[CANCER RESEARCH 61, 5090–5101, July 1, 2001]

Acquired Resistance to the Antitumor Effect of Epidermal Growth FactorReceptor-blocking Antibodies in Vivo: A Role for AlteredTumor Angiogenesis1

Alicia Viloria-Petit, Tania Crombet, Serge Jothy, Daniel Hicklin, Peter Bohlen, Jean Marc Schlaeppi, Janusz Rak,and Robert S. Kerbel2

Molecular and Cellular Biology Research [A. V-P., R. S. K.] and Department of Anatomic Pathology [S. J.], Sunnybrook and Women’s College Health Sciences Centre, Toronto,Ontario M4N 3M5, Canada; Clinical Immunology Division, Center of Molecular Immunology, Havana, Cuba [T. C.]; ImClone Systems, Inc., New York, New York [D. H., P. B.];Core Technology Department, Novartis Pharmaceuticals, Novartis Limited, Basel, Switzerland [J. M. S.]; and Hamilton Civic Hospitals Research Centre, Ontario, Canada [J. R.]

ABSTRACT

Inhibitors of epidermal growth factor receptor (EGFR) signaling areamong the novel drugs showing great promise for cancer treatment in theclinic. However, the possibility of acquired resistance to such drugs be-cause of tumor cell genetic instabilities has not yet been explored. Here wereport the experimental derivation and properties of such cell variantsobtained from recurrent tumor xenografts of the human A431 squamouscell carcinoma, after two consecutive cycles of therapy with one of threedifferent anti-EGFR monoclonal antibodies: mR3, hR3, or C225. Initialresponse to a 2-week period of treatment was generally total tumorregression and was not significantly different among the three antibodygroups. However, tumors often reappeared at the site of inoculation,generally after prolonged latency periods, and most of the tumors becamerefractory to a second round of therapy. Cell lines established from suchresistant tumors retained high EGFR expression, normal sensitivity toanti-EGFR antibody or ligand, and unaltered growth rate when comparedwith the parental line in vitro. In contrast, the A431 cell variants exhibitedan accelerated growth rate and a significantly attenuated response toanti-EGFR antibodies in vivo relative to the parental line. Because of thereported suppressive effect of EGFR inhibitors on vascular endothelialgrowth factor (VEGF) expression, and the demonstrated role of VEGF inthe angiogenesis and growth of A431 tumor xenografts, relative VEGFexpression was examined. Five of six resistant variants expressed in-creased levels of VEGF, which paralleled an increase in both angiogenicpotential in vitro and tumor angiogenesisin vivo. In addition, elevatedexpression of VEGF in variants of A431 cells obtained by gene transfec-tion rendered the cells significantly resistant to anti-EGFR antibodiesinvivo. Taken together, the results suggest that, at least in the A431 system,variants displaying acquired resistance to anti-EGFR antibodies canemergein vivo and can do so, at least in part, by mechanisms involving theselection of tumor cell subpopulations with increased angiogenic potential.

INTRODUCTION

Among the most successful of the new molecular-targeted drugs forthe treatment of cancer are the signal transduction inhibitors (1).These drugs, in general, target specific molecular alterations that arethought vital for such functions as cancer cell proliferation, survival,and ability to induce angiogenesis. Examples include humanizedmAbs3 such as Herceptin, directed against the overexpressed EGFR

family member erbB2/Her2/neu (2, 3); chimeric mAbs such as C225;or small molecule antagonists such as ZD1839 (Iressa) directed to theEGF (erbB1) receptor (2, 4), ST1571, which is highly specific for thebcr-abl kinase (5), and to Ras farnesyltransferase inhibitors (6). Someof these drugs are already clinically approved,e.g., Herceptin andST1571, or have shown impressive effects in clinical trials, either ontheir own or in combination with standard treatments such as radiationor chemotherapy,e.g., C225 in several types of solid tumors (7).Indeed, a recurring theme of signal transduction inhibitors is theability of such drugs to function as effective radiation or chemosen-sitizers, thereby rendering tumor cells more vulnerable to the cyto-toxic effects of standard therapies (8–12). This may occur by variousmechanisms such as lowering the threshold for apoptosis or impairingDNA repair processes (12, 13).

A surprising feature of the preclinical research undertaken onanticancer signal transduction therapy is the almost total lack ofinformation dealing with the issue of acquired resistance to suchdrugs. This stands in conspicuous contrast to conventional therapeu-tics such as all classes of cytotoxic chemotherapeutic drugs. Becausegenetic instabilities of cancer cells are a major driving force ofacquired drug resistance (14, 15), it would be expected that acquiredresistance would eventually develop to signal transduction inhibitorsthat target exclusively a cancer cell-associated genetic or phenotypicalteration. An example of this possibility is the emergence of variantresistant cells to a Ras farnesyltransferase inhibitor after prolongedexposure of drug-sensitive tumor cellsin vitro (16). Another is thedevelopment of resistance to ST1571in vivo in bcr-abl-positivechronic myelogenous leukemia cells grown in nude mice, as a resultof the binding ofa1 acid glycoprotein to the drug (17). Similar studiesundertaken in anin vivo context with other signal transduction inhib-itors, including those targeting EGFR, are lacking. With this in mind,we embarked on a long-term study to isolate and to begin character-izing tumor cell variants resistant to EGFR-targeting drugs such asmonoclonal neutralizing Abs. Our approach was based on the obser-vation that a small number of human tumor cell lines,e.g.,the A431squamous cell carcinoma or the 253J B-V bladder cell carcinoma,when grown as established xenografts, can be induced to substantiallyor even totally regress when treated with anti-EGFR Abs such asC225 (18, 19). We reasoned that if similarly treated tumors were thenfollowed for long periods, variants might eventually emerge, and if so,such recurrent tumors could represent the outgrowth of the progeny ofrare variants no longer fully responsive to the EGFR-targeting drug.

The rationale for undertaking such experiments was not only for theobvious intrinsic value of deriving such variants but also as a meansof exploring in greater depth the hypothesis that EGFR-targetingagents functionin vivo, at least in part, by blocking angiogenesis (19,20). For example, C225 treatment can block the production of severalproangiogenic growth factors in treated EGFR-positive tumor cells,including VEGF, interleukin-8, and basic fibroblast growth factor(19–22). We hypothesized that this effect could contribute to itstherapeutic efficacyin vivo (20). If this assumption was correct,

Received 3/13/01; accepted 5/02/01.The costs of publication of this article were defrayed in part by the payment of page

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

1 Supported primarily by Grant MT-5815 from the Medical Research Council ofCanada (to R. S. K.) and NIH Grant CA-41233 (to R. S. K.).

2 To whom requests for reprints should be addressed, at Molecular and CellularBiology Research, Sunnybrook and Women’s College Health Sciences Centre, S-218Research Building, 2075 Bayview Avenue, Toronto, ON M4N 3 M5, Canada. Phone:(416) 480-5711; Fax: (416) 480-5703; E-mail: robert.kerbel@swchsc.on.ca.

