Acquired Resistance to the Antitumor Effect of ... - Cancer Research

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

Acquired Resistance to the Antitumor Effect of Epidermal Growth Factor Receptor-blocking Antibodies in Vivo: A Role for Altered Tumor 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 are among the novel drugs showing great promise for cancer treatment in the clinic. However, the possibility of acquired resistance to such drugs because of tumor cell genetic instabilities has not yet been explored. Here we report the experimental derivation and properties of such cell variants obtained from recurrent tumor xenografts of the human A431 squamous cell carcinoma, after two consecutive cycles of therapy with one of three different anti-EGFR monoclonal antibodies: mR3, hR3, or C225. Initial response to a 2-week period of treatment was generally total tumor regression and was not significantly different among the three antibody groups. However, tumors often reappeared at the site of inoculation, generally after prolonged latency periods, and most of the tumors became refractory to a second round of therapy. Cell lines established from such resistant tumors retained high EGFR expression, normal sensitivity to anti-EGFR antibody or ligand, and unaltered growth rate when compared with the parental line in vitro. In contrast, the A431 cell variants exhibited an accelerated growth rate and a significantly attenuated response to anti-EGFR antibodies in vivo relative to the parental line. Because of the reported suppressive effect of EGFR inhibitors on vascular endothelial growth factor (VEGF) expression, and the demonstrated role of VEGF in the angiogenesis and growth of A431 tumor xenografts, relative VEGF expression was examined. Five of six resistant variants expressed increased levels of VEGF, which paralleled an increase in both angiogenic potential in vitro and tumor angiogenesis in vivo. In addition, elevated expression of VEGF in variants of A431 cells obtained by gene transfection rendered the cells significantly resistant to anti-EGFR antibodies in vivo. Taken together, the results suggest that, at least in the A431 system, variants displaying acquired resistance to anti-EGFR antibodies can emerge in vivo and can do so, at least in part, by mechanisms involving the selection of tumor cell subpopulations with increased angiogenic potential.

INTRODUCTION Among the most successful of the new molecular-targeted drugs for the treatment of cancer are the signal transduction inhibitors (1). These drugs, in general, target specific molecular alterations that are thought vital for such functions as cancer cell proliferation, survival, and ability to induce angiogenesis. Examples include humanized mAbs3 such as Herceptin, directed against the overexpressed EGFR 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 marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported primarily by Grant MT-5815 from the Medical Research Council of Canada (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 Cellular Biology Research, Sunnybrook and Women’s College Health Sciences Centre, S-218 Research Building, 2075 Bayview Avenue, Toronto, ON M4N 3 M5, Canada. Phone: (416) 480-5711; Fax: (416) 480-5703; E-mail: [email protected]. 3 The abbreviations used are: Ab, antibody; mAb, monoclonal Ab; EGF, epidermal growth factor; EGFR, EGF receptor; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; CM, conditioned medium; CMV, cytomegalovirus; TGF, tumor growth 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).

family member erbB2/Her2/neu (2, 3); chimeric mAbs such as C225; or small molecule antagonists such as ZD1839 (Iressa) directed to the EGF (erbB1) receptor (2, 4), ST1571, which is highly specific for the bcr-abl kinase (5), and to Ras farnesyltransferase inhibitors (6). Some of these drugs are already clinically approved, e.g., Herceptin and ST1571, or have shown impressive effects in clinical trials, either on their own or in combination with standard treatments such as radiation or chemotherapy, e.g., C225 in several types of solid tumors (7). Indeed, a recurring theme of signal transduction inhibitors is the ability of such drugs to function as effective radiation or chemosensitizers, thereby rendering tumor cells more vulnerable to the cytotoxic effects of standard therapies (8 –12). This may occur by various mechanisms such as lowering the threshold for apoptosis or impairing DNA repair processes (12, 13). A surprising feature of the preclinical research undertaken on anticancer signal transduction therapy is the almost total lack of information dealing with the issue of acquired resistance to such drugs. This stands in conspicuous contrast to conventional therapeutics such as all classes of cytotoxic chemotherapeutic drugs. Because genetic instabilities of cancer cells are a major driving force of acquired drug resistance (14, 15), it would be expected that acquired resistance would eventually develop to signal transduction inhibitors that target exclusively a cancer cell-associated genetic or phenotypic alteration. An example of this possibility is the emergence of variant resistant cells to a Ras farnesyltransferase inhibitor after prolonged exposure of drug-sensitive tumor cells in vitro (16). Another is the development of resistance to ST1571 in vivo in bcr-abl-positive chronic myelogenous leukemia cells grown in nude mice, as a result of the binding of ␣1 acid glycoprotein to the drug (17). Similar studies undertaken in an in vivo context with other signal transduction inhibitors, including those targeting EGFR, are lacking. With this in mind, we embarked on a long-term study to isolate and to begin characterizing tumor cell variants resistant to EGFR-targeting drugs such as monoclonal neutralizing Abs. Our approach was based on the observation that a small number of human tumor cell lines, e.g., the A431 squamous cell carcinoma or the 253J B-V bladder cell carcinoma, when grown as established xenografts, can be induced to substantially or even totally regress when treated with anti-EGFR Abs such as C225 (18, 19). We reasoned that if similarly treated tumors were then followed for long periods, variants might eventually emerge, and if so, such recurrent tumors could represent the outgrowth of the progeny of rare variants no longer fully responsive to the EGFR-targeting drug. The rationale for undertaking such experiments was not only for the obvious intrinsic value of deriving such variants but also as a means of exploring in greater depth the hypothesis that EGFR-targeting agents function in vivo, at least in part, by blocking angiogenesis (19, 20). For example, C225 treatment can block the production of several proangiogenic 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 its therapeutic efficacy in vivo (20). If this assumption was correct,

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RESISTANCE TO EGFR ABs

resistant variants could arise in vivo by such mechanisms as increased VEGF production (23) or by activating one or more alternative proangiogenic growth factors. The present experiments were performed using three different monoclonal anti-EGFR Abs and the A431 human tumor cell line. The Abs included an unmodified mouse mAb (mR3), a humanized version of this Ab (hR3), and an independent, chimeric, Ab (C225). We found that variants could indeed arise, usually only after a protracted period of time posttreatment, and not in all mice. All of the six independent variants that we analyzed, with the exception of one, displayed increased VEGF production. These in vivo selected variants should be useful tools to investigate the role of the EGFR in cancer growth and angiogenesis, as well as for studies designed to improve the use of anticancer drugs that target tumor cell-associated receptor tyrosine kinases, particularly the EGFR.

