Anti-epidermal Growth Factor Receptor Antibody C225...

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Anti-epidermal Growth Factor Receptor Antibody C225 InhibitsAngiogenesis in Human Transitional Cell CarcinomaGrowing Orthotopically in Nude Mice 1

Paul Perrotte, Takashi Matsumoto, Keiji Inoue,Hiroki Kuniyasu, Beryl Y. Eve, Daniel J. Hicklin,Robert Radinsky, and Colin P. N. Dinney2

Departments of Urology [P. P., C. P. N. D.] and Cancer Biology[T. M., K. I., H. K., B. Y. E., R. R.], The University of Texas M. D.Anderson Cancer Center, Houston, Texas 77030, and ImCloneSystems, New York, New York [D. J. H.]

AbstractEpidermal growth factor receptor (EGFR) regulates

the growth and progression of human transitional cell car-cinoma (TCC) of the bladder. We have shown that therapytargeting EGFR inhibited the growth of human TCC estab-lished orthotopically in nude mice. The purpose of this studywas to evaluate whether EGFR-directed therapy affects an-giogenesis associated with the growth and metastasis ofhuman TCC. We determined the cytostatic effect and theeffect on production of angiogenic factors after in vitrotreatment of the human TCC cell line 253J B-V with MAbC225, a chimerized monoclonal anti-EGFR antibody. The253J B-V cells were implanted orthotopically into athymicnude mice, and established tumors (4 weeks) were treatedwith i.p. MAb C225. Expression of the angiogenic factorsvascular endothelial growth factor (VEGF), interleukin-8(IL-8), and basic fibroblast growth factor (bFGF) was eval-uated by immunohistochemistry andin situ mRNA hybrid-ization analyses and correlated with microvessel densityevaluated after immunohistochemical staining with anti-CD31. In vitro treatment with MAb C225 inhibited mRNAand protein production of VEGF, IL-8, and bFGF by 253JB-V cells in a dose-dependent manner. MAb C225 therapyof nude mice with established TCCs growing orthotopicallyresulted in inhibition of growth and metastasis comparedwith controls (P <0.0005). VEGF, IL-8, and bFGF expres-sion was significantly lower in treated tumors than in con-trols. The down-regulation of these angiogenic factors pre-ceded the involution of blood vessels. These studies indicate

that therapy with anti-EGFR MAb C225 has a significantantitumor effect mediated, in part, by inhibition of angio-genesis.

IntroductionAngiogenesis is crucial for the growth and metastasis of

human TCC3 (1–4). Human TCC overexpresses the angiogenicfactors bFGF, VEGF, and platelet-derived endothelial growthfactor, which are important in modulating tumor-host interac-tions that result in tumor neovascularization. Urine from patientswith bladder cancer stimulates endothelial cell proliferation andmigration (5, 6). Furthermore, the prognosis of patients withadvanced TCC correlates with microvessel density (7). EGFR-mediated signaling mechanisms are also fundamental to thetumorigenicity of human TCC and compose an important path-way regulating cell division. The level of expression of EGFRcorrelates with stage, grade, and progression of human TCC(7–16).

EGFR signaling and angiogenesis have been independentlyevaluated as targets for therapy, but the link between has onlyrecently been identified (17–24). Both EGF and TGF-a, whichare ligands for EGFR, induce angiogenesis. Therefore, we hy-pothesized that down-regulating EGFR signaling pathways mayinhibit tumor growth by inhibiting tumor-mediated angiogene-sis, independent of any direct cytostatic effect on tumor growth.We recently reported that therapy with either protein tyrosinekinase inhibitors (20) or anti-EGFR MAb C2254 inhibited thegrowth of established human TCC growing orthotopically inathymic nude mice. We now report that therapy with MAb C225reduces TCC neovascularization by down-regulating the tumorcell expression of the angiogenic factors VEGF, IL-8, andbFGF, resulting in abrogation of tumor growth and metastasis.