3 The abbreviations used are: Ab, antibody; mAb, monoclonal Ab; EGF, epidermalgrowth factor; EGFR, EGF receptor; FBS, fetal bovine serum; HUVEC, human umbilicalvein endothelial cell; CM, conditioned medium; CMV, cytomegalovirus; TGF, tumorgrowth factor; SCID, severe combined immunodeficient; TSP, thrombospondin; VEGF,vascular endothelial growth factor; hVEGF, human VEGF; mVEGF, murine VEGF;ADCC, Ab-dependent cell-mediated cytotoxicity; A431P, parental A431 (cells).

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resistant variants could arisein vivo by such mechanisms as increasedVEGF production (23) or by activating one or more alternativeproangiogenic growth factors.

The present experiments were performed using three differentmonoclonal anti-EGFR Abs and the A431 human tumor cell line. TheAbs included an unmodified mouse mAb (mR3), a humanized versionof this Ab (hR3), and an independent, chimeric, Ab (C225). We foundthat variants could indeed arise, usually only after a protracted periodof time posttreatment, and not in all mice. All of the six independentvariants that we analyzed, with the exception of one, displayed in-creased VEGF production. Thesein vivo selected variants should beuseful tools to investigate the role of the EGFR in cancer growth andangiogenesis, as well as for studies designed to improve the use ofanticancer drugs that target tumor cell-associated receptor tyrosinekinases, particularly the EGFR.

MATERIALS AND METHODS

Abs. The neutralizing anti-EGFR mAb C225, a human-mouse chimericform of the original mouse monoclonal 225, initially described by Kawamotoet al. (24) was manufactured by ImClone Systems, Inc. (New York, NY). Thehumanized anti-EGFR mAb hR3 as well as its murine form mR3 (25, 26) wereprovided by York Medical (YM) BioSciences, Inc. (Mississauga, Ontario,Canada). The anti-hVEGF mAb 4301-42-35 was described previously (27).

Cell Lines and Culture Conditions. The human epidermoid carcinomacell line A431 was obtained from the American Type Culture Collection(ATCC, Rockville, Maryland). Independent variants of A431 cells resistant toEGFR-blocking Abs (R cells) were derived during this study (see “Derivationof EGFR Ab-resistant Variants” below). Both the parental and A431 cellvariants were cultured in DMEM supplemented with 5% FBS (Life Technol-ogies, Inc., Grand Island, NY). HUVECs were cultured on gelatin-coateddishes as described previously (28). Cultures were maintained at 37°C and 5%CO2 in a humidified incubator.

Determination of in Vitro Cell Growth. The effect of different conditionson anchorage-dependent growth of parental and variant A431 cells was meas-ured by a [3H]thymidine incorporation assay in monolayer cultures. Briefly,recently trypsinized cells were resuspended at a density of 33 103 per 100mlof DMEM (Life Technologies, Inc.) containing 1% FBS, and plated in a96-well plate (Nalge Nunc Inc., Naperville, IL). Plates were incubated understandard conditions for 24 h, at which point, 100ml of the given treatment (23concentrated C225, hR3, or TGFa in 1% FBS/DMEM) was added. Cells wereincubated for an additional 48 h, and 2mCi/well [3H]thymidine (Amersham)were added for the last 4–6 h. The incorporated radioactivity was determinedusing a Betaplate liquid scintillation counter (Pharmacia). The same assay wasperformed to test the effect of CM from A431 or R cells on the growth ofHUVECs. In this case, cells were plated on gelatin-coated wells in completegrowth medium, which was replaced with CM 24 h later. To compare thegrowth properties of the Ab-resistant variants and A431 parental cells, 104

cells/0.5 ml/well were plated on a 24-well plate in regular growth medium(DMEM 1 5% FBS). Cells were incubated under standard conditions andcounted every day to day 6 using a Coulter counter. Growth medium wasreplaced at day 3. Cell number after 24-h incubation was taken as baseline. Forthe clonogenic survival assay, cells were plated under the same conditions,except for the lower density (103/well). The colonies formed after 1 week werestained with Cristal Violet and counted by naked eye.

Evaluation of the in Vivo Antitumor Activity of Three Anti-EGFRmAbs. A431 cells (53 106/200ml of PBS) were inoculated s.c. into the rightflank of 6–8-week-old SCID mice (average weight, 23 g). After 10 days(tumor average size, 300 mm3) the animals were randomly separated intoseven groups (four to five mice per group). “High-dose” groups were treatedwith 1 mg of C225, hR3, or mR3 administered i.p. every 48 h. “Low-dose”groups were treated with one-fourth of this dose,i.e.,0.25 mg of the given Abfollowing the same schedule. Control group was treated with PBS. Treatmentwas stopped after a total of eight injections.

Derivation of EGFR Ab-resistant Variants of A431 Cells. After 2 weeksof continuous treatment with either the higher dose (1 mg per injection) or alower dose (0.25 mg per injection) of the Abs C225, hR3, or mR3, most tumors

regressed, with only a few exceptions. Mice were then maintained and mon-itored for tumor recurrences, which were first observed in a proportion of micein the 0.25-mg-dose-per-injection groups, 2 months after the beginning oftreatment (see “Results” for details). As soon as the recurrent tumors reacheda size similar to that of the initially treated tumors (i.e.,200–600 mm3), asecond round of treatment was initiated using the same Ab, dose, and scheduleinitially applied to treat that tumor-bearing mouse (e.g.,0.25 mg of mR3 every48 h for 2 weeks, if the recurrent tumor was in a mouse from the mR3lower-dose treatment group). Tumors that did not respond during the 1st weekof treatment with the 0.25-mg dose were then treated with 1 mg per injectionaccording to the same schedule (i.e.,every 48 h) during the next 2 weeks orless, depending on tumor response. Tumors initially treated with a dose of 1mg per injection were treated only with the same dose and schedule. Tumorsthat did not regress or remain dormant (as in the first round of treatment) afterthe second treatment were considered resistant. The corresponding mouse wasthen euthenized, and the tumor cells were recoveredin vitro by enzymatictreatment (29) under aseptic conditions and were grown in tissue culture. Cellswere passaged in culture at least three times before any further testing.

DNA Fingerprinting. Genomic DNA (10mg) was digested withHinfI(New England Biolabs Inc., Mississauga, Ontario, Canada), resolved in a 0.6%agarose gel, and transferred onto a nylon membrane. Banding pattern wasevaluated after hybridization with a NICE-labeled 33.15 multilocus probe(Cellmark Diagnostic, United Kingdom) used in combination with CDP-Startchemiluminescent substrate (Tropic Inc., Bedford, MA) following the manu-facturer’s instructions.