regressed, with only a few exceptions. Mice were then maintained and monitored for tumor recurrences, which were first observed in a proportion of mice in the 0.25-mg-dose-per-injection groups, 2 months after the beginning of treatment (see “Results” for details). As soon as the recurrent tumors reached a size similar to that of the initially treated tumors (i.e., 200 – 600 mm3), a second round of treatment was initiated using the same Ab, dose, and schedule initially applied to treat that tumor-bearing mouse (e.g., 0.25 mg of mR3 every 48 h for 2 weeks, if the recurrent tumor was in a mouse from the mR3 lower-dose treatment group). Tumors that did not respond during the 1st week of treatment with the 0.25-mg dose were then treated with 1 mg per injection according to the same schedule (i.e., every 48 h) during the next 2 weeks or less, depending on tumor response. Tumors initially treated with a dose of 1 mg per injection were treated only with the same dose and schedule. Tumors that did not regress or remain dormant (as in the first round of treatment) after the second treatment were considered resistant. The corresponding mouse was then euthenized, and the tumor cells were recovered in vitro by enzymatic treatment (29) under aseptic conditions and were grown in tissue culture. Cells were passaged in culture at least three times before any further testing. DNA Fingerprinting. Genomic DNA (10 ␮g) was digested with HinfI MATERIALS AND METHODS (New England Biolabs Inc., Mississauga, Ontario, Canada), resolved in a 0.6% Abs. The neutralizing anti-EGFR mAb C225, a human-mouse chimeric agarose gel, and transferred onto a nylon membrane. Banding pattern was form of the original mouse monoclonal 225, initially described by Kawamoto evaluated after hybridization with a NICE-labeled 33.15 multilocus probe et al. (24) was manufactured by ImClone Systems, Inc. (New York, NY). The (Cellmark Diagnostic, United Kingdom) used in combination with CDP-Start humanized anti-EGFR mAb hR3 as well as its murine form mR3 (25, 26) were chemiluminescent substrate (Tropic Inc., Bedford, MA) following the manuprovided by York Medical (YM) BioSciences, Inc. (Mississauga, Ontario, facturer’s instructions. Western Blotting. To detect the expression of EGFR and HER-2, cell Canada). The anti-hVEGF mAb 4301-42-35 was described previously (27). Cell Lines and Culture Conditions. The human epidermoid carcinoma lysates were obtained as described previously (28). Proteins were resolved by cell line A431 was obtained from the American Type Culture Collection SDS-PAGE and transferred onto an Immobilon-P membrane (Millipore, Bed(ATCC, Rockville, Maryland). Independent variants of A431 cells resistant to ford, MA). After blocking in 1% casein/TBST, the membrane was incubated EGFR-blocking Abs (R cells) were derived during this study (see “Derivation with either the rabbit polyclonal anti-EGFR Ab SC-003 or the anti-HER-2/Neu of EGFR Ab-resistant Variants” below). Both the parental and A431 cell Ab SC-284 (Santa Cruz Biotechnologies, San Francisco, CA) at 0.2 ␮g/ml, variants were cultured in DMEM supplemented with 5% FBS (Life Technol- then washed and incubated with a peroxidase-conjugated goat antirabbit Ab ogies, Inc., Grand Island, NY). HUVECs were cultured on gelatin-coated (Jackson Immunoresearch Laboratories Inc., West Grove, PA). The loading dishes as described previously (28). Cultures were maintained at 37°C and 5% was verified by ␣-actin probing with A5441 Ab (Sigma Chemical Co.) at 0.22 ␮g/ml. Phosphorylated EGFR was determined in lysates from TGF-␣-stimuCO2 in a humidified incubator. Determination of in Vitro Cell Growth. The effect of different conditions lated cells using the mouse anti-pEGFR no. 324864 (Calbiochem) at 0.1 on anchorage-dependent growth of parental and variant A431 cells was meas- ␮g/ml. The peroxidase-conjugated antimouse W402B Ab (Promega) at 0.2 ured by a [3H]thymidine incorporation assay in monolayer cultures. Briefly, ␮g/ml was used as secondary to detect mouse-derived primary Abs. Blots were visualized using the ECL chemiluminescence kit (Amersham Corp., Arlington recently trypsinized cells were resuspended at a density of 3 ⫻ 103 per 100 ␮l of DMEM (Life Technologies, Inc.) containing 1% FBS, and plated in a Heights, IL). Measurement of Human and Mouse VEGF Protein Levels in CM. A 96-well plate (Nalge Nunc Inc., Naperville, IL). Plates were incubated under commercially available human or mouse VEGF ELISA kit (R&D Systems, standard conditions for 24 h, at which point, 100 ␮l of the given treatment (2⫻ concentrated C225, hR3, or TGF␣ in 1% FBS/DMEM) was added. Cells were Inc., Minneapolis, MN) was used to determine VEGF protein levels in CM incubated for an additional 48 h, and 2 ␮Ci/well [3H]thymidine (Amersham) obtained from A431, Ab-resistant variants, or mVEGF-transfected clones. were added for the last 4 – 6 h. The incorporated radioactivity was determined Briefly, cells were plated at a density of 105 cells/0.5 ml/well in a 24-well plate using a Betaplate liquid scintillation counter (Pharmacia). The same assay was under normal serum conditions and allowed to reach 80% confluency, at which performed to test the effect of CM from A431 or R cells on the growth of point, the medium was replaced by DMEM/1% FBS with or without C225. HUVECs. In this case, cells were plated on gelatin-coated wells in complete Medium was collected after 24 h and cells were counted as described previgrowth medium, which was replaced with CM 24 h later. To compare the ously (20). Northern Blot Analysis. Approximately 107 cells were used for the exgrowth 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 traction of total RNA using Trizol (Life Technologies, Inc., Gaithersburg, MD) (DMEM ⫹ 5% FBS). Cells were incubated under standard conditions and following the manufacturer’s protocol. VEGF and TSP-1 Northern blots were performed as described previously(30). Equal RNA loading and the efficiency counted every day to day 6 using a Coulter counter. Growth medium was replaced at day 3. Cell number after 24-h incubation was taken as baseline. For of the transfer were visualized by ethidium bromide staining of both the gel the clonogenic survival assay, cells were plated under the same conditions, and the membrane. Derivation of A431 Variants Overexpressing VEGF. An expression except for the lower density (103/well). The colonies formed after 1 week were plasmid encoding mVEGF164 (981 bp) driven by a CMV promoter, a generous stained with Cristal Violet and counted by naked eye. Evaluation of the in Vivo Antitumor Activity of Three Anti-EGFR gift from Dr. Kevin Claffey (Beth Israel Deaconess Medical Center-Research mAbs. A431 cells (5 ⫻ 106/200 ␮l of PBS) were inoculated s.c. into the right North, Boston, MA), was transfected into A431 cells using SuperFect reagent flank of 6 – 8-week-old SCID mice (average weight, 23 g). After 10 days (Qiagen Inc., Valencia, CA) following the manufacturer’s instructions. Control (tumor average size, 300 mm3) the animals were randomly separated into clones resulted from transfection with an empty vector. Positive clones were seven groups (four to five mice per group). “High-dose” groups were treated selected with increasing concentrations (500 – 800 ␮g/ml) of Geneticin (Life with 1 mg of C225, hR3, or mR3 administered i.p. every 48 h. “Low-dose” Technologies, Inc., Grand Island, NY) and were maintained in the presence of groups were treated with one-fourth of this dose, i.e., 0.25 mg of the given Ab 800 ␮g/ml of the drug. Three of the clones (mVEGF.S1, mVEGF.S2, and following the same schedule. Control group was treated with PBS. Treatment mVEGF.S3) were chosen for an EGFR Ab resistance test in vivo, which was performed after the low-dose-treatment regimen described in a previous secwas stopped after a total of eight injections. Derivation of EGFR Ab-resistant Variants of A431 Cells. After 2 weeks tion. VEGF-neutralizing Treatment in Vivo. VEGF-induced angiogenesis was of continuous treatment with either the higher dose (1 mg per injection) or a examined in both R and A431 parental cells after a short VEGF-neutralizing in lower dose (0.25 mg per injection) of the Abs C225, hR3, or mR3, most tumors 5091


vivo experiment. Briefly, SCID mice (five per group) bearing A431, R1, and R5 s.c. tumor xenografts (average tumor volume, 250 mm3) were treated with two injections of either PBS (control) or 200 ␮g of anti-hVEGF Ab 4201– 42-35 (Novartis). Tumors were recovered for immunohistochemical analysis after 1 week (average tumor volume: 321 mm3, 452 mm3, and 423 mm3 for A431, R1, and R5, respectively). Immunohistochemistry. VEGF staining was performed on formalin-fixed and paraffin-embedded specimens as described previously (20). Blood vessels were visualized by CD31 staining, which was performed on 7-␮m-thick cryosections using the rat antimouse CD31 no 01951D (PharMingen) at a concentration of 1 ␮g/ml 1:500. Biotinylated rabbit anti-rat (Jackson Immunoresearch) and Histostain kit (Zymed Laboratories, San Francisco, CA) reagents were used to reveal antigen as a red signal. Contrast was provided with Harris Hematoxylin (Surgipath Canada, Inc., Winnipeg, Manitoba, Canada). Statistics. Statistical analysis was performed using the GraphPAD InStat software, version 1.14 (GraphPAD Inc., San Diego, CA). In vivo experiments were analyzed by a one-way ANOVA test coupled to a Bonferroni test for multiple comparisons. In vitro data were compared by a t test.

