-17 than in untransfected and controlvector-transfected cells (Fig. 1C).

Effect of vascular endothelial growth factor small interferingRNA on vascular endothelial growth factor– induced endothelialcell proliferation and migration. TC71 cells secrete significantamounts of VEGF (ref. 12; Fig. 1C). Because VEGF has beenshown to induce the migration and proliferation ofendothelial cells, we hypothesized that VEGF siRNA wouldreduce the chemotactic and mitogenic effect of TC71 culturedsupernatants on endothelial cells. We collected culturedsupernatants from TC71, TC/si, and TC/VEGFsi clones 7-1and 7-17. As shown in Fig. 2A, the culture supernatants fromuntransfected and control vector-transfected cells inducedrobust HMVEC cell migration. By contrast, few HMVEC cellsmigrated when conditioned medium from TC/VEGFsi clone7-1 or 7-17 was used as the chemotactic stimulus. HMVEC

cell proliferation was also significantly lower when cells werecultured for 48 hours in conditioned medium from TC/VEGFsi clone 7-1 and 7-17 cells rather than in conditionedmedium from untransfected or control vector-transfected cells(Fig. 2B).

To determine whether VEGF165 inhibition by siRNA influ-enced TC71 cell growth in vitro, the doubling time of each cellline was quantified. There was no difference in growth ratesbefore and after VEGFsi transfection. Both Flt-1 and Flk-1 weredetected on the TC71 cell membrane by immunohistochemicalanalysis (data not shown).

Effect of vascular endothelial growth factor small interferingRNA on HER-2/neu and topoisomerase IIa expression. We haveshown previously that, in addition to VEGF, TC71 cellsoverexpress HER-2/neu (22). Transfection of E1A resulted inthe down-regulation of both HER-2/neu and VEGF and theup-regulation of topo IIa (22). We therefore determinedwhether VEGF siRNA also affected HER-2/neu or topo IIaexpression. As shown in Fig. 3, there was no significant changein either HER-2/neu protein levels or topo IIa expressionfollowing transfection with VEGFsi. Furthermore, clone 7-1and 7-17 cells were as sensitive to herceptin as control vector-transfected cells (data not shown). These data show thespecificity of our VEGF siRNA.


Fig. 5. Effect of VEGFsi on tumor vessel density, tumor cell apoptosis, necrosis, tumor bFGF, and PDGF-h expression. A , vessel density was assessed usingimmunohistochemistry for CD31. Tumors from (A1) untreated mice, (A2) mice inoculated with TC/si, or (A3) TC/VEGFsi clone 7-1 cells and mice treated with (A4)polyethylenimine/si or (A5) polyethylenimine/VEGFsi-7. B, apoptosis and necrosis was analyzed using terminal deoxynucleotidyl transferase�mediated dUTP nick endlabeling. Tumors from (B1) untreated mice, (B2) mice inoculated with TC/si, or (B3) TC/VEGFsi clone 7-1 cells and mice treated with (B4) polyethylenimine/si or (B5)polyethylenimine/VEGFsi-7. C, immunohistochemical staining for bFGF. Tumors from (C1) untreated mice, (C2) mice inoculated withTC/si, or (C3) TC/VEGFsi clone 7-1cells and mice treated with (C4) polyethylenimine/si or (C5) polyethylenimine/VEGFsi-7. D, immunohistochemical staining for PDGF-h. Tumors from (D1) untreated mice,(D2) mice inoculated with TC/si, or (D3) TC/VEGFsi clone 7-1 cells and mice treated with (D4) polyethylenimine/si or (D5) polyethylenimine/VEGFsi-7.


Effect of vascular endothelial growth factor small interferingRNA on Ewing’s sarcoma tumor growth in vivo. To determinewhether inhibition of VEGF165 by siRNA had an effect ontumor growth, TC71, TC/si, or TC/VEGFsi clone 7-1 cells wereinoculated s.c. into nu/nu mice. TC71 and TC/si cells grewrapidly, resulting in palpable tumors 3 to 4 days followinginjection (Fig. 4A). By contrast, tumor formation was signifi-cantly slower after inoculation of TC/VEGFsi clone 7-1. The TC/VEGFsi clone 7-1 tumors were significantly smaller than thosein both control groups. Survival time was also significantlylonger for mice inoculated with TC/VEGFsi clone 7-1 cells(Fig. 4B). TC/VEGFsi clone 7-1 tumors were pale, with amassively necrotic center and a thin layer of tumor cells inthe periphery. Similar findings were seen with TC/VEGFsi clone7-17 tumors (data not shown). No significant difference ineither tumor growth or macroscopic appearance was detectedbetween the tumors induced by the TC/si control cells and theparental TC71 cells.

To determine the VEGF status in these tumors, RNA wasextracted from tumor tissue and Northern blotting for VEGF wasdone. Both VEGF165 and VEGF189 were detected in TC71 andTC/si tumors, but only VEGF189 was expressed in TC/VEGFsiclone 7-1 tumors (data not shown), indicating that inhibition ofVEGF165 by VEGFsi transfection was stable in vivo .