Western Blotting. To detect the expression of EGFR and HER-2, celllysates were obtained as described previously (28). Proteins were resolved bySDS-PAGE and transferred onto an Immobilon-P membrane (Millipore, Bed-ford, MA). After blocking in 1% casein/TBST, the membrane was incubatedwith either the rabbit polyclonal anti-EGFR Ab SC-003 or the anti-HER-2/NeuAb SC-284 (Santa Cruz Biotechnologies, San Francisco, CA) at 0.2mg/ml,then washed and incubated with a peroxidase-conjugated goat antirabbit Ab(Jackson Immunoresearch Laboratories Inc., West Grove, PA). The loadingwas verified bya-actin probing with A5441 Ab (Sigma Chemical Co.) at 0.22mg/ml. Phosphorylated EGFR was determined in lysates from TGF-a-stimu-lated cells using the mouse anti-pEGFR no. 324864 (Calbiochem) at 0.1mg/ml. The peroxidase-conjugated antimouse W402B Ab (Promega) at 0.2mg/ml was used as secondary to detect mouse-derived primary Abs. Blots werevisualized using the ECL chemiluminescence kit (Amersham Corp., ArlingtonHeights, IL).

Measurement of Human and Mouse VEGF Protein Levels in CM.Acommercially available human or mouse VEGF ELISA kit (R&D Systems,Inc., Minneapolis, MN) was used to determine VEGF protein levels in CMobtained from A431, Ab-resistant variants, or mVEGF-transfected clones.Briefly, cells were plated at a density of 105 cells/0.5 ml/well in a 24-well plateunder normal serum conditions and allowed to reach 80% confluency, at whichpoint, the medium was replaced by DMEM/1% FBS with or without C225.Medium was collected after 24 h and cells were counted as described previ-ously (20).

Northern Blot Analysis. Approximately 107 cells were used for the ex-traction of total RNA using Trizol (Life Technologies, Inc., Gaithersburg, MD)following the manufacturer’s protocol. VEGF and TSP-1 Northern blots wereperformed as described previously(30). Equal RNA loading and the efficiencyof the transfer were visualized by ethidium bromide staining of both the geland the membrane.

Derivation of A431 Variants Overexpressing VEGF. An expressionplasmid encoding mVEGF164 (981 bp) driven by a CMV promoter, a generousgift from Dr. Kevin Claffey (Beth Israel Deaconess Medical Center-ResearchNorth, Boston, MA), was transfected into A431 cells using SuperFect reagent(Qiagen Inc., Valencia, CA) following the manufacturer’s instructions. Controlclones resulted from transfection with an empty vector. Positive clones wereselected with increasing concentrations (500–800mg/ml) of Geneticin (LifeTechnologies, Inc., Grand Island, NY) and were maintained in the presence of800 mg/ml of the drug. Three of the clones (mVEGF.S1, mVEGF.S2, andmVEGF.S3) were chosen for an EGFR Ab resistance testin vivo, which wasperformed after the low-dose-treatment regimen described in a previous sec-tion.

VEGF-neutralizing Treatment in Vivo. VEGF-induced angiogenesis wasexamined in both R and A431 parental cells after a short VEGF-neutralizingin

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vivo experiment. Briefly, SCID mice (five per group) bearing A431, R1, andR5 s.c. tumor xenografts (average tumor volume, 250 mm3) were treated withtwo injections of either PBS (control) or 200mg of anti-hVEGF Ab 4201–42-35 (Novartis). Tumors were recovered for immunohistochemical analysisafter 1 week (average tumor volume: 321 mm3, 452 mm3, and 423 mm3 forA431, R1, and R5, respectively).

Immunohistochemistry. VEGF staining was performed on formalin-fixedand paraffin-embedded specimens as described previously (20). Blood vesselswere visualized by CD31 staining, which was performed on 7-mm-thickcryosections using the rat antimouse CD31 no 01951D (PharMingen) at aconcentration of 1mg/ml 1:500. Biotinylated rabbit anti-rat (Jackson Immu-noresearch) and Histostain kit (Zymed Laboratories, San Francisco, CA)reagents were used to reveal antigen as a red signal. Contrast was providedwith Harris Hematoxylin (Surgipath Canada, Inc., Winnipeg, Manitoba, Can-ada).

Statistics. Statistical analysis was performed using the GraphPAD InStatsoftware, version 1.14 (GraphPAD Inc., San Diego, CA).In vivo experimentswere analyzed by a one-way ANOVA test coupled to a Bonferroni test formultiple comparisons.In vitro data were compared by at test.

RESULTS

Antitumor Activity of hR3 and C225 mAbs. One of the two mainaims of this study was to evaluate the possibility that the use ofEGFR-blocking Absin vivo might result in a selection of drug-resistant variants. We decided to explore this subject using at least twodifferent EGFR-neutralizing Abs, C225 and hR3, which possess sim-ilar binding affinities but different specificities (24, 26). We chose thehuman squamous cell carcinoma A431 based on its extensive use asa model over the past 15 years in the study of the efficacy of manydifferent antagonists of EGFR, including C225 (18, 31, 32). We havepreviously found ( by using [3H]Thymidine incorporation assays) thatthe maximum growth inhibitory effect of either C225 or hR3in vitrois 40%. This was achieved on monolayer cultures after 48-h exposureto doses ranging between 50 and 100mg/ml.4 To investigate therelative efficacy of the Abs (C225, mR3, and hR3)in vivo, we testedtheir antitumor properties on A431 s.c. tumor xenografts, thus resem-bling the orthotopic location for this tumor, implanted into SCIDmice. Under such conditions, the three Abs seemed equally efficientin causing total regression of well-established tumors after 2 weeks oftreatment (eight injections) with a low- or a high-dose regimen (Fig.1), at least during the first 3 weeks of follow-up. No significantdifference was observed among the groups when compared by dose orby Ab type (P. 0.05 in all cases).

Sporadic Tumor Recurrence after Treatment with Anti-EGFRAbs. After the tumor regressions became evident in the first respond-ers, treatment was terminated, and the mice were monitored for tumorrecurrence. The first such recurrences occurred 1.5 months aftertermination of the first round of treatment in the 0.25-mg-dose-per-injection groups injected with mR3 or hR3 (Fig. 1,A andC). In eachof these groups, one tumor did not regress completely after the eightinjections, which perhaps suggests a low level of intrinsic resistanceto EGFR inhibition. These tumors were the first to regrow (as ex-pected) over a period as short as 18 days after the maximum antitumoreffect was achieved (Fig. 1,A and C). In the 0.25-mg-dose-per-injection C225 treatment group, one tumor recurrence was observedafter a latency period of 2 months (Fig. 1E). Recurrence of tumors inthe higher-dose groups took longer and was first evident after a4.5-month latency period in the mR3-treatment group and after 1.5-months in the hR3-treatment group (Fig. 1,B andD). Recurrence alsotook a long period to occur in the higher-dose C225 group,e.g.,after3.5 months (Fig. 1F). The frequencies of tumor recurrence for C225,

hR3, and mR3, respectively, were 20, 40, and 80% for the 0.25-mg-dose treatment groups and 25, 25, and 80% for the 1-mg-dose groups.Mice with recurrent tumors in all of the groups subsequently receiveda second round of treatment (Table 1).