RESULTS Antitumor Activity of hR3 and C225 mAbs. One of the two main aims of this study was to evaluate the possibility that the use of EGFR-blocking Abs in vivo might result in a selection of drugresistant variants. We decided to explore this subject using at least two different EGFR-neutralizing Abs, C225 and hR3, which possess similar binding affinities but different specificities (24, 26). We chose the human squamous cell carcinoma A431 based on its extensive use as a model over the past 15 years in the study of the efficacy of many different antagonists of EGFR, including C225 (18, 31, 32). We have previously found ( by using [3H]Thymidine incorporation assays) that the maximum growth inhibitory effect of either C225 or hR3 in vitro is 40%. This was achieved on monolayer cultures after 48-h exposure to doses ranging between 50 and 100 ␮g/ml.4 To investigate the relative efficacy of the Abs (C225, mR3, and hR3) in vivo, we tested their antitumor properties on A431 s.c. tumor xenografts, thus resembling the orthotopic location for this tumor, implanted into SCID mice. Under such conditions, the three Abs seemed equally efficient in causing total regression of well-established tumors after 2 weeks of treatment (eight injections) with a low- or a high-dose regimen (Fig. 1), at least during the first 3 weeks of follow-up. No significant difference was observed among the groups when compared by dose or by Ab type (P ⬎ 0.05 in all cases). Sporadic Tumor Recurrence after Treatment with Anti-EGFR Abs. After the tumor regressions became evident in the first responders, treatment was terminated, and the mice were monitored for tumor recurrence. The first such recurrences occurred 1.5 months after termination of the first round of treatment in the 0.25-mg-dose-perinjection groups injected with mR3 or hR3 (Fig. 1, A and C). In each of these groups, one tumor did not regress completely after the eight injections, which perhaps suggests a low level of intrinsic resistance to EGFR inhibition. These tumors were the first to regrow (as expected) over a period as short as 18 days after the maximum antitumor effect was achieved (Fig. 1, A and C). In the 0.25-mg-dose-perinjection C225 treatment group, one tumor recurrence was observed after a latency period of 2 months (Fig. 1E). Recurrence of tumors in the higher-dose groups took longer and was first evident after a 4.5-month latency period in the mR3-treatment group and after 1.5months in the hR3-treatment group (Fig. 1, B and D). Recurrence also took a long period to occur in the higher-dose C225 group, e.g., after 3.5 months (Fig. 1F). The frequencies of tumor recurrence for C225, 4

T. Crombet, unpublished observations.

hR3, and mR3, respectively, were 20, 40, and 80% for the 0.25-mgdose treatment groups and 25, 25, and 80% for the 1-mg-dose groups. Mice with recurrent tumors in all of the groups subsequently received a second round of treatment (Table 1). A431 Recurrent Tumors Escape a Second Round of AntiEGFR Treatment: Derivation of Variants. While administering the second round of treatment, we observed that in most cases the tumors did not respond to the same extent as they did originally. Tumors regrew rapidly during, or immediately after, this cycle of therapy. Cell lines established from these tumors were designated as follows: R1 and R2 [two morphologically different subpopulations of a resistant tumor from the mR3, 0.25-mg-dose group (Fig. 1A)]; R3 and R4, from two 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 of the resistant variants are summarized in Table 1. A common cellular origin for the A431 R variants was demonstrated using a DNA fingerprinting analysis (Fig. 2A). Identical banding patterns suggest that all of the cell lines are A-431-derived and are not comprised of contaminating human or mouse tumor cells. Having established this, two resistant variants (R1 and R5) were chosen for a final in vivo resistance test based on a pilot screening. R1 and R5 were both representative of the similar in vivo growth properties found in the 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 per treatment group), and tumor growth was monitored every 4 days to compare tumor take rates. Treatment was initiated when tumors reached an average size of 200 –300 mm3 and was administered as described for the 0.25-mg/injection group. Both R1 and R5 manifested an accelerated growth in vivo (Fig. 2B), which became evident 18 days after tumor cell injection and which became significant by day 22. The increased growth rate of these variants was even more significant after a 30-day period of growth, as observed in an independent tumor-take experiment (data not shown). These in vivo results stand in marked contrast to the in vitro data, in which similar growth rates between the R variants and the A431P cells were observed (Table 2). When the variants were tested for their response to EGFRblocking Abs (e.g., C225) in vivo, both R1 and R5 showed a similar and significant delayed response. (Fig. 2C). By day 45, all of the five tumors included in the A431 group had totally regressed, whereas in the R1 and R5 groups, only one and two such total regressions were observed, respectively. Maximum average responses for R tumors were registered at day 45. The remaining R1 and R5 tumors did not regress by day 86, at which point at least one of the tumors in each group (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 was seen by day 86 in any of the animals included in the A431 group treated with C225. Similar results were obtained after hR3 treatment (data not shown). Absence of Changes in EGFR Status in A431 Variants Resistant to EGFR-blocking Abs in Vivo. As a second step in the characterization of the A431-resistant variants, we examined the level of EGFR expression, because resistance to treatment with EGFR-blocking agents could have obviously resulted from loss or reduction in the level of expression of the receptor itself. However, we did not detect evidence to implicate such a mechanism, because five of six variants showed levels of EGFR similar to those expressed by A431 parental cells (Fig. 3A). To demonstrate that EGFR was equally functional in A431 and the variants, we stimulated the different cell lines with increasing concentrations of TGF-␣ and measured the level of phospho-active EGFR. Activation of EGFR was found to be equivalent

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Fig. 1. In vivo derivation 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 2 weeks. 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 included in 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 treated with 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 immediately afterward (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-mg dose 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 of treatment. 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.

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

This conclusion is reinforced by the similar growth responses of A431 cells and the R variants to the inhibitory effects of C225 (50 ␮g/ml) and TGF-␣ (100 ng/ml) treatment in vitro (Table 2). High concentrations of EGFR-specific ligands, i.e., EGF and TGF-␣, have been reported to cause a potent growth inhibition of A431 cells, a phenomenon associated with their considerably elevated expression of EGFR (33, 34). The virtually identical pattern of growth inhibition of A431

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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 I

mAb (dosing)a mR3 (0.25 mg)

II

mR3 (1 mg)

Mice per Recurrent Tumor group tumors (%) I.D.b 5

5

4 (80)

4 (80)

III

hR3 (0.25 mg)

5

2 (40)

IV V VI

hR3 (1 mg) C225 (0.25 mg) C225 (1 mg)

4 5 4

1 (25) 1 (20) 1 (25)

Second cycled (no. of injections ⫻ dose)

Initial size first cyclec

Initial size second cycle

T2 T3 T4 T5 T1 T3 T4 T5

144 113 107 208 270 352 304 298

550 225 239 587 600 575 451 368

5 8 8 7 1 7 7 8

T2 T5 T3 T1 T2

270 352 225 221 288

320 446 361 496 304

8 ⫻ 0.25 mg ⫹ 8 ⫻ 1 mg same as T2 8 ⫻ 1 mg 2 ⫻ 0.25 mg 8 ⫻ 1 mg-break- 8 ⫻ 1 mg

⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻

0.25 mg 0.25 mg 0.25 mg ⫹ 8 ⫻ 1 mg 0.25 mg 1 mg 1 mg 1 mg 1 mg

Recurrent variant obtained no R1, R2 no no no R6 no no R3 R4 R5 no no

Observations Mice 2 and 5 euthanized Cells from T4 did not plate. Mouse 1 euthanized (open tumor) Cells from T4 did not plate. Mouse 5 was found dead after eighth injection of second cycle.

