in vivo enhancement of tumor radioresponse by c225 ... · egfr-mediated signaling pathway with...

In Vivo Enhancement of Tumor Radioresponse by C225Antiepidermal Growth Factor Receptor Antibody 1

Luka Milas, 2 Kathy Mason, Nancy Hunter,Sven Petersen, Michitaka Yamakawa, Kian Ang,John Mendelsohn,3 and Zhen FanDepartments of Experimental Radiation Oncology [L. M., K. M.,N. H., S. P., M. Y.], Radiation Oncology [K. A.], and ClinicalInvestigation [J. M., Z. F.], The University of Texas M. D. AndersonCancer Center, Houston, Texas 77030

ABSTRACTOverexpression of epidermal growth factor receptor

(EGFR) has been correlated with tumor resistance to cyto-toxic agents, including radiation (T. Akimoto et al., Clin.Cancer Res.,5: 2884–2890, 1999), and thus is a candidatetarget for anticancer treatment. This study investigatedwhether treatment with C225 anti-EGFR antibody wouldimprove tumor response to radiotherapy. Nude mice bear-ing 8-mm-diameter A431 tumor xenografts in the hind legwere treated with C225 antibody, 18 Gy of single-dose localtumor irradiation, or both. C225 was given i.p. at a dose of1 mg/mouse 6 h before irradiation or 6 h before and 3 and6 days after irradiation. Delay in tumor growth was thetreatment end point. C225 dramatically improved the effi-cacy of local tumor irradiation, particularly when multipleinjections of C225 were administered. Tumor radioresponsewas enhanced by a factor of 1.59 by a single dose and by afactor of 3.62 by 3 doses of C225. Histological analyses oftumors revealed that C225 caused a striking increase incentral tumor necrosis associated with hemorrhage and vas-cular thrombosis when combined with radiotherapy. In ad-dition, C225 induced heavy tumor infiltration with granu-locytes, increased tumor cell terminal differentiation, andinhibited tumor angiogenesis. We conclude that C225 anti-EGFR antibody enhances tumor radioresponse by multiplemechanisms that may involve direct and indirect actions ontumor cell survival.

INTRODUCTIONEGFR4 is a transmembrane protein involved in signaling

pathways essential for cell survival. Overexpression of this receptoroften accompanies development and growth of malignant tumors.There is increasing evidence that high expression of EGFR iscorrelated with aggressive tumor growth, as well as with poorclinical outcome of common cancers in humans, including breast,cervix, lung, and head and neck carcinomas (1–5).

There is also increasing evidence that links EGFR withresistance to chemotherapeutic drugs and radiation. Suggestionsfor a causal relationship are the observations that transfection ofEGFR into human breast cancer cells increased the resistance ofthese cells to cytotoxic drugs (6) and that the blockade of theEGFR-mediated signaling pathway with antibodies to EGFRenhanced the sensitivity of tumor cells to a number of chemo-therapeutic agents (1, 7–9). Recent studies have shown thatanti-EGFR antibodies can be highly effective in the treatment ofhuman tumor xenografts when combined with chemotherapeuticdrugs (1, 7–11).

A number of studies have shown a positive relationshipbetween EGFR expression and cell or tumor resistance to radi-ation (12, 13). Sheridanet al. (13) found that cell culturesderived from head and neck carcinoma patients expressing highlevels of EGFR were more radioresistant than those expressinglow levels of EGFR. Our study demonstrated that the expressionof EGFR varied greatly, by 21-fold, among murine carcinomasof different histology and that the magnitude of the EGFRexpression positively correlated with increased tumor radiore-sistance (14). Addition of EGF to cell cultures protected cellsagainst radiation (15), whereas treatment of cell cultures withantibodies to EGFR yielded the opposite effect,i.e., sensitiza-tion to radiation (16). Huanget al. (17) reported that treatmentof cultured human head and neck carcinoma cells with C225antibody, a mouse-human chimeric anti-EGFR mAb, enhancedthe radioresponse of these cellsin vitro; the enhancement wasattributed to increased radiation-induced apoptosis.