A431 Recurrent Tumors Escape a Second Round of Anti-EGFR Treatment: Derivation of Variants. While administering thesecond round of treatment, we observed that in most cases the tumorsdid not respond to the same extent as they did originally. Tumorsregrew rapidly during, or immediately after, this cycle of therapy. Celllines established from these tumors were designated as follows: R1and R2 [two morphologically different subpopulations of a resistanttumor from the mR3, 0.25-mg-dose group (Fig. 1A)]; R3 and R4, fromtwo individual resistant tumors of the hR3, 0.25-mg-dose group (Fig.1C); R5, recovered from a tumor in the hR3, 1-mg-dose group (Fig.1D); and R6, which was recovered from a resistant tumor in the mR3,1-mg-dose-group (Fig. 1B). More detailed data on the derivation ofthe resistant variants are summarized in Table 1.

A common cellular origin for the A431 R variants was demon-strated using a DNA fingerprinting analysis (Fig. 2A). Identical band-ing patterns suggest that all of the cell lines are A-431-derived and arenot comprised of contaminating human or mouse tumor cells. Havingestablished this, two resistant variants (R1 and R5) were chosen for afinal in vivo resistance test based on a pilot screening. R1 and R5 wereboth representative of the similarin vivo growth properties found inthe majority of the resistant cells. For the final test, 106 cells (A431,R1, and R5) were inoculated s.c. into 6-week-old nude mice (five pertreatment group), and tumor growth was monitored every 4 days tocompare tumor take rates. Treatment was initiated when tumorsreached an average size of 200–300 mm3 and was administered asdescribed for the 0.25-mg/injection group. Both R1 and R5 mani-fested an accelerated growthin vivo (Fig. 2B), which became evident18 days after tumor cell injection and which became significant byday 22. The increased growth rate of these variants was even moresignificant after a 30-day period of growth, as observed in an inde-pendent tumor-take experiment (data not shown). Thesein vivo resultsstand in marked contrast to thein vitro data, in which similar growthrates between the R variants and the A431P cells were observed(Table 2). When the variants were tested for their response to EGFR-blocking Abs (e.g.,C225) in vivo, both R1 and R5 showed a similarand significant delayed response. (Fig. 2C). By day 45, all of the fivetumors included in the A431 group had totally regressed, whereas inthe R1 and R5 groups, only one and two such total regressions wereobserved, respectively. Maximum average responses for R tumorswere registered at day 45. The remaining R1 and R5 tumors did notregress by day 86, at which point at least one of the tumors in eachgroup (R1 or R5) regrew to the maximum allowed size (1700 mm3)according to the guidelines of the Canadian Council on Animal Care(CCAC), and the experiment was terminated. No sign of tumor wasseen by day 86 in any of the animals included in the A431 grouptreated with C225. Similar results were obtained after hR3 treatment(data not shown).

Absence of Changes in EGFR Status in A431 Variants Resist-ant to EGFR-blocking Abs in Vivo. As a second step in the char-acterization of the A431-resistant variants, we examined the level ofEGFR expression, because resistance to treatment with EGFR-block-ing agents could have obviously resulted from loss or reduction in thelevel of expression of the receptor itself. However, we did not detectevidence to implicate such a mechanism, because five of six variantsshowed levels of EGFR similar to those expressed by A431 parentalcells (Fig. 3A). To demonstrate that EGFR was equally functional inA431 and the variants, we stimulated the different cell lines withincreasing concentrations of TGF-a and measured the level of phos-pho-active EGFR. Activation of EGFR was found to be equivalent4 T. Crombet, unpublished observations.

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between A431 and all of the R variant cells, with the exception of R6,which showed less phospho-active receptor (data not shown) in pro-portion to a lower amount of EGFR expressed by these cells. Only theresults obtained with R1 and R5 are shown (Fig. 3A). The similarpattern in expression level and ligand-induced activation of EGFRbetween A431 and the R variants suggests that major changes in theEGFR status are unlikely to be responsible for the resistant phenotype.

This conclusion is reinforced by the similar growth responses of A431cells and the R variants to the inhibitory effects of C225 (50mg/ml)and TGF-a (100 ng/ml) treatmentin vitro (Table 2). High concentra-tions of EGFR-specific ligands,i.e., EGF and TGF-a, have beenreported to cause a potent growth inhibition of A431 cells, a phenom-enon associated with their considerably elevated expression of EGFR(33, 34). The virtually identical pattern of growth inhibition of A431

Fig. 1.In vivoderivation of anti-EGFR Ab-resistant variants of A431. The effect of Ab treatment on tumor growth as well as the kinetics of tumor relapse in:A, mice initially treated(1st) with 0.25 mg (Low dose) of mR3 every other day for 2 weeks. Four of five tumors relapsed. A431 variants R1 and R2 were recovered from nonresponding tumor 3 after a second(2nd) round of treatment. No tumor cells could be isolated from tumor 4 (NI, not isolated).B, mice initially treated with a dose of 1 mg (High dose) of mR3 every other day for 2weeks. Four of five tumors relapsed after this treatment. A431 variant R6 was recovered from tumor 3 in this group. Cells from tumor 4 could not be recovered (NI).C, mice includedin the 0.25-mg-dose group of hR3 treatment. Two of five tumors relapsed and both were unresponsive to a second round of treatment (variants R3 and R4).D, A431 tumors treatedwith hR3 on a 1-mg dose schedule. In this group, only one of four tumors relapsed. Although this tumor seemed to respond to a second treatment, it started to regrow immediatelyafterward (variant R5).E, mice treated with C225 on a 0.25-mg dose schedule. Only one of five tumors relapsed, but the cells could not be recovered.F, group of mice in the 1-mgdose group of C225 treatment. One of four tumors relapsed. This tumor partially responded to a second round of the same treatment but began to regrow during a third round oftreatment. No cells could be obtained (NI). Other abbreviations: “D,” mouse dead during the experiment; “E,” mouse euthanized and discarded by animal colony staff without notice.

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and the R variants R1-R5 by TGF-a is consistent with their equivalentlevels of EGFR expression. Similarly, we did not find significantchanges in the levels of expression of HER-2 (Fig. 3A), the secondmember of the EGFR family also known to be involved in celltransformation and tumor progression (35). HER-2 has been shown toheterodimerize with EGFR, altering the patterns of downstream cel-lular signaling (36, 37). With the exception of R6, which appears toexpress lower levels of HER-2, none of the variants expressed levelsof HER-2 that were significantly different from that of the parentalcells, which suggests that changes in HER-2 expression are not acommon feature of the resistant variants. In agreement with thedecreased expression of EGFR and HER-2 found in R6, this cell lineshowed an unusually slow growth rate (data not shown).