Mouse was euthanized Tumor did not regress or grow back after second cycle. It began to regrow during a third cycle. Cells could not be recovered.

a

This dose of the given Ab was injected every other day for 2 weeks during the first cycle of treatment. 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. b

and the R variants R1-R5 by TGF-␣ is consistent with their equivalent levels of EGFR expression. Similarly, we did not find significant changes in the levels of expression of HER-2 (Fig. 3A), the second member of the EGFR family also known to be involved in cell transformation and tumor progression (35). HER-2 has been shown to heterodimerize with EGFR, altering the patterns of downstream cellular signaling (36, 37). With the exception of R6, which appears to express lower levels of HER-2, none of the variants expressed levels of HER-2 that were significantly different from that of the parental cells, which suggests that changes in HER-2 expression are not a common feature of the resistant variants. In agreement with the decreased expression of EGFR and HER-2 found in R6, this cell line showed an unusually slow growth rate (data not shown). Constitutive VEGF Up-Regulation in Anti-EGFR-resistant Variants of A431. Because of the clear discrepancy between the in vitro findings (in which resistance was not seen) and in vivo findings (in which resistance was detected), we considered the possibility that the R variants possess increased survival properties under restricted growth conditions or, alternatively, an increased angiogenic ability, which could translate into both superior growth and prolonged survival in vivo. Because neither C225 nor hR3 is able to induce cell death of A431 cells under restricted culture conditions, e.g., threedimensional/low serum cultures,4 we could not compare differences in survival in response to Ab treatment under such conditions. An alternative experiment, a clonogenic survival analysis, failed to show a survival advantage in the resistant variants (Table 2), which suggests that an improved basal cellular survival is unlikely to explain the in vivo growth advantage. To evaluate the possibility of enhanced proangiogenic capacity in the R variants, we measured the levels of the proangiogenic factor VEGF, at both the RNA and protein levels in all of the cell lines. The importance of this growth factor for tumor angiogenesis has been established for A431 cells (38), and, furthermore, we have reported previously that the EGFR-neutralizing Ab C225 is able to down-regulate VEGF protein up to 50% in vitro and to an even greater extent in vivo in these cells (20), which we hypothesized could account for the reduced vessel counts found in this and other tumor models after treatment with EGFR-blocking agents (19, 20). We found that five of the six resistant variants, R6 being the exception, expressed at least 2-fold more VEGF mRNA and protein than the A431P cells (Fig. 3, B and C). Although VEGF levels were still down-regulated in vitro by 50% as a result of treatment with C225 (50 ␮g/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 VEGF expression was merely the result of in vivo passaging of A431 cells, by comparing the expression of VEGF with that in A431 parental cells passaged in vivo for 14 days. These cells expressed levels of VEGF that were similar to those of the parental cells and also showed an equivalent growth rate in vivo (data not shown). In addition, we also observed an increase in the growth response of HUVECs to the CM of R variants 1 to 5 (Fig. 3D), but not to that from variant R6 (data not shown), suggesting an increased angiogenesis-inducing capacity in the VEGF-overexpressing cells. The absence of an increase in VEGF expression in addition to the reduced levels of EGFR, HER-2, and the delayed growth rate found in R6 cells suggests that this variant escaped the antitumor effect of anti-EGFR Ab treatment by a different mechanism from that in the other variants. In contrast to the results with VEGF, no common pattern was observed in the levels of TSP-1 (Fig. 3B), a naturally occurring angiogenesis inhibitor previously shown to be down-regulated by activation 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-1 transcript levels. We have no explanation for this increased TSP-1 and its biological impact because it is obvious from the in vivo data that increased TSP-1 does not confer a growth disadvantage to R5 (Fig. 2B). In Vivo Resistance to EGFR-neutralizing Ab of A431 Cell Lines Engineered to Overexpress VEGF. Because of the apparent increase in VEGF in the resistant versus A431P cells, we decided to test whether or not such increased VEGF levels could account, at least in part, for the resistant phenotype to EGFR-blocking agents in vivo. We reasoned that the resistance of these variants could result from the inability of EGFR-blocking Abs to down-regulate VEGF levels to the same extent that they do in the parental cells. This would limit the ability of these agents to achieve the same degree of antiangiogenic effect as in the case of A431 parental tumors. Consequently, an endogenous EGFR-independent increase in VEGF expression could act as a de facto resistance mechanism for EGFR inhibitors. To test this hypothesis, we decided to boost VEGF production in A431 parental cells using a gene transfection procedure. An expression vector was used that encoded the murine form of VEGF164 (equivalent to human isoform VEGF165), a secretable form of VEGF that appears to be overexpressed in the R variants of A431 cells. After

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

preliminary screening, three clones (mVEGF.S1, mVEGF.S2, and mVEGF.S3), expressing varying amounts of VEGF ranging between 800 and 12,000 pg/ml, were selected for further analysis. The levels of mVEGF produced by these clones were completely refractory to treatment with C225 (Fig. 4A). This was expected because the expression of exogenous mVEGF164 is under the control of the constitutively active and strong CMV promoter. In addition, the clones were found to maintain an intact in vitro response to C225 in terms of growth inhibition (data not shown) and expression of endogenous

hVEGF (Fig. 4A). We compared the in vivo responsiveness of VEGFoverexpressing (mVEGF.S) and A431P cells to the C225 Ab. As previously found, a low-dose regimen was sufficient to cause total tumor regression of well-established A431 parental and control (vector-transfected) tumors but was unable to do so in the case of the VEGF transfectants (Fig. 4, B and C), which showed significant (P ⬍ 0.05) resistance compared with the control groups. No significant difference was found (P ⬎ 0.05) among the three VEGFoverexpressing clones, although a tendency of a direct correlation between the level of VEGF produced and the degree of resistance was observed. In this regard, it is important to mention that the clone expressing the lowest amount of exogenous VEGF (mVEGF.S1) secretes an overall quantity of this protein that is in close similarity to that secreted by some of the resistant variants themselves. The fact that this clone still exhibits a significant resistance to EGFR-blocking treatment in vivo reinforces the idea of an involvement of VEGF in the resistance mechanism. Histopathological analysis of 3-week-old s.c. tumors of parental and mVEGF164-transfected A431 cells revealed no differences in cellular morphology but did reveal a slightly larger central area of necrosis in the mVEGF164 tumors, probably reflecting their more accelerated growth rate. In addition, tumors derived from clones S2 and S3 exhibited focal areas of hemorrhage that were particularly large in the S2 tumors (data not shown), which suggests that the extremely nonphysiological amount of VEGF164 produced by this clone could be responsible for this characteristic. Staining of blood vessels with the endothelial-specific marker CD31 did not show an increase in the microvessel density of mVEGF-overexpressing tumors but showed a striking difference in the size of the vascular channels found in mVEGF164 transfectants compared with those found in A431 parental tumors (Fig. 5). Vessels in mVEGF-expressing tumors were highly enlarged, consisting in many cases of a complex structures suggestive of splitting of the vessel lumen by endothelial intravessel walls (Fig. 5, B and C). The overall appearance of these vessels seem to resemble that found in the previously described “mother vessels” (40), which result from increased mVEGF164-dependent angiogenesis. The functionality and impact of these enlarged vessels on tumor biology is still unclear. However, in the context of the s.c. A431 model, these vessels appear to be a hallmark of VEGF164-dependent angiogenesis, which did not result, in this case, in an increase in microvessel density. This altered angiogenesis could account for the reduced response of A431 tumors to EGFR-blocking Abs, such as C225 (Fig. 4, B and C). The general appearance of tumor tissue from mVEGF164-transfectant and parental tumors after C225 treatment would appear to support this hypothesis. A431 tumors appear almost totally necrotic, with only a rim of viable tissue in the periphery (Fig. 5E), which virtually lacks blood vessels (Fig. 5F). In contrast, C225treated mVEGF164 transfectant tumors are comprised of a mixture of