Materials and MethodsTumor Cell Line. The highly metastatic human TCC

cell line 253J B-V was maintained as a monolayer in modifiedEMEM supplemented with 10% FBS, vitamins, sodium pyru-vate, L-glutamine, and nonessential amino acids, as describedpreviously (25).

Received 8/6/98; accepted 11/9/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported in part by Cancer Center Core Grant CA 16672, NIH GrantsCA 67952 (P.P.) and CA 67914 (C.P.N.D.), and the Gustavus andLouise Pfeiffer Research Foundation.2 To whom requests for reprints should be addressed, at The Universityof Texas M. D. Anderson Cancer Center, Department of Urology–Box110, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713)792-3250; Fax: (713) 794-4824; E-mail: [email protected].

3 The abbreviations used are: TCC, transitional cell carcinoma; VEGF,vascular endothelial growth factor; bFGF, basic fibroblast growth fac-tor; IL-8, interleukin-8; ISH,in situ hybridization; MAb, monoclonalantibody; EGF, epidermal growth factor; EGFR, EGF receptor; EMEM,Eagle’s MEM; FBS, fetal bovine serum; TGF, transforming growthfactor.4 T. Matsumoto, P. Perrotte, D. J. Hicklin, C. P. M. Dinney, and R.Radinsky. Inhibition of human transitional cell carcinoma growing inthe bladder of nude mice by anti-EGFR andibody C225: a biomarkeranalysis. Submitted for publication.

257Vol. 5, 257–264, February 1999 Clinical Cancer Research

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Animals. Male athymic BALB/c nude mice were ob-tained from the Animal Production Area of the National CancerInstitute, Frederick Cancer Research Facility (Frederick, MD).The mice were maintained in a laminar air-flow cabinet underpathogen-free conditions and used at 8–12 weeks of age. Allfacilities were approved by the American Association for Ac-creditation of Laboratory Animal Care in accordance withUnited States Department of Agriculture, Department of Healthand Human Services, and NIH standards.

Antibodies. Chimeric anti-EGFR MAb C225 was gener-ously provided by ImClone Systems, Inc. (New York, NY).Human IgG (Sigma Biosciences, St. Louis, MO) was used as acontrol.

In Vitro Therapy of Tumor Cells. The in vitro dose-dependent antiproliferative effect of MAb C225 was evaluatedby incubating 253J B-V cells for 24 h in serum-free medium,then exchanging the medium for 0%, 1%, or 10% FBS-supple-mented EMEM containing increasing concentrations of MAbC225 (0.08–10mg/ml). Growth inhibition was determined by[3H]thymidine incorporation after a 72-h incubation period. Toevaluate expression of VEGF, IL-8, and bFGF after therapywith MAb C225, 53 103 cells were plated in a 96-well plate in200ml of EMEM supplemented with 10% FBS, then treated thenext day with 1mg/ml or 10mg/ml of MAb C225. Ten percentFBS-supplemented EMEM without MAb C225 and EGFR-irrelevant human IgG (10mg/ml) served as controls. Aftertreatment, cells were counted, and both the supernatant and thecells were collected and stored at -20°C for protein quantifica-tion. To evaluate the influence of EGFR activation on theexpression of the angiogenic factors, exogenous EGF (50 ng/ml)was added to stimulate EGFR signaling pathways. Cell-associ-ated bFGF and supernatant VEGF and IL-8 protein levels weremeasured using the commercially available Quantine ELISA kit(R&D Systems, Inc., Minneapolis, MN). Steady-state mRNAexpression was determined by Northern blot analysis.

Northern Blot Analysis. Polyadenylated mRNA was ex-tracted from 108 cells growing in culture with the FastTrackmRNA isolation kit (Invitrogen Co., San Diego, CA). ThemRNA was electrophoresed on 1% denaturing formaldehyde/agarose gel and electrotransferred to Genescreen nylon mem-

brane (DuPont Co., Boston, MA), using a UV Stratalinker 1800cross-linked with 120,000mJ/cm2. Filters were washed at 55°Cwith 30 mM sodium citrate and 0.1% SDS (w/v). The mem-branes were then hybridized and probed for VEGF, bFGF, IL-8,andb-actin as a control. The cDNA probes used were: (a) a Jirdactin 1.2-kb DNA fragment (26); (b) a 1.4-kb cDNA fragmentof bovine bFGF (27); (c) a 204-bpBamHI EcoRI fragment ofhuman VEGF cDNA (28); and (d) a 0.5-kb EcoRI cDNAfragment corresponding to human IL-8 (a gift from Dr. K.Matsushima, Kanazawa, Japan; Ref. 29). The probes were ra-diolabeled by a random primer technique and [a-32P]dCTP(Amersham Corp., Arlington Heights, IL). Autoradiography ofthe membrane was performed after washing. Densitometry scan-ning permitted quantitation of the bands.

Orthotopic Implantation of Tumor Cells. For the invivo portion of the study, cultured 253J B-V cells (70% conflu-ent) were prepared for injection, as previously described (25).Mice were anesthetized with methoxyflurane, a lower midlineincision was made, and the bladder was exposed. Viable tumorcells (13 106/0.05 ml of HBSS) were injected into the wall ofthe bladder. The formation of a bulla was the sign of a satis-factory injection. The bladder was returned into the abdominalcavity, and the abdominal wall was closed in a single layer withmetal clips.

Therapy of Established Human TCC Tumors Growingin the Bladders of Athymic Nude Mice. Treatment com-menced 28 days after tumor implantation. The presence andvolume of tumors were confirmed using ultrasound or palpation.At the time of therapy, tumor weights were between 200 and400 mg. Mice were randomly separated into two groups. Thefirst group (eight mice) was treated with 1 mg of anti-EGFRchimeric antibody MAb C225 i.p. twice a week for 5 weeks.Eight control mice received an equivalent volume of PBS on thesame schedule. Treated mice were closely monitored for anysigns of progressive disease and sacrificed if they became mor-ibund. Control mice were sacrificed at or about 5 weeks aftertumor implantation because they became moribund. To evaluatethe temporal effects of MAb C225 on angiogenic factor expres-sion and microvessel density, mice were sacrificed 1, 3, and 5weeks after initiation of therapy.

Necropsy. Necropsy was performed, and local tumor-igenicity and distant metastases (lymph node and lung nod-ules) were determined. The bladders were harvested,weighed, and either mechanically dissociated forin vitroculture, flash-frozen in liquid nitrogen for mRNA extraction,or cut in two and either embedded in Optimal Cutting Tem-perature gel (Sokera Inc., Torrence, CA) for frozen sectionsor fixed in 10% buffer and formalin for paraffin sections. Thelymph nodes were fixed in formalin. Lungs were mechani-cally dissociated for reestablishment in culture to evaluate thepresence of micrometastases or fixed in Bouin’s fluid forhistological analysis.

Immunohistochemical Determination of bFGF, VEGF,and IL-8. The expression of angiogenic factors was detectedin cryostat (IL-8) or paraffin sections (bFGF, VEGF) of tumorstreated with either MAb C225 or PBS (30). bFGF proteinexpression was detected using primary rabbit anti-bFGF (SigmaChemical Co., St. Louis, MO), which reacts with residues 147–153 and shows no cross-reaction with acidic fibroblast growth

Fig. 1 In vitro growth inhibition of 253J B-V cells treated with MAbC225 evaluated by [3H]thymidine incorporation assay. There is a dose-dependent inhibition of growth with maximum cytostasis of 55% at 10mg/ml. Lines represent varying concentrations of FBS.

258 Inhibition of Angiogenesis in TCC by EGFR Blockade



factor. VEGF and IL-8 proteins were detected using primaryrabbit antibodies (Santa Cruz Biotechnology, Santa Cruz, CA)at 1:1200 dilution. Thea-immunoperoxidase technique for im-munohistochemical staining was used with a second peroxidase-conjugated goat antirabbit antibody (IgG, F[ab]2 fragment; Jack-son ImmunoResearch Laboratory, Inc., West Grove, PA; Ref.30).

Quantification of Microvessel Density. Cryostat sec-tions of bladder tumor were fixed with acetone and chloroformsolutions for 15 min and then washed twice with PBS. Endog-enous peroxidase was blocked with 3% hydrogen peroxide inmethanol, and the sections were washed with PBS and incubatedovernight in a protein-blocking solution. The excess blockingsolution was removed, and the samples were incubated with therat antimouse CD31 antibody that stains endothelial cells

(PharMingen, San Diego, CA). Swine peroxidase conjugatedantirabbit antibody was applied for 30 min after the primaryantibody was removed. The samples were rinsed with PBS anddeveloped with 3-amino-9-ethylcarbazole at room temperaturefor 20 min. The sections were counterstained with aqueoushematoxylin. A positive reaction was indicated by a brownishprecipitate (30).

Two investigators counted the microvessels independentlyin a blinded fashion. The tissues were examined at low power(340), and the three3200 fields of highest microvessel densitywere identified for vessel counts. The three selected fields(high-power field,320 objective and310 ocular, 0.739 mm2/field) were recorded using a computer-linked cooled CCD Op-totronics Tec 470 camera (Optotronics Engineering, Goletha,CA) to ensure that both investigators would count the same

Fig. 2 A, in vitro inhibitionof VEGF, IL-8, and bFGFprotein production by C225MAb. The 253J B-V cellswere cultured in the presenceor absence of C225.B, inhibi-tion of VEGF, IL-8, and bFGFprotein production by MAbC225 after EGF stimulation(50 ng/ml; P , 0.05). IL-8and cell-associated bFGFwere measured by ELISA.The protein production ofthese angiogenic factors is ex-pressed in relation to the pro-duction of untreated cells,which was assumed to be100%. Values are the mean6SD of triplicate cultures andhave been normalized to ac-count for cell number.

259Clinical Cancer Research



areas within the specimens. Microvessels were quantitated ac-cording to the method described by Weidneret al.(31). Clustersof stained endothelial cells distinct from adjacent microvessels,tumor cells, or other stromal cells were counted as one mi-crovessel. The results were expressed as the highest number ofmicrovessels identified within a single3200 field.

In Situ mRNA Hybridization. Paraffin sections of fixedtissue (3–5mm) were mounted on ProbOn slides (Fisher Scien-tific, Pittsburgh, PA). The slides were dewaxed and prepared,and ISH was performed using the Microprobe system (FisherScientific) as described previously (32, 33). Slides were rinsedthree times in Tris buffer for 30 s; hybridization of the probeswas then performed at 45°C for 45 min. The slides were washedwith 2 3 SSC three times for 2 min each time at 45°C. Thesamples were then incubated with avidin labeled with phospha-tase for 30 min at 45°C, rinsed in 50 mM Tris buffer (pH 7.6),

and then briefly (1 min) rinsed in alkaline phosphatase. Thesamples were then incubated with chromogen substrate for 20min at 45°C. If necessary, additional incubation was performedwith fresh chromogen to enhance a weak reaction. The sampleswere then covered with Universal Mount fixative (ResearchGenetics, Huntsville, AL), heat-dried, and examined. A positivereaction in this assay stained red. To control for endogenousalkaline phosphatase, additional samples were treated in theabsence of biotinylated probe. No immunoreactivity was ob-served in the controls.

Densitometry Quantification of Immunohistochemicaland In Situ mRNA Hybridization. The intensity of immu-nohistochemical staining andin situ mRNA hybridization wasevaluated in five fields representing areas of highest stainingintensity. Each field was evaluated using the ImageQuant ana-lyzer and Optimas software program (Bioscan, Edmonds, WA).Immunohistochemical staining intensity of each specimen wascompared with the staining intensity of the normal urothelium inthe same sample and expressed as a ratio (tumor cells:normalurothelium). In situ mRNA hybridization quantification wasperformed in the same manner on corresponding sections. Nor-mal urothelium served as the internal control for mRNA expres-sion, and poly-dT staining controlled for mRNA preservation.Results were expressed as the ratio of the intensity of tumor ISHstaining to normal urothelium staining and normalized forpoly-dT expression.

Statistical Analysis. Bladder tumor weights were com-pared by the Mann-Whitney test. Expression of VEGF, IL-8,and bFGF and microvessel density quantification were com-pared by Student’st test.

ResultsIn Vitro Cell Growth Inhibition by MAb C225. In vitro

treatment of 253J B-V cells with MAb C225 resulted in dose-

Fig. 3 In vitro inhibition ofVEGF (A), IL-8 (A), and bFGF(B) steady-state mRNA expres-sion by C225 MAb. Cells werecultured in the presence of orabsence of MAb C225. mRNAwas extracted after 48 h oftreatment and analyzed byNorthern blotting. Using imagedensitometry, the mRNA ex-pression of untreated cells wasassumed to be 100% of poten-tial expression for these cells.Treatment with C225 down-regulated VEGF, IL-8, andbFGF mRNA expression in adose-dependent fashion.b-actin served as a control forloading (exposure, 30 min).

Table 1 Systemic MAb C225 therapy of 253J B-V bladder tumorsin nude mice

1 3 106 253J B-V cells were implanted into the bladder walls ofnude mice. Therapy began on day 28 when palpable tumors were felt.Tumors were 200–400 mg at initiation of therapy. MAb C225 (1 mg)was injected i.p. twice a week for 5 weeks. Tumor weight and metas-tases were evaluated at necropsy. One representative experiment ofthree.

Treatment

No. micewith tumors/total no. mice

Median tumorweight, mg

(range)

No. mice withspontaneous

metastasis/totalno. mice

Lung Lymph node

Control (PBS) 8/8 834 (193–1127)a 3/8b 8/8C225 8/8 67 (31–109) 0/8 0/8

a P 5 0.0005.b P , 0.005.




dependent cytostasis, as measured by [3H]thymidine incorpora-tion assay. The maximum inhibition observed was 55% after72 h of continuous exposure at the highest dose (10mg/ml) inserum-free medium (Fig. 1). Treatment with MAb C225 in 10%serum-containing medium resulted in minimum cytostasis(,20%).

Down-regulation of bFGF, VEGF, and IL-8 in HumanTCC Cells after in Vitro Therapy with MAb C225. Usingan ELISA, we observed dose-dependent down-regulation ofVEGF, IL-8, and bFGF after continuous exposure to MAb C225

for 48 h. Secreted VEGF and IL-8 protein production andcell-associated bFGF protein production by the 253J B-V cellline was significantly inhibited by MAb C225 at 10mg/ml (Fig.2A). Northern blot analysis directly correlated with the ELISAresults (Fig. 3). A decrease in the steady-state gene expressionof VEGF, IL-8, and bFGF was observed. After treatment, VEGFsteady-state mRNA expression was reduced 2-fold, IL-8 expres-sion was reduced 10-fold, and bFGF expression was reduced2-fold when cells were treated with the high dose of MAb C225at 10mg/ml.

Fig. 4 ISH of VEGF, IL-8, andbFGF steady-state mRNA of hu-man TCC tumors growing in thebladder walls of athymic nudemice. Bladder tumors harvestedfrom control mice (A, C, E, andG)and MAb C225-treated mice (B, D,F, andH) were processed for ISH.Note the increased staining inten-sity for the three angiogenic factorsin the control tumors comparedwith that of the treated ones.Poly-dT served as an inner controlfor mRNA integrity. Note that allsamples had an intense colorimetricreaction, indicating that mRNA waswell preserved.




When the cells were treated with EGF (50 ng/ml), in-creased overexpression of all three angiogenic factors was ob-served by ELISA (Fig. 2B). Treatment of 253J B-V cells withMAb C225 after pretreatment with EGF resulted in inhibition ofthe production of VEGF, IL-8, and bFGF (Fig. 2B). These datasupport that EGFR blockade by MAb C225 in TCC cells resultsin decreased expression and protein production of VEGF, IL-8,and bFGF.

Inhibition of Growth and Metastasis of EstablishedHuman TCC Tumors in the Bladders of Mice. Therapy ofestablished 253J B-V tumors commenced 28 days after tumorcells implantation into the bladder wall of athymic nude mice.Therapy of mice with 1 mg of MAb C225 i.p. twice a week for5 weeks resulted in dramatic tumor regression compared withcontrol with significant reductions in tumor weight and inhibi-tion of metastasis (Table 1).4 All control mice harbored lymphnode metastases, and approximately 40% demonstrated lungmetastases at the time of death. In contrast, none of the MAbC225-treated mice harbored lymph node or lung metastasis atnecropsy (P, 0.005). Median tumor weight was reduced from834 mg in control animals to 67 mg after therapy with MAbC225. This difference in tumor weight represents tumor regres-sion and inhibition of tumor growth because the treated tumorswere smaller after completion of therapy than when therapy wasinitiated. In contrast, in the control group, all mice showed signsof progressive disease and became moribund and were sacri-ficed within 2–4 weeks of initiating therapy.

Inhibition of bFGF, VEGF, and IL-8 Production andMicrovessel Density in Vivo after EGFR Blockade. Be-cause treatment with MAb C225 down-regulated bFGF, VEGF,and IL-8 production by 253J B-V cellsin vitro, we analyzed thepotential for this agent to inhibit angiogenesisin vivo. Theexpression of VEGF, IL-8, and bFGF and the microvesseldensity of 253J B-V tumors growing in the bladders of nudemice was analyzed in both MAb C225-treated and controlanimals. Protein and mRNA expression of VEGF, IL-8, andbFGF was evaluated by immunohistochemistry andin situmRNA hybridization, respectively, after 1, 3, and 5 weeks ofEGFR blockade therapy and compared with expression by con-trol tumors. Representative tumor sections analyzed for expres-sion of mRNA of VEGF, IL-8, and bFGF usingin situ mRNAhybridization are shown in Fig. 5. After completion of therapyof tumors, the intensity of the expression of VEGF, IL-8, andbFGF message as evaluated byin situ mRNA hybridization inthe treated tumors was lower than in control tumors (Fig. 4).Computer image analysis of representative sections confirmed asignificant reduction in the steady-state mRNA expression forbFGF and IL-8 by week 1 and VEGF by week 3 after initiationof therapy (Fig. 4, Table 2). Using immunohistochemical stain-ing, we observed that the immunoreactivity of VEGF, IL-8, andbFGF protein in 253J B-V tumors growing in the bladders ofathymic nude mice followed the same pattern of down-regula-tion after therapy with MAb 225 (Fig. 5, Table 3).

Microvessel density was significantly lower in tumorstreated for 5 weeks with MAb C225 than in control tumors(84 6 4 versus185 6 30, P , 0.005). This reduction inmicrovessel density was not observed 1 week (2006 16) or 3weeks (1936 15) after initiation of therapy. The down-regula-tion of VEGF, IL-8, and bFGF mRNA and protein between 1

and 5 weeks of therapy preceded the reduction in microvesseldensity (Tables 2, 3). This temporal sequence suggests that theinhibition in the expression of VEGF, IL-8, and bFGF is re-sponsible for the reduction in neovascularity observed in thetreated tumors. No significant difference in microvessel densityor VEGF, IL-8, and bFGF expression was observed over timewithin the control tumors.

DiscussionIn the studies described herein, we have identified a new

mechanism that contributes to the antitumor effect of EGFRblockade therapy with MAb C225: inhibition of angiogenesis.Our data suggest that the reduction in bladder cancer vascular-ization is secondary to down-regulation of VEGF, IL-8, andbFGF expression by EGFR blockade therapy with MAb C225.Our data indicate that the observed antiangiogenic effect is notan artifact of the antiproliferation secondary to blockade ofEGFR signaling pathways: (a) immunohistochemical stainingand in situ mRNA hybridization studies demonstrate that aftertherapy viable bladder tumors were less vascular than control-treated tumors; and (b) the down-regulation of angiogenic fac-tors precedes the involution of microvessels. We hypothesizethat selective down-regulation of VEGF, IL-8, and bFGF by thetumor cells after MAb C225 therapy leads to involution oftumor vessels, contributing to the growth inhibition and regres-sion of the primary tumors and, hence, reduction in spontaneousmetastases from these highly metastatic tumors. This premise isstrengthened by the observation of Benjamin and Keshet (34),who reported that down-regulation of VEGF in hemangioblas-toma directly resulted in eventual endothelial cell apoptosis andblood vessel regression.

Previously, we reported that the EGFR tyrosine kinaseinhibitor CGP 54211 inhibited the growth of 253J B-V tumorsestablished in the bladders of athymic nude mice (20). Althoughthe mechanism of this drug was presumed to be cytostatic, weobserved fibrosis in the treated tumor consistent with somedegree of cytotoxicity. Similarly, our results suggest that theinvivo effect of C225 is a result of cytotoxic activity as well asantiproliferative activity becausein vitro maximum cytostasiswas only 55% andin vivo there was actual regression of thetumors. The regression of life-threatening hemangiomas of in-fancy after antiangiogenic therapy with IFN-ais a clinical

Table 2 In situmRNA hybridization analysis of VEGF, IL-8, andbFGF after therapy with MAb C225

VEGFa IL-8a bFGFa

Control 1286 28 1986 24 2086 29MAb C225

Week 1 1096 8 1176 11b 1646 6c

Week 3 846 26c 936 28b

1216 43c

Week 5 646 17c 716 21b 976 45c

a VEGF, IL-8, and bFGF cytoplasmic staining was evaluated bycomputer-assisted image analysis and is expressed as a ratio of tumorexpression to normal urothelium expression, normalized by poly-dTexpression.

b P , 0.005.c P 5 0.05.




example of the cytotoxic effect of angiogenesis-directed therapy(35).

The mechanisms by which EGFR signaling pathways reg-ulate VEGF, IL-8, and bFGF are unclear, but it is established

that up-regulation of these factors follows activation of theEGFR signaling pathways by EGF or TGF-a. Transcription ofVEGF is potentiated by activation of the four AP-1 binding siteswithin its promoter; the bFGF and IL-8 promoter have one AP-1

Fig. 5 Immunohistochemical staining of human TCC tumors growing in the bladders of athymic nude mice. MAb C225-treated tumors (after 3weeks and 5 weeks of therapy) and control tumors were stained with CD31 antibodies directed against mouse endothelial cells (A-C). Note theincreased vessel density in the control tumors (A) and the 3-week C225-treated tumors (B) compared with that of the 5-week C225-treated specimen(C). Immunostaining for VEGF (D-F), IL-8 (G-I), and bFGF (J-L) is higher in the control tumors (D,G, andJ) than in the MAb C225-treatedspecimens (E,F, H, I, K, and L) at 3 weeks and 5 weeks. Note that the down-regulation of VEGF, IL-8, and bFGF precedes the inhibition ofangiogenesis.




site each (36–39). After activation of EGFR signaling pathways,ras and raf are activated, resulting in phosphorylation of c-fosand c-jun, leading to increased AP-1 activity (36, 40–43). Thisincrease in AP-1 activity leads to transcription of genes withAP-1 sites in their promoter (41, 42, 44). Because VEGF, IL-8,and bFGF all share AP-1 binding sites, they are potential targetsfor therapies that down-regulate EGFR signaling pathways,which results in reduced AP-1 activity. Present studies analyz-ing the promoters of these genes will directly clarify this issue.5

The results of our study support the involvement of EGFRsignaling pathways in the regulation of angiogenesis. Inductionof EGFR signaling pathways with EGF resulted in overexpres-sion of VEGF, IL-8, and bFGF. Furthermore, EGFR blockadewith C225 down-regulated the expression of these factors afterEGF induction. Both TGF-aand EGF are recognized as potentangiogenic factors. It has been established that EGFR regulatesthe in vitro expression of VEGF, bFGF, and specified metallo-proteinases. Grugelet al. (36) showed thatras and raf, down-stream effectors in the EGFR pathway, stimulate the expressionof VEGF and metalloproteinase by NIH 3T3 cells. EGFR acti-vation has also been shown to stimulate VEGF expression byhuman glioblastoma cells (45) and to regulate tumor invasion byDU-145 prostate cancer cells (46). Petitet al. (17) reported thatin vitro treatment of the human epidermoid carcinoma cell lineA431 with MAb C225 down-regulated VEGF and that afterinvivo therapy tumors showed reduction in microvessel densitycounts. Similar observations were reported by Ciardelloet al.(47) using a human colon cancer model.

Collectively, our studies confirm that systemic administra-tion of the chimeric anti-EGFR MAb C225 inhibits growth andmetastasis of human TCCs established in the bladder wall ofathymic nude mice. We have shown that therapy with MAbC225 has a significant antitumor effect mediated, in part, byinhibition of angiogenesis. The down-regulation of these angio-genic factors produced by the tumor restores the balance be-

tween stimulating and inhibitory factors that keeps angiogenesisdormant under normal conditions. The observation that down-regulation of the angiogenic stimulus of the tumor cells inhibitsthe host angiogenic response emphasizes the complexity oftumor-host interactions. These experiments demonstrate thatinhibition of angiogenesis characterizes in part the antitumoreffect of therapy directed at inhibiting EGFR signaling path-ways in human TCC cells. Furthermore, these experimentsindicate that normalization of angiogenesis-related biomarkerssuch as VEGF, IL-8, bFGF, or microvessel density are clinicallyuseful to demonstrate regression or eradication of cancer afterEGFR-directed therapy. Analysis of these biomarkers should beincluded in clinical trials for evaluating this form of therapy.Finally, combination of this approach with standard chemother-apy may provide increased benefit in patients with advancedTCC.

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Table 3 Immunochemical analysis of microvessel density andVEGF, IL-8, and bFGF protein production after therapy with

MAb C225

Microvesselcount VEGFa IL-8a bFGFa

Controlb 1856 30 2596 20 1556 29 906 10MAb C225

Week 1 2006 16 2356 62 836 5 766 6c

Week 3 1936 15 1746 30 216 3d 336 14c

Week 5 846 4d 566 15d 346 1d 146 9c

a VEGF, IL-8, and bFGF cytoplasmic staining was evaluated bycomputer-assisted image analysis and is expressed as a ratio of tumorexpression to normal urothelium expression.

b Control mice were sacrificed when they became moribund about4–6 weeks after tumor implantation. No significant difference in mi-crovessel density or VEGF, IL-8, and bFGF expression was observed incontrol mice sacrificed between 4 and 6 weeks.

c P , 0.05.d P , 0.005.




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1999;5:257-264. Clin Cancer Res Paul Perrotte, Takashi Matsumoto, Keiji Inoue, et al. Orthotopically in Nude MiceAngiogenesis in Human Transitional Cell Carcinoma Growing Anti-epidermal Growth Factor Receptor Antibody C225 Inhibits

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