Constitutive VEGF Up-Regulation in Anti-EGFR-resistantVariants of A431. Because of the clear discrepancy between theinvitro findings (in which resistance was not seen) andin vivo findings(in which resistance was detected), we considered the possibility thatthe R variants possess increased survival properties under restrictedgrowth conditions or, alternatively, an increased angiogenic ability,which could translate into both superior growth and prolonged sur-vival in vivo. Because neither C225 nor hR3 is able to induce celldeath of A431 cells under restricted culture conditions,e.g., three-dimensional/low serum cultures,4 we could not compare differences insurvival in response to Ab treatment under such conditions. Analternative experiment, a clonogenic survival analysis, failed to showa survival advantage in the resistant variants (Table 2), which suggeststhat an improved basal cellular survival is unlikely to explain theinvivogrowth advantage. To evaluate the possibility of enhanced proan-giogenic capacity in the R variants, we measured the levels of theproangiogenic factor VEGF, at both the RNA and protein levels in allof the cell lines. The importance of this growth factor for tumorangiogenesis has been established for A431 cells (38), and, further-more, we have reported previously that the EGFR-neutralizing AbC225 is able to down-regulate VEGF protein up to 50%in vitro andto an even greater extentin vivo in these cells (20), which wehypothesized could account for the reduced vessel counts found in thisand other tumor models after treatment with EGFR-blocking agents(19, 20). We found that five of the six resistant variants, R6 being theexception, expressed at least 2-fold more VEGF mRNA and proteinthan the A431P cells (Fig. 3,B andC). Although VEGF levels werestill down-regulatedin vitro by 50% as a result of treatment with C225(50 mg/ml), it should be noted that, even under such conditions,

variant cells still produce 2- to 4-fold more VEGF than does A431(Fig. 3C). We discarded the possibility that the elevated VEGFexpression was merely the result ofin vivo passaging of A431 cells,by comparing the expression of VEGF with that in A431 parental cellspassagedin vivo for 14 days. These cells expressed levels of VEGFthat were similar to those of the parental cells and also showed anequivalent growth ratein vivo (data not shown). In addition, we alsoobserved an increase in the growth response of HUVECs to the CMof R variants 1 to 5 (Fig. 3D), but not to that from variant R6 (data notshown), suggesting an increased angiogenesis-inducing capacity inthe VEGF-overexpressing cells. The absence of an increase in VEGFexpression in addition to the reduced levels of EGFR, HER-2, and thedelayed growth rate found in R6 cells suggests that this variantescaped the antitumor effect of anti-EGFR Ab treatment by a differentmechanism from that in the other variants.

In contrast to the results with VEGF, no common pattern wasobserved in the levels of TSP-1 (Fig. 3B), a naturally occurringangiogenesis inhibitor previously shown to be down-regulated byactivation of Ras, one of the downstream effectors of EGFR (39).Whereas A431 cells appear to express very low amounts of TSP-1,two variants, R2 and R5, exhibit a paradoxical increase in TSP-1transcript levels. We have no explanation for this increased TSP-1 andits biological impact because it is obvious from thein vivo data thatincreased TSP-1 does not confer a growth disadvantage to R5(Fig. 2B).

In Vivo Resistance to EGFR-neutralizing Ab of A431 Cell LinesEngineered to Overexpress VEGF.Because of the apparent in-crease in VEGF in the resistantversusA431P cells, we decided to testwhether or not such increased VEGF levels could account, at least inpart, for the resistant phenotype to EGFR-blocking agentsin vivo. Wereasoned that the resistance of these variants could result from theinability of EGFR-blocking Abs to down-regulate VEGF levels tothe same extent that they do in the parental cells. This would limit theability of these agents to achieve the same degree of antiangiogeniceffect as in the case of A431 parental tumors. Consequently, anendogenous EGFR-independent increase in VEGF expression couldact as ade factoresistance mechanism for EGFR inhibitors. To testthis hypothesis, we decided to boost VEGF production in A431parental cells using a gene transfection procedure. An expressionvector was used that encoded the murine form of VEGF164 (equiva-lent to human isoform VEGF165), a secretable form of VEGF thatappears to be overexpressed in the R variants of A431 cells. After

Table 1 Data summary of the in vivo generation of A431 recurrent variants displaying a resistant phenotype to a second cycle of treatment with anti-EGFR Abs

Group mAb (dosing)aMice per

groupRecurrent

tumors (%)TumorI.D.b

Initial sizefirst cyclec

Initial sizesecond cycle

Second cycled

(no. of injections3 dose)Recurrent variant

obtained Observations

I mR3 (0.25 mg) 5 4 (80) T2 144 550 53 0.25 mg no Mice 2 and 5 euthanizedT3 113 225 83 0.25 mg R1, R2T4 107 239 83 0.25 mg1 8 3 1 mg no Cells from T4 did not plate.T5 208 587 73 0.25 mg no

II mR3 (1 mg) 5 4 (80) T1 270 600 13 1 mg no Mouse 1 euthanized (open tumor)T3 352 575 73 1 mg R6T4 304 451 73 1 mg no Cells from T4 did not plate.T5 298 368 83 1 mg no Mouse 5 was found dead after

eighth injection of second cycle.III hR3 (0.25 mg) 5 2 (40) T2 270 320 83 0.25 mg1 8 3 1 mg R3

T5 352 446 same as T2 R4IV hR3 (1 mg) 4 1 (25) T3 225 361 83 1 mg R5V C225 (0.25 mg) 5 1 (20) T1 221 496 23 0.25 mg no Mouse was euthanizedVI C225 (1 mg) 4 1 (25) T2 288 304 83 1 mg-break- 83 1 mg no Tumor did not regress or grow back

after second cycle. It began toregrow during a third cycle. Cellscould not be recovered.

a This dose of the given Ab was injected every other day for 2 weeks during the first cycle of treatment.b Tumor I.D., the number assigned to a particular tumor in a group of treatment.c Initial size, the size of the tumor (in mm3) at time 0 in either first or second cycle of treatment.d Injections in second cycle of treatment were given every other day.

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preliminary screening, three clones (mVEGF.S1, mVEGF.S2, andmVEGF.S3), expressing varying amounts of VEGF ranging between800 and 12,000 pg/ml, were selected for further analysis. The levelsof mVEGF produced by these clones were completely refractory totreatment with C225 (Fig. 4A). This was expected because the ex-pression of exogenous mVEGF164 is under the control of the consti-tutively active and strong CMV promoter. In addition, the clones werefound to maintain an intactin vitro response to C225 in terms ofgrowth inhibition (data not shown) and expression of endogenous

hVEGF (Fig. 4A). We compared thein vivo responsiveness of VEGF-overexpressing (mVEGF.S) and A431P cells to the C225 Ab. Aspreviously found, a low-dose regimen was sufficient to cause totaltumor regression of well-established A431 parental and control (vec-tor-transfected) tumors but was unable to do so in the case of theVEGF transfectants (Fig. 4,B and C), which showed significant(P , 0.05) resistance compared with the control groups. No signifi-cant difference was found (P. 0.05) among the three VEGF-overexpressing clones, although a tendency of a direct correlationbetween the level of VEGF produced and the degree of resistance wasobserved. In this regard, it is important to mention that the cloneexpressing the lowest amount of exogenous VEGF (mVEGF.S1)secretes an overall quantity of this protein that is in close similarity tothat secreted by some of the resistant variants themselves. The factthat this clone still exhibits a significant resistance to EGFR-blockingtreatmentin vivo reinforces the idea of an involvement of VEGF in theresistance mechanism.