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

Cell type A431 R1 R3 R4 R5

TGF-␣dependent C225-dependent growth growth inhibition inhibition a (% ⫾ SD) (% ⫾ SD)a 33 ⫾ 5.6 41 ⫾ 10.0 43 ⫾ 5.3 33 ⫾ 11.4 35 ⫾ 8.1

91 ⫾ 5.2 80 ⫾ 3.2 88 ⫾ 7.9 85 ⫾ 7.5 84 ⫾ 9.4

Doubling time (h ⫾ SD)b

Clonogenic survivalc (colony, n ⫾ SD)b

30.2 ⫾ 2.3 28.7 ⫾ 0.6 30.6 ⫾ 1.1 32.4 ⫾ 1.1 35.4 ⫾ 1.6

186 ⫾ 4.5 189 ⫾ 1.5 111 ⫾ 6.0 176 ⫾ 13.2 58 ⫾ 7.1

a 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.

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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-induced activation 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 A431P cells, 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-␣. 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). ␤-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 more VEGF 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 (bottom panel) 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 thymidine incorporation 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 measurements and are standardized to cell number at the time of CM collection. Error bars, SD.

necrotic and viable tissue (Fig. 5G), with highly enlarged vascular channels supporting the growth of tumor cell “cuffs” (Fig. 5H). Taken together, the results suggest that increased VEGF expression, which is refractory 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 Vascularity of A431 Variants. To determine whether the elevated VEGF expression of the resistant variants could result in an altered VEGF-dependent angiogenesis phenotype, we analyzed tumor sections obtained from a short in vivo experiment, in which mice bearing A431, R1, and R5 tumors were treated with either PBS (control) or anti-hVEGF Ab for 1 week, after which a moderate antitumor effect was observed in each group (treated:control ratio: 0.66, 0.61, and 0.81 for A431, R1, and R5, respectively; see “Materials and Methods” for details). Both VEGF expression and vascularity were evaluated by immunostaining. We observed that in both A431P and R tumor sections, VEGF staining was 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 welldifferentiated squamous morphology. Slightly smaller cells with higher nuclear/cytoplasmic ratio surrounding these islands were negative for VEGF staining (Fig. 6A). In contrast, VEGF staining in the R1 and R5 variants was more diffuse and present in almost all of the

viable cells, which showed a relatively high nuclear:cytoplasmic ratio and a moderately differentiated morphology (Fig. 6, D and G). Staining for CD31 showed no obvious increase in the vessel density of R1 and R5 tumors compared with those of A431P cells. Instead, the size of the vessels appeared significantly larger in the R variant tumors (Fig. 6, B, E, and H). A majority of blood vessels in the R1 tumors were more than 2-fold larger than those detected in A431, whereas R5 tumors appeared to possess even more expanded and complex vascular 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 be VEGF-dependent, because they became less common or smaller after 1 week of treatment with the anti-hVEGF Ab 4301-42-35 (Ref. 27; Fig. 6, F and I). In contrast, the same treatment caused a reduction in the microvascular density in A431 P tumors (Fig. 6, C versus B) but no 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 advanced clinical development status of others, such as IMC-225 (7, 41) and Iressa/ZD1839, both of which block EGFR function, highlight the

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Fig. 4. Exogenous expression of VEGF by mVEGF164 transfectants and its impact on the 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 mouse protein, as expected. Horizontal black bar, the average level of hVEGF165 protein expressed by A431P cells as well as mVEGF164 transfectants. C225 down-regulates only the endogenous human protein (hVEGF165) but not the exogenous mVEGF164, as expected. The data represent the average from two independent determinations. B, antitumor effect of C225 Abs on the in vivo growth of s.c. tumor xenografts derived from A431P and vector or mVEGF164-transfectant clones S1–S3. C225 Ab treatment (arrow, day of injection) causes tumor regression only in A431P and vector control tumors but not in the mVEGF164 transfectants. C, tumor-bearing mice after treatment with C225. Tumor size is representative of the average for each treatment group.

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

long-term-observation experiments that we detected the emergence of resistant variants. Second, it should be noted that the only possible target of the Abs in our model was the EGFR expressed by the implanted, genetically unstable human A431 tumor cells growing in the SCID mice. In contrast, in the clinical situation, such Abs would have the potential opportunity to block EGFRs expressed by normal (genetically stable) host endothelial cells of newly formed tumor blood vessels (42, 43) particularly in TGF-␣-expressing tumors, in which TGF-␣ itself may up-regulate EGFR expression in activated endothelial cells (44, 45). As a result, EGFR-blocking agents might conceivably block angiogenesis not only indirectly but also directly, especially when such drugs are combined with chemotherapy. This was shown recently by Bruns et al. (22, 46), who reported significant induction of endothelial cell apoptosis in human pancreatic or prostate carcinoma xenograft microvessels by combination treatment with either a small molecule EGFR antagonist or C225 and gemcitabine. Third, acquired resistance may also be delayed by more protracted treatment with the Abs, i.e., continuing the treatment in tumors that do not regress after eight injections and even after tumors have regressed, or by combining the Ab treatment with other therapeutic modalities such as chemotherapy or radiation, which is currently the way EGFRblocking drugs are used clinically (7, 41). In this regard, another interesting feature of our results is the necessity of undertaking very-long-term (e.g., up to one year) in vivo experiments to detect the emergence of resistant variants, an approach lacking in virtually all previous studies using such therapeutic agents. We must also acknowledge the possible influence on the results as a consequence of using exclusively the A431 cell line for our studies, a cell line that expresses a very high number of EGFRs and that is known for its hypersensitivity to EGFR-blocking drugs in vivo. Analysis of other cell lines that have fewer numbers of EGFR may not reveal evidence of acquired resistance or, alternatively, may reveal evidence for such resistance but involving different mechanisms. Possible Impact of Altered VEGF Expression and Tumor Angiogenesis in the Resistant Phenotype. One of the most interesting (and unexpected) findings of this study was the absence of an obvious alteration in the EGFR status in the R variants, which we established by different means, including Western blot analysis of EGFR expression and evaluation of the growth response of the variants to EGFR-blocking Abs (C225 and hR3), as well as the determination of levels of phospho-active EGFR after stimulation with increasing concentrations of TGF-␣. We reasoned that major changes in the level of expression of EGFR and/or its ligands could translate into a different biological response to ligand-induced stimulation and/or phosphorylation (i.e., activation). However, we did not find any evidence for such changes. Of considerable importance in this regard was the virtually identical in vitro growth characteristics and inhibitory responses to treatment with the anti-EGFR blocking Abs of the variants, compared with the parental cell line. Thus, interestingly, the resistant phenotype manifested itself only in vivo. One possible explanation for this would be a reduced sensitivity to undergoing Fc receptor-mediated ADCC, a host process that can take place only in vivo and that has been implicated as a major determinant of response to certain therapeutic Abs such as Herceptin and Rituxin (47). However, ADCC does not appear to be similarly important for the antitumor activity of C225, because F(ab⬘)2 fragments of this Ab, which cannot activate an ADCC response, still retain a significant growth inhibitory activity against A431 tumor xenografts (48). Hence, we reasoned that an alternative host-dependent mechanism, namely angiogenesis (a process required in vivo but not in vitro for tumor growth), could conceivably contribute, at least in part, to the resistant phenotype.