Effect of VEGFsi gene therapy on Ewing’s sarcoma tumorgrowth. We next investigated whether VEGF siRNA can beused as a gene therapy. We elected to use polyethylenimineas our gene delivery system because of our previousexperience with this nonviral vector (20). TC71 cells wereinjected s.c. into nude mice. Three days later, the palpabletumors were injected with polyethylenimine/VEGFsi-7 orpolyethylenimine/si control. As shown in Fig. 4C, polyethy-lenimine/VEGFsi-7 gene therapy significantly inhibited tumorgrowth in mice compared with the polyethylenimine/sicontrol. Survival time for the polyethylenimine/VEGFsi-treated mice was also significantly longer (Fig. 4D).Polyethylenimine/VEGFsi-7-treated tumors grew slowly, withulceration appearing when the tumor reached f6 mm indiameter.

Immunohistochemical findings. Tumor tissue from micewas excised and subjected to histologic staining. As shownin Fig. 5, CD31-positive vessels were abundant in TC71 andTC/si tumors (A1 and A2). Vessel density was significantlydecreased in tumors formed by TC/VEGFsi clone 7-1,although numerous vessels were seen in the normal tissue

surrounding the tumor (A3). A similar phenomenon wasseen in tumors treated with polyethylenimine/VEGFsi-7 (A5),whereas the vessel density in tumors treated with polyethy-lenimine/si control was similar to that observed in theuntreated TC71 tumors (A4). Terminal deoxynucleotidyltransferase�mediated dUTP nick end labeling assay revealedthe presence of massive apoptotic and necrosis cells in miceinoculated with TC/VEGFsi clone 7-1 and in wild-typetumors treated with polyethylenimine/VEGFsi-7 (B3 andB5). By contrast, TC71, TC/si, and polyethylenimine/si-treated tumors showed only small areas of necrosis andapoptosis (B1 , B2 , and B4). Four other important angiogenicfactors (bFGF, PDGF-h, TGF-h, and IL-8) remained un-changed in the TC/si, TC/VEGFsi clone 7-1, and polyethyle-nimine/VEGFsi-7-treated tumors (Fig. 5C and D; data notshown). These data indicated that the antiangiogenic andantitumor effect was secondary to the inhibition of VEGF165.

Effect of VEGFsi on bone tumor formation. The data aboveshowed that VEGFsi-7 inhibited s.c. growth of TC71 cells. Wenext evaluated whether VEGFsi influenced tumor formation inthe bone. TC71, TC/si, and TC/VEGFsi clone 7-1 cells wereinjected into the tibias of nude mice. After 3 weeks, 70% ofmice injected with TC71 cells and 80% of mice injected withTC/si cells had developed osteolytic bone tumors, as assessedby radiography, compared with only 29% of mice injectedwith TC/VEGFsi clone 7-1 cells (Fig. 6; Table 1; P < 0.01). Thebone damage was quantified using a numerical gradingsystem defined by Weber et al. (21). The average level of


Fig. 6. Effect ofVEGFsi in tumor-induced bonelysis.TC71,TC/si, orTC/VEGFsi clone 7-1cells(2� 105) were injected into the right tibia of nudemice. Radiographic images were taken 3 weekslater.Themost severe bone tumors from each groupare shown.

Table 1. Effect ofVEGFsi on tumor-induced bone lysis

Tumor cellinjected

Bone tumorincidence*

Average lyticlevelc

TC71 7/10 (70%) 1.8TC/si 8/10 (80%) 2.5TC/VEGFsi clone 7-1 2/7 (29/%)b 0.3

NOTE: TC71,TC/si, orTC/VEGFsi clone7-1cells were inoculated into tibias ofnude mice.Three weeks later, radiograph images were obtained.*Mice with bone tumors/total number of mice (%with tumors).cOsteolytic bone lesionswere graded from0 to 4 (with 4 being themost lytic)as defined in Materials andMethods.bP < 0.01, compared with eitherTC71orTC/si.


bone lysis in TC71 and TC/si injected animals was 1.8 and2.5, respectively, compared with 0.3 in animals injected withTC/VEGFsi clone 7-1.