Histopathological analysis of 3-week-old s.c. tumors of parentaland mVEGF164-transfected A431 cells revealed no differences incellular morphology but did reveal a slightly larger central area ofnecrosis in the mVEGF164 tumors, probably reflecting their moreaccelerated growth rate. In addition, tumors derived from clones S2and S3 exhibited focal areas of hemorrhage that were particularlylarge in the S2 tumors (data not shown), which suggests that theextremely nonphysiological amount of VEGF164 produced by thisclone could be responsible for this characteristic. Staining of bloodvessels with the endothelial-specific marker CD31 did not show anincrease in the microvessel density of mVEGF-overexpressing tumorsbut showed a striking difference in the size of the vascular channelsfound in mVEGF164transfectants compared with those found in A431parental tumors (Fig. 5). Vessels in mVEGF-expressing tumors werehighly enlarged, consisting in many cases of a complex structuressuggestive of splitting of the vessel lumen by endothelial intravesselwalls (Fig. 5,B andC). The overall appearance of these vessels seemto resemble that found in the previously described “mother vessels”(40), which result from increased mVEGF164-dependent angiogene-sis. The functionality and impact of these enlarged vessels on tumorbiology is still unclear. However, in the context of the s.c. A431model, these vessels appear to be a hallmark of VEGF164-dependentangiogenesis, which did not result, in this case, in an increase inmicrovessel density. This altered angiogenesis could account for thereduced response of A431 tumors to EGFR-blocking Abs, such asC225 (Fig. 4,B andC). The general appearance of tumor tissue frommVEGF164-transfectant and parental tumors after C225 treatmentwould appear to support this hypothesis. A431 tumors appear almosttotally necrotic, with only a rim of viable tissue in the periphery (Fig.5E), which virtually lacks blood vessels (Fig. 5F). In contrast, C225-treated mVEGF164 transfectant tumors are comprised of a mixture of

Table 2 Growth properties of in vivo-derived recurrent variants of A431

Cell type

C225-dependentgrowth inhibition

(% 6 SD)a

TGF-a-dependent

growthinhibition

(% 6 SD)aDoubling time

(h 6 SD)bClonogenic survivalc

(colony,n 6 SD)b

A431 336 5.6 916 5.2 30.26 2.3 1866 4.5R1 416 10.0 806 3.2 28.76 0.6 1896 1.5R3 436 5.3 886 7.9 30.66 1.1 1116 6.0R4 336 11.4 856 7.5 32.46 1.1 1766 13.2R5 356 8.1 846 9.4 35.46 1.6 586 7.1a Values represent the average for two independent experiments, each one including

triplicates.b Calculated from triplicate cultures.c The ability of cells to form clonal colonies was analyzed as described previously (58).

See “Materials and Methods” for details.

Fig. 2. Phenotypic profile of EGFR Ab-resistant variants of A431.A, DNA finger-printing analysis showing identical banding pattern of resistant variants and parental A431cells.B, growth of control tumors showing increased growth rate of R1 and R5 comparedwith the parental cells.C, treatment of R1 and R5 tumor xenografts with C225 in newhosts results in growth inhibition, but this is significantly delayed compared with theimmediate antitumor response observed with the A431P tumors (see “Results” section fordetails).Error bars, SD.

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necrotic and viable tissue (Fig. 5G), with highly enlarged vascularchannels supporting the growth of tumor cell “cuffs” (Fig. 5H). Takentogether, the results suggest that increased VEGF expression, which isrefractory to the effects of C225, could be responsible, at least in part,for the resistance of the variant A431 tumors to EGFR-blocking Abs.

Evidence of a Causal Role of VEGF in the Altered Vascularityof A431 Variants. To determine whether the elevated VEGF expres-sion of the resistant variants could result in an altered VEGF-depend-ent angiogenesis phenotype, we analyzed tumor sections obtainedfrom a shortin vivoexperiment, in which mice bearing A431, R1, andR5 tumors were treated with either PBS (control) or anti-hVEGF Abfor 1 week, after which a moderate antitumor effect was observed ineach group (treated:control ratio: 0.66, 0.61, and 0.81 for A431, R1,and R5, respectively; see “Materials and Methods” for details). BothVEGF expression and vascularity were evaluated by immunostaining.We observed that in both A431P and R tumor sections, VEGF stainingwas mostly localized at the tumor periphery. In the A431 tumors,VEGF expression was usually seen in large patches or “islands” (Fig.6A) composed mainly of large cells with a moderately- to well-differentiated squamous morphology. Slightly smaller cells withhigher nuclear/cytoplasmic ratio surrounding these islands were neg-ative for VEGF staining (Fig. 6A). In contrast, VEGF staining in theR1 and R5 variants was more diffuse and present in almost all of the

viable cells, which showed a relatively high nuclear:cytoplasmic ratioand a moderately differentiated morphology (Fig. 6,D andG). Stain-ing for CD31 showed no obvious increase in the vessel density of R1and R5 tumors compared with those of A431P cells. Instead, the sizeof the vessels appeared significantly larger in the R variant tumors(Fig. 6, B, E, andH). A majority of blood vessels in the R1 tumorswere more than 2-fold larger than those detected in A431, whereas R5tumors appeared to possess even more expanded and complex vascu-lar channels, consisting in some cases of several compartments (Fig.6H) resembling those found in mVEGF164 A431 transfectants (Fig.5). These large vessels (40) in the R variant tumors appeared to beVEGF-dependent, because they became less common or smaller after1 week of treatment with the anti-hVEGF Ab 4301-42-35 (Ref. 27;Fig. 6,F andI). In contrast, the same treatment caused a reduction inthe microvascular density in A431 P tumors (Fig. 6,C versus B) butno apparent change in vessel size.

