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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Experimental Radiation Oncology [L. M., K. M., N. H., S. P., M. Y.], Radiation Oncology [K. A.], and Clinical Investigation [J. M., Z. F.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is also increasing evidence that links EGFR with resistance to chemotherapeutic drugs and radiation. Suggestions for a causal relationship are the observations that transfection of EGFR into human breast cancer cells increased the resistance of these cells to cytotoxic drugs (6) and that the blockade of the EGFR-mediated signaling pathway with antibodies to EGFR enhanced the sensitivity of tumor cells to a number of chemotherapeutic agents (1 , 7, 8, 9) . Recent studies have shown that anti-EGFR antibodies can be highly effective in the treatment of human tumor xenografts when combined with chemotherapeutic drugs (1 , 7, 8, 9, 10, 11) .
A number of studies have shown a positive relationship between EGFR expression and cell or tumor resistance to radiation (12 , 13) . Sheridan et al. (13) found that cell cultures derived from head and neck carcinoma patients expressing high levels of EGFR were more radioresistant than those expressing low levels of EGFR. Our study demonstrated that the expression of EGFR varied greatly, by 21-fold, among murine carcinomas of different histology and that the magnitude of the EGFR expression positively correlated with increased tumor radioresistance (14) . Addition of EGF to cell cultures protected cells against radiation (15) , whereas treatment of cell cultures with antibodies to EGFR yielded the opposite effect, i.e., sensitization to radiation (16) . Huang et al. (17) reported that treatment of cultured human head and neck carcinoma cells with C225 antibody, a mouse-human chimeric anti-EGFR mAb, enhanced the radioresponse of these cells in vitro; the enhancement was attributed to increased radiation-induced apoptosis.
Recently, C225 antibody has undergone extensive exploration of its therapeutic potential (1 , 11 , 18, 19, 20) . In vitro studies demonstrated that the antibody can be either inhibitory for cell growth or cytotoxic (1 , 20 , 21) and can enhance the cytotoxic effect of chemotherapeutic drugs (1 , 8 , 9) or ionizing radiation (17) . The antibody is also highly effective against tumor xenografts, particularly when combined with chemotherapeutic drugs (8 , 9 , 11) . Because no information is available on the in vivo efficacy of C225 when combined with radiotherapy, the present study investigated whether C225 affects the response of human tumor xenografts to local tumor irradiation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C225 Monoclonal Antibody.
Human mouse chimeric anti-EGFR mAb C225 was derived from murine mAb 225
clone and was provided by Dr. Harlan Warksal from ImClone Systems, Inc.
(New York, NY). C225 fully retains the activity of murine mAb 225 in
blocking EGF or transforming growth factor 
for receptor binding and
produces a similar spectrum of antitumor activity on a variety of
cultured and xenografted human cell lines (22
, 23)
. The
dose of C225 used in the present experiments was 1 mg/mouse and was
administered i.p. in a volume of 0.45 ml.
Tumor Irradiation.
Unanesthetized mice were immobilized in a jig, and tumors were centered
in a 3-cm diameter circular field. A single 18-Gy dose of gamma
radiation was locally delivered using a dual-source
137Cs unit at a dose rate of 6.25 Gy/min. The
tumor-bearing mice were treated with either C225 or local tumor
irradiation alone, or with C225 6 h before or 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 radiation alone, C225 alone, and the combination of the
two treatments on tumor response was assessed by tumor growth delay.
Three orthogonal tumor diameters were measured using Vernier calipers
at 2–3-day intervals until the tumors grew to at least 12 mm in mean
diameter. Tumor growth was measured for up to 120 days. The degree of
growth delay was expressed either as the absolute or normalized growth
delay. Absolute growth delay was defined as the time in days for
tumors in the treatment arms to grow from 8 to 12 mm in diameter minus
the time in days for the tumors in the untreated control group to reach
the same size. Normalized growth delay was defined as the time for
tumors in groups treated with a combined regimen to grow from 8 to 12
mm minus the time to reach the same size in mice treated with C225
alone.
Analysis of Apoptosis and Necrosis.