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Fig. 5. Increased vessel size and complexity in tumors derived from mVEGF164 transfectants cells as compared with A431P tumors. A, A431P. B, mVEGF.S1. C, mVEGF.S2. D, mVEGF.S3. All of the three mVEGF transfectant tumors exhibit highly enlarged vascular channels compared with parental tumors. The widely enlarged lumen of these vessels appears often with a complex structure, divided into smaller compartments by CD31positive intravascular septae (B and C, insets top right). E, low-power (⫻2.5) magnification of A431P tumor after treatment with C225 Ab. Most tumor tissue appears necrotic, with only a small rim of viable tissue (blue) surrounding the large (brown), dead central area. F, at a higher (⫻10) magnification, very few small vessels are seen in the viable tissue area of the C225-treated A431P tumors. G, mVEGF.S3 tumor after treatment with C225 Ab. Tumor tissue appears as a mosaic of necrotic (brown) and viable (blue) areas. H, higher magnification (⫻10) of the same tumor section showing a cuff of viable cells supported by a very large central vessel. A–D, ⫻10; highlighted areas in right insets, ⫻20. Tumors (E–H) were treated with four doses of 0.25 mg C225, given i.p every other day.

We, therefore, examined the R variants for changes in the expression of VEGF, not only because VEGF is a potent inducer of tumor angiogenesis in general but also because A431 cells have been shown to be highly dependent on VEGF for angiogenesis and, hence, in vivo growth (38). We found that five of six of the resistant variants express a significantly higher level of VEGF than does the parental line. Relative VEGF levels appeared to correlate with the in vivo growth of the variants as well as with their angiogenic profiles. Staining of VEGF on sections derived from R1 and R5 s.c. tumor xenografts suggests 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 is unknown, but it is likely that anti-EGFR Ab treatment selects for A431 subpopulations with an elevated degree of VEGF expression. In addition to altered patterns of VEGF expression, we also found that variants R1 and R5 possessed conspicuously large blood vessels, which we suggest is the result of increased VEGF-dependent angiogenesis, because blocking VEGF signaling with a VEGFneutralizing Ab reduces the size of these vessels (Fig. 6). The presence of such large vessels is a hallmark of pathological VEGF-

dependent angiogenesis (40, 49). A recent study in which the properties of newly formed vessels induced by VEGF/VPF164 have been investigated in detail showed that large vessels are commonly found in situations of VEGF overexpression and are referred to as “mother vessels” (40). We found a striking resemblance between these 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 transfected with the mVEGF164 gene. Our results raise the interesting possibility that, when applicable, assessment of changes in the number of such mother vessels (as opposed to changes in microvessel counts or density) could be a useful surrogate marker to evaluate and monitor the activity of certain anti-angiogenic drugs. However, measurement of vascular density seems inappropriate, as it is difficult to establish the margins of an individual vessel. Because of their compartmentalization, it is difficult to determine whether the structures that we detected correspond to several vessels or to just one vessel, subdivided into several compartments. For this reason, we did not undertake vessel counts in the present study.

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Fig. 6. Different patterns of VEGF staining and enlarged vascular channels in tumors derived from A431 R variants. A, D, and G, VEGF staining (red) of A431P, R1, and R5 tumor sections, 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 negative for VEGF protein expression. In both R1 and R5 (D and G) tumors, VEGF staining appears diffuse, with the majority of the cells being strongly positive. B, C, E, F, H, and I, staining of the endothelial cell-associated antigen CD31 (red) in A431P (B and C), R1 (E and F), and R5 (H and I) tumor sections. Tumor-bearing mice were either treated with PBS (B, E, and H) or with the VEGF-neutralizing Ab 4301-42-35 (C, F, and I) for 1 week prior to tumor recovery (see “Materials and Methods” for details). No significant differences in vascular density were found among control tumors of A431P, R1, or R5 (B, E, and H, respectively). However, vascular channels are highly enlarged in both R1 (E) and R5 (H) tumors compared with A431P (B) tumors. In R5 tumors, these vascular channels seem highly complex, showing abnormally enlarged lumens divided into several compartments (H). Anti-VEGF treatment causes 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.

Possible Mechanisms of Increased VEGF Production in the R Variants. An obvious and important question raised by our results concerns the mechanism responsible for the elevated VEGF levels detected in the R variants. At present, we do not know the answer, but there are a number of possibilities. For example, several different oncogenes, when activated, are known to induce or up-regulate VEGF (39). These include ras, src, and erbB2/neu (39, 50), among others. Likewise certain tumor suppressor genes, such as p53, VHL, or PTEN, when mutated/inactivated, can result in elevated VEGF (39, 51). Thus the variants may express elevated VEGF levels as a result of the selection of cells possessing one or more such genetic changes during the EGFR Ab-mediated therapy. Alternatively, aberrations in signaling pathways downstream of EGFR activation that are known to affect VEGF expression could conceivably be involved. Such changes, for example, could include phosphatidylinositol 3⬘-kinase (PI3 kinase; Refs. 51, 52), and/or src kinase (39, 53, 54) overactivation, and/or ras mutation (30, 39). Preliminary data suggest that neither increased src nor ras activity are obvious in the resistant variants of A431 (data not shown). Whatever the mechanism(s), the magnitude of the increase in VEGF that we detected in the variants could significantly increase

tumor angiogenesis, based on results reported in several previous studies, using gene transfection approaches (55, 56). Our results demonstrate that, in principle, acquired resistance to agents that block tumor cell EGFR function can develop in vivo. However, the extent of this resistance, and the rate at which it develops, appear encouraging for use of such drugs in the clinic, especially because they are frequently used as chemotherapy or radiation sensitizers, rather than single agents. The basis of acquired resistance in the A431 system appears, at least in part, to have a link with tumor angiogenesis, an observation that strengthens the putative linkage established between oncogene function, including that of oncogenic receptor tyrosine kinases, and tumor angiogenesis (39). Finally, we do not wish to infer that acquired resistance to EGFR inhibitors, when it occurs, will always involve the latter type of mechanism. Tumor cell resistance mechanisms with respect to chemotherapeutic drugs and other agents, e.g., ST1571 (17, 57), are usually highly pleiotropic, and the same is likely to be true for inhibitors of EGFR signaling. Indeed, the basis of acquired resistance of one of the six A431 variants that we analyzed, R6, is different from that of variants R1–R5 and may not involve altered angiogenesis. It

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would be of interest, in this respect, to test other EGFR-positive cell lines for the possibility of additional mechanisms of acquired resistance, and to use different antagonists of EGFR function for their selection. ACKNOWLEDGMENTS

22.

23.

We are grateful for the excellent secretarial assistance of Lynda Woodcock and Cassandra Cheng. We thank Joanne Yu and Dr. Guido Bocci for their critical review of the manuscript and YM BioSciences Inc., Toronto, for the R3 and hR3 monoclonal antibodies to EGFR.

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25.