DISCUSSION

The clinical approval of drugs that block receptor tyrosine kinases,such as Herceptin, for the treatment of cancer, and the advancedclinical development status of others, such as IMC-225 (7, 41) andIressa/ZD1839, both of which block EGFR function, highlight the

Fig. 3. Absence of significant changes in EGFR receptor status and increased VEGF expression in resistant variants of A431.A, expression of EGFR, HER-2 and ligand-inducedactivation of EGFR as determined by Western blots. No significant quantitative differences were detected in the EGFRs expressed by the A431 R variants compared with the A431Pcells, with the exception of R6, which showed reduced levels of both EGFR and HER-2. In addition, A431P and R variants respond similarly to increasing concentrations of TGF-a.The results shown are those obtained with R1 and R5, which are representative of all of the variants, except for R6 (see “Results” for details).b-actin is shown as loading control.B, levels of VEGF and TSP-1 RNA expressed by variants R1 to R5, and A431P cells. All of the variant cells, with the exception of R6 (data not shown), express at least 2-fold moreVEGF mRNA than parental cells. C225 treatment down-regulates VEGF in all of the cell lines. No trend is observed in the levels of TSP-1 mRNA in the R variants. rRNA (bottompanel) was used as loading control.C, increased production of VEGF protein in the A431 R variants as determined by ELISA. The results are consistent with the Northern blot analysis,showing a 2- to 3-fold increase in VEGF protein in the R variants compared with A431.D, response of HUVECs to the CM produced by either A431 or R cells. An increase in thymidineincorporation is seen only with the CM from VEGF-overexpressing cells. VEGF (50 ng/ml) was used as a positive control. The results are the average of quadruplicate measurementsand are standardized to cell number at the time of CM collection.Error bars, SD.

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surprising fact that no preclinical studies have been undertaken toevaluate whether, and how, acquired resistance to such drugs maydevelop. Our results show that acquired resistance can indeed developin vivo to EGFR blocking Abs. However, it is encouraging that theefficiency with which such resistance develops appears to be rathermodest. First, in our study, recurrent tumors did not develop in all ofthe mice, and when they did occur, it was usually after prolongedlatency periods. Indeed, it was only as a result of undertaking such

long-term-observation experiments that we detected the emergence ofresistant variants. Second, it should be noted that the only possibletarget of the Abs in our model was the EGFR expressed by theimplanted, genetically unstable human A431 tumor cells growing inthe SCID mice. In contrast, in the clinical situation, such Abs wouldhave the potential opportunity to block EGFRs expressed by normal(genetically stable) host endothelial cells of newly formed tumorblood vessels (42, 43) particularly in TGF-a-expressing tumors, inwhich TGF-a itself may up-regulate EGFR expression in activatedendothelial cells (44, 45). As a result, EGFR-blocking agents mightconceivably block angiogenesis not only indirectly but also directly,especially when such drugs are combined with chemotherapy. Thiswas shown recently by Brunset al. (22, 46), who reported significantinduction of endothelial cell apoptosis in human pancreatic or prostatecarcinoma xenograft microvessels by combination treatment witheither a small molecule EGFR antagonist or C225 and gemcitabine.Third, acquired resistance may also be delayed by more protractedtreatment with the Abs,i.e.,continuing the treatment in tumors that donot regress after eight injections and even after tumors have regressed,or by combining the Ab treatment with other therapeutic modalitiessuch as chemotherapy or radiation, which is currently the way EGFR-blocking drugs are used clinically (7, 41). In this regard, anotherinteresting feature of our results is the necessity of undertakingvery-long-term (e.g.,up to one year)in vivo experiments to detect theemergence of resistant variants, an approach lacking in virtually allprevious studies using such therapeutic agents.

We must also acknowledge the possible influence on the resultsas a consequence of using exclusively the A431 cell line for ourstudies, a cell line that expresses a very high number of EGFRs andthat is known for its hypersensitivity to EGFR-blocking drugsinvivo. Analysis of other cell lines that have fewer numbers of EGFRmay not reveal evidence of acquired resistance or, alternatively,may reveal evidence for such resistance but involving differentmechanisms.

Possible Impact of Altered VEGF Expression and TumorAngiogenesis in the Resistant Phenotype.One of the most interest-ing (and unexpected) findings of this study was the absence of anobvious alteration in the EGFR status in the R variants, which weestablished by different means, including Western blot analysis ofEGFR expression and evaluation of the growth response of the vari-ants to EGFR-blocking Abs (C225 and hR3), as well as the determi-nation of levels of phospho-active EGFR after stimulation with in-creasing concentrations of TGF-a. We reasoned that major changes inthe level of expression of EGFR and/or its ligands could translate intoa different biological response to ligand-induced stimulation and/orphosphorylation (i.e.,activation). However, we did not find anyevidence for such changes. Of considerable importance in this regardwas the virtually identicalin vitro growth characteristics and inhibi-tory responses to treatment with the anti-EGFR blocking Abs of thevariants, compared with the parental cell line. Thus, interestingly, theresistant phenotype manifested itself onlyin vivo. One possible ex-planation for this would be a reduced sensitivity to undergoing Fcreceptor-mediated ADCC, a host process that can take place onlyinvivo and that has been implicated as a major determinant of responseto certain therapeutic Abs such as Herceptin and Rituxin (47). How-ever, ADCC does not appear to be similarly important for the antitu-mor activity of C225, because F(ab9)2 fragments of this Ab, whichcannot activate an ADCC response, still retain a significant growthinhibitory activity against A431 tumor xenografts (48). Hence, wereasoned that an alternative host-dependent mechanism, namely an-giogenesis (a process requiredin vivo but not in vitro for tumorgrowth), could conceivably contribute, at least in part, to the resistantphenotype.

Fig. 4. Exogenous expression of VEGF by mVEGF164 transfectants and its impact onthe in vivo response to EGFR-neutralizing Ab C225.A, mVEGF164 (mVEGF164 isoform)protein produced by clones S1 to S3. The human A431P cells do not express the mouseprotein, as expected.Horizontal black bar, the average level of hVEGF165 proteinexpressed by A431P cells as well as mVEGF164 transfectants. C225 down-regulates onlythe endogenous human protein (hVEGF165) but not the exogenous mVEGF164, as ex-pected. The data represent the average from two independent determinations.B, antitumoreffect of C225 Abs on thein vivogrowth of s.c. tumor xenografts derived from A431P andvector or mVEGF164-transfectant clones S1–S3. C225 Ab treatment (arrow, day ofinjection) causes tumor regression only in A431P and vector control tumors but not in themVEGF164 transfectants.C, tumor-bearing mice after treatment with C225. Tumor size isrepresentative of the average for each treatment group.

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We, therefore, examined the R variants for changes in the expres-sion of VEGF, not only because VEGF is a potent inducer of tumorangiogenesis in general but also because A431 cells have been shownto be highly dependent on VEGF for angiogenesis and, hence,in vivogrowth (38). We found that five of six of the resistant variants expressa significantly higher level of VEGF than does the parental line.Relative VEGF levels appeared to correlate with thein vivogrowth ofthe variants as well as with their angiogenic profiles. Staining ofVEGF on sections derived from R1 and R5 s.c. tumor xenograftssuggests that these tumors express a higher overall amount of VEGF,mainly attributable to an increase in the ratio of VEGF expressing:nonexpressing cells. The mechanism responsible for this change isunknown, but it is likely that anti-EGFR Ab treatment selects forA431 subpopulations with an elevated degree of VEGF expression.