Recently, C225 antibody has undergone extensive explo-ration of its therapeutic potential (1, 11, 18–20).In vitro studiesdemonstrated that the antibody can be either inhibitory for cellgrowth or cytotoxic (1, 20, 21) and can enhance the cytotoxiceffect of chemotherapeutic drugs (1, 8, 9) or ionizing radiation(17). The antibody is also highly effective against tumor xe-nografts, particularly when combined with chemotherapeuticdrugs (8, 9, 11). Because no information is available on theinvivo efficacy of C225 when combined with radiotherapy, thepresent study investigated whether C225 affects the response ofhuman tumor xenografts to local tumor irradiation.

Received 9/20/99; revised 10/26/99; accepted 10/28/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 This work was supported by NIH Research Grants CA-06294, CA-16672, and CA-42060.2 To whom requests for reprints should be addressed, at Department ofExperimental Radiation Oncology 066, The University of Texas M. D.Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,TX 77030-4095. Phone: (713) 792-3263; Fax: (713) 794-5369; E-mail:[email protected] Dr. Mendelsohn is on the Board of Directors of ImClone Systems, Inc.,and also holds stock options.

4 The abbreviations used are: EGFR, epidermal growth factor receptor;EGF, epidermal growth factor; VEGF, vascular endothelial growthfactor; mAb, monoclonal antibody.

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MATERIALS AND METHODSMice and Tumors. Male nude (nu/nu) mice were bred

and maintained in our own specific pathogen-free mouse colonyand housed five in each cage. Animals used in this study weremaintained in facilities approved by the American Associationfor Accreditation of Laboratory Animal Care and in accordancewith current regulations and standards of the United StatesDepartment of Agriculture and Department of Health and Hu-man Services. Solitary tumors were produced by inoculation of106 cells into the muscle of the right hind leg of 3–4-month-oldmice. Tumor cell suspensions were prepared from A431 cellsgrown as monolayersin vitro. Experiments were initiated whenthe tumors had grown to 8 mm in diameter.

C225 Monoclonal Antibody. Human mouse chimericanti-EGFR mAb C225 was derived from murine mAb 225 cloneand was provided by Dr. Harlan Warksal from ImClone Sys-tems, Inc. (New York, NY). C225 fully retains the activity ofmurine mAb 225 in blocking EGF or transforming growth factora for receptor binding and produces a similar spectrum ofantitumor activity on a variety of cultured and xenograftedhuman cell lines (22, 23). The dose of C225 used in the presentexperiments was 1 mg/mouse and was administered i.p. in avolume of 0.45 ml.

Tumor Irradiation. Unanesthetized mice were immobi-lized in a jig, and tumors were centered in a 3-cm diametercircular field. A single 18-Gy dose of gamma radiation waslocally delivered using a dual-source137Cs unit at a dose rate of6.25 Gy/min. The tumor-bearing mice were treated with eitherC225 or local tumor irradiation alone, or with C225 6 h beforeor 6 h before and 3 and 6 days after local tumor irradiation.Untreated tumor-bearing mice served as controls.

Measures of Tumor Response. The effect of radiationalone, C225 alone, and the combination of the two treatments ontumor response was assessed by tumor growth delay. Threeorthogonal tumor diameters were measured using Vernier cali-pers at 2–3-day intervals until the tumors grew to at least 12 mmin mean diameter. Tumor growth was measured for up to 120days. The degree of growth delay was expressed either as theabsolute or normalized growth delay. Absolute growth delaywas defined as the time in days for tumors in the treatment armsto grow from 8 to 12 mm in diameter minus the time in days forthe tumors in the untreated control group to reach the same size.Normalized growth delay was defined as the time for tumors ingroups treated with a combined regimen to grow from 8 to 12mm minus the time to reach the same size in mice treated withC225 alone.