REFERENCES 1. Klohs, W. D., Fry, D. W., and Kraker, A. J. Inhibitors of tyrosine kinase. Curr. Opin. Oncol., 9: 562–568, 1997. 2. Fan, Z., and Mendelsohn, J. Therapeutic application of anti-growth factor receptor antibodies. Curr. Opin. Cell Biol., 10: 67–73, 1998. 3. Holliger, P., and Hoogenboom, H. Antibodies come back from the brink. Nat. Biotechnol., 16: 1015–1016, 1998. 4. Woodburn, J. R. The epidermal growth factor receptor and its inhibition in cancer therapy. Pharmacol. Ther., 82: 241–250, 1999. 5. Heinrich, M. C., Griffith, D. J., Druker, B. J., Wait, C. L., Ott, K. A., and Zigler, A. J. Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood, 96: 925–932, 2000. 6. Kohl, N. E., Omer, C. A., Conner, M. W., Anthony, N. J., Davide, J. P., deSolms, S. J., Giuliani, E. A., Gomez, R. P., Graham, S. L., Hamilton, K., Handt, L. K., Hartman, G. D., Koblan, K. S., Kral, A. M., Miller, P. J., Mosser, S. D., O’Neill, T. J., Rands, E., Schaber, M. D., Gibbs, J. B., and Oliff, A. Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat. Med., 1: 792–797, 1995. 7. Stephenson, J. Researchers describe findings for targeted cancer therapies [news]. JAMA, 284: 293–295, 2000. 8. Pietras, R. J., Pegram, M. D., Finn, R. S., Maneval, D. A., and Slamon, D. J. Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs. Oncogene, 17: 2235–2249, 1998. 9. Pegram, M. D., Lipton, A., Hayes, D. F., Weber, B. L., Baselga, J. M., Tripathy, D., Baly, D., Baughman, S. A., Twaddell, T., Glaspy, J. A., and Slamon, D. J. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized antip185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J. Clin. Oncol., 16: 2659 –2671, 1998. 10. Milas, L., Mason, K., Hunter, N., Petersen, S., Yamakawa, M., Ang, K., Mendelsohn, J., and Fan, Z. In vivo enhancement of tumor radioresponse by C225 antiepidermal growth factor receptor antibody. Clin. Cancer Res., 6: 701–708, 2000. 11. Ciardiello, F., Caputo, R., Bianco, R., Damiano, V., Pomatico, G., De Placido, S., Bianco, A. R., and Tortora, G. Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor. Clin. Cancer Res., 6: 2053–2063, 2000. 12. Huang, S. M., and Harari, P. M. Modulation of radiation response after epidermal growth factor receptor blockade in squamous cell carcinomas: inhibition of damage repair, cell cycle kinetics, and tumor angiogenesis. Clin. Cancer Res., 6: 2166 –2174, 2000. 13. Baselga, J., and Mendelsohn, J. The epidermal growth factor receptor as a target for therapy in breast carcinoma. Breast Cancer Res. Treat, 29: 127–138, 1994. 14. Kerbel, R. S. Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents. BioEssays, 13: 31–36, 1991. 15. Folkman, J., Hahnfeldt, P., and Hlatky, L. Cancer: looking outside the genome. Nat. Rev., 1: 76 –79, 2000. 16. Prendergast, G. C., Davide, J. P., Lebowitz, P. F., Wechsler-Reya, R., and Kohl, N. E. Resistance of a variant ras-transformed cell line to phenotypic reversion by farnesyl transferase inhibitors. Cancer Res., 56: 2626 –2632, 1996. 17. Gambacorti-Passerini, C., Barni, R., le Coutre, P., Zucchetti, M., Cabrita, G., Cleris, L., Rossi, F., Gianazza, E., Brueggen, J., Cozens, R., Pioltelli, P., Pogliani, E., Corneo, G., Formelli, F., and D’Incalci, M. Role of ␣1 acid glycoprotein in the in vivo resistance of human BCR-ABL(⫹) leukemic cells to the abl inhibitor STI571. J. Natl. Cancer Inst. (Bethesda), 92: 1641–1650, 2000. 18. Goldstein, N. I., Prewett, M., Zuklys, K., Rockwell, P., and Mendelsohn, J. Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin. Cancer Res., 1: 1311–1318, 1995. 19. Perrotte, P., Matsumoto, T., Inoue, K., Kuniyasu, H., Eve, B. Y., Hicklin, D. J., Radinsky, R., and Dinney, C. P. Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clin. Cancer Res., 5: 257–265, 1999. 20. Viloria-Petit, A. M., Rak, J., Hung, M.-C., Rockwell, P., Goldstein, N., and Kerbel, R. S. Neutralizing antibodies against EGF and ErbB-2/neu receptor tyrosine kinases down-regulate VEGF production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am. J. Pathol., 151: 1523–1530, 1997. 21. Ciardiello, F., Bianco, R., Damiano, V., Fontanini, G., Caputo, R., Pomatico, G., De Placido, S., Bianco, A. R., Mendelsohn, J., and Tortora, G. Antiangiogenic and

26.

27.

28.

29.

30.

31.

32.

33.

34.

35. 36.

37.

38.

39. 40.

41.

42.

43.

44.

45.