In addition to altered patterns of VEGF expression, we alsofound that variants R1 and R5 possessed conspicuously large bloodvessels, which we suggest is the result of increased VEGF-depend-ent angiogenesis, because blocking VEGF signaling with a VEGF-neutralizing Ab reduces the size of these vessels (Fig. 6). Thepresence of such large vessels is a hallmark of pathological VEGF-

dependent angiogenesis (40, 49). A recent study in which theproperties of newly formed vessels induced by VEGF/VPF164 havebeen investigated in detail showed that large vessels are commonlyfound in situations of VEGF overexpression and are referred to as“mother vessels” (40). We found a striking resemblance betweenthese mother vessels and the large vessels found in R variants,particularly R5. We also found similar, but even more enlarged,vascular channels in the tumors formed from A431 cells trans-fected with the mVEGF164 gene.

Our results raise the interesting possibility that, when applicable,assessment of changes in the number of such mother vessels (asopposed to changes in microvessel counts or density) could be auseful surrogate marker to evaluate and monitor the activity of certainanti-angiogenic drugs. However, measurement of vascular densityseems inappropriate, as it is difficult to establish the margins of anindividual vessel. Because of their compartmentalization, it is difficultto determine whether the structures that we detected correspond toseveral vessels or to just one vessel, subdivided into several compart-ments. For this reason, we did not undertake vessel counts in thepresent study.

Fig. 5. Increased vessel size and complexity intumors derived from mVEGF164 transfectants cellsas compared with A431P tumors.A, A431P. B,mVEGF.S1.C, mVEGF.S2.D, mVEGF.S3. All ofthe three mVEGF transfectant tumors exhibithighly enlarged vascular channels compared withparental tumors. The widely enlarged lumen ofthese vessels appears often with a complex struc-ture, divided into smaller compartments by CD31-positive intravascular septae (Band C, insets topright). E, low-power (32.5) magnification ofA431P tumor after treatment with C225 Ab. Mosttumor tissue appears necrotic, with only a small rimof viable tissue (blue) surrounding the large(brown), dead central area.F, at a higher (310)magnification, very few small vessels are seen inthe viable tissue area of the C225-treated A431Ptumors.G, mVEGF.S3 tumor after treatment withC225 Ab. Tumor tissue appears as a mosaic ofnecrotic (brown) and viable (blue) areas.H, highermagnification (310) of the same tumor sectionshowing a cuff of viable cells supported by a verylarge central vessel.A–D, 310; highlighted areasin right insets,320. Tumors (E–H) were treatedwith four doses of 0.25 mg C225, given i.p everyother day.

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Possible Mechanisms of Increased VEGF Production in the RVariants. An obvious and important question raised by our resultsconcerns the mechanism responsible for the elevated VEGF levelsdetected in the R variants. At present, we do not know the answer, butthere are a number of possibilities. For example, several differentoncogenes, when activated, are known to induce or up-regulate VEGF(39). These includeras, src, anderbB2/neu(39, 50), among others.Likewise certain tumor suppressor genes, such asp53,VHL, or PTEN,when mutated/inactivated, can result in elevated VEGF (39, 51). Thusthe variants may express elevated VEGF levels as a result of theselection of cells possessing one or more such genetic changes duringthe EGFR Ab-mediated therapy. Alternatively, aberrations in signal-ing pathways downstream of EGFR activation that are known to affectVEGF expression could conceivably be involved. Such changes, forexample, could include phosphatidylinositol 39-kinase (PI3 kinase;Refs. 51, 52), and/or src kinase (39, 53, 54) overactivation, and/or rasmutation (30, 39). Preliminary data suggest that neither increased srcnor ras activity are obvious in the resistant variants of A431 (data notshown). Whatever the mechanism(s), the magnitude of the increase inVEGF that we detected in the variants could significantly increase

tumor angiogenesis, based on results reported in several previousstudies, using gene transfection approaches (55, 56).

Our results demonstrate that, in principle, acquired resistance toagents that block tumor cell EGFR function can developin vivo.However, the extent of this resistance, and the rate at which itdevelops, appear encouraging for use of such drugs in the clinic,especially because they are frequently used as chemotherapy or radi-ation sensitizers, rather than single agents. The basis of acquiredresistance in the A431 system appears, at least in part, to have a linkwith tumor angiogenesis, an observation that strengthens the putativelinkage established between oncogene function, including that ofoncogenic receptor tyrosine kinases, and tumor angiogenesis (39).Finally, we do not wish to infer that acquired resistance to EGFRinhibitors, when it occurs, will always involve the latter type ofmechanism. Tumor cell resistance mechanisms with respect to che-motherapeutic drugs and other agents,e.g., ST1571 (17, 57), areusually highly pleiotropic, and the same is likely to be true forinhibitors of EGFR signaling. Indeed, the basis of acquired resistanceof one of the six A431 variants that we analyzed, R6, is different fromthat of variants R1–R5 and may not involve altered angiogenesis. It

Fig. 6. Different patterns of VEGF staining and enlarged vascular channels in tumors derived from A431 R variants.A, D, andG, VEGF staining (red) of A431P, R1, and R5 tumorsections, respectively. In the A431P tumors (A) the staining is localized to subgroups of moderately- to well-differentiated cells. Some tumor cells, usually smaller in size, are negativefor VEGF protein expression. In both R1 and R5 (DandG) tumors, VEGF staining appears diffuse, with the majority of the cells being strongly positive.B, C, E, F, H, andI, stainingof the endothelial cell-associated antigen CD31 (red) in A431P (BandC), R1 (EandF), and R5 (Hand I) tumor sections. Tumor-bearing mice were either treated with PBS (B,E,andH) or with the VEGF-neutralizing Ab 4301-42-35 (C,F, andI) for 1 week prior to tumor recovery (see “Materials and Methods” for details). No significant differences in vasculardensity were found among control tumors of A431P, R1, or R5 (B, E, andH, respectively). However, vascular channels are highly enlarged in both R1 (E) and R5 (H) tumors comparedwith A431P (B) tumors. In R5 tumors, these vascular channels seem highly complex, showing abnormally enlarged lumens divided into several compartments (H). Anti-VEGF treatmentcauses a reduction in blood vessel counts in A431P tumors (C versus B) and a remarkable reduction in both vessel size and complexity, in both R1 (F versus E) and R5 (I versus H)tumors.

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would be of interest, in this respect, to test other EGFR-positive celllines for the possibility of additional mechanisms of acquired resist-ance, and to use different antagonists of EGFR function for theirselection.

ACKNOWLEDGMENTS

We are grateful for the excellent secretarial assistance of Lynda Woodcockand Cassandra Cheng. We thank Joanne Yu and Dr. Guido Bocci for theircritical review of the manuscript and YM BioSciences Inc., Toronto, for the R3and hR3 monoclonal antibodies to EGFR.

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2001;61:5090-5101. Cancer Res   Alicia Viloria-Petit, Tania Crombet, Serge Jothy, et al.   for Altered Tumor Angiogenesis

: A Rolein VivoGrowth Factor Receptor-blocking Antibodies Acquired Resistance to the Antitumor Effect of Epidermal

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