Mice were killed by CO2 inhalation at different
times after treatment, and the tumors were immediately excised and
placed in neutral buffered formalin. The tissues were embedded in
paraffin blocks, and 4-µm sections were cut and stained with H&E. The
apoptotic cells were scored on coded slides at x400 magnification. The
morphological features used to identify apoptosis in murine tumors have
been previously described, illustrated, and associated with positive
terminal deoxynucleotidyl transferase-mediated nick end labeling
staining (24
, 25)
. Five fields of nonnecrotic 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 after treatment. The percentage of
cells that were necrotic was determined using a Chalkey point counter
with 25 random points (26)
. At a magnification of x200,
the number of points that fell on necrosis were counted in 20 fields
distributed evenly across the area of the tumor section. Thus, the
percentage of necrosis was based on scoring 500 points per section as
either necrotic or nonnecrotic. The light-microscopic features used to
identify necrosis included increased cell size, indistinct cell border,
eosinophilic cytoplasm, loss or condensation of the nucleus, and
associated inflammation.
Tumor Angiogenesis.
An intradermal assay (27
, 28)
was used to assess the
effect of C225 on tumor angiogenesis. Mice were anesthetized with
Nembutal (0.06 mg/g body weight), and a triangular skin flap was
constructed at the right abdominal region by making a skin incision
along the midline of the abdomen and extending it to the right groin.
The skin flap was separated from the s.c. tissue by a gentle pull
laterally and then was examined for the area with the fewest tiny blood
vessels using a dissecting microscope with a magnification of x20.
After the number of blood vessels was recorded at the tumor cell
injection site, 106 A431 cells were injected
intradermally in a volume of 0.03 ml of PBS using a 30-gauge needle.
The skin flap was then brought back to the midline and closed using
surgical clips. One day after the injection of tumor cells, the mice
were given C225 at an i.p. dose of 1 mg/mouse. The number of blood
vessels as well as tumor size was determined at 2, 4, 6, 8, and 10 days
after tumor cell injection. This was performed under a dissecting
microscope (x20) in anesthetized animals in which the skin flap was
reopened by removing the surgical clips and pulling the flap laterally.
The tumor volume was calculated using the formula for elliptical mass
(1/6 
abc).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Fig. 1
show that the combined treatment with
C225 and 18 Gy delayed tumor growth more than was the additive effect
of individual treatments. The growth of tumors from 8 to 12 mm in
diameter was delayed by 5.5 ± 2.8 days after one dose of C225
(P = 0.17), by 7.4 ± 3.5 days after three doses
of C225 (P = 0.07), by 19.3 ± 3.4 days after 18
Gy (P < 0.01), by 36.2 ± 3.3 days after one dose
of C225 plus 18 Gy (P < 0.01), and by 77.3 ±
10.1 days after three injections of C225 plus 18 Gy (P < 0.01). In this latter group, one of seven tumors was permanently
eradicated and was excluded from the analysis. Thus, treatment with
C225 greatly potentiated the efficacy of local tumor irradiation, with
three doses of C225 being more effective than one dose. The enhancement
factors, estimates of the additional effect of C225 over and above the
effect of radiation alone, were 1.59 for a single dose of C225 plus 18
Gy and 3.62 for three doses of C225 plus 18 Gy.
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It was recently reported that C225 could enhance in vitro
radioresponse of head and neck carcinoma cells; this effect was
attributed to an increase in cell sensitivity to radiation-induced
apoptosis (17)
. To determine whether this mechanism was
involved in the observed enhancement of A431 tumor radioresponse
in vivo, apoptotic indices were determined in untreated
tumors and in tumors treated with a single dose of C225, 18 Gy of local
tumor irradiation, or C225 plus radiation, in which the antibody was
administered 6 h before irradiation of 8-mm-diameter A431 tumors.
Apoptosis and necrosis were quantified at 4 h, 1 day, 3 days, and
7 days after local tumor irradiation. The results presented in Table 2
show that the mean background value of
apoptosis was less than 1% and that neither C225, radiation, nor their
combination increased this value significantly. The apoptotic index in
the treated groups in no case exceeded 2%. These findings suggest that
the induction of apoptosis may not account for the observed tumor
radioenhancement of A431 tumor in vivo.