5100

antitumor activity of anti-epidermal growth factor receptor C225 monoclonal antibody in combination with vascular endothelial growth factor antisense oligonucleotide in human GEO colon cancer cells. Clin. Cancer Res., 6: 3739 –3747, 2000. Bruns, C. J., Harbison, M. T., Davis, D. W., Portera, C. A., Tsan, R., Mcconkey, D. J., Evans, D. B., Abbruzzese, J. L., Hicklin, D. J., and Radinsky, R. Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms. Clin. Cancer Res., 6: 1936 –1948, 2000. Jain, R. K., Safabakhsh, N., Sckell, A., Chen, Y., Jiang, P., Benjamin, L., Yuan, F., and Keshet, E. Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: role of vascular endothelial growth factor. Proc. Natl. Acad. Sci. USA, 95: 10820 –10825, 1998. Kawamoto, T., Sato, J. D., Le, A., Polikoff, J., Sato, G. H., and Mendelsohn, J. Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc. Natl. Acad. Sci. USA, 80: 1337–1341, 1983. Fernandez, A., Spitzer, E., Perez, R., Boehmer, F. D., Eckert, K., Zschiesche, W., and Grosse, R. A new monoclonal antibody for detection of EGF-receptors in western blots and paraffin-embedded tissue sections. J. Cell. Biochem., 49: 157–165, 1992. Mateo, C., Moreno, E., Amour, K., Lombardero, J., Harris, W., and Perez, R. Humanization of a mouse monoclonal antibody that blocks the epidermal growth factor receptor: recovery of antagonistic activity. Immunotechnology (Shannon), 3: 71– 81, 1997. Schlaeppi, J. M., Siemeister, G., Weindel, K., Schnell, C., and Wood, J. Characterization of a new potent, in vivo neutralizing monoclonal antibody to human vascular endothelial growth factor. J. Cancer Res. Clin Oncol, 125: 336 –342, 1999. Tran, J., Rak, J., Sheehan, C., Saibil, S. D., LaCasse, E., Korneluk, R. G., and Kerbel, R. S. Marked induction of the IAP family anti-apoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells. Biochem. Biophys. Res. Commun., 264: 781–788, 1999. Bani, M. R., Rak, J., Adachi, D., Wiltshire, R., Trent, J. M., Kerbel, R. S., and Ben-David, Y. Multiple features of advanced melanoma recapitulated in tumorigenic variants of early stage (radial growth phase) human melanoma cell lines: evidence for a dominant phenotype. Cancer Res., 56: 3075–3086, 1996. Rak, J., Mitsuhashi, Y., Sheehan, C., Tamir, A., Viloria-Petit, A. M., Filmus, J., Mansour, S. J., Ahn, N. G., and Kerbel, R. S. Oncogenes and tumor angiogenesis: differential modes of VEGF up-regulation in ras-transformed epithelial cells and fibroblasts. Cancer Res., 60: 490 – 498, 2000. Lydon, N. B., Mett, H., Mueller, M., Becker, M., Cozens, R. M., Stover, D., Daniels, D., Traxler, P., and Buchdunger, E. A potent protein-tyrosine kinase inhibitor which selectively blocks proliferation of epidermal growth factor receptor-expressing tumor cells in vitro and in vivo. Int. J. Cancer, 76: 154 –163, 1998. Yang, X. D., Jia, X. C., Corvalan, J. R., Wang, P., Davis, C. G., and Jakobovits, A. Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy. Cancer Res., 59: 1236 –1243, 1999. King, I. C., and Sartorelli, A. C. The relationship between epidermal growth factor receptors and the terminal differentiation of A431 carcinoma cells. Biochem. Biophys. Res. Commun., 140: 837– 843, 1986. Fan, Z., Shang, B. Y., Lu, Y., Chou, J. L., and Mendelsohn, J. Reciprocal changes in p27(Kip1) and p21(Cip1) in growth inhibition mediated by blockade or overstimulation of epidermal growth factor receptors. Clin. Cancer Res., 3: 1943–1948, 1997. Ross, J. S., and Fletcher, J. A. The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy. Stem Cells, 16: 413– 428, 1998. Olayioye, M. A., Graus-Porta, D., Beerli, R. R., Rohrer, J., Gay, B., and Hynes, N. E. ErbB-1 and ErbB-2 acquire distinct signaling properties dependent upon their dimerization partner. Mol. Cell. Biol., 18: 5042–5051, 1998. Hsieh, S. S., Malerczyk, C., Aigner, A., and Czubayko, F. ERbB-2 expression is rate-limiting for epidermal growth factor-mediated stimulation of ovarian cancer cell proliferation. Int. J. Cancer, 86: 644 – 651, 2000. Millauer, B., Longhi, M. P., Plate, K. H., Shawver, L. K., Risau, W., Ullrich, A., and Strawn, L. M. Dominant-negative inhibition of Flk-1 suppresses the growth of many tumor types in vivo. Cancer Res., 56: 1615–1620, 1996. Kerbel, R. S., Viloria-Petit, A. M., Okada, F., and Rak, J. W. Establishing a link between oncogenes and tumor angiogenesis. Mol. Med., 4: 286 –295, 1998. Pettersson, A., Nagy, J. A., Brown, L. F., Sundberg, C., Morgan, E., Jungles, S., Carter, R., Krieger, J. E., Manseau, E. J., Harvey, V. S., Eckelhoefer, I. A., Feng, D., Dvorak, A. M., Mulligan, R. C., and Dvorak, H. F. Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/ vascular endothelial growth factor. Lab. Investig., 80: 99 –115, 2000. Baselga, J., Pfister, D., Cooper, M. R., Cohen, R., Burtness, B., Bos, M., D’Andrea, G., Seidman, A., Norton, L., Gunnett, K., Falcey, J., Anderson, V., Waksal, H., and Mendelsohn, J. Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin. J. Clin. Oncol., 18: 904 –914, 2000. Shiurba, R. A., Eng, L. F., Vogel, H., Lee, Y. L., Horoupian, D. S., and Urich, H. Epidermal growth factor receptor in meningiomas is expressed predominantly on endothelial cells. Cancer (Phila.), 62: 2139 –2144, 1988. Bohling, T., Hatva, E., Kujala, M., Claesson-Welsh, L., Alitalo, K., and Haltia, M. Expression of growth factors and growth factor receptors in capillary hemangioblastoma. J. Neuropathol. Exp. Neurol., 55: 522–527, 1996. Kim, S. J., Uehara, H., Karashima, T., Luo, L., and Fidler, I. J. Blockade of EGF-R signaling inhibits resorption of bone and growth of human prostate cancer cells implanted into the tibias of nude mice. Proc. Am. Assoc. Cancer Res., in press, 2001. Baker, C. M., McCarty, M. F., Tsan, R., and Fidler, I. J. Phenotypic diversity of organ specific endothelial cells. Proc. Am. Assoc. Cancer Res., 42: 406, 2001.


46. Bruns, C. J., Solorzano, C. C., Harbison, M. T., Ozawa, S., Tsan, R., Fan, D., Abbruzzese, J., Traxler, P., Buchdunger, E., Radinsky, R., and Fidler, I. J. Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma. Cancer Res., 60: 2926 –2935, 2000. 47. Clynes, R. A., Towers, T. L., Presta, L. G., and Ravetch, J. V. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat. Med., 6: 443– 446, 2000. 48. Fan, Z., Masui, H., Altas, I., and Mendelsohn, J. Blockade of epidermal growth factor receptor function by bivalent and monovalent fragments of C225 anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res., 53: 4322– 4328, 1993. 49. Detmar, M., Velasco, P., Richard, L., Claffey, K. P., Streit, M., Riccardi, L., Skobe, M., and Brown, L. F. Expression of vascular endothelial growth factor induces an invasive phenotype in human squamous cell carcinomas. Am. J. Pathol., 156: 159 – 167, 2000. 50. Yen, L., You, X. L., Al Moustafa, A. E., Batist, G., Hynes, N. E., Mader, S., Meloche, S., and Alaoui-Jamali, M. A. Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis. Oncogene, 19: 3460 –3469, 2000. 51. Zhong, H., Chiles, K., Feldser, D., Laughner, E., Hanrahan, C., Georgescu, M. M., Simons, J. W., and Semenza, G. L. Modulation of hypoxia-inducible factor 1␣ expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/ FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res., 60: 1541–1545, 2000. 52. Maity, A., Pore, N., Lee, J., Solomon, D., and O’Rourke, D. M. Epidermal growth factor receptor transcriptionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells via a pathway involving phosphatidylinositol

53.

54.

55.

56.

57.

58.

5101

3⬘-kinase and distinct from that induced by hypoxia. Cancer Res., 60: 5879 –5886, 2000. Karni, R., and Levitzki, A. pp60(cSrc) is a caspase-3 substrate and is essential for the transformed phenotype of A431 cells. Mol. Cell. Biol. Res. Commun., 3: 98 –104, 2000. Sato, K. I., Kimoto, M., Kakumoto, M., Horiuchi, D., Iwasaki, T., Tokmakov, A. A., and Fukami, Y. Adaptor protein shc undergoes translocation and mediates upregulation of the tyrosine kinase c-Src in EGF-stimulated A431 cells. Genes Cells, 5: 749 –764, 2000. Okada, F., Rak, J., St. Croix, B., Lieubeau, B., Kaya, M., Roncari, L., Sasazuki, S., and Kerbel, R. S. Impact of oncogenes on tumor angiogenesis: mutant K-ras upregulation of VEGF/VPF is necessary but not sufficient for tumorigenicity of human colorectal carcinoma cells. Proc. Natl. Acad. Sci. USA, 95: 3609 –3614, 1998. Cheng, S. Y., Huang, H. J., Nagane, M., Ji, X. D., Wang, D., Shih, C. C., Arap, W., Huang, C. M., and Cavenee, W. K. Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor. Proc. Natl. Acad. Sci. USA, 93: 8502– 8507, 1996. Mahon, F. X., Deininger, M. W., Schultheis, B., Chabrol, J., Reiffers, J., Goldman, J. M., and Melo, J. V. Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood, 96: 1070 –1079, 2000. le Coutre, P., Tassi, E., Varella-Garcia, M., Barni, R., Mologni, L., Cabrita, G., Marchesi, E., Stupino, R., and Gambacorti-Passerini, C. Induction of resistance to the Abelson inhibitor ST1571 in human leukemic cells through gene amplification. Blood, 95: 1758 –1766, 2000.