Analysis of Apoptosis and Necrosis. Mice were killedby CO2 inhalation at different times after treatment, and thetumors were immediately excised and placed in neutral bufferedformalin. The tissues were embedded in paraffin blocks, and4-mm sections were cut and stained with H&E. The apoptoticcells were scored on coded slides at3400 magnification. Themorphological features used to identify apoptosis in murinetumors have been previously described, illustrated, and associ-ated with positive terminal deoxynucleotidyl transferase-medi-ated nick end labeling staining (24, 25). Five fields of nonne-crotic areas were selected randomly across each tumor section,and in each field, apoptotic cells were expressed as a percentage

based on the scoring of 1500 nuclei at each time interval aftertreatment. The percentage of cells that were necrotic was deter-mined using a Chalkey point counter with 25 random points(26). At a magnification of3200, the number of points that fellon necrosis were counted in 20 fields distributed evenly acrossthe area of the tumor section. Thus, the percentage of necrosiswas based on scoring 500 points per section as either necrotic ornonnecrotic. The light-microscopic features used to identifynecrosis included increased cell size, indistinct cell border,eosinophilic cytoplasm, loss or condensation of the nucleus, andassociated inflammation.

Tumor Angiogenesis. An intradermal assay (27, 28) wasused to assess the effect of C225 on tumor angiogenesis. Micewere anesthetized with Nembutal (0.06 mg/g body weight), anda triangular skin flap was constructed at the right abdominalregion by making a skin incision along the midline of theabdomen and extending it to the right groin. The skin flap wasseparated from the s.c. tissue by a gentle pull laterally and thenwas examined for the area with the fewest tiny blood vesselsusing a dissecting microscope with a magnification of320.After the number of blood vessels was recorded at the tumor cellinjection site, 106 A431 cells were injected intradermally in avolume of 0.03 ml of PBS using a 30-gauge needle. The skinflap was then brought back to the midline and closed usingsurgical clips. One day after the injection of tumor cells, themice were given C225 at an i.p. dose of 1 mg/mouse. Thenumber of blood vessels as well as tumor size was determinedat 2, 4, 6, 8, and 10 days after tumor cell injection. This wasperformed under a dissecting microscope (320) in anesthetizedanimals in which the skin flap was reopened by removing thesurgical clips and pulling the flap laterally. The tumor volumewas calculated using the formula for elliptical mass (1⁄6 p abc).

RESULTSTo determine the effect of C225 on tumor radioresponse, mice

bearing 8-mm-diameter A431 tumor xenografts were treated withC225, 18 Gy of local tumor irradiation, or both. C225, at a dose of1 mg/mouse, was given i.p. either as a single injection 6 h beforetumor irradiation or in three injections administered 3 days apart,the first one preceding tumor irradiation by 6 h. Tumor growthdelay was the treatment end point. The results presented in Table 1and Fig. 1 show that the combined treatment with C225 and 18 Gydelayed tumor growth more than was the additive effect of indi-vidual treatments. The growth of tumors from 8 to 12 mm indiameter was delayed by 5.56 2.8 days after one dose of C225(P 5 0.17), by 7.46 3.5 days after three doses of C225 (P5 0.07),by 19.36 3.4 days after 18 Gy (P, 0.01), by 36.26 3.3 days afterone dose of C225 plus 18 Gy (P, 0.01), and by 77.36 10.1 daysafter three injections of C225 plus 18 Gy (P , 0.01). In this lattergroup, one of seven tumors was permanently eradicated and wasexcluded from the analysis. Thus, treatment with C225 greatlypotentiated the efficacy of local tumor irradiation, with three dosesof C225 being more effective than one dose. The enhancementfactors, estimates of the additional effect of C225 over and abovethe effect of radiation alone, were 1.59 for a single dose of C225plus 18 Gy and 3.62 for three doses of C225 plus 18 Gy.

In this study, a single dose of 18 Gy was used because itcaused significant tumor growth delay in a previous pilot study.

702 C225 Enhances Tumor Radioresponse



The dose of 1 mg of C225 per mouse was selected based on ourprevious studies on its antitumor efficacy (8, 29). This dosecaused no mouse morbidity. A 6-h interval for C225 adminis-tration before tumor irradiation was used to allow sufficient timefor the antibody to redistribute within the tumor. The half-life ofthe antibody in mice is 72 h (29).

It was recently reported that C225 could enhancein vitroradioresponse of head and neck carcinoma cells; this effect wasattributed to an increase in cell sensitivity to radiation-inducedapoptosis (17). To determine whether this mechanism was in-volved in the observed enhancement of A431 tumor radiore-sponsein vivo, apoptotic indices were determined in untreated

tumors and in tumors treated with a single dose of C225, 18 Gyof local tumor irradiation, or C225 plus radiation, in which theantibody was administered 6 h before irradiation of 8-mm-diameter A431 tumors. Apoptosis and necrosis were quantifiedat 4 h, 1 day, 3 days, and 7 days after local tumor irradiation.The results presented in Table 2 show that the mean backgroundvalue of apoptosis was less than 1% and that neither C225,radiation, nor their combination increased this value signifi-cantly. The apoptotic index in the treated groups in no caseexceeded 2%. These findings suggest that the induction ofapoptosis may not account for the observed tumor radioen-hancement of A431 tumorin vivo.

In contrast to apoptosis, an increase in the extent of necro-sis was a dominant histological feature after tumor treatmentwith C225 plus irradiation (Table 2). In untreated tumors of 8mm in diameter, the percentage of necrosis was 10.46 2.1%.The amount of necrosis in tumors treated with C225 alone washigher within 3 days after the antibody administration, but notsignificantly. It ranged from 13.36 4.6% (P5 0.57) at 1 dayto 26.9 6 10.0% (P5 0.16) at 3 days after the injection ofC225. However, on day 7 after the treatment with C225, thepercentage of necrosis was 55.56 8.3% (P5 0.04). Tumorstreated with radiation only showed a progressive increase in theamount of necrosis within 7 days after irradiation: 21.76 5.0%at 4 h (P 5 0.11), 41.16 3.5% at 1 day (P, 0.01), 64.86 8.3%at 3 days (P5 0.01), and 74.86 5.5% at 7 days (P, 0.01). Thelargest increase in the amount of necrosis resulted from thecombined C225 plus radiation treatment. The percentage ofnecrosis was 59.86 6.9% at 4 h after irradiation (P5 0.03),57.86 2.0% at 1 day (P, 0.01), 80.16 6.2% at 3 days (P,0.01), and 85.96 4.0% at 7 days (P, 0.01) after irradiation.The presence of massive necrosis by 4 h after the combinedtreatment suggests that the treatment severely disrupted tumorvascularity very quickly. Fig. 2 illustrates that at 7 days after thecombined treatment, tumor tissue remained viable only at thetumor periphery.

There were other histological changes in tumors treatedwith C225, particularly in combination with local tumor irradi-

Table 1 Effect of C225 on radioresponse of A431 tumor xenografts in nude mice

Treatmenta

Time in days that tumorsrequired to grow from 8

to 12 mmAbsolute

growth delaybNormalized

growth delaycEnhancement

factorsd

No treatment 6.56 0.4e

C225 (single) 12.06 2.8 5.56 2.8C225 (multiple) 13.96 3.5 7.46 3.518 Gy 25.86 3.4 19.36 3.4C225 single plus 18 Gy 42.76 3.3 36.26 3.3 30.76 3.3 1.59C225 multiple plus 18 Gy 83.86 10.1 77.36 10.1 69.96 10.1 3.62a Mice bearing 8-mm tumors in the right hind leg were given i.p. 1 mg of C225 antibody once or three times, local tumor irradiation, or the

antibody plus local tumor irradiation. Three injections of C225 were given 3 days apart. When the two agents were combined, C225 was given 6 hbefore or 6 h before and 3 and 6 days after local tumor irradiation. Groups consisted of 6–8 mice each.

b Absolute tumor growth delay caused by radiation, C225, or both agents is defined as the time in days tumors required to reach 12 mm fromthe time of treatment initiation minus the time in days untreated tumors required to grow from 8 to 12 mm.

c Normalized tumor growth delay is defined as the time in days for tumors to reach 12 mm in the mice treated with the combination of C225and radiation minus the time in days to reach 12 mm in mice treated with C225 only.

d Enhancement factors were obtained by dividing normalized tumor growth delay in mice treated by C225 plus radiation with the absolute growthdelay in mice treated with radiation only.

e Mean6 SE.

Fig. 1 Effect of C225 antibody on radioresponse of A431 tumor xe-nograft:E, no treatment;M, treated with a single injection of C225;L,treated with three injections of C225;F, 18 Gy of local tumor irradia-tion; f, single dose of C225 plus 18 Gy of local tumor irradiation;l,three doses of C225 plus 18 Gy. For details, see legend of Table 1. Oneof seven tumors in the group treated with three doses of C225 andradiation permanently regressed and was excluded from the regrowthanalysis.z z z z, leg thickness of 5 mm, below which it is not possible tomeasure tumor size accurately.

703Clinical Cancer Research



ation. These include tumor infiltration with granulocytes, hem-orrhage, vascular thrombosis, and increased cell differentiation.The histological appearance of untreated tumors is shown in Fig.3A. In treated tumors, the infiltration with granulocytes, mainlyneutrophils, took place primarily in perivascular areas but waspresent throughout the tumor tissue (Fig. 3B). Some degree ofgranulocyte accumulation was present in untreated tumors aswell but was confined to the stromal capsule surrounding thesexenografts. The occlusion of blood vessels with intravascularthrombi (Fig. 3C) was frequently associated with hemorrhageinto the surrounding tumor areas. Cell differentiation was morefrequent and involved larger areas than in untreated tumors (Fig.3D). These changes increased progressively during the 7-dayobservation period.

Because inhibition of tumor angiogenesis was recentlyfound to increase tumor radioresponse (30, 31) and becauseC225 was suggested to possess antiangiogenic activity (32, 33),we tested whether C225 inhibits the formation of blood vessels

induced by A431 tumor cells. We used an intradermal assay,developed in our laboratory (27), for studying angiogenesis inmice. The mice were injected intradermally with 106 A431 cells,and the number of vessels at the injection site was determined 2,4, 6, 8, and 10 days later. C225, at a dose of 1 mg/mouse, wasgiven i.p. 1 day after tumor cell inoculation. Fig. 4 shows thatC225 significantly reduced the number of newly formed bloodvessels (P5 0.02) and that this reduction was associated withsignificant tumor growth retardation (P 5 0.03). TheseP valuesare for the day 10 point only.

DISCUSSIONAberrant signal transduction pathways, including the path-

way in which EGFR is involved, are being increasingly exploredas promising targets in cancer therapy. One of these approachesconsists of blocking EGFR by such anti-EGFR antibodies asC225 (1, 8, 9, 11). As elaborated on in the Introduction, C225

Table 2 Effect of C225, radiation, or both on apoptosis and necrosis in A431 tumor xenograftsa

Time afterirradiationb

No treatment C225 treatment 18 Gy irradiation C2251 18 Gy irradiation

Apoptosis(%)

Necrosis(%)

Apoptosis(%)

Necrosis(%)

Apoptosis(%)

Necrosis(%)

Apoptosis(%)

Necrosis(%)

0 h 0.76 0.4c 10.46 2.1 0.86 0.2 19.96 6.24 h 0.76 0.2 19.76 8.0 2.26 0.6 21.76 5.0 0.96 0.6 59.86 6.91 day 1.26 0.4 13.36 4.6 1.96 0.1 41.16 3.5 1.06 0.4 57.86 2.03 days 0.96 0.5 26.96 10.0 1.56 0.3 64.86 8.3 1.36 0.4 80.16 6.27 days 0.56 0.1 55.56 8.3 0.26 0.1 74.86 5.5 0.36 0.2 85.96 4.0

a Mice bearing 8-mm tumors in the right hind leg were given i.p. 1 mg of C225 antibody, 18 Gy of local tumor irradiation or the antibody plus18 Gy. When the two agents were combined, C225 was given 6 h before tumor irradiation. Groups consisted of 3–7 mice each.

b The times indicated are in relation to the time of local tumor irradiation. In the case of C225 treatment only, 6 h needs to be added to thesetimes. For example, 0 h analysis for apoptosis or necrosis is actually 6 h after administration of C225.

c Mean6 SE.

Fig. 2 Histological appear-ance of A431 xenografts, un-treated or 7 days after treatmentwith C225 antibody (1 mg i.p.per mouse) plus 18 Gy. Thetreated xenograft shows centralnecrosis; viable tumor tissue re-mained only at the tumor pe-riphery (H&E staining; magni-fication, 312).




Fig. 3 Histological features (H&E staining) of A431 xenografts treated with C225 antibody (1 mg i.p. per mouse). Untreated tumors (A) are denselycellular, and mitotic figures can be seen (long arrow), as well as rare microareas of terminal cell differentiation and necrotic disintegration (shortarrow; magnification,3250).B, tumor infiltration with granuluocytes (magnification,3250).C, intravascular thrombosis (magnification,3400).D,terminal differentiation (magnification,3250).




can be cytotoxic or inhibit proliferation of tumor cells (1, 8, 9,20–22), and it can enhance the cytotoxicity of chemotherapeuticdrugs (1, 8, 9, 11) and of ionizing radiation (17). In this report,we demonstrate that C225 greatly enhanced thein vivo tumorresponse to radiation. The effect was greater than the sum ofgrowth delays caused by the individual treatments.

The combined antibody-radiation treatment consisted of 18Gy of single-dose tumor irradiation and systemic application ofa single dose or multiple (three) doses of C225. Whereas asingle dose of C225 on its own caused some, but not significant,growth delay of a relatively large tumor mass (8 mm in diam-eter), it greatly enhanced tumor radioresponse. The enhance-ment factor was 1.59. Treatment with C225 preceded radiationdelivery by 6 h. However the augmentation of tumor radiore-sponse was particularly dramatic, reaching an enhancementfactor of 3.62, when three doses of C225 were combined withirradiation. One dose of C225 was given 6 h before irradiation,one 3 days after irradiation, and one 6 days after irradiation.

A preliminary assessment of potential mechanisms of in-teraction revealed several findings. We first considered a pos-sibility that C225 increased susceptibility of cells to radiation-induced apoptosis. Induction of apoptosis is commonly regardedas underlying the cytotoxicity of agents that inhibit EGFRtyrosine kinase (34, 35) or block EGFR (16, 36, 37), includingC225 antibodies (21). Also, the ability of anti-EGFR antibodiesto enhancein vitro response of tumor cells to cytotoxic drugs (1,8, 9, 11) or ionizing irradiation (16, 17) was reported to be dueto augmentation of drug- or radiation-induced apoptosis. How-ever, our present findings (Table 2) do not support induction ofapoptosis as an underlying mechanism of antitumor action ofeither C225, irradiation, or the combination of the two in thisparticular tumor. The percentage of apoptosis within 7 days afterall of these treatments was not significantly increased above thebackground level of 1%.

Second, C225 induced a significant increase in the amountof tumor necrosis (Table 2), which progressed within the obser-vation period of 7 days. In some tumors, necrosis was veryextensive, occupying the whole tumor except for a tiny periph-eral rim (illustrated in Fig. 2). Cell death by necrosis in treatedtumors could have resulted from direct damage to tumor cells,indirectly through the damage of tumor vascularization, or morelikely both. The involvement of the vascular effect was sup-ported by histological features that included intravascularthrombosis, tumor hemorrhage, and massive necrosis positionedmore centrally within the tumor.

Similar results were observed using DiFi colon adenocar-cinoma cells (21). Exposure of DiFi cells to C225 antibodyresulted in typical apoptosis in tissue culture: the antibodyactivated the caspase cascade, induced DNA laddering, andcaused cell membrane blebbing (21).5 Also, treatment of nudemice bearing DiFi cell xenografts resulted in complete regres-sion of these xenografts. However, no signs of increased apop-tosis were observed in the xenograft tissue specimens. Instead,the xenografts were found to be necrotic and infiltrated withinflammatory cells.4 It appears that there might be differences ineither induction or detection of C225-induced apoptosis be-tweenin vitro andin vivo settings, and the possibility that C225increases tumor radioresponsein vivoby enhancing induction ofapoptosis followed by necrosis cannot be ruled out. It may bethat massive necrosis induced by radiation masked the apoptoticresponse. Whether these effects are specific to the A431 tumorxenografts is being currently explored by using other tumorsexpressing EGFR.

A third possibility considered was that C225 exerted anti-tumor effects and enhanced tumor radioresponse by interferingwith tumor neovascularization. EGFR ligands, EGF, and trans-forming growth factora were reported to play a role in tumorangiogenesis, directly (38) or indirectly by interaction withVEGF (39). Our present study demonstrated that C225 signifi-cantly inhibited formation of new vessels at the site of A431tumor cell inoculation (Fig. 4). This inhibition of neoangiogen-esis was associated with significant delay in tumor growth. Thisdelay could have resulted either from a direct effect of C225 ontumor angiogenesis or from inhibition of tumor cell prolifera-tion, thus indirectly inhibiting angiogenesis. The direct inhibi-tion effect of C225 is supported by a recent study showing thatthe antibody inhibited mRNA and protein production of theVEGF, interleukin 8, and basic fibroblast growth factor angio-genic factors in cultured bladder cancer cells, as well as inxenografts derived from these cells (33).

Thus, the observed C225 antitumor effect is mediated, atleast in part, by inhibition of tumor angiogenesis. However, it isnot clear whether this inhibition underlies the C225-inducedenhancement of A431 tumor radioresponse. That this mecha-nism was likely involved is suggested by a number of recentreports that treatment with such antiangiogenesis agents asangiostatin and TNP-470 increases tumor radioresponse in pre-clinical tumor models (30, 31, 40–42). The effect was attributedprimarily to the damage of endothelial cells (31, 40–42). Inter-

5 Z. Fan and J. Mendelsohn, unpublished data.

Fig. 4 Effect of C225 antibody on tumor angiogenesis. The mice weregiven intradermal inoculation of 106 A431 tumor cells, and the numberof vessels at the injection site was determined in C225-treated (l) andcontrol (F) mice. Tumor volumes in C225-treated (L) and control (E)mice were also plotted.Bars, SE of the mean values. Treatment withC225 (1 mg i.p.) was given 1 day after tumor cell inoculation.




estingly, VEGF was recently reported to be radioprotective forendothelial cells (30). This protection was abolished by ananti-VEGF antibody, and furthermore, treatment of tumor-bear-ing mice with this antibody enhanced tumor radioresponse (30).Because C225 inhibits VEGF production (32, 33), it is possiblethat it enhanced tumor radioresponse of A431 tumors by amechanism similar to that reported by Gorskiet al. (30).

Another interesting histological feature associated withC225 treatment was tumor infiltration with granulocytes. Theinfiltrating cells were present throughout tumor tissue but wereparticularly pronounced in the perivascular region. Most likely,the granulocytic response was related to clearing dead tumorcells, but a possibility exists that these infiltrating cells wereinvolved in tumor cell kill on their own. This possibility issuggested by a recent observation of Stockmeyeret al. (43) thatgranulocytes primed with the granulocyte colony-stimulatingfactor were cytolytic to a number of breast cancer cell linesexpressing HER-2/neu in the presence of anti-HER-2/neumonoclonal antibodies. It is then also possible that heavyperivascular infiltration with granulocytes could damage vascu-lar walls and indirectly contribute to tumor cell kill and devel-opment of the severe necrosis discussed above.

Treatment with C225 was associated with increased termi-nal cell differentiation. Microregions of differentiation wereseen throughout the tumor and were especially pronouncedwhen the antibody was combined with irradiation. Terminal celldifferentiation was recently reported to occur in cultured tumorcells when exposed to anti-EGFR antibodies (ICR63 or ICR80)or tyrosine kinase inhibitors (37). It is also possible that C225delayed the onset of cell repopulation in irradiated tumors andthus greatly postponed the regrowth of treated tumors. Thisrepopulation-inhibitory effect could account for the dramaticretardation in tumor growth when C225 was administered bothbefore and after radiation treatment.

Overall, our observations showed that C225 antibodygreatly increased tumor response to local tumor irradiation.Multiple administrations of C225 were more effective than asingle dose of C225 given several hours before irradiation. Thestudy also showed that a number of mechanisms were likelyinvolved, including inhibition of tumor angiogenesis, vasculardamage, and tumor infiltration with polymorphonuclear cells.These findings suggest that anti-EGFR antibodies have highpotential to improve the efficacy of radiotherapy.

ACKNOWLEDGMENTSWe thank Dr. Harlan Waksal at Imclone Systems, Inc. for provid-

ing anti-EGF receptor mAb C225 for this study. We are also grateful toLane Watkins and his staff for the supply and care of the mice used inthese studies. We also thank Kuriakose Abraham for preparation ofhistology slides and Patricia Norfleet for assistance in the preparation ofthe manuscript.

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2000;6:701-708. Clin Cancer Res Luka Milas, Kathy Mason, Nancy Hunter, et al. Antiepidermal Growth Factor Receptor Antibody

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