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. In untreated tumors of 8 mm in diameter, the
percentage of necrosis was 10.4 ± 2.1%. The amount of necrosis
in tumors treated with C225 alone was higher within 3 days after the
antibody administration, but not significantly. It ranged from
13.3 ± 4.6% (P = 0.57) at 1 day to
26.9 ± 10.0% (P = 0.16) at 3 days after the
injection of C225. However, on day 7 after the treatment with C225, the
percentage of necrosis was 55.5 ± 8.3% (P =
0.04). Tumors treated with radiation only showed a progressive increase
in the amount of necrosis within 7 days after irradiation: 21.7 ±
5.0% at 4 h (P = 0.11), 41.1 ± 3.5% at 1
day (P < 0.01), 64.8 ± 8.3% at 3 days
(P = 0.01), and 74.8 ± 5.5% at 7 days
(P < 0.01). The largest increase in the amount of
necrosis resulted from the combined C225 plus radiation treatment. The
percentage of necrosis was 59.8 ± 6.9% at 4 h after
irradiation (P = 0.03), 57.8 ± 2.0% at 1 day
(P < 0.01), 80.1 ± 6.2% at 3 days
(P < 0.01), and 85.9 ± 4.0% at 7 days
(P < 0.01) after irradiation. The presence of massive
necrosis by 4 h after the combined treatment suggests that the
treatment severely disrupted tumor vascularity very quickly. Fig. 2
illustrates that at 7 days after the
combined treatment, tumor tissue remained viable only at the tumor
periphery.
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. In treated tumors, the
infiltration with granulocytes, mainly neutrophils, took place
primarily in perivascular areas but was present throughout the tumor
tissue (Fig. 3B)
. Some degree of granulocyte accumulation
was present in untreated tumors as well but was confined to the stromal
capsule surrounding these xenografts. The occlusion of blood vessels
with intravascular thrombi (Fig. 3C)
was frequently
associated with hemorrhage into the surrounding tumor areas. Cell
differentiation was more frequent and involved larger areas than in
untreated tumors (Fig. 3D)
. These changes increased
progressively during the 7-day observation period.
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shows that C225 significantly reduced
the number of newly formed blood vessels (P = 0.02) and
that this reduction was associated with significant tumor growth
retardation (P = 0.03). These P values are
for the day 10 point only.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The combined antibody-radiation treatment consisted of 18 Gy of single-dose tumor irradiation and systemic application of a single dose or multiple (three) doses of C225. Whereas a single dose of C225 on its own caused some, but not significant, growth delay of a relatively large tumor mass (8 mm in diameter), it greatly enhanced tumor radioresponse. The enhancement factor was 1.59. Treatment with C225 preceded radiation delivery by 6 h. However the augmentation of tumor radioresponse was particularly dramatic, reaching an enhancement factor of 3.62, when three doses of C225 were combined with irradiation. 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 interaction
revealed several findings. We first considered a possibility that C225
increased susceptibility of cells to radiation-induced apoptosis.
Induction of apoptosis is commonly regarded as underlying the
cytotoxicity of agents that inhibit EGFR tyrosine kinase (34
, 35)
or block EGFR (16
, 36
, 37)
, including C225
antibodies (21)
. Also, the ability of anti-EGFR antibodies
to enhance in vitro response of tumor cells to cytotoxic
drugs (1
, 8
, 9
, 11)
or ionizing irradiation (16
, 17)
was reported to be due to augmentation of drug- or
radiation-induced apoptosis. However, our present findings (Table 2)
do
not support induction of apoptosis as an underlying mechanism of
antitumor action of either C225, irradiation, or the combination of the
two in this particular tumor. The percentage of apoptosis within 7 days
after all of these treatments was not significantly increased above the
background level of 1%.
Second, C225 induced a significant increase in the amount of tumor
necrosis (Table 2)
, which progressed within the observation period of 7
days. In some tumors, necrosis was very extensive, occupying the whole
tumor except for a tiny peripheral rim (illustrated in Fig. 2
). Cell
death by necrosis in treated tumors could have resulted from direct
damage to tumor cells, indirectly through the damage of tumor
vascularization, or more likely both. The involvement of the vascular
effect was supported by histological features that included
intravascular thrombosis, tumor hemorrhage, and massive necrosis
positioned more centrally within the tumor.
Similar results were observed using DiFi colon adenocarcinoma cells (21) . Exposure of DiFi cells to C225 antibody resulted in typical apoptosis in tissue culture: the antibody activated the caspase cascade, induced DNA laddering, and caused cell membrane blebbing (21) .5 Also, treatment of nude mice bearing DiFi cell xenografts resulted in complete regression of these xenografts. However, no signs of increased apoptosis were observed in the xenograft tissue specimens. Instead, the xenografts were found to be necrotic and infiltrated with inflammatory cells.4 It appears that there might be differences in either induction or detection of C225-induced apoptosis between in vitro and in vivo settings, and the possibility that C225 increases tumor radioresponse in vivo by enhancing induction of apoptosis followed by necrosis cannot be ruled out. It may be that massive necrosis induced by radiation masked the apoptotic response. Whether these effects are specific to the A431 tumor xenografts is being currently explored by using other tumors expressing EGFR.
A third possibility considered was that C225 exerted antitumor effects
and enhanced tumor radioresponse by interfering with tumor
neovascularization. EGFR ligands, EGF, and transforming growth factor
were reported to play a role in tumor angiogenesis, directly
(38)
or indirectly by interaction with VEGF
(39)
. Our present study demonstrated that C225
significantly inhibited formation of new vessels at the site of A431
tumor cell inoculation (Fig. 4)
. This inhibition of neoangiogenesis was
associated with significant delay in tumor growth. This delay could
have resulted either from a direct effect of C225 on tumor angiogenesis
or from inhibition of tumor cell proliferation, thus indirectly
inhibiting angiogenesis. The direct inhibition effect of C225 is
supported by a recent study showing that the antibody inhibited mRNA
and protein production of the VEGF, interleukin 8, and basic fibroblast
growth factor angiogenic factors in cultured bladder cancer cells, as
well as in xenografts derived from these cells (33)
.
Thus, the observed C225 antitumor effect is mediated, at least in part, by inhibition of tumor angiogenesis. However, it is not clear whether this inhibition underlies the C225-induced enhancement of A431 tumor radioresponse. That this mechanism was likely involved is suggested by a number of recent reports that treatment with such antiangiogenesis agents as angiostatin and TNP-470 increases tumor radioresponse in preclinical tumor models (30 , 31 , 40, 41, 42) . The effect was attributed primarily to the damage of endothelial cells (31 , 40, 41, 42) . Interestingly, VEGF was recently reported to be radioprotective for endothelial cells (30) . This protection was abolished by an anti-VEGF antibody, and furthermore, treatment of tumor-bearing mice with this antibody enhanced tumor radioresponse (30) . Because C225 inhibits VEGF production (32 , 33) , it is possible that it enhanced tumor radioresponse of A431 tumors by a mechanism similar to that reported by Gorski et al. (30) .
Another interesting histological feature associated with C225 treatment was tumor infiltration with granulocytes. The infiltrating cells were present throughout tumor tissue but were particularly pronounced in the perivascular region. Most likely, the granulocytic response was related to clearing dead tumor cells, but a possibility exists that these infiltrating cells were involved in tumor cell kill on their own. This possibility is suggested by a recent observation of Stockmeyer et al. (43) that granulocytes primed with the granulocyte colony-stimulating factor were cytolytic to a number of breast cancer cell lines expressing HER-2/neu in the presence of anti-HER-2/neu monoclonal antibodies. It is then also possible that heavy perivascular infiltration with granulocytes could damage vascular walls and indirectly contribute to tumor cell kill and development of the severe necrosis discussed above.
Treatment with C225 was associated with increased terminal cell differentiation. Microregions of differentiation were seen throughout the tumor and were especially pronounced when the antibody was combined with irradiation. Terminal cell differentiation was recently reported to occur in cultured tumor cells when exposed to anti-EGFR antibodies (ICR63 or ICR80) or tyrosine kinase inhibitors (37) . It is also possible that C225 delayed the onset of cell repopulation in irradiated tumors and thus greatly postponed the regrowth of treated tumors. This repopulation-inhibitory effect could account for the dramatic retardation in tumor growth when C225 was administered both before and after radiation treatment.
Overall, our observations showed that C225 antibody greatly increased tumor response to local tumor irradiation. Multiple administrations of C225 were more effective than a single dose of C225 given several hours before irradiation. The study also showed that a number of mechanisms were likely involved, including inhibition of tumor angiogenesis, vascular damage, and tumor infiltration with polymorphonuclear cells. These findings suggest that anti-EGFR antibodies have high potential to improve the efficacy of radiotherapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 of Experimental 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: lmilas{at}mdanderson.org
3 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 growth factor; mAb, monoclonal antibody.
5 Z. Fan and J. Mendelsohn, unpublished data.
Received 9/20/99; revised 10/26/99; accepted 10/28/99.
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, no treatment; 
, treated with a single
injection of C225; 
, treated with three injections of C225; •, 18
Gy of local tumor irradiation; 
, single dose of C225 plus 18 Gy of
local tumor irradiation; 
, three doses of C225 plus 18 Gy. For
details, see legend of Table 1