Discussion

We showed previously that compared with normal humanosteoblasts three of four Ewing’s sarcoma cell lines over-express VEGF and that there is a shift in isoform productionfrom the membrane VEGF189 to the more soluble VEGF165

(12, 23). We have also shown that VEGF is abundantlyexpressed in the primary tumors of Ewing’s sarcoma patients(data not shown). In the present study, we show thatVEGF165 plays a critical role in the growth of Ewing’ssarcoma. This was done by selectively inhibiting VEGF165

expression and protein production using RNA-mediatedinterference by siRNA. Delivery of siRNA can be achievedthrough exogenous application of synthetic siRNA or throughendogenous expression using plasmid or vector delivery tothe target cell. Chemically or enzymatically synthesizedsiRNA is costly and has been shown to have a relativelyshort half-life with only transient inhibition of the targetgene (24). To overcome these shortcomings, we constructedseveral vector-based expression systems in which sense andantisense strands of short VEGF sequences were transcribedinto hairpin structures under the control of a U6 promoterand then processed into functional siRNAs by doublestrand–specific RNase called Dicer inside the cells (25). Wedesigned four different VEGFsi plasmids targeting fourdifferent VEGF sequences in different regions of the VEGFmRNA. All were located between exons 1 and 5. VEGFsi-7specifically blocked VEGF165 expression in both Ewing’ssarcoma and breast cancer cells. By contrast, VEGF189

expression was unchanged in the transfected cells. VEGFprotein secretion was similarly inhibited by VEGFsi-7 asquantitated by ELISA. Reduction in VEGF protein productionwas also documented by the finding that HMVEC cellproliferation and migration induced by TC71 conditionedmedium was almost completely abolished following trans-fection with VEGFsi-7 but not affected by the VEGFsi controlplasmid. The exact mechanism of this selective VEGF165

inhibition is unclear but may involve an effect of the siRNAat the post-transcriptional level (16).

One of the drawbacks of siRNA is that other nontargetedgenes with as few as 11 continuous nucleotides similar tothose of the targeted gene can be affected (26). To confirmthe specificity of VEGFsi-7, we examined its effect on theexpression of other structurally or functionally related genes,including bFGF, PDGF-h, TGF-h1, IL-8, HER-2/neu , and topoIIa. None were affected following transfection. HER-2/neu isan upstream regulator of VEGF (27), and we have shownpreviously a link between HER-2/neu and VEGF (12). Down-regulation of HER-2/neu in Ewing’s sarcoma cells byherceptin also resulted in decreased VEGF expression and

protein production.5 E1A transfection led to a decrease inVEGF and HER-2/neu expression and an increase in topo IIa.By contrast, the inhibition of VEGF165 by VEGFsi-7 had noeffect on either HER-2/neu or topo IIa expression, againindicating the specificity of this particular siRNA.

Transfection of VEGFsi-7 into TC71 cells did not alter cellgrowth in vitro . However, when these cells were injected eithers.c. or into the bone of nude mice, tumor growth was slowerthan in parental and TC/si control-transfected cells. TC/VEGFsicells produced small tumors that were avascular in appearancewith decreased vessel density. Because other proteins have beenshown to be involved in tumor angiogenesis, we examined thetumors by immunohistochemical analysis and found nochange in bFGF, PDGF-h, TGF-h1, or IL-8. Together, these dataconfirm once again the specificity of our VEGFsi and indicatethat VEGF165 plays a pivotal role in Ewing’s sarcomaangiogenesis and tumor growth.

Our data also indicate that gene therapy targeting VEGF165

may have therapeutic benefit. In this study, we elected to usepolyethylenimine as the vector delivery system because of ourprior experience and success with polyethylenimine/IL-12 genetherapy (20). Polyethylenimine is a cationic polymer, which isnontoxic when delivered in vivo . This polymer retains itscationic state at physiologic pH levels, prevents endosomalbuffering, and does not elicit a significant immune response.The injection of polyethylenimine/VEGFsi into palpable TC71murine tumors resulted in the inhibition of tumor growth,increased animal survival, decreased tumor vessel density,decreased tumor VEGF expression, and increased tumorapoptosis and necrosis compared with tumor injected withpolyethylenimine/si control vector. As seen with the TC/VEGFsi-transfected tumors, the levels of bFGF, PDGF-h, TGF-h1, and IL-8 were unchanged and similar in the polyethylenimine/VEGFsiand polyethylenimine/si control-treated tumors. Once again,these data indicate that the antiangiogenic and antitumor effectseen was secondary to the inhibition of VEGF165.

In summary, we have shown that siRNA technology can beused to specifically inhibit one VEGF isoform. Both celltransfection and delivery by polyethylenimine resulted inselective inhibition of VEGF165 expression, leading todecreased tumor vascularity and growth in vivo . These dataindicate that VEGF165 plays a central role in Ewing’s sarcomaangiogenesis, because PDGF-h, bFGF TGF-h1, and IL-8 wereall unchanged. Therefore, targeting VEGF with specific small-molecule inhibitors may have therapeutic benefit. The curerate for patients with Ewing’s sarcoma, particularly those whopresent with large tumors or metastatic disease, is poor, witha disease-free survival rate of 40% to 50% at 2 years (3, 4).Survival rates have remained stagnant over the past 20 yearsdespite aggressive dose-intensive chemotherapy combinedwith radiation therapy and surgery. Therefore, it behoovesus to consider novel therapeutic approaches in an effort toimprove the outcomes of these patients.


5 Unpublished data.

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A Small Interfering RNATargetingVascular Endothelial ...wing’s sarcoma is a primitive neuroectodermal tumor that most often affects children and young adults in the